NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272

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 va...

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Published in:BMC pharmacology 2001-12, Vol.1 (1), p.13-13, Article 13
Main Authors: Becker, E M, Alonso-Alija, C, Apeler, H, Gerzer, R, Minuth, T, Pleiss, U, Schmidt, P, Schramm, M, Schröder, H, Schroeder, W, Steinke, W, Straub, A, Stasch, J P
Format: Article
Language:English
Subjects:
Animals
Cells, Cultured
Enzyme Activation
Enzyme Activators - pharmacology
Guanylate Cyclase - genetics
Guanylate Cyclase - metabolism
Indazoles - pharmacology
Insecta - cytology
Nitric Oxide - metabolism
Photoaffinity Labels
Pyrazoles - pharmacology
Pyridines - pharmacology
Recombinant Proteins - drug effects
Recombinant Proteins - metabolism
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Summary: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. 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. 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.
ISSN:1471-2210
1471-2210
DOI:10.1186/1471-2210-1-13