From Phenotypic Hit to Chemical Probe: Chemical Biology Approaches to Elucidate Small Molecule Action in Complex Biological Systems

Abstract

Biologically active small molecules have a central role in drug development, and as chemical probes and tool compounds to perturb and elucidate biological processes. Small molecules can be rationally designed for a given target, or a library of molecules can be screened against a target or phenotype of interest. Especially in the case of phenotypic screening approaches, a major challenge is to translate the compound-induced phenotype into a well-defined cellular target and mode of action of the hit compound. There is no “one size fits all” approach, and recent years have seen an increase in available target deconvolution strategies, rooted in organic chemistry, proteomics, and genetics. This review provides an overview of advances in target identification and mechanism of action studies, describes the strengths and weaknesses of the different approaches, and illustrates the need for chemical biologists to integrate and expand the existing tools to increase the probability of evolving screen hits to robust chemical probes.

Accelerating reaction generality and mechanistic insight through additive mapping

“…This approach has the potential to simultaneously uncover and modulate hitherto unrecognized biomolecular mechanisms. It has proven invaluable in developing powerful therapeutic approaches that leverage unappreciated mechanisms of action and improved indications recalcitrant to traditional drug discovery techniques (20)(21)(22)(23). We reasoned that complex reactions can be likened to complex biological systems, so there would be enormous benefit to applying this concept to the study and improvement of the former.…”

Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog Pathway Inhibitor-1

“…Target deconvolution of small molecule hits from phenotypic screening campaigns remains a major challenge and there is unfortunately no one-size fits all approach. 25 Here, we report a PROTAC-based strategy for target identification which we applied to discover the cellular targets of Hedgehog pathway inhibitor HPI-1. Since their discovery, PROTACs have found widespread use due to their unique mechanism of action, as probes for target validation and off-target discovery, and as drugs.…”

“…24 The majority of reported molecules have been identified through phenotypic screens for the Hedgehog pathway, and it has proven highly challenging to unravel the cellular target and molecular mechanisms of these hit compounds, severely limiting their use as chemical probes or therapeutic leads. 25–32…”

“…24 The majority of reported molecules have been identified through phenotypic screens for the Hedgehog pathway, and it has proven highly challenging to unravel the cellular target and molecular mechanisms of these hit compounds, severely limiting their use as chemical probes or therapeutic leads. [25][26][27][28][29][30][31][32] Exemplary of this is Hedgehog Pathway Inhibitor (HPI-1), a dihydropyridine molecule discovered by Hyman et al as a robust downstream Hh signaling inhibitor, with anti-cancer properties. 26,33,34 Often referred to as a GLI inhibitor, [35][36][37] its cellular target has remained elusive for many years.…”

Induction of Genes Implicated in Stress Response and Autophagy by a Novel Quinolin-8-yl-nicotinamide QN523 in Pancreatic Cancer

“…Phenotypic screening campaigns are especially suited to produce multi-targeted agents with polypharmacology. − A disadvantage of phenotypic screen lies in the challenges in establishing the true direct target(s) of the identified lead inhibitors. − Numerous technologies have been established throughout the years for target identification. Unfortunately, all these methods demand extensive time, resources, and sophisticated instrumentation .…”