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
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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.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.155432.
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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.
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↵1 Current affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin.
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↵2 Current affiliation: Research and Development, Cardiovascular Diseases, Sanofi-Aventis, Frankfurt/Main, Germany.
- Received April 22, 2009.
- Accepted June 2, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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