Human caspase-3 inhibition by Z- tLeu-Asp-H: tLeu(P 2) counterbalances Asp(P 4) and Glu(P 3) specific inhibitor truncation

Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z- tLeu-Asp-H, Z- tLeu-Val-Asp-H, and Z-Val- tLeu-Asp-H are reported. Z- tLeu-Asp-H, Z- tLeu-Val-Asp-H, and Z-Val- tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with K i 0 = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 and 25 °C). Moreover, Z- tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z- tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P 2 position.