RESUMO
BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in multiple inflammatory and non-inflammatory diseases, including liver injury induced by acetaminophen (APAP) overdose. Multiple small molecule inhibitors of MIF have been described, including the clinically available anti-rheumatic drug T-614 (iguratimod); however, this drug's mode of inhibition has not been fully investigated. METHODS: We conducted in vitro testing including kinetic analysis and protein crystallography to elucidate the interactions between MIF and T-614. We also performed in vivo experiments testing the efficacy of T-614 in a murine model of acetaminophen toxicity. We analyzed survival in lethal APAP overdose with and without T-614 and using two different dosing schedules of T-614. We also examined MIF and MIF inhibition effects on hepatic hydrogen peroxide (H2O2) as a surrogate of oxidative stress in non-lethal APAP overdose. RESULTS: Kinetic analysis was consistent with a non-competitive type of inhibition and an inhibition constant (Ki) value of 16 µM. Crystallographic analysis revealed that T-614 binds outside of the tautomerase active site of the MIF trimer, with only the mesyl group of the molecule entering the active site pocket. T-614 improved survival in lethal APAP overdose when given prophylactically, but this protection was not observed when the drug was administered late (6 h after APAP). T-614 also decreased hepatic hydrogen peroxide concentrations during non-lethal APAP overdose in a MIF-dependent fashion. CONCLUSIONS: T-614 is an allosteric inhibitor of MIF that prevented death and decreased hepatic hydrogen peroxide concentrations when given prophylactically in a murine model of acetaminophen overdose. Further studies are needed to elucidate the mechanistic role of MIF in APAP toxicity.
Assuntos
Benzopiranos , Doença Hepática Induzida por Substâncias e Drogas , Cromonas , Fatores Inibidores da Migração de Macrófagos , Sulfonamidas , Camundongos , Animais , Acetaminofen/efeitos adversos , Peróxido de Hidrogênio/metabolismo , Modelos Animais de Doenças , Cinética , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Estresse Oxidativo , Fígado/metabolismoRESUMO
Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (T(4)) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma T(4) concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-T(4) (or its hormonally inert isomer D-T(4)) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-T(4) significantly improved survival in mice with severe sepsis. To examine the specificity of the MIFT(4) interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-T(4) or vehicle. D-T(4) significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo T(4) may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for T(4) as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma T(4) concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and T(4) may be beneficial in improving outcome from sepsis.
Assuntos
Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Tiroxina/fisiologia , Animais , Sítios de Ligação , Humanos , Inflamação , Fatores Inibidores da Migração de Macrófagos/sangue , Camundongos , Camundongos Knockout , Sepse/sangue , Sepse/tratamento farmacológico , Taxa de Sobrevida , Hormônios Tireóideos/sangue , Tiroxina/sangue , Tiroxina/uso terapêuticoRESUMO
Previous studies have shown that amyloid beta protein (Abeta ), the essential molecule for the formation of toxic oligomers and, subsequently, Alzheimer plaques, has been associated in vivo with the immune modulator, macrophage migration inhibitory factor (MIF) (17). To further investigate this association in vivo we used the APP transgenic mouse model. Serial brain sections of transgenic APP mice were stained for Abeta plaques and MIF and we observed MIF immunolabeling in microglial cells in association with Abeta plaques in the transgenic mouse brain sections. In addition, functional studies in murine and human neuronal cell lines revealed that Abeta-induced toxicity could be reversed significantly by a small molecule inhibitor of MIF (ISO-1). Finally, to elucidate the role of MIF in Alzheimer's Disease (AD) we measured MIF levels in the brain cytosol and cerebrospinal fluid (CSF) of AD patients and age-matched controls. Our results demonstrate a marked increase of MIF levels within the CSF of AD patients compared with controls. Combined, our results indicate a strong role for MIF in the pathogenesis of AD and furthermore suggest that inhibition of MIF may provide a valuable avenue of investigation for the prevention of disease onset, progression and/or severity.
Assuntos
Doença de Alzheimer/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Idoso , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Encéfalo/metabolismo , Química Encefálica , Sobrevivência Celular/efeitos dos fármacos , Feminino , Histocitoquímica , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/líquido cefalorraquidiano , Isoxazóis/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/líquido cefalorraquidiano , Masculino , Camundongos , Camundongos Transgênicos , Neuroblastoma , Nexinas de Proteases , Receptores de Superfície Celular/genética , Estatísticas não Paramétricas , Células Tumorais CultivadasRESUMO
A series of phenolic hydrazones were synthesized and evaluated for their inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity. Compound 7 emerged as a potent inhibitor of MIF with an IC50 of 130 nM. Compound 7 dose-dependently suppressed TNFalpha secretion from lipopolysaccharide stimulated macrophages. The therapeutic importance of the MIF inhibition by 7 is demonstrated by the significant protection from the lethality of sepsis when administration of the compound was initiated in a clinically relevant time frame.
Assuntos
Hidrazonas/síntese química , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fenóis/síntese química , Sepse/tratamento farmacológico , Animais , Linhagem Celular , Hidrazonas/química , Hidrazonas/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fenóis/química , Fenóis/farmacologia , Sepse/metabolismo , Sepse/mortalidade , Relação Estrutura-Atividade , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The pro-inflammatory mediator macrophage migration inhibitory factor (MIF) is produced by immune and endocrine cells and inhibits the antiinflammatory activities of glucocorticoids. MIF also catalyzes the tautomerization of the non-naturally occurring D-isomer of dopachrome, phenylpyruvate, and certain catecholamines, suggesting that MIF might exert its biological effects via enzymatic action on a substrate. However, no physiologically relevant substrate for MIF has been identified. Site-directed mutagenesis studies have not consistently supported a requirement for an intact, functional catalytic site as a prerequisite for MIF bioactivity. We hypothesized that the catalytically active site, but not the enzymatic activity per se, nevertheless plays a critical role in MIF pro-inflammatory activity. Accordingly, we designed small druglike molecules that bind at the catalytically active tautomerase site of MIF and tested the complex for MIF bioactivity. We describe herein the rational design and synthesis of a class of imine conjugates produced by coupling amino acids to a range of benzaldehyde derivatives that inhibit MIF tautomerase and biological activities. We found that aromatic amino acid Schiff bases were better inhibitors of MIF enzymatic and bioactivities compared to the aliphatic ones. For instance, the IC(50) inhibition of MIF tautomerase activity by aromatic amino acid Schiff base methyl esters was achieved at a concentration between 1.65 and 50 microM, suggesting a critical role for the additional binding of the aromatic residues within the vicinity of the active site. The most potent inhibitor of MIF tautomerase activity was 2-[(4-hydroxybenzylidene)amino]-3-(1H-indol-3-yl)propionic acid methyl ester (8), with an IC(50) of 1.65 microM. We found that compound 8 binding to MIF active site resulted in the inhibition of MIF bioactivity in three established bioassays: ERK-1/2 MAP kinase activation, p53-dependent apoptosis, and proliferation of serum-starved cells. Compound 8 inhibited MIF interaction with its as yet unidentified cognate cell surface receptor as shown by flow cytometry, concluding a critical role for the tautomerase active site in receptor binding. Thus the inhibitory effect of compound 8 on MIF bioactivities strongly correlated with the inhibition of MIF tautomerase activity, a connection not made previously through use of small-molecule MIF inhibitors. The inhibitory activity of amino acid-benzaldehyde Schiff base-type MIF antagonists is the first step toward a meaningful structure/function analysis of inhibitors of MIF cellular bioactivities.
Assuntos
Inibidores Enzimáticos/síntese química , Iminas/síntese química , Indóis/síntese química , Isomerases/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Bases de Schiff/síntese química , Animais , Apoptose/efeitos dos fármacos , Domínio Catalítico , Células Cultivadas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Iminas/química , Iminas/farmacologia , Indóis/química , Indóis/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/síntese química , Mitógenos/química , Mitógenos/farmacologia , Modelos Moleculares , Fosforilação , Ligação Proteica , Bases de Schiff/química , Bases de Schiff/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Alzheimer's disease (AD) is characterized by neuronal atrophy caused by soluble amyloid beta protein (Abeta) peptide "oligomers" and a microglial-mediated inflammatory response elicited by extensive amyloid deposition in the brain. We show that CNI-1493, a tetravalent guanylhydrazone with established antiinflammatory properties, interferes with Abeta assembly and protects neuronal cells from the toxic effect of soluble Abeta oligomers. Administration of CNI-1493 to TgCRND8 mice overexpressing human amyloid precursor protein (APP) for a treatment period of 8 wk significantly reduced Abeta deposition. CNI-1493 treatment resulted in 70% reduction of amyloid plaque area in the cortex and 87% reduction in the hippocampus of these animals. Administration of CNI-1493 significantly improved memory performance in a cognition task compared with vehicle-treated mice. In vitro analysis of CNI-1493 on APP processing in an APP-overexpressing cell line revealed a significant dose-dependent decrease of total Abeta accumulation. This study indicates that the antiinflammatory agent CNI-1493 can ameliorate the pathophysiology and cognitive defects in a murine model of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/biossíntese , Anti-Inflamatórios não Esteroides/farmacologia , Hipocampo/metabolismo , Hidrazonas/farmacologia , Microglia/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Animais , Relação Dose-Resposta a Droga , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Camundongos , Camundongos Transgênicos , Microglia/patologia , Fatores de TempoRESUMO
Macrophage migration inhibitory factor (MIF) is a ubiquitous cytokine that functions in reproduction and plays an important role in sperm maturation and motility. Here we reveal a correlation between MIF levels in human seminal fluid and fertility status. We identify an abnormal biphasic profile of MIF in the seminal fluid of patients with impaired sperm parameters. Our findings may be of interest for the development of a diagnostic method for fertility status.
Assuntos
Infertilidade Masculina , Fatores Inibidores da Migração de Macrófagos/metabolismo , Sêmen/metabolismo , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Indicadores e Reagentes/metabolismo , Fatores Inibidores da Migração de Macrófagos/farmacologia , Masculino , Propídio/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
Pharmacophores are chemical scaffolds upon which changes in chemical moieties (R-groups) at specific sites are made to identify a combination of R-groups that increases the therapeutic potency of a small molecule inhibitor while minimizing adverse effects. We developed a pharmacophore based on a carbonyloxime (OXIM) scaffold for macrophage migration inhibitory factor (MIF), a protein involved in the pathology of sepsis, to validate that inhibition of a catalytic site could produce therapeutic benefits. We studied the crystal structures of MIF.OXIM-based inhibitors and found two opposite orientations for binding to the active site that were dependent on the chemical structures of an R-group. One orientation was completely unexpected based on previous studies with hydroxyphenylpyruvate and (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1). We further confirmed that the unexpected binding mode targets MIF in cellular studies by showing that one compound, OXIM-11, abolished the counter-regulatory activity of MIF on anti-inflammatory glucocorticoid action. OXIM-11 treatment of mice, initiated 24 h after the onset of cecal ligation and puncture-induced sepsis, significantly improved survival when compared with vehicle-treated controls, confirming that inhibition of the MIF catalytic site could produce therapeutic effects. The crystal structures of the MIF inhibitor complexes provide insight for further structure-based drug design efforts.
Assuntos
Química Farmacêutica/métodos , Fatores Inibidores da Migração de Macrófagos/metabolismo , Oximas/química , Anti-Inflamatórios/química , Sítios de Ligação , Carbono/química , Cristalografia por Raios X/métodos , Desenho de Fármacos , Glucocorticoides/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Fatores Inibidores da Migração de Macrófagos/química , Modelos Químicos , NF-kappa B/metabolismo , Oximas/farmacologia , Ligação Proteica , Proteínas Recombinantes/químicaRESUMO
We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.
Assuntos
Amiloide/síntese química , Amiloide/ultraestrutura , Fatores Inibidores da Migração de Macrófagos/química , Animais , Sítios de Ligação , Dimerização , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação ProteicaRESUMO
MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.
Assuntos
Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Oxirredutases Intramoleculares/química , Isoxazóis/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Sepse/prevenção & controle , Animais , Sítios de Ligação , Ceco/efeitos dos fármacos , Ceco/metabolismo , Sobrevivência Celular/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Endotoxemia/tratamento farmacológico , Endotoxemia/etiologia , Isoxazóis/química , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Punções , Sepse/tratamento farmacológico , Sepse/patologia , Taxa de Sobrevida , Fatores de Necrose Tumoral/metabolismoRESUMO
Macrophage migration inhibitory factor (MIF) is an immunoregulatory protein that is a potential therapeutic target for a number of inflammatory diseases. Evidence exists that an unexpected catalytic active site of MIF may have a biological function. To gain further insight into the role of the catalytic active site, a series of mutational, structural, and biological activity studies were performed. The insertion of an alanine between Pro-1 and Met-2 (PAM) abolishes a non-physiological catalytic activity, and this mutant is defective in the in vitro glucocorticoid counter-regulatory activity of MIF. The crystal structure of MIF complexed to (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF d-dopachrome tautomerase activity, reveals that ISO-1 binds to the same position of the active site as p-hydroxyphenylpyruvic acid, a substrate of MIF. ISO-1 inhibits several MIF biological activities, further establishing a role for the catalytic active site of MIF.