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 work 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 ex- ploit 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. We are still working to understand the conformational transitions of caspase-6 between the helical and strand state and to understand the impact of our caspase-6 inhibitor on that conformational equilibrium. These stud- ies are only possible by using high-field NMR experiments. We have begun many of these studies by use of NMR spectrometers at national facilities and the results are extremely promising. We have recently been awarded funds from the Massachusetts Life Science Center for purchasing a new 800 MHz NMR for UMass Amherst that is appropriate for our ongoing experiments. This will dramatically improve our ability to assess the conformational sate of caspase-6 and ultimately of other caspases. The robust and reliable function of the 800 MHz NMR is dependent on cooling of the magnet with liquid Helium (He). Unfortunately, due to changes in manufacturing of and availability of He-recovery systems, we are not able to purchase the expected He- recovery system to support the functioning of the new UMass 800 MHz Bruker spectrometer and the NMR experiments in the Hardy lab. This proposal is requests partial funding for a PICASSO He-recovery system from Quantum Technologies Corp.