RESUMEN
c-Jun N-terminal kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bidentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC(50) = 18 nM; K(i) = 1.5 nM) and JNK/substrate association in a displacement assay (IC(50) = 46 nM; K(i) = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 successfully inhibits JNK in a variety of cell-based experiments, as well as in vivo where it is shown to protect against Jo-2 induced liver damage and improve glucose tolerance in diabetic mice.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Péptidos/química , Secuencias de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Secuencia de Consenso , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diseño de Fármacos , Femenino , Intolerancia a la Glucosa/tratamiento farmacológico , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/química , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Imitación Molecular , Péptidos/síntesis química , Péptidos/farmacología , Fosforilación , Unión Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Antibody microarrays, one emerging class of proteomic technologies, have broad applications in proteome analysis, disease diagnostics and quantitative analysis. Compared to DNA microarrays, protein targets have significantly more complex interactions with their ligands such as antibodies. To introduce antibody microarrays for clinical diagnostics and thus to complement or replace conventional immunoassays, several new developments are addressed. We discuss different microarray surfaces, immobilization techniques, detection systems and advantages and disadvantages of antibody microarrays compared to standard clinical techniques. Currently, the probes with highest specificity, well-characterized binding properties, and the possibility of large-scale production using display libraries are recombinant antibodies.
Asunto(s)
Anticuerpos/genética , Anticuerpos/inmunología , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Animales , Anticuerpos/química , Humanos , Análisis por Matrices de Proteínas/tendencias , Proteínas Recombinantes/química , Sensibilidad y EspecificidadRESUMEN
The authors describe a microarray system for disease diagnosis based on antibody-antigen interactions. Either biotinylated antibodies or antigens are coupled via streptavidin linkers onto a gold surface. This platform has been used to establish recombinant antibody-antigen interactions and to detect specific IgM antibodies in sera of patients suspected of Lyme borreliosis. Therefore, this microarray system can be adapted for further applications.
Asunto(s)
Biotina/metabolismo , Inmunoensayo/métodos , Análisis por Matrices de Proteínas/métodos , Estreptavidina/metabolismo , Animales , Antígenos/metabolismo , Oro/metabolismo , Humanos , Inmunoensayo/instrumentación , Fragmentos de Inmunoglobulinas/metabolismo , Inmunoglobulina M/sangre , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/inmunología , Péptidos/metabolismo , Reproducibilidad de los ResultadosRESUMEN
This review focuses on the application of novel technologies for generating biocompatible surfaces for high-throughput screening (HTS) of proteins. Various methods of coupling and spotting proteins on self-assembled monolayer (SAM) surfaces will be described along with the protein chip challenges pertaining to spot homogeneity, morphology, biocompatibility and reproducibility.
Asunto(s)
Análisis por Matrices de Proteínas/métodos , Materiales Biocompatibles/química , Células Inmovilizadas , Dextranos/química , Electroforesis en Gel Bidimensional/instrumentación , Electroforesis en Gel Bidimensional/métodos , Diseño de Equipo , Vidrio , Membrana Dobles de Lípidos/química , Ensayo de Materiales , Análisis por Matrices de Proteínas/instrumentación , Propiedades de SuperficieRESUMEN
We report a novel high-throughput (HTP) protein chip platform, constructed on gold using self-assembly techniques, for conducting high quality antigen-antibody interactions. Biotinylated monolayers were used to immobilize a streptavidin surface with high packing density. This biocompatible platform was then used for detection of serum IgM antibodies. Serum samples of patients suspected to suffer from Lyme borreliosis were used to validate the protein chip platform using biotinylated peptide AAOspC8 molecules as the test probes. Various experimental parameters such as the effect of concentration of probes, targets, temperature of incubation, and their effect on the resulting signal-to-noise ratio are described in detail. Highly specific protein interaction data with a high signal-to-noise ratio were obtained with serum sample solutions as low as 1 microL/spot (1/10 diluted).