RESUMEN
Human coagulation factor XIa (FXIa), a serine protease activated by site-specific cleavage of factor XI by thrombin, FXIIa, or autoactivation, is a critical enzyme in the amplification phase of the coagulation cascade. To investigate the potential of FXIa inhibitors as safe anticoagulants, a series of potent, selective peptidomimetic inhibitors of FXIa were designed and synthesized. Some of these inhibitors showed low nanomolar FXIa inhibitory activity with >1000-fold FXa selectivity and >100-fold thrombin selectivity. The X-ray structure of one of these inhibitors, 36, demonstrates its unique binding interactions with FXIa. Compound 32 caused a doubling of the activated partial thromboplastin time in human plasma at 2.4 microM and was efficacious in a rat model of venous thrombosis. These data suggest that factor XIa plays a significant role in venous thrombosis and may be a suitable target for the development of antithrombotic therapy.
Asunto(s)
Anticoagulantes/farmacología , Diseño de Fármacos , Factor XIa/antagonistas & inhibidores , Inhibidores del Factor Xa , Fragmentos de Péptidos/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Animales , Anticoagulantes/síntesis química , Anticoagulantes/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Masculino , Estructura Molecular , Tiempo de Tromboplastina Parcial , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Relación Estructura-Actividad , Trombina/antagonistas & inhibidores , Trombosis de la Vena/tratamiento farmacológicoRESUMEN
A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal structures of the protein-inhibitor complexes led to the design and synthesis of second generation compounds showing single digit micromolar inhibition against FXIa and selectivity against thrombin, trypsin, and FXa.
Asunto(s)
Anticoagulantes/síntesis química , Ácidos Borónicos/síntesis química , Ácidos Borónicos/farmacología , Factor XIa/antagonistas & inhibidores , Anticoagulantes/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Unión Proteica , Conformación Proteica , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-ActividadRESUMEN
Using an alpha-ketothiazole arginine moiety as a key recognition element, a series of small peptidomimetic molecules was designed and synthesized, and their co-crystal structures with factor XIa were studied in an effort to develop smaller, less peptidic inhibitors as antithrombotic agents.
Asunto(s)
Arginina/química , Arginina/farmacología , Factor XIa/antagonistas & inhibidores , Tiazoles/química , Tiazoles/farmacología , Amidas/química , Arginina/análogos & derivados , Arginina/síntesis química , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Tiazoles/síntesis químicaRESUMEN
Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370-607 (rhFXI370-607). Here we report the first crystal structure of rhFXI370-607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 A resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2' in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI370-607-ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas de Escherichia coli/química , Factor XIa/química , Proteínas Periplasmáticas/química , Anticoagulantes/química , Sitios de Unión , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Dimerización , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Riesgo , Serina/química , Estereoisomerismo , TrombosisRESUMEN
The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the zeta subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 A resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N- and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas/química , Estaurosporina/farmacología , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Baculoviridae/metabolismo , Sitios de Unión , Células COS , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Estereoisomerismo , Factores de Tiempo , Transfección , Tirosina/química , Proteína Tirosina Quinasa ZAP-70 , Dominios Homologos srcRESUMEN
PURPOSE: Primary uveal melanocytes and many ocular melanoma cells are resistant to interferon (IFN)-gamma-mediated induction of major histocompatibility complex (MHC) class II molecule expression. This suppression of class II MHC induction is considered to be one of the ways in which the eye is able to inhibit inflammatory responses. However, the mechanism(s) of this suppression is unknown. In this study, we have probed the molecular basis of this phenotype and report two distinct mechanisms underlying this phenotype. METHODS: Primary ocular melanocytes and ocular melanoma cell lines (retaining this IFN-gamma-resistant class II MHC phenotype) were examined for the expression of class II MHC molecules on the cell surface by flow cytometry. Class II MHC gene expression was further examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. RESULTS: The IFN-gamma signal-transduction pathway was found to be intact by electrophoretic mobility shift assay (EMSA) and transfection of reporter constructs. The lack of class II MHC gene expression appears to result from at least two mechanisms: (1) a specific inhibition of CIITA (class II transactivator) gene expression (reminiscent of trophoblasts), and (2) posttranscriptional regulation of class II MHC genes. CONCLUSIONS: The inability of primary uveal melanocytes and ocular melanoma cells to express class II MHC molecules after treatment with IFN-gamma has been found to map to two distinct points in the class II MHC biosynthetic pathway. The predominant mechanism appears to involve the silencing of the endogenous gene encoding the class II transactivator (CIITA). Here, the blockade does not involve signal transduction from the IFN-gamma receptor, but rather involves a specific silencing of the CIITA gene. A second mechanism involves the posttranscriptional regulation of class II MHC genes.