Differential inhibition of various adenylyl cyclase isoforms and soluble guanylyl cyclase by 2′,3′-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5′-triphosphates

Srividya Suryanarayana, Martin Göttle, Melanie Hübner, Andreas Gille, Tung Chung Mou, Stephen R. Sprang, Mark Richter, Roland Seifert

Research output: Contribution to journalArticlepeer-review

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 (K i, 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G s- 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.

Original languageEnglish
Pages (from-to)687-695
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume330
Issue number3
DOIs
StatePublished - 2009

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