Abstract
ORAL ABSTRACTS
O-1: SynBio-Driven Potential for Reviving Natural Products Legend
Synthetic biology combines biology and engineering technology to create new metabolic pathways, cells or organisms for useful purposes by designing and constructing new biological devices and systems. In this respect, synthetic microbiology seems leading the field, as many engineered microorganisms including Escherichia coli, Yeasts, or other microbes has now started to emerge for the successful production of fine chemicals and pharmaceuticals. In this talk, I will introduce some experimental examples of making new fine chemicals and natural products or their structural analogues in our laboratories by using the engineered microorganisms. An engineered E. coli strain has been constructed by combining Synechococcus elnogatus fatty acyl-ACP reductase and E. coli endogenous AdhP and can efficiently produce fatty alcohols—components of surfactants and cosmetic products—directly from glycerol. Furthermore, the high production of fatty acid short-chain esters, has been achieved also by using engineering an E. coli strain through combination of fatty acid and 2-keto acid pathways, which is now regarded as renewable and biodegradable biofuel molecules, some of which with the property of low freezing temperature. The mutant also produced esters with fruit perfume smell. When the same strategy was applied to S. cerevisia in a similar way for the reconstitution of the mevalonate pathway, terpenoid derived biofuel farnesene was also successfully overproduced with a titer up to 1.1 g/L in shake flask. We also tried to engineer the iterative polyketide pathway from the secondary microbial metabolism for targeted pentadecane overproduction in E. coli, successfully and interestingly, a single form of alkane rather than a mixture of the reported fatty acids-derived pathway, was generated. Actinomycetes produce many natural products with bioactive diversities, the structural diversities of which, however, are not sufficient for the discovery of new pharmaceuticals and agrochemicals. By engineering Actinomycetes, producing strains of many clinically used antibiotics, natural products with greater chemo-diversity have been produced in our laboratories. Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics by inhibiting fungal cell wall biosynthesis. An engineered strain of Streptomyces aureochromogenes polyoxin producer constructed by combination of some biosynthetic genes of polyoxin and nikkomycin can produce polyoxin-nikkomycin hybrid compounds, some of which showed higher antifungal activity. The inhibitor of microbial protein biosynthesis, Chlorotetracycline (CTC) is always industrially produced by S. aureofaciens along with tetracycline (TC). An engineered S. aureofaciens with the inactivation of the halogenase CtcP exclusively produced TC, while the engineered S. aureofaciens with overexpression of the halogenase CtcP significantly improved the CTC production. Streptonigrin is a potent anticancer agent produced by S. flocculus and some engineered strains by the inactivation of specific biosynthetic genes can produce several streptonigrin analogues, such as more active and low-toxic 10-demethoxystreptonigrin. These successful events suggested that synthetic microbiology would undoubtly show greater potential for structural modification and production of fine chemicals and natural products.
O-2: Is Induction of Proteotoxic Crisis a Broadly Applicable Approach to Therapy of Cancer?
Genomic alterations may render cancer cells more dependent on mechanisms of proteostasis (including protein folding and degradation) than normal cells. Thus, agents that block these processes may induce a ‘proteotoxic crisis’ in cancer cells at concentrations that are tolerated by normal cells. This hypothesis is supported by the clinical use of the proteasome inhibitors bortezomib/VelcadeTM and carfilzomib/KyprolisTM to treat multiple myeloma and mantle cell lymphoma. However, use of these agents has not expanded beyond these indications, calling into question the general applicability of the proteotoxic crisis concept. Here, I discuss aspects of proteasome inhibitors that may limit their broad utility, and describe alternative strategies for testing whether induction of proteotoxic crisis might be more broadly useful in cancer therapy.
O-3: Debranching Ubiquitin Chains with Ubiquitin C-Terminal Hydrolases
The human genome encodes approximately 100 deubiquitinating enzymes (also known as DUBs). These enzymes regulate a broad swath of cell and organismal biology by removing the small protein ubiquitin (Ub) from target proteins or trimming Ub oligomers. Despite the importance of DUBs, there are fundamental gaps in our knowledge regarding how they work. The family of DUBs known as the Ub C-terminal hydrolases (UCHs) embodies this situation. Biochemical data suggests UCHs catalyze the removal of small C-terminal adducts from Ub, whereas data from cellular studies implicates these enzymes in the disassembly of Ub oligomers. Recently, our laboratory developed a straightforward chemical approach towards the synthesis of a wide array of ubiquitin oligomers. Using these oligomers to probe the function of DUBs, we discovered two members of the UCH family, UCH37 and UCHL3, selectively hydrolyze Ub chains in which a single Ub subunit is modified with two Ub molecules through two lysine residues (herein referred to as branched Ub chains). Considering the importance of UCHL3 and UCH37 in cellular differentiation, development, and motility, our results suggest branched Ub chains play far more important roles in biology than ever appreciated. In this talk, I will discuss our efforts to understand the molecular details of this unique biochemical activity.
O-4: Lenalidomide Promotes CRBN-Mediated Ubiquitination and Degradation of IKZF1 and IKZF3
Lenalidomide (len) is a highly effective drug for treatment of multiple myeloma but the precise molecular mechanism of action still remained unknown. Previous work found that thalidomide, an analogue of len, binds and modulates the activity of the CRBN-DDB1 E3 ligase. Applying multiple quantitative proteomic workflows we found that transcription factors (TFs) IKZF1 and IKZF3 have altered abundance and ubiquitination status upon len treatment in multiple myeloma cells. Protein interaction analyses performed in the presence and absence of len showed that IKZF1 and IKZF3 bind to CRBN and importantly that len stabilized these interactions. In vitro ubiquitination assays demonstrated that IKZF3 is a substrate of the CRBN-DDB1 ligase. A degron region of IKZF3 was identified to be sufficient for mediating len-induced effects on protein stability and a single amino acid substitution in this region conferred resistance to len. Multiple myeloma cells were found to be dependent on IKZF1 and IKZF3 expression. Knockdown of these proteins inhibited cell growth while expression of the len-resistant IKZF3 conferred len resistance of MM1S cells. IKZF1 and IKZF3 levels markedly decreased in primary T cells upon len treatment. Since IKZF3 is a known transcriptional repressor at the IL2 locus we investigated the effect of IKZF3 shRNAs on IL2 production. Len induced IL2 expression in cells expressing a control shRNA, and this induction was blocked by IKZF3 knockdown. These studies reveal that len modulates the CRBN-DDB1 ligase to increase ubiquitination of two TFs that would otherwise be considered “undruggable.” Our findings reveal a novel mechanism of action for a therapeutic agent; alteration of ligase activity leading to selective degradation of specific targets.
O-5: Targeting Protein-Protein Interactions in the Heat Shock Protein 70 (Hsp70) Complex
Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein quality control. Hsp70 helps in virtually every phase of each protein's ‘lifecycle,’ including its folding, transport and turnover. In these tasks, Hsp70 is assisted by co-chaperones, including J proteins and nucleotide exchange factors (NEFs), which bind Hsp70 and shape its activities. For example, some NEFs are thought to link Hsp70 to the proteasomal degradation pathway. To better understand how Hsp70 operates in concert with its co-chaperones, we have developed high throughput screening (HTS) methods to identify drug-like small molecules that interrupt protein-protein interactions (PPIs) between Hsp70 and its co-chaperones. We hypothesized that these PPIs are involved in the key decisions to fold or degrade proteins, thus establishing a critical balance in protein quality control. Indeed, we have found that interrupting specific interactions in the Hsp70 complex can “tune” quality control decisions. Using these molecules, we are exploring ways of rebalancing protein homeostasis in diseases of protein misfolding. Lessons learned from this process will be discussed.
O-6: The Hsp70-40-Nucleotide Exchange Factor Folding Pathway Hastens Transthyretin Folding and Alters the Resulting Structure Rendering it Kinetically More Stable
The cellular protein homeostasis, or proteostasis network, regulates proteome function by controlling ribosomal protein synthesis, chaperone and chaperonin mediated protein folding, protein trafficking, protein degradation and the like. Transthyretin (TTR) is a 55 kDa tetrameric protein that is folded in the endoplasmic reticulum and subsequently secreted into the blood, mainly by the liver. Rate-limiting TTR tetramer dissociation and subsequent monomer misfolding leads to TTR aggregation–which is genetically and pharmacologically strongly linked to a variety of degenerative diseases, including polyneuropathies and cardiomyopathies. A comprehensive biological and biochemical data package will be presented that demonstrates that the composition of the cellular protein homeostasis network strongly effects the stability and structure of the resulting TTR tetramer, relative to unassisted folding in buffer. Evidence will be presented that shows that the reconstituted Hsp70-40-Nucleotide exchange factor folding pathway hastens TTR folding and assembly and importantly alters the packing of aliphatic sides rendering the tetramer more kinetically stable. To the best of our knowledge this is the first demonstration that the Hsp70-40-Nucleotide exchange factor pathway can hasten folding and assembly while also altering the structure of the resulting folded TTR tetramer.
O-7: Design, Development, and Functionalization of Chemical Probes for the Lysine Methyltransferases EZH2 and G9a
In eukaryotic genomes, DNA is wrapped around histone proteins to form repeating units known as nucleosomes, which are further condensed into chromosomes. This high level of structure creates a barrier to transcription, which is maintained or reversed via modifications to the N-terminal tails of histone proteins. Histone lysine methyltransferases catalyze the mono-, di-, and/or trimethylation of lysine residues within histone tails and the methylation state of histone tails has profound effects on transcription. For example, polycomb repressive complex 2 (PRC2) is responsible for regulating the methylation status of histone 3 lysine 27 (H3K27) via the catalytic subunit EZH1 or EZH2, and lysine methyltransferases G9a and GLP catalyze mono- and dimethylation of histone 3 lysine 9 (H3K9). These methyltransferases are of great interest, because trimethylation of H3K27 and dimethylation of H3K9 are transcriptionally repressive marks that play a key role in the progression of many diseases. Chemical probes that selectively inhibit the methyltransferase of interest are valuable tools to drive further understanding of the biological function of these proteins and assess their potential as therapeutic targets. Here we describe the design and development of chemical probes for both EZH2 (UNC1999) and G9a/GLP (UNC0638). These probes were then functionalized into biotinylated tools which allow for chemiprecipitation of EZH2 (UNC2399) and G9a (UNC0965). We detail the in vitro and in vivo use of these tools in the chemical-based chromatin immunoprecipitation (chem-ChIP) assay for studying chromatin occupancy. Additionally, we are developing UNC1999 derivatives that are pan active versus EZH1 and EZH2 as a potential therapeutic option for the treatment of MLL-rearranged leukemias.
O-8: Development of Small Molecule Menin-MLL Inhibitors for Epigenetic Regulation in Cancer
Menin is a protein that directly interacts with the Mixed Lineage Leukemia 1 (MLL1) and MLL2 histone methyltransferases, and is required for their recruitment to the target genes to drive H3K4 methylation. Menin also binds to MLL fusion proteins and this interaction is required for development of acute leukemias with translocations of the MLL gene. Accumulating evidences strongly suggest that menin complexes with MLL1 and MLL2 methyltransferases play a role in development of solid cancers, including liver, colon and breast cancers. Therefore, targeting the protein-protein interaction between menin and MLL fusion proteins or MLL1/MLL2 wild type proteins with small molecules should result in new therapeutic strategies for acute leukemia and possibly solid cancers. By applying the high throughput screening followed by extensive medicinal chemistry combined with structure-based design we developed very potent small molecules that specifically bind to menin with low nanomolar affinities and inhibit the menin-MLL protein-protein interaction in vitro and in human cells. We have also optimized the drug-like properties of these compounds making them suitable for in vivo studies. The menin-MLL inhibitors we developed impair the recruitment of menin, MLL1 and MLL fusion protein complexes to the chromatin of target genes, resulting in epigenetic changes, such as decreased H3K4 trimethylation and H3K79 dimethylation. These effects are associated with a substantial downregulation of HOXA and MEIS1 genes expression, which play a critical oncogenic role in the pathogenesis of the MLL-rearranged leukemias. Gene expression and epigenetic alterations induced by the menin-MLL inhibitors are associated with the phenotypic changes in the MLL leukemia cells, including inhibition of cell proliferation, induced apoptosis and differentiation. More importantly, the menin-MLL inhibitors we developed block development and progression of acute leukemia in vivo in different mice models of MLL leukemia, validating their therapeutic potential. Currently, broader applications of these compounds in various cancer cell lines and in vivo models of solid cancers are being explored. These efforts should result in new potential anti-cancer agents for hematologic and solid cancers as well as new chemical tools to explore the role of menin and MLL in biological processes and human diseases. Our work provides another example of successful targeting of protein-protein interactions with small molecules for therapeutic applications.
O-9: Chemical Probes to Antagonize Readers and Writers Methyl Marks
We are taking a protein family approach to understand the network of human proteins that deposit and recognize specific histone tail sequences and their posttranslational modifications to regulate chromatin structure and gene expression. A variety of biophysical techniques (peptide arrays, ITC, fluorescence polarization, differential scanning fluorimetry and enzymatic assays) together with structural studies are used to characterize substrates and identify the mechanisms of binding selectivity for these proteins. Using structure-guided medicinal chemistry via a network of collaborators in academia and the pharmaceutical industry we are generating potent, selective and cell-active inhibitors of protein methyltransferases and antagonists of protein-histone interactions of reader domains. I will discuss the discovery and applications of such bioactive small molecules to explore the functional role and therapeutic potential of methyl readers and writers.
O-10: Small Molecule Probes to Quantify the Functional Fraction of a Specific Protein in a Cell with Minimal Folding Equilibrium Shifts
Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Historically, the folding of individual proteins in buffers has been studied spectroscopically. However, the majority of these methods (NMR and fluorescence excluded) rely on the structural compaction or change of a protein-of-interest (POI) to report on protein folding. The extent to which they shift folding equilibria and faithfully quantify the folded and functional fraction has not been well established. Herein, we introduce a chemical tool, named protein folding probes, which when used in cell lysates with sufficient holdase activity, faithfully quantify the folded and functional fraction of a POI at a time point of interest in a cell by selectively reacting with that state to afford a fluorescent signal. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone / chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a none-enzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Our work provides a blueprint for how to convert enzyme inhibitors, ligands for nonenzyme proteins, etc. into protein folding probes to efficiently and specifically investigate how a protein's intracellular function is controlled by the proteostasis network as a function of cellular perturbations.
O-11: From Computational Chemogenomics to Target Deconvolution of Phenotypic Screens
One of the main goals of chemogenomics is prediction of the activities of compounds against a comprehensive set of protein targets, rather than against a single protein target of interest. Most of the published computational chemogenomics models (CGM) use a 2D representation of the ligands to model their activity to specific targets. These methods achieve good predictive capabilities if sufficiently diverse activity datasets are available [1]. If the 3D structure of ligands and their protein targets is available, the 2D methods can be complemented by so-called structural computational chemogenomics that use 3D shape- or pharmacophore of the bioactive conformation of the ligands and also protein binding sites [2]. To achieve the broadest possible coverage of targets with optimal models at an industrial scale, we have set up a framework to deploy 2D and 3D CGMs in a standardized benchmark that enabled us to integrate published academic methods, but also commercial and internally developed approaches. Combining the structural and non-structural CGMs we improved prediction quality for a significant amount of protein targets. Finally, we present a novel approach to harness the predictive power of CGMs for the target deconvolution of phenotypic screens.
1. van Westen, Gerard JP, et al. “Proteochemometric modeling as a tool to design selective compounds and for extrapolating to novel targets.” MedChemComm 2.1 (2011): 16–30.
2. Chupakhin V, Marcou G, et al. “Predicting Ligand Binding Modes from Neural Networks Trained on Protein” “Ligand Interaction Fingerprints.” Journal of chemical information and modeling 53.4 (2013): 763–772.
O-12: Activity-Based Proteomics – Applications for Enzyme and Inhibitor Discovery
Genome sequencing projects have revealed that eukaryotic and prokaryotic organisms universally possess a huge number of uncharacterized enzymes. The functional annotation of enzymatic pathways thus represents a grand challenge for researchers in the genome era. To address this problem, we have introduced chemical proteomic and metabolomic technologies that globally profile enzyme activities in complex biological systems. These methods include activity-based protein profiling (ABPP), which utilizes active site-directed chemical probes to determine the functional state of large numbers of enzymes in native proteomes. In this lecture, I will describe the application of ABPP and complementary proteomic methods to discover and functionally annotate enzyme activities in mammalian physiology and disease. I will also present competitive ABPP platforms for developing selective inhibitors for poorly characterized enzymes and discuss ongoing challenges that face researchers interested in assigning protein function using chemoproteomic methods.
O-13: A Chemical Biology Approach to Understanding Protein Quality Control Surveillance Mechanisms in Eukaryotic Cells
Cellular proteins tend to adopt folded conformations in which most hydrophobic amino acids are buried and most hydrophilic amino acids are exposed to the aqueous environment, and proteins typically need to be properly folded to carry out their functions. A variety of cellular stresses can affect protein folding ranging from a global scale (heat shock) to a protein-specific scale (e.g., point mutations and translation errors). These stresses can lead to misfolded or unfolded proteins that have the potential to aggregate and acquire new, sometimes pathological functions. Cells try to deal with aberrantly folded proteins by refolding them using chaperones or degrading them using proteasomes or autophagy. However, we do not understand the basic mechanisms by which cells target cytosolic and nuclear proteins for degradation when the proper folded state cannot be achieved. Our lack of knowledge of the cellular machinery responsible for quality control surveillance is a critical barrier to progress in this field. We have developed a unique collection of engineered proteins called destabilizing domains that allow us to interrogate the cellular protein quality control machinery in eukaryotic cells with an unprecedented level of control. Our destabilizing domains are properly folded and metabolically stable when bound to a cell-permeable small molecule, but they become unfolded and are rapidly degraded by the proteasome when the stabilizing ligand is withdrawn. This technology provides a general method for exerting small molecule control of the stability of any protein of interest. Furthermore, we are using these domains to learn more about how eukaryotic cells recognize and ultimately degrade unfolded proteins.
O-14: Modulating Signaling via Polypharmacology of Kinases and RA
Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to directly target this oncogene have faced difficulties due to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent upon relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. I will discuss the development of small molecules that irreversibly bind to a common oncogenic mutant, K-RasG12C. These compounds rely on the mutant cysteine for binding and therefore do not affect the wild type protein (WT). Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. These data provide structure-based validation of a novel allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
O-15: Labeling the Bacterial Outer Membrane Transporter LptD Using an Antimicrobial Peptide by Chemical Cross-Linking
The increasing resistance of bacteria to conventional antibiotics has resulted in a considerable effort to develop new antimicrobial compounds with new mechanisms of action. A new family of antimicrobial ß-hairpin-shaped peptidomimetics show antimicrobial activity in the nanomolar range specifically against Gram-negative Pseudomonas sp. Interestingly the outer membrane protein LptD, required for LPS transport to the cell surface, has been identified as the target by a forward genetic screen for resistance in Pseudomonas aeruginosa and by photo-affinity labelling experiments with a photo-active peptide analogue (1). The aim of this research is the optimization of the labelling in order to investigate the interaction between LptD and the peptide antibiotic in more detail. Recently, peptides containing 3,4-dihydroxyphenylalanine (DOPA) have been shown after oxidation to covalently cross-link to target proteins. This method is based on the known oxidation chemistry of DOPA-containing proteins common in mollusks (2,3).
In this work, analogues of the antimicrobial ß-hairpin antibiotics were chemically synthesized, with DOPA substituting for a tryptophan residue, and with a biotin tag linked to a glutamic acid side chain for detection of cross-linking to the target LptD. The antimicrobial activity of the DOPA-derivatives were typically about one hundred fold lower than that of the parent antibiotic, and one-tenth of that of the previously used photo-probe as determined by minimum inhibitory concentration assays.
Despite the decreased activity compared to the parent compound, the DOPA-derivative could be successfully cross-linked to LptD. Cross-linking was inhibited by excess antimicrobial peptide, proving the specificity of the labelling. The labelling efficiency was estimated to be higher than cross-linking using the photoaffinity technique. The labelling with the DOPA-derivative was also carried out on P. aeruginosa PAO1 cells. The DOPA-peptide labels LptD in vivo with a reduced selectivity compared to the photo-active peptide. In conclusion, a new labeling probe was successfully developed, which may be a valuable tool for future investigations on the binding site of the antimicrobial peptide in the target protein LptD.
1. N. Srinivas, J. A. Robinson, “Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa”, Science 2010, 327, 1010.
2. Burdine, L., Gillette, T. G., Lin, H. J., and Kodadek, T. “Periodate-triggered cross linking of DOPA containing peptideprotein complexes”. J. Am. Chem. Soc. 2004, 126, 11442–11443.
3. B. Liu, L. Burdine, T. Kodadek, “Chemistry of Periodate-Mediated Cross-Linking of 3,4-Dihydroxylphenylalanine-Containing Molecules to Proteins”, J. A. Chem. Soc. 2006, 128, 15228; Burdine, L., Gillette, T. G., Lin, H. J., and Kodadek, T. (2004) “Periodate-triggered cross linking of DOPA containing peptideprotein complexes”. J. Am. Chem. Soc. 126, 11442–11443.
O-16: Chemical Biology that Controls DNA Structure and Function: DNA Origami and Artificial Genetic Switch
DNA is one of the most promising molecules for the creation of various self-assembled components and scaffolds to prepare complicated patterns, and for selective placement of functional molecules and nanomaterials. The DNA origami method developed for the preparation of fully addressable two-dimensional (2-D) structures has been utilized for the selective positioning of the functional molecules and nanoparticles and for the design of various 3-D architectures. The method is valuable for the preparation of predesigned 2D DNA structures, which leads to the defined assembly of meso-scaled structures. We reported the design of “DNA frame” using the DNA origami method to examine enzymatic action. We newly developed the tension-controlled dsDNA substrates in the DNA frame and showed the importance of DNA strand relaxation in allowing double helix bending during enzymatic reaction.1)
Recently, epigenetic modification has been revealed to play an important role in gene expression, which controls the gene expression through DNA methylation and histone modification. This is closely related to the cell reprogramming and differentiation. We have been undertaking original research on the molecular recognition of DNA by antitumor antibiotics, and the analysis of atom-specific chemical reaction toward DNA with these agents. By reconstituting such knowledge, various functionalized sequence-specific DNA binders were synthesized as an artificial genetic switch. We have successfully developed synthetic genetic switches that could trigger cellular reprogramming by switching “ON” the transcriptional machinery conferring to pluripotency. Unlike other small molecules that can control the fate of stem cells, PIP conjugates can be programmed to bind with predetermined DNA sequences and could perturb the architecture of the packed chromatin. Studies on the specificity landscapes of DNA binding molecules revealed PIPs are relatively superior to the natural DNA binding proteins. Therefore, strategies to expand our tunable SAHA-PIPs could create an epoch-making approach in cellular reprogramming and regenerative medicine to modulate the desired genes.2) Recent progress of DNA origami for single molecular imaging technique and regulation of the epigenetic gene expression using designed molecules, will be discussed.
1) (a) DNA Origami Based Visualization System for Studying Site-Specific Recombination Events Suzuki, Y. et al. J. Am. Chem. Soc. In Press. (b) HIV-1 Nucleocapsid Proteins as Molecular Chaperones for Tetramolecular Antiparallel G-Quadruplex Formation Rajendran, A. et al. J. Am. Chem. Soc. 2013, 135, 18575–18585. (c) State-of-the Art High Speed Atomic Force Microscopy for the Investigation of Single-Molecular Dynamics of Proteins Rajendran, A. et al. Chem. Rev. In Press.
2) (a) A Synthetic Small Molecule Enforces Targeted Transcriptional Activation of Germ Cell Genes in a Human Somatic Cell Han, L. et al. Angew. Chem. Int. Ed. 2013, 52, 13410–13413. (b) Distinct DNA-based Epigenetic Switches Trigger Transcriptional Activation of Silent Genes in Human Dermal Fibroblast Pandian, G. N. et al. Sci. Rep. 2014, 4, 3843.
O-17: Pneumococcal Neuraminidase Substrates Identified Through Chemoenzymatic Labeling
Streptococcus pneumoniae (pneumococcus) is a highly infectious pathogen and the main cause of bacterial meningitis in children and older adults. Additionally, pneumococcus is increasingly resistant to antibiotics, highlighting the need for more effective therapeutics. To facilitate host infection, pneumococcus utilizes three neuraminidases as virulence factors. Neuraminidases cleave sialic acid from glycans on host cell surfaces, a required step in blood-brain barrier binding and transcytosis. However, precise identity of pneumococcal neuraminidase substrates and how neuraminidase activity facilitates infection remain unknown. To answer these questions, I adapted chemoselective glycan labeling reactions to identify both glycan and protein components of substrates desialylated by pneumococcal neuraminidases. To elucidate glycan specificity of pneumococcal neuraminidases, I treated a variety of immobilized sialylated glycans with neuraminidase followed by oxidation and oxime ligation of a fluorophore to remaining sialic acids. Using this technique, I discovered that pneumococcal neuraminidases prefer the sialic acid NeuAc over NeuGc, which is not naturally synthesized in humans. This may be due to pneumococcus being primarily a human pathogen, and may explain its ability to infect the brain, where NeuGc incorporation is excluded. I also found that all three pneumococcal neuraminidases differ in their glycan specificity, suggesting each may play a unique role in infection. I am applying this approach to a microarray of naturally occurring glycans, which should provide further insight into classes of glycans preferred by each neuraminidase. To identify proteins de-sialylated by pneumococcal neuraminidases in meningitis infection, I have adapted the chemoselective labeling technique to the blood-brain barrier model cell line, hCMEC/D3. Upon neuraminidase treatment, exposed galactose residues are oxidized before oxime ligation of a biotin tag. Proteins subsequently pulled down by streptavidin are subjected to mass spectrometry for identification. Using this technique, I have detected enrichment of ∼60 proteins in neuraminidase-treated cells compared to untreated cells. Of particular interest are numerous genes involved in cell adhesion and barrier integrity as well as proteins associated with ß-catenin signaling, which is implicated in pneumococcal lung infections. I am currently assessing how desialylation of neuraminidase targets may facilitate blood-brain barrier crossing. Information gained from these two techniques could reveal potential therapeutic targets for pneumococcal meningitis as well as mechanistic insight into blood-brain barrier passage by pathogens or small molecules.
O-18: Engineering Light-Responsive Proteins
A major goal of modern cell biology involves correlating biochemical action with cell behavior. However, the current arsenal of intracellular probes display properties that cannot be modulated in a manner analogous to signaling pathways, namely in terms of time or space. There is widespread interest in what is now commonly referred to as optogenetics, which has been hailed as “Method of the Year” and “Insight of the Decade”. The ability to control biochemical behavior in an instant using a flash of light has important implications in the study of the biochemical basis of behavior from organelles to organisms. An inkling of the ramifications of optogenetics is most obvious in the field of neuroscience, where light-activated ion channels have been employed to address a wide variety of questions across species boundaries. However, the ready translation of light-responsive ion channels to the proteome in general has not been forthcoming. In short, a recent review described the “protein engineering strategies” in this field unfortunately remaining in the “development stage” since they “suffer from non-negligible background activation at the dark state, which requires meticulous control of expression levels”. Furthermore, proteins commonly perform more than one function and the consequences of their activity are commonly dependent upon their intracellular site of action. Therefore, any light-mediated strategy must not only be able to activate the target protein rapidly and robustly, but must also do so in a fashion that often exceeds the spatial resolution of light. An optogenetic engineering approach that addresses these issues by drawing on the 100 year old Michaelis-Menten equation will be discussed.
O-19: A Chemical Approach to Controlling Cell Fate
Recent advances in stem cell biology may make possible new approaches for treatment of a number of diseases. A better understanding of molecular mechanisms that control stem cell fate/function as well as an improved ability to manipulate them are required. Toward these goals, we have developed and implemented high throughput cell-based screens of arrayed chemical libraries to identify and further characterize small molecules that can control stem cell fate in various systems. This talk will provide latest examples of discovery efforts in my lab that have advanced our ability and understanding toward controlling stem cell fate, including self-renewal, survival, differentiation and reprogramming of cells.
O-20: Targeting Sensors and Molecular Probes: Oxidative Stress and Aging
The lecture will provide our answers to two key questions in chemical biology: How can we find out what is happening inside living cells and whole organisms? How can we control a biological process at a particular place and time within a cell? I will show how the chemistry of synthesized small molecules can unravel some of the mysteries of mitochondrial biology and provide new drug strategies for dealing with mitochondrial dysfunction. Two types of molecule will be considered: sensors that report on events in cells, and probes that can instruct cellular processes. The latter will also exemplify new prodrug strategies. Living cells are not at equilibrium: there are fluctuations in concentrations of ions and other signalling molecules, so that regions within the cell have different concentrations of these. Even the simplest of biomolecules can have different effects depending on its local environment and concentration e.g. reactive oxygen species (ROS) are important in signalling, but they are also responsible for the oxidative stress involved heart attacks, stroke, neurodegeneration, diabetes and cancer. The lecture will concentrate mostly on oxidative stress and the mitochondria, which are the powerhouses of the cell. Mitochondria are vital to the healthy functioning of the cell: they are central to metabolism and signalling, but also play a key role in the process of apoptosis.[1] Small molecules can be physicochemically targeted to the mitochondrial matrix by a lipophilic cation, e.g. the non-toxic alkyltriphenylphosphonium (TPP) group. Mitochondria-targeted molecular probes can be used as exomarkers[2] to elucidate the processes that occur in the mitochondria, e.g. to quantify the reactive oxygen species (ROS) produced in the mitochondria by reduction of oxygen by electrons leaked from the electron transport chain,[3–5] or the results of oxidative damage.[6] The advantage of this chemical biological approach is the small molecule probes can in theory be used in any living system. Therapeutics can also be targeted to the mitochondria using TPP, and a range of strategies will be explained: (i) drugs using a TPP targeting group, (ii) prodrugs activated by something in the mitochondria[7] and (iii) prodrugs activated by external light shining onto the mitochondria.[8]
[1] Smith et al. Trends Pharmacol. Sci. 2012, 33, 341–352.
[2] Logan et al. Biochim. Biophys. Acta 2014, 1840, 923–930.
[3] Cochemé et al. Cell Metabolism 2011, 13, 340–350 and Nature Protocols 2012, 7, 946–958.
[4] Cairns et al. Chem. Eur. J. 2014, 20, in press (doi: 10.1002/chem.201304241)
[5] Chouchani et al. Nature Medicine 2013, 19, 753–759
[6] Pun et al. Free Radic. Biol. Med. 2014, 67, 437–450.
[7] McQuaker et al. ChemBioChem 2013, 14, 901–1013
[8] Chalmers et al. J. Am. Chem. Soc. 2012, 134, 758–761
O-21: Long-Term Activity Imaging in Cells and Mice by Transposon-Mediated Stable-Expression of FRET Biosensors
Genetically-encoded biosensors based on the principle of Foerster resonance energy transfer (FRET) have been widely used to visualize spatio-temporal dynamics of intracellular signaling molecules in living cells. However, due to high homology between the nucleic acid sequences of the donor and acceptor fluorescent proteins, establishment of cell lines and transgenic mice stably-expressing bright FRET biosensors had been a difficult task. We established protocols to express FRET biosensors stably in cell lines and mice by transposon-mediated gene transfer. Either piggyBac or Tol2 transposase could be used satisfactorily. By long-term fluorescent microscopy with cell lines expressing FRET biosensors, we found that activity of Rac1 small GTPase fluctuates with a time scale much longer than cell cycle. Meanwhile, it was also revealed that ERK MAP kinase exhibits stochastic activity pulses with 10 to 20 minutes during replication. The stochastic ERK activity pulses could be laterally propagated in a manner dependent on EGF and EGF receptors. Furthermore, transgenic mice expressing FRET biosensors for ERK and PKA revealed that PKA negatively regulates ERK during neutrophil recruitment from vein to inflamed tissues. These observations proved that the transposon-mediated stable expression of FRET biosensors has opened a new window to observe dynamic activity change of signaling molecules in living tissues.
O-22: Deprotection Chemistry-Mediated Protein Activation in Living Cells
Employing small molecules or chemical reagents to modulate the function of an intracellular protein of interest, particularly in a gain-of-function fashion, remains a challenge. In this talk, I will briefly introduce a “chemical uncaging” strategy that relies on a palladium-mediated deprotection reaction to control protein activation in living cells. We identified biocompatible and efficient palladium catalysts for propargylcarbamate cleavage, which allowed the liberation of a free lysine from its propargyloxycarbonyl-protected lysine analogue that was genetically and site-specifically incorporated into proteins. This “bioorthogonal protection group-catalyst pair” was utilized for caging and subsequent releasing of a crucial lysine residue from a bacterial TypeIII effector protein that modulates MAPK signaling pathway within host cells. Our strategy extended the rapidly emerging palladium-mediated intracellular chemistry from small molecules to proteins, which may be generally applicable for chemically rescuing an essential lysine residue from a given protein, thus manipulating its activity within a native cellular context.
O-23: New Chemical Probes for Imaging Glycolipid Trafficking
Invariant Natural Killer T (iNKT) cells have been described as the ‘˜Swiss army knife of the immune system’ owing to their major role in a wide range of immune surveillance mechanisms and their ability to link the adaptive and innate arms of the immune response; the identification of small molecules that can harness selectively the various roles of these cells has therefore become an area of intense investigation.
iNKT cells are restricted by the non-polymorphic, MHC Class I-like molecule CD1d. This highly conserved antigen-presenting protein is found on the surface of dendritic cells, B cells and other cells of the hematopoietic lineage, and binds and presents glycolipid antigens to T cell receptors (TCRs) located on the surface of iNKT cells. This presentation event acts as the trigger for iNKT cells to secrete a range of regulatory and pro-inflammatory cytokines, the exact composition of which dictates the resulting immune response.
We, [1] and others, [2] have shown that it is possible to bias the cytokine profile resulting from iNKT cell activation in therapeutically useful ways, by changing the structure of the glycolipid that is presented by CD1d molecules. While CD1d and TCR binding affinity likely play a role in the varying biological activities of these different glycolipids, there is increasing evidence that other factors, such as the cellular location of glycolipid loading on the CD1d molecule, also play a critical role.
The trafficking behavior of glycolipid CD1d agonists and the proteins involved in their uptake by professional antigen-presenting cells remain poorly understood. [3] To this end, we sought methods for labeling CD1d glycolipid agonists in order to probe how these mechanisms contribute to the different modes of iNKT cell activation. We will report how analysis of X-ray structures of TCR-glycolipid-CD1d ternary complexes identified possible locations for incorporating labels into a range of glycolipids, and will describe the development of general synthetic strategies that allow the introduction of a selection of chemical probes (fluorophores, biotin labels) into a range of CD1d glycolipid agonists. [4] We will also show how the position of label attachment is crucial for providing a labeled analogue that displays similar in vivo functional activity to the CD1d agonist under investigation. These labeled glycolipid analogues are serving as useful chemical tools for probing the cellular behavior of glycolipid CD1d agonists that may find therapeutic application in treating a diverse range of diseases from cancer and flu through to autoimmune diseases such as type I diabetes and lupus.
1. J. Wojno, J.-P. Jukes, H. Ghadbane, D. Shepherd, G. S. Besra, V. Cerundolo, L. R. Cox, ACS Chem. Biol., 2012, 7, 847–855.
2. A. Banchet-Cadeddu, E. Heńon, M. Dauchez, J.-H. Renault, F. Monneaux, A. Haudrechy, Org. Biomol. Chem., 2012, 9, 3080–3104.
3. M. Salio, H. Ghadbane, O. Dushek, D. Shepherd, J. Cypen, U. Gileadi, M. C. Aichinger, G. Napolitani, X. Qi, P. A. van der Merwe, J. Wojno, N. Veerapen, L. R. Cox, G. S. Besra, W. Yuan, P. Cresswell, V. Cerundolo, Proc. Natl. Acad. Sci. USA, 2013, 110, E4753-E4761.
4. P. J. Jervis, P. Polzella, J, Wojno, J.-P. Jukes, H. Ghadbane, Y. R. Garcia Diaz, G. S. Besra, V. Cerundolo, L. R. Cox, Bioconjugate Chem., 2013, 24, 586–594.
O-24: Small Fluorogen-Activating Protein Tags for Protein Imaging in Living Cells
The most popular method to observe a protein in living cells is to fuse it to GFP. The success of this approach results from the specificity of the genetic encoding. However, the use of GFP can be thwarted by its size, its limited photostability and the slow and O2-dependent fluorophore maturation. To circumvent these issues, new fluorescent reporters were developed. They rely on engineered protein tags binding endogenous (natural) or exogenous (synthetic) fluorogens, which are not fluorescent (i.e. invisible) when free in solution but brighten up upon binding. Fluorescence activation results from rigidifying the fluorogen upon binding, which excludes non-radiative deexcitation. We developed a new family of such fluorogen-activating proteins (FAPs). As chromophore, we sought for fluorogens that could undergo a specific red-shift in absorption upon binding to a designed tag and display fluorescence enhancement. We anticipated that this double discrimination would permit to increase the contrast by enabling specific excitation of the fluorescent complex. By screening donor-acceptor conjugated chromophores bearing an electron-donating phenol conjugated to different electron-withdrawing head groups, we identified an attractive fluorogen hit. We next engineered a FAP scaffold by remodeling the binding site of a small chromophore-binding protein to selectively recognize and activate the fluorogen. Protein engineering combining yeast display and fluorescence-activated cell sorting (FACS) enabled us to generate fluorogen-activating variants. The best FAPs shows high fluorescence quantum yield comparable with cofactor-based fluorescent proteins, enabling to image proteins in eukaryotic cells.
O-25: Development of a Red Fluorescence Probe for Monitoring Dynamics of Cytoplasmic Calcium Ion
The development of sophisticated fluorescence sensor probes has contributed to elucidation of the molecular mechanisms of many complex biological phenomena. In particular, calcium ion (Ca2+) is a pivotal second messenger inside cells, and fluctuation of intracellular Ca2+ works together with various biomolecules in biological systems. So, we expect that simultaneous visualization of Ca2+ and other biomolecules, i.e., multicolor imaging, brings us many biological findings. However, color choices are not sufficient at present, that is, reported long wavelength fluorescence probes for Ca2+ have some disadvantages. For example, AM ester form of Rhod-2, one of the most widely used red fluorescence probe for Ca2+, often localizes into mitochondria and monitors mitochondrial Ca2+ concentration change, although cytoplasmic Ca2+ is much more important for the research of Ca2+ signaling. Thus, we set out to develop a red fluorescence probe for Ca2+ with excellent properties including the cytoplasmic distribution to elucidate cytoplasmic Ca2+-related biological phenomena.
First, we developed a novel fluorescein analogue, TokyoMagenta (TM) (Chem. Commun. 2011, 47, 4162) in which the O atom at the 10 position of the xanthene chromophore of fluorescein is replaced with a Si atom. The absorption and emission wavelengths of TM were about 90 nm longer than those of a fluorescein derivative. We further introduced chlorine into the fluorophore and developed dichloro TokyoMagenta (DCTM). Then, by utilizing DCTM, we developed a red fluorescence probe for Ca2+, CaTM-2 (Angew. Chem. Int. Ed. 2013, 52, 38740), and its activation ratio of the fluorescence intensity reaching 16-fold was practically useful. The chlorination of the fluorophore was also advantageous, and the pKa value of CaTM-2 was greatly shifted to the acidic region compared with that of TM, and was sufficiently low (pKa=5.1) for practical use. For cellular application, we synthesized CaTM-2 AM, an AM ester form of CaTM-2. CaTM-2 AM diffused into cytosol uniformly in living cells, and showed the change in its fluorescence intensity by the histamine stimulus, monitoring the change of the cytoplasmic Ca2+ concentration. As a further demonstration of the usefulness of CaTM-2 AM, we confirmed that it could be applied to rat hippocampal slice cultures for monitoring activities of neurons. Thus, CaTM-2 and CaTM-2 AM would provide an innovative approach for researchers to work on many challenges related to Ca2+.
O-26: A Spontaneously Blinking Fluorophore Based on Intramolecular Spirocyclization for Live-Cell Super-Resolution Imaging
Single-molecule localization microscopy (SLM), including (f)PALM, STORM, and GSDIM, is used to construct super-resolution images by repeated detection and high-precision localization of individual fluorophores. However, to achieve an initial dark state and subsequent blinking of conventional fluorophores for SLM, additives such as thiols, an oxygen scavenging system, and high-intensity laser irradiation prior to measurement are needed, and these requirements are unfavorable for live-cell imaging. From this point of view, fluorophores that spontaneously blink in the absence of any additive and without the need for special conditions are highly desirable. Herein, we report a new class of spontaneously blinking fluorophore, utilizing intramolecular spirocyclization, which is suitable for live-cell SLM without additives or special conditions. We focused on the phenomenon that rhodamine derivatives bearing an intramolecular nucleophile exist in thermal equilibrium between a fluorescent open form and a non-fluorescent spirocyclic form in the ground state. In order to utilize this thermal equilibrium to achieve spontaneous fluorescence blinking suitable for SLM, we optimized two parameters, an equilibrium constant of intramolecular spirocyclization (pKcycl) and a lifetime of the open form (the duration until the open form reverts to the closed form,tau). First, we prepared a series of rhodamine derivatives bearing various intramolecular nucleophiles and/or fluorophores (electrophiles), and evaluated the influence of chemical structure on the equilibrium and kinetics of intramolecular spirocyclization. As a result, increased nucleophilicity of the intramolecular nucleophile and the electrophilicity of the fluorophore stabilized the spirocyclic structure, thereby shifting the pKcycl value to acidic pH. Furthermore, results of laser flash photolysis indicated that the lifetime of open form was greatly influenced by the intramolecular nucleophiles. Among rhodamine candidates, we selected HMSiR, which has the pKcycl value of 5.8 and the life time of 245 ms. HMSiR exists in almost non-fluorescent at pH 7.4 and the fluorescent state lasts for several camera frames to enable detection of sufficient photons for accurate localization. Next, we performed SLM of RecA filaments polymerized on a plasmid DNA in vitro using HMSiR. The super-resolution image of RecA filaments revealed their circular structure with greatly increased spatial resolution compared with the projection image. Then, we performed live-cell SLM of microtubules in HeLa cells with HMSiR. b-Tubulin-SNAP was expressed and labeled with benzylguanine derivative of HMSiR, and SLM was carried out in culture medium after washing. Spontaneous blinking of HMSiR was observed in the absence of any additive in the intracellular environment and microtubule structures were successfully constructed at far higher resolution than the projection image. In conclusion, HMSiR can be used for live-cell SLM without the any additive and high-intensity laser irradiation to convert the fluorophore to the dark state. Thus, SLM with HMSiR has substantial advantages over existing methodology for super-resolution imaging in live cells.
O-27: Chemical Biology of Ligand-Gated Ion Channels for Causal Neuroscience
Ionic flux through ligand-gated ion channels (LGICs) mediates electrical activity in the brain, which underlies animal behavior. To remotely control behavior in mice, we combined chemistry, protein engineering, and neurobiology to systematically create a toolbox of cell type-selective pharmacological probes for manipulating neuron activity and cellular function. This includes LGICs with orthogonal pharmacological selectivity and divergent functional properties which can be used to selectively manipulate neuron activity in mammalian brains in vivo. The diversity of ion channel tools accessible from these approaches has been useful for examining causal relationships between neuronal activity, circuit function, and animal behavior.
O-28: An Unbiased Multidimensional Profiling Method to Study the Biology of Parkinson's Disease
Parkinson's disease (PD) is an ageing-related neurodegenerative disorder that arises from interactions between environmental risk factors and genetic predispositions. This multifactorial aspect of the disease makes it difficult to find a therapy that benefits patients. Here, we developed an unbiased multidimensional profiling method to examine the activity of a set of natural products (NPs) on human olfactory neurosphere-derived (hONS) cells derived from a PD patient. The biological profile of NPs was examined through a series of phenotypic responses to cell components implicated in PD such as mitochondria, lysosomes, endosomes, apoptosis and autophagy. The compounds were clustered based on their biological signature. We used this to select a sub-set of compounds to screen against a panel of hONS cells from different PD patients and controls as a tool for generating a response profile for each patient. By exploring the response of patients to different chemical probes, we can gain a valued knowledge about the biology of PD.
O-29: A Chemical Biology Approach Identified a New Inhibitor of LIM Kinases
The emergence of tumor resistance to conventional drugs restricts their clinical use. Our team uses phenotypic screens and chemical biology strategies to identify new drugs with new mechanism of action. Using a cell-based assay that recognizes microtubule polymerization status to screen for chemicals that interact with regulators of microtubule dynamics, we have identified LIM-Pyr1, a cell permeable inhibitor of LIM Kinases (LIMK1/2). LIMK1/2 regulates actin polymerization through cofilin phosphorylation. Cofilin is an actin-depolymerizing factor and its phosphorylation inactivates its actin severing activity. LIMK1/2 also regulates microtubule dynamics, through a yet not known mechanism. LIM-Pyr1 reversibly stabilized microtubules and blocked actin microfilament dynamics. We have showed that the microtubule stabilizing effect of LIM-Pyr1 was independent of any direct effects on the actin cytoskeleton. Thus, LIM Kinase functions as a signaling node that controls both actin and microtubule dynamics. LIMK1/2 may therefore represent a targetable enzyme for cancer treatment Indeed, it has been shown that LIMK1/2 is overexpressed in many invasive cancers and appears to be a relevant target for anticancer therapy. We have shown that LIM-Pyr1 is toxic on cell lines resistant to conventional chemotherapy. Moreover, in vitro, LIMK1/2 inhibition led to anti-migratory and anti-invasive effects. On different models of mice tumor xenografts, LIM-Pyr1 shows potent antitumor activity, with no detectable undesirable side effects. Thus LIM-Pyr1 and its derivatives could represent a pharmacological alternative to overcome the resistances often observed when tumors are treated with microtubule targeting agents.
O-30: The Discovery and Early Development of the PI3K Inhibitor GDC-0032
PI3K kinase is an important signaling enzyme in all cells. Many cancers have co-opted this pathway to ensure their uncontrolled growth and survival and a large percentage of them have developed activating mutations in PI3K-alpha. We have discovered a structurally unique inhibitor of PI3K (GDC-0032) that spares the beta form of the enzyme and has increased potency against cells harboring activating mutants of PI3K-alpha. We will describe the discovery and early development of this molecule and its analogues.
O-31: A Chemoselective Tool for Lys-Phosphorylation
Phosphorylation of proteins is one of the most important post-translational modifications (PTMs) that occur in proteins controlling different processes in cells. Although phosphorylation on amino acids such as serine, threonine and tyrosine and also recently histidine and arginine are well-established and studied, the extent and biological significance of lysine phosphorylation has remained elusive. In our group, we deal with a possible phosphorylation on Lysine, which is assumed to occur in nature,[1] but often gets missed in phosphoproteomic studies due to the acid lability of the P-N bond. Research in this area is particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) using acid-labile resins due to the intrinsic acid-lability of the P-N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine-(pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. [2] [3] The phosphoramidate esters can be converted into the natural modification via UV irradiation or basic deprotection. We also demonstrated that electron transfer dissociation (ETD) tandem mass spectrometry can be used for unambiguous assignment of phosphorylated lysine-residues within peptides. This new tagging method is expected to be an essential tool to evaluate the biological relevance of lysine phosphorylation.
[1] Besant, P. G.; Attwood, P. V.; Piggott, M. J. Curr Protein Pept Sci. 2009, 10, 536
[2] Serwa, R.; Wilkening, I.; Del Signore, G.; Mühlberg, M.; Claussnitzer, I.; Weise, C.; Gerrits, M.; Hackenberger, C. P. R. Angew. Chem. Int. Ed. 2009, 48, 8234
[3] Bertran-Vicente, J.; Serwa, R.; Schümann, M.; Krause, E.; Hackenberger, C. P. R.; submitted
O-32: Heterocycles as Tuning Forks to Modulate Cell Signaling
G protein-coupled receptors are cell surface proteins that have been the targets for around one third of pharmaceuticals due to the pivotal roles of these proteins in mediating signal transduction in human physiology and disease. Switching GPCRs on (agonists) or off (antagonists) has been the goal of numerous medicinal chemistry programs aimed at controlling physiological function. We have been investigating how to switch on these receptors on immune and cancer cells, while switching off one or more of the intracellular signalling pathways linked to the GPCR and, in turn, linking this fine-tuning control over cell function to the treatment of specific diseases in vivo. Here we use different heterocycles to modulate molecular conformation, fine-tune intracellular signalling profiles, and impart strikingly different pharmacological properties.
O-33: Chemical Tools for Imaging Cellular Fatty Acylation
Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Due to a lack of tools, studying the behavior of Wnts and other post-translationally palmitoylated proteins has proven challenging. Over the past few years, there have been considerable advances in developing biochemical techniques for detecting palmitoylated cellular proteins with good sensitivity. Yet, methods that enable tracking the palmitoylated form of an individual protein in single cells are currently lacking. Such approaches will be invaluable as they provide an extra layer of spatial information about the palmitoylated state of a protein and its intracellular dynamics. Here we describe a strategy which integrates click chemistry, proximity ligation and fluorescence microscopy to visualize the palmitoylated form of proteins with subcellular resolution. We applied the system to image Wnt proteins, thereby revealing new biological insights about Wnt trafficking and its biochemical regulation. The system is also adaptable to a high throughput format, making it of potential use in drug discovery to screen for or validate modulators of Wnt palmitoylation that may interfere with Wnt-driven cancers. Because the method developed here is modular in nature, it can be applied to imaging the palmitoylation of cellular proteins as well as other protein post-translational modifications that are detectable by clickable chemical reporters
O-34: Fragment-Based FTMS Screening of a Unique Natural Product Library
Chemoinformatic analysis of natural products using
• Atom type analysis using radial fingerprint
• Atom function analysis using pharmacophore fingerprint
• Ring scaffold analysis
• Consideration of non-flat molecules
has been used to analysis natural products and design a diversity set of natural products with molecular weight ≤250 Da that can be used in fragment-based screening.
Structural genomics consortia and structure-based drug discovery based on structures obtained by X-ray crystallography and NMR have been very successful. Even greater advances in cloning, expression and purification of proteins has underpinned this success. One outcome has been large numbers of proteins, in purified form, that have defied crystallization or solution structural studies. A technique that would allow identification of ligands binding to these proteins would facilitate lead identification.
We use direct observation of ligand-protein complexes in the ICR cell of a FTMS. We have validated the hits from bioaffinity mass spectrometry by demonstrating cellular and biochemical activity. We have discovered naturally occurring fragments that inhibit Plasmodium gametocytes.1 We are generating a Heat Map with multiple proteins against the fragment library. The lecture will present the diversity analysis and the Hits identified in the Heat Map.
1. Vu, H.; Roullier, C.; Campitelli, M.; Trenholme, K. R.; Gardiner, D. L.; Andrews, K. T.; Skinner-Adams, T.; Crowther, G. J.; Van Voorhis, W. C.; Quinn, R. J. Plasmodium gametocyte inhibition Identified from a natural-product-based fragment library. ACS Chem. Biol. 2013, 8, 2654–2659.
O-35: Anti-Cancer Effects and Mechanisms of Piperlongumine in Brain and Liver Tumors
Piperlongumine (PL), a natural product from long peppers, is recently reported to be a selective anti-cancer drug by targeting to stress response to reactive oxygen species (ROS). Here, we demonstrated that PL was effective in killing glioblastoma multiforme (GBM) and hepatocellular carcinoma (HCC) cells in vitro and in vivo via ROS-mediated mechanisms. The half maximal inhibitory concentration (IC50) of PL was among 10–20 μM. PL elevated ROS, reduced glutathione levels, activated p38 and JNK in GBM and HCC cells. Antioxidants N-acetyl-L-cysteine (NAC) completely reversed PL-induced cell death and p38 or JNK activation. In GBM cells, PL suppressed cell migration via ROS-p38/JNK-NFκB pathway. In HCC cell, PL activated the endoplasmic reticulum stress responses depending on ROS. Our data first suggests that PL may have clinical application for treating the highly malignant and refractory tumors such as GBM and HCC. (Supported by National Nature Science Foundation of China, No. 81070937, 81172397; the Fundamental Research Funds for the Central Universities, HUST, No. 2013ZHYX017; China Postdoctoral Science Foundation, No. 2013M542026).
O-36: Anti-Infective and Anti-Tumor Natural Products Discovery and Biosynthesis from Marine Actinomycetes
Natural products remain the best sources for drugs or drug leads. Marine microorganisms, the underexplored ecological niches, have emerged as an exciting resource for novel bioactive natural products discovery. Recent advances in deciphering and engineering the biosynthetic pathway governing natural product biosynthesis provide an alternative strategy to optimize the structures of complex natural products. Selected examples from our current research will be discussed on i) discovery of anti-infective and cytotoxic natural products as promising drug leads from marine actinomycetes to highlight structural diversity, and ii) genetic engineering and heterologous expression of the biosynthetic pathways of polyketide, polypeptide and alkaloid natural products to make new analogues and characterize novel enzymes capable of catalyzing new chemistry.
1. Ju, J.* et al. Angew. Chem. Int. Ed., 2013, 52, 9980–9984; 2011, 50, 7797–7802.
2. Ju, J.* et al. J. Am. Chem. Soc. 2012, 134, 2844–2847.
3. Ju, J.* et al. Org. Lett. 2013, 15, 3254–3257, 1278–1281; 2011, 13, 2212–2215.
4. Ju, J.* et al. ChemBioChem, 2014, doi: 10.1002/cbic.201400062; 2012, 13, 547–552, 2745–2757.
5. Ju, J.* et al. J. Nat. Prod. 2013, 76, 2263–2268; 2012, 75, 202–208, 1215–1219, 1346–1352, 2251–2255; 2011, 74, 2122–2127.
O-37: Data Reproducibility in the Post Genomics Era: Death of a Noble Technology
The Nobel Prize in Physiology or Medicine (2006) was jointly awarded to Fire and Mello for the discovery of RNA interference (RNAi), a phenomenon believed to bear the potential of revolutionizing the field of functional genomics. Since then, the technology was rapidly adapted for high throughput screening and hailed as the second genomics wave, and in combination with the human genome-sequencing projects, would constitute the holy grail of modern genetics. More than a decade later, the field finds itself surrounded by controversy questioning breakthrough discoveries of gene(s) allegedly identified through this random process e.g. PLK1, BRD4, STK33 and TBK1. This has led us and many others to ask two very simple questions: 1) Can RNAi screening truly identify high-value gene target(s) through a random process? If so, where is the concrete evidence; and 2) What are the scientific merits of pooled RNAi screens? especially shRNA based screens because they simply violate the basic law of averages for each cell to be infected only by one viral particle.
My talk will take you through a fascinating journey of RNAi data reproducibility issues with a special emphasis of what we have termed “ghost” hits - these published hits were allegedly obtained through this random screening process of pooled shRNA libraries but turned out that their targeting sequences/plasmids were totally absent and not represented in the screened libraries. I will leave it to the audience to reflect and decide whether RNAi technology is dying or not; genome editing technologies, though may have similar issues, are by far taking over this noble technology and becoming as popular as ever but the jury is out as to their true specificity and performance in delivering novel gene targets to fight global health problems.
O-38: Reproducible Research - What Should You Look for, and Who Can You Trust?
Science is knowledge gained through repeated observation or experiment. It is communicated through publication of papers in journals. Science can only flourish if journals maintain minimal standards, and papers are read critically. Yet John Ioannidis has used statistics to prove that most claimed research findings are false, and pharmaceutical companies have shown that few research findings can be reproduced. The number of retractions is rapidly increasing, and Ferric Fang's analysis suggests the main reason for retractions is research misconduct. In this talk I will provide some suggestions for things to look at when critically reviewing papers, and discuss the roles of researchers, editors, and institutions in the fostering of responsible, reproducible, research.
O-39: How to Run a Research Laboratory from the Beach
O-40: A European research infrastructure for Chemical Biology – EU-OPENSCREEN
EU-OPENSCREEN is a pan-European research infrastructure initiative that aims at enabling academic chemical biology research to develop novel small molecule research ‘tools' for studies in all areas of the Life Sciences. EU-OPENSCREEN offers to researchers open access to its shared resources, including the latest screening technologies, a unique compound collection composed of commercial and proprietary compounds, and medicinal chemistry support. Chemists are invited to include their compounds into this jointly-used collection and they receive back rich information about the biological activities from the screening against a wide range of bio-assays. EU-OPENSCREEN builds on national networks of chemists and biologists in 16 European partner countries and takes advantage of their expertise and specialized facilities with many years of proven high quality services. This distributed network of partner sites embedded in their excellent research environments will serve as a truly pan-European infrastructure of open-access technology platforms with a broad, collaborative purpose.
EU-OPENSCREEN has no bias towards target families, biological topics or models. The chemical tools developed within EU-OPENSCREEN will foster a wider use of the pharmacological approach to biology and help to enter new fields beyond the parent themes of pharmacology, human and veterinary medicine, and toxicology. EU-OPENSCREEN explicitly advocates wide-ranging projects that will enhance cross-fertilisation between disciplines. By testing systematically the chemical collection of EU-OPENSCREEN in a multitude of highly standardized assays originating from very different biological themes, the screening process will generate enormous valuable information about the structure-activity relations of the substances and thereby steadily enrich our understanding of how and where they act. Collecting these data in a central database will help to visualize the pleiotropic effects of chemicals and to overcome the narrow view of ‘one-compound-one-target', leading to a broader assessment of polypharmacology and toxicology. By comparing small-molecule activities across a broad spectrum of assays, biological fingerprints will be obtained which reflect the true, and often complex, activities of small molecules.
EU-OPENSCREEN is listed on the roadmap for large research infrastructures of ESFRI, the European Strategy Forum on Research Infrastructures established in 2002 by the EU Competitiveness Council and the research ministers of the European member states. Inclusion on the roadmap grants Chemical Biology a high priority along with 12 other research fields in the thematic area of biological and medical sciences, ranging from bioinformatics, structural biology and bio-banking to ecosystems, marine and microbial diversity. Currently in its Preparatory Phase and funded by the European Commission, EU-OPENSCREEN is expected to start full operations in late 2015. It interacts with similar consortia of other continents to advance mutual exchange of compound collections, linkage of databases, agreement on standards, and exchange of best practice.
O-41: Systems Chemical Biology of Drug Action and Drug Transport
The profile of expression of the 23,000 or so genes in the human genome characterizes the state of a cell in an unequivocal way. Yet it is the activity of ensuing proteins, with their respective modifications, that execute the encoded program and achieve meaningful biological function. Even more: it is the integration of the activity of the thousands of molecular machines that result from the cooperation on individual proteins in terms of cellular structures, signaling and the resulting metabolites that best represent the physiological state of a biological system such as a cell. And as soon as we abandon the individual cell level and consider cell-cell interactions in an organ and then organs in organisms and organisms in ecosystems, we need to take the exchange of chemical matter across membranes into account. We have been studying networks of protein complexes and drug action for some time, by a number of approaches: 1) chemical proteomics (affinity purifications with drug matrices / mass spectrometry), 2) chemical genetics (random mutagenesis of genome of near-haploid CML cells), 3) functional proteomics (affinity purification / mass spectrometry), 4) transcriptional profiling, 5) phosphoproteomics, 6) computational network analysis and modeling, and 7) validation by focused gene inactivation (RNAi and genome editing). We have identified: 1) new targets for known drugs, 2) previously unknown mechanisms of drug resistance, 3) “effector” genes for the compounds (genes required for the drug to exert its action), 4) mechanisms of synergy between compounds, 5) mechanisms of drug transport, and in a few cases 6) new medical use of existing drugs. This “systems-level” characterization of chemical entities should help understanding the biology of drug action better, taking chemical exchange over membranes into account and allow the development of improved drugs.
POSTER ABSTRACTS
P-42: Discovery of Antibiotics Targeting Protein Homeostasis
A rise in antibiotic resistance has provided an urgent need for new drug targets. The prokaryotic heat shock protein 70 (Hsp70), DnaK, is a highly conserved molecular chaperone that maintains normal protein homeostasis by limiting protein aggregation and favoring folding. Deletion of dnaK produces strains of Staphylococcus aureus that have diminished survival in host infection models, suggesting that this chaperone might be a new anti-bacterial target. However, few selective inhibitors of Hsp70/DnaK are known, so this hypothesis has been under-explored. Our group has recently developed potent, new Hsp70/DnaK inhibitors based on the rhodacyanine MKT-077. These compounds bind to a conserved allosteric site in Hsp70/DnaK and inhibit its chaperone functions. In this study, we synthesized ∼300 analogues of MKT-077 and tested them for activity against six different strains, including Bacillus anthracis and S. aureus. We found that several MKT-077 derivatives had promising activity in both gram-positive and gram-negative bacteria, with minimal inhibitory concentration (MIC) values ranging from 16 μg/mL to 1 μg/mL. Based on these initial results, MKT-077 analogs appear to be useful chemical probes for studying the role of Hsp70/DnaK in bacterial pathogenesis. Further, these compounds might serve as leads for the development of antibiotics with a new target and a new mechanism-of-action.
P-43: Role of Collaboration in Chemical Biology and Drug Discovery
Collaborative innovation is uniquely able to realize the economics of well-integrated specialization required for chemical biology and drug discovery. Particularly in the neglected infectious disease areas lacking a profit motive, better collaborative tools are fundamentally important to catalyze faster progress. Layering unique collaborative capabilities upon requisite drug discovery database functionality unlocks and amplifies synergy between biologists and chemists. Researchers need to have tools that balance individual needs for robust, intuitive registration and bioactivity analyses while at the same time facilitating collaborations with secure data partitioning, communication, and group engagement. Recent results shared publicly for Tuberculosis, Malaria, and Kinetoplastids, as well as an unusual collaboration among hundreds of undergraduates students developing novel compounds for Neglected Disease applications amply demonstrate these bold suppositions are true and general. Since collaborative technology is “therapeutic area agnostic” it has general been proven equally applicable for commercial applications. Representative case studies include:
• NIH Neuroscience Blueprint consortia with AMRI, CDD, Southern Research Institute, and SRI International to support seven leading academic biology laboratories and the NIH to advance new CNS drugs into the clinic.
• MM4TB EU funded collaboration with 25 partners in 13 countries including two large pharmas working together as if one organization.
• The Bill & Melinda Gates Foundation multi-year CDD TB Database Project: 250 users, 58 labs, 20 collaborations leading to 3 projects partnered with 7 big pharmas.
• UCLA campus-wide and Rockefeller University for secure inter-campus collaborations.
• Acetyton Pharmaceuticals: A Harvard spinout company managing academic-industry and China CRO collaborations advancing a selective HDAC inhibitor into the clinic.
P-44: Identification of the Biosynthetic Gene Cluster of A-102395, a Novel Nucleoside Antibiotic Inhibiting Cell Wall Formation
A-102395 was isolated from the culture broth of Amycolatopsis sp. SANK 60206 in 2007 based on potent inhibitory activity of bacterial translocase I (EC 2.7.8.13) with an IC50 of 11nM. A-102395 shares structural features with capuramcyins, nucleoside antibiotics also known to inhibit translocase I, but contain a unique aromatic polyamide side chain in place of the aminocaprolactam characteristic of the latter. We identified the biosynthetic gene cluster of A-102395, including 35 putative open reading frames responsible for biosynthesis and resistance. A series of gene inactivations abolished the production of A-102395, thus confirming the gene cluster was indeed cloned. Two genes, cpr17 and cpr19 were heterologously expressed in E. coli, and the purified gene products were assigned as an A-102395 phosphotransferase conferring self-resistance and a non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase that initiates the biosynthesis of the modified nucleoside, respectively. Additional in vivo and in vitro studies have allowed us to propose a biosynthetic pathway for the unusual aromatic side chain of A-102395.
P-45: Photoactivated Labeling for Individual Mitochondria
Mitochondria contain much of the metabolic machinery of the cell and are usually the main site for ATP production[1]. They are also communication hubs for redox signaling, for the control of chemical energy production and for apoptosis. The mitochondria within each cell can be heterogeneous and their exact locations appear to be important to the roles they fulfill. This has led our group to explore controlling individual mitochondria through photoactivated probes[2], and correcting only dysfunctional mitochondria through probes that respond to reactive oxygen species (ROS)[3]. Mitochondria can be extremely dynamic undergoing fusion and fission, and ultimately being subjected to mitophagy. This movement appears to be function, cell-type and cell cycle dependent, but to better understand such processes probes are needed to track mitochondria in native cells that have been unaffected by cell culture conditions. We present an approach to doing this.
P-46: Establishing Structure-Activity Relationships for Inhibitors of Human O-GlcNAc Hydrolase
O-linked N-acetylglucosamine (O-GlcNAc), an abundant post-translational modification of intracellular proteins that is found on serine and threonine residues, is regulated by the anabolic O-GlcNAc transferase (OGT) and catabolic O-GlcNAc hydrolase (OGA) enzymes. Recently, it was shown that the inhibition of OGA was a useful tool for manipulating the levels of O-GlcNAc in cells and animals. Additionally, OGA-inhibition was demonstrated to have potential utility as a disease modifying approach for Alzheimer's disease. The mechanism of OGA involves the neighbouring-group participation of the 2-acetamido group of the substrate which displaces the glycosidically-linked leaving group, resulting in the formation of an oxazoline intermediate. The aim of this work was to (i) synthesize a panel of amino-thiazoline-containing OGA inhibitors which mimic the oxazoline intermediate and contain varying chain lengths attached to an exo-cyclic amine (ii) to determine the Ki values for each compound, (iii) to establish the selectivity ratios for OGA over the lysosomal β-Hexosaminidase and iv) to examine whether these inhibitors are transition state mimicks of oxazoline formation using linear free energy relationships. Following synthesis, we clarified the structure-activity relationships for this series of OGA inhibitors, identifying several picomolar and nanomolar inhibitors which are highly selective for human OGA over human β-Hexosaminidase. Recent preliminary data suggests that these inhibitors mimic the oxazoline formation transition state which could partially account for their potencies. This information could be utilized to potentially further the investigation of the progression of neurodegenerative diseases by examination of elevated O-GlcNAc levels in cells and animals.
P-47: Design, Synthesis, and Evaluation of Novel LDHA Inhibitors
Human lactate dehydrogenase A (LDHA) is involved in the conversion of glucose to lactate under anaerobic conditions. Knockdown of LDHA expression in cell lines decreases cell proliferation under hypoxic conditions, making LDHA a potential anti-tumor target. From a HTS screen, a series of 3-hydroxy-2-mercaptocyclohex-2-enones LDHA inhibitors was identified. Subsequently, versatile synthesis was developed to enable generation of a large variety analogs for further SAR studies and led to the identification of inhibitors with sub-micromolar IC50s potency against the enzyme. Crystal structures of inhibitors with LDHA were obtained and provide valuable structural information for further optimization.
P-48: Engineering the Substrate Specificity of ADP-Ribosyltransferases for Identifying Direct Protein Targets
ADP-ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans makes the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. In this presentation I will describe the rational design of orthogonal NAD analog-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogs containing substituents at the C-5 position, we synthesized the orthogonal NAD variant 5-Et-6-a-NAD. We found that 5-Et-6-a-NAD is used as a substrate for several engineered ARTDs (ARTD1, 2, and 6), but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This results in a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.
P-49: HTS of IDH1 R132H Leads to Identification of a Potent Inhibitor of IDH1 R132H Capable of Reducing 2-hydroxyglutarate Production in U87-MG Glioblastoma Cells
Mutations (R132H or R132C) in the active site of isocitrate dehydrogenase (IDH1) have been associated with a number of cancers, including acute myeloid leukemia and glioblastoma. These mutations result in loss of activity for metabolism of isocitrate, but confer gain-of-function for the production of the oncometabolite 2-hydroxyglutarate (2-HG). Herein we describe the quantitative high-throughput screening of IDH1 R132H. A diverse library of 387,602 compounds was screened as six-point dilution series, yielding several validated inhibitory series. Results from the screen will be presented, along with the characterization of a phenylglycine probe. A member of the series, ML309, is a potent and selective inhibitor of mutant IDH1 and effectively lowers cell-based production of 2-HG in a U87-MG IDH1 R132H glioblastoma cell line. ML309 is competitive with respect to α-KG and uncompetitive with respect to NADPH, which had also been reported for another member of the phenylglycine series.
P-50: Simultaneous Imaging of Protonated and Deprotonated Carbonylcyanide p-TrifluoromethoxyPhenylhydrazone (FCCP) in Live Cells by Raman Microscopy
We previously reported alkyne-tag Raman imaging (ATRI) as a promising approach for visualizing small molecules in live cells. Small and bioorthogonal alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous molecules. Therefore alkyne-tagged molecules are selectively detected by Raman microscopy without bulky fluorescent tags which tend to decrease the biological activity of the parent compound. To expand the utility of this methodology, we examined the structure-Raman shift/intensity relationships of a series of alkynes and other functional groups. Based on these structure activity relationships, we succeeded in multicolor Raman imaging of small molecules. In addition to alkyne, nitrile and deuterium were found to be suitable as a Raman tag for small molecules. In this study, we report the Raman imaging of carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) molecules in live cells by using nitrile as a Raman tag. FCCP is a typical mitochondrial uncoupler, which is thought to exist as an equilibrium mixture of protonated and deprotonated forms in cells. Nitriles of FCCP were found to show different Raman peaks between protonated and deprotonated forms. Based on these Raman peaks, we tried to discriminate between the protonated and deprotonated forms of FCCP in live cells. As a result, we succeeded in the simultaneous Raman imaging of protonated and deprotonated forms of FCCP, which showed different cellular localization patterns. These results indicate the potential of Raman microscopy for structure-based imaging of bioactive small molecules in live cells.
1. Yamakoshi, H.; Dodo, K.; Okada, M.; Ando, J.; Palonpon, A.; Fujita, K.; Kawata, S.; Sodeoka, M. J. Am. Chem. Soc. 2011, 133, 6102.
2. Yamakoshi, H.; Dodo, K.; Palonpon, A.; Ando, J.; Fujita, K.; Kawata, S.; Sodeoka, M. J. Am. Chem. Soc. 2012, 134, 20681.
3. Yamakoshi, H.; Palonpon, A.; Dodo, K.; Ando, J.; Kawata, S.; Fujita, K.; Sodeoka, M. Chem. Comm. 2014, 50, 1341.
P-51: Novel Approach of Dual Targeting of the Catalytic and Allosteric Sites of the Ras GEF, SOS1
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Ras GTPases regulate multiple intracellular signaling processes involved in gene expression, cell proliferation, and cell survival. SOS1 is a major RasGEF that transduces receptor tyrosine kinase signals to Ras and associated ERK/Akt pathways. SOS1 gain-of-function mutations have been found in Noonan syndrome patients. In addition, SOS1 has been shown to be differentially expressed in prostate and breast cancer cells, and is involved in malignancies dependent upon aberrant Ras signaling. Previous biochemical and structural studies have elucidated that SOS1 catalysis of Ras activation involves two compounding mechanisms: a direct GEF catalysis of Ras GTP/GDP exchange through the Cdc25 catalytic domain, and an allosteric regulation by active Ras, Ras-GTP, at a distal site of the REM-Cdc25 domains. In the current studies, we undertook structure-based virtual screening coupled with experimental screening of a 118,500 compound subset of the NCI/DTP Open Chemical Repository to identify lead chemical inhibitors targeting the Ras catalytic site and distal Ras allosteric site of SOS1. As a result of the virtual screen, 36 and 37 compounds were selected from the catalytic site and allosteric site candidates for experimental screening by a guanine nucleotide exchange assay, i.e. FL-GDP dissociation of Ras under catalysis by SOS1. Two compounds targeting the catalytic site (NSC-674954 and NSC-658497) and allosteric site (NSC-45186 and NSC-70220) were identified as true hits which displayed micromolar efficacy in inhibiting normal or allosteric SOS1 catalysis of FL-GDP dissociation from Ras. Additionally, NSC-658497 displayed a micromolar target binding affinity, along with dose-dependently disrupting the SOS1 interaction with Ras as assayed by microscale thermophoresis. Mutagenesis studies mapped the NSC-658497 action site specifically to residues I825, T828, T829, and Y912 in the SOS1 Cdc25 catalytic domain. In fibroblast cells, NSC-658497 inhibited EGF-stimulated Ras and ERK/AKT activities. Furthermore, NSC-658497 inhibited fibroblast and DU-145 prostate cancer cell proliferation along with corresponding downstream Ras-dependent ERK/AKT activities. Thus, we have established a new approach of dual targeting of an allosteric site and the catalytic site of the Ras activating SOS1 enzyme, and identified SOS1 inhibitors of several chemical spaces that are useful tools and therapeutic leads for Rasopathies.
P-52: High-Throughput Screening (HTS) Approaches for Protein-Protein Interaction (PPI) Inhibitors: Identifying and Confirming Inhibitors of the NRF2:Keap1 complex
Although PPIs underlie numerous cellular processes from extracellular signaling to regulation of gene transcription, these interactions are difficult to target with small molecules. In addition, screening for PPIs with small molecule collections can be fraught with misleading ‘œhits’ that result from aggregation of compound and/or protein or from assay specific artifacts. Here we describe a multipronged approach to identify and characterize on-target hits from PPI inhibitor screens. We present a case study focused on Nrf2:Keap1, a transcriptional regulator binding to a member of a protein degradation complex involved in a number of therapeutically relevant processes, including cancer, neurodegeneration and inflammation. We used orthogonal high-throughput approaches (HTRF and Alphalisa) for identifying initial hits, and we incorporated more rigorous methods to characterize specific PPI inhibitiors in Biacore (GE) surface plasmon resonance and EPIC (Corning) biosensor assays. We also used the EPIC to identify false positives due to aggregation. Lastly, we employed the PathHunter (DiscoveRx) technology to detect disruption of the Nrf2:Keap1 complex in a cellular context. Collectively, these approaches provide potential chemical leads with true PPI inhibition for further development.
P-53: Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity Against Nav1.5
The NaV1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding NaV1.7 resulting in either loss- (congenital analgesia) or gain- (paroxysmal extreme pain disorder) of-function pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of NaV1.7. A full-deck high-throughput screen was developed for both NaV1.7 and cardiac NaV1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve (AUC) which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in NaV1.7 relative to 1.5, only the AUC measurement and a subsequent refinement of this measurement were able to extract compounds with novel Nav1.7 pharmacological selectivity over Nav1.5 as confirmed in electrophysiology.
P-54: Selective Production of Superoxide within the Mitochondrial Matrix by a Mitochondria-Targeted Redox Cycler
The human body uses oxygen in the process of respiration. As a result, reactive oxygen species (ROS) such as superoxide (O2•−) are produced as side products. Superoxide is hypothesized to play a unique role in the development of pathology associated with oxidative stress, and in the propagation of redox signals arising from the mitochondria. There are robust tools to detect or neutralize mitochondria-derived superoxide but there are no exogenous agents that could rapidly and specifically increase superoxide production within the matrix. We present the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Due to its lipophilic nature MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix MitoPQ acts as a redox cycler at the flavin site of complex I leading to a dramatic increase in superoxide production. The ability of MitoPQ to achieve rapid and specific increases in matrix superoxide will be of use in the further exploration of mitochondrial ROS in cells and in vivo.
P-55: Targeting the Switch II Pocket of K-Ras
The small GTPase K-Ras is the most frequently mutated oncogene in cancer. Despite the prevalence of K-Ras mutations, its high nucleotide affinity and lack of druggable pockets have made direct inhibitors unavailable until recently. Our lab has identified covalent inhibitors of K-Ras G12C that bind a novel inhibitory pocket, which we have termed the switch-II pocket (S-IIP). This pocket can be exploited to allosterically alter nucleotide affinity towards the inactive GDP-bound state and interfere with effector interactions. These inhibitors are specific for the GDP state, have relatively low binding affinity, and rely on covalent attachment to cysteine-12. These limitations are problematic since a majority of Ras-driven cancers express other non-cysteine mutations at positions 12, 13, or 61, which are predominately in the active, GTP-state. Using the wealth of crystallographic data obtained in our original investigation, we have developed new targeted screening methods designed to select for compounds that are free of these limitations. Particularly, the introduction of unnatural cysteine residues near the S-IIP for tethering and covalent docking have yielded new and exciting scaffolds. These new leads will provide structural insight into the development of reversible S-IIP inhibitors that are independent of nucleotide state and do not rely on a cysteine for covalent attachment.
P-56: Peptides with Constrained Irregular Secondary Structure as Inhibitors of Protein-Protein Interactions
Bioactive conformations of peptides can be stabilized by macrocyclization resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein-protein interactions (PPI). Most peptide-derived PPI inhibitors involve stabilized α-helices[1,2], leaving a large number of secondary structures unaddressed. The virulence factor exoenzyme S (ExoS) secreted by Pseudomonas aeruginosa binds the human protein 14-3-3 in an irregular and mostly extended conformation. The stabilization of the irregular peptide structure using hydrophobic cross-links, that replace residues crucially involved in target binding was envisioned. A small rational library of macrocyclic peptides was synthesized, resulting in a cross-linked peptide capable to inhibit the interaction between 14-3-3 and ExoS.[3] The molecular basis of this interaction was elucidated by fluorescence polarization, X-ray crystallography and isothermal titration calorimetry.
[1] V. Azzarito, K. Long, N. S. Murphy, A. J. Wilson, Nat. Chem. 2013, 5, 161–173.
[2] Y.W. Kim, T. N. Grossmann, G. L. Verdine, Nat. Protoc. 2011, 6, 761–771.
[3] A. Glas, D. Bier, G. Hahne, C. Rademacher, C. Ottmann, T. N. Grossmann, Angew. Chem. Int. Ed.. 2014, 53, 2489–2493.
P-57: Multicolor Imaging of N-glycan Processing Enzymes in Cultured Cells
The majority of secretory proteins are co-translationally labeled with asparagine-linked oligosaccharides (N-glycans). N-glycans and N-glycan processing enzymes are considered to play important roles in protein quality control during protein synthesis and folding. These functions are essential for cellular health. However, despite their importance in cellular health, imaging technologies to measure the biological activity of N-glycan processing enzymes at the cellular level have yet to be fully established. Our group has previously described specific, multicolor fluorescent probes for the detection of the activity of an ER-localized carboxylesterase using the quinone methide cleavage process (W. Hakamata et al, ACS Med. Chem. Lett. 2014, 5, 321–325). This esterase is well known as a prodrug-activating enzyme. Multicolor imaging of N-glycan processing enzymes at the cellular level is clearly of interest and is feasible based on our previous research. The following N-glycan processing enzymes were targeted in our first trail : glucosidase II in the endoplasmic reticulum, mannosidase I in the golgi body, and fucosidase in the lysosome. To develop probes for multicolor imaging of these N-glycan processing enzymes in cultured cells, resorufin, 4-trifluoromethylumbelliferone, and 2-methyl TokyoGreen were chosen as the red, blue, and green fluorophores, respectively. These fluorophores each have distinct excitation and emission wavelengths, excellent fluorescence quantum yields, and low pH-dependent change in fluorescence. Here we describe the design, synthesis, and photochemical and biological properties of activity-based fluorescence probes for glucosidase II, mannosidase, and fucosidase activity. These probes allowed rapid and clear visualization of activity with high specificity for organelles, based on the localization of parent enzymes in three cell lines: HT-1080 human fibrosarcoma cell line, HeLa human cervical cancer cell line, and SK-N-SH human neuroblastoma cell line.
P-58: The Discovery of Vacquinol-1: Phenotypic Identification of Acquired Vulnerabilities in Patient-Derived Tumor-Initiating Glioblastoma Cells
Glioblastoma multiforme (GBM) is the most aggressive and lethal of all tumors to affect the CNS, resulting in marginal life expectancy in patients. Current treatment options are limited and tumor recurrence frequent. Due to the diverse genetic landscape of gliomas, the success of targeted therapies focusing on specific tumor-driving mutations has been disappointing. Thus, novel methods are required for successfully identifying new therapies. Based on a hypothesis that mutations in GBM lead to acquired cellular functions not only involved in the tumorigenic process, we performed an unbiased phenotypic screen of a small molecule library on patient-derived, tumor-initiating GBM cells to identify specific cellular responses which could be selectively modulated by small molecules. An identified quinoline derivative and subsequent synthesized analogs (termed Vacquinols) reliably compromised GBM viability through a non-apoptotic mechanism associated with massive induction of macropinocytosis which was not induced in other cell types investigated. Activating this unique cellular response with our lead compound Vacquinol-1 results in marked membrane ruffling, cell rounding, accumulation of large intracellular macropinosomes and eventually caspase-independent cell lysis at low micromolar concentrations. Though the primary pharmacological target as of yet remains unknown, shRNA-induced Vacquinol-1 resistant mutants revealed the MAP-kinase MKK4 as a critical signaling node. Vacquinol-1 displays excellent in vitro and in vivo pharmacokinetics with good exposure into the CNS. Oral administration in a mouse xenograft model of GBM dramatically reduces tumor size, attenuates disease progression and significantly prolongs survival.1 The project is part of an ongoing collaboration between Chemical Biology Consortium Sweden, Karolinska Institutet and Uppsala University. This presentation will summarize these exciting discoveries and describe current efforts focused on further advancing the Vacquinol series towards clinical development, identification of the primary target(s) of Vacquinol-1 and elucidation of the underlying cellular pathways involved in the response.
P-59: Linking Arms: The Design of Triazolyl-Bridged Peptides as Inhibitors of Epidermal Growth Factor Receptor Dimerization
Receptor tyrosine kinases play a key role in the regulation of signaling pathways that mediate cellular processes such as proliferation, migration and apoptosis. Thus, alterations in the expression or regulation of these receptors can result in aberrant signaling found in various disease states. Epidermal growth factor receptor is a well-studied receptor tyrosine kinase whose overexpression promotes oncogenic activity in a variety of carcinomas. Small molecule tyrosine kinase inhibitors and monoclonal antibodies that target the receptor have proven to be effective at inhibiting EGFR. However, the serious side effects, the high cost of antibody production, and the development of acquired resistance demonstrate a need for alternative targeting strategies. Recent efforts have been placed in the development of peptide-based inhibitors of EGFR dimerization, a protein-protein interaction required for activation. The EGFR dimerization arm presents a promising foundation for the design of peptide inhibitors, as this short β-loop sequence contributes a majority of the energy required for formation of the EGFR dimer. We sought to generate a stable dimerization arm peptide by incorporating an intrastrand triazolyl-crosslink. A panel of cyclic peptides was designed by altering the length and orientation of the asymmetric triazolyl-crosslink. Our work demonstrates that the introduction of the triazolyl-crosslink not only generates a proteolytically stable peptide, but also allows for maintenance of secondary structure at the acidic pH representative of the tumor microenvironment, whereas the disulfide control loses its secondary structure at acidic pH. Within our panel, one notable peptide was capable of inhibiting EGFR dimerization and phosphorylation, as well as phosphorylation of the downstream substrate, AKT. Upon further investigation, we also observed that this peptide was internalized with EGFR after stimulation with EGF, providing evidence for binding of the peptide to EGFR. Thus, we have successfully synthesized a stable triazolyl-bridged dimerization arm mimic capable of binding EGFR and inhibiting receptor dimerization and kinase activation.
P-60: Synthesis And Isolation of New Analogues of Amphotericin B; 16-Descarboxyl-16-Methyl-19-(O)-Perosaminyl-amphoteronolide and 7-Hydroxy-amphotericin B
Amphotericin B is a gold standard antifungal drug used against systemic and tropical fungal infections as well as leishmaniasis. However the drug has several limitations such as limited dosage due to its high nephrotoxicity levels and poor solvent solubility (blood serum, water etc.). The drugs efficiency could be improved if non- toxic amphotericin derivatives could be produced economically on large scale. Semi-synthetic attempts have been made to produce several derivatives including MFAME, which showed the greatest promise due to reduced toxicity and increased antifungal activity in comparison to amphotericin B. However due to its complex synthesis, it could not be carried forward for economic reasons. Genetic manipulation of the natural biosynthesis of Streptomyces nodosus, has previously lead to the production and isolation of various derivatives including 16- descarboxyl-16-methyl-amphotericin B, 16-descarboxyl-16-methyl-19-(O)-perosaminyl- amphoteronolide, 8-deoxy amphotericin B and 7-Oxo amphotericin B. Here we report the synthesis and isolation of a new analogue 16-descarboxyl-16-methyl-19-(O)-perosaminyl- amphoteronolide16-descarboxyl-16-methyl-19-(O)-perosaminyl-amphoteronolide and 7- Hydroxy amphotericin B. New production and purification protocols of 16-descarboxyl-16- methyl-19-(O)-perosaminyl-amphoteronolide have been achieved. 16-descarboxyl-16- methyl-19-(O)-perosaminyl-amphoteronolide has been partially characterised. 16- descarboxyl-16-methyl-19-(O)-perosaminyl-amphoteronolide will probably show therapeutic potential and haemolytic activity. In addition to this, 7-Hydroxy amphotericin B has been successfully produced, isolated, purified and characterised, and also showed less haemolytic activity.
P-61: Development of a Multiplexed Proteomic Platform to Quantitate Deubiquitinase Activity Across Biological Samples
Deubiquitinases (DUBs) are important regulators of cell physiology and emerging drug targets due to their critical roles in protein homeostasis, DNA repair, cell cycle regulation, and cell signaling. However, the exact function or regulation of the majority of the estimated 100 human DUBs remains largely enigmatic. DUB activity-based probes are engineered ubiquitin variants conjugated to a cysteine-reactive chemical group. We envisioned that DUB activity-based probes coupled with mass spectrometry may provide a highly sensitive and unbiased means to aid our understanding of DUB function and regulation in healthy and diseased cell states and to identify novel routes of therapeutic intervention in DUB-controlled processes that are relevant to disease. Recent developments in mass spectrometry and chemistry now allow for powerful multiplexing and quantitation of proteins in single runs, thus enabling the throughput needed to efficiently assess the statistical significance of changes in DUB activity. However, current protocols are still limited by the number of samples that can be quantitatively compared, or sacrifice throughput for unlimited sample comparison. Devising a methodology to quantitatively compare the activity of DUBs across theoretically unlimited sample numbers that are assayed in separate experiments would be useful in order to better understand complex biological processes that are regulated by DUBs and to leverage DUB-based therapies. To this end, we here combine DUB probes with chemical multiplexing and targeted mass spectrometry, employ internal reaction standards, and devise a novel data analysis and visualization platform that allows us to quantitate changes in DUB activity across a theoretically unlimited number of samples in a high-throughput manner. We provide case studies that illustrate the efficacy of this technology to facilitate the discovery of disease-relevant functions and the regulation of this highly versatile enzyme class. We also discuss current challenges and potential improvements of our methods to increase the robustness, sensitivity and versatility of our technology.
P-62: Towards the Discovery of a Selective EPHA2 Inhibitor
The receptor tyrosine kinase EPHA2 is implicated in various cancer related processes and, thus, is emerging as a potential target for cancer therapy. However, investigation of its distinct functions in cancerogenesis and cancer progression has been so far impaired by the current lack of selective inhibitors. Dasatinib is an approved small molecule kinase inhibitor which inhibits EPHA2 in the nanomolar range, but at the same time features a very poor selectivity profile. Based on Dasatinib as a lead structure, we have designed analogs with improved selectivity but similar potency against EPHA2. Selectivity profiles of the inhibitors have been obtained by the KinobeadsTM technology coupled to quantitative mass spectrometry, which allows for the simultaneous detection of up to 300 kinases from complex cell lysates. This technology is a chemical proteomics approach, which facilitates the identification of targets as well as the determination of their binding constants in competition experiments. Moreover, assays addressing biochemical activity and cell viability have revealed insight into the potency of the new compounds as well as the ability to cross the membrane. Promising candidates have been further characterised by protein NMR to shed light on their binding mode as type I inhibitor targeting the active kinase conformation. We have identified an EPHA2 inhibitor with an improved selectivity profile and consistent potency compared to the lead structure of Dasatinib. Our EPHA2 inhibitor proves to be a valuable tool to elucidate EPHA2 contribution to cancerogenesis and other signaling processes. Given the potential of EPHA2 as a therapeutic target, this inhibitor class might also become interesting in the frame of cancer treatment.
P-63: Enzymatic Tagging to Identify the Protein Targets of Bioactive Small Molecules
Bioactive small molecules are a major class of therapeutics used for the treatment of human diseases. The development of new drugs is critical for the treatment of many diseases and aberrant phenotypes. In the last 15–20 years there has been a large focus on targeted drug discovery in which small molecules are developed to modulate the activity of a specific protein in a cellular pathway. While this method has had some success, it has not proven as facile as originally hoped. For this reason, there has been a renewed interest in phenotypic drug screening. In this method, compounds are screened against a cell or animal model until a desired phenotype is observed. Unfortunately, the target of any phenotype-inducing compounds must then be identified. This step is often slow, causing a bottleneck in the drug-development process. For this reason, new methods to rapidly identify the direct protein targets of bioactive small molecules are of great importance. We will present an engineered enzymatic tagging method that enables specific labeling and enrichment of protein targets from complex lysates. This method couples the binding of a small molecule to a proximity-based labeling event. Labeled target proteins are enriched and then identified using LC-MS/MS. We will discuss several variations of this method and highlight some of our recent successes in target identification and validation.
P-64: Development of Fluorescent Sensors with its Functions Regulated by Chemical or Enzymatic Reaction
Fluorescent sensors, whose fluorescence properties are changed by each analyte, enable the estimation of its concentration or activity. The localization of the sensor to specific cells, like cancer cells, or subcellular compartments enable high-fidelity tracking of each analyte, and could be useful for the elucidation of its physiological functions and clinical diagnosis. To date, several sensors, which could localize to specific site due to the characteristics of conjugated chemical moiety such as peptide or protein, have been developed. However, in most of these sensors, the fluorescent properties of the sensors localized in target site were not changed, compared with those located in other site, which means that wash-out process would be necessary for selective detection. Thus, our group have tried to develop fluorescent sensors, whose function could be changed by chemical or enzymatic reaction that would specifically occur in target site. Previously, our group constructed the library of fluorescent compounds based on coumarin structure, and several fluorescent sensors were found from this library, including multi-analyte sensor, viscosity sensor and fluorescent ligands for progesterone receptor. In this study, fluorescent properties of this library compounds were comprehensively investigated, especially focusing on functional group at 7-positon of coumarin. As a result, 7-substituent could regulate the property of coumarins as various sensors. For example, in the coumarins bearing the 6-aryl group as the recognition site for analyte cation, the mode of fluorescent change could be varied by 7-substituent, that is, in 7-dietrhylamino derivatives, the change of fluorescent intensity occurred by binding to the analyte cation, whereas, in 7-methoxy or 7-hydroxy derivatives, the shift of fluorescent maximum wavelength occurred. In the case of coumarins bearing the recognition site at the 3-positon via triazole ring, 7-substituent could regulate the availability as sensor. For example, in the compounds with dipicolyamine moiety as the recognition site, 7-amido compound could work as zinc sensor, whereas 7-amino compound did not work. These results suggested that introduction of peptide to 7-position would afford the activatable zinc sensor that can function only after the removal of the peptide moiety by specific peptidase. In addition, we also introduced ethynyl group to coumarin structure, whose function could be changed by Huisgen 1,3-cycloadditon with azido compounds, which could also be used as selectively activatable sensor.
P-65: Investigation of Struture-Activity Relationships, DNA Intercalation and Redox Cycling of Dioxonaptho[2,3-d]imidazolium Analogs as Chemotherapeutics for the Treatment of Renal Cell Carcinoma and Non-Small Cell Lung Carcinoma
Renal cell carcinoma (RCC) and Non-small cell lung carcinoma (NSCLC) highly chemoresistant cancers with very poor prognoses in late stages. Current chemotherapeutics such as multi-kinase inhibitor sunitinib for RCC and EGFR inhibitor gefitinib for NSCLC have not improved overall survival rates significantly and are plagued with multiple toxicities. YM155 is the first dioxonaphthoimidazolium compound exhibiting anti-cancer properties, with nanomolar potencies on several cancer cell lines. However, there is a lack of knowledge on the structure-activity relationships of this compound, and its relatively planar structure and quinone moiety has raised questions about its toxicity (Glaros et al., 2012). We systematically designed and synthesized several analogs of YM155 to investigate the importance of each portion of the molecular structure of YM155. These analogs were assessed for anti-proliferative activity on 2 RCC cell lines and 2 NSCLC cell lines. Additionally, a non-malignant cell line IMR-90 was included to assess selectivity. A3-3 showed improved activity on 786-0 (RCC, IC50=26.7 nM) and H1299 (NSCLC, IC50=17.0 nM) over YM155 with good selectivity for malignant cells. YM155 and analogs were also investigated for their propensity to intercalate DNA and were subsequently found to be 4–20 times weaker than doxorubicin, a known DNA intercalator. Although these analogs were discovered to undergo redox cycling with similar ability as menadione, poor correlation of their anti-proliferative activities with DNA intercalation and redox cycling ability suggests that these compounds are unlikely to be exerting their potent anti-cancer activities via non-specific DNA damage. Finally, YM155 and AB-1 were found to promote cleavage of caspase-3, indicating induction of apoptosis. This work supports the development of dioxonaphthoimidazolium analogs as chemotherapeutic agents for RCC with potent activities and good safety profile.
P-66: Improving Paclitaxel Production by Exploring Transcriptional Regulation During Methyl Jasmonate Elicitation
Paclitaxel is a key anti-cancer drug isolated from the bark of Taxus spp. Demand for paclitaxel is high and plant cell culture is an attractive production route. Cambial meristematic cells provide a good platform from which to increase drug production as they possess superior growth properties on an industrial scale compared to typical dedifferentiated cell culture. Elicitors, such as methyl-jasmonate (MeJA), can up regulate paclitaxel production in plant cell culture however the effect is only transient. Roche454 and Solexa sequencing were used to identify transcription factors (TFs) that were highly induced at an early time point (0.5 h) after MeJA elicitation. Subsequent analysis identified 19 TFs from five distinct families as candidate regulators of paclitaxel biosynthesis. The function of these TFs was explored by investigating their binding with eight promoters of important paclitaxel biosynthetic genes, which are rich in cognate binding sites for the candidate TFs. Interaction was examined in vitro using electromobility shift assays and in vivo by transient protoplast assays. One TF has been identified which binds to seven of the promoters tested indicating it maybe a potential overall regulator of paclitaxel biosynthesis.
P-67: Target Identification and Mode of Action Studies of Potent Mycobacterium Tuberculosis Inhibitors Based on an Aminothiazole Scaffold
Tuberculosis remains a serious global health concern, affecting nearly 2 billion people worldwide and is responsible for the deaths of 2 million people annually. The problem is compounded by the increasing identification of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb). While development of new anti-mycobacterial drugs is desperately needed, a better understanding of the mode of action of bactericidal compounds and the identification of vulnerable pathways containing potential novel drug targets is equally critical. 2-aminothiazoles (AT) were recently identified in a high throughput screen to have activity against actively growing Mtb. A series of AT analogs were synthesized and evaluated for structure activity relationship (SAR) determination. A number of these compounds possess potent activity against Mtb and display rapid kill kinetics. The mode of action of this series is unknown and analogs demonstrated poor pharmacokinetic (PK) properties. Identification of the bacterial target of AT compounds would aid in structure-based design of novel AT analogs with improved PK properties. Thus, we have employed AT-based molecular probes to elucidate the mycobacterial target and/or cellular pathways linked to bactericidal activity and mode of action. We have also conducted metabolomics studies of both wild-type and AT resistant mutants to examine the cellular response to compound exposure.
P-68: Tethering: Site-Directed Fragment Discovery for Challenging Targets
Tethering is a site-directed method that employs disulfide exchange between a library of thiol-containing Monophores and native or introduced cysteine residues. Tethering is a unique technology for biological tool discovery and early hit identification, enabling highly focused exploration of protein surface ligand-ability. Fragment adduct formation has historically been assayed through LC-MS, which gives a positive binding signal and target/ligand stoichiometry. We have expanded Tethering to use traditional plate-based assays, which read out biological activity under orthogonal conditions. These bring together the complementary elements of site-directed, mechanism-based discovery and functional biochemistry. We will present developments in Monophore library synthesis, screening approaches and data mining tools employed in their analysis, together with recent examples of ligands found to control protein function through allosteric sites and protein-protein interfaces.
P-69: O-GlcNAc Transferase Makes the Cut
O-GlcNAc Transferase (OGT) is an essential human enzyme that catalyzes the addition of a single N-acetylglucosamine to serine or threonine residues of intracellular proteins. This unique post-translational modification modulates a wide range of signaling pathways linked to glucose metabolism and its abnormality has been linked to many human diseases. Recently, OGT has also been suggested to participate in the proteolytic maturation of Host Cell Factor 1 (HCF-1) – an important regulator of the cell cycle. However, the role of OGT in the proteolysis of HCF-1 remains largely unknown. This poster will present the elucidation of this unique activity of OGT by using biochemistry, mass spectrometry and X-ray crystallography. This study solves a long-standing biological mystery, while expanding our knowledge of the already impressive regulatory mechanisms of OGT in different life processes.
P-70: Unraveling the Mechanism of Cell Death Induced by Chemical Fibrils
We previously discovered a small-molecule inducer of cell death, named 1541, that non-covalently self-assembles into chemical fibrils (“chemi-fibrils”) and activates procaspase-3 in vitro. We report here that 1541-induced cell death is caused by the fibrillar, rather than the soluble form of the drug. An shRNA screen reveals that knockdown of genes involved in endocytosis, vesicle trafficking, and lysosomal acidification causes partial 1541 resistance. We confirm the role of these pathways using pharmacological inhibitors. Microscopy shows that the fluorescent chemi-fibrils accumulate in punctae inside cells that partially co-localize with lysosomes. Notably, the chemi-fibrils bind and induce liposome leakage in vitro, suggesting they may do the same in cells. The chemi-fibrils induce extensive proteolysis including caspase substrates, yet modulatory profiling reveals that chemi-fibrils form a distinct class from existing inducers of cell death. The chemi-fibrils share similarities to proteinaceous fibrils and may provide insight into their mechanism of cellular toxicity.
P-71: A Two-Photon NIR Fluorescent Probe for Amyloid-β Plaque in Alzheimer's Disease Model
Two-photon (excitation) microscopy (TPM) combined with the laser excitation technology has arrested recent attention in the bioimaging area because it shows several advantageous features over one-photon excitation microscopy (OPM). TPM is most often emphasized to be “less photo-toxic, less photo-bleaching/-damaging”. In addition, TPM enables deeper tissue (700–1100 nm) imaging with high spatio temporal resolution, and thus is particularly useful for tissue imaging where auto-fluorescence becomes serious in the case of OPM [1]. The aim of this research is to develop new two-photon probe which show desirable photo-physical properties for bioimaging, overcoming some drawbacks of known dyes such as DDNP and DANIR derivatives which are molecular probes for amyloid-bata(Aβ) plaque in Alzheimer's disease(AD) [2]. AD is the most common form of dementia, a disorder characterized by progressive impairment of episodic memory and language deficits. As the pathogenesis of Alzheimer's disease is associated with formation of insoluble aggregates of amyloid-beta peptide (Aβ plaque), approaches allowing the direct and non-invasive visualization of plaque growth in vivo would be beneficial for biomedical research [3]. We have developed novel many π-extended benzocoumarin and other derivatives, which show promising properties in two-photon imaging applications [4]. In this research, we are focused on the studies of two-photon excitable and NIR emissive fluorescent probe for deep-tissue imaging in the AD mouse model [5].
[1] D. Kim, H. G. Ryu, K. H. Ahn. Org. Biomol. Chem. 2014, 12, 4550.
[2] M. Cui, M. Ono, H. Watanabe, H. Kimura, B. Liu, H. Saji. J. Am. Chem. Soc. 2014, 136, 3388.
[3] C. H. Heo, K. H. Kim, H. J. Kim, S. H. Baik, H. Song, Y. S. Kim, J. Lee, I. Mook-jung, H. M. Kim. Chem. Commun. 2013, 49, 1303.
[4] (1) I. Kim, D. Kim, S. Sambasivan, K. H. Ahn. Asian J. Org. Chem. 2012, 1, 60–64. (2) D. Kim, S. Sambasivan, H. Nam, K. H. Kim, J. Y. Kim, T. Joo, K. Lee, K. Kim, K. H. Ahn. Chem. Commun. 2012, 48, 6833–6835; (3) D. Kim, S. Singha, T. Wang, E. Seo, J. H. Lee, S.-J. Lee, K. H. Kim, K. H. Ahn. Chem. Commun. 2012, 48, 10243.
[5] D. Kim, S. H. Baik, I. Mook-Jung, K. H. Ahn. 2014, to be published.
P-72: Mitochondria-Targeting Nitric Oxide Releasers Controllable with Visible Light
Nitric oxide (NO) is a small-molecular and gaseous mediator that plays important roles in various physiological processes. Since NO is unstable under ambient conditions, compounds that release NO in situ, NO releasers, have been developed and employed for NO research, and photocontrollable NO releasers seem particularly attractive as a means to achieve spatiotemporally controlled intracellular NO release. We have developed 2,6-dimethylnitrobenzene (2,6-DNB) derivatives as photocontrollable NO releasers. In this study, we degined and synthesized two rhodamine-based 2,6-DNB derivatives, Rol-DNB-mor (1) and Rol-DNB-pyr (2). The ESR spin trapping method showed that these compounds released NO upon exposure to the non-cytotoxic visible light in the absorption wavelength of the rhodamine (530–590 nm). These compounds were applicable to living cells and localized in mitochondria owing to the cationic property of rhodamine. The photocontrolled intracellular NO release was detected by DAR-FM DA, a green fluorescence probe for NO, and the localization of the compounds were confirmed by costaining with MitoTrackerGreen FM, a green fluorescence marker for mitochondria. Moreover, it was suggested that Rol-DNB-mor (1) caused visible-light dependent cancer cell death which was presumably induced by released NO. In conclusion, we developed mitochondria-targeting NO releasers controllable with non-cytotoxic visible light, Rol-DNB-mor (1) and Rol-DNB-pyr (2), which would be useful for NO research and potentially applicable to photodynamic therapy.
P-73: Photoimmunotherapy: Basis, and Applications
Photo-immunotherapy (PIT) is a newly developed, molecularly-targeted cancer photo-theranostics (imaging diagnosis plus therapy) using conjugates of a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (MAb) thereby targeting specific cell-surface molecules. When exposed to NIR light, the conjugate induces a highly-selective necrotic cell death only in receptor-positive MAb-IR700-bound cancer cells (Nature Med 2011). Necrosis occurs as early as 1 minute after exposure to NIR light and results in irreversible morphologic changes including cellular swelling, bleb formation, and rupture of vesicles due to membrane damage based on photo-reactive structural changes of associated proteins with photo-immunoconjugates induced by photo-chemical cleavage of silica-phthalocyanine dye. Meanwhile, immediately adjacent receptor-negative cells are unharmed. Due to the concentration gradient of MAb-IR700 leaking from vessels, PIT first causes necrosis in perivascular cancer cells resulting in dramatically enhanced vascular permeability with enhanced nano-particle delivery to cancer tissue, an effect termed “super-enhanced permeability and retention (SUPR)” (ACS Nano 2013). The combination of PIT and SUPR effects can effectively treat a variety of solid cancers including inhomogeneous cancers and cancer stem-like cells by employing different targeting molecules (including but not limited to MAbs) and nano-sized anti-cancer drugs. Preclinical examples of successful PIT, employing a variety of single- and multi-molecular target-PIT (EGFR, HER2, CD25, PSMA), combined with nano-sized anti-cancer agents (DaunoXome, Ablaxane), will be shown. The combination of PIT and nano-sized systemic therapies is especially well adapted for real world heterogeneous tumors containing both receptor positive and receptor negative cells.
P-74: Discovering Enzymes with Desired Biochemical Activities with Use of Small Molecular Fluorescent Substrate Probes
In this presentation, we introduce a proteomic method to characterize the enzymes with desired biochemical activities by using fluorescent substrate probes. Fluorescent substrate probe, which acts as the substrates of certain enzymes to report their activities, is one of the powerful chemical biological tools that enables us to study protein functions in living cells. However, in using the fluorescent substrate probes in complex biological systems, it is often difficult to identify the true target of the synthesized substrates in proteome, since thousands of enzymes are present in living systems. We here developed the method to find the target protein of fluorescent probes in proteomic samples, which is useful for the characterization of enzymes with desired biochemical activities (J. Am. Chem. Soc. 135 (2013) 6002–6005). The method is based on zymography, a colorimetric assay performed on non-denaturing electrophoresis gel. The conventional zymography has been applied to studying functions of more than 400 enzymes, but the method was hardly applicable to the study with fluorescent probes (c.f. colorimetric probes), since the fluorescent product of the enzymatic reaction readily diffuses in the gel, making it difficult to determine the precise location of the target protein. Thus, we came up with a novel method that can overcome the problem of diffusion by dicing the electrophoresis gel into small pieces that are separately loaded into wells of multiwell plates for fluorometric assay. With use of the method, we newly identified two enzymes that are in charge of metabolic inactivation of bioactive formyl peptides. The function of one of the characterized enzymes, acylamino acid-releasing enzyme (APEH) was further studied in in cellulo and in vivo systems. We believe that the overall method is expected to serve as the useful platform to discover novel enzymatic functions by the help of fluorescent substrate probes, and the scope of its application in discovering a novel biomarker of lung inflammation will be discussed, too.
P-75: Discovery of Novel SENP inhibitors Utilizing Structure Based Virtual Screening
Sumoylation is a post-translational modification that involves the reversible modification of target proteins with small ubiquitin-like modifier (SUMO) protein. Sumoylation is one of the important mechanism regulating the activities of various proteins involved in cellular processes like DNA replication and repair, chromosome packing and dynamics, genome integrity, nuclear transport, signal transduction and cell proliferation. Four SUMO paralogs (SUMO1-4) have been identified in humans, which are expressed as precursors. These proteins need to be proteolytically processed by specific proteases known as SUMO specific proteases (SENPs). SENPs cleave pro or inactive form of SUMO at C-terminus using its hydrolase activity to expose two glycine residues. SENPs also possess isopeptidase activity that is crucial for recycling of SUMO from substrate proteins. SENPs are attractive targets for drug discovery due to their crucial role in development of various diseases like cancer, atherosclerosis and heart diseases. However, until now the SENPs inhibitor discovery efforts were limited and only a few inhibitors or activity based probes have been identified. Here in this study, we used the combination of structure based virtual screening and quantitative FRET based assay to identify several inhibitors of SENP2 that could be used in chemical biology and therapeutic studies. Our virtual screening protocol initially involves the identification of small molecules that have similar shape and electrostatic properties with SUMO1 C-terminal residues. Molecular docking was then used to prioritize these small molecules for FRET based assay that quantifies SENP2 endopeptidase activity. The result of biological assay and subsequent similarity search resulted in the identification of several classes of small molecules. Most of the compounds also inhibited closely related isoform SENP1 while no detectable inhibition on other proteases, such as papain and trypsin was observed. Our study presents starting points for the development of novel therapeutic agents against various diseases targeting SENPs.
P-76: Enhanced Intracellular Delivery and Gene Silencing Efficacy Triggered by Chemically Modified Cell Penetrating Asymmetric siRNA
Small interfering RNA (siRNA) is a class of double-stranded RNA molecules that mediate target gene silencing in a sequence-specific manner and can be designed to target genes for therapeutic applications. Existing siRNA therapeutics, however, have drawbacks such as off-target effect, instability and poor delivery efficacy. To overcome these limitations, we previously developed novel siRNA structural variants including cell penetrating asymmetric siRNA(cp-asiRNA) structures which could enter into cells and trigger RNAi without transfection reagent in many cell lines. In this study, we present further optimized the cp-asiRNA structures. Alternated molecular patterns were applied and we observed that the renewed cp-asiRNA structures retained enhanced gene silencing activity. Further optimization of asiRNA structure is underway and this will provide a clear insight into the potential of cp-asiRNA for diverse applications including siRNA-based therapeutics.
P-77: Small Molecules Mediated Protein Activation in Living Cells
Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain- of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Recently we developed a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.
P-78: Discovery of Small Molecule Inhibitors of Lin28a-let-7 Interaction
MicroRNAs (miRNAs) are small noncoding RNAs that play crucial roles in regulating gene expression. Among them, let-7 miRNA family members have been the subject of intense researches owing to their tumor suppressor functions. They are downregulated in many different cancers, and restoration of their expression effectively inhibited growth of cancers in mouse models. Lin28a is a specific and post-transcriptional inhibitor of let-7 biogenesis. Lin28a binds to the primary and precursor let-7 miRNAs, thereby blocking their processing by Dicer and recruiting TUTase that adds uridines to the 3'end of miRNA for degradation. Small molecule inhibitors of Lin28-let-7 interaction could be a valuable tool for studying let-7 biogenesis and potential anticancer agents. We have developed a FRET-based assay to monitor binding between Lin28 and let-7, and applied it to a high-throughput screening to identify a small molecule inhibitor of Lin28-let-7 interaction. Several lead compounds were discovered from our privileged diversity-oriented synthesis (pDOS) library. Their bioacitivity and mode of action will be presented.
P-79: Insights into the Function and Inhibition of the HER3 Pseudokinase Receptor from the HER3/EGFR Heterodimer Kinase Structure
Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor tyrosine kinase that lacks catalytic activity but is essential for cellular homeostasis due to its ability to allosterically activate its heterodimeric partners, EGFR and HER2 (also known as HER1/ErbB1 and ErbB2, respectively). HER3 is an important therapeutic cancer target because the signaling emanating from HER3 heterodimers is critical for the progression and maintenance of several human epithelial malignancies. Although body of structural work has focused on understanding the molecular basis for activation of the catalytically active HER receptors, not much is known about the allosteric activity of HER3. To gain insight into the function of HER3, we have solved a crystal structure of the heterodimeric complex between the HER3 pseudokinase domain and the catalytically active kinase domain of EGFR. This is to our knowledge the first crystal structure of a heterodimeric complex between any members of the HER family of receptor tyrosine kinases. The structure reveals a first unbiased conformation of the HER3 pseudokinase in its functional state as an allosteric activator, and reveals the structural features of the HER3 pseudokinase that have not been previously observed. Our structure also provides first visualization of the juxtamembrane segment-mediated interactions between two different HER receptors providing insights into the conservation of this regulatory step of the HER receptor activation mechanism. The structure of the HER3/EGFR heterodimer also provides molecular explanation for the activating effect of several mutations recently reported in HER3 in human cancers, and constitutes an invaluable platform for designing inhibitors that could target the deregulated HER3 function in human diseases.
P-80: The Morphologies of Chitinibacter tainanensis in Conditional Medium
Chitin is the second abundant polysaccharide on the earth. Chitin and its derivatives are the major components of most fungal cell walls, insect exoskeletons and the shells of crustaceans. They are also the main components of the cell wall of prokaryotes. Chitin is the polymer and the major monomer is N-acetyl-D-glucosamine (NAG). Derivatives of chitin, inclusive of oligomers, monomers and their modifications, can be applied versatile and ubiquitously. Recently, Chitinibacter tainanensis was isolated from the soil in Southern Taiwan. The final unique product of chitin treated with the fermentate of C. tainanensis is NAG. The yield was 0.75 g/g with α-chitin as substrate, while that was 0.98 g/g with β-chitin as substrate. With the electron microscope, some particles adhered on the surface of the bacteria. In the work, different conditions of medium were applied in inoculation and the bacteria were examined with electron microscope. The morphologies will be presented and interpreted.
P-81: Labelling of GPCR Based on a New Peptide-Templated Acyl Transfer Reaction
Live cell imaging plays an important role in understanding and characterization of G protein coupled receptors (GPCR) in their native environment. To achieve this fluorescent moieties have to be attached to the protein of interest. Widely used strategies such as fusion proteins e.g. fusion of GFP or specific labelling tags such as SNAP-tag or Halo-tag have their advantages. But they also have certain drawbacks, for example the size, the labelling time and in case of the fusion protein its permanent fluorescence after folding. To overcome these disadvantages a new covalent labelling approach based on a coiled-coil interaction is presented. The de novo designed E3/K3 coiled-coil is characterized by a small size with 5–6 kDa and a high binding affinity with a KD of 60–70 nM [1]. The GPCR of interest is genetically fused to the Cys-E3 peptide (acceptor) at its N-terminus which is capable to interact with the thioester-armed K3 peptide (donor). After interaction of the donor and acceptor peptide an acyl transfer reaction takes place, which transfers only the reporter group from the K3 peptide to the Cys-E3 tagged GPCR [2]. The advantages of this labelling strategy are high target specificity, fast reaction time within seconds to minutes and the free choice of the reporter group. Moreover the mass increase during the acyl transfer of the reporter group is minimal compared to the Cys-E3 tagged GPCR. This new chemical method for labelling GPCR in living cells is suitable for e.g. pulse-chase experiments. First experiments showed a successful labelling for several class A GPCR in HEK293 cells. Signal transduction assays demonstrate that the Cys-E3 tagged GPCR are still fully functional.
P-82: Streamlining ATPase and Kinase Assay Development using Direct ADP Detection with the Transcreener ADP2 Assays
Biochemical HTS assays are a critical component of drug discovery programs focused on kinases and other types of ATP-dependent enzymes, such as chaperonins and helicases. Assembling, evaluating and optimizing high quality enzymes and assay reagents can be a costly and time consuming undertaking. The shift toward screening focused libraries against multiple targets in parallel can add to the assay development burden substantially. BellBrook's Transcreener ADP Assay provides an extensively validated, generic HTS platform for ATPase and kinase assays based on homogenous immunodetection of ADP with a choice of FP, TR-FRET or FI readouts. In this study, we outline a streamlined approach for optimizing conditions to measure enzyme initial velocity for kinases and ATPases that leverages some of the differentiating characteristics of the Transcreener assay including direct detection of ADP without the use of coupling enzymes and low nanomolar sensitivity. Key steps include tuning the dynamic range to accommodate enzymes with different ATP concentrations, optimizing enzyme concentration to produce a good signal, and determining IC50 values for inhibitors. We illustrate how monitoring ADP production in real time allows optimization of multiple assay parameters using a single reaction and show that IC50 values can be determined directly from raw fluorescence data, eliminating the need to use a standard curve to quantify product formation. These approaches reduce the number of experiments required to incorporate new enzymes into a screening or profiling campaign, saving time and reducing reagent costs. Further, we demonstrate a straightforward approach for minimizing enzyme usage by finding the right balance between ATP concentration and enzyme velocity. Taken together, these methods provide a rapid, user friendly solution for screening or profiling kinases and other types of ATP dependent enzymes with minimal pilot experimentation and reagent usage.
P-83: Targeting Mcl-1 for the Therapy of Breast Cancer
Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family anti-apoptotic proteins that are important regulators of programmed cell death (apoptosis). Overexpression of Mcl-1 is associated with high tumor grade, resistance to chemotherapy and poor prognosis in many types of cancers. Amplification of the gene encoding the anti-apoptotic protein Mcl-1 is a common genetic aberration in breast cancer. Preclinical evidence suggests that Mcl-1 is a promising target for the treatment of breast cancers including the highly aggressive triple negative breast cancer (TNBC) subtype. In this study genetic and chemical genetic approach was employed to understand the role of Mcl-1 in regulating the cell death pathway using a panel of breast cancer cells and to further validate Mcl-1 as potential therapeutic target. Several TNBC cell lines showed susceptibility to novel class of small molecule Mcl-1 inhibitors developed in our laboratory, followed by induction of early apoptosis. Importantly, the sensitive cell lines were found to be addicted to Mcl-1 for survival using BH3 profiling assay where Noxa was able to trigger mitochondrial outer membrane depolarization. Furthermore, the Mcl-1 dependence was confirmed by cell transfection with small interfering RNA (siRNA) targeting Mcl-1 and inducing cell death and apoptosis. These promising results demonstrate the therapeutic potential of Mcl-1 inhibitors against TNBC and development of novel targeted drugs against this subtype of breast cancer is a major unmet need.
P-84: Modeling Mammalian Nonapoptotic Cell Death in Yeast
Ferroptosis is a novel form of non-apoptotic cell death. One hallmark of ferroptosis is the accumulation of toxic lipid reactive oxygen species (L-ROS), which is likely linked to the oxidative fragmentation of plasma membrane lipids containing polyunsaturated fatty acids. Our previous studies showed that in both human cells and budding yeast, L-ROS toxicity is rescued by co-treatment with lipophilic antioxidants; however, the genetic network underlying L-ROS production, toxicity and detoxification is not well understood. With the genome-wide tools available for budding yeast, we are using this model organism to identify genes that modulate L-ROS toxicity. We will present preliminary results from genetic screens designed to identify enhancers and suppressors of oxidative lipid fragmentation. Importantly, genes identified from our screens that are conserved across eukaryotes may provide novel targets for the development of small molecule probes and drugs that can alter L-ROS production or accumulation, and consequently cell fate, in diseased cells and tissues.
P-85: Chemical Tools to Monitor Neuronal Activity with High Spatiotemporal Fidelity
Optical imaging has become an indispensable method for interrogating biological systems, driven in large part by the development of new chemical probes for measuring and monitoring cellular events in real time. Charting neuronal activity with high spatial and temporal fidelity remains a pressing challenge to the scientific community. To address this challenge we aim to investigate new chemical tools to overcome limitations of existing approaches and to complement current efforts to map the activity of multiple neurons. This presentation will describe our most recent efforts on the development and application of optical approaches to monitoring neuronal activity with high spatial and temporal resolution.
P-86: Cooperative Activation of PPARγ by Combination of Irreversible Antagonist and Partial Agonists: Implication for Novel Activation Mechanism Based on Covalent Modification
PPARγ is a ligand-dependent transcriptional factor whose activation regulates adipocyte differentiation and function. PPARγ has a large ligand-binding pocket (LBP) with multiple sub-pocket, where plural ligands can simultaneously bind to. GW9662 (1) is a widely-used PPARγ antagonist which irreversibly inhibits agonist-mediated transactivation through covalent modification of Cys285 residue in PPARγ ligand-binding domain (LBD). However, we found here the novel function of 1, which cooperatively activate PPARγ in combination with plant-derived partial agonists such as ethyl-p-methoxycinnamate (2) or ethyl-m,p-methoxycinnamate (3).
Luciferase reporter gene assay showed that the weak transactivation potential of 2 and 3 was notably enhanced by co-treatment with 1 to the same extent as full agonists. Although full agonists represented by thiazolidinedione are known to bind to and activate AF-2 regions in LBD, molecular docking studies predicted that both 2 and 3 bind to Ω-loop region of LBD in the presence of 1. 1 also exhibited the cooperative activation in combination with ligands that bind to not AF-2 but Ω-loop region, suggesting that cooperative effect is mediated by conformational change near Ω-loop region. As opposed to thiazolidinedione, adipocyte differentiation of 3T3-L1 cells was inhibited by combination of 1 and 2 in a PPARγ-dependent manner. Furthermore, cooperative transactivation and adipocyte phenotype were abolished by either mutation at Cys285 residue or non-covalent GW9662 analogues.
These findings implicate the presence of a novel mechanism for PPARγ activation based on covalent modification of Cys285 residue, where different cellular phenotype is induced. Our results also provide a potential approach to new PPARγ ligand design with anti-obesity effect or reduced side effects.
P-87: High-Throughput Mapping of Dynamic Protein-Protein Interactions in Living Cells with a NanoLuc-Based Bioluminescence Resonance Energy Transfer Technology
The bioluminescence resonance energy transfer (BRET) technique was initially developed to monitor protein-protein interactions (PPIs) in living cells. The broad emission spectrum and weak luminescence signal of conventional luciferases, however, hinder its broad applications. In this study, a recently reported luciferase, NanoLuc, with a relatively narrow emission spectrum and high signal intensity, has been utilized to develop a BRET technology that we termed BRETn, which enables monitoring of PPIs in an uHTS format. In our design, NanoLuc as a luminescence energy donor is coupled with proteins tagged with Venus, an enhanced fluorescence protein. We carried out a series of optimization studies using several known PPI pairs, and have achieved conditions for the robust performance of BRETn to allow a miniaturized assay in a 1536-well uHTS format. The utility of the BRETn was examined by mapping the binary PPIs between a panel of oncogenic proteins. We have achieved a robust BRETn assay with significantly enhanced signal-to-background ratio up to three fold, and excellent Z' values of >0.7. To further evaluate its applications with this uHTS format, we monitored the dynamic interaction between YAP1 and TEAD2. Upon serum stimulation, we observed significantly increased BRETn signal of YAP1/TEAD2 interaction in a dose and time dependent manner. Thus, this BRETn technology is applicable for live-cell based high-throughput mapping of dynamic PPIs and for uHTS campaigns to discover small molecule PPI modulators.
P-88: Inducible Cellular Proteolysis: Engineering Selective Cleavage of Apoptotic Substrates
Proteolysis is a fundamental process in biology; it plays a crucial role across development of multicellular organisms, aids in maintaining tissue homeostasis, and is integral in cell signaling. Intracellular proteolysis frequently focuses on proteasome mediated protein degradation, however the tightly regulated and selective proteolysis mediated by the cysteine-aspartyl specific proteases, caspases, often leave their substrates intact. Caspase proteolysis results in dramatic modification of the substrate's function. A key unmet question in the field is to characterize how individual substrate cleavages, a growing list of 1500 proteins, lead to the profound morphological transformations that are the hallmark of apoptotic cells. In order to investigate the functional consequence of site-specific protein cleavage, we have developed an orthogonal system to induce and selectively cleavage single cellular substrates. Our system combines an inducible and orthogonal protease with a novel lentiviral based knockdown/knockin vector. Several aspects of the project will be discussed, including vector design and optimization, cellular engineering, and resulting cellular biology will be discussed.
P-89: Lipase-Catalyzed Optical Resolution and Biological Activity of Boron-Cluster-Based Progesterone Receptor Ligands
Carboranes (dicarba-closo-dodecaborane) are the icosahedral boron clusters having ten boron atoms and two carbon atoms as their vertices. Carboranes have characteristic properties such as remarkable thermal and chemical stability, and an exceptionally hydrophobicity, and can be applied as hydrophobic core structures of bioactive molecules in the field of medicinal chemistry. In this study, we investigated the lipase-catalyzed asymmetric acylation of carborane-containing secondary alcohols in order to develop the efficient methods for optical resolution of bioactive carborane derivatives. Lipase-catalyzed asymmetric acetylation of racemic 1-(7-phenyl-m-carboranyl)ethanol (1) was investigated as the model reaction. As the result of lipase screening, lipase TL isolated from Pseudomonas stutzeri exhibited exceptionally high enantioselectivity with moderate reaction rate. The absolute configuration of unreacted enantiomer of 1 was determined to be S by X-ray crystallography of its conjugate with chiral calboxylic acid. Thus, lipase catalyzed R-1 selectively in the same manner as other general secondary alcohols. Our previous studies showed that C-(cyanophenyl)carborane bearing proper substituent on another carbon atom of the carborane is the key structure of progesterone receptor (PR) ligand. Therefore, we designed 1-(7-(4-cyanophenyl)-m-carboranyl)ethanol (2) as a novel PR ligand candidate, and examined the lipase-catalyzed kinetic resolution of 2. By the same procedure as for 1, lipase TL catalyzed-acetylation of racemic 2 proceeded with high enantioselectivity and efficiently resolved rac-2 into both enantiomers. The progesterone activities of the purified R- and S-enantiomers of 2 were examined by alkaline phosphatase assay using T47D breast-carcinoma cell line. Both enantiomers of 2 acted as PR partial agonists and S-2 exhibited more potent agonistic activity than R-2. In the presence of 2 nM progesterone, compound 2 acted as the PR antagonist. The effect of chirality on the antagonist activity of 2 was relatively small, compared with that on the agonistic activity. The methodology and information in this study would be useful for the structure-activity relationship studies of chiral carborane derivatives.
P-90: Developing Conditionally Activated Apoptotic Cell Models via an Engineered RNA-Guided Nuclease
Genome editing is becoming a widely used approach, employing sequence-specific DNA nucleases to manipulate specific genes in diverse cell types and organisms. Cas9-based RNA-guided nuclease (RGN) has emerged to be the most versatile method for genome editing due to the ease of construction of RGN reagents for targeted genomic sequences. Here we aim to engineer Cas9 for conditional activation and employ the engineered RGN in genome editing to create cell lines to study the apoptotic pathways. This study will hopefully provide insights on the roles of individual signaling nodes in apoptosis.
P-91: Marine Natural Products Targeting Cell Membrane
Cell membrane is one of the most challenging research subjects, partly because lipid functions at a molecular level are largely unknown. We are conducting chemical genetics approaches using compounds targeting lipids to elucidate the structure and function of cell membranes. Here we report that heronamides, polyene macrolactams from a marine-derived Streptomyces sp., are a unique class of membrane binders.1 We have constructed a screening system to find natural products targeting membrane lipids, using fission yeast as a model organism. During the course of the screening, we isolated heronamides from a marine-derived Streptomyces sp. The chemical structures of heronamides including 8-deoxyheronamide C (8-dHC), a new 20-membered polyene macrolactam, were determined by chemical conversion and spectroscopic analysis. Among heronamides, 8-dHC exhibited unique cell growth inhibition pattern that was typical for membrane binders. To demonstrate the affinity between heronamides and lipid membrane, we examined surface plasmon resonance (SPR) analysis. The sensorgrams indicated that 8-dHC and heronamide C strongly bound lipid membranes consisting of lipids with saturated hydrocarbon chains. In microscopic analysis, drastic morphological changes were observed when fission yeast cells were exposed to heronamides. Curiously, this phenomenon was critically similar to that exhibited by theonellamides, antifugal peptides from marine sponges, that target ergosterol.2 These results suggest that heronamides modulate some functions of specific membrane domains, for example, lipid rafts.
1. J. Am. Chem. Soc. 2014, 136, 5209.
2. Nat. Chem. Biol. 2010, 6, 519.
P-92: From Peptidic Substrates to Inhibitors of Human Sirtuin 5 - Small Lysine Modifications Make the Difference
The (de)acetylation of lysine residues is one of the most abundant posttranslational modification modulating protein activity and stability. The acetylation level in the cell is regulated through lysine-acetyltransferases and lysine-deacetylases. The NAD+-dependent lysine-deacetylases (Sirtuins) are acting as sensors in metabolic pathways, stress response and aging processes. In mammalia there are seven Sirtuin isoforms known which emerged as potential therapeutic targets due to molecular links between cell metabolism and human disorders. The mitochondrial Sirtuin 5 (Sirt5) is a weak deacetylase but is known to be a very efficient desuccinylase and demalonylase [1]. To date, the substrate acyl specificity of Sirt5 has not been systematically analyzed. We therefore investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity. Besides, we designed potent peptide based inhibitors interacting with the NAD+ binding pocket. To elucidate the molecular basis of substrate and inhibitor properties we determine several x-ray crystal structures of Sirt5. Our results provide insights into the Sirt5 acyl selectivity and its molecular basis and enable the design of high affinity mechanism-based inhibitors for Sirt5.
[1] Du, J., Zhou, Y., Su, X., Yu, J.J., Khan, S., Jiang, H., Kim, J., Woo, J., Kim, J.H., Choi, B.H., He, B., Chen, W., Zhang, S., Cerione, R.A., Auwerx, J., Hao, Q., Lin, H., 2011. Sirt5 Is a NAD-Dependent Protein Lysine Demalonylase and Desuccinylase. Science 334, 806–809.
P-93: Development of a Fluorescence Probe for Folate Receptors on the Cell Membrane
Background: Folate receptors (FRs) are membrane proteins which have a role in taking in folic acid. It is reported that they are overexpressed in ovarian and endometrium cancer, and also expressed at the stage of neural tube closure. Therefore, FRs attracts attention in both fields of life science and clinical medicine. In this study, we developed fluorescence probes selective for FRs on the cell membrane to visualize them by fluorescence imaging.
Method and Results: We selected four xanthene-based fluorophores (rhodamines and fluorescein), and synthesized four fluorescence probes by conjugating these fluorophores thorough the peptide linker to folate. We succeeded in the visualization of FRs on cell membrane when each of them was applied to KB cells expressing FRs. However, we also observed the fluorescence signal in OVCAR-3 cells which don't express FRs when rhodamine-based probes were applied, therefore it was considered that they were taken into cells, irrespective of FRs. In the experiments, the case that we didn't observe nonspecific uptake of folate into cells was only when fluorescein was used as a fluorophore. Based on these results, we tried to develop a red fluorescence probe with DichloroTokyoMagenta (DCTM), which was the red fluorescein analogue. When DCTM-based probe was applied to KB cells, the FR-independent uptake of folate was not observed. Thus, we succeeded in visualizing FRs on the cell membrane by red fluorescence. On the other hand, when we investigated photophysical properties of fluorescence probes for FRs, we also found that these probes showed lower fluorescence quantum yield and shorter fluorescence lifetime than fluorophores themselves. We hypothesized that this shortening of fluorescence lifetime was eliminated by weakening interaction between folate and fluorophore when the probe binds to FR. So, we applied the probe to fluorescence lifetime imaging microscopy (FLIM). As a result, we observed a longer fluorescence lifetime in the cell membrane than that in the extracellular region as expected, and succeeded in the imaging of FRs on the cell membrane by monitoring the fluorescence lifetime change of the probe. Aiming at the application to the animal experiment, we will perform the further improvement of the probe.
P-94: ProMs: A Modular Toolkit to Inhibit Proline-Rich Motif Mediated Protein-Protein Interactions
Small molecules that compete with native protein-protein interaction interfaces are in urgent demand for fully capitalizing on large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets are domains specialised in recognizing proline-rich segments (PRS), including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace PRS. As proof-of-principle we developed a small, selective, non-peptidic inhibitor against Ena/VASP EVH1 domains. Highly invasive MDA MB 231 breast cancer cells treated with this ligand showed displacement of VASP from focal adhesions as well as from the front of lamellipodia and yielded a two-thirds reduction in cell invasion. Our design strategy appears to be general for all PRDs, as illustrated by an ErbB4-derived ligand containing ProM-1 for the YAP1-WW domain with an 5-fold increase in affinity.
P-95: Oligo-Alanine-Modified Pluronic-F127 Nanocarriers for the Delivery of Curcumin with Enhanced Entrapment Efficiency
Curcumin is a naturally occurring compound that has been shown to have anti-oxidant, anti-inflammatory, and anti-carcinogenic activities. However, its pharmaceutical potential has been limited due to its low solubility in water. The use of amphiphilic nanocarriers is an attractive and simple method to solubilize curcumin. In this study, we modified Pluronic F-127 [poly(ethylene glycol)100-block-poly(propylene glycol)65-blockpoly(ethylene glycol)100] (PF-127) with oligomers of alanine, an amino acid, to increase the drug entrapment efficiency of curcumin through core stabilization. Alanine-modified PF-127 exhibited lower critical micelle concentration (CMC) and decreased molecular motion in both the hydrophilic and hydrophobic segments (1H NMR). Nanocarriers in the size range of 54.2 nm to 68.4 nm were observed. Entrapment efficiency of curcumin increased by at most 66% (from 25.3% to 91.3%) and the difference in solubility was clearly visualized by increased transparency of the nanocarrier solutions. Curcumin was released continuously up to 120 hours from modified carriers while drug release from unmodified carriers plateaued within 24 hours. These modified nanocarriers exhibited no cytotoxicity and more efficiently delivered drugs to HeLa cells as confirmed by fluorescent microscopy. This study demonstrated that alanine modification of FDAapproved PF-127 affects copolymer nano-assembly and has a profound impact on curcumin loading and possibly on other hydrophobic drugs as well.
P-96: Re-Purposing the Ubiquitination System for Extracellular Interactomics
Despite tremendous progress in developing technologies for unbiased analysis of protein-protein interactions for soluble proteins (interactomics), technologies for interactomics among membrane proteins has poorly lagged. It is estimated that ∼30% of mammalian genes encode for membrane proteins and ∼50% of all drug targets are at the membrane, so the ability to explore protein-protein and drug-protein interactions in this context has significant therapeutic importance. Membrane protein interactomics has been challenging largely due to a lack of good proteomic tools for identifying weak, transient, or membrane-bound complexes. Here we seek to develop a proximity tagging tool that can label in a minimally biased fashion with great sensitivity. This tool, which we call the ubiquitron, utilizes components of the endogenous ubiquitination system to label targets on the surface of intact cells with biotinylated ubiquitin. Targets can then be identified by AP-MS/MS. We envision using the ubiquitron to rapidly identify the complete interactome for orphan ligands.
P-97: Small-Molecule Mimics of a Peptide Allosteric Effector Disrupt Kinase Function in Cells
Small-molecule inhibitors of protein kinases have revolutionized biomedical science. In the clinic, protein kinase inhibitors have ushered in the era of personalized medicine by transforming the standard of care for a variety of cancers. At the bench, the widespread use of protein kinase inhibitors as research tools has significantly advanced our knowledge of the roles of protein kinases in signaling pathways. However, the molecular mechanisms that regulate the function of individual kinases remain poorly understood. We are developing chemical tools to understand how an allosteric site found in many kinases regulates their substrate specificity, subcellular localization, and catalytic activity. In this study, we have focused on the protein kinase PDK1 where this allosteric site, known as the PIF pocket, is required for the phosphorylation of most PDK1 substrates. Our lab previously demonstrated that PDK1 could be activated or inhibited by directly targeting the PIF pocket with disulfide-trapped small molecules (Sadowsky et al. 2011, PNAS). We recently identified molecules from high-throughput screening that bind this allosteric site and can activate PDK1 up to 6-fold. We determined the cocrystal structure of PDK1 bound to its native peptide agonist (PIFtide) and compare this to structures of PDK1 bound to two small-molecule allosteric activators. Finally, we show that these molecules alter PDK1 substrate specificity in vitro and in cells. Paradoxically, these compounds inhibit phosphorylation of downstream kinases despite being activators of peptide substrate phosphorylation in vitro. This work identifies cell active allosteric inhibitors of PDK1 and provide a structural framework for targeting the analogous allosteric site in other protein kinases.
P-98: Carrying Therapeutic Antibodies Across the Blood-Brain Barrier to Treat Glioblastoma
Glioblastoma is the most common and aggressive brain tumor. In Europe alone, 13000 new cases are diagnosed every year, and current therapies increase life expectancy no more than a few months.[1] Some of these treatments rely on the administration of antibodies,[2] which show low permeability across the blood-brain barrier (BBB) and therefore cannot reach the invasive front of the tumor. Our approach envisages the use of peptides that actively cross the BBB (BBB shuttles) to carry antibodies through this barrier.[3] We hypothesize that these conjugates will be distributed throughout the brain, thereby enhancing the effect of the therapeutic antibody. Two main parameters, namely the location and the number of the peptide shuttles, need to be controlled in order to achieve maximal permeability with the lowest perturbation of binding affinity and pharmacokinetics. Here we explore several conjugation strategies to link selected BBB shuttles to two therapeutic antibodies (Avastin® and Erbitux®) currently used to treat glioblastoma. Each of these methods allows the attachment of a controlled average number of peptides to different parts of the antibody. Among the selected methods, classical derivatization of the solvent exposed lysines through different chemistries and modification of the oligosaccharidic chain are explored. In addition, more selective modification of cysteines of interchain disulfide bridges or trans-amination of the N-terminal residue has been employed. We also present the effect on the binding affinity of all these conjugates and their transport on a human BBB cellular model. The in vivo evaluation of the more promising candidates in healthy mouse to show BBB pasage together with the evaluation on a glioblastoma animal model will be presented.
[1] In
[2] Scott, A. M.; Wolchok, J. D.; Old, L. J. Nat. Rev. Cancer, 2012, 12, 278.
[3] Malakoutikhah, M.; Teixido, M.; Giralt, E. Angew. Chem. Int. Ed. 2011, 50, 7998
P-99: Biological Effects of Glyceric Acid and Glucosylglyceric Acid on Skin Cells
Glyceric acid (GA; 2,3-dihydroxypropanoic acid) is found in some plants as a phytochemical, and its biological activities like liver stimulation in dogs and acceleration of ethanol metabolism in rats were reported. We have reported that GA can be produced from glycerol by acetic acid bacterial fermentation, and novel applications of GA and its derivatives can be developed. Of the GA derivatives, glucosylglyceric acid (GGA) is a compatible solute in some bacterial cells, and is considered to protect cells from environmental stresses. Thus these compounds are expected to have further biological activities. In this study, biological effects of GA and GGA on skin cells were evaluated. It was found that GA showed a positive effect on the proliferation of human dermal fibroblasts, whereas GGA increased collagen production by fibroblasts. These results demonstrated that GA and GGA have a variety of functions as a cellular stimulant toward human dermal cells.
P-100: Development of Allosteric Heat Shock Protein 70 (Hsp70) Inhibitors that Disrupt its Protein-Protein Interactions
Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein homeostasis. Hsp70 collaborates with a number of co-chaperones to promote cell survival, especially in response to stress. Because of this activity, Hsp70 has been suggested as a promising anti-cancer target. However, it has proven challenging to develop molecules that target the nucleotide binding cleft on this chaperone. We have recently taken a different approach and characterized a molecule, MKT-077, that binds an allosteric site. We found that this molecule traps Hsp70 in its ADP-bound state by preventing protein-protein interactions with a key nucleotide-exchange factor (NEF). Despite this interesting mechanism, MKT-077 has relatively modest potency in cancer cells (2 to 5 μM) and poor metabolic stability (lifetime of <5 min in liver microsomes). In this project, we aimed to design more potent and stable analogues. Using knowledge of the binding site and the results of a metabolite identification study, we synthesized a novel series of analogues and developed preliminary structure-activity relationships. The most potent molecules had EC50 values of 30 to 200 nM in MDA-MB-231 and MCF7 breast cancer cells. Further, the molecules had lifetimes of >30 minutes in liver microsome studies. The lead compound reduced chaperone clients, such as Akt1 and Raf1, and induced rapid apoptosis. These studies suggest that an allosteric site on Hsp70 can be used to develop selective and potent inhibitors. These studies should aid the development of new anti-cancer therapeutics.
P-101: Mass Spectrometry: A Tool to Expedite Secondary Metabolite Discovery and Functional Characterization
Natural products exhibit a broad range of biological properties and have been a crucial source of therapeutic agents and novel scaffolds. Significant compounds have been discovered from myriad biological sources, making continued exploration an important goal. Although bacterial secondary metabolomes are widely studied, they remain incompletely characterized with a significant portion being elusive to current isolation and characterization strategies. To delve deeper into this untapped chemical space, we have generated an integrated discovery platform that combines bacterial growth perturbation, accurate mass spectrometry (MS and MS2), and informatics for the identification of low abundant and structurally novel compounds from complex biological matrices. In this investigation, we analyzed the secreted metabolome of the extensively studied Actinomycete, Streptomyces coelicolor M145, and discovered a suite of 15 low abundant secondary metabolites. Our MS-based characterization approach was then integrated with microbial imaging mass spectrometry for the identification of spatially resolved signaling molecules. The discovered compounds represent the remaining metabolic potential of both well-studied and new organisms of interest that could be uncovered with this sensitive and robust approach
P-102: Fe(II) Triggered Drug Release Systems and their Application in Cancer
Iron is of central importance to many critical processes in living systems. As such, iron homeostasis is rigorously regulated. This regulation is known to be disturbed in a variety of disease states including malaria and cancer, resulting in aberrantly high levels of reactive iron species. These high levels of Fe(II) are targeted by well known endoperoxide bearing drugs such as artemisinin and arterolane used in the treatment of malaria. This class of molecules functions by producing cytotoxic radical species as a result of selective cleavage of the endoperoxide bond by free Fe(II)-heme (a byproduct of hemoglobin degradation in the digestive vacuole of the parasite). These compounds have exceptional safety profiles, indicating highly selective fragmentation in biological systems. Our group has thus used the selective reactivity of 1,2,4-trioxolanes to develop a novel class of Fe(II) activated drug delivery agents for use in malaria and, potentially, cancer.
1. Vennerstrom JL, Arbe-barnes S, Brun R., Identification of an antimalarial synthetic trioxolane drug development candidate. Nature. (2004)
2. Valecha N, et al. Clin. infect. dis. (2010)
3. Hartwig CL, et al. Investigating the antimalarial action of 1,2,4-trioxolanes with fluorescent chemical probes. J. Med. Chem. (2011)
4. Mahajan SS, et. al. A fragmenting hybrid approach for targeted delivery of multiple therapeutic agents to the malaria parasite. Chem Med Chem (2011)
5. Deu, E., et al. (2013) Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo. PNAS (2013).
P-103: Hydrocarbon Stapled Peptides Targeting Protein-Protein Interactions of Small GTPases
Small GTPases constitute a family of GDP-/GTP-regulated conformational switches which play an important role in controlling a wide range of essential cellular processes. [1] Their aberrant function and regulation are implicated in numerous human diseases, rendering members of this protein family attractive targets in drug discovery. For instance, Rab (Ras-related in brain) GTPases are key regulators of intracellular membrane trafficking, and malfunctions of Rab proteins have increasingly been implicated in a variety of human pathologies including neurodegenerative diseases and various forms of cancer. [2] The direct targeting of small GTPases has frequently proven difficult, as their signaling and regulation is mediated by extensive and relatively flat protein interfaces. We designed a library of hydrocarbon stapled peptides based on crystal structures of Rab proteins bound to their interaction partners to develop inhibitors for Rab protein-protein interactions (PPIs). [3] The resulting peptides show significantly increased Rab affinities including a stapled peptide which selectively binds activated Rab8a and is able to inhibit a Rab8a-effector interaction in vitro. Our results provide the proof of concept for direct targeting of Rab GTPases and suggest that peptide stapling might also enable the development of inhibitors for other small GTPases.
[1] J. Cherfils, M. Zeghouf, Regulation of Small GTPases by GEFs, GAPs, and GDIs, Physiol. Rev. 2013, 93, 269–309.
[2] S. Mitra, K. W. Cheng, G. B. Mills, Rab GTPases implicated in inherited and acquired disorders, Semin. Cell Dev. Biol. 2011, 22, 57–68.
[3] J. Spiegel, P. M. Cromm, A. Itzen, R. S. Goody, T. N. Grossmann, H. Waldmann, Direct Targeting of Rab-GTPase-Effector Interactions, Angew. Chem. Int. Ed. 2014, 53, 2498–2503.
P-104: Chan-Lam Coupling: Synthesis of 1,4-Dihydroquinazolines with Various Boronic Acids Catalyzed by Copper an “Cross-Coupling Reaction”
We have developed copper-catalyzed cross-coupling reactions for the formation of carbon-sulfur bonds. These newly developed methods demonstrate that the conditions of the traditional chan-lam coupling can be improved. We describe the synthesis of S-substituted quinazoline through the cross-coupling of aryl boronic acid and1,4-dihydroquinazoline using [Cu(oAc)2]as the catalyst. All of these methods afford the desired product in good to excellent yields without the use of transition metal or expensive / air sensitive additives. These derivatives were prepared by conventional method. Structure of the compounds has been established by means of 1H-NMR, 13C-NMR, LC-MS, IR.
P-105: De-orphanization of Nuclear Receptors for Drug Discovery
Nuclear receptors are an important and successful drug target class. There are 48 receptors in the human genome and about half are orphans, with no known endogenous or synthetic ligands. We devised screening strategies to de-orphanize and validate RORγ and SF-1 (steroidogenic factor-1). Natural and synthetic antagonists to RORγ inhibit both the formation and the function of Th-17 cells, a central T helper cell involved in autoimmune disease pathogenesis. Ligand discovery at RORγ has therefore provided validation of the target for small molecule discovery for treatment of autoimmune disease. Synthetic SF-1 antagonists inhibit the synthesis of cortisol in primary human adrenal cells and the human adrenocortical cancer cell line H295R. In addition, SF-1 antagonists block proliferation and DNA synthesis in the rat Leydig cell carcinoma line, R2C. Since SF-1 is pathogenically upregulated in adrenocortical tumor cells and is expressed in gonadal steroidogenic cells, SF-1 antagonists have therapeutic potential for treatment of Cushing's syndrome, adrenocortical cancer and sex cord stromal tumors. Our findings demonstrate the power of ligand discovery in positioning orphan nuclear receptors for further drug discovery through exploratory characterization of therapeutic potential.
P-106: Some like it Hot: Determination of Biomolecular Interactions using MicroScale Thermophoresis
The analysis of bio-molecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, not only helps to develop therapeutics or diagnostics techniques, but it also provides important insights into cellular processes. Here we present a novel tether-free technology to analyze the affinity of biomolecular interactions, which is based on the method Microscale Thermophoresis (MST). MST analyzes the directed movement of molecules in optically generated microscopic temperature gradients. This thermophoretic movement is determined by the entropy of the hydration shell around the molecules. Almost all interactions and also any biochemical process relating to a change in size, charge and conformation of molecules alters this hydration shell and is thus detectable by MST. Here we show examples how MST can be used to quantify interactions in a labeled or label-free manner by using non-intrinsic fluorescence (labeled) and the intrinsic tryptophane fluorescence of proteins (label-free). In addition, examples are shown how MST can measure interactions with high selectivity in complex bioliquids like cell lysate or blood serum using a non-intrinsic source of fluorescence.
P-107: Low-Dose Curcumin Stimulates Proliferation, Migration and Phagocytic Activity of Olfactory Ensheathing Cells
One of the promising strategies for neural repair therapies is the transplantation of olfactory ensheathing cells (OECs) that are the glial cells of the olfactory system. We evaluated the effects of low-dose curcumin on the behaviour of OECs to determine if it could enhance the therapeutic potential of OECs. Curcumin, a natural polyphenol compound found in the spice turmeric, is known for its anti-cancer properties via activation of MAP kinases, particularly p38 and ERKs at doses over 10 μM, and often at 50 μM, in cancer cells. In contrast, low-dose curcumin (0.5 μM) applied to OECs strikingly modulated the dynamic morphology, promoted significant proliferation of the OECs and increased the rate of migration up to 4-fold. Most dramatically, low-dose curcumin stimulated a 10-fold increase in the phagocytic activity of OECs. All of these potently stimulated behavioural characteristics of OECs are favourable for neural repair therapies. Importantly, low-dose curcumin gave a transient activation of p38 and ERK MAP kinase pathways, which is in contrast to the high dose curcumin effects on cancer cells in which the MAP kinases tend to undergo prolonged activation. Thus, the low-dose curcumin'“mediated effects on OECs demonstrate cell-type specific activation of MAP kinases. These results constitute the first evidence that low-dose curcumin can modulate the behaviour of olfactory glia into a phenotype potentially more favourable for neural repair and thereby may improve the therapeutic use of OECs for transplantation therapies.
P-108: Targeting Drug-Resistant Bacteria by Genetic Medicinal Chemistry of Thiopeptide Natural Products
With the emergence of life-threatening drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), the need for new antibiotics is greater than ever. Yet, the development of new antibiotics has remained stagnant for nearly 50 years. A promising new class of natural products, called thiopeptides, inhibits the growth of these gram-positive bacteria at nanomolar concentrations. Thiopeptides are exciting as potential antibiotics, because their site of action, the interface between ribosomal protein L11 and 23S rRNA, is distinct from all existing classes of antibiotics. Importantly, these natural products are derived from genetically encoded peptides, which allows for the generation of new thiopeptide analogs by simple mutagenesis. The goal of this project is to develop a continuous and genetically encoded source of new antibiotics against drug-resistant bacterial pathogens by combining the power of recombinant DNA technology with the biosynthesis of natural product antibiotics. We have chosen to study the thiopeptide thiocillin as a model system, because unlike other thiopeptides, thiocillin is produced in the genetically tractable Bacillus cereus strain and is known to tolerate a variety of mutations. If successful, this project will have a significant impact on public health because the development of new antibiotic drugs is necessary to combat the rising problem of emerging drug resistance. Moreover, it will lay the groundwork for generating a new diverse class of natural products to screen for many other therapeutic applications.
P-109: Metabolic Labeling of Cellular DNA with Bio-Orthogonal Functional Groups to Address the Immortal Strand Hypothesis
The distribution of genetic material from parental chromosomes of a stem cell into new daughter cells is the subject of the controversial “immortal strand” hypothesis first proposed by John Cairns in 1975.[1] This hypothesis states that stem cells can minimize mutations in their genomes by dividing their DNA non-randomly. By retaining the same set of template DNA strands, a subset of stem cells might provide a “true copy” of genetic code that is protected from accumulated mutations due to DNA replication. Evidence for and against this hypothesis have been reported for single stem cell populations in vivo,[2] but the immortal strand hypothesis has not yet been tested in a global manner in a whole animal. To evaluate the immortal strand hypothesis, a pulse-chase strategy is employed in zebrafish. Thereby a short pulse of the non-toxic nucleoside analog F-ara-EdU ((2′2S)-2′2-deoxy-2′2-fluoro-5-ethynyluridine)[3] is used to uniformly label DNA strands of all embryonic stem cells during early development. A short pulse of BrdU (5-bromo-2′2-deoxyuridine) is applied during homeostatis to label all actively dividing cells. Visualization of the nucleosides by means of azide-alkyne Huisgen cycloaddition and immunolabeling allows us to track the flow of embryonic versus late-replicated DNA in adult zebrafish. We found that the F-ara-EdU label is metabolically stable in the fish over prolonged time periods in areas containing quiescent and senescent cells. In organs with high cell proliferation we observed F-ara-EdU label dilution over time and no label retention in stem cells. High-resolution imaging provided further evidence of random chromosome segregation in stem cells of zebrafish, arguing against the immortal strand hypothesis in vivo.
[1] J. Cairns, Nature 1975, 255, 197–200.
[2] a) P. Karpowicz, C. Morshead, A. Kam, E. Jervis, J. Ramuns, V. Cheng, D. van der Kooy, J Cell Biol 2005, 170, 721–732; b) M. J. Kiel, S. H. He, R. Ashkenazi, S. N. Gentry, M. Teta, J. A. Kushner, T. L. Jackson, S. J. Morrison, Nature 2007, 449, 238-U210; c) P. M. Lansdorp, Cell 2007, 129, 1244–1247.
[3] A. B. Neef, N. W. Luedtke, PNAS 2011.
P-110: Mitochondrial Complex II Inhibitors/mKATP Activators: Chemical Tools to Probe the Mitochondria and a New Target of Action for Anticancer Drug Discovery?
Pharmacological activation of mitochondrial ATP-sensitive potassium channels (mKATP) result in a range of therapeutically significant effects such as neuroprotection and cardiprotection. Despite intense study the molecular identity of the channel is yet to be determined and no specific chemical tools have been developed to probe the underlying chemical biology of this protein. Significant pharmacological overlap exists between mKATP and mitochondrial complex II (itself a novel target for anticancer drug design). Many small molecule activators of mKATP also inhibit complex II, with suggestions that mKATP itself is composed of mitochondrial proteins such as complex II. The lack of information on the molecular composition of mKATP and the poor specificity of known pharmacological agents (1–300 μM) coupled with its broad relevance to many disease states, highlight the need for more potent small molecule activators to elucidate mKATP molecular features and its relationship to complex II. This presents an opportunity to use chemical tools and synthesis to address a significant unsolved problem at the chemistry, biology, and medicine interface. The natural product atpenin A5 is a potent complex II inhibitor (IC50=10 nM) which is the most potent mKATP activator (1 nM) known. However, its suitability as a chemical probe is hindered by its insolubility in water, low natural abundance, complex structure, and lack of a defined structure-activity relationship (SAR). We describe the development of a synthetic route suitable for the generation of structurally diverse derivatives of atpenin to elucidate SARs for affinity probe design. A simplified analogue suitable for biotinylation that retains activity in the range required of a chemical probe has been identified and preliminary SAR elucidated. Initial bioactivity assay shows potent cytotoxic effects against DU-145 prostate cancer cells.
P-111: Orthogonal Thiol Functionalization for Profiling Ubiquitin and Ubiquitin-Like Activating Enzymes
Transthiolation is a fundamental biological reaction and is utilized by many enzymes involved in the conjugation of ubiquitin and ubiquitin-like proteins. However, tools that enable selective profiling of this activity are lacking. Transthiolation requires cysteine-cysteine juxtaposition therefore a method that enables irreversible “stapling” of proximal thiols would facilitate the development of novel probes that could be used to profile this activity. We demonstrate biocompatible chemistry that enables the orthogonal functionalization of cysteines within proteins. We use our method to develop a new class of activity-based probe that profiles transthiolation activity of human ubiquitin E1 activating enzyme. The probe can be used in vitro, in situ and is compatible with competitive activity-based protein profiling. Furthermore, we anticipate that this method of orthogonal thiol functionalization will have broad utility by enabling simple redox-stable cross linking of proximal cysteines in general.
P-112: Probing Covalent Regulatory Enzymes using Homogenous Detection of Nucleotide Products
Post-translational modifications such as phosphorylation, methylation and glycosylation embellish most eukaryotic proteins, controlling their localization, enzymatic activity, and interaction with other proteins in signaling networks. The enzymes that catalyze covalent modification reactions comprise a significant fraction of the human proteome and, not surprisingly they are proving to be a rich source of drug targets. Protein kinases are one of the most intensively screened target classes, methyltransferases are being intensively targeted for diverse diseases with an epigenetic component and glycosyltransferases are emerging targets for cancer and metabolic diseases. Development of high-throughput screening assays for these enzymes can be challenging because of the diversity of acceptor modified products. To enable more efficient probing of covalent regulatory enzymes, we developed highly selective antibodies for the invariant nucleotide products of key group transfer reactions; e.g., ADP for kinases. We incorporated these antibodies into homogenous, “mix-and-read” assay formats with commonly used fluorescent readouts (FP, TR-FRET, FI) and commercialized them, starting in 2006, as the Transcreener® HTS assay platform. Transcreener Assays have facilitated screening and profiling of hundreds of drug targets by allowing the use of a single assay for all members of a family of group transfer enzymes, regardless of the acceptor substrates used. They are designed to allow initial velocity detection, with a dynamic range that can be tuned to accommodate different donor substrate concentrations. Here we summarize recent developments with the Transcreener platform, including formatting the UDP and GDP assays for TR-FRET and validation with therapeutically relevant glycosyltransferases. We also provide examples of how the platform has been extended to other types of enzymes, including hydrolases (e.g., GTPases, phosphodiesterases) and synthetases/ligases (e.g., SUMO ligases).
P-113: Discovery and Validation of Novel Human L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) Inhibitors
Human L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) is the first and rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is a potential target to help prevent secondary complications of type II diabetes. GFAT catalyzes the irreversible reaction between L-glutamine and D-fructose-6-phosphate to produce L-glutamate and D-glucosamine-6-phosphate. There are only a handful of known inhibitors available to probe the enzyme and the majority of these are toxic, non-specific, or substrate analogs. Following a high-throughput screening campaign of three-thousand nine-hundred and fifty bioactive compounds assayed for GFAT inhibitory activity, three novel classes of compounds were identified as GFAT inhibitors: aminothiazoles, pyridinones and quinones. To validate the lead compounds as inhibitors of native GFAT and determine their cell permeability, a cell based assay was developed. The end-product of the HBP, UDP-GlcNAc, was detected in cell culture with UPLC-TOF-MS utilizing a ZIC®-HILIC column. The leads, alloxan, lapachol and amrinone all displayed a significant decrease in UDP-GlcNAc in cell culture.
P-114: Preparation of RAFT-Based Fluorescent Glycopolymers and Their Use in Profiling Pseudomonas aeruginosa's Carbohydrate-Binding Patterns
As a critical step of the persistent Pseudomonas aeruginosa infection in cystic fibrosis lungs, the Gram-negative bacteria use two lectins, LecA and LecB, together with other adhesins to bind specific carbohydrates on the surface of respiratory tract to start colonization. Blocking this binding with inhaled anti-adhesive carbohydrates offers a promising treatment for the infection. Due to the low binding affinities of many lectins towards singular sugar epitopes, multivalent glycoconjugates are often required to investigate bacterial binding with carbohydrates. Here we present the syntheses of a group of fluorescent tri-component glycopolymers and their use in studying the carbohydrate-binding of P. aeruginosa's. Prepared via reversible addition- fragmentation chain-transfer (RAFT)-based polymerizations, the resultant glycopolymers contained three different monomers: N-(2-hydroxyethyl) acrylamide, N-(2-aminoethyl) methacrylamide hydrochloride, and N-(2-glyconamidoethyl)-methacrylamides possessing different pendant monosaccharides. Fluorescent glycopolymers with controlled molecular weight distribution were obtained after post-modification with fluorescence tags. Using the synthesized polymers together with commercial glycopolymers, the monosaccharide-binding pattern of a panel of P. aeruginosa strains and clinical isolates was profiled. As revealed by fluorescence microscopy, fluorimetry, flow cytometry, among the nine monosaccharides tested, α-D-galactose, β-D-N-acetylgalactosamine, and β-D-galactose-3-sulfate demonstrated strong binding with all strains and isolates. However, within an isogenic bacterial population, only a small percentage (<2%) showed observable binding with the glycopolymers, suggesting a dramatic phenotypic heterogeneity of the bacteria. The localizations of the bacterial lectins were also visualized by transmission electron microscopy. This strategy also holds promise for investigating the carbohydrate-binding of other human pathogens, like Staphylococcus aureus, Helicobacter pylori, and Escherichia coli.
P-115: Bivalent Compounds for Selective Enzyme Inhibition
Chemical approaches for modulating protein function utilize small molecules that alter the function of target proteins. The most prominent examples of chemical approaches for modulating protein function incorporate small molecules to inhibit enzymatic activity. However, a prevailing challenge in the development of targeted inhibitors is achieving selective enzyme inhibition. For example, the active site of protein families such as kinases and histone deacetylases, are highly conserved between family members and consequently are difficult to selectively target. Methods to produce selective enzyme inhibitors would be invaluable for furthering understanding of protein function and manipulating proteins for therapeutic applications. We have explored two chemical genetic approaches that utilize bivalent compounds for selective enzyme inhibition, each uniquely utilizing the SNAP-tag. The bivalent strategies incorporate an active-site competitive ligand tethered to a secondary ligand binding domain. Fluorometric kinetic assays and chemical proteomic analyses were used to evaluate binding. The bivalent display of ligands was found to increase potency and selectivity of inhibition, though this phenomenon was ligand-dependent. We seek to develop bifunctional molecules as a new class of selectively binding enzyme inhibitors and as tools for studying signal transduction pathways.
P-116: Development of a Caged HDAC Inhibitor with 7-diethylaminocoumarin-Type Photolabile Protecting Group
Histone deacetylases (HDACs) are known to catalyze hydrolysis of acetyl groups from histone lysine residues, and play roles in the regulation of gene expression. Histone acetylation is considered to be involved in the differentiation of stem cells. Therefore HDACs are expected as a new target for the regenerative medicine. However HDACs are ubiquitous in the human body. To study regulation mechanisms of HDACs in detail, the technique that can control the HDAC activity on demand is indispensable. In this study, we designed and synthesized a caged HDAC inhibitor, AC-SAHA, by introducing a photolabile protecting group derived from 7-diethylaminocoumarin into the activity site of suberoylanilide hydroxamic acid (SAHA/ ZOLINZA®), a HDAC inhibitor used in clinical. By an HPLC analysis, it was confirmed that AC-SAHA released SAHA upon exposure to visible light (400–430 nm). The photolytic quantum yield (φ) and φɛ (ɛ, extinction coefficient) of AC-SAHA was determined as 0.0048 and 56, respectively. As a result, it was suggested that AC-SAHA would be a HDAC inhibitor that is spatially and temporally controllable from the outside of the system. Spatiotemporal control of histone acetylation potentially leads to the development of cell manufacture technologies by regulation of stem cell differentiation.
P-117: Chemical Rescue of Malaria Parasites Lacking an Essential Plastid Organelle
Malaria caused by Plasmodium spp parasites has an enormous disease burden that disproportionately affects the world's poorest and youngest. New anti-malarials with novel drug mechanisms are desperately needed in the face of existing or emerging drug resistance to all available therapies. Investigation of Plasmodium biology offers both the potential for important biomedical impact and an opportunity to explore fascinating eukaryotic biology. Given the challenges of genetic and other approaches to studying this complex organism, the development of chemical tools will be especially critical in pushing forward basic research. Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development.
P-118: Comparison of Water Soluble Perylene-PEG Dye with Typical Labeling Strategies on Tracking Mesenchyme Stem Cells
With the development of stem cell transplantation and intravital microscopy, there is a demand for low toxicity cell-labeling probes for in vitro and in vivo studies. Water-soluble perylene tetracarboxylic acid bisimides (PBIs) with terminally linked polyethylene glycol have been synthesized as a low toxicity cytoplasm probe. These PBI probes have bright fluorescent yields and have outstanding water solubility. In order to assess the feasibility of PBI-PEG for stem cell tracking, PBI-PEG labeling was applied on human mesenchymal stem cells. In comparison to CFSE labeling and EGFP transfection, PBI-PEGs shows better MSC-labeling rates determined by Flow Cytometry Analysis. Furthermore, PBI-PEG probes were found to have lower cell toxicity on cell proliferation and lower retention in MSCs. Only 2.62% of cells were detected with fluorescence at 72 h after labeling with an initial efficiency as high as 98.5%. These results suggest that PBI-PEG probes are effective cytoplasm dye and suitable for short-term adult stem cell tracking.
P-119: Probing Lipid Membranes with the Quartz-Crystal Microbalance and Dual-Polarization Interferometry
The adsorption, fusion, and rupture of lipid vesicles on charged surfaces provide a useful way to study clean lipid bilayers at the solid-aqueous interface. These solid-supported bilayers provide a convenient platform for mimicking biological cell membranes. We use the Quartz-Crystal Microbalance with Dissipation monitoring (QCM-D) along with Dual-Polarization Interferometry (DPI) to characterize the formation of these systems in real time. In particular, we show that probing the bilayers with both of these techniques allows us to indirectly gauge the degree the hydration within these assemblies. We present some data on our recent work using QCM-D and DPI in understanding the fusion and rupture process on silica surfaces, in addition to some preliminary work on the interaction and integration of small molecular probes (fluorescent dyes) within the membrane for applications in live cell imaging.