RESUMEN
BACKGROUND: Macrophage migration inhibitory factor (MIF) exerts a protective effect on ischemic myocardium by activating AMP-activated protein kinase (AMPK). Small molecules that increase the affinity of MIF for its receptor have been recently designed, and we hypothesized that such agonists may enhance AMPK activation and limit ischemic tissue injury. METHODS AND RESULTS: Treatment of cardiomyocytes with the candidate MIF agonist, MIF20, augmented AMPK phosphorylation, increased by 50% the surface localization of glucose transporter, and enhanced by 25% cellular glucose uptake in comparison with MIF alone. In mouse hearts perfused with MIF20 before no-flow ischemia and reperfusion, postischemic left ventricular function improved commensurately with an increase in cardiac MIF-AMPK activation and an augmentation in myocardial glucose uptake. By contrast, small-molecule MIF agonism was not effective in cells or tissues genetically deficient in MIF or the MIF receptor, verifying the specificity of MIF20 for MIF-dependent AMPK signaling. The protective effect of MIF20 also was evident in an in vivo regional ischemia model. Mice treated with MIF20 followed by left coronary artery occlusion and reperfusion showed a significant reduction in infarcted myocardium. CONCLUSIONS: These data support the pharmacological utility of small-molecule MIF agonists in enhancing AMPK activation and reducing cardiac ischemic injury.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Histocompatibilidad Clase II/genética , Oxidorreductasas Intramoleculares/farmacología , Factores Inhibidores de la Migración de Macrófagos/farmacología , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antígenos de Diferenciación de Linfocitos B/metabolismo , Cardiotónicos/farmacología , Células Cultivadas , Glucosa/farmacocinética , Antígenos de Histocompatibilidad Clase II/metabolismo , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/genética , Isoxazoles/farmacología , Factores Inhibidores de la Migración de Macrófagos/agonistas , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Isquemia Miocárdica/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiologíaRESUMEN
The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also promotes AMPK activation with potential benefits for response to myocardial infarction and ischemia-reperfusion. Structure-based molecular design has led to the discovery of not only antagonists, but also the first agonists of MIF-CD74 binding. The compounds contain a triazole core that is readily assembled via Cu-catalyzed click chemistry. The agonist and antagonist behaviors were confirmed via study of MIF-dependent ERK1/2 phosphorylation in human fibroblasts.
Asunto(s)
Diseño de Fármacos , Receptores Inmunológicos/agonistas , Triazoles/farmacología , Células Cultivadas , Química Clic , Cobre , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Relación Estructura-ActividadRESUMEN
The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto-enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 µM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC(50) values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF-CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.
Asunto(s)
Benzoxazoles/química , Receptores Inmunológicos/antagonistas & inhibidores , Antígenos de Diferenciación de Linfocitos B/química , Benzoxazoles/síntesis química , Benzoxazoles/farmacología , Sitios de Unión , Simulación por Computador , Fibroblastos/enzimología , Antígenos de Histocompatibilidad Clase II/química , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Receptores Inmunológicos/metabolismoRESUMEN
Macrophage migration inhibitory factor (MIF) is a cytokine that is involved in the regulation of inflammation as well as cell proliferation and differentiation. Deactivation of MIF by antibodies or inhibition of MIF binding to its receptor, CD74, attenuates tumor growth and angiogenesis. To discover small-molecule inhibitors of MIF's biological activity, virtual screening was performed by docking 2.1 million compounds into the MIF tautomerase active site. After visual inspection of 1200 top-ranked MIF-ligand complexes, 26 possible inhibitors were selected and purchased and 23 of them were assayed. The in vitro binding assay for MIF with CD74 revealed that 11 of the compounds have inhibitory activity in the micromolar regime, including four compounds with IC(50) values below 5 microM. Inhibition of MIF tautomerase activity was also established for many of the compounds with IC(50) values as low as 0.5 microM; Michaelis-Menten analysis was performed for two cases and confirmed the competitive inhibition.