title: (D)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Ialpha.
abstract: The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.
authors.affiliation: Department of Pharmacology, University of Vermont, College of Medicine, HSRF 330, 149 Beaumont Avenue, Burlington, VT 05405, USA.
authors.forename: Christian K
authors.initials: CK
authors.lastname: Nickl
authors.forename: Shiv Kumar
authors.initials: SK
authors.lastname: Raidas
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Matthias
authors.initials: M
authors.lastname: Sausbier
authors.forename: Peter
authors.initials: P
authors.lastname: Ruth
authors.forename: Werner
authors.initials: W
authors.lastname: Tegge
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Brayden
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Dostmann
mesh_terms: D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D002460:Cell Line; D003324:Coronary Artery Disease / Q000188:drug therapy / Q000201:enzymology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000494:pharmacology* / Q000627:therapeutic use; D006973:Hypertension / Q000188:drug therapy / Q000201:enzymology; D008297:Male; D008638:Mesenteric Arteries / Q000201:enzymology; D051379:Mice; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology; D010446:Peptide Fragments / Q000494:pharmacology* / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D018411:Spodoptera; D014661:Vasoconstriction / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects*
chemical_list: C513505:DT-2 compound; D005452:Fluoresceins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; C016276:8-bromocyclic GMP; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569720:Prkg1 protein, mouse; D006152:Cyclic GMP