title: NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272.
abstract: BACKGROUND
  The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.
  
  
  RESULTS
  We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.
  
  
  CONCLUSIONS
  Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.
authors.affiliation: Pharma Research Center, Bayer AG, Wuppertal, Germany. eva.becker@gmx.net
authors.forename: E M
authors.initials: EM
authors.lastname: Becker
authors.forename: C
authors.initials: C
authors.lastname: Alonso-Alija
authors.forename: H
authors.initials: H
authors.lastname: Apeler
authors.forename: R
authors.initials: R
authors.lastname: Gerzer
authors.forename: T
authors.initials: T
authors.lastname: Minuth
authors.forename: U
authors.initials: U
authors.lastname: Pleiss
authors.forename: P
authors.initials: P
authors.lastname: Schmidt
authors.forename: M
authors.initials: M
authors.lastname: Schramm
authors.forename: H
authors.initials: H
authors.lastname: Schröder
authors.forename: W
authors.initials: W
authors.lastname: Schroeder
authors.forename: W
authors.initials: W
authors.lastname: Steinke
authors.forename: A
authors.initials: A
authors.lastname: Straub
authors.forename: J P
authors.initials: JP
authors.lastname: Stasch
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D007191:Indazoles / Q000494:pharmacology*; D007313:Insecta / Q000166:cytology; D009569:Nitric Oxide / Q000378:metabolism*; D019920:Photoaffinity Labels; D011720:Pyrazoles / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism
chemical_list: C423582:3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo(3,4-b)pyridine; D020536:Enzyme Activators; D007191:Indazoles; D019920:Photoaffinity Labels; D011720:Pyrazoles; D011725:Pyridines; D011994:Recombinant Proteins; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009569:Nitric Oxide; D006162:Guanylate Cyclase