Differential inhibition of various adenylyl cyclase isoforms and soluble guanylyl cyclase by 2',3'-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5'-triphosphates.
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.
Suryanarayana
Srividya
S
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
Göttle
Martin
M
Hübner
Melanie
M
Gille
Andreas
A
Mou
Tung-Chung
TC
Sprang
Stephen R
SR
Richter
Mark
M
Seifert
Roland
R
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008958:Models, Molecular; D009711:Nucleotides / Q000138:chemical synthesis* / Q000494:pharmacology*; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D009711:Nucleotides; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase