Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

  1. Srividya Suryanarayana1,
  2. Martin Göttle,
  3. Melanie Hübner,
  4. Andreas Gille2,
  5. Tung-Chung Mou,
  6. Stephen R. Sprang,
  7. Mark Richter and
  8. Roland Seifert
  1. Department of Molecular Biosciences (S.S., M.R.) and Department of Pharmacology and Toxicology (A.G.), the University of Kansas, Lawrence, Kansas; Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany (M.G., M.H.); Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana (T.-C.M., S.R.S.); and Institute of Pharmacology, Medical School of Hannover, Hannover, Germany (R.S.)
  1. Address correspondence to:
    Dr. Roland Seifert, Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: seifert.roland{at}mh-hannover.de

Abstract

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70:878–886, 2006), we reported that 2′,3′-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5′-triphosphates (TNP-NTPs) are potent inhibitors (Ki values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (Ki, 3.7 nM) and sGC (Ki, 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (Ki, 7.1 nM) and ACII (Ki, 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by Gs- and Gi-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.

Footnotes

  • This work was supported in part by the Deutsche Forschungsgemeinschaft [Grants Se529/5-1] (to R.S.); a predoctoral fellowship of the Studienstiftung des Deutschen Volkes (to A.G.); a predoctoral fellowship from the Elite Graduate Student Program of the Free State of Bavaria (to M.H.); and the National Institutes of Health [Grant R01-DK46371] (to S.R.S.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • doi:10.1124/jpet.109.155432.

  • ABBREVIATIONS: AC, adenylyl cyclase; mAC, membranous adenylyl cyclase; sAC, soluble adenylyl cyclase; β2AR, β2-adrenoceptor; Giα, inhibitory G-protein α-subunit; GsαS, short splice variant of the stimulatory G-protein α-subunit; GsαL, long splice variant of the stimulatory G-protein α-subunit; Gαolf, olfactory G-protein α-subunit; sGC, soluble guanylyl cyclase; FPR, formyl peptide receptor; FS, forskolin; GPCR, G-protein-coupled receptor; DMB-FS, 7-acetyl-7-[O-(N-methylpiperazino)-γ-butyryl]-forskolin; MANT, 2′(3′)-O-(N-methylanthraniloyl); TNP, 2′,3′-O-(2,4,6-trinitrophenyl); VC1 and IIC2, the N- and C-terminal catalytic domains from canine type V mAC and rat type II mAC, respectively, expressed as soluble proteins; GTPγS, guanosine 5′-[γ-thio]triphosphate.

  • ↵1 Current affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin.

  • ↵2 Current affiliation: Research and Development, Cardiovascular Diseases, Sanofi-Aventis, Frankfurt/Main, Germany.

    • Received April 22, 2009.
    • Accepted June 2, 2009.
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  1. Published online before print June 3, 2009, doi: 10.1124/jpet.109.155432 JPET September 2009 vol. 330 no. 3 687-695
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