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

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.

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.