In vitro activity: Clemizole hydrochloride (also known as AL 20, P 48, and/or Reactrol), is a potent inhibitor of transient receptor potential channel TRPC5 (Canonical transient receptor potential channel 5) and also an H1 histamine receptor antagonist. It is found to substantially inhibit HCV replication. The IC50 of Clemizole for RNA binding by NS4B is 24±1 nM, whereas its EC50 for viral replication is 8 μM. Clemizole efficiently blocks TRPC5 currents and Ca(2+) entry in the low micromolar range (IC50 = 1.0-1.3 μM). Clemizole was not only effective in blocking heterologously expressed TRPC5 homomers but also TRPC1:TRPC5 heteromers as well as native TRPC5-like currents in the U-87 glioblastoma cell line.

Kinase Assay: The IC50 of Clemizole for RNA binding by NS4B is 24±1 nM, whereas its EC50 for viral replication is 8 μM. Clemizole efficiently blocks TRPC5 currents and Ca(2+) entry in the low micromolar range (IC50 = 1.0-1.3 μM).

Cell Assay: Clemizole hydrochloride is found to inhibit HCV RNA replication in cell culture that is mediated by its suppression of NS4B’s RNA binding, with little toxicity for the host cell. The EC50 of Clemizole on the W55R mutant J6/JFH RNA is ~18 μM (2.25 times the EC50 of the wild-type RNA). Clemizole is a novel inhibitor of TRPC5 channels. Clemizole efficiently blocks TRPC5 currents and Ca2+ entry in the low micromolar range (IC50=1.0-1.3 μM). Clemizole exhibits a six-fold selectivity for TRPC5 over TRPC4β (IC50=6.4 μM), the closest structural relative of TRPC5, and an almost 10-fold selectivity over TRPC3 (IC50=9.1 μM) and TRPC6 (IC50=11.3 μM). Clemizole hydrochloride as a novel blocker of TRPC5 with a half-maximal inhibitory concentration of 1.1 μM. The concentration-response curves confirmed a concentration-dependent block of TRPC5 by Clemizole and revealed an apparent IC50 of 1.1±0.04 μM.