Research

The concept of a “chemical probe” emerged from sequencing of the human genome, which revealed a large number of uncharacterized potential therapeutic targets. Coupled with advances in the high throughput screening (HTS) of compound libraries and technology-enabled medicinal chemistry to rapidly follow-up on hits, as well as the emergence of chemical biology, the discovery and development of individual small molecule modulators of uncharacterized protein targets in academia became more realistic.1.

In 2005, the NIH launched the Molecular Libraries Program with the goal of developing quality small molecules probes to interrogate biological pathways and new therapeutic hypotheses.2

During the course of this decade-long program much was learned, often by trial and error, regarding which chemical, pharmacological, biological, and physical properties and characteristics determine chemical probe quality. Vanderbilt was a participant in the Molecular Library Screening Center Network (MLSCN) and Molecular Library Probe Production Centers Network (MLPCN) phases of this NIH initiative and contributed to the development of a number of quality chemical probes, which in some instances led to pre-clinical leads for further drug development (see Section 2. Chemical probes and drug discovery at Vanderbilt University).

A high quality chemical probe, which is typically a suitable small molecule, enables the unbiased interpretation of biological experiments in cell and animal models (for example to elucidate the role of a protein target disease and healthy cells or tissue). To best achieve informative experimental outcomes, a probe requires high potency (typically <100 nM), protein target selectivity (typically >100-fold over other proteins) to avoid off-target effects, and robust evidence of target engagement using chemical biology tools (Figure 1).[3]

Ideally, an optimized chemical probe is only one member of a series of structurally related compounds of varying potency and biological effects. An associated tractable Structure-Activity Relationship (SAR) provides further confidence in the on-target nature of compound action, and also often produces a structurally related negative control molecule. Ex vivo experiments such as experiments in cells, tissue samples, or organoids further require meeting minimum requirements for compound solubility and permeability. In vivo animal studies typically require the small molecule to be further evaluated and/or optimized for additional drug-like properties. Overall, the process of chemical probe development usually requires collaboration between pharmacologists, biologists, and chemists to produce a high-quality molecule.

While many useful drugs can also function as effective probes of their respective biological systems, the goals behind the discovery of a chemical probe molecule versus discovery of a drug can be quite different (Figure 2).[1]

Indeed, the criteria to which they are held can vary, since these criteria are carefully aligned to their respective purposes. For example, the therapeutic effect of a drug may rely on polypharmacology (e.g., by modulating multiple protein receptors), meaning that they do not necessarily need to exhibit the same selectivity profile as a chemical probe used to examine and validate a new biological effect. Conversely, chemical probes do not necessarily need to fully meet the rigorous and stringent pharmacokinetic and toxicological requirements needed for drugs intended for clinical studies with human subjects. Overall, chemical probe discovery often precedes and informs more costly and financially incentivized drug development, which ultimately aims to achieve extremely high confidence in efficacy and safety windows before declaring a single entity an Investigative New Drug (IND) for clinical development.

Since the early NIH-supported probe development initiatives, much has been learned and shared amongst and between academic and industrial investigators.[4-5]

Most notable is the demonstrated impact that a quality probe, and the development of associated chemical biology techniques and methods to characterize the probe in cell and animal models, can have on drug discovery. Additional confidence in, and validation of, a new target or therapeutic concept using a probe of very high quality often serves to de-risk a novel approach, enabling and encouraging additional investment of resources. A key asset at Vanderbilt is provided by accumulated genetic data associated with disease phenotypes and maintained in BioVU. When associated with a gene-based phenotype, the validation of targets with a chemical probe can position a team of investigators well for a successful drug discovery and development campaign.

While the requirements for drug development and eventual clinical assessment are well-defined and ultimately require FDA approval, the requirements of a chemical probe can be flexible and determined by investigator’s purpose of a “chemical probe”. Not all chemical probes are destined to test a therapeutic in an animal model. For example, staurosporine (a natural product) is a non-selective kinase inhibitor used by investigators in specific situations. Early consideration of purpose/function of any small molecule is critical to developing a lead optimization program whether it’s a pre-clinical lead optimization or chemical probe for proof-of-concept in animal studies or use in a structural biology experiment (see Section 3. Examples of development pathways for Chemical Probe and Drug Development).

1. Arrowsmith, C. H.; Audia, J. E.; Austin, C.; Baell, J.; Bennett, J.; Blagg, J.; Bountra, C.; Brennan, P. E.; Brown, P. J.; Bunnage, M. E.; Buser-Doepner, C.; Campbell, R. M.; Carter, A. J.; Cohen, P.; Copeland, R. A.; Cravatt, B.; Dahlin, J. L.; Dhanak, D.; Edwards, A. M.; Frye, S. V.; Gray, N.; Grimshaw, C. E.; Hepworth, D.; Howe, T.; Huber, K. V. M.; Jin, J.; Knapp, S.; Kotz, J. D.; Kruger, R. G.; Lowe, D.; Mader, M. M.; Marsden, B.; Mueller-Fahrnow, A.; Muller, S.; O’Hagan, R. C.; Overington, J. P.; Owen, D. R.; Rosenberg, S. H.; Roth, B.; Ross, R.; Schapira, M.; Schreiber, S. L.; Shoichet, B.; Sundstrom, M.; Superti-Furga, G.; Taunton, J.; Toledo-Sherman, L.; Walpole, C.; Walters, M. A.; Willson, T. M.; Workman, P.; Young, R. N.; Zuercher, W. J., The promise and peril of chemical probes. Nature Chemical Biology 2015, 11, 536-541.
2. Schreiber, S. L.; Kotz, J. D.; Li, M.; Aube, J.; Austin, C. P.; Reed, J. C.; Rosen, H.; White, E. L.; Sklar, L. A.; Lindsley, C. W.; Alexander, B. R.; Bittker, J. A.; Clemons, P. A.; De Souza, A.; Foley, M. A.; Palmer, M.; Shamji, A. F.; Wawer, M. J.; McManus, O.; Wu, M.; Zou, B. Y.; Yu, H. B.; Golden, J. E.; Schoenen, F. J.; Simeonov, A.; Jadhav, A.; Jackson, M. R.; Pinkerton, A. B.; Chung, T. D. Y.; Griffin, P. R.; Cravatt, B. F.; Hodder, P. S.; Roush, W. R.; Roberts, E.; Chung, D. H.; Jonsson, C. B.; Noah, J. W.; Severson, W. E.; Ananthan, S.; Edwards, B.; Oprea, T. I.; Conn, P. J.; Hopkins, C. R.; Wood, M. R.; Stauffer, S. R.; Emmitte, K. A., Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes. Cell 2015, 161, 1252-1265.
3. Bunnage, M. E.; Chekler, E. L. P.; Jones, L. H., Target validation using chemical probes. Nature Chemical Biology 2013, 9, 195-199.
4. Bunnage, M. E.; Gilbert, A. M.; Jones, L. H.; Hett, E. C., Know your target, know your molecule. Nature Chemical Biology 2015, 11, 368-372.
5. Garbaccio, R. M.; Parmee, E. R., The Impact of Chemical Probes in Drug Discovery: A Pharmaceutical Industry Perspective. Cell Chemical Biology 2016, 23, 10-17.