Ac-tLeu-ASP-H is the minimal and highly effective human caspase-3 inhibitor: Biological and in silico studies

Anna Ferrucci, Loris Leboffe, Mariangela Agamennone, Antonella Di Pizio, Marco Fiocchetti, Maria Marino, Paolo Ascenzi, Grazia Luisi

Research output: Contribution to journalArticlepeer-review


Caspase-3 displays a pivotal role as an executioner of apoptosis, hydrolyzing several proteins including the nuclear enzyme poly(ADP-ribose)polymerase (PARP). Ac-Asp-Glu-Val-Asp-H (K i° = 2.3 × 10-10 M at pH 7.5 and 25.0 °C), designed on the basis of the cleavage site of PARP, has been reported as a highly specific human caspase-3 inhibitor. Here, di- and tri-peptidyl aldehydes 11-13 and 27-29 have been synthesized to overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of the current inhibitors. Compounds 11-13, 27-29 inhibit in vitro human caspase-3 competitively, values of K i° ranging between 6.5 (±0.82) × 10-9 M and 1.1 (±0.04) × 10-7 M (at pH 7.4 and 25.0 °C). Moreover, the most effective caspase-3 inhibitor 11 impairs apoptosis in human DLD-1 colon adenocarcinoma cells. Furthermore, the binding mode of 11-13 and 27-29 to human caspase-3 has been investigated in silico. The comparative analysis of human caspase-3 inhibitors indicates that (1) aldehyde 11 is the minimal highly effective inhibitor, (2) the tLeu-Asp sequence is pivotal for satisfactory enzyme inhibition, and (3) the occurrence of the tLeu residue at the inhibitor P2 position is fundamental for enzyme/inhibitor recognition. Moreover, calculations suggest that the tLeu residue reduces the conformational flexibility of the inhibitor that binds to the enzyme with a lower energetic penalty.

Original languageEnglish
Pages (from-to)153-162
Number of pages10
JournalAmino Acids
Issue number1
Publication statusPublished - Jan 1 2015


  • Apoptosis
  • Enzyme competitive inhibition
  • Human caspase-3
  • Human DLD-1 colon adenocarcinoma cells
  • Peptidyl aldehyde inhibitors

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Organic Chemistry
  • Medicine(all)

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