Direct stimulation of adenylyl cyclase 9 by the fungicide imidazole miconazole
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In mammals, nine genes encode trans-membrane adenylyl cyclase (tmAC) isoforms that synthesize the intracellular messenger compound cAMP from ATP. As cAMP is produced in virtually all types of cell, isoform-selective modulators of tmAC would have major research and therapeutic potential. This study investigated the effects of fungicide imidazoles previously shown to suppress cAMP production in various tissues on the activities of tmAC isoforms AC1, 2, or 9 stably expressed in human embryonic kidney 293 cells. Intact cells, as well as crude membranes, were exposed to various imidazoles or known stimulators of tmAC and the ensuing changes in the production of cAMP analyzed. In crude membranes, the activity of AC9 in the presence of GDP-β-S was enhanced by miconazole with an EC50 of ~ 8 μM, while AC1 and AC2 were inhibited with an IC50 of ~ 20 μM. Clotrimazole (10–100 μM) was an inhibitor of all the ACs tested. Substrate saturation analysis indicated that miconazole increased the Vmax of AC9 by 3-fold while having no effect on the Km. In intact cells, the effect of miconazole on cAMP production through AC9 was additive with that of isoproterenol. The stimulation of cAMP production by miconazole was inhibited by Ca2+, and this could be prevented by the calcineurin blocker FK506. In sum, activation of AC9 by miconazole is through a mechanism distinct from that of forskolin, activated G proteins, or the COOH-terminal mediated autoinhibition. However, it is subject to the AC9 isoform-specific inhibition by Ca2+/calcineurin. Differential modulation of mammalian tmAC paralogs appears to be achievable by an imidazole with phenylated side chains. Optimization of the lead compound and exploration of the underlying mechanism(s) of action in more detail could exploit this further.
KeywordsAdenosine 3′5′monophosphate Trans-membrane adenylyl cyclase Adenylyl cyclase Ca2+/calcineurin
We thank Dr. Janice Paterson and Mrs. Susan M. Smith for HEK293 cells expressing human AC9, Dr. A. Friedrich, Fujisawa GmbH, Munich, Germany for FK506, Dr. William Parsons, Merck and Co, Rahway N.J., for L685,818, and Dr. H. Wachtel, Schering AG., Berlin, Germany for rolipram.
AP and JS carried out experiments, collated and analyzed data, FAA designed and carried out experiments, analyzed and collated data, wrote the manuscript. All authors have agreed on the text of the submitted manuscript.
This work was supported by the Medical Research Council, UK and the Servier-Egis research co-operation agreement.
Compliance with ethical standards
Conflict of interest
AP and FAA were employees of Egis Pharmaceuticals PLC, Budapest, Hungary.
- Antoni FA (2016) ADCY9 (Adenylyl cyclase 9). In: Choi S (ed.) Encyclopedia of Signaling Molecules. Springer International Publishing, New York, pp. https://doi.org/10.1007/1978-1001-4614-6438-1009
- Baldwin TA, Dessauer CW (2018) Function of adenylyl cyclase in heart: the AKAP connection. J Cardiovasc Dev Dis 5. https://doi.org/10.3390/jcdd5010002
- Defer N, Best-Belpomme M, Hanoune J (2000) Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol 279:F400–F416Google Scholar
- Dumont FJ, Staruch MJ, Koprak SL, Siekierka JJ, Lin CS, Harrison R, Sewell T, Kindt VM, Beattie TR, Wyratt M, Sigal NH (1992) The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin. J Exp Med 176:751–760CrossRefGoogle Scholar
- Kerwin JF Jr (1994) Adenylate cyclase subtypes as molecular drug targets. Annu Rep Med Chem 29:287–296Google Scholar
- Kratochwil NA, Gatti-McArthur S, Hoener MC, Lindemann L, Christ AD, Green LG, Guba W, Martin RE, Malherbe P, Porter RH, Slack JP, Winnig M, Dehmlow H, Grether U, Hertel C, Narquizian R, Panousis CG, Kolczewski S, Steward L (2011) G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey. Curr Top Med Chem 11:1902–1924CrossRefGoogle Scholar
- Sargeant P, Farndale RW, Sage SO (1994) The imidazole antimycotics econazole and miconazole reduce agonist-evoked protein-tyrosine phosphorylation and evoke membrane depolarisation in human platelets: cautions for their use in studying Ca2+ signalling pathways. Cell Calcium 16:413–418CrossRefGoogle Scholar
- Watson PA (1990) Direct stimulation of adenylate cyclase by mechanical forces in S49 mouse lymphoma cells during hyposmotic swelling. J Biol Chem 265:6569–6575Google Scholar