期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 13, 页码 5383-5388出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0812756106
关键词
fluorescence; high-througput screening; Na-K-2Cl cotransporter; thallium
资金
- National Institutes of Health [R21 NS53658, U54 MH074427, U54 MH084659]
- Vanderbilt Specialized Chemistry Center for Accelerated Probe Development
KCC2, a neuronal-specific K-CI 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-2CI 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 [CI-].
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