Characterization of a Novel CLC-ec1 Inhibitor

CLC chloride channels and transporters are vital to many physiological processes. Defects in these proteins can lead to diseases that affect muscle, cardiovascular system, kidneys, and bones. Historically, high affinity inhibitors have played crucial roles in the characterization of ion transporter structure and function and in the treatment of disease. However, there is a dearth of known CLC inhibitors. To search for improved inhibitors, we synthesized derivatives of DIDS (4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid), a small molecule that inhibits several CLC proteins with low affinity and specificity. We found that linking DIDS and octanoic acid generated a CLC-ec1 inhibitor (OADS) that inhibits CLC-ec1 with low micromolar affinity. We are characterizing the mechanism of inhibition using flux assays, electrophysiology, isothermal titration calorimetry, photoaffinity labeling, and mutagenesis. Given that OADS and OADS derivatives that inhibit ClC-ec1 are rather hydrophobic, we also explored their potential to alter lipid bilayer properties using a gramicidin based fluorescence assay.