Discovery and Characterization of Small Molecule Potentiators of KCC2 Activity

Abstract

Deficits in inhibitory neurotransmission, and the resulting excitatory/inhibitory (E/I) imbalance, are a feature of a host of neurological disorders, including: epilepsy, chronic pain, schizophrenia, and autism. Increased intracellular Cl− ([Cl−]i) has been implicated as an underlying cause of the E/I imbalance via disruption of fast inhibitory neurotransmission. The K+‐Cl− cotransporter 2 (KCC2) is a principal exporter of neuronal Cl−. Owing to the effect of KCC2 on [Cl−]i, pharmacological potentiation of KCC2 activity has become a promising therapeutic approach for these neurological disorders. While there has been substantial progress in discovering pharmacological potentiators of KCC2 activity in recent years, current pharmacological tools targeting KCC2 either act by indirect mechanisms or have some controversy surrounding their activity at KCC2. Although these compounds represent major improvements to KCC2 potentiator pharmacology, the discovery of direct‐acting KCC2 potentiators would still be a substantial contribution to the field because: (a) direct‐acting KCC2 potentiators would be better chemical tools to assess the contribution of KCC2 to a given phenotype, since they would lack the off‐target effects associated with indirect KCC2 potentiators, and (b) de‐risk KCC2 itself as a therapeutic target for drug discovery. To discover direct‐acting KCC2 potentiators, we have developed and validated a suite of high throughput‐compatible assays to measure KCC2 activity. Using a primary assay measuring Cl− export via a genetically‐encoded Cl− sensor, we have iteratively screened over 30,000 compounds for their effect on KCC2 activity. Our iterative screening approach maximized our ability to explore chemical scaffolds that were active across primary and orthogonal assays – allowing us to discover a series of putative KCC2 potentiators, despite screening a relatively small chemical library. From these efforts, we have discovered compounds capable of decreasing [Cl−]i in a KCC2 inhibitor‐sensitive manner, potentiating transport of KCC2 substrate congeners in a KCC2‐dependent fashion, and blocking epileptiform calcium oscillations in cultured neurons. We are currently investigating the mechanism by which these compounds affect KCC2 activity. The compound class we have discovered may be useful as a pharmacological tool for evaluating the therapeutic potential of KCC2 in a host of neurological disorders which feature E/I imbalance.Support or Funding InformationVanderbilt Institute for Clinical and Translational Research Award (VR52556)Chemistry‐Biology Interface T32 (5T32GM065086‐15)