Caspase-6 is a critical factor in the development of several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Transgenic mice in which the caspase-6 cleavage sites in Amyloid Precursor Protein or Huntingtin Protein were mutated to prevent caspase-6 cleavage are protected from neurodegeneration, making caspase-6 an attractive target for treatment of Alzheimer's and Huntington's Diseases. Caspase-6 is also a member of the family of apoptotic proteins that control apoptotic cell death. To date it has been impossible to enumerate the specific roles caspase-6 plays in the cell that are related to or unique from other family members. This information is of central importance because blocking these additional roles could potentially lead to negative side-effects if caspase-6 were targeted in treatments for Huntington's and Alzheimer's long term. No caspase-specific probes exist because all available probes for use in cell or animal models function at the active sites of caspases and all caspases have very similar active sites. This screen makes use of a recently discovered allosteric site in caspase-6 that is not present in any other caspase. This allosteric site makes it possible to achieve caspase-6 specific inhibition for the very first time. The goal of this project is to develop a probe that specifically targets this unique allosteric site in caspase-6. A chemical probe that is specific an selective for caspase-6 will allow the native biological role of caspase-6 to be distinguished from other apoptotic caspases for the first time. This will allow validation of caspase-6 as a novel target for treatment of neurodegeneration. In addition to validating the allosteric site for drug discovery, the probes will also serve as lead compounds for discovery of novel drugs for neurodegeneration. This screen uses a robust screening protocol for identifying caspase-6 allosteric inhibitors at an allosteric site we have identified by crystallography and mutagenesis. This allosteric site is unique to caspase-6 and is not present in any other caspase. In the pilot screen, a hit rate of 2.9% with a cutoff of 30% inhibition was observed. The hit-rate can be adjusted to the desired level by using more stringent cutoffs. A Z'score of 0.8 was also observed, suggesting that caspase-6 is a tractable target for this type of screening. A panel of 61 hits was retested in the primary assay and subjected to two secondary assays. Two additional secondary assays have also been established and validated. This screen also utilizes a novel tertiary assay based on biophysical and spectroscopic observations. The allosterically inhibited conformation targeted in this screen exhibits a unique signature in the circular dichroism spectra. Thus using the circular dichroism assay provides mechanistic information about both the mode and location of probe binding. This assay is the first and only assay available that allows allosteric site inhibitors to be distinguished from active site inhibitors inany caspase. As this is the first screen enabling identification of caspase-6 selective compounds, our screen will allow discovery of probes to control this important protease.
Caspase-6 is a member of a family of enzymes that control how healthy cells die by apoptosis and is also a critical factor in the development of Alzheimer's and Huntington's Diseases. We have discovered an allosteric site in caspase-6 that is not present in any other caspase that can be used to achieve caspase-6-specific inhibition. The probe we will develop for this new allosteric site will allow dissection of the unique roles of caspase-6 within the larger family of caspases and will predict the efficacy of allosteric caspase-6 drugs in the treatment of Huntington's and Alzheimer's Diseases. ! !
