Project Summary Abstract Caspase-2 has been implicated in neurological indications such as stroke, Alzheimer's, Parkinson's, and Huntington's diseases, neuroblastoma, neuro-ophthalomology, and frontotemporal dementia. The broad, long- term objective of our work is the development of specific caspase-2 inhibitors as neuro-therapeutic agents. This long-term objective hinges on first developing caspase-2 probes that will allow us to show that specific pharmacological reduction of caspase-2 activity by small molecule probes leads to the amelioration of disease phenotype, initially in animal models. Our specific aims all target the discovery and development of caspase-2 probes but each has a different starting point. We will use measurement of ?tau314 levels, a specific therapeutically- and clinically-relevant biomarker, as part of our testing funnel, to gauge the efficacy of our probes in cells. Our probe design and development will feature three parallel paths of compound characterization and optimization, each of which will inform the other as to caspase-2 binding that influences specificity and potency. Our three aims are to develop (1) probes from proteins that are specifically cleaved by caspase-2, (2) probes from HTS follow-on, and (3) probes from known selective caspase-2 inhibitors. Our goal in each of these is to produce a probe or tool compound which has in vitro potency <100 nM at the target protein, possesses >30-fold selectivity relative to sequence-related targets in the same family, has been profiled against an ?industry- standard? panel of pharmacologically-relevant off-targets, and has demonstrated on-target effect in cells of <1 M. To demonstrate in vivo relevance, we will test these probes in rTg4510 mice using the Morris water maze, a well-established model of cognition. We expect that these probes that specifically target caspase-2 will restore cognition in these aged mice without observable side effects. Ultimately, we will use these specific caspase-2 probes to investigate the broad range of neurological disorders in which caspase-2 activity has been implicated. This will constitute and important vertical advancement and spur the pharmaceutical development of therapeutics that will positively impact human health.