Human caspase-3 inhibition by Z-tLeu-Asp-H: tLeu(P2) counterbalances Asp(P4) and Glu(P3) specific inhibitor truncation |
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Authors: | Colantonio Patrizia Leboffe Loris Bolli Alessandro Marino Maria Ascenzi Paolo Luisi Grazia |
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Institution: | a Department of Drug Sciences, University ‘Gabriele D’Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy b Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy c National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italy |
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Abstract: | 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 = 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 P2 position. |
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Keywords: | Ac-Asp-Glu-Val-Asp-H N-acetyl-Asp-Glu-Val-Asp-aldehyde Ac-Asp-Glu-Val-Asp-AMC N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin AcOH acetic acid DCC dicyclohexylcarbodiimide DCM dichloromethane DMSO dimethylsulfoxide DTT dithiothreitol EtOH ethanol HOBt 1-hydroxybenzotriazole MeOH methanol OBut tert-butyl ester derivative OSu N-hydroxysuccinimide derivative PARP poly(ADP-ribose) polymerase TFA trifluoroacetic acid tLeu (S)-tert-leucine Z benzyloxycarbonyl group Z-Glu-Leu-Asp-H N-benxylozycarbonyl-Glu-Leu-Asp-aldehyde Z-tLeu-Asp-H N-benxylozycarbonyl-tLeu-Asp-aldehyde Z-tLeu-Val-Asp-H N-benxylozycarbonyl-tLeu-Val-Asp-aldehyde Z-Val-tLeu-Asp-H N-benxylozycarbonyl-Val-tLeu-Asp-aldehyde |
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