A target enablement package for modulating the activity of PLCγ2 as a therapeutic strategy for the treatment of Alzheimer’s disease
Brent Clayton, Stephanie J Bissel, Timothy I Richardson, Steven M Massey, Daniel E Beck, Karson S Putt, Logan M Bedford, Tada Utsuki, Ramya Visvanathan, Andrew D Mesecar, Emma K Lendy, Bridget L Kaiser, Shaoyou Chu, Emily R Mason, Sara K Quinney, Murugesh Kandasamy, Andrea R Masters, Bruce T Lamb, Alan D Palkowitz, John Sondek, Qisheng Zhang- Psychiatry and Mental health
- Cellular and Molecular Neuroscience
- Geriatrics and Gerontology
- Neurology (clinical)
- Developmental Neuroscience
- Health Policy
- Epidemiology
Abstract
Background
Genome‐wide association studies, whole genome sequencing, and gene‐expression network analyses comparing normal to Alzheimer’s disease (AD) brain tissue have identified risk and protective variants in genes essential to microglial function. Among these genes is phospholipase C gamma2 (PLCγ2). The P522R variant of PLCγ2 is associated with reduced risk for AD and has been characterized as a functional hypermorph. Carriers of P522R with mild cognitive impairment exhibited a slower cognitive decline rate. Therefore, activation of the protein PLCγ2 with small molecules has been proposed as a therapeutic strategy to reduce the rate of disease progression and cognitive decline in AD patients.
Method
We evaluated analogs of a reported phospholipase activator and performed a high‐throughput screen using full‐length PLCγ2 and affinity selection mass spectrometry (ASMS). Soluble substrate and liposomal‐based biochemical assays were developed to evaluate activation and inhibition of the enzyme. A Cellular Thermal Shift Assay (CETSA) was developed to confirm target engagement in cells. A high‐content imaging assay measuring phagocytosis, cell number, and nuclear intensity was implemented using the BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Results were recapitulated in primary murine microglia. Active compounds predicted to have drug‐like properties were subjected to assays measuring solubility, cellular permeability, and mouse microsomal stability.
Result
Structurally distinct, novel PLCγ2 activators have been discovered. In preparation for in vivo studies in mice, in vitro absorption, distribution, metabolism, and excretion (ADME) assays have been carried out for activators with sufficient cellular potency and drug‐like properties.
Conclusion
Activation of PLCγ2 is a novel therapeutic strategy for treatment of AD. We identified structurally distinct molecular scaffolds with varying degrees of enzyme activation and cellular activity. Recommendations for use of probe molecules in target validation studies and the development of lead‐like molecules for clinical studies will be made.