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

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, small-molecule 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 spectrometry-based 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.