title: Structure-activity relationships for the interactions of 2'- and 3'-(O)-(N-methyl)anthraniloyl-substituted purine and pyrimidine nucleotides with mammalian adenylyl cyclases.
abstract: Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that 2',3'-(O)-(N-methylanthraniloyl) (MANT)-substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic structure-activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5. (M)ANT-nucleotides inhibited fully activated VC1:IIC2 in the order of affinity for bases hypoxanthine>uracil>cytosine>adenine∼guanine≫xanthine. Omission of a hydroxyl group at the 2' or 3'-position reduced inhibitor potency as did introduction of a γ-thiophosphate group or omission of the γ-phosphate group. Substitution of the MANT-group by an ANT-group had little effect on affinity. Although all nucleotides bound to VC1:IIC2 similarly according to the tripartite pharmacophore model with a site for the base, the ribose, and the phosphate chain, nucleotides exhibited subtle differences in their binding modes as revealed by fluorescence spectroscopy and molecular modelling. MANT-nucleotides also differentially interacted with the VC1:VC1 homodimer as assessed by fluorescence spectroscopy and modelling. Similar structure-activity relationships as for VC1:IIC2 were obtained for recombinant ACs 1, 2 and 5, with AC2 being the least sensitive AC isoform in terms of inhibition. Overall, ACs possess a broad base-specificity with no preference for the "cognate" base adenine as verified by enzyme inhibition, fluorescence spectroscopy and molecular modelling. These properties of ACs are indicative for ligand-specific conformational landscapes that extend to the VC1:VC1 homodimer and should facilitate development of non-nucleotide inhibitors.
authors.affiliation: Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS, USA.
authors.forename: Cibele
authors.initials: C
authors.lastname: Pinto
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Lushington
authors.forename: Mark
authors.initials: M
authors.lastname: Richter
authors.forename: Andreas
authors.initials: A
authors.lastname: Gille
authors.forename: Jens
authors.initials: J
authors.lastname: Geduhn
authors.forename: Burkhard
authors.initials: B
authors.lastname: König
authors.forename: Tung-Chung
authors.initials: TC
authors.lastname: Mou
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008322:Mammals; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D011685:Purine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D011742:Pyrimidine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D013329:Structure-Activity Relationship; D062367:ortho-Aminobenzoates / Q000737:chemistry / Q000378:metabolism
chemical_list: C518935:3'-O-(N-methylanthraniloyl) guanosine 5'-triphosphate; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases