Expanding Chemical Probe Space: Quality Criteria for Covalent and Degrader Probes

Abstract

Within druggable target space, new small-molecule modalities, particularly covalent inhibitors and targeted degraders, have expanded the repertoire of medicinal chemists. Molecules with such modes of action have a large potential not only as drugs but also as chemical probes. Criteria have previously been established to describe the potency, selectivity, and properties of smallmolecule probes that are qualified to enable the interrogation and validation of drug targets. These definitions have been tailored to reversibly acting modulators but fall short in their applicability to other modalities. While initial guidelines have been proposed, we delineate here a full set of criteria for the characterization of covalent, irreversible inhibitors as well as heterobifunctional degraders ("proteolysis-targeting chimeras", or PROTACs) and molecular glue degraders. We propose modified potency and selectivity criteria compared to those for reversible inhibitors. We discuss their relevance and highlight examples of suitable probe and pathfinder compounds. ■ SIGNIFICANCE• High-quality chemical probes are important tools which allow generation of robust and reproducible insights into the cellular function of proteins of interest. • Covalently acting small molecules and small-molecule protein degraders extend druggable space beyond that fraction of the proteome which is targetable with reversibly acting ligands. • When applied, the proposed set of quality criteria increases the likelihood that cell biology studies provide robust insights of high basic and translational relevance.

Structural Fusion of Natural and Synthetic Ligand Features Boosts RXR Agonist Potency

“…24 In systematically probing the SAR of 17 as an RXR agonist, we previously found that a 3,5-bis-trifluoromethyl substitution pattern was highly favored on the 5-phenyl residue and boosted RXR agonism to double-digit nanomolar potency (24, 24 Table 2). Incorporation of the acrylic acid motif in the corresponding derivative 25 did not alter activity on RXR compared to 24, while additional introduction of the α-methyl substituent (26) was accompanied by a slight 2-fold gain in potency. Fusion with the favored trans-cyclopropanecarboxylic acid of rac-22 in rac-27, however, remarkably enhanced potency by >10-fold compared to that of 24 and yielded an RXR agonist with an unprecedented single-digit nanomolar activity in cellular setting.…”

“…This activity and property profile of rac-27 is superior to available RXR agonist tools and meets quality criteria for chemical probes. 26 rac-27 (JP3000) and the negative control compound rac-28 (JP3001) are available through the chemical probe program of the Structural Genomics Consortium (SGC). 27 ■ CONCLUSIONS RXR favors fatty acid mimetic 29 ligands, mimicking the Lshaped geometry of the natural ligand 9-cis retinoic acid and related retinoids, which is, for example, established in the drugapproved RXR agonist bexarotene.…”

“…Based on the acrylic acid’s PAINS character, we also evaluated its bioisosteric replacement, which eventually resulted in the RXR agonist rac -27 exhibiting a remarkable single-digit nanomolar potency on all RXRs in cellular setting. Favorable physicochemical properties, low toxicity, and >100-fold selectivity in the nuclear receptor family further highlighted rac -27 as a chemical probe , to study RXR in health and disease.…”

Structure-Guided Design of a Domain-Selective Bromodomain and Extra Terminal N-Terminal Bromodomain Chemical Probe

“…The utility of a chemical probe in target validation is directly related to its quality, with important publications detailing requirements to aid researchers. − Taking inspiration from this, the BET BD1 chemical probe requirements were defined as follows: Structural differentiation to internally developed (GSK778 ( 8 ) and GSK789 ( 9 )) and reported BET BD1 chemical series (Figure ) was critical. Activity against BET BD1 as judged by a TR-FRET assay pIC 50 > 7.5 was required, together with an aspirational target of 1000-fold selectivity over BET BD2.…”

Targeting LC3/GABARAP for degrader development and autophagy modulation

“…33-36 Recently also first standards for covalent inhibitors and degrader molecules have been defined. 37…”