RESUMEN
Prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) are generated by the action of cyclooxygenase-2 on the endocannabinoids 2-arachidonylglycerol (2-AG) and arachidonylethanolamide, respectively. These novel eicosanoids may have unique pharmacological properties and/or serve as latent sources of prostaglandins at sites remote from their tissue of origin. Therefore, we investigated the metabolism of PG-Gs and PG-EAs in vitro and in vivo. PGE(2)-G was rapidly hydrolyzed in rat plasma to generate PGE(2) (t(1/2) = 14 s) but was only slowly metabolized in human plasma (t(1/2) > 10 min). An intermediate extent of metabolism of PGE(2)-G was observed in human whole blood (t(1/2) approximately 7 min). The parent arachidonylglycerol, 2-AG, and the more stable regioisomer, 1-AG, also were much more rapidly metabolized in rat plasma compared with human plasma. PGE(2)-EA was not significantly hydrolyzed in plasma, undergoing slow dehydration/isomerization to PGB(2)-EA. Both PGE(2)-G and PGE(2)-EA were stable in canine, bovine, and human cerebrospinal fluid. Human 15-hydroxyprostaglandin dehydrogenase, the enzyme responsible for the initial step in PG inactivation in vivo, oxidized both PGE(2)-G and PGE(2)-EA less efficiently than the free acid. The sterically hindered glyceryl prostaglandin was the poorest substrate examined in the E series. Minimal 15-hydroxyprostaglandin dehydrogenase oxidation of PGF(2 alpha)-G was observed. PGE(2)-G and PGE(2)-EA pharmacokinetics were assessed in rats. PGE(2)-G was not detected in plasma 5 min following an intravenous dose of 2 mg/kg. However, PGE(2)-EA was detectable up to 2 h following an identical dose, displaying a large apparent volume of distribution and a half-life of over 6 min. The results suggest that endocannabinoid-derived PG-like compounds may be sufficiently stable in humans to exert actions systemically. Furthermore, these results suggest that the rat is not an adequate model for investigating the biological activities of 2-arachidonylglycerol or glyceryl prostaglandins in humans.
Asunto(s)
Dinoprostona/análogos & derivados , Etanolaminas/metabolismo , Glicéridos/metabolismo , Prostaglandinas/metabolismo , Animales , Moduladores de Receptores de Cannabinoides , Dinoprostona/metabolismo , Dinoprostona/farmacocinética , Estabilidad de Medicamentos , Ésteres/metabolismo , Etanolaminas/farmacocinética , Glicéridos/farmacocinética , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Masculino , Plasma/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H2, undergoes rearrangement to the highly reactive gamma-keto aldehydes, levuglandin (LG) E2 and D2. We previously have demonstrated that LGE2 reacts with the epsilon-amine of lysine to form both the lysyl-levuglandin Shiff base and the pyrrole-derived lysyl-levuglandin lactam adducts. We now demonstrate that these lysyl-levuglandin adducts are formed on the PGHSs following the oxygenation of arachidonic acid; after reduction of the putative Schiff base, proteolytic digestion of the enzyme, and isolation of the adducted amino acid residues, these adducts were identified by liquid chromatography-tandem mass spectrometry. The reactivity of the LGs is reflected by the finding that virtually all of the LG predicted to be formed from PGH2 can be accounted for as adducts of the PGH-synthase and that oxygenation of arachidonic acid by PGH-synthases also leads to the formation of adducts of other proteins present in the reaction solution. The reactivity of the PGH-synthase adducts themselves is demonstrated by the formation of intermolecular cross-links.
Asunto(s)
Ácido Araquidónico/metabolismo , Lisina/química , Lisina/metabolismo , Oxígeno/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Catálisis , Electroforesis en Gel de Poliacrilamida , Cinética , Ratones , Prostaglandina D2/metabolismo , Prostaglandinas E/metabolismo , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Nitric oxide (( small middle dot)NO) plays a central role in vascular homeostasis via regulation of smooth muscle relaxation and platelet aggregation. Although mechanisms for ( small middle dot)NO formation are well known, removal pathways are less well characterized, particularly in cells that respond to ( small middle dot)NO through activation of soluble guanylate cyclase. Herein, we report that ( small middle dot)NO is catalytically consumed by prostaglandin H synthase-1 (PGHS-1) through acting as a reducing peroxidase substrate. With purified ovine PGHS-1, ( small middle dot)NO consumption requires peroxide (LOOH or H(2)O(2)), with a K(m)( (app)) for 15(S)hydroperoxyeicosatetraenoic acid (HPETE) of 3. 27 +/- 0.35 microm. During this, 2 mol ( small middle dot)NO are consumed per mol HPETE, and loss of HPETE hydroperoxy group occurs with retention of the conjugated diene spectrum. Hydroperoxide-stimulated ( small middle dot)NO consumption requires heme incorporation, is not inhibited by indomethacin, and is further stimulated by the reducing peroxidase substrate, phenol. PGHS-1-dependent ( small middle dot)NO consumption also occurs during arachidonate, thrombin, or activation of platelets (1-2 microm.min(-1) for typical plasma platelet concentrations) and prevents ( small middle dot)NO stimulation of platelet soluble guanylate cyclase. Platelet sensitivity to ( small middle dot)NO as an inhibitor of aggregation is greater using a platelet-activating stimulus () that does not cause ( small middle dot)NO consumption, indicating that this mechanism overcomes the anti-aggregatory effects of ( small middle dot)NO. Catalytic consumption of ( small middle dot)NO during eicosanoid synthesis thus represents both a novel proaggregatory function for PGHS-1 and a regulated mechanism for vascular ( small middle dot)NO removal.
Asunto(s)
Plaquetas/fisiología , Óxido Nítrico/metabolismo , Agregación Plaquetaria/fisiología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Ácido Araquidónico/farmacología , Catálisis , Bovinos , Ciclooxigenasa 1 , Glutatión/análogos & derivados , Glutatión/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Isoenzimas/metabolismo , Cinética , Leucotrienos/metabolismo , Peróxidos Lipídicos/metabolismo , Proteínas de la Membrana , Compuestos Nitrosos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , S-Nitrosoglutatión , Ovinos , Especificidad por Sustrato , Trombina/farmacologíaRESUMEN
Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Indometacina/análogos & derivados , Indometacina/síntesis química , Isoenzimas/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Humanos , Indometacina/química , Indometacina/farmacología , Isoenzimas/química , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Proteínas de la Membrana , Ratones , Prostaglandina-Endoperóxido Sintasas/química , Ovinos , Relación Estructura-ActividadRESUMEN
We investigated the effects of targeted deletion of the inducible NO synthase (iNOS) gene on the formation of prostaglandins in vivo and ex vivo. Peritoneal macrophages were obtained from control and iNOS-deficient mice, and prostaglandin E(2) (PGE(2)) was quantified after stimulation with gamma-interferon and lipopolysaccharide to induce COX-2. Total nitrate and nitrite production was completely abolished in cells from iNOS-deficient animals compared with control cells. PGE(2) formation by cells from iNOS-deficient animals was decreased compared with cells from control animals 80% at 12 h (0.85 +/- 0.90 ng/10(6) cells versus 15.4 +/- 2.1 ng/10(6) cells, p < 0.01) and 74% at 24 h (9.4 +/- 4.3 ng/10(6) cells versus 36.8 +/- 4.1 ng/10(6) cells, p < 0.01). COX-2 protein expression was not significantly different in cells from control or knockout animals. Levels of PGE(2) in the urine of iNOS-deficient mice were decreased 78% (0.24 +/- 0.14 ng/mg of creatinine versus 1.09 +/- 0.66 ng/mg of creatinine, p < 0.01) compared with control animals. In addition, the levels of urinary F(2)-isoprostanes, an index of endogenous oxidant stress, were significantly decreased in iNOS-deficient animals. In contrast, the levels of thromboxane B(2) derived from platelets allowed to aggregate ex vivo were significantly increased in iNOS-deficient mice compared with wild-type mice. These studies support the hypothesis that NO and/or NO-derived species modulate cyclooxygenase activity and eicosanoid production in vivo.
Asunto(s)
Dinoprostona/biosíntesis , Macrófagos Peritoneales/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , Ciclooxigenasa 2 , Eliminación de Gen , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/biosíntesis , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Acetilación , Animales , Ácido Araquidónico/química , Aspirina/farmacología , Sitios de Unión , Cromatografía Líquida de Alta Presión , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Isoenzimas/genética , Proteínas de la Membrana , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandinas/metabolismo , SpodopteraRESUMEN
All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isozymes to different extents, which accounts for their anti-inflammatory and analgesic activities and their gastrointestinal side effects. We have exploited biochemical differences between the two COX enzymes to identify a strategy for converting carboxylate-containing NSAIDs into selective COX-2 inhibitors. Derivatization of the carboxylate moiety in moderately selective COX-1 inhibitors, such as 5,8,11,14-eicosatetraynoic acid (ETYA) and arylacetic and fenamic acid NSAIDs, exemplified by indomethacin and meclofenamic acid, respectively, generated potent and selective COX-2 inhibitors. In the indomethacin series, esters and primary and secondary amides are superior to tertiary amides as selective inhibitors. Only the amide derivatives of ETYA and meclofenamic acid inhibit COX-2; the esters are either inactive or nonselective. Inhibition kinetics reveal that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Site-directed mutagenesis of murine COX-2 indicates that the molecular basis for selectivity differs from the parent NSAIDs and from diarylheterocycles. Selectivity arises from novel interactions at the opening and at the apex of the substrate-binding site. Lead compounds in the present study are potent inhibitors of COX-2 activity in cultured inflammatory cells. Furthermore, indomethacin amides are orally active, nonulcerogenic, anti-inflammatory agents in an in vivo model of acute inflammation. Expansion of this approach can be envisioned for the modification of all carboxylic acid-containing NSAIDs into selective COX-2 inhibitors.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/farmacología , Prostaglandina-Endoperóxido Sintasas/farmacología , Ácido 5,8,11,14-Eicosatetrainoico/análogos & derivados , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Amidas , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Diseño de Fármacos , Edema/prevención & control , Ésteres , Enfermedades Gastrointestinales/inducido químicamente , Miembro Posterior , Humanos , Indometacina/efectos adversos , Indometacina/análogos & derivados , Indometacina/farmacología , Cinética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ácido Meclofenámico/análogos & derivados , Ácido Meclofenámico/farmacología , Proteínas de la Membrana , Ratones , Ratas , Ratas Sprague-Dawley , OvinosRESUMEN
OBJECTIVE: To determine CSF eicosanoid concentrations and brain cyclo-oxygenase activity in AD patients and age-matched control subjects. BACKGROUND: Nonsteroidal anti-inflammatory drugs may benefit AD patients by inhibiting cyclo-oxygenases and thereby reducing prostaglandin (PG) production or oxidant stress in the CNS. METHODS: CSF eicosanoid and F2-isoprostane (IsoP) levels were determined in seven probable AD patients and seven age-matched control subjects. Cyclo-oxygenase activity was determined in microsomes prepared from the hippocampus of 10 definite AD patients and 8 age-matched control subjects. All measurements were made using gas chromatography/mass spectrometry. RESULTS: CSF concentrations of prostaglandin (PG) E2 were increased fivefold (p < 0.01) and 6-keto-PGF1alpha was decreased fourfold (p < 0.01) in probable AD patients. There was no change in total CSF eicosanoid concentration in probable AD patients. CSF F2-IsoP, a quantitative marker of lipid peroxidation in vivo, was increased in probable AD patients (p < 0.05). Cyclo-oxygenase activity in the hippocampus from definite AD patients was not different from age-matched control subjects. CONCLUSIONS: These data suggest that cyclo-oxygenase activity may not contribute significantly to CNS oxidative damage in AD. Increased CSF PGE2 concentration in probable AD patients suggest that cyclo-oxygenase inhibitors may benefit AD patients by limiting PG production.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Dinoprostona/líquido cefalorraquídeo , Anciano , Animales , Encéfalo/enzimología , Eicosanoides/líquido cefalorraquídeo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Ratones , Persona de Mediana Edad , Prostaglandina-Endoperóxido Sintasas/líquido cefalorraquídeo , Valores de ReferenciaRESUMEN
Cyclooxygenase-2 (COX-2) inhibitors reduce angiogenic responses to a variety of stimuli, suggesting that products of COX-2 may mediate critical steps. Here, we show that thromboxane A2 (TXA2) is one of several eicosanoid products generated by activated human microvascular endothelial cells. Selective COX-2 antagonists inhibit TXA2 production, endothelial migration, and fibroblast growth factor-induced corneal angiogenesis. Endothelial migration and corneal angiogenesis are similarly inhibited by a TXA2 receptor antagonist, SQ29548. A TXA2 agonist, U46619, reconstitutes both migration and angiogenesis responses under COX-2-inhibited conditions. These findings identify TXA2 as a COX-2 product that functions as a critical intermediary of angiogenesis.
Asunto(s)
Córnea/irrigación sanguínea , Inhibidores de la Ciclooxigenasa/farmacología , Endotelio Vascular/fisiología , Isoenzimas/metabolismo , Neovascularización Fisiológica/fisiología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tromboxano A2/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprost/farmacología , Dinoprostona/farmacología , Endotelio Vascular/efectos de los fármacos , Ácidos Grasos Insaturados , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Hidrazinas/farmacología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Microcirculación , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , Circulación Renal , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The chemical mandates for arachidonic acid conversion to prostaglandin G(2) within the cyclooxygenase (COX) active site predict that the substrate will orient in a kinked or L-shaped conformation. Molecular modeling of arachidonic acid in sheep COX-1 confirms that this L-shaped conformation is possible, with the carboxylate moiety binding to Arg-120 and the omega-end positioned above Ser-530 in a region termed the top channel. Mutations of Gly-533 to valine or leucine in the top channel of mCOX-2 abolished the conversion of arachidonic acid to prostaglandin G(2), presumably because of a steric clash between the omega-end of the substrate and the introduced side chains. A smaller G533A mutant retained partial COX activity. The loss of COX activity with these mutants was not the result of reduced peroxidase activity, because the activity of all mutants was equivalent to the wild-type enzyme and the addition of exogenous peroxide did not restore full COX activity to any of the mutants. However, the Gly-533 mutants were able to oxidize the carbon 18 fatty acid substrates linolenic acid and stearidonic acid, which contain an allylic carbon at the omega-5 position. In contrast, linoleic acid, which is like arachidonic acid in that its most omega-proximal allylic carbon is at the omega-8 position, was not oxidized by the Gly-533 mutants. Finally, the ability of Gly-533 mutants to efficiently process omega-5 allylic substrates suggests that the top channel does not serve as a product exit route indicating that oxygenated substrate diffuses from the cyclooxygenase active site in a membrane proximal direction.
Asunto(s)
Ácido Araquidónico/metabolismo , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Sitios de Unión , Ciclooxigenasa 2 , Isoenzimas/química , Isoenzimas/genética , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/genética , Conformación Proteica , OvinosAsunto(s)
Inhibidores de la Ciclooxigenasa/clasificación , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/farmacología , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/farmacología , Acetileno/análogos & derivados , Acetileno/química , Acetileno/farmacología , Alquinos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Dominio Catalítico , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Humanos , Técnicas In Vitro , Espectrometría de Masas , Proteínas de la Membrana , Ovinos , Relación Estructura-Actividad , Sulfuros/química , Sulfuros/farmacologíaRESUMEN
All of the selective COX-2 inhibitors described to date inhibit the isoform by binding tightly but noncovalently at the substrate binding site. Recently, we reported the first account of selective covalent modification of COX-2 by a novel inactivator, 2-acetoxyphenyl hept-2-ynyl sulfide (70) (Science 1998, 280, 1268-1270). Compound 70 selectively inactivates COX-2 by acetylating the same serine residue that aspirin acetylates. This paper describes the extensive structure-activity relationship (SAR) studies on the initial lead compound 2-acetoxyphenyl methyl sulfide (36) that led to the discovery of 70. Extension of the S-alkyl chain in 36 with higher alkyl homologues led to significant increases in inhibitory potency. The heptyl chain in 2-acetoxyphenyl heptyl sulfide (46) was optimum for COX-2 inhibitory potency, and introduction of a triple bond in the heptyl chain (compound 70) led to further increments in potency and selectivity. The alkynyl analogues were more potent and selective COX-2 inhibitors than the corresponding alkyl homologues. Sulfides were more potent and selective COX-2 inhibitors than the corresponding sulfoxides or sulfones or other heteroatom-containing compounds. In addition to inhibiting purified COX-2, 36, 46, and 70 also inhibited COX-2 activity in murine macrophages. Analogue 36 which displayed moderate potency and selectivity against purified human COX-2 was a potent inhibitor of COX-2 activity in the mouse macrophages. Tryptic digestion and peptide mapping of COX-2 reacted with [1-14C-acetyl]-36 indicated that selective COX-2 inhibition by 36 also resulted in the acetylation of Ser516. That COX-2 inhibition by aspirin resulted from the acetylation of Ser516 was confirmed by tryptic digestion and peptide mapping of COX-2 labeled with [1-14C-acetyl]salicyclic acid. The efficacy of the sulfides in inhibiting COX-2 activity in inflammatory cells, our recent results on the selectivity of 70 in attenuating growth of COX-2-expressing colon cancer cells, and its selectivity for inhibition of COX-2 over COX-1 in vivo indicate that this novel class of covalent modifiers may serve as potential therapeutic agents in inflammatory and proliferative disorders.
Asunto(s)
Acetileno/análogos & derivados , Antiinflamatorios no Esteroideos/química , Antineoplásicos/química , Inhibidores de la Ciclooxigenasa/química , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sulfuros/química , Acetilación , Acetileno/síntesis química , Acetileno/química , Acetileno/farmacología , Alquinos , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/antagonistas & inhibidores , Exudados y Transudados/efectos de los fármacos , Exudados y Transudados/metabolismo , Humanos , Técnicas In Vitro , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Proteínas de la Membrana , Ratones , Ratas , Ovinos , Relación Estructura-Actividad , Sulfuros/síntesis química , Sulfuros/farmacología , Tromboxano B2/sangre , Células Tumorales CultivadasRESUMEN
Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.
Asunto(s)
Acetileno/análogos & derivados , Antiinflamatorios no Esteroideos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sulfuros/síntesis química , Acetilación , Acetileno/síntesis química , Acetileno/química , Acetileno/farmacología , Alquinos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Aspirina/química , Aspirina/farmacología , Sitios de Unión , División Celular/efectos de los fármacos , Línea Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/biosíntesis , Diseño de Fármacos , Humanos , Indometacina/farmacología , Isoenzimas/química , Isoenzimas/genética , Macrófagos/enzimología , Proteínas de la Membrana , Mutagénesis Sitio-Dirigida , Prostaglandina D2/biosíntesis , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/genética , Ratas , Ratas Endogámicas Lew , Sulfuros/química , Sulfuros/farmacología , Tromboxano B2/biosíntesis , Células Tumorales CultivadasRESUMEN
Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.
Asunto(s)
Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/metabolismo , Óxido Nítrico/metabolismo , Tirosina , Tirosina/metabolismo , Secuencia de Aminoácidos , Ácido Araquidónico/metabolismo , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Indometacina/farmacología , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Mapeo Peptídico , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tetranitrometano/metabolismo , Tirosina/análogos & derivadosRESUMEN
Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.
Asunto(s)
Isoenzimas/química , Peroxidasas/química , Prostaglandina-Endoperóxido Sintasas/química , Animales , Ácido Araquidónico/metabolismo , Sitios de Unión , Catálisis , Ciclooxigenasa 2 , Activación Enzimática , Ácidos Grasos/metabolismo , Glutamina/química , Histidina/química , Peróxido de Hidrógeno/metabolismo , Imidazoles/farmacología , Leucotrienos/metabolismo , Peróxidos Lipídicos/metabolismo , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Oxígeno/metabolismo , Peróxidos/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Recombinantes , Spodoptera , Relación Estructura-ActividadAsunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Maleimidas/farmacología , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Caprilatos/farmacología , Inhibidores de la Ciclooxigenasa/química , Humanos , Maleimidas/química , Peroxidasas/antagonistas & inhibidores , Especificidad por Sustrato , Tripsina/farmacologíaRESUMEN
Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes' peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Nitratos/farmacología , Óxido Nítrico/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/biosíntesis , Superóxidos/metabolismo , Animales , Ácido Araquidónico/metabolismo , Línea Celular , Activación Enzimática , Cinética , Macrófagos , Metaloporfirinas/farmacología , Molsidomina/análogos & derivados , Molsidomina/farmacología , Nitratos/síntesis química , Nitratos/química , Óxido Nítrico/química , Óxido Nítrico Sintasa/metabolismo , Salicilatos/farmacología , Superóxidos/químicaRESUMEN
Many nonsteroidal antiinflammatory agents (NSAIDs) bind to prostaglandin endoperoxide synthase (PGHS) and induce a conformational change in the PGHS apoprotein that renders it resistant to cleavage by trypsin at Arg277. In the present study, the trypsin protection assay was modified to permit detection of conformational changes at times as short as 5 s after the addition of inhibitor. The kinetics of the induction and reversal of trypsin resistance in apoPGHS-1 by a series of NSAIDs and isozyme-specific PGHS-1 and PGHS-2 inhibitors were determined. All compounds induced resistance to trypsin cleavage at a rapid rate. The conformational change induced by competitive inhibitors was reversed on prolonged incubation with trypsin (approximately 5 min). In contrast, the resistance induced by irreversible inhibitors was not lost during a 5 min incubation with trypsin. All of the selective PGHS-2 inhibitors protected against tryptic cleavage of apoPGHS-1 but did not inhibit the protein's cyclooxygenase activity. The results suggest that induction of trypsin resistance is a reflection of the initial association of reversible as well as irreversible inhibitors with the apoprotein.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Antiinflamatorios no Esteroideos/metabolismo , Ácido Araquidónico/farmacología , Inhibidores de la Ciclooxigenasa/metabolismo , Hemina/farmacología , Ibuprofeno/farmacología , Cinética , Estructura Molecular , Nitrobencenos/farmacología , Prostaglandina-Endoperóxido Sintasas/química , Ácidos Prostanoicos/farmacología , Unión Proteica , Conformación Proteica , Sulfonamidas/farmacología , Tiazoles/farmacología , Tripsina/farmacologíaRESUMEN
N-(Carboxyalkyl)maleimides are rapid as well as time-dependent inhibitors of prostaglandin endoperoxide synthase (PGHS). The corresponding N-alkylmaleimides were only time-dependent inactivators of PGHS, suggesting that the carboxylate is critical for rapid inhibition. Several N-substituted maleimide analogs containing structural features similar to those of the nonsteroidal anti-inflammatory drug aspirin were synthesized and evaluated as inhibitors of PGHS. Most of the aspirin-like maleimides inactivated the cyclooxygenase activity of purified ovine PGHS-1 in a time- and concentration-dependent manner similar to that of aspirin. The peroxidase activity of PGHS was also inactivated by the maleimide analogs. The cyclooxygenase activity of the inducible isozyme, i.e., PGHS-2, was also inhibited by these compounds. The corresponding succinimide analog of N-5-maleimido-2-acetoxy-1-benzoic acid did not inhibit either enzyme activity, suggesting that inactivation was due to covalent modification of the protein. The mechanism of inhibition of PGHS-1 by N-(carboxyheptyl)maleimide was investigated. Incubation of apoPGHS-1 with 2 equiv of N-(carboxyheptyl)[3,4-14C]maleimide led to the incorporation of radioactivity in the protein, but no adduct was detected by reversed-phase HPLC, suggesting that it was unstable to the chromatographic conditions. Furthermore, hematin-reconstituted PGHS-1, which was rapidly inhibited by N-(carboxyheptyl)maleimide, displayed spontaneous regeneration of about 50% of the cyclooxygenase and peroxidase activities, suggesting that the adduct responsible for the inhibition breaks down to regenerate active enzyme. ApoPGHS-1, inhibited by N-(carboxyheptyl)maleimide, did not display regeneration of enzyme activity, but addition of hematin to the inhibited apoenzyme led to spontaneous recovery of about 50% of cyclooxygenase activity. These results suggest that addition of heme leads to a conformational change in the protein which increases the susceptibility of the adduct toward hydrolytic cleavage. ApoPGHS-1, pretreated with N-(carboxyheptyl)maleimide, was resistant to trypsin cleavage, suggesting that the carboxylate functionality of the maleimide binds in the cyclooxygenase channel. A model for the interaction of N-(carboxyheptyl)maleimide in the cyclooxygenase active site is proposed.
Asunto(s)
Inhibidores de la Ciclooxigenasa/síntesis química , Maleimidas/síntesis química , Animales , Cromatografía Líquida de Alta Presión , Inhibidores de la Ciclooxigenasa/farmacología , Diseño de Fármacos , Hemina/farmacología , Masculino , Maleimidas/farmacología , Ovinos , Relación Estructura-ActividadRESUMEN
To test the hypothesis that a large portion of the bait region of human alpha 2-macroglobulin (alpha 2M) can be removed without adversely affecting the protein's structural and functional properties, we expressed two human alpha 2M variants with truncated bait regions and examined whether these variants folded normally and functioned as proteinase inhibitors. Each variant contains sites that are normal bait region cleavage sites in wild-type alpha 2M, including the primary trypsin cleavage site. The truncated bait regions are shorter by 23 and 27 residues, respectively, and lack the C-terminal portion as well as different parts of the N-terminal section of the bait region. We found that such bait region truncation permitted normal folding of the monomers as well as formation of the thiol ester and dimerization by disulfide cross-linking, although the resulting species bound 6-(p-toluidino)-2-naphthalenesulfonic acid in a manner more like thiol ester-cleaved alpha 2M than native alpha 2M. The variants' thiol esters reacted with nucleophiles at rates identical to wild-type alpha 2M. Surprisingly, however, the truncations prevented the noncovalent association of the covalent 360-kDa dimers that normally gives tetrameric alpha 2M, decoupled bait region cleavage from thiol ester activation, and resulted in the inability of the two variants to "trap" proteinase. This was despite apparent cleavage of the bait region by proteinase, albeit at very much reduced rates relative to wild-type tetrameric alpha 2M. The kinetics of thiol ester cleavage-dependent protein conformational changes also changed from sigmoidal to exponential. These findings indicate that residues in the bait region appear to be necessary for noncovalent association of 360-kDa disulfide-linked dimers to give tetrameric alpha 2M and suggest a role for the bait region in normal alpha 2M in coupling bait region cleavage to the sequence of conformational changes that result in thiol ester activation and ultimately proteinase trapping.