title: 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 are therefore 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 (Ki 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. In this study, 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'-(2,4,6-trinitrophenyl) (TNP)-ATP. TNP-substituted purine and pyrimidine nucleotides inhibited VC1:IIC2 with moderately high potency (Ki 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 are 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 and VC1:IIC2 relative to MANT-GTP. Our data are indicative of a three-site mAC pharmacophore; the 2',3'-O-ribosyl substituent and the polyphosphate chain have the largest impact on inhibitor affinity and the nucleotide base has 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.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9050, USA.
authors.forename: Tung-Chung
authors.initials: TC
authors.lastname: Mou
authors.forename: Andreas
authors.initials: A
authors.lastname: Gille
authors.forename: Srividya
authors.initials: S
authors.lastname: Suryanarayana
authors.forename: Mark
authors.initials: M
authors.lastname: Richter
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain / Q000187:drug effects; D018360:Crystallography, X-Ray; D004583:Electrons; D005453:Fluorescence; D008958:Models, Molecular; D013379:Substrate Specificity / Q000187:drug effects; D062367:ortho-Aminobenzoates / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D062367:ortho-Aminobenzoates; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases