Received for publication May 9, 2006.
Revised June 8, 2006.
Accepted for publication June 8, 2006.

Broad Specificity of Mammalian Adenylyl Cyclase for Interaction with 2’,3’-Substituted Purine- and Pyrimidine Nucleotide Inhibitors

Abstract

Membrane adenylyl cyclases (mACs) play an important role in signal transduction and therefore are potential drug targets. Earlier, we identified 2',3'-O-(N-methylanthraniloyl) (MANT)-substituted purine nucleotides as a novel class of highly potent competitive mAC inhibitors (K_i values in the 10 nM range). MANT-nucleotides discriminate among various mAC isoforms through differential interactions with a binding pocket localized at the interface between the C1- and C2 domains of mAC. Here we examine the structure/activity relationships for 2',3'-substituted nucleotides and compare the crystal structures of mAC catalytic domains (VC1:IIC2) bound to MANT-GTP, MANT-ATP and 2',3'-92,4,6-trinitrophenyl) (TNP)-ATP. TNP-substituted purine and pyrimidine nucleotides inhibited VC1:IIC2 with moderately high potency (K_i values in the 100 nM range). Elongation of the linker between the ribosyl group and the MANT group and substitution of N-adenine atoms with MANT reduces inhibitory potency. Crystal structures show that MANT-GTP, MANT-ATP and TNP-ATP reside in the same binding pocket in the VC1:IIC2 protein complex, but there were substantial differences in interactions of base, fluorophore and polyphosphate chain of the inhibitors with mAC. Fluorescence emission and resonance transfer spectra also reflect differences in the interaction between MANT-ATP with VC1:IIC2 relative to MANT-GTP. Our data are indicative of a three-site mAC pharmacophore, with the 2',3'-O-ribosyl substituent and the polyphosphate chain having the largest impact on inhibitor affinity and the nucleotide base having the least. The mAC binding site exhibits broad specificity, accommodating various bases and fluorescent groups at the 2',3'-O-ribosyl position. These data should greatly facilitate the rational design of potent, isoform-selective mAC inhibitors.

Key words: Adrenergic, Adenylyl cyclases, Structure determinations, Structure-activity relationships and modeling, Optical spectroscopy (fluorescence, DC, etc.), X-ray crystallography, Enzymology, Structure/function/mechanism, Nucleoside/Nucleotide derivatives

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