THE CALPAIN PROTEASE ACTIVE SITE: A TARGET FOR INHIBITOR AND ACTIVITY-BASED PROBE DESIGN

ii The calpain family of intracellular Ca2+-dependent cysteine proteases is involved in a number of intracellular signaling processes. Calpain hyperactivity has also been implicated in ischemic injury, neurodegenerative diseases and cataract formation. However, the specific function of calpains in these normal and diseased states remains unclear. Competitive inhibition of calpain is useful for studying their functions and can lead to pharmacological treatments, while monitoring their activity with activity-based probes (ABPs) can reveal how calpain is regulated and be applied to screen for inhibitors in vivo. But these strategies are complicated by the similarity of the calpain active-site when compared to other intracellular cysteine proteases. Therefore, there is a need to design inhibitors and ABPs that selectively target calpain. Using X-ray crystallography, the interactions between the calpain active-site and each of two reversible inhibitors was studied. This led to the discovery of novel non-covalent aromatic stacking and hydrogen bonding interactions between the primed-side adenine group of one inhibitor and indol