Small ligands modulating the activity of mammalian adenylyl cyclases: a novel mode of inhibition by calmidazolium.

Molecular cloning of membrane-spanning mammalian adenylyl cyclases (ACs) has led to the discovery of nine different isotypes, making ACs potentially useful therapeutic targets. This study investigated the mechanism by which fungicidal nitroimidazole compounds modulate AC activity. Current evidence indicates that biological control of AC activity occurs through the cytosolic domains. Hence, full-length ACII, ACIX, and recombinant fusion proteins composed of the cytoplasmic loops of human ACIX or the first and second cytoplasmic loops of rat ACV and ACII, respectively, were expressed in human embryonic kidney 293 cells. The AC activities of the respective proteins were characterized, and their modulation by nitroimidazoles was investigated. Calmidazolium inhibited the activities of both full-length ACs and soluble fusion proteins (IC(50), approximately 10 microM). Inhibition of ACIX by calmidazolium was mediated by direct interaction with the catalytic core in a noncompetitive fashion. ACIX was essentially insensitive to 2'-deoxyadenosine 3'-monophosphate, a known blocker of AC activity. The ACV-ACII fusion protein was inhibited by calmidazolium (IC(50), approximately 20 microM) as well as by 2'-deoxyadenosine 3'-AMP (IC(50), approximately 2 microM), in a manner indicating independent mechanisms of action. Taken together, the data demonstrate that ACIX is insensitive to adenosine analogs and that calmidazolium inhibits AC activity by a novel, noncompetitive mechanism.