Sequence: electroneutral potassium-chloride cotransporter KCC2 [Homo sapiens]

Gene:SLC12A5     Conserved Domain     Related Protein 3D Structures

Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters
Assay Provider: Eric Delpire
Assay Provider Affliation: Vanderbilt University
Grant Title: Identification of Novel Modulators of Cl- dependent Transport Process via HTS
Grant Number: R21NS053658-01
Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, David Weaver
Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig Lindsley


Cation-chloride cotransporters such as K-Cl cotransport and Na-K-2Cl cotransport play major roles in a variety of physiological settings, including the modulation of GABAergic synaptic transmission. For instance, KCC2, a neuronal-specific K-Cl cotransporter is up-regulated in the brain during postnatal development, and is responsible for lowering the intracellular Cl- concentration in neurons, thus promoting GABA inhibition. Reduction in KCC2 expression results in brain hyperexcitability, as demonstrated by animal models. Furthermore, KCC2 expression is decreased in brain tissue isolated from epileptic patients.

There are very few pharmacological agents that affect K-Cl cotransporters. First, there are no specific inhibitors of K-Cl cotransporters. Furosemide is mostly used to inhibit K-Cl cotransporter function, but the diuretic is not very potent and is not specific as it inhibits the Na-K-2Cl cotransporter (diuretic effect), many Cl- channels including the GABAA receptor. Finding new inhibitors will provide important tools for the study of KCC2 in modulating inhibitory neurotransmission.

To identify novel molecules that affect the activity of KCC2, we developed a novel fluorescence-based method appropriate for high throughput screening. Indeed, all traditional methods used to assess cation-chloride cotransporter activity are not adequate for HTS. The method makes use of a fluorescent dye sensitive to thallium, a cation that is transported by cation-chloride cotransporter. Thallium, in combination with dye, was previously used to assay uptakes of K+ though K+ channels(Weaver, et al). Using this method, we screened a library of 234,000 compounds with KCC2 as a target, and identified a number of compounds that affect the activity of the cotransporter.

The screen was conducted in multiple 384-well plates containing HEK293 cells over-expressing KCC2. For each plate, 4 columns were dedicated to controls: 2 columns (32 wells) using standard HBSS conditions with ouabain and 2 columns using HBSS + 2 mM bumetanide. These wells allowed for internal quality assessment. Each of the remaining 320 wells was dedicated to one compound tested at a concentration of nominally 10 uM. Utilizing 733 plates, we screened 234,560 compounds with an average Z' value of 0.686 +/- 0.090, indicating both high fidelity and good separation between the two control signals (plus and minus bumetanide). Compounds that decreased or stimulated fluorescence signals more than three standard deviations from the test compound population measured on a per-plate basis were counted as primary hits (AID 1456). We pursued only antagonists.

After elimination of duplicates from the primary hits, we preformed secondary assays. Each compound was tested in duplicate at a concentration of 10 uM using both KCC2-overexpressing cells (AID 1717) and naive HEK293 cells (AID 1716) in the presence of ouabain. With each cell line, a secondary screen was also performed in the absence of ouabain (HEK, AID 1718 and KCC2, AID 1715). KCC2 expressing cells were also tested in sodium-free buffer (AID 1713) and in the presence of bumetanide (AID 1714). The compounds of interest where then tested at various concentrations (ranging from 0.5 nM to 30 uM) in triplicate against KCC2 and HEK cells in the presence of ouabain (AID 1736, AID 1753), in the presence of bumetanide (AID 1723, AID 1735), and in sodium-free buffer (AID 1737, AID 1738). Using the potency and and structure of the compound in the dose-dependent testing, we selected 26 compounds that inhibited thallium-induced fluorescence increase and tested them on KCC2 and NKCC1 functions using 86Rb uptakes in HEK293 cells (AID 1734). For KCC2, we used N-ethylmaleimide pre-treatment in KCC2 over-expressing HEK293 cells. The alkylating agent not only stimulates K-Cl cotransport, but abrogates the function of the native Na-K-2Cl cotransporter. In these assays furosemide inhibited 93% of the uptake at a concentration of 2 mM and had no effect at a concentration below 30 uM. For NKCC1, we used a hyperosmotic solution to stimulate the transporter in naive HEK293 cells. 86Rb uptake was 95% inhibited by a low dose (20 uM) of bumetanide. Compounds like D4 (SID 24814385) inhibit KCC2 with a measured IC50 in the submicromolar range while minimally affecting NKCC1. In contrast, other compounds like D8 (17512842) acted equally well on KCC2 and NKCC1. Two rounds of synthesis, afforded the probe molecule (CID 25064704), the most potent and the only selective KCC2 antagonist described. CID 25067404 has an IC50 of 537nM for inhibition of KCC2 in Rb+ flux assays, a greater than 3 orders of magnitude improvement over bumetanide (IC50 = 655uM). Moreover, it was found to be highly selective for KCC2 versus NKCC1 (IC50>>50uM). A lead profiling screen from MDS Pharma (The Triad 2200 Renaissance Blvd, Suite 400, King of Prussia, PA 19406-2755) revealed that the probe molecule had an excellent ancillary profile with no activities >50% at 10uM (AID 1793). Furthermore, the compound is soluble in pure DMSO at >50mM and in saline at 10mg/mL.

The combination of KCC2 potency, selectivity versus NKCC1, broad panel selectivity, favorable calculated physiochemical properties and observed solubility profile, provides a best in class KCC2 antagonist probe which can be used in vitro to study KCC2 function with confidence.

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 Mar 31;106(13):5383-8. Epub 2009 Mar 11. PMID: 19279215

A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells. Weaver CD, Harden D, Dworetzky SI, Robertson B, Knox RJ.J Biomol Screen. 2004 Dec;9(8):671-7.PMID 15634793

Grant Number: R21NS053658-01