RESUMEN
Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ â¼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
Asunto(s)
Descubrimiento de Drogas , Glutamato Deshidrogenasa/ultraestructura , Isocitrato Deshidrogenasa/ultraestructura , L-Lactato Deshidrogenasa/ultraestructura , Aminoquinolinas/química , Aminoquinolinas/farmacología , Animales , Bovinos , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/química , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Modelos Moleculares , Conformación Proteica , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.
Asunto(s)
Endosomas , Fosfohidrolasa PTEN , Fosfatidilinositoles , Vacuolas , Vacuolas/metabolismo , Vacuolas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/efectos de los fármacos , Humanos , Fosfatidilinositoles/metabolismo , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Ratones , Morfolinas/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , Citoplasma/metabolismo , Células HeLa , Aminopiridinas , Compuestos Heterocíclicos con 3 AnillosRESUMEN
BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.
Asunto(s)
Trombocitopenia , Humanos , Ratones , Animales , Trombocitopenia/inducido químicamente , Heparina/efectos adversos , Plaquetas , Anticoagulantes/efectos adversos , Ratones Transgénicos , Fenotipo , Fibrina/genética , Factor Plaquetario 4/genéticaRESUMEN
Increased phosphoinositide signaling is commonly associated with cancers. While "one-drug one-target" has been a major drug discovery strategy for cancer therapy, a "one-drug multi-targets" approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIß) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kß, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3 h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3,5)P2, and PI(3,4,5)P3 levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways.
RESUMEN
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Asunto(s)
Neoplasias Colorrectales/patología , Glucosa/metabolismo , Glutamina/metabolismo , Complejos Multiproteicos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Glucólisis , Células HT29 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Trasplante de NeoplasiasRESUMEN
Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.
Asunto(s)
Clobetasol/farmacología , Miconazol/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Células Madre Pluripotentes/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Cerebelo/patología , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Estratos Germinativos/efectos de los fármacos , Estratos Germinativos/metabolismo , Estratos Germinativos/patología , Humanos , Lisofosfatidilcolinas , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Esclerosis Múltiple/patología , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Fenotipo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores de Glucocorticoides/metabolismo , Regeneración/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
Classic Galactosemia is a potentially lethal autosomal recessive metabolic disorder caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-p) in cells. Galactokinase (GALK1) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK1 is hypothesized to be therapeutic strategy for treating galactosemia by reducing production of gal-1-p. In this study, we report the discovery of novel series of GALK1 inhibitors by structure-based virtual screening (VS). Followed by an extensive structural modeling and binding mode analysis of the active compounds identified from quantitative high-throughput screen (qHTS), we developed an efficient pharmacophore-based VS approach and applied for a large-scale in silico database screening. Out of 230,000 compounds virtually screened, 350 compounds were cherry-picked based on multi-factor prioritization procedure, and 75 representing a diversity of chemotypes exhibited inhibitory activity in GALK1 biochemical assay. Furthermore, a phenylsulfonamide series with excellent in vitro ADME properties was selected for downstream characterization and demonstrated its ability to lower gal-1-p in primary patient fibroblasts. The compounds described herein should provide a starting point for further development of drug candidates for the GALK1 modulation in the Classic Galactosemia.
Asunto(s)
Galactoquinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Dominio Catalítico/efectos de los fármacos , Diseño de Fármacos , Descubrimiento de Drogas , Galactoquinasa/química , Galactoquinasa/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.
Asunto(s)
Caspasa 1/genética , Inflamasomas/metabolismo , Cuerpos de Lewy/metabolismo , Neuronas/metabolismo , Agregado de Proteínas/genética , alfa-Sinucleína/genética , Compuestos de Alumbre/farmacología , Caspasa 1/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dipéptidos/farmacología , Regulación de la Expresión Génica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Cuerpos de Lewy/efectos de los fármacos , Cuerpos de Lewy/patología , Lipopolisacáridos/farmacología , Neuronas/efectos de los fármacos , Neuronas/patología , Nigericina/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Vitamina K 3/farmacología , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , para-Aminobenzoatos/farmacologíaRESUMEN
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
Asunto(s)
Carbono/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/biosíntesis , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Carbono/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Femenino , Glucólisis/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/metabolismo , Purinas/biosíntesis , Serina/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Timidina/biosíntesis , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016).
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Piperazinas/farmacología , Tiourea/análogos & derivados , Tiourea/farmacología , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Piperazinas/síntesis química , Piperazinas/química , Relación Estructura-Actividad , Tiourea/síntesis química , Tiourea/químicaRESUMEN
Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Ciclina D1/metabolismo , Endopeptidasas/metabolismo , Linfoma de Células del Manto/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Ciclina D1/genética , Daño del ADN , Reparación del ADN , Endopeptidasas/genética , Humanos , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/metabolismo , Proteínas de Neoplasias/genética , Inhibidores de Proteasas/química , Ubiquitina TiolesterasaRESUMEN
The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/inmunología , Linfoma de Células B Grandes Difuso/patología , Pirazoles/farmacología , Pirimidinas/farmacología , Adenina/análogos & derivados , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , PiperidinasRESUMEN
Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as â¼1 µM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.
Asunto(s)
Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Hexoquinasa/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/biosíntesis , Antimaláricos/química , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Expresión Génica , Genes Reporteros , Glucólisis/efectos de los fármacos , Células HEK293 , Células HeLa , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Relación Señal-Ruido , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 µm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 µm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.
Asunto(s)
Acetamidas/farmacología , Bencimidazoles/farmacología , Fenómenos Biofísicos , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Mutación , Acetamidas/metabolismo , Acetamidas/farmacocinética , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacocinética , Línea Celular Tumoral , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.
Asunto(s)
Galactoquinasa/antagonistas & inhibidores , Animales , Benzoxazoles/síntesis química , Benzoxazoles/química , Benzoxazoles/metabolismo , Cristalografía por Rayos X , Galactoquinasa/genética , Galactoquinasa/metabolismo , Galactosafosfatos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Ratones , Microsomas Hepáticos/metabolismo , Conformación Molecular , Unión Proteica , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.
Asunto(s)
Biopolímeros/metabolismo , Transformación Celular Neoplásica , Activadores de Enzimas/farmacología , Piruvato Quinasa/metabolismo , Animales , Biopolímeros/química , Western Blotting , Proliferación Celular , Humanos , Ratones , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Piruvato Quinasa/químicaRESUMEN
Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing ß cells and involves an interplay between ß cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in ß cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our findings demonstrate that VLX-1005, a potent 12-LOX inhibitor, effectively delays the onset of autoimmune diabetes in human gene replacement non-obese diabetic (NOD) mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecules PD-L1 and PD-1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets from inhibitor-treated mice revealed significant alteration of cytokine-responsive pathways. RNA sequencing of polarized proinflammatory macrophages showed that VLX-1005 significantly reduced the interferon response. Our studies demonstrate that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.
RESUMEN
Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC(1280))) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.
Asunto(s)
Apomorfina/química , Azoles/química , Benzofenantridinas/química , Glutaminasa/antagonistas & inhibidores , Compuestos de Organoselenio/química , Complejo SIDA Demencia/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Glutaminasa/química , Glutaminasa/metabolismo , Humanos , Concentración 50 Inhibidora , Isoindoles , Neoplasias/tratamiento farmacológico , ARN Interferente Pequeño/metabolismo , Sensibilidad y EspecificidadRESUMEN
The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a-c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region.