title: Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation-induced degradation.
abstract: OBJECTIVE
  In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and subsequent loss of the soluble guanylyl cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272).
  
  
  METHODS
  The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism underlying the effects observed.
  
  
  RESULTS
  Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast, the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the constitutively haem-free sGC mutant beta(1)H105F was stabilized by BAY 58-2667 and Zn-PPIX.
  
  
  CONCLUSIONS
  The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC stimulators such as HMR 1766 and BAY 41-2272, respectively.
authors.affiliation: Pharma Research Centre, Bayer HealthCare, Aprather Weg 18a, Wuppertal, Germany.
authors.forename: L S
authors.initials: LS
authors.lastname: Hoffmann
authors.forename: P M
authors.initials: PM
authors.lastname: Schmidt
authors.forename: Y
authors.initials: Y
authors.lastname: Keim
authors.forename: S
authors.initials: S
authors.lastname: Schaefer
authors.forename: H H H W
authors.initials: HH
authors.lastname: Schmidt
authors.forename: J P
authors.initials: JP
authors.lastname: Stasch
mesh_terms: D000331:Aequorin / Q000235:genetics; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D017930:Genes, Reporter; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D010069:Oxadiazoles / Q000494:pharmacology; D010084:Oxidation-Reduction; D011524:Protoporphyrins / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology; D013552:Swine; D014162:Transfection; D062367:ortho-Aminobenzoates / Q000494:pharmacology
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; C423582:3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo(3,4-b)pyridine; D001565:Benzoates; D054815:Cyclic Nucleotide-Gated Cation Channels; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D010069:Oxadiazoles; D011524:Protoporphyrins; D011720:Pyrazoles; D011725:Pyridines; D011810:Quinoxalines; D018160:Receptors, Cytoplasmic and Nuclear; D013449:Sulfonamides; D062367:ortho-Aminobenzoates; C017803:zinc protoporphyrin; C480588:BAY 58-2667; D006418:Heme; D000331:Aequorin; D000242:Cyclic AMP; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; C515616:5-chloro-2-(5-chlorothiophene-2-sulfonylamino)-N-(4-(morpholine-4-sulfonyl)phenyl)benzamide