Ours is an enzymology laboratory that works at the interface of chemistry, physiology, and pharmacology. We have been involved in the study of transmembrane signal transduction systems emphasizing cyclic nucleotide cascades and focusing on the adenylyl cyclase-cAMP-protein kinase A pathway. Adenylyl cyclases are a family of enzymes that catalyze the formation of adenosine 3':5'-monophosphate (cAMP) from 5'ATP and are central to signaling pathways in virtually all prokaryotes and eukaryotes. cAMP acts to control cell function by activation of protein kinase A, select ion channels, and Epacs, small G-proteins. Membrane-bound forms of mammalian adenylyl cyclases are regulated by hormones via G protein-coupled cell-surface receptors (GPCRs), whereas a soluble form is regulated by bicarbonate. These systems play diverse roles in cell regulation, mediating effects of hormones, neurotransmitters, odorants, tastes, pathogens, and other agents.
The lab has created sets of the most potent inhibitors of adenylyl cyclases that are respectively selective for pre- and post-transition enzyme states. These were used to solve structures of the enzyme catalytic core and these structures offer a basis for the design and development of isozyme selective agents. Protected nucleotides (pro-nucleotides), with all the hallmarks of prodrugs, were created from precursor nucleoside monophosphates. The most potent of these prodrugs inhibit cAMP formation in intact cells and tissues at low nanomolar concentrations. In addition, we have created fluorophore-tagged prodrugs that are being used to quantify their uptake and intracellular metabolism and are also being used to aid in the design and synthesis of tissue-selective inhibitors. Because of the importance of these enzymes in physiologic and pathophysiologic cell function, the prodrugs we have created have potential therapeutic use in the treatment of type II diabetes, some types of congestive heart failure, in the treatment and/or prevention of several infectious diseases, specifically malaria, anthrax, and cholera, and, in combination therapy as anti-fungal agents or for treatment of some cancers. These potent, cell-permeable inhibitors are being used in intact cells to test their pharmacologic usefulness in controlling cell and tissue function. The lab has two areas of immediate interest. One, is the use of these inhibitors in cardiac tissue to block the effects of catecholamines to increase heart rate and force of contraction, that is, a beta-blocker type of action, but acting from within the cell. And second, is to use these inhibitors to block the formation of cAMP in Candida albicans, in which cAMP formation is essential for virulence. Candida albicans is the most frequently isolated fungal pathogen in humans, is the third most common of hospital acquired bloodstream infections in the US, and can cause life-threatening systemic infections in immune-compromised patients. Despite antifungal treatment, mortality due to C. albicans is increasing. It is therefore important to identify additional targets for antifungal therapy that lack or are structurally distinct from their human homologs. The adenylyl cyclase of C. albicans is unique, is structurally distinct from human forms, and is essential to fungal morphological transitions and to virulence. Neither potent inhibitors nor structures of the C. albicans adenylyl cyclase have been reported. Thus an overall orientation of the lab is to explore the inhibition of adenylyl cyclases by unique compounds, originated in the laboratory, and the translational application of these agents to intact cell and tissue systems.
Shoshani, I., Taussig, R., Iyengar, I., and Johnson, R.A. (2000) Synthesis and use of 3'-(azidoiodosalicyl)-derivatives of 2',5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase. Arch. Biochem. Biophys. 374: (March, In Press).
Shoshani, I., Bianchi, G., Désaubry, L., Dessauer, C.W., and Johnson, R.A. (2000) Lys-Ala mutations of type I adenylyl cyclase result in altered susceptibility to inhibition by adenine nucleoside 3'-polyphosphates. Arch Biochem. Biophys. 374: 389-394.
Shoshani, I., Boudou, V., Pierra, C., Gosselin, G., and Johnson, R.A. (1999) Enzymatic synthesis of unlabeled and [b-32P]-labeled b-L-2',3'-dideoxyadenosine-5'-triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand. J. Biol. Chem. 274: 34735-34741.
Shoshani, I., Laux, W.H.G., Périgaud, C., Gosselin, G., and Johnson, R.A. (1999) Inhibition of adenylyl cyclase by acyclic nucleoside phosphonate anti-viral agents. J. Biol. Chem. 274: 34742-34744 .
Doronin, S., Murray, L., Dessauer, C.W., and Johnson, R.A. (1999) Covalent labeling of adenylyl cyclase cytosolic domains with 2',5'-dideoxy-[g-32P]-3'-ATP and the mechanism for P-site-mediated inhibition. J. Biol. Chem. 274: 34745-34750 .
Tesmer, J.J.G., Sunahara, R.K., Johnson, R.A., Gosselin, G., Gilman, A.G., and Sprang, S.R. (1999) Two metal ion catalysis in adenylyl cyclase revealed by its complexes with ATP analogs, Mg2+, Mn2+, and Zn2+. Science 285: 756-760.
Ibrahimi, A., Abumrad, N., Maghareie, H., Golia, M., Shoshani, I., Désaubry, L., and Johnson, R.A. (1999) Adenylyl cyclase P-site inhibitors accelerated differentiation in Ob-1771 preadipocytes. Am J. Physiol. 276 (Cell Physiol. 45): C487-C496.
Szczepanik, M., Désaubry, L., and Johnson, R.A. (1998) One-pot synthesis of deoxy-adenosine 3'-thiophosphates. Tetrahedron Lett. 39: 7455-7458.
Doronin, S., Dessauer, C., and Johnson, R.A. (1998) Direct photoaffinity labeling of individual cytosolic domains of adenylyl cyclase by [32P]-2'-d-3'AMP and [a-32P]-5'ATP. J. Biol. Chem. 273: 32416-32420 .
Désaubry, L., and Johnson, R.A. (1998) Adenine nucleoside-3'-tetraphosphates are novel and potent inhibitors of adenylyl cyclases. J. Biol. Chem. 273: 24972-24977 .
Johnson, R.A., Désaubry, L., Bianchi, G.,, Shoshani, I., Lyons, E., Jr., Taussig, R., Watson, P.A., Cali, J.J., Krupinski, J., Pieroni, J.P., and Iyengar, R. (1997) Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside-3'-polyphosphates. J. Biol. Chem. 272: 8962-8966.
Désaubry, L., Shoshani, I., and Johnson, R.A. (1996) Inhibition of adenylyl cyclase by a family of newly synthesized adenine nucleoside 3'-polyphosphates suggests new regulatory pathways. J. Biol. Chem. 271: 14028-14034.
Désaubry, L., Shoshani, I., and Johnson, R.A. (1996) 2',5'-dideoxyadenosine-3'-polyphosphates are potent inhibitors of adenylyl cyclases. J. Biol. Chem. 271: 2380-2383.
Désaubry, L., and Johnson, R.A. (1996) Conjugation of nucleoside triphosphates to an amino linker. Bioorg. & Med. Chem. Lett. 7: 123-126.
Theil, F., Ballschuh, Sibylle, von Janta-Lipinski, M., and Johnson, R.A. (1996) Chemoenzymatic synthesis of carbocyclic nucleoside analogues with bicyclo[3.1.0]hexyl residues. J. Chem. Soc., Perkin Trans. 1: 255-258.
Désaubry, L., , Shoshani, I., and Johnson, R.A. (1995) Synthesis of 2'-deoxy- and 2',5'-dideoxy-adenosine-3'-di- and -3'-triphosphate. Tetrahedron Lett. 36: 995-996.
Désaubry, L., Shoshani, I., and Johnson, R.A. (1995) Synthesis of 2',5'-dideoxyadenosine-3'-monophosphate derivatives as allosteric inhibitors of adenylyl cyclase. Nucleosides and Nucleotides 14: 1453-1460.
Shoshani, I., Qui, H., Johnson, F., Taussig, R., and Johnson, R.A. (1995) Azido-iodo-phenyl- analogs of 2',5'-dideoxyadenosine as affinity ligands for adenylyl cyclase. Biochim. Biophys. Acta 1245: 37-42.
Shoshani, I., Qiu, H., Johnson, F., and Johnson, R.A. (1994) Synthesis of iodo-aryl-azido adenosine analogs as affinity ligands for adenylyl cyclase. Nucleosides and Nucleotides 13: 1977-1989.
Bushfield, M., Shoshani, I., and Johnson, R.A. (1990) Tissue levels, source, and regulation of 3'AMP: An intracellular inhibitor of adenylyl cyclase. Molec. Pharmacol. 38: 848-853.
Johnson, R.A., and Shoshani, I. (1990) Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and "P"-site agonists. J. Biol. Chem. 265: 19035-19039.
Yeung, S.-M.H., and Johnson, R.A. (1990) Irreversible inactivation of adenylate cyclase by the P-site agonist 2',5'-dideoxy-3'-p-fluorosulfonyl-benzoyl-adenosine. J. Biol. Chem. 265: 16745-16750.
Johnson, R.A., and Shoshani, I. (1990) Kinetics of "P"-site-mediated inhibition of adenylyl cyclase and the requirements for substrate. J. Biol. Chem. 265: 11595-11600.
Johnson, R.A., Yeung, S.M.H., Stübner, D., Bushfield, M., and Shoshani, I. (1989) Cation and structural requirements for "P"-site-mediated inhibition of adenylate cyclase. Molec. Pharmacol. 35: 681-688.