Applications of Biophysics in Early Drug Discovery

This special collection originated from a desire to illustrate the existing and developing use of biophysical methods in the identification and characterization of molecules in early drug discovery. Many of these methods can be applied to understanding the binding interactions of target proteins with small molecules and other modalities such as peptides, proteins, antibodies, sugars, and nucleic acids.

Biophysical methods complement traditional biochemical approaches, with these methods ideally positioned to (1) help define protein function prior to assay development, (2) characterize tool ligands identified from literature sources, (3) identify hits in focused screens, (4) evaluate hits in an orthogonal manner post primary hit finding, and (5) contribute to full mechanistic characterization of prioritized hits and leads.

Access to a toolbox of biophysical methods provides the ability to obtain structural, kinetic, and thermodynamic profiles using highly sensitive detection methods, which can often be applied rapidly and efficiently during early drug discovery.

The application of biophysical methods has been utilized following high-throughput screening (HTS),^1,2 for fragment-based lead generation,^3,4 and not only for isolated protein assays but also in cellular systems. ⁵ The increasing use of cryo-electron microscopy ⁶ has also provided access to structural information on large protein complexes that may have been problematic using more traditional x-ray methods.

In this issue, we have tried to capture articles exemplifying the adaptation of existing technologies, alongside examples of developing technologies that have been applied successfully to identify or characterize hits.

Almeida et al. ⁷ provide examples of the use of nuclear magnetic resonance (NMR) reporter assays to characterize weak binding, often associated with fragment-based drug discovery. They provide guidelines on establishing these assays, as well as comparing the performance with other commonly used methods.

At the opposite end of the size scale, Martinez et al. ⁸ describe the use of a developing method, employing graphene bioelectronic sensing technology, to measure anti-MCL1 antibody binding to monocarboxylate transporter 1 (MCT1) captured on the graphene biosensor. This represents an important development in detecting binding to native integral membrane protein targets in a physiologically relevant membrane environment.

Gradl et al. ⁹ demonstrate a biophysical method that can truly be high-throughput, illustrating the application of thermal shift assays to screen more than 400,000 compounds. This work led to the identification of a novel series of SMYD3 inhibitors and illustrates the range of additional structural, biophysical, and biochemical methods employed to fully characterize a selective nanomolar compound.

Increasing throughput is also the objective in the work described by Kartal et al. ¹⁰ They describe a new grating-coupled interferometry-based approach consisting of an injection method using a single sample, which potentially offers increased throughput compared with sequential injection methods.

Fluorescence-based methods have been an established tool in the biophysical and biochemical toolbox for many years. It has been relatively recent, however, that temperature-related intensity changes associated with fluorescent signals have themselves been used to monitor binding events. Jeridi et al. ¹¹ illustrate the application of this method to screen fragments versus the target MAPK/ERK kinase 1 (Mek1). They demonstrate the utility of the method to identify compounds that could be mistaken as false-positive hits in other methods.

The theme of utilizing light continues in the article from Cho and Dalby. ¹² They provide an overview of the use of luminescence methods, including NanoBRET (nano-bioluminescence resonance energy transfer) and ALPHA (Amplified Luminescent Proximity Homogeneous Assay) technologies, in detecting and analyzing the molecular biophysical properties of complexes in environments such as lysates and cells.

We move away from light and embrace the detection of mass in the articles from Simon et al. ¹³ and Scholle et al. ¹⁴ showing the high potential of mass spectrometry (MS)-based applications in HTS activity and affinity screens. Simon et al. ¹³ describe the use of acoustic droplet ejection (ADE)–open port interface (OPI)–MS. They present the optimization of the method and show results from a single-concentration screen using a test set of more than 5000 compounds alongside comparison with previous MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) methods. Scholle and colleagues ¹⁴ applied a novel SAMDI-ASMS method (self-assembled monolayers of alkanethiolates affinity selection MS) enabling them to run a 100,000-ligand screen for human rhinovirus 3C protease (HRV3C) within 8 h. Generated hits were validated by additional biophysical and functional protease assays.

We would like to thank everyone who has contributed to this special collection and hope that it inspires opportunities to combine biophysical methods alongside traditional biochemical or cell-based approaches, as we believe the synergy derived from working in this way is a valuable addition to early drug discovery.