Trends in Pharmacological Sciences
ReviewThe interactions of adenylate cyclases with P-site inhibitors
Section snippets
Historical context
Adenosine was first shown to stimulate cAMP accumulation in brain slices by Sattin and Rall11, while others later demonstrated inhibition of cAMP synthesis by adenosine, or even biphasic stimulation and inhibition of adenylate cyclase activity in certain tissues12, 13, 14, 15, 16, 17, 18, 19, 20. Based on studies of structure-activity relationships, Londos and Wolff identified two distinct sites of action for adenosine: those with strict requirements for the ribose moiety, designated R sites,
Mechanisms of catalysis and inhibition
In view of the structural relationship between substrate ATP and P-site compounds, it is surprising that these inhibitors typically act noncompetitively or uncompetitively with respect to ATP, depending on the identity of the divalent cation cofactor. Inhibition is typically noncompetitive in the presence of Mn2+ and uncompetitive when Mg2+ is present17, 21, 27, 28, 29, 30. Furthermore, the apparent potency of P-site inhibitors increases when adenylate cyclase is activated20, 27, 28, 29, 31.
P-site inhibitors bind to the active site
Equilibrium dialysis of the reconstituted fragments of adenylate cyclase revealed single, distinct binding sites for both forskolin and the substrate analogue Ap(CH2)pp; both C1 and C2 are required for substrate binding35. Addition of high concentrations of the P-site inhibitors 2′-deoxyadenosine or 2′-deoxy-3′-AMP has no effect on substrate binding35. Kinetic analysis of the forward reaction showed that 2′-deoxyadenosine inhibits the Gsα-activated enzyme uncompetitively with respect to MgATP
Structure of bound inhibitor
High resolution crystal structures of the Gsα–C1a–C2a–forskolin complex have also provided great insight into mechanism of P-site inhibitions. These structures, with the results of mutagenesis37, 38 and kinetic analysis36, conclusively demonstrate that most if not all P-site inhibitors bind to the active site of adenylate cyclase. The following is a brief description of the structure of the catalytic core of adenylate cyclase in complexes with ATP and various P-site inhibitors.
The catalytic
Structural determinants of P-site inhibitor affinities
The more potent P-site inhibitors contain 3′-polyphosphates23. In the C1a–C2a-2′-deoxy-3′-AMP–PPi complex, the 3′-α-phosphate is ligated by ‘metal A’, the metal that binds the 3′-hydroxyl of ATP and activates it for nucleophilic attack. The 3′-α-phosphate is not precisely in the position expected for the 5′-α-phosphate of 5′-ATP (Fig. 3c). When additional phosphates are added to the 3′-position of the P-site inhibitor, apparent affinities of the compounds for adenylate cyclase are higher,
P-site inhibitors as therapeutic agents
The ubiquitous distribution of adenylate cyclase and its crucial role in a host of biological processes, up to and including cognition, have apparently precluded consideration of this important enzyme as a target for drug development. Recent appreciation of the large number of mammalian isoforms and the possibility of isoform-specific inhibitors of the enzyme suggest that the question should be reopened. Despite the fact some isoform selectivity has been observed among P-site inhibitors
Acknowledgements
Work from the authors' laboratories was supported by NIH grants DK46371 and GM34497, Welch Foundation Grants I-1229 and I-1271 and the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology.
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