title: Ac-tLeu-Asp-H is the minimal and highly effective human caspase-3 inhibitor: biological and in silico studies.
abstract: 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.
authors.affiliation: Department of Pharmacy, University "Gabriele d'Annunzio", Via dei Vestini 31, 66013, Chieti, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Ferrucci
authors.forename: Loris
authors.initials: L
authors.lastname: Leboffe
authors.forename: Mariangela
authors.initials: M
authors.lastname: Agamennone
authors.forename: Antonella
authors.initials: A
authors.lastname: Di Pizio
authors.forename: Marco
authors.initials: M
authors.lastname: Fiocchetti
authors.forename: Maria
authors.initials: M
authors.lastname: Marino
authors.forename: Paolo
authors.initials: P
authors.lastname: Ascenzi
authors.forename: Grazia
authors.initials: G
authors.lastname: Luisi
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000737:chemistry*; D061945:Caspase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D045744:Cell Line, Tumor; D003198:Computer Simulation; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D061945:Caspase Inhibitors; D010455:Peptides; D053148:Caspase 3