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Open Access Highly Accessed Research article

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

Atsushi Yoshimori12, Junichi Sakai23, Satoshi Sunaga23, Takanobu Kobayashi4, Satoshi Takahashi1, Naoyuki Okita2, Ryoko Takasawa3 and Sei-ichi Tanuma23*

Author Affiliations

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

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

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

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

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

Published: 27 June 2007



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.


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.


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.