Journal
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Volume 330, Issue 3, Pages 687-695Publisher
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/jpet.109.155432
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Funding
- Deutsche Forschungsgemeinschaft [Se529/5-1]
- Studienstiftung des Deutschen Volkes
- Elite Graduate Student Program of the Free State of Bavaria
- National Institutes of Health [R01-DK46371]
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK046371] Funding Source: NIH RePORTER
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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 (K-i 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 (K-i, 3.7 nM) and sGC (K-i, 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K-i, 7.1 nM) and ACII (K-i, 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G(s)- and G(i)-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.
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