Development of a functional cell-based assay and cellular thermal shift for evaluation of drug target interactions with Phospholipase C-gamma 2, PLCɣ2.

Abstract: Phosphoinositide-specific phospholipase C (PLC) enzymes are expressed in all mammalian cells and are key elements in signal transmission networks. Specifically, PLCg2 is predominantly expressed in the immune and haematopoietic cells and differentially regulated by receptor tyrosine kinases (RTKs) or non-receptor tyrosine kinases. It selectively hydrolyses 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate to secondary messengers’ inositol-3-phosphate (IP3) and diacylglycerol. Genome Wide Association Studies (GWAS) have identified a novel coding variant (P522R) in the immune gene PLCG2 that is protective against the cognitive decline associated with late onset Alzheimer’s Disease (LOAD). The protective PLCg2 variant has a mild hypermorphic effect on enzymatic activity, and therefore identifying small molecules that mimic this moderate potentiation represents a novel therapeutic strategy for Alzheimer’s Disease. To facilitate the identification of modulators of PLCg2 we developed a B cell RAMOS assay, since the PLCg2 pathway downstream of the B cell receptor is well characterised and complemented this with the Cellular Thermal Shift Assay, CETSA® technology in the THP-1 macrophage cell line.
PLCg2 is the predominant PLC isoform in the RAMOS B cells and the protocol was optimised for use with cells in suspension. Reaction conditions were optimised to identify small molecule modulators of the B cell pathway using a biochemical IP1 HTRF endpoint as a readout of IP3 production.
In the THP-1 cells, where PLCg2 is highly expressed, the remaining intact PLCg2 protein was measured after heat shock in the presence of compounds to determine that the compounds were either on target or proximal to PLCg2 whilst eliciting the desired pharmacological profile. Alpha® reagents were used to for the detection of stabilised intact protein.
We used these approaches in parallel to screen a library of 20,000 diversity compounds and identified 62 potentiators that were concordant. The compounds were screened in single-point and selected compounds were run to determine EC50. Cut-off criteria for failing plates were Z prime < 0.5 and CV>10%. B scoring normalisation was used to correct for plate and positional effects. % Effect was calculated over DMSO controls. These approaches identified novel modulators with an overall hit rate of 0.31%.
We used functional and biophysical cell-based approaches to identify positive modulators of PLCg2. This offered the advantage of identifying compounds engaging with the activated form of PLCg2 in the cellular environment where the membrane, native substrate and other interacting partners of the complex are present. It is unclear how the PLCg2 P522R variant exerts its protective effect but the higher PLC activity could be achieved by adopting a more stable active form or by enhanced interactions with regulatory proteins or membrane. The combination of these approaches allowed identification of novel modulators thus demonstrating the feasibility of identifying small molecules that mimic the hypermorphic effect of the P522R variant.