Design of a potent and selective inhibitor of the intermediate-conductanceCa2+-activated K+ channel, IKCa1: A potential immunosuppressant

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conduc tance calcium-activated K+ channel, IKCa1, is in clinical trials for the tr eatment of sickle cell disease and diarrhea and is effective in amelioratin g the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P 450 enzymes by clotrimazole limits its therapeutic value. We have used a ra tional design strategy to develop a clotrimazole analog that selectively in hibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triar ylmethanes revealed the pharmacophore for channel block to be different fro m that required for cytochrome P450 inhibition. The "IKCa1-pharmacophore" c onsists of a (2-halogenophenyl)diphenylmethane moiety substituted by an uns ubstituted polar pi-electron-rich heterocycle (pyrazole or tetrazole) or a -C=N group, whereas cytochrome P450 inhibition absolutely requires the imid azole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synth esized and found to selectively block IKCa1. TRAM-34 (1-[(2-chlorophenyl)di phenylmethyl]-1H-pyrazole) inhibits the cloned and the native IKCa1 channel in human T lymphocytes with a K-d of 20-25 nM and is 200- to 1,500-fold se lective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppre ssion of mitogen-stimulated [H-3]thymidine incorporation of preactivated ly mphocytes with EC50-values of 100 nM-1 mu M depending on the donor. Combina tions of TRAM-34 and cyclosporin A are more effective in suppressing lympho cyte mitogenesis than either compound alone. Our studies suggest that TRAM- 34 and related compounds may hold therapeutic promise as immunosuppressants .