Design, Synthesis And Biological Activity Of Novel Caspase-3 Inhibitors

Apoptosis, or programmed cell death, is the result of cellular biochemical events carried out by several mediators, especially, the family of caspases. The caspases family comprises a family of highly homologous cysteine proteases that play key roles in apoptosis. Among several different groups of caspase enzymes, caspase-3 lies at a key junction in the apoptotic cascade, mediating apoptosis from both the intrinsic and extrinsic activation pathways. So caspase-3 is an attractive target for therapeutic intervention in several diseases because of the central role played by apoptosis in some conditions. Excessive, uncontrolled apoptosis appears to be related to several diseases that currently lack suitable treatment such as neurodegenerative disease, ischemia-reperfusion injury and autoimmune diseases. In the recently been demonstrated that tumors cause apoptotic death of key immune cells. Therefore, inhibitors of caspase-3 as promising lead drugs may believe to be a valuable therapeutic approach to treating these diseases.The most of potent caspase-3 inhibitors reports to-date have peptide or peptidomimetic nature. However such inhibitors often possess poor metabolic stability and poor cell penetration, which result in a search for non-peptidic inhibitors of caspase-3. To find small-molecular caspase-3 inhibitors with a novel structure directed against diseases involving abnormally up-regulated caspase-3-dependent apoptosis. Our work was initially based on screening of a small-molecule library of selected compounds using recombinant caspase-3. As part of our ongoing efforts of discovering the small-molecules, we found that the ninhydrin had a low activity of inhibiting caspase-3 enzyme assay in vitro (IC5o=55μM). In order to verify the universality of this new structure, we designed and synthesized series of novel, small non-peptidic molecule inhibitors of caspase-3 based on ninhydrin scaffold. The ninhydrin-based inhibitors have been tested for their ability to inhibit caspase-3 proteolytic activity to breakdown its fluorogenic substrate, DEVD-AMC and displayed activity in vitro caspase-3 inhibition assay. Based on the structure of ninhydrin, our initial series of compounds were designed to explore the hydrophilic interaction of inhibitors with caspase-3 S1 (Cys163)subsite. First, we modified one of the ninhydrin 2-hydroxyl by installing substituent, but the results were unsatisfactorily (Fig4.11).As recently report, high variable hydrophobic residues in the S2 subsite (Trp206, Phe256, and Tyr204) are found in caspase-3. Examination of these residues reveals that those which make up the S2 subsite are likely responsible for the observed inhibitor selectivity. In order to adjoin the S2 subsite, we designed and synthesized the compound 8. As expected, compound 5 was in the active center of caspase-3(ICso=2.7μM)Interestingly, substituent's chirality would significantly influence the inhibitory activity for the pair of individual stereo isomers. One unexpected observation was the high potency of the compound 9 (IC50=0.15μM). As shown in Fig4.12, the two stereo isomers 8 and 9 were prepared and evaluated for caspase-3 inhibitory activity. In contrast, compound 9 appeared to be 18-fold more active than compound 8. The result also suggested that compound 9 is likely to be a novel inhibitor of caspase-3 with pharmaceutical application potential.In order to improve our understanding of the interaction between compound 9 and caspase-3, we analyzed the binding mode of compound 9 through a computational docking simulation. Compound 9 was located at the active site of caspase-3 and indene ring tightly bound to the S2 subsite based on hydrophobic interactions. In compound 9, it formed seven hydrogen bonds with Arg 64, Gin 161, Ser120, His 121, Ser 205and Arg 207. The results of molecular docking suggested that compound 9 could interact with the active site.As shown in Fig.4.15, the distance between one ketone group (R2) and thiol of Cys163 was 3.65 A, while the distance between the same group position of compound 8 and the Cysl63 thiol was 6.50 A. These dates suggested that the specificity and potency of compound 9 was caused by the specific interactions with the active site of caspase-3. Probably, the binding of compound 9 to caspase-3 provided a good structural basis to explain the efficient inhibitory action.In summary, here we have discovered a novel class of potent caspase-3 inhibitors based on ninhydrin scaffold. The results of this study have extended the structure-activity relationships and further insight to the development of non-peptide-based inhibitors of caspase-3. From our work described in this paper, we feel there is potential for further optimization of this series and subsequent work will be reported in due course.