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.