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

Becker EM, Alonso-Alija C, Apeler H, Gerzer R, Minuth T, Pleiss U, Schmidt P, Schramm M, Schröder H, Schroeder W, Steinke W, Straub A, Stasch JP - BMC Pharmacol. (2001)

Bottom Line: The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharma Research Center, Bayer AG, Wuppertal, Germany. eva.becker@gmx.net

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.

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Synthesis of the ortho-, meta- and para-PAL compounds.
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Figure 1: Synthesis of the ortho-, meta- and para-PAL compounds.

Mentions: We developed the first photoaffinity label for direct and NO-independent sGC stimulators. Coming from YC-1 and BAY 41-2272 as lead structures of direct and NO-independent sGC stimulators we synthesized the ortho- (BAY 50-6038), meta- (BAY 51-9491) and para-PAL (BAY 50-8364) compounds (Fig. 1) and tested their influence on sGC activity. The meta- and para-PAL showed on the one hand a direct sGC stimulation comparable to YC-1 and on the other hand in combination with NO distinct synergistic effects on sGC activity. The ortho-PAL showed the lowest sGC stimulation and in combination with NO no synergistic effect on sGC activity (Tab. 1). Both under reducing conditions and in the absence of DTT, in the sGC assay a similar sGC activating profile of the three PALs is given (Tab. 1). Because of the more dominant stimulation of sGC by the single compound both in the presence and absence of DTT the meta-PAL compound was chosen as photoaffinitylabel for the following studies (Tab. 1).


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

Becker EM, Alonso-Alija C, Apeler H, Gerzer R, Minuth T, Pleiss U, Schmidt P, Schramm M, Schröder H, Schroeder W, Steinke W, Straub A, Stasch JP - BMC Pharmacol. (2001)

Synthesis of the ortho-, meta- and para-PAL compounds.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC64637&req=5

Figure 1: Synthesis of the ortho-, meta- and para-PAL compounds.
Mentions: We developed the first photoaffinity label for direct and NO-independent sGC stimulators. Coming from YC-1 and BAY 41-2272 as lead structures of direct and NO-independent sGC stimulators we synthesized the ortho- (BAY 50-6038), meta- (BAY 51-9491) and para-PAL (BAY 50-8364) compounds (Fig. 1) and tested their influence on sGC activity. The meta- and para-PAL showed on the one hand a direct sGC stimulation comparable to YC-1 and on the other hand in combination with NO distinct synergistic effects on sGC activity. The ortho-PAL showed the lowest sGC stimulation and in combination with NO no synergistic effect on sGC activity (Tab. 1). Both under reducing conditions and in the absence of DTT, in the sGC assay a similar sGC activating profile of the three PALs is given (Tab. 1). Because of the more dominant stimulation of sGC by the single compound both in the presence and absence of DTT the meta-PAL compound was chosen as photoaffinitylabel for the following studies (Tab. 1).

Bottom Line: The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharma Research Center, Bayer AG, Wuppertal, Germany. eva.becker@gmx.net

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.

Show MeSH
Related in: MedlinePlus