Proteome-wide covalent ligand discovery in native biological systems

Abstract

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered “undruggable” 1,2. Fragment-based ligand discovery (FBLD) can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries 1,3. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes 4–10, including those that can access regions of proteins that are difficult to access through binding affinity alone 5,10,11. In this manuscript, we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T-cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and −10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer

“…We next investigated the ligandability of cysteines in NRF2-regulated proteins by performing competitive isoTOP-ABPP of proteomes from three KEAP1 -mutant (H2122, H460 and A549) and three KEAP1 -WT (H1975, H2009 and H358) NSCLC lines with two electrophilic fragments – 2 and 3 (Figure 2A) – that showed broad cysteine reactivity in previous studies (Backus et al, 2016). We refer to these compounds as ‘scout’ fragments capable of providing a global portrait of covalent small molecule-cysteine interactions in native biological systems.…”

“…Both catalytic and non-catalytic cysteines in a wide range of proteins have been targeted with electrophilic small molecules to create covalent inhibitors for use as chemical probes (Ostrem et al, 2013; Johnson et al, 2010; Liu et al, 2013) and therapeutic agents, including ibrutinib, which targets Bruton’s tyrosine kinase BTK for treatment of B-cell cancers (Pan, 2008) and afatinib and AZD9291, which target mutant forms of EGFR for treatment of lung cancer (Finlay et al, 2014). Recently, our lab performed a global analysis of cysteine ligandability in human cancer cell proteomes, revealing a rich content of cysteines amenable to modification by electrophilic small molecules (Backus et al, 2016). Some of these cysteines were found in proteins that have been historically considered undruggable, such as transcription factors and adaptor proteins, suggesting that cysteine-reactive covalent chemistry may substantially expand the portion of the human proteome that can be targeted by small-molecule probes.…”

High‐Throughput Kinetic Analysis for Target‐Directed Covalent Ligand Discovery

“…Fragment‐based ligand discovery (FBLD) is a complementary technique that is able to generate ligands against many difficult‐to‐target proteins 2. Early strategies focused on non‐covalent fragments; however, recently FBLD has begun to incorporate electrophilic molecules 3, 4, 5, 6, 7, 8, 9, 10, 11. These fragments form covalent bonds with nucleophilic amino acids on target proteins and consist of a specificity‐determining element and a reactive warhead.…”

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery

“…Some of these approaches are limited to certain target classes, because they require probes that are known to bind the target (13)(14)(15)(16)18) or target properties to allow detection [e.g., the stability of the target needs to be affected by the compound (8,9)]. Furthermore, exposure can only be detected in cell types that express the target, limiting their use when studying offtarget effects and drug toxicity.…”

“…Currently, indirect estimates of intracellular drug levels are extrapolated from transcellular permeability experiments (7) or cellular target engagement data (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Some of these approaches are limited to certain target classes, because they require probes that are known to bind the target (13)(14)(15)(16)18) or target properties to allow detection [e.g., the stability of the target needs to be affected by the compound (8,9)].…”