Research article

Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO

Atsushi Yoshimori^1,2 , Junichi Sakai^2,3 , Satoshi Sunaga^2,3 , Takanobu Kobayashi⁴ , Satoshi Takahashi¹ , Naoyuki Okita² , Ryoko Takasawa³ and Sei-ichi Tanuma^2,3

¹Institute for Theoretical Medicine, Inc., Venture Kanda 504, 1-1-5 Uchikanda Chiyoda, Tokyo 101-0047, Japan

²Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki Noda, Chiba 278-8510, Japan

³Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki Noda, Chiba 278-8510, Japan

⁴Department of Molecular Biology, Faculty of Pharmaceutical Science, Tokushima Bunri University, 1314-1 Shido Sanuki, Kagawa 769-2193, Japan

author email corresponding author email

BMC Pharmacology 2007, 7:8doi:10.1186/1471-2210-7-8

Published:	27 June 2007

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 K_i^app 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 K_i^app 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.

Conclusion

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.