Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2.

Delpire E, Days E, Lewis LM, Mi D, Kim K, Lindsley CW, Weaver CD
Proc Natl Acad Sci U S A. 2009 106 (13): 5383-8

PMID: 19279215 · PMCID: PMC2654392 · DOI:10.1073/pnas.0812756106

KCC2, a neuronal-specific K-Cl cotransporter, plays a major role in maintaining intracellular Cl(-) concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K-Cl cotransporter is dominated by loop diuretics such as furosemide and bumetanide, molecules used in clinical medicine because they inhibit the loop of Henle Na-K-2Cl cotransporter with much higher affinity. To identify molecules that affect KCC2 activity, we developed a fluorescence-based assay suitable for high-throughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. Here, we report the characterization of a small number of inhibitors, some of which inhibit KCC2 activity in the submicomolar range without substantially affecting NKCC1 activity. Using medicinal chemistry, we synthesized a number of variants, tested their effect on KCC2 function, and provide an analysis of structure/activity relationships. We also used one of the compounds to demonstrate competitive inhibition in regard to external [K(+)] versus noncompetitive inhibition in respect to external [Cl(-)].

MeSH Terms (8)

Binding, Competitive Cell Line Drug Discovery Humans Neurons Small Molecule Libraries Structure-Activity Relationship Symporters

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