title: Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2.
abstract: 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(-)].
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Delpire
authors.forename: Emily
authors.initials: E
authors.lastname: Days
authors.forename: L Michelle
authors.initials: LM
authors.lastname: Lewis
authors.forename: Dehui
authors.initials: D
authors.lastname: Mi
authors.forename: Kwangho
authors.initials: K
authors.lastname: Kim
authors.forename: Craig W
authors.initials: CW
authors.lastname: Lindsley
authors.forename: C David
authors.initials: CD
authors.lastname: Weaver
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D009474:Neurons; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship; D027981:Symporters / Q000037:antagonists & inhibitors*; D000096922:K Cl- Cotransporters
chemical_list: D054852:Small Molecule Libraries; D027981:Symporters; D000096922:K Cl- Cotransporters