A high-throughput assay platform to discover small molecule activators of the phospholipase PLC-γ2 to treat Alzheimer's disease.

A naturally occurring missense variant of the phospholipase C isozyme, PLC-γ2, harboring a single substitution (P522R) protects against several neurodegenerative diseases, including Alzheimer's disease. The phospholipase activity of PLC-γ2 (P522R) is slightly elevated relative to its WT counterpart, and the general consensus is that this increased activity in microglia confers protection against neurodegeneration. In order to phenocopy this protection, we have developed a high-throughput assay to identify small molecule activators of PLC-γ2. The assay takes advantage of the fluorescent reporter, XY-69, embedded in lipid vesicles to readout the allosteric activation of PLC-γ2. The assay is highly reproducible and capable of identifying compounds with a large range of efficacies. A series of secondary assays have been established to define the selectivity of compounds for PLC-γ2, establish relevant activation of PLC-γ2 by compounds in a microglia cell line, and measure affinities between PLC-γ2 and hit compounds. The established workflow was prototyped using approximately 6000 compounds to produce several promising hits, but more importantly, enables screens of much larger chemical libraries to identify selective activators of PLC-γ2 to be used as chemical probes and drug leads.