Optimizing brain penetrance of caspase-6 inhibitors to treat neurodegenerative diseases

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PROJECT SUMMARY/ABSTRACT The cysteine protease caspase-6 (Casp6) has been associated with neurodegenerative diseases for over fifteen years. In Alzheimer’s disease (AD), Casp6 is colocalized with amyloid plaques and tau tangles in human brain, and both human and animal model data indicate that activated Casp6 contributes to neuronal inflammation, neurodegeneration, and cleavage of proteins to toxic forms. Humans and mice lacking Casp6 are healthy and resistant to inflammation; furthermore, Casp6 knockout in 5xFAD mice bearing familial AD mutations in presenilin1 and amyloid beta precursor protein (APP) show reduced levels in neuronal degeneration and inflammation. Thus, selective inhibition of Casp6 could be safe and effective in treatment of AD. To this end, we propose to design, synthesize and profile potent and highly selective inhibitors of the activated form of Casp6 (aCasp6) based on our initial SAR established for SU110 and SU134. These novel compounds gain selectivity by binding covalently to a unique cysteine residue not found in other members of the caspase family. While both SU110 and SU134 have promising drug-like properties, including low nanomolar inhibition of aCasp6 in biochemical and cell-based assays, low toxicity, few off- targets in proteome-wide studies, serum exposures 100-fold higher than cellular IC50, 50-77% oral bioavailability, and up to 23% brain/plasma exposure, we will further explore the SAR to identify compounds with improved PK and brain penetration properties (Aim 1). We will conduct standard mouse PK experiments to determine oral bioavailability and to define structure–brain exposure relationships. For Aim 2, we have developed a click-based probe of aCasp6 binding that demonstrates sufficient potency and selectivity to use as an ex vivo activity-based probe of aCasp6. We will use this probe, in conjunction with aCasp6 protein half-life determination and measurement of the aCasp6 substrate tau, to determine the lifetime of target inhibition by optimized aCasp6 inhibitors in cells; these data will be used in Phase II of the project to assess target engagement in animals. From these SAR, PK and tissue distribution studies, we will identify the most appropriate compounds to conduct PD assays and disease models to be run during Phase II of the project. In the subsequent Phase II application, we will develop biomarker assays and evaluate changes in these biomarkers after treatment with optimized lead compound in the 5xFAD model and additional animal models of AD. The conclusion of Phase II studies will be refinement of the Target Candidate Profile and will anticipate the selection of a development candidate for IND-enabling studies.