An electrophilic fragment screening for the development of small molecules targeting caspase-2

Recent Alzheimer's research has shown increasing interest in the caspase-2 (Casp2) enzyme. However, the available Casp2 inhibitors, which have been pentapeptides or peptidomimetics, face challenges for use as CNS drugs. In this study, we successfully screened a 1920-compound chloroacetamide-based, electrophilic fragment library from Enamine. Our two-point dose screen identified 64 Casp2 hits, which were further evaluated in a ten-point dose-response study to assess selectivity over Casp3. We discovered compounds with inhibition values in the single-digit micromolar and sub-micromolar range, as well as up to 32-fold selectivity for Casp2 over Casp3. Target engagement analysis confirmed the covalent-irreversible binding of the selected fragments to Cys320 at the active site of Casp2. Overall, our findings lay a strong foundation for the future development of small-molecule Casp2 inhibitors. [Display omitted] •Electrophilic fragment screening for caspase-2 (Casp2) identified 64 hit compounds.•Single-digit micromolar affinity for Casp2 and up to 32-fold selectivity over Casp3.•Successful hit validation for selected fragments.•Target engagement study using mass spectrometry peptide sequencing (MSPS).•Covalent-irreversible fragment binding to cysteine-320 at the active site of Casp2.