Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors

Abstract

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, smallmolecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometrybased chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

Silyl Ether Enables High Coverage Chemoproteomic Interaction Site Mapping

“…Whether or not a defined small molecule binding site is present in RTN4 is still unclear, as the protein remains structurally unresolved in the PDB and is highly disordered in the AlphaFold model (Figure S18; ID: AF-Q9NQC3-F1). Notably, C1101 exhibits hyperreactivity towards the cysteinereactive probe iodoacetamide alkyne (IAA) [84][85][86] , which is suggestive that cysteine reactive may be a central driver of RTN4 labeling by our asciminib probes.…”

“…We also observed that STING (TMEM173), an important regulator of innate immunity which elicits anti-tumor immune responses and a target of a number of chemical probe and drug development campaigns 82,83 , was also enriched and competed only in K562 cells (Figure 5C). Given our interest in mapping labeling sites we were also intrigued to observe reticulon-4 (RTN4) as a target of asciminib, due to the presence of a hyperreactive cysteine residue in this key regulator of endoplasmic reticulum structure (Figure 5A-C) [84][85][86][87][88] . Strikingly absent from the proteins significantly enriched by 5b was the bona fide target of asciminib ABL1 kinase-of note, several peptides from BCR were detected as modestly enriched, albeit below the threshold of significance (Figure 5D).…”