title: Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO.
abstract: BACKGROUND
  The rational design of peptide-based specific inhibitors of the caspase family members using their X-ray crystallographies is an important strategy for chemical knockdown to define the critical role of each enzyme in apoptosis and inflammation. Recently, we designed a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 using a new computational screening system named the Amino acid Positional Fitness (APF) method (BMC Pharmacol. 2004, 4:7). Here, we report the specificity of the DNLD sequence against caspase-3 over other major caspase family members that participate in apoptosis by computational docking and site-directed mutagenesis studies.
  
  
  RESULTS
  Ac-DNLD-CHO inhibits caspases-3, -7, -8, and -9 activities with Kiapp values of 0.68, 55.7, >200, and >200 nM, respectively. In contrast, a well-known caspase-3 inhibitor, Ac-DEVD-CHO, inhibits all these caspases with similar Kiapp values. The selective recognition of a DNLD sequence by caspase-3 was confirmed by substrate preference studies using fluorometric methylcoumarin-amide (MCA)-fused peptide substrates. The bases for its selectivity and potency were assessed on a notable interaction between the substrate Asn (N) and the caspase-3 residue Ser209 in the S3 subsite and the tight interaction between the substrate Leu (L) and the caspase-3 hydrophobic S2 subsite, respectively, in computational docking studies. Expectedly, the substitution of Ser209 with alanine resulted in loss of the cleavage activity on Ac-DNLD-MCA and had virtually no effect on cleaving Ac-DEVD-MCA. These findings suggest that N and L residues in Ac-DNLD-CHO are the determinants for the selective and potent inhibitory activity against caspase-3.
  
  
  CONCLUSIONS
  On the basis of our results, we conclude that Ac-DNLD-CHO is a reliable, potent and selective inhibitor of caspase-3. The specific inhibitory effect on caspase-3 suggests that this inhibitor could become an important tool for investigations of the biological function of caspase-3. Furthermore, Ac-DNLD-CHO may be an attractive lead compound to generate novel effective non-peptidic pharmaceuticals for caspase-mediated apoptosis diseases, such as neurodegenerative disorders and viral infection diseases.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki Noda, Chiba, Japan. yosimori@rs.noda.tus.ac.jp <yosimori@rs.noda.tus.ac.jp>
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yoshimori
authors.forename: Junichi
authors.initials: J
authors.lastname: Sakai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sunaga
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Okita
authors.forename: Ryoko
authors.initials: R
authors.lastname: Takasawa
authors.forename: Sei-ichi
authors.initials: S
authors.lastname: Tanuma
mesh_terms: D001665:Binding Sites; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D053148:Caspase 3