Abstract
BackgroundThe 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.ResultsAc-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.ConclusionOn 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.
Highlights
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
We investigated the structural and functional relevance to potency and selectivity of this rationally designed peptide by enzyme kinetic analyses, computational docking studies and site-directed mutagenesis analysis
Ac-DEVD-CHO inhibits caspase-7 (Kiapp = 4.48 nM), -8 (Kiapp = 0.597 nM), and -9 (Kiapp = 1.35 nM) to similar extents. This observation is consistent with the previous report that Ac-DEVD-CHO inhibits caspases-3, -7, and -8 [31]
Summary
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. 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. Apoptotic caspases can be subdivided into initiator and executioner caspases They are normally expressed as proenzymes that mature to their fully functional form through proteolytic cleavage [4,5,6,7,8,9]. More than 60 caspase substrates have been identified to date [12]
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