1. Small-molecule screen identifies inhibitors of the neuronal K-CI cotransporter KCC2
- Author
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Delpire, Eric, Days, Emily, Lewis, L. Michelle, Mi, Dehui, Kim, Kwangho, Lindsley, Craig W., and Weaver, C. David
- Subjects
Biological transport -- Physiological aspects ,Biological transport -- Research ,Enzyme inhibitors -- Physiological aspects ,Enzyme inhibitors -- Research ,Carrier proteins -- Physiological aspects ,Carrier proteins -- Research ,Science and technology - Abstract
KCC2, a neuronal-specific K-CI cotransporter, plays a major role in maintaining intracellular CI- concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K-CI cotransporter is dominated by loop diuretics such as furosemide and bumetanide, molecules used in dinical medicine because they inhibit the Ioop of Henle Na-K-2CI cotransporter with much higher affinity. To identify molecules that affect KCC2 activity, we developed a fluorescence-based assay suitable for highthroughput 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.sup.+]] versus noncompetitive inhibition in respect to external [CI-]. fluorescence | high-througput screening | Na-K-2CI cotransporter | thallium
- Published
- 2009