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.

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.

Figure 1

Inhibitory effects of caspase-3 inhibitors on caspases. Human recombinant caspase-3 (A), caspase-7 (B), caspase-8 (C), and caspase-9 (D) were preincubated for 10 min with indicated concentrations of Ac-DNLD-CHO (●), Ac-DEVD-CHO (○), Ac-DQTD-CHO (△), or Ac-DMQD-CHO (□), and then the activities of the caspases were measured with each substrate as described in ''Methods''. The kinetics data presented are the means of three independent experiments.

Figure 2

Hydrolysis of Ac-DNLD-MCA by caspases. The time course of the abilities of caspases-3 (A), -7 (B), -8, (C) and -9 (D) to cleave the fluorometric caspase substrates Ac-DNLD-MCA (●) and Ac-DEVD-CHO (○) were compared. The cleavage assay was performed as described in "Methods" The y-axis represents the concentration of MCA production (pmol) and the x-axis represents incubation period. Data indicate the mean of three independent experiments.

Figure 3

Sequence alignments and structural superpositions of caspases. A, Polypeptide sequence alignments of human caspases were performed using the Clustal W program [44] and then adjusted manually. Amino acid residues are numbered to the right of each sequence. Active site residues are highlighted in red, and S₁ (■), S₂ (□), S₃ (●), and S₄ (○) subsites on the active sites are indicated. B, Structural superpositions of Cα atoms of caspases-3 (blue), -7 (pink), -8 (green), and -9 (orange) are presented in line representation, and active site residues on the three-dimensional structures of caspase-3 are red in space-fill representation. The superpositions were performed using the McLaghlan algorithm [47] as implemented in the ProFit program [48].

Figure 4

Definition of active site residues of caspases. A, Alignments of active site residues of caspases. The residues were identified by inspecting hydrogen bonding and van der Waals interactions in the X-ray structures of caspases (codes 1PAU, 1F1J, 1F9E, and 1JXQ) and using examples as described [27], and then assigning the particular subsites (S_x1-x2, where x2 is the position in the S_x1 subsite). B, Schematic representation showing the locations of the subsites on the active site with Ac-DEVD-CHO. The underlined subsite has a conserved residue except the S_1–5(S/A) subsite.

Figure 5

Plot of the least squares fit plot of the predicted binding free energies (ΔG_calc) (kcal/mol) vs experimentally observed K_i^app values. Least squares fit analysis performed over set of the data points in Table 2 yielded the following equation, log K_i^app = +0.56ΔG_calc - 1.43.

Figure 6

Phylogenetic relationships of caspases. Phylogenetic relationships were constructed by the neighbor-joining algorithm [35]. Bootstrap analysis was performed on 1000 random samples and analyzed by the Clustal W program [44]. The numbers at branches were determined by the bootstrap analysis, indicating the times in 1000 repeat samples. The relationships are based on full-length caspases (A) and the active site residues according to our definition (B).

Figure 7

Binding interactions for Ac-DNLD-CHO and Ac-DEVD-CHO on the active site of caspase-3. Nitrogen, oxygen, and carbon atoms of the inhibitors are illustrated in blue, red, and green, respectively. Hydrogen bonds are shown as dashed lines. Hydrophobic interactions are shown as thick broken lines schematically. A, The binding mode of Ac-DNLD-CHO was obtained from docking simulations. B, The binding mode of Ac-DEVD-CHO was obtained from the X-ray crystal structure (1PAU). C, The time courses of liberation of fluorescence (MCA) from Ac-DNLD-MCA catalyzed by wild-type caspase-3 (●) and substituted (S209A) caspase-3 (▲). D, The time courses of liberation of fluorescence (MCA) from Ac-DEVD-MCA catalyzed by wild-type caspase-3 (○) and substituted (S209A) caspase-3 (△). The cleavage assays were performed as described in ''Methods''. Data indicate the mean of three independent experiments. E, Amounts of wild-type (lane 2) and substituted (S209A) (lane 3) active caspase-3 proteins generated by coexpression of HA-p17 and HA-p12 subunits in in vitro translation system were analyzed by Western blotting as described under ''Methods''. In this experiment, empty vector was used as control (lane 1).

Figure 8

Comparison of the binding modes of Ac-DNLD-CHO, Ac-DEVD-CHO, Ac-DQTD-CHO, and Ac-DMQD-CHO with caspases-3, -7, -8, and -9. Nitrogen, oxygen, and carbon atoms of the inhibitors are illustrated in blue, red, and green, respectively. Hydrogen bonds with the S3 subsite are shown as blue circles and dashed lines. Hydrophobic interactions with the S2 subsite are shown as red circles.