RESUMEN
Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases.
Asunto(s)
Guanilato Ciclasa/química , Óxido Nítrico/química , Secuencia de Aminoácidos , Animales , Antihipertensivos/uso terapéutico , Sitios de Unión , Presión Sanguínea/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Cisteína/química , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Guanilato Ciclasa/metabolismo , Hemo/química , Humanos , Imidazoles/farmacología , Técnicas In Vitro , Indazoles/farmacología , Datos de Secuencia Molecular , Etiquetas de Fotoafinidad , Inhibidores de Agregación Plaquetaria/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Ratas , SolubilidadRESUMEN
SARs around a novel type of guanylate cyclase stimulator which act by a mechanism different from classical NO-donors are described. Several pyrazolopyridinylpyrimidines are shown to relax aortic rings and revealed a long-lasting blood pressure lowering effect in rats after oral application.
Asunto(s)
Activadores de Enzimas/farmacología , Guanilato Ciclasa/metabolismo , Indazoles/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Activadores de Enzimas/síntesis química , Activadores de Enzimas/química , Indazoles/síntesis química , Indazoles/química , Pirazoles/química , Pirazoles/farmacología , Piridinas/química , Piridinas/farmacología , Conejos , Relación Estructura-Actividad , Vasodilatación/efectos de los fármacosRESUMEN
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.
