RESUMEN
Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Indoles/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Tiofenos/química , Sitio Alostérico , Cristalografía por Rayos X , Descubrimiento de Drogas , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/síntesis química , Indoles/metabolismo , Estructura Molecular , NAD/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , Espermidina/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismoRESUMEN
BACKGROUND: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. METHODS: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. RESULTS: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. CONCLUSION: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Dihidroorotato Deshidrogenasa , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.
RESUMEN
Mutations in succinate dehydrogenase B (SDHB) gene are frequently observed in several tumors and associated with poor prognosis in these tumors. Therefore, drugs effective for SDHB-deficient tumors could fulfill an unmet medical need. In addition, such drugs would have an advantage in that selection of patients with SDHB-mutant cancer could increase the probability of success in clinical trials. Currently, however, the characteristics of SDHB-deficient cancers are not completely understood. Here, we established SDHB knockout cancer cell lines from human colon cancer HCT116 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout system, and clarified its metabolic characteristics.In the SDHB knockout cells, succinate was accumulated and fumarate was decreased. The oxygen consumption rate was decreased while the extracellular acidification rate was increased in the SDHB knockout cells. Accordingly, an enhanced glycolysis pathway in the SDHB knockout cells was demonstrated by metabolomics analysis. Tracer experiments showed bidirectional metabolic flow in the tricarboxylic acid (TCA) cycle, possibly to maintain the necessary amounts of metabolites in the SDHB knockout cells. The proliferation of SDHB knockout cells was suppressed by a glycolysis inhibitor but not by a mitochondrial inhibitor. Additionally, partial dependence on glutaminolysis was observed in the SDHB knockout cells. Compound screening revealed that a bromodomain and extra-terminal (BET) inhibitor, which downregulated c-Myc, suppressed the growth of the SDHB knockout cells more potently than that of control cells. These findings provide an understanding of the metabolic characteristics of SDHB-deficient cancer and its vulnerabilities, which may lead to new therapeutic options.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Succinato Deshidrogenasa/genética , Antineoplásicos/uso terapéutico , Azepinas/farmacología , Azepinas/uso terapéutico , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Sistemas CRISPR-Cas , Ciclo del Ácido Cítrico , Deshidroepiandrosterona/farmacología , Fumaratos/metabolismo , Técnicas de Inactivación de Genes , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucólisis , Células HCT116 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Isoenzimas/antagonistas & inhibidores , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Lactato Deshidrogenasa 5 , Metabolómica , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Neoplasias/patología , Consumo de Oxígeno , Fenformina/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ácido Succínico/metabolismo , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Most cancer cells are characterized by elevated lipid biosynthesis. The rapid proliferation of cancer cells requires de novo synthesis of fatty acids. Stearoyl-CoA desaturase-1 (SCD1), a key enzyme for lipogenesis, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. Therefore, it has been studied as a candidate target for cancer therapy. In this study, we demonstrate the pharmacological properties of T-3764518, a novel and orally available small molecule inhibitor of SCD1. T-3764518 inhibited stearoyl-CoA desaturase-catalyzed conversion of stearoyl-CoA to oleoyl-CoA in colorectal cancer HCT-116 cells and their growth. Further, it slowed tumor growth in an HCT-116 and a mesothelioma MSTO-211H mouse xenograft model. Comprehensive lipidomic analyses revealed that T-3764518 increases the membrane ratio of saturated: unsaturated fatty acids in various lipid species such as phosphatidylcholines and diacylglycerols in both cultured cells and HCT-116 xenografts. Treatment-associated lipidomic changes were followed by activated endoplasmic reticulum (ER) stress responses such as increased immunoglobulin heavy chain-binding protein expression in HCT-116 cells. These T-3764518-induced changes led to an increase in cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptosis. Additionally, bovine serum albumin conjugated with oleic acid, an SCD1 product, prevented cell growth inhibition and ER stress responses by T-3764518, indicating that these outcomes were not attributable to off-target effects. These results indicate that T-3764518 is a promising new anticancer drug candidate.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Inhibidores Enzimáticos/farmacología , Oxadiazoles/farmacología , Oxadiazoles/farmacocinética , Piridazinas/farmacología , Piridazinas/farmacocinética , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Ácidos Grasos/metabolismo , Células HCT116 , Humanos , Ratones , Oxadiazoles/administración & dosificación , Oxadiazoles/metabolismo , Piridazinas/administración & dosificación , Piridazinas/metabolismo , Estearoil-CoA Desaturasa/metabolismoRESUMEN
BACKGROUND: Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. METHODS: High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and ß-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. RESULTS: We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of ß-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of ß-catenin signaling. AR-V7 signaling and ß-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular ß-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. CONCLUSIONS: Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos/metabolismo , beta Catenina/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Masculino , Próstata , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/terapia , Transducción de Señal/efectos de los fármacosRESUMEN
Metabolic alteration constitutes a hallmark of cancer. Glycolysis and antioxidant pathways in kidney cancer are elevated, with frequent mutation of the VHL gene. Intratumor genetic heterogeneity has been recently demonstrated in kidney cancer. However, intratumor metabolic heterogeneity has not been investigated. Here, we used global metabolomics analysis and tissue slice tracer studies to demonstrate that different portions of a human primary kidney tumor possess different metabolic characteristics and drug sensitivity. Pyruvate levels were elevated and pyruvate metabolism was altered in some tumor sections. These observations indicated that pyruvate metabolism may constitute a possible vulnerability of kidney cancer; indeed, pyruvate stimulated the growth of primary kidney cancer cells and pharmacological inhibition of pyruvate transporters slowed the growth of patient-derived kidney tumors in mice. These findings deepen our understanding of the intratumor metabolic heterogeneity of kidney cancer and may inform novel therapeutic approaches in human kidney cancer.
Asunto(s)
Neoplasias Renales/metabolismo , Ácido Pirúvico/metabolismo , Acrilatos/farmacología , Animales , Antineoplásicos/uso terapéutico , Células Cultivadas , Femenino , Glucólisis , Humanos , Neoplasias Renales/tratamiento farmacológico , Metabolómica , Ratones , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vacuolar (H+ )-ATPases (V-ATPases) have important roles in the supply of nutrients to tumors by mediating autophagy and the endocytic uptake of extracellular fluids. Accordingly, V-ATPases are attractive therapeutic targets for cancer. However, the clinical use of V-ATPase inhibitors as anticancer drugs has not been realized, possibly owing to their high toxicity in humans. Inhibition of V-ATPase may be an appropriate strategy in highly susceptible cancers. In this study, we explored markers of V-ATPase inhibitor sensitivity. V-ATPase inhibitors led to pH impairment in acidic intracellular compartments, suppression of macropinocytosis, and decreased intracellular amino acid levels. The sensitivity of cells to V-ATPase inhibitors was correlated with low cathepsin D expression, and cancer cells showed increased sensitivity to V-ATPase inhibitors after pretreatment with a cathepsin D inhibitor and siRNA targeting the cathepsin D gene (CTSD). In addition, V-ATPase inhibitor treatment led to the induction of the amino acid starvation response, upregulation of endoplasmic reticulum stress markers, and suppression of mammalian target of rapamycin (mTOR) signaling in cells expressing low levels of cathepsin D. Some colorectal cancer patients showed the downregulation of cathepsin D in tumor tissues compared with matched normal tissues. These findings indicate that V-ATPase inhibitors are promising therapeutic options for cancers with downregulated cathepsin D.
Asunto(s)
Catepsina D/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Autofagia/genética , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Células HCT116 , Humanos , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.