SLAS DISCOVERY: Advancing the Science of Drug Discovery

A typical modern high-throughput screening (HTS) operation consists of testing thousands of chemical compounds to select active ones for future detailed examination. The authors describe 3 clustering techniques that can be used to improve the selection of active compounds (i.e., hits). They are designed to identify quality hits in the observed HTS measurements. The considered clustering techniques were first tested on simulated data and then applied to analyze the assay inhibiting Escherichia coli dihydrofo-late reductase produced at the HTS laboratory of McMaster University.

An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CL^pro) by virtual screening correlating with surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) technologies-based assays. The authors screened 81,287 small molecular compounds against SPECS database by virtual screening; 256 compounds were subsequently selected for biological evaluation. Through SPR technology-based assay, 52 from these 256 compounds were discovered to show binding to SARS-CoV 3CL^pro. The enzymatic inhibition activities of these 52 SARS-CoV 3CL^pro binders were further applied to FRET-based assay, and IC₅₀ values were determined. Based on this integrated assay platform, 8 new SARS-CoV 3CL^pro inhibitors were discovered. The fact that the obtained IC₅₀ values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants (K_Ds) assayed by SPR implied the reliability of this platform. Our current work is hoped to supply a powerful approach in the discovery of potent SARS-CoV 3CL^pro inhibitors, and the determined inhibitors could be used as possible lead compounds for further research.

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

Many companies possess a compound collection consisting of purified compounds and of unpurified products from combinatorial libraries. Using commercial and proprietary compounds as examples, this report provides clear examples of the significant impact purification can have on the activity observed for a compound and highlights the need to retest the purified compounds prior to creating structure-activity relationships. Crude mixtures made with commercial compounds led to an increase in the number of false positives in the SXR-GAL4 assay as compared with their pure and purified counterparts. An examination of proprietary compounds in an HIV assay resulted in the purification of 61 active crude synthetic mixtures. Of these 61 compounds, 32 were 5-fold less active and 2 were 5-fold more active after purification. This report details a semiautomated process developed and implemented for cherry-picking, tracking, and selectively purifying compounds found active in high-throughput screening campaigns.

Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.

Fluorescence polarization is a screening technology that is radioactivity free, homogeneous, and ratiometric. The signal measured with this technology is a weighted value of free and bound ligand. As a consequence, saturation curves are accessible only after calculation of the corresponding concentrations of free and bound ligand. To make this technology more accessible to assay development, the authors propose a simple mathematical model that predicts fluorescence polarization values from ligand and receptor total concentrations, depending on the corresponding dissociation constant. This model was validated using data of Bodipy-NDP-αMSH binding to MC₅, obtained after either ligand saturation of a receptor preparation or, conversely, receptor saturation of a ligand solution. These experimental data were also used to calculate the actual concentration of free and bound ligand and receptor and to obtain pharmacological constants by Scatchard analysis. A general method is proposed, which facilitates the design of fluorescence polarization binding assays by relying on the representation of theoretical polarization values. This approach is illustrated by the application to 2 systems of very different affinities.

The nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-γ) and to inflammatory processes by deacetylating the transcription factor NF-κB. First, modulation of SIRT1 activity affects lipid accumulation in adipocytes, which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second, activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening, the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes, as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents, reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition, the same compounds have anti-inflammatory properties, as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). In contrast, a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes, a feature often linked to insulin sensitization.

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (γ-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of γ-AmNS-PPi, which has higher intrinsic fluorescence than (γ-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 μM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC₅₀ below 100 μM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.

The authors have designed high-throughput screens to identify compounds that promote or inhibit terminal differentiation of primary human epidermal keratinocytes. Eleven known inhibitors of signaling pathways and approximately 4000 compounds of diverse structure were screened using an In-Cell Western system based on immunofluorescent staining of the terminal differentiation marker, involucrin. Staurosporine, a nonspecific protein kinase C inhibitor, and H89, a protein kinase A inhibitor, promoted expression of involucrin. Conversely, U0126, a MEK inhibitor, and SAHA or SBHA, 2 histone deacetylase inhibitors, reduced the expression of involucrin during calcium-induced stratification. In addition, the authors found 1 novel compound that induced keratinocyte differentiation and 2 novel compounds that were inhibitory to calcium-induced differentiation. The differentiation-inducing compound also inhibited growth of a human squamous cell carcinoma line by stimulating both differentiation and apoptosis. Because the compound affected the tumor cells at a lower concentration than primary keratinocytes, it may have potential as an antitumor therapy.

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The routine use of generic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in our hands led to the discovery of very similar and often not very selective molecules. Therefore, to increase the chances to discover novel chemical scaffolds, a side-by-side comparison between the DiFMUP assay and a chip-based mobility shift assay with a specific phosphopeptide was performed, on 1 PTP, using a focused set of compounds. Assay robustness and sensitivity were comparable for both the DiFMUP and mobility shift assays. The off-chip mobility shift assay required a longer development time because of identification, synthesis, and characterization of a specific peptide, and its cost per point was higher. However, although most potent scaffolds found with the DiFMUP assay were confirmed in the mobility shift format, the off-chip mobility shift assay led to the identification of previously unidentified chemical scaffolds with improved druglike properties.

The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the β-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un-cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un-cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, followed by removal of unbound ampicillin. The substrate for the TP, un-cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin scintillation proximity assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1bcatalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme assay, it has the potential to detect novel, non-β-lactam TP inhibitors and could lead to the discovery of new antibacterial agents.

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.

Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC₅₀ values close to those reported in the literature for tritiated serotonin uptake. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Ź of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a “classical” assay for serotonin uptake are all reported. The assay can also be read in “high-content” mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The K_m values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH₄Cl, 3.25 mM for MgCl₂, and 2.5 mM for adenosine triphosphate. The K_m value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25° C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z′ factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 μg/ml concentration. The IC₅₀ values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 μM and 30 μM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z′ and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.