Abstract
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.
Similar content being viewed by others
References
Antoni FA (2000) Molecular diversity of cyclic AMP signaling. Front Neuroendocrinol 21:103–132
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
Antoni FA, Barnard RJO, Shipston MJ, Smith SM, Simpson J, Paterson JM (1995) Calcineurin feedback inhibition of agonist-evoked cAMP formation. J Biol Chem 270:28055–28061
Antoni FA, Palkovits M, Simpson J, Smith SM, Leitch AL, Rosie R, Fink G, Paterson JM (1998a) Ca2+/calcineurin-inhibited adenylyl cyclase highly abundant in forebrain regions important for learning and memory. J Neurosci 18:9650–9661
Antoni FA, Smith SM, Simpson J, Rosie R, Fink G, Paterson JM (1998b) Calcium control of adenylyl cyclase — the calcineurin connection. Adv Second Messenger Phosphoprotein Res 32:153–172
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
Bassler J, Schultz JE, Lupas AN (2018) Adenylate cyclases: receivers, transducers, and generators of signals. Cell Signal 46:135–144
Beltz S, Bassler J, Schultz JE (2016) Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases. Elife 5:e13098
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of dye-binding. Anal Biochem 72:248–254
Brust TF, Alongkronrusmee D, Soto-Velasquez M, Baldwin TA, Ye Z, Dai M, Dessauer CW, van Rijn RM, Watts VJ (2017) Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties. Sci Signal 10:eaah5381
Coan KE, Maltby DA, Burlingame AL, Shoichet BK (2009) Promiscuous aggregate-based inhibitors promote enzyme unfolding. J Med Chem 52:2067–2075
Defer N, Best-Belpomme M, Hanoune J (2000) Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol 279:F400–F416
Dessauer CW, Gilman AG (1996) Purification and characterization of a soluble form of mammalian adenylyl cyclase. J Biol Chem 271:16967–16974
Dessauer CW, Watts VJ, Ostrom RS, Conti M, Dove S, Seifert R (2017) International Union of Basic and Clinical Pharmacology. CI. Structures and small molecule modulators of mammalian adenylyl cyclases. Pharmacol Rev 69:93–139
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 176:751–760
Feinstein PG, Schrader KA, Bakalyar HA, Tang WJ, Krupinski J, Gilman AG, Reed RR (1991) Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain. Proc Natl Acad Sci U S A 88:10173–10177
Haunsø A, Simpson J, Antoni FA (2003) Small ligands modulating the activity of mammalian adenylyl cyclases: a novel mode of inhibition by calmidazolium. Mol Pharmacol 63:624–631
Husebye ES, Flatmark T (1988) Phosphatidylinositol kinase of bovine adrenal chromaffin granules. Modulation by hydrophilic and amphiphilic cations. Biochem Pharmacol 37:4149–4156
Kerwin JF Jr (1994) Adenylate cyclase subtypes as molecular drug targets. Annu Rep Med Chem 29:287–296
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 11:1902–1924
Krupinski J, Cali JJ (1998) Molecular diversity of the adenylyl cyclases. Adv Second Messenger Phosphoprotein Res 32:53–79
Krupinski J, Coussen F, Bakalyar HA, Tang WJ, Feinstein PG, Orth K, Slaughter C, Reed RR, Gilman AG (1989) Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244:1558–1564
MacNeil S, Griffin M, Cooke AM, Pettett NJ, Dawson RA, Owen R, Blackburn GM (1988) Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry. Biochem Pharmacol 37:1717–1723
Miller PS, Scott S, Masiulis S, De Colibus L, Pardon E, Steyaert J, Aricescu AR (2017) Structural basis for GABAA receptor potentiation by neurosteroids. Nat Struct Mol Biol 24:986–992
Nury H, Van Renterghem C, Weng Y, Tran A, Baaden M, Dufresne V, Changeux JP, Sonner JM, Delarue M, Corringer PJ (2011) X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. Nature 469:428–431
Pálvölgyi A, Simpson J, Bodnár I, Bíró J, Palkovits M, Radovits T, Skehel P, Antoni FA (2018) Auto-inhibition of adenylyl cyclase 9 (AC9) by an isoform-specific motif in the carboxyl-terminal region. Cell Signal 51:266–275
Paterson JM, Smith SM, Harmar AJ, Antoni FA (1995) Control of a novel adenylyl cyclase by calcineurin. Biochem Biophys Res Commun 214:1000–1008
Paterson JM, Smith SM, Simpson J, Grace OC, Sosunov AA, Bell J, Antoni FA (2000) Characterisation of human adenylyl cyclase IX reveals inhibition by Ca2+/calcineurin and differential mRNA polyadenylation. J Neurochem 75:1358–1367
Pierre S, Eschenhagen T, Geisslinger G, Scholich K (2009) Capturing adenylyl cyclases as potential drug targets. Nat Rev Drug Discov 8:321–335
Raker VK, Becker C, Steinbrink K (2016) The cAMP pathway as therapeutic target in autoimmune and inflammatory diseases. Front Immunol 7:123
Renaud J-P, Chari A, Ciferri C, W-t L, Rémigy H-W, Stark H, Wiesmann C (2018) Cryo-EM in drug discovery: achievements, limitations and prospects. Nat Rev Drug Discov 17:471–492
Rosethorne EM, Turner RJ, Fairhurst RA, Charlton SJ (2010) Efficacy is a contributing factor to the clinical onset of bronchodilation of inhaled β2-adrenoceptor agonists. Naunyn Schmiedeberg's Arch Pharmacol 382:255–263
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 16:413–418
Scholich K, Barbier AJ, Mullenix JB, Patel TB (1997) Characterisation of soluble forms of non-chimeric type V adenylyl cyclases. Proc Natl Acad Sci U S A 94:2915–2920
Seebacher T, Linder JU, Schultz JE (2001) An isoform-specific interaction of the membrane anchors affects mammalian adenylyl cyclase type V activity. Eur J Biochem 268:105–110
Seifert R, Lushington GH, Mou TC, Gille A, Sprang SR (2012) Inhibitors of membranous adenylyl cyclases. Trends Pharmacol Sci 33:64–78
Serezani CH, Ballinger MN, Aronoff DM, Peters-Golden M (2008) Cyclic AMP: master regulator of innate immune cell function. Am J Respir Cell Mol Biol 39:127–132
Stalla GK, Stalla J, Huber M, Loeffler JP, Hollt V, von Werder K, Muller OA (1988) Ketoconazole inhibits corticotropic cell function in vitro. Endocrinology 122:618–623
Stalla GK, Stalla J, von Werder K, Muller OA, Gerzer R, Hollt V, Jakobs KH (1989) Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme. Endocrinology 125:699–706
Tang WJ, Gilman AG (1995) Construction of a soluble adenylyl-cyclase activated by G(s)a and forskolin. Science 268:1769–1772
Taussig R, Gilman AG (1995) Mammalian membrane-bound adenylyl cyclases. J Biol Chem 270:1–4
Tesmer J, Sprang S (1998) The structure, catalytic mechanism and regulation of adenylyl cyclase. Curr Opin Struct Biol 8:713–719
Tesmer JJ, Sunahara RK, Johnson RA, Gosselin G, Gilman AG, Sprang SR (1999) Two-metal-ion catalysis in adenylyl cyclase. Science 285:756–760
Visel A, Alvarez-Bolado G, Thaller C, Eichele G (2006) Comprehensive analysis of the expression patterns of the adenylate cyclase gene family in the developing and adult mouse brain. J Comp Neurol 496:684–697
Watson PA (1990) Direct stimulation of adenylate cyclase by mechanical forces in S49 mouse lymphoma cells during hyposmotic swelling. J Biol Chem 265:6569–6575
Whisnant RE, Gilman AG, Dessauer CW (1996) Interaction of the 2 cytosolic domains of mammalian adenylyl-cyclase. Proc Natl Acad Sci U S A 93:6621–6625
Willoughby D, Cooper DM (2007) Organization and Ca2+ regulation of adenylyl cyclases in cAMP microdomains. Physiol Rev 87:965–1010
Yan S-Z, Huang Z-H, Andrews RK, Tang W-J (1998) Conversion of forskolin-insensitive to forskolin-sensitive (mouse-type IX) adenylyl cyclase. Mol Pharmacol 53:182–187
Acknowledgements
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.
Funding
This work was supported by the Medical Research Council, UK and the Servier-Egis research co-operation agreement.
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Conflict of interest
AP and FAA were employees of Egis Pharmaceuticals PLC, Budapest, Hungary.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Supplementary Figure 1
(DOCX 250 kb)
Rights and permissions
About this article
Cite this article
Simpson, J., Pálvölgyi, A. & Antoni, F.A. Direct stimulation of adenylyl cyclase 9 by the fungicide imidazole miconazole. Naunyn-Schmiedeberg's Arch Pharmacol 392, 497–504 (2019). https://doi.org/10.1007/s00210-018-01610-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00210-018-01610-1