title: The commonly used cGMP-dependent protein kinase type I (cGKI) inhibitor Rp-8-Br-PET-cGMPS can activate cGKI in vitro and in intact cells.
abstract: Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany.
authors.forename: Nadejda
authors.initials: N
authors.lastname: Valtcheva
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Nestorov
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Beck
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Russwurm
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hillenbrand
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany.
authors.forename: Pascal
authors.initials: P
authors.lastname: Weinmeister
authors.affiliation: Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany. Electronic address: robert.feil@uni-tuebingen.de.
authors.forename: Robert
authors.initials: R
authors.lastname: Feil
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D007527:Isoenzymes; D008840:Microfilament Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D000098662:Vasodilator-Stimulated Phosphoprotein; D011494:Protein Kinases; D006152:Cyclic GMP