Discovering and Exploiting Caspase Regulatory, Allosteric and Exosites

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PROJECT SUMMARY Caspases are cysteine proteases that control apoptotic cell death. Caspases are activated to kill cancerous cells, but inhibiting caspases can prevent deleterious cell death in diseases like heart attack and stroke. Thus, there has been significant interest in caspases as drug targets. This interest heightened further with the finding that caspase-6 plays a central role in neurodegeneration. Unfortunately, no caspase-directed therapies are on the market, primarily because research has focused on the active site, the most conserved region of the family. It is clear that each caspase is regulated in a unique manner. A comprehensive understanding of which is essential to achieve caspase-specific inhibition. Thus, our long-term project goal has been to define and exploit unique regulatory features for each apoptotic caspase. Our pursuit of that goal has been successful. Due to our understanding of the unique features of each apop- totic caspase, we developed an allosteric caspase-6 inhibitor that is more potent than any reported (33 nM) and is also by far the most selective, preferring caspase-6 500-fold or more over all other caspases. This selectivity was possible because the allosteric site we targeted is unique to caspase-6, locking it into a helical conformation not attainable by any other caspase. It is gratifying that our intense and systematic study of caspase regulation - cleavage state, conformational change, zinc binding and phosphorylation - culminated in a structural understanding that enabled us to meet our goal of caspase-selective allosteric inhibition. Thus, we propose research that will further extend our understanding of the regulation of the apoptotic caspases in new key areas - substrate selectivity by caspase isoforms, core unfolding and aggregation of caspase-9 by phosphorylation, and interactions between caspase core and prodomains to achieve substrate selection. In addition, we are currently moving this caspase-6 inhibitor forward toward clinical use. While we are thrilled at the success of developing an inhibitor that can block the function of just one of the twelve human caspases, the substrate-selective inhibitors proposed here promise to be even more invaluable. For example, preventing caspase-6 cleavage of Tau would have a major impact on preventing the formation of neurofibrillary tangles in Alzheimer’s Disease. On the other hand, cleavage of DJ-1 (PARK7) by caspase- 6 is important for preventing Parkinson’s Disease. Thus, an ideal caspase-6 inhibitor for treatment of Alz- heimer’s disease would block Tau cleavage, but would still cleave apoptotic substrates and DJ-1, without promoting cancer or Parkinson’s disease. The proposed studies identifying exosites for Tau and DJ-1 will enable us to develop substrate-selective inhibitors (nanobodies) that can block cleavage of Tau, but not of DJ-1. Together this work plan increases both our fundamental understanding of the biology and regulation of caspases and enables development of a novel highly tuned class of caspase-directed therapies.