RESUMEN
Screening of a fragment library identified 2-hydrazinobenzothiazole as a potent inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme expressed by tumours that suppresses the immune system. Spectroscopic studies indicated that 2-hydrazinobenzothiazole interacted with the IDO1 haem and in silico docking predicted that the interaction was through hydrazine. Subsequent studies of hydrazine derivatives identified phenylhydrazine (IC50=0.25 ± 0.07 µM) to be 32-fold more potent than 2-hydrazinobenzothiazole (IC50=8.0 ± 2.3 µM) in inhibiting rhIDO1 and that it inhibited cellular IDO1 at concentrations that were noncytotoxic to cells. Here, phenylhydrazine is shown to inhibit IDO1 through binding to haem.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Hidrazinas/farmacología , Sistema Inmunológico/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Hidrazinas/química , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Modelos Moleculares , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC(50) values determined using the new assay for three known IDO1 inhibitors-1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole-were consistent with their literature values, further validating the new assay for measuring IDO1 activity.
Asunto(s)
Pruebas de Enzimas/métodos , Colorantes Fluorescentes/química , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Quinurenina/análogos & derivados , Mediciones Luminiscentes/métodos , Evaluación Preclínica de Medicamentos , Pruebas de Enzimas/instrumentación , Inhibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Quinurenina/química , Mediciones Luminiscentes/instrumentaciónRESUMEN
The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-alpha production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.
Asunto(s)
Etiquetas de Fotoafinidad/farmacología , Proteínas/metabolismo , Coloración y Etiquetado/métodos , Xantonas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Electroforesis en Gel Bidimensional , Células Endoteliales/química , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Oxidación-Reducción , Etiquetas de Fotoafinidad/química , Etiquetas de Fotoafinidad/metabolismo , Fotoquímica , Proteínas/análisis , Proteínas/química , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/fisiología , Xantonas/químicaRESUMEN
PURPOSE: SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a DNA intercalating drug that binds selectively to GC-rich DNA and shows curative activity against the Colon 38 adenocarcinoma in mice. We wished to investigate the roles of topoisomerase (topo) I, topo II and RNA transcription in the action of SN 28049. METHODS: We used clonogenic assays to study the cytotoxicity of SN 28049; RNA interference and enzyme assays to examine the role of topo I in SN 28049 action; 3H uridine incorporation and reporter assays to study its effects on transcription; and RT-PCR to examine its ability to reduce endogenous h-TERT expression. RESULTS: In clonogenic assays, SN 28049 showed a biphasic cytotoxic dose response curve in H460 cells typical of acridine derivatives such as N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) although it was approximately 16-fold more potent. Down-regulation of topo IIalpha in HTETOP cells reduced the cytotoxicity of SN 28049, establishing its action as a topo IIalpha poison. Surprisingly, down-regulation of topo I in H460 cells by RNA interference sensitised them to the actions of SN 28049 and other topo II poisons. SN 28049 also inhibited topo I-mediated relaxation of supercoiled plasmid DNA. SN 28049 was also an inhibitor of transcription in HEK293 cells and was more potent at reducing luciferase expression from a GC-rich SP-1 binding promoter than from a non-GC-rich AP-1 binding promoter. The drug also reduced luciferase reporter gene expression driven by the SP-1-binding survivin promoter as well as reducing endogenous h-TERT expression in HEK293 cells whose promoter also contains SP-1 binding sites. CONCLUSION: We conclude that SN 28049 has a complex action that may involve poisoning of topo IIalpha, suppression of topo I and inhibition of gene transcription from promoters with SP-1 sites. These actions may contribute to the promising experimental solid tumour anticancer activity of SN 28049.