title: Structural basis for the high-affinity inhibition of mammalian membranous adenylyl cyclase by 2',3'-o-(N-methylanthraniloyl)-inosine 5'-triphosphate.
abstract: 2',3'-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K(i), 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the G(s)α- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.
authors.affiliation: Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Hübner
authors.forename: Anshuman
authors.initials: A
authors.lastname: Dixit
authors.forename: Tung-Chung
authors.initials: TC
authors.lastname: Mou
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Lushington
authors.forename: Cibele
authors.initials: C
authors.lastname: Pinto
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: Stephen R
authors.initials: SR
authors.lastname: Sprang
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007293:Inosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D008322:Mammals; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; C561028:2',3'-O-(N-methylanthraniloyl)inosine 5'-triphosphate; D007293:Inosine Triphosphate; D000262:Adenylyl Cyclases