Covalent Labeling of Adenylyl Cyclase Cytosolic Domains with γ-Methylimidazole-2′,5′-dideoxy-[γ-32P]3′-ATP and the Mechanism for P-site-mediated Inhibition

Doronin, Sergey V.; Murray, Leyton; Dessauer, Carmen W.; Johnson, Roger A.

doi:10.1074/jbc.274.49.34745

Cited by 5 publications

(3 citation statements)

References 19 publications

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“…Binding of 2′,5′-dd-[γ-32 P]-3′-ATP required both C 1a and C 2a and was not observed with the individual domains at the protein concentrations tested. Previous covalent labeling experiments with the irreversibly binding affinity probe, 2′,5′-dideoxyadenosine 3′-[γ-(1methylimidizole)-triphosphate], suggested that the inhibitor interacts with individual C 1a and C 2a domains (17). However, no binding of 2′,5′-dd-[γ-32 P]-3′-ATP could be detected by equilibrium dialysis using the individual domains, despite the inclusion of Mg 2+ , Mn 2+ or Mg 2+ ‚PP i .…”

Section: Crystal Structures Of 2′-d-3′mentioning

confidence: 99%

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Molecular Basis for P-Site Inhibition of Adenylyl Cyclase^,

et al. 2000

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P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.

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Section: Crystal Structures Of 2′-d-3′mentioning

confidence: 99%

“…Recently, adenosine-3‘-polyphosphates have been developed as a new class of P-site inhibitors with enhanced potency ( , ). Extrapolating from the structure of 2‘-d-3‘-AMP·PP i in complex with adenylyl cyclase, it has been proposed that the β and γ phosphates of adenine nucleoside-3‘-triphosphates occupy the PP i binding site ( , ).…”

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Molecular Basis for P-Site Inhibition of Adenylyl Cyclase^,

et al. 2000

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“…as reversibly and irreversibly binding ligands (6,8) and for resolving aspects of the three-dimensional structure of the enzyme (9, 10), but they are not useful for studies in intact cells because they do not cross cell membranes intact. In general, the problem of membrane permeability has been circumvented by the use of nucleosides in studies with intact cell systems.…”

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Pro-nucleotide Inhibitors of Adenylyl Cyclases in Intact Cells

Laux

Pande

Shoshani

et al. 2004

Journal of Biological Chemistry

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9-Substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3-phosphoryl derivatives of 2,5-dideoxyadenosine (2,5-dd-Ado) and ␤-L-2,5-ddAdo, protected 5-phosphoryl derivatives of ␤-L-2,3-ddAdo, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl-or three S-acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [ 3 H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [ 3 H]adenine. A striking selectivity for 2,5-dd-Ado-3-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC 50 ϳ2 M) were much less potent than S-acyl-2-thioethyl-derivatives. Best studied of these was 2,5-dd- Ado-3-O-bis(S-pivaloyl-2-thioethyl)-phosphate, which blocked [3 H]cAMP formation in preadipocytes (IC 50 ϳ30 nM) and suppressed opening of cAMP-dependent Cl ؊ channels in cardiac myocytes (IC 50 ϳ800 nM). None of the pro-nucleotides inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.

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