Abstract

Evaluation of drug-like properties in vitro is critical in identifying viable candidates for new central nervous system (CNS) drugs. Excitatory amino acid transporter 2 (EAAT2) is the primary glutamate transporter in the brain, responsible for approximately 90% of synaptic glutamate clearance. Its dysfunction leads to excessive glutamate accumulation and excitotoxic neuronal death, contributing to disorders including epilepsy, amyotrophic lateral sclerosis, and Alzheimer’s disease. Positive allosteric modulation of EAAT2 represents a promising therapeutic strategy to restore glutamate homeostasis. This study aimed to evaluate in vitro drug-like properties of two novel positive allosteric modulators of EAAT2, (R)-AS-78 and (R)-PC-2, and to assess their potential for CNS drug development. PAMPA assay and bidirectional Caco-2 assay were used to evaluate membrane permeability and P-glycoprotein efflux liability. Rapid equilibrium dialysis was used to determine plasma protein binding. Metabolic stability and metabolite identification were assessed by incubation with human liver microsomes and LC-MS/MS analysis. CYP3A4 inhibitory potential was evaluated using the P450-Glo luminescence assay, and cytotoxicity was assessed using the MTS assay in HepG2 and SH-SY5Y cell lines. ADMET Predictor and MetaSite software predictions supplemented the experimental results. The results indicate that both compounds demonstrate drug-like properties suitable for further development as CNS drug candidates, with acceptable permeability, plasma protein binding and metabolic stability, no significant CYP3A4 inhibition, and no cytotoxicity. This is important as inadequate drug-like properties remain a leading cause of failure in CNS drug development.

By Mugisha Sambazi Kevin