Discovery of potent CLC chloride channel inhibitors

Anion-transport proteins are. central to all of physiology, for processes ranging from regulating bone-density, muscle excitability; and blood pressure, to facilitating extreme-acid survival of pathogenic bacteria. 4,4-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has-been used as an anion-transport inhibitor for decades. In this study, We demonstrate that polythiourea products derived from DIDS hydrolysis inhibit three different CLC chloride-transport proteins, ClC-ec1, ClC-0, and ClC-Ka, more effectively than DIDS itself. The structures of the five major products were determined, by NMR spectroscopy, mass spectrometry, and chemical synthesis. These compounds bind directly to the CLC proteins, as evidenced, by the fact that inhibition of ClC-0 occurs only from the intracellular side and inhibition of ClC-Ka is prevented by,the point mutation N68D. These polythioureas are the highest affinity inhibitors known for the CLCs and provide a new class,of chemical probes for dissecting the molecular mecha- nisms of chloride transport.