RESUMO
The active metabolite (2) of the novel immunosuppressive agent leflunomide (1) has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogues of the active metabolite 2 have been synthesized. Their in vivo biological activity determined in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compound (3) has shown activity in rat and mouse collagen (II)-induced arthritis models (ED50 = 2 and 31 mg/kg, respectively) and has shown a shorter half-life in man when compared with leflunomide. Clinical studies in rheumatoid arthritis are in progress.
Assuntos
Acrilamidas/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Inibidores Enzimáticos/síntese química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/antagonistas & inibidores , Acrilamidas/farmacocinética , Acrilamidas/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Feminino , Hipersensibilidade Tardia , Cinética , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos DBA , Ratos , Ratos Wistar , Espectrofotometria Infravermelho , Relação Estrutura-AtividadeRESUMO
HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'(trifluoromethyl)-phenyl]-propenamide) is an immunomodulatory compound through pyrimidine biosynthesis inhibition with antiproliferative properties which was derived from the isoxazol compound A77 1726 [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide]. During studies of the effects on early signal transduction events of this type of compound, it was found that HR325 dose-dependently inhibited adenosine 3',5'-cyclic monophosphate (cAMP) synthesis by Jurkat cells stimulated with prostaglandin E(2), (PGE(2)), cholera toxin (CTX), or forskolin (FKN). The potency of inhibition by HR325 of FKN-stimulated cells (IC(50) 30.4 microM) was approximately 3-fold higher than that of the other agonists (11.6 and 11.7 microM) and was independent of time of preincubation for both PGE(2) and FKN. Interestingly, A77 1726, an analogue of HR325, displayed a markedly different profile of stimulus-dependent potencies. The inhibition of cAMP synthesis by HR325 when stimulated by both PGE(2) and FKN was unaffected by glucose supplementation, in contrast to HR325-inhibited ATP levels, which were restored under such conditions. Further studies revealed that HR325 reduced intracellular ATP levels by uncoupling oxidative phosphorylation, albeit with a 1000-fold lower potency than the antihelmintic drug niclosamide. In addition, glucose supplementation experiments showed that, in contrast to HR325, the niclosamide-mediated reduction of ATP levels was wholly responsible for its inhibition of PGE(2)- and FKN-stimulated cAMP synthesis.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Anilina/farmacologia , AMP Cíclico/metabolismo , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Interações Medicamentosas , Glucose/farmacologia , Humanos , Células Jurkat , Mitocôndrias/metabolismo , Oxirredução , Fosforilação/efeitos dos fármacosAssuntos
Imunossupressores/farmacologia , Isoxazóis/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/imunologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Di-Hidro-Orotato Desidrogenase , Humanos , Membranas Intracelulares/enzimologia , Leflunomida , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Microssomos/enzimologia , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ratos , Homologia de Sequência de Aminoácidos , Baço/imunologiaRESUMO
Leflunomide is currently in phase-III clinical trials for the treatment of rheumatoid arthritis. In this study, we have focused our efforts on the study of the mechanism of action of the active metabolite of leflunomide, A77 1726, in cells and tissue of human origin. The human high-affinity binding protein for radiolabelled A77 1726 was purified from solubilized U937 membranes by following the binding activity through the purification process and was characterized as the mitochondrial enzyme dihydro-orotate dehydrogenase (DHO-DH). The human and murine enzyme displayed identical pI and molecular mass values on SDS/PAGE (43 kDa), which contrasts notably with previous reports suggesting a molecular mass of 50 kDa for the human enzyme. DHO-DH activity was inhibited by A77 1726 and its analogue HR325 with similar potency in U937 and human spleen membrane preparations. HR325 was found to be anti-proliferative for phytohaemagglutinin-stimulated human peripheral blood mononuclear cells, at the same concentrations that caused accumulation of DHO and depletion of uridine. Supplementation of the cultures with exogenous uridine led to partial abrogation of the anti-proliferative effect. This is in line with our recent demonstration that the anti-proliferative effect in vitro of A77 1726 on lipopolysaccharide-stimulated mouse spleen cells was mediated by DHO-DH inhibition [Williamson, Yea, Robson, Curnock, Gadher, Hambleton, Woodward, Bruneau, Hambleton, Moss et al., (1995) J. Biol. Chem. 270, 22467-22472]. Thus, DHO-DH inhibition by A77 1726 and its analogues is responsible for the anti-proliferative effects in vitro of the compounds on human cells and is likely to be responsible for some of its effects in vivo.
Assuntos
Compostos de Anilina/farmacologia , Hidroxibutiratos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/antagonistas & inibidores , Oxirredutases/isolamento & purificação , Compostos de Anilina/metabolismo , Sítios de Ligação , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Crotonatos , Di-Hidro-Orotato Desidrogenase , Humanos , Hidroxibutiratos/metabolismo , Linfoma/enzimologia , Nitrilas , Oxirredutases/metabolismo , Baço/enzimologia , Toluidinas , Células Tumorais Cultivadas , Uridina/metabolismo , Uridina/farmacologiaRESUMO
A protein with high affinity (Kd 12 nM) for the immunomodulatory compound A77 1726 has been isolated from mouse spleen and identified as the mitochondrial enzyme dihydroorotate dehydrogenase (EC 1.3.3.1). The purified protein had a pI 9.6-9.8 and a subunit Mr of 43,000. Peptides derived from the mouse protein displayed high microsequence similarity to human and rat dihydroorotate dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate dehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthesis. The in vitro antiproliferative effects of A77 1726 are mediated by enzyme inhibition and can be overcome by addition of exogenous uridine. The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to counteract inhibition of the murine acute graft versus host response.