Design of a potent and selective inhibitor of the intermediate-conductance Ca²⁺-activated K⁺channel,<i>IKCa1</i>: A potential immunosuppressant

TLDR

Clotrimazole, a potent IKCa1 inhibitor used in trials for sickle cell disease, diarrhea, and rheumatoid arthritis, is limited by its inhibition of cytochrome P450 enzymes. The study aimed to design a clotrimazole analog that selectively blocks IKCa1 without affecting cytochrome P450 activity. By screening 83 triarylmethanes, the authors identified an IKCa1 pharmacophore comprising a (2‑halogenophenyl)diphenylmethane core with a polar heterocycle (pyrazole or tetrazole) or N‑substituent, distinct from the imidazole ring required for CYP450 inhibition, and synthesized pyrazole, acetonitrile, and tetrazole derivatives that selectively blocked IKCa1. TRAM‑34, a 1‑[(2‑chlorophenyl)diphenylmethyl]‑1H‑pyrazole, potently and selectively inhibits IKCa1 (Kd 20–25 nM, 200–1,500× selectivity), suppresses lymphocyte proliferation (EC50 100 nM–1 µM), and synergizes with cyclosporin A, suggesting therapeutic promise as an immunosuppressant.

Abstract

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K + channel, IKCa1 , is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The “ IKCa1 -pharmacophore” consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar π-electron-rich heterocycle (pyrazole or tetrazole) or a −C 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 N group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively block IKCa1 . TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1 H -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 selective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [ 3 H]thymidine incorporation of preactivated lymphocytes with EC 50 -values of 100 nM-1 μM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants.