Genuine Selective Caspase-2 Inhibition with new Irreversible Small Peptidomimetics

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases including nonalcoholic steatohepatitis (NASH) and Alzheimer’s disease (AD). However, the design of active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics shows that these new compounds, such as LJ2 or its specific isomer LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. According to Casp2 role in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~ 5 500 000 M−1s−1) and the most selective inhibitor, LJ3a, has a near to 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that spatial configuration of Cα at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with β-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment. ### Competing Interest Statement E.J. is inventor of a patent titled: Novel derivatives and their use as selective inhibitors of Caspase-2 (WO/2017/162674). E.J. and E.B. are co-inventors of a patent titled: Novel compounds and their use as selective inhibitors of Caspase-2 WO/2019/068538. MK hold a patent on mouse models for the treatment of NASH, is a founder of Elgia Pharmaceuticals and had received research support from Jansen and Gossamer Bio.