Structural basis for the high-affinity inhibition of mammalian membranous adenylyl cyclase by 2',3'-o-(N-methylanthraniloyl)-inosine 5'-triphosphate.

Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany.
Molecular pharmacology (Impact Factor: 4.13). 07/2011; 80(1):87-96. DOI: 10.1124/mol.111.071894
Source: PubMed


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.

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    • "and the TNP or (M)ANT group projects into a pocket adjacent to the catalytic site, conferring high affinity of the ligands for mACs and stabilizing inactive mAC conformations between the open and closed states (Mou et al., 2005, 2006; Hübner et al., 2011; Seifert et al., 2012). Hydrophobic interactions of the TNP-or (M)ANT-nucleotides were also explored to monitor conformational changes in mACs by fluorescence spectroscopy, providing major new insights into the dynamics of enzyme regulation (Mou et al., 2005, 2006; Pinto et al., 2009, 2011; Suryanarayana et al., 2009; Hübner et al., 2011). "
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