RESUMO
Indoleamine 2,3-dioxygenase (IDO1) catalyzes the first step in tryptophan breakdown along the kynurenine pathway. Therapeutic inhibition of IDO1 is receiving much attention due to its proposed role in the pathogenesis of several diseases including cancer, hypotension and neurodegenerative disorders. A related enzyme, IDO2 has recently been described. We report the first purification and kinetic characterization of human IDO2 using a facile l-tryptophan consumption assay amenable to high throughput screening. We found that the K(m) of human IDO2 for l-tryptophan is much higher than that of IDO1. We also describe the identification and characterization of a new IDO1 inhibitor compound, Amg-1, by high throughput screening, and compare the inhibition profiles of IDO1 and IDO2 with Amg-1 and previously described compounds. Our data indicate that human IDO1 and IDO2 have different kinetic parameters and different inhibition profiles. Docking of Amg-1 and related analogs to the known structure of IDO1 and to homology-modeled IDO2 suggests possible rationales for the different inhibition profiles of IDO1 and IDO2.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/isolamento & purificação , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/isolamento & purificação , Descoberta de Drogas/métodos , Inibidores Enzimáticos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
From the recombinant human leukocyte antigen (HLA)-DR1/H2-E(k) major histocompatibility complex (MHC) class II-transgenic mice, we have generated two CD4(+) T-cell hybridomas specific for peptides which were derived from human prostatic acid phosphatase (PAP) complexed to the human class II molecule HLA-DR1. Both hybridomas strongly react to PAP-pulsed antigen-presenting cells (APC) from transgenic mice. Interestingly, these hybridomas also responded to PAP antigen presented by HLA-DR1-positive human APC. The species-mismatched T-cell stimulation occurs despite the biologic discordance in participating accessory molecules, which are required for the optimal T-cell-APC interaction. Our results demonstrate various degrees of functional interaction between coreceptors, costimulatory molecules, and integrins, which are expressed on the surface of T-cell hybridomas and heterologous APC.
Assuntos
Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Antígenos Heterófilos/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos H-2/imunologia , Antígeno HLA-DR1/imunologia , Hibridomas/imunologia , Fosfatase Ácida , Animais , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular/imunologia , Epitopos de Linfócito T/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Antígenos H-2/genética , Antígeno HLA-DR1/genética , Humanos , Interleucina-2/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Tirosina Fosfatases/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Especificidade da EspécieRESUMO
Trop2 is a cell-surface glycoprotein reported to be overexpressed in various types of adenocarcinomas with minimal expression in normal tissues. Recent findings that Trop2 expression correlates with tumor aggressiveness have increased interest in Trop2 as a potential target for cancer immunotherapy. The goal of this study was to extensively evaluate Trop2 expression at the transcript and protein levels in normal and tumor tissues. It was determined that Trop2 is overexpressed on some carcinomas relative to the corresponding normal tissue. However, in human and mouse, Trop2 is highly expressed at both the transcript and protein levels on several essential normal tissues. The findings suggest that the development of therapeutic agents to target Trop2 may require strategies that target Trop2 on malignant tissues in order to minimize potential toxicities to essential normal tissues that also express high levels of Trop2.