RESUMEN
BACKGROUND AND PURPOSE: As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT(1A) receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT(1A) receptor antagonist activities, was evaluated in preclinical models. EXPERIMENTAL APPROACH: Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models. KEY RESULTS: WAY-211612 inhibited 5-HT reuptake (K(i) = 1.5 nmol.L(-1); K(B) = 17.7 nmol.L(-1)) and exhibited full 5-HT(1A) receptor antagonist activity (K(i) = 1.2 nmol.L(-1); K(B) = 6.3 nmol.L(-1); I(max) 100% in adenyl cyclase assays; K(B) = 19.8 nmol.L(-1); I(max) 100% in GTPgammaS). WAY-211612 (3 and 30 mg.kg(-1), po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT(1A) receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3-30 mg.kg(-1), po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg.kg(-1), s.c.) and a 5-HT(1A) antagonist (WAY-100635; 0.3 mg.kg(-1), s.c). WAY-211612 (3.3-30 mg.kg(-1), s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3-30 mg.kg(-1), i.p. and 10-56 mg.kg(-1), po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg.kg(-1), i.p.) in the rat scheduled-induced polydipsia model. CONCLUSIONS AND IMPLICATIONS: These findings suggest that WAY-211612 may represent a novel antidepressant.
Asunto(s)
Antidepresivos de Segunda Generación/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas de la Serotonina/farmacología , Animales , Conducta Animal/efectos de los fármacos , Cromatografía Líquida de Alta Presión , AMP Cíclico/metabolismo , Masculino , Ratones , Microdiálisis , Ratas , Ratas Sprague-DawleyRESUMEN
A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.
Asunto(s)
Asparagina/síntesis química , Endopeptidasas/metabolismo , Ácidos Hidroxámicos/síntesis química , Inhibidores de Proteasas/síntesis química , Administración Oral , Animales , Asparagina/análogos & derivados , Asparagina/química , Asparagina/farmacocinética , Asparagina/farmacología , Disponibilidad Biológica , Perros , Diseño de Fármacos , Endopeptidasas/química , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacocinética , Ácidos Hidroxámicos/farmacología , Metaloproteinasa 1 de la Matriz/química , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 8 de la Matriz/química , Metaloproteinasa 9 de la Matriz/química , Modelos Moleculares , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Unión Proteica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.
Asunto(s)
Compuestos Heterocíclicos con 1 Anillo/síntesis química , Ácidos Hidroxámicos/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas ADAM , Proteína ADAM17 , Sitios de Unión , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/farmacología , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Lipopolisacáridos/farmacología , Modelos Moleculares , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión Proteica , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of =0.2 microM in whole blood assay (WBA). Although the P3' area can accommodate a broad array of structurally diversified functional groups including polar residues, hydrophobic residues, and amino and carboxylic acid moieties, in both the cyclophane series and the cyclic carbamate series, a glycine residue at P3' was identified as a critical structural component to achieve both good in vitro potency and good oral activity. With a glycine residue at P3', an N-methylamide at P4' provided the best cyclophane analogue, SL422 (WBA IC(50) = 0.22 microM, LPS-mouse ED(50) = 15 mg/kg, po), whereas a morpholinylamide at P4' afforded the most potent and most orally active cyclic carbamate analogue, SP057 (WBA IC(50) = 0.067 microM, LPS-mouse ED(50) = 2.3 mg/kg, po). Further profiling for SL422 and SP057 showed that these macrocyclic compounds are potent TACE inhibitors, with K(i) values of 12 and 4.2 nM in the porcine TACE assay, and are broad-spectrum MMP inhibitors. Pharmacokinetic studies in beagle dogs revealed that SL422 and SP057 are orally bioavailable, with oral bioavailabilities of 11% and 23%, respectively.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Ácidos Hidroxámicos/síntesis química , Lactamas/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas ADAM , Proteína ADAM17 , Administración Oral , Animales , Disponibilidad Biológica , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacocinética , Carbamatos/farmacología , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacocinética , Ácidos Hidroxámicos/farmacología , Técnicas In Vitro , Lactamas/química , Lactamas/farmacocinética , Lactamas/farmacología , Masculino , Ratones , Estereoisomerismo , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.
Asunto(s)
Antiinflamatorios/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Animales , Butadienos/farmacología , Butadienos/uso terapéutico , Edema/prevención & control , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , Masculino , Ratones , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Nitrilos/uso terapéutico , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.
Asunto(s)
Endopeptidasas/genética , Metaloendopeptidasas/genética , Proteínas ADAM , Proteína ADAMTS5 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Humanos , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.
Asunto(s)
Cartílago/enzimología , Endopeptidasas/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Animales , Cartílago/efectos de los fármacos , Bovinos , Endopeptidasas/genética , Interleucina-1/farmacología , Cinética , Metaloproteinasa 3 de la Matriz/genética , Metaloendopeptidasas/metabolismo , Tabique Nasal , Técnicas de Cultivo de Órganos , Factores de TiempoRESUMEN
We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.
Asunto(s)
Proteínas de la Matriz Extracelular , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Proteínas ADAM , Proteína ADAMTS1 , Proteína ADAMTS4 , Agrecanos , Secuencia de Aminoácidos , Artritis/tratamiento farmacológico , Cartílago/metabolismo , Dominio Catalítico , Clonación Molecular , Desintegrinas/química , Desintegrinas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Interleucina-1/farmacología , Lectinas Tipo C , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Procolágeno N-Endopeptidasa , Inhibidores de Proteasas/farmacología , Señales de Clasificación de Proteína , Proteoglicanos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de SecuenciaRESUMEN
The discovery of terphenyl derivatives as highly selective COX-2 inhibitors resulted from our efforts to overcome poor pharmacokinetics demonstrated by the COX-2 selective diarylthiophene DuP 697 [2-bromo-4-(4'-sulfonylmethyl)phenyl-5-(4'-fluoro)phenylthiophe ne]. Detailed SAR related to the ortho-biphenyls and variants of the central ring are described herein.
Asunto(s)
Inhibidores de la Ciclooxigenasa/química , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacocinética , Isoenzimas/efectos de los fármacos , Estructura Molecular , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Relación Estructura-Actividad , Tiofenos/farmacocinéticaRESUMEN
Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However, the importance of this event for monocyte function is not clear. To address this issue, we used the novel MAP/ERK kinase (MEK) inhibitor, U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK, Jun NH2-terminal kinase (JNK), and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However, the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound, confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function, we measured production of the cytokines IL-1, IL-8, and TNF, as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1, IL-8, TNF, or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS.
Asunto(s)
Citocinas/biosíntesis , Dinoprostona/biosíntesis , Lipopolisacáridos/farmacología , Proteínas Quinasas Activadas por Mitógenos , Monocitos/enzimología , Monocitos/metabolismo , Inhibidores de Proteínas Quinasas , Butadienos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Citocinas/antagonistas & inhibidores , Citocinas/genética , Dinoprostona/antagonistas & inhibidores , Activación Enzimática/inmunología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Proteínas de la Membrana , Quinasas de Proteína Quinasa Activadas por Mitógenos , Monocitos/inmunología , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Brequinar is an immunosuppressant with the potential to be combined with cyclosporine in synergistic combination therapy. The drug tends to accumulate when given daily per os, and pharmacokinetic interaction with cyclosporine appears to enhance toxicity. Analogues with similar immunosuppressive activity have been identified at Du Pont Merck Pharmaceutical Co., that do not accumulate upon daily oral dosing in rats, and hence could have an improved potential in combination treatment with cyclosporine. We performed a toxicity study with brequinar and two brequinar analogues, administered orally once daily for 4 weeks, either alone or in combination with cyclosporine (Neoral, Novartis Pharma AG). In a first study relatively high doses were evaluated with cyclosporine at non-toxic doses of 5 and 10 mg/kg/d. The maximum tolerated dose of brequinar alone was estimated between 5 and 10 mg/kg/d; that of the analogues was estimated between 10 and 20 mg/kg/d, and above 20 mg/kg/d, respectively. In combination with cyclosporine at 5 and 10 mg/kg/d, approximately a 2-fold reduction in the maximum tolerated dose was observed. In a second study lower doses were evaluated in combination with cyclosporine at 2.5 and 5 mg/kg/d. Also this study revealed increased toxicity of brequinar (analogues) when given in combination with cyclosporine. The side effects observed were typical for drugs in the brequinar class and included leukocytopenia and thrombocytopenia, reduced body weight gain or body weight loss, thymic atrophy, cellular depletion of bone marrow and splenic white pulp, and villous atrophy in jejunum. Concentrations of brequinar (analogues) were determined in blood sampled 4 h after administration at day 1, 14 and 21-28 of the experiment. There was a tendency for drug accumulation in some groups treated with brequinar and cyclosporine. For one of the analogues at a low dose, higher concentrations were measured in groups treated with combinations of this compound and cyclosporine. We conclude that a potential synergism in immunosuppression using combinations of brequinar (analogues) and cyclosporine can be complicated by enhanced toxicity of the compounds. This indicates the need for a careful evaluation of the therapeutic window in a combined treatment together with detailed pharmacokinetics.
Asunto(s)
Compuestos de Bifenilo/toxicidad , Ciclosporina/toxicidad , Inmunosupresores/toxicidad , Administración Oral , Animales , Compuestos de Bifenilo/administración & dosificación , Compuestos de Bifenilo/sangre , Recuento de Células Sanguíneas , Peso Corporal/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Ciclosporina/administración & dosificación , Ciclosporina/sangre , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inmunosupresores/administración & dosificación , Inmunosupresores/sangre , Yeyuno/efectos de los fármacos , Yeyuno/patología , Leucopenia/inducido químicamente , Masculino , Ratas , Ratas Endogámicas Lew , Bazo/efectos de los fármacos , Bazo/patología , Trombocitopenia/inducido químicamente , Timo/efectos de los fármacos , Timo/patología , Pruebas de Toxicidad , Aumento de Peso/efectos de los fármacosRESUMEN
The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.
Asunto(s)
Butadienos/farmacología , Inhibidores Enzimáticos/farmacología , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas , Animales , Butadienos/química , Células COS , ADN/metabolismo , Inhibidores Enzimáticos/química , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Cinética , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Nitrilos/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismoRESUMEN
Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.
Asunto(s)
Activación de Linfocitos , Proteínas Quinasas/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD28/inmunología , Complejo CD3/inmunología , División Celular/inmunología , Células Clonales , Interleucina-2/inmunología , Ratones , Ratones Endogámicos BALB C , Quinasas de Proteína Quinasa Activadas por Mitógenos , Linfocitos T/citologíaRESUMEN
Structure-activity relationships were explored for some analogs of Brequinar having a linking atom between the 2-biphenyl substituent and the quinoline ring. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to the overall shape and lipophilicity of the 2-substituent.
Asunto(s)
Inmunosupresores/química , Inmunosupresores/farmacología , Compuestos de Bifenilo/química , Carbono/química , Prueba de Cultivo Mixto de Linfocitos , Relación Estructura-ActividadRESUMEN
In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.
Asunto(s)
Butadienos/química , Inhibidores Enzimáticos/química , Nitrilos/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Biotransformación , Butadienos/farmacocinética , Butadienos/farmacología , Ciclización , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacocinética , Nitrilos/farmacología , Ratas , Factor de Transcripción AP-1/antagonistas & inhibidoresRESUMEN
Examination of the S1 area of the active site of pro-stromelysin has led us to the design of novel and potent inhibitors of matrix metalloproteinases containing constrained quaternary-hydroxy group at P1. The synthesis and biological activity of these compounds with variations at P1', P2', and P3' will be described.
Asunto(s)
Ácidos Hidroxámicos/química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Dominio Catalítico , Colagenasas/química , Simulación por Computador , Diseño de Fármacos , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/química , Enlace de Hidrógeno , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Indicadores y Reactivos , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 3 de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/química , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The structure-activity relationships of some tetracyclic heterocycles related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction are related to ring system, heteroatom placement, and pendant ring substitution.
Asunto(s)
Compuestos de Bifenilo/química , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Inmunosupresores/química , Inmunosupresores/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Prueba de Cultivo Mixto de Linfocitos , Oxidorreductasas/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen. Studies following chymotrypsin activation of proMMP-3 by SDS/PAGE indicated that altered processing of the 57 kDa zymogen to the active form occurred in the presence of compound. The 53 kDa intermediate seen on normal activation was not formed; instead a different intermediate appeared with a molecular mass of approx. 46 kDa. N-terminal sequence analysis indicated that this intermediate was formed by cleavage at the putative 4-aminophenylmercuric acid cleavage site. Importantly the 45 kDa active MMP-3 species formed in the presence of compound was one amino acid residue shorter than the native MMP-3. These results suggest that the inhibition of cartilage proteoglycan degradation by isothiazolones might be due to their ability to bind to the Cys75 in the propeptide region of the MMP zymogen and interfere with its normal activation process.
Asunto(s)
Cartílago/metabolismo , Precursores Enzimáticos/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Inhibidores de Proteasas/farmacología , Proteoglicanos/metabolismo , Tiazoles/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Activación Enzimática , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/química , Cinética , Metaloproteinasa 3 de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz , Datos de Secuencia Molecular , Nariz , Técnicas de Cultivo de Órganos , Procesamiento Proteico-Postraduccional , Relación Estructura-ActividadRESUMEN
A series of 2,5-diarylisothiazolones is reported that inhibit the IL-1 beta-induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, nonpeptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.
Asunto(s)
Cartílago/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Interleucina-1/metabolismo , Tiazoles/farmacología , Animales , Cartílago/metabolismo , Bovinos , Hidrólisis , Interleucina-1/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética , Técnicas de Cultivo de Órganos , Osteoartritis/metabolismo , Osteoartritis/prevención & control , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
The active metabolite of leflunomide. A771726, is a novel immunosuppressive compound that has been shown to be a powerful antiproliferative agent for mononuclear and T-cells. The molecular mechanism of action for this compound has not been clearly established. In vitro cellular and enzymatic assays, however, demonstrate that leflunomide is an inhibitor of several protein tyrosine kinases, with IC50 values between 30 and 100 microM. The in vivo properties of A771726 are reminiscent of another immunosuppressive agent, brequinar sodium, which has been shown to be a nonomolar inhibitor (Ki = 10-30 nM) of the enzyme dihydroorotate dehydrogenase (DHODase). On the basis, we have investigated the effects of leflunomide and A771726 on the activity of purified recombinant human DHODase. We find that A771726 is a potent inhibitor of DHODase (Ki = 179 +/- 19 nM), while the parent compound, leflunomide, had no inhibitory effect at concentrations as high as 1 microM. Studies of the dependence of inhibition on the concentrations of the substrates ubiquinone and dihydroorotate demonstrate that A771726 is a competitive inhibitor of the ubiquinone binding site and is noncompetitive with respect to dihydroorotate. The potency of A771726 as a DHODase inhibitor is thus 100-100-fold greater than that reported for its inhibition of protein tyrosine kinases. These data suggest that an alternative explanation for the immunosuppressive efficacy of A771726 may be the potent inhibition of DHODase by this compound.