Chloride extrusion enhancers as novel therapeutics for neurological diseases

Journal name:
Nature Medicine
Year published:
Published online

The K+-Cl cotransporter KCC2 is responsible for maintaining low Cl concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABAA and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl transport10, 11, 12. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl]i. CLP257 restored impaired Cl transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

At a glance


  1. Screening for, and improvement of KCC2-dependent intracellular Cl- lowering compounds.
    Figure 1: Screening for, and improvement of KCC2-dependent intracellular Cl lowering compounds.

    (a) Expression of KCC2 in NG108 cells stably transfected with Clomeleon (NG108-cl). Top, immunoblots of KCC2 in HEK293-cl cells (negative control), hippocampal neurons (positive control) and NG108-cl cells (total lysates versus after KCC2 immunoprecipitation; IP). Asterisk indicates a nonspecific band not observed after IP. Bottom, immunoblot of NKCC1 in total lysates of all cell types. Input 1 and 2: β-actin used as loading control. The top band in Input 1 corresponds to IgGs serving as control for the amount of antibodies used for IP. MW, molecular weight. (b) Relationship between [Cl] and the ratio of Clomeleon YFP to CFP fluorescence. [Cl]i was clamped to [Cl]e by membrane permeabilization using 0.15% Triton X-100 (means ± s.d.; n = 4 assays). Inset, effect of pH on fluorescence ratio (open circles: with 0.15% Triton X-100, closed squares: without Triton X-100; means ± s.d.; n = 4 assays). (c) Chemical structures of the CL-058 analogs tested in d. (d) Concentration-response curves of the CL-058 hit compound and selected analogs. In c and d, [Cl]i was measured after a 5-h exposure to compounds. (e) Concentration-response curves of CLP257 in NG108-cl and HEK293-cl cells (means ± s.e.m.; n = 4 assays). Inset shows [Cl]i response of NG108-cl cells to 1.25 μM CLP257 over time (means ± s.e.m.; n = 4 assays). (f) Effect of CLP257 pretreatment on 45-min Rb+ influx assays in CCC-expressing X. laevis oocytes (means ± s.e.m.; n = 12–20 oocytes. Kruskall-Wallis H = 41; ***P < 0.001). (g) Effect of the KCC2 antagonist VU0240551 on response to CLP257. Shown are [Cl]i in NG108-cl cells after 5-h exposure to CLP257 500 nM + DMSO vehicle or VU0240551 (means ± s.e.m.; n = 4 assays).

  2. CLP257 restores Cl- transport in adult spinal cord slices with impaired KCC2 function.
    Figure 2: CLP257 restores Cl transport in adult spinal cord slices with impaired KCC2 function.

    (a) Diagram illustrating inversion of KCC2 transport upon raising [K+]e. (b) Color-coded lifetime image of lamina II cells loaded with the Cl indicator MQAE in slices treated with BNDF or BDNF with 15 mM [K+]e; lower lifetime values correspond to high [Cl]. Scale bars, 50 μm. (c) Time-lapse recording of Cl accumulation in the cell bodies of neurons upon extracellular application of 15 mM KCl. Measurements taken every 10 s. Scale bars: vertical, 50 ps; horizontal, 20 s. Insets show examples of photon-distribution histograms fitted to extract the lifetimes shown in b (arrows), during the control period (top) and after Cl equilibration in 15 mM KCl (bottom). Scale bars: vertical, 40 photons; horizontal, 2 ns. (d) Effects of CLP257 on the efficacy of Cl transport in lamina II cells. Slope of the change in fluorescence 2 h after addition of CLP257 (100 μM) to a slice pretreated with BDNF for 2 h. (e) Comparison of the rate of Cl changes measured in SDH neurons in spinal cord slices treated with BDNF or taken from PNI rats, before and 2 h after addition of CLP257 (means ± s.e.m.; n = 4–12, H: 32; *P < 0.05; ***P < 0.001). Ctrl represents the rate of Cl transport measured in naive animals. (f,g) Effect of CLP257 (25 μM, >1 h) on EGABA in SDH neurons in slices from rats after PNI (f) or BDNF (g) treatment. Left, responses to 30-ms GABA puffs (black arrowheads) in lamina II neurons in the presence of a Cl load (30 mM in the recording pipette). Right, I-V relationships from representative cells. (h) Pooled EGABA of neurons from PNI rats under control conditions (n = 11 cells) or treated with CLP257 (n = 8 cells; Mann-Whitney U = 9, **P < 0.01) and from BDNF-treated slices with (n = 8 cells) or without CLP257 (n = 7 cells; U: 7, *P < 0.05). (i) Quantification of GABAA responses (normalized to baseline) in neurons after 5 min of CLP257 incubation (25 μM, n = 7 cells, Wilcoxon W = −4, P > 0.05; 100 μM, n = 5 cells, W: −5, P > 0.05) using 120 mM CsCl–filled pipettes and in the presence of the KCC2 antagonist VU0240551. Inset shows representative responses of SDH neurons to GABA puffs before and after application of CLP257 (100 μM).

  3. CLP257 increases plasmalemmal KCC2 protein in BDNF-treated adult rat spinal cord slices.
    Figure 3: CLP257 increases plasmalemmal KCC2 protein in BDNF-treated adult rat spinal cord slices.

    (a) Two cell surface biotinylation and total immunoblots (Exp. 1 and Exp. 2) of KCC2 monomers and dimers after BDNF + 100 μM CLP257 treatment. Input 1: total biotinylated cell surface proteins used as loading control. Input 2: overexposed film of a β-actin immunoblot showing no signal. Input 3: total β-actin protein used as loading control. (b) Quantification of the ratio of surface to total KCC2 monomer and dimer expression in response to treatment of slices with BDNF + 100 μM CLP257 versus control BDNF + DMSO; each spinal cord served as its own control (means ± s.e.m.; n = 16 pairs from 16 rats; Wmonomers: 9; Wdimers: 26; *P < 0.05; ***P < 0.001).

  4. In vivo assessment of the efficacy and pharmacokinetics of CLP257 and its prodrug CLP290.
    Figure 4: In vivo assessment of the efficacy and pharmacokinetics of CLP257 and its prodrug CLP290.

    (a) Input/output relationship between the field electrophysiological response recorded in the superficial layers of the spinal dorsal horn and the strength of mechanical stimuli applied to the receptive field (footpad) after local spinal administration of vehicle (saline) or CLP257 in normal animals. Inset: representative example of field responses upon local spinal administration of saline, CLP257 or tetrodotoxin (TTX). (means ± s.e.m., n = 7 rats). (b) Same analysis as in a but in PNI rats (means ± s.e.m.; W: 21, *P < 0.05). (c) Effect of CLP257 on the normalized maximal field response in control animals (Ctrl, n = 7 rats) and animals with peripheral nerve injury (PNI, n = 6 rats, W: 28, *P < 0.05). (d) Black bars: mean (± s.e.m.) maximal field responses to mechanical stimulation of the receptive field in control (Ctrl, n = 7 rats) and PNI rats (PNI, n = 6 rats, U: 4, *P < 0.05). Red bars: effect of CLP257 application in control (n = 7 rats) and PNI rats (n = 6 rats, W: 27, *P < 0.05). (e) Effect of CLP257 on paw withdrawal threshold in PNI rats. CLP257 was dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (HPCD) and administered i.p. (means ± s.e.m.; n = 8–10; *P < 0.05, **P < 0.01; ***P < 0.001). BW, body weight. (f) Pharmacokinetic profile of CLP257 and the carbamate prodrug CLP290 in rats after intravenous (i.v.), i.p. or oral (p.o.) administration (means ± s.e.m.; n = 3 animals per time point). Inset: structure of CLP290. (g) Analgesic effect of CLP290 administered p.o. in a PNI rat (means ± s.e.m.; n = 7–32; *P < 0.05). (h) Effect of CLP290 and pregabalin (at equipotent analgesic dose) on motor performance in rats as measured by time spent on an accelerating rotorod (means ± s.e.m.; n = 4–12; U: 6; *P < 0.05).


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Author information

  1. These authors contributed equally to this work.

    • Martin Gagnon &
    • Marc J Bergeron


  1. Institut Universitaire en Santé Mentale de Québec, Québec, Québec, Canada.

    • Martin Gagnon,
    • Marc J Bergeron,
    • Guillaume Lavertu,
    • Annie Castonguay,
    • Robert P Bonin,
    • Jimena Perez-Sanchez,
    • Dominic Boudreau,
    • Karine Bachand,
    • Christian Tardif &
    • Yves De Koninck
  2. Department of Psychiatry & Neuroscience, Université Laval, Québec, Québec, Canada.

    • Martin Gagnon,
    • Marc J Bergeron,
    • Guillaume Lavertu,
    • Annie Castonguay,
    • Robert P Bonin,
    • Jimena Perez-Sanchez,
    • Dominic Boudreau,
    • Karine Bachand &
    • Yves De Koninck
  3. Chlorion Pharma, Laval, Québec, Canada.

    • Martin Gagnon,
    • Sasmita Tripathy,
    • Bin Wang,
    • Lionel Dumas,
    • Isabelle Valade,
    • Irenej Kianicka,
    • Giorgio Attardo &
    • Jeffrey A M Coull
  4. Graduate Program in Biophotonics, Université Laval, Québec, Québec, Canada.

    • Christian Tardif &
    • Yves De Koninck
  5. Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Canada.

    • Mariève Jacob-Wagner &
    • Paul Isenring


M.G., J.A.M.C. and Y.D.K. conceived of and designed the project. M.G., J.A.M.C., P.I., I.K. and Y.D.K. supervised the experiments. M.G., M.J.B., G.L., A.C., R.P.B., J.P.-S., D.B., K.B. and M.J.-W. performed the experiments. G.A., S.T., B.W., L.D. and I.V. designed and synthesized analogs of CL-058. M.G., M.J.B., G.L., A.C., R.P.B., J.P.-S., D.B., M.J.-W., C.T. and I.K. analyzed the data. M.G., M.J.B., G.L., A.C., R.P.B. and Y.D.K. wrote the manuscript. All of the authors read and discussed the manuscript.

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