Fragment-based covalent ligand discovery

Abstract

Targeted covalent inhibitors have regained widespread attention in drug discovery and have emerged as powerful tools for basic biomedical research. Fueled by considerable improvements in mass spectrometry sensitivity and sample processing, chemoproteomic strategies have revealed thousands of proteins that can be covalently modified by reactive small molecules. Fragment-based drug discovery, which has traditionally been used in a target-centric fashion, is now being deployed on a proteome-wide scale thereby expanding its utility to both the discovery of novel covalent ligands and their cognate protein targets. This powerful approach is allowing 'high-throughput' serendipitous discovery of cryptic pockets leading to the identification of pharmacological modulators of proteins previously viewed as "undruggable". The reactive fragment toolkit has been enabled by recent advances in the development of new chemistries that target residues other than cysteine including lysine and tyrosine. Here, we review the emerging area of covalent fragment-based ligand discovery, which integrates the benefits of covalent targeting and fragment-based medicinal chemistry. We discuss how the two strategies synergize to facilitate the efficient discovery of new pharmacological modulators of established and new therapeutic target proteins.

Fig. 1. The roadmap for fragment-based covalent ligand discovery. (A) State-of-the-art chemoproteomic strategies helps to systematically unveil potential ligandable sites in disease-associated targets. Cells are treated with reactive fragments (biased towards thiols (Cys), amines (lysine) and phenols (tyrosine)) and then chemoproteomics allows identification of proteins and reaction sites. (B) Fragment-based covalent ligand screening identified covalent fragment hits, which can be evolved into a more potent, selective, biocompatible and drug-like ligands by iterative elaboration and optimization. (C) Fragment-based ligand discovery pipeline holds great promise as an initial ligand-discovery approach that can be elaborated to make bivalent molecules that can recruit other enzymes including E3s for PROTACs etc.

Fig. 2. The structures of representative well-characterized electrophilic fragments identified from target-based screening strategies in recent years. (A) KRAS-G12C allele-specific covalent fragment (6H05) identified from tethering screen, which was further elaborated to compound 12. This inspired numerous groups to develop further optimized inhibitors, within which AMG510³³ and MRTX849 successfully entered clinical trials. (B) Compound 5 targets the active cysteine (C885) of HOIP. (C) OTUB2-COV-1 targets the active cysteine (C51) of OTUB2 and NUDT7-COV-1 target C73 of NUDT7. (D) Sulfopin targets the active cysteine of Pin1 (C113). (E) Representative covalent fragment scaffolds target the active cysteine (C145) of SARS-COV-2 main protease (M^pro).

Fig. 3. State-of-the-art chemoproteomic approaches for reimaging druggable proteome and selectivity profiling of covalent fragments. (A) Activity-based protein profiling (IsoTOP-ABPP and TMT-ABPP) using isotope-labeled probes or isotopic TMT labeling agents for multiplex quantitative chemoproteomics. (B) Covalent Inhibitor Target-site Identification (CITe-Id), another complementary chemoproteomic platform to understand the proteome-wide on/off-target effect in covalent fragment development program, in which desthiobiotinylated covalent inhibitor is used in lieu of non-selective iodoacetamide probe to directly monitor target engagement. (C) Cysteine-Reactive Phosphate Tags (CPTs) developed recently can be applied as a chemoproteomic using phosphate-tagged iodoacetamide for global cysteine profiling with high coverage of the cysteine proteome.

Fig. 4. The structures of representative ABPP probes applied in state-of-the-art chemoproteomic strategies. (A) Iodoacetamide-alkyne is a widely applied cysteine-reactive ABPP probes. (B) NHS-ester-alkyne is a versatile covalent ABPP probe for protein nucleophiles (cysteines, lysines, tyrosines, serines, threonines, and arginines). (C) STP-alkyne is an amine-reactive covalent ABPP probe. (D) Latent electrophiles as exemplified by SuFEx-based arylfluorosulfates for lysine/tyrosine targeting. (E) HHS-465 and HHS-475 are SuTEx-chemistry based ABPP probes for tyrosine profiling. (F) Carbon nucleophiles help to explore ligandability of sulfenylated (oxidized) cysteines.

Fig. 5. Schematic of photoactivation-assisted fragment discovery. Photoreactive fragments contains a photoactivable diazirine, which could generate the carbene intermediate upon UV activation that can crosslink to proximal protein residues. Photoactivation-assisted fragment discovery has broad applicability in both target-based and cell-based screening campaigns.