{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,28]],"date-time":"2026-10-28T01:35:52Z","timestamp":1793151352251,"version":"4.5.0"},"reference-count":41,"publisher":"National Academy of Sciences","issue":"13","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2009,3,31]]},"abstract":"<jats:p>\n                    KCC2, a neuronal-specific K-Cl cotransporter, plays a major role in maintaining intracellular Cl\n                    <jats:sup>\u2212<\/jats:sup>\n                    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\n                    <jats:sup>+<\/jats:sup>\n                    ] versus noncompetitive inhibition in respect to external [Cl\n                    <jats:sup>\u2212<\/jats:sup>\n                    ].\n                  <\/jats:p>","DOI":"10.1073\/pnas.0812756106","type":"journal-article","created":{"date-parts":[[2009,3,11]],"date-time":"2009-03-11T22:17:28Z","timestamp":1236809848000},"page":"5383-5388","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":148,"title":["Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2"],"prefix":"10.1073","volume":"106","author":[{"given":"Eric","family":"Delpire","sequence":"first","affiliation":[{"name":"Department of Anesthesiology,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Days","sequence":"additional","affiliation":[{"name":"Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, Vanderbilt University Medical Center, Nashville, TN 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"L. Michelle","family":"Lewis","sequence":"additional","affiliation":[{"name":"Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, Vanderbilt University Medical Center, Nashville, TN 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dehui","family":"Mi","sequence":"additional","affiliation":[{"name":"Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, Vanderbilt University Medical Center, Nashville, TN 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kwangho","family":"Kim","sequence":"additional","affiliation":[{"name":"Vanderbilt Specialized Chemistry Center, and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Craig W.","family":"Lindsley","sequence":"additional","affiliation":[{"name":"Department of Pharmacology,"},{"name":"Vanderbilt Specialized Chemistry Center, and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C. David","family":"Weaver","sequence":"additional","affiliation":[{"name":"Department of Pharmacology,"},{"name":"Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, Vanderbilt University Medical Center, Nashville, TN 37232"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2009,3,31]]},"reference":[{"key":"e_1_3_3_1_2","doi-asserted-by":"publisher","DOI":"10.1002\/hipo.10014"},{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.eplepsyres.2008.02.005"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.neulet.2008.04.032"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature01868"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.neures.2006.08.012"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pain.2008.07.005"},{"key":"e_1_3_3_7_2","article-title":"Reduced potassium-chloride co-transporter expression in spinal cord dorsal horn neurons contributes to inflammatory pain hypersensitivity in rats","author":"Zhang W","year":"2008","unstructured":"W Zhang, L-Y Liu, T-L Xu, Reduced potassium-chloride co-transporter expression in spinal cord dorsal horn neurons contributes to inflammatory pain hypersensitivity in rats. Neuroscience, 2008).","journal-title":"Neuroscience"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pain.2007.10.016"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1186\/1744-8069-4-36"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0030-6665(20)30770-2"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0378-5955(97)00009-9"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1038\/9713"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF01871100"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1152\/ajpcell.1997.273.5.C1516"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M003112200"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF00233463"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI111867"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bb.18.060189.002145"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.physiol.62.1.493"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1177\/1087057104268749"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1085\/jgp.200409059"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1124\/mol.107.043208"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.27.16245"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.20-21-08069.2000"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.4390-07.2007"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1113\/jphysiol.2008.157024"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(01)00297-5"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1152\/jn.00616.2004"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.4998-05.2006"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.3791-07.2008"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1136\/bmj.2.5425.1640"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1136\/bmj.1.5794.211"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.77.3.1711"},{"key":"e_1_3_3_34_2","first-page":"221","article-title":"A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes","volume":"70","author":"Lauf PK","year":"1980","unstructured":"PK Lauf, BE Theg, A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes. Biochem Biophys Res Commun 70, 221\u2013242 (1980).","journal-title":"Biochem Biophys Res Commun"},{"key":"e_1_3_3_35_2","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1016\/S0026-895X(25)13396-8","article-title":"Demonstration of a [K+Cl\u2212]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: Regulation of cell swelling and distinction from the bumetanide-sensitive [Na+, K+, Cl\u2212]-cotransport system","volume":"33","author":"Garay RP","year":"1988","unstructured":"RP Garay, C Nazaret, PA Hannaert, EJ Cragoe, Demonstration of a [K+Cl\u2212]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: Regulation of cell swelling and distinction from the bumetanide-sensitive [Na+, K+, Cl\u2212]-cotransport system. Mol Pharmacol 33, 696\u2013701 (1988).","journal-title":"Mol Pharmacol"},{"key":"e_1_3_3_36_2","first-page":"309","article-title":"Cation-chloride cotransporters in neuronal communication","volume":"15","author":"Delpire E","year":"2000","unstructured":"E Delpire, Cation-chloride cotransporters in neuronal communication. News Physiol Sci 15, 309\u2013312 (2000).","journal-title":"News Physiol Sci"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncpneuro0883"},{"key":"e_1_3_3_38_2","unstructured":"IH Segel Enzyme Kinetics. Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems . (Wiley New York 1975)."},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1085\/jgp.97.2.173"},{"key":"e_1_3_3_40_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF00370803"},{"key":"e_1_3_3_41_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)81739-8"}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.0812756106","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,2,8]],"date-time":"2025-02-08T04:44:58Z","timestamp":1738989898000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/full\/10.1073\/pnas.0812756106"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2009,3,31]]},"references-count":41,"journal-issue":{"issue":"13","published-print":{"date-parts":[[2009,3,31]]}},"alternative-id":["10.1073\/pnas.0812756106"],"URL":"https:\/\/doi.org\/10.1073\/pnas.0812756106","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2009,3,31]]},"assertion":[{"value":"2008-12-16","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2009-03-31","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}