Forskolin decreases sensitivity of brain adenylate cyclase to inhibition by 2′,5′‐dideoxyadenosine

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The synthesis of a number of adenine nucleoside 3'-polyphosphates has been devised via a phosphotriester approach that combines the method of alkoxide activation with the use of 2,2,2-tribromoethyl phosphoromorpholinochloridate as a phosphorylating agent. The family of compounds included 3'ADP, 3'ATP, 2'-deoxy-3'ADP, 2'-deoxy-3'ATP, 2',5'-dideoxy-3'ADP, and 2',5'-dideoxy-3'ATP. Potency as inhibitors of adenylyl cyclases followed the order: 3'-mono- < 3'-di- < 3'-triphosphate and adenosine (Ado) < 2'-d-Ado < 2',5'-dd-Ado derivatives, with 2',5'-dideoxy-3'ATP exhibiting an IC50 of approximately 40 nM. This order was maintained with purified and recombinant forms of the type I enzyme. The nucleoside 3'-phosphates caused noncompetitive inhibition of the type I adenylyl cyclase from bovine brain, consistent with inhibition via the P-site. Inhibition was not due to hydrolytic products because this was minimal and inhibition kinetics by inorganic polyphosphates were inconsistent with those caused by the nucleoside 3'-polyphosphates. Only 3'ATP underwent cation-catalyzed, nonenzymatic hydrolysis, with the primary product being 2':3'-cAMP. Because 3'-ADP and 3'-ATP are naturally occurring, this class of compounds may physiologically regulate adenylyl cyclases and possibly other enzymes, mediating responses that include a reduction in 3':5'-cAMP levels and consequent reductions in protein kinase A-activated pathways.

Section: Resultsmentioning

confidence: 99%

Inhibition of Adenylyl Cyclase by a Family of Newly Synthesized Adenine Nucleoside 3′-Polyphosphates

Désaubry

Shoshani

Johnson

1996

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“…It has also been reported that the IC 50 values of P-site ligands differ in the presence of various stimulators (25,27). Forskolin stimulation leads to increased sensitivity to inhibition by 2Ј-d-3Ј-AMP with type VI but decreased sensitivity with types I and II, and forskolin decreases the sensitivity of brain adenylyl cyclase to inhibition by 2Ј,5Ј-dideoxyadenosine (42). We also examined the IC 50 values of THFA when either forskolin or G s ␣⅐GTP␥S was used as the stimulator (Figs.…”

Section: Effects Of Modifying Different Positions Of Forskolin-asmentioning

confidence: 99%

Type-specific Regulation of Adenylyl Cyclase

Onda

Hashimoto

Nagai

et al. 2001

103

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2-deoxyadenosine 3-monophosphate, a classical and 3-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel nonnucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.The G protein 1 -sensitive, membrane-bound form of adenylyl cyclase consists of a large family; nine isoforms have been isolated and extensively studied (1-3). These isoforms are characterized by distinct biochemical properties and tissue distribution. For example, calcium-inhibitable type V is expressed in the heart as a major isoform (4); protein kinase C-sensitive type II is expressed in lungs (5); calmodulin-sensitive type I and type VIII are expressed exclusively in neuronal tissues (6); type III is expressed abundantly in olfactory tissues (7, 8) but also in other tissues including lungs (9), atria (10), and adipose tissue (11); type IV and VII are widely expressed (12, 13). Therefore, it is now accepted that the content and mixture of adenylyl cyclase isoforms provide a biochemical signature of tissue cyclic AMP generation. Forskolin, like digitalis, is a natural plant extract that has been used in traditional medicine (14). Forskolin directly activates adenylyl cyclase to increase the concentration of intracellular cyclic AMP. This mechanism for activation is now explained as follows. Forskolin binds to the catalytic core at the opposite end of the same ventral cleft that contains the active site and activates the enzyme by gluing together the two cytoplasmic domains in the core (C 1 and C 2 ) using a combination of hydropho...

“…Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5,6), inhibition kinetics (7,8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain.…”

mentioning

confidence: 98%

“…Brain-Both detergent-solubilized and particulate preparations of adenylyl cyclase from rat and bovine brains were prepared and assayed as described previously (4,5,8,9,17). Enzyme assays were conducted in duplicate or triplicate.…”

Section: Preparation and Assay Of Adenylyl Cyclases From Rat And Bovinementioning

confidence: 99%

Isozyme-dependent Sensitivity of Adenylyl Cyclases to P-site-mediated Inhibition by Adenine Nucleosides and Nucleoside 3′-Polyphosphates

Johnson

Désaubry

Bianchi

et al. 1997

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Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3-polyphosphates (Dé saubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028 -14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2,5-dideoxyadenosine (2,5-ddAdo) > ␤-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3-polyphosphates inhibitory potency followed the order Ado < 2-dAdo < 2,5-ddAdo and 3-mono-< 3-di-< 3-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2,5-dd-3-ATP with IC 50 values of 40 -300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a domain referred to as the P-site from its requirement for an intact purine moiety (1-4). Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5, 6), inhibition kinetics (7, 8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain. The observation that purified native and recombinant Type I adenylyl cyclases are inhibited by P-site ligands, although exhibiting decreased sensitivity to inhibition (4 -6, 11), establishes the locus of the P-site on the enzyme per se and that inhibition is not via cell surface receptors or G-proteins.P-site-mediated inhibition has been characterized pharmacologically (1, 2, 4, 12-16). Inhibition requires an intact adenine moiety, and potency of inhibition is increased substantially for deoxyribose and especially 3Ј-phosphorylribose adenine nucleosides. Inhibitory potency follows the order: 3Ј-mono-Ͻ 3Ј-di Ͻ 3Ј-triphosphate and adenosine (Ado) Ͻ 2Ј-deoxy (d) 1 -Ado Ͻ 2Ј,5Ј-ddAdo, with 2Ј,5Ј-dd-3Ј-ATP being the most potent ligand and exhibiting an IC 50 ϳ40 nM (15). In addition, tolerance for large substitutions at the 3Ј-position and for other ribose modifications has been demonstrated (1, 2, 4).We reported previously that levels of 2Ј-d-3Ј-AMP and 3Ј-AMP varied considerably in different tissues and were dependent on the metabolic state of the animal (17). Moreover, sensitivity o...