Activation of caspases is one of the most widely recognized features of apoptosis. Caspases are cysteine-dependent, aspartate-specific proteases. They exist as latent precursors, which, when activated by limited proteolysis, initiate the death program by destroying key components of the cellular infrastructure and activating factors that mediate damage to the cells. The mechanism of caspase activation appears to be conserved in evolution. Thus far, 14 members of the caspase family have been identified, 11 of which are present in humans. Caspases have been categorized into upstream initiators and downstream executioners. A distinctive feature of caspases is the absolute requirement of an aspartic acid residue in the substrate P1 position. The P4 residue is important in substrate recognition and specificity. Generally, catalysis involves a cysteine protease mechanism. The tetrapeptide corresponding to the substrate P4 - P1 residues is sufficient to recognize both caspase-1 and caspase-3. This also forms the basis of designing novel inhibitors of caspases.
Caspase activation is generally considered as the "point of no return” in apoptotic pathways. Caspases are activated by two major pathways: the receptor-mediated (Fas ligand or TNF-α-mediated) pathway and the mitochondrial pathway. The receptor-mediated pathway leads to the activation of pro-caspase-8. In the mitochondrial pathway, pro-apoptotic members of the Bcl-2 family associate with mitochondria and direct the release of cytochrome c (Cyt c) and other proteins, which activate pro-caspase-9.
Caspase inhibitors act by binding to the active site of caspases either in a reversible or irreversible manner. Inhibitor design includes a peptide recognition sequence attached to a functional group such as an aldehyde (CHO), chloromethylketone (CMK), or fluoromethylketone (FMK). The peptide recognition sequence corresponding to that found in endogenous substrates determines the specificity of a particular caspase. For example, compounds with the Ac-YVAD-CHO sequence are potent inhibitors of caspases-1 (Ki ≈ 10 nM), and exhibit very weak inhibitory effect on caspases-3 and -7 (Ki ≈ 50 µM). Exclusion of the tyrosine residue from the inhibitor peptide results in a potent but less specific inhibitor. For example, Z-VAD-FMK inhibits not only caspases-1 and -4, but also caspases-3 and -7.