Discovery of allosteric activators of phospholipase C-gamma2 to treat Alzheimer's disease

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ABSTRACT Current therapies for Alzheimer’s disease (AD) do not reverse, or even slow, progression of the disease. This situation is dire and exacerbated by the failure of antibodies directed toward two of the more promising targets—phosphorylated tau and beta-amyloids—to treat the disease. Clearly, new treatments are urgently needed. In 2017, a large genome-wide study associated a naturally-occurring variant (P522R) of PLCG2, the gene encoding PLC-2, with protection from late onset AD. In follow-up studies, this genetic association has remained strong and highly reproducible. Even more encouraging, in clinical studies of patients with mild cognitive impairment, people that carried PLCG2 (P522R) had slower rates of cognitive decline compared to non-carriers. Protection was observed even for patients homozygous for ApoE4, a biomarker strongly linked to AD. In the brain, PLC-2 is primarily expressed in microglial cells where it controls phagocytic and neuroinflammatory processes. It is more highly expressed in pathological areas of patients with AD. In microglia, PLC-2 is activated downstream of both TREM2 (which uses ApoE4 as a ligand) and CSF1R, two transmembrane receptors that are strongly linked to AD. Similarly, PLC-2 activates PKC, which is also linked to AD. Thus, genetic and cellular data strongly support PLC-2 as a novel therapeutic target for treatment of AD. The phospholipase activity of PLC-2 (P522R) is modestly elevated relative to its wild-type counterpart and it is this increased activity in microglia that is generally accepted to protect against AD. We propose to identify and optimize small molecules that selectively activate PLC-2 to reproduce the neuroprotective effects of PLC- 2 (P522R) and treat AD. The research plan relies on complementary high-throughput assays enabled by two fluorogenic substrates for eukaryotic PLCs that we invented explicitly for this research. Consequently, we will pursue three Aims. In Aim 1, in-house collections totaling ~300,000 compounds will be screened for activators of PLC-2 and primary hits verified for activity, selectivity, composition, and purity; cheminformatics will be used to structurally classify hits. In Aim 2, a high-quality model of full-length PLC-2 coupled with molecular dynamics simulations will be used for computational screens of tens of millions of compounds. In Aim 3, a suite of biochemical, biophysical, and cell biological studies will be used to prioritize allosteric activators of PLC-2 with favorable chemical and pharmacological properties. These novel small molecules will be invaluable tools to further understand how PLC-2 (P522R) reduces the risk of AD. The small molecules will also be used as leads for the development of novel therapeutics to treat AD.