NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272.
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.
Becker
E M
EM
Pharma Research Center, Bayer AG, Wuppertal, Germany. eva.becker@gmx.net
Alonso-Alija
C
C
Apeler
H
H
Gerzer
R
R
Minuth
T
T
Pleiss
U
U
Schmidt
P
P
Schramm
M
M
Schröder
H
H
Schroeder
W
W
Steinke
W
W
Straub
A
A
Stasch
J P
JP
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
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