The cysteine protease caspase-6 (Casp6) has been associated with neurodegenerative diseasesfor over fifteen years. In Alzheimer's disease (AD), Casp6 is colocalized with amyloid plaques andtau tangles in human brain, and both human and animal model data indicate that activated Casp6contributes to neuronal inflammation, neurodegeneration, and cleavage of proteins to toxic forms.Humans and mice lacking Casp6 are healthy and resistant to inflammation; furthermore, Casp6knockout in 5xFAD mice bearing familial AD mutations in presenilin1 and amyloid beta precursorprotein (APP) show reduced levels in neuronal degeneration and inflammation. Thus, selectiveinhibition of Casp6 could be safe and effective in treatment of AD. To this end, we propose todesign, 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 compoundsgain selectivity by binding covalently to a unique cysteine residue not found in other members ofthe caspase family. While both SU110 and SU134 have promising drug-like properties, includinglow 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% oralbioavailability, and up to 23% brain/plasma exposure, we will further explore the SAR to identifycompounds with improved PK and brain penetration properties (Aim 1). We will conduct standardmouse PK experiments to determine oral bioavailability and to define structure-brain exposurerelationships. For Aim 2, we have developed a click-based probe of aCasp6 binding thatdemonstrates sufficient potency and selectivity to use as an ex vivo activity-based probe ofaCasp6. We will use this probe, in conjunction with aCasp6 protein half-life determination andmeasurement of the aCasp6 substrate tau, to determine the lifetime of target inhibition byoptimized aCasp6 inhibitors in cells; these data will be used in Phase II of the project to assesstarget engagement in animals. From these SAR, PK and tissue distribution studies, we will identifythe most appropriate compounds to conduct PD assays and disease models to be run duringPhase II of the project. In the subsequent Phase II application, we will develop biomarker assaysand evaluate changes in these biomarkers after treatment with optimized lead compound in the5xFAD model and additional animal models of AD. The conclusion of Phase II studies will berefinement of the Target Candidate Profile and will anticipate the selection of a developmentcandidate for IND-enabling studies.
Public Health Relevance Statement: PROJECT NARRATIVE
The protease caspase-6 contributes to inflammation, neurodegeneration, and protein aggregation
in neurodegenerative diseases like Alzheimer's (AD). We have developed highly potent inhibitors
of caspase-6 that gain exquisite selectivity by binding covalently to a unique cysteine residue in
the substrate-binding groove. This Phase I STTR project will further develop this novel series of
inhibitors and will utilize PK and tissue distribution studies to select the most promising
compounds for AD disease-specific animal models. With potent inhibitors in hand, we will be able
to elucidate target half-life and engagement with an established click-based probe of aCasp6
binding. This work will enable design of a future studies that will determine whether caspase-6
inhibitors are effective at blocking disease progression in animal models.
Project Terms: <Ï Proteins><Ï aggregation> 