RESUMEN
The oncogenic role of KRAS in colorectal cancer (CRC) progression is well-established. Despite this, identifying effective therapeutic targets for KRAS-mutated CRC remains a significant challenge. This study identifies pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) as a previously unrecognized yet crucial regulator in the progression of KRAS mutant CRC. A substantial upregulation of PDP1 expression is observed in KRAS mutant CRC cells and tissues compared to wild-type KRAS samples, which correlates with poorer prognosis. Functional experiments elucidate that PDP1 accelerates the malignance of KRAS mutant CRC cells, both in vitro and in vivo. Mechanistically, PDP1 acts as a scaffold, enhancing BRAF and MEK1 interaction and activating the MAPK signaling, thereby promoting CRC progression. Additionally, transcription factor KLF5 is identified as the key regulator for PDP1 upregulation in KRAS mutant CRC. Crucially, targeting PDP1 combined with MAPK inhibitors exhibits an obvious inhibitory effect on KRAS mutant CRC. Overall, PDP1 is underscored as a vital oncogenic driver and promising therapeutic target for KRAS mutant CRC.
Asunto(s)
Neoplasias Colorrectales , Progresión de la Enfermedad , MAP Quinasa Quinasa 1 , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Animales , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Ratones , Línea Celular Tumoral , Mutación , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Femenino , Proliferación Celular , Ratones Desnudos , Masculino , Regulación hacia ArribaRESUMEN
Polypharmacology drugs-compounds that inhibit multiple proteins-have many applications but are difficult to design. To address this challenge we have developed POLYGON, an approach to polypharmacology based on generative reinforcement learning. POLYGON embeds chemical space and iteratively samples it to generate new molecular structures; these are rewarded by the predicted ability to inhibit each of two protein targets and by drug-likeness and ease-of-synthesis. In binding data for >100,000 compounds, POLYGON correctly recognizes polypharmacology interactions with 82.5% accuracy. We subsequently generate de-novo compounds targeting ten pairs of proteins with documented co-dependency. Docking analysis indicates that top structures bind their two targets with low free energies and similar 3D orientations to canonical single-protein inhibitors. We synthesize 32 compounds targeting MEK1 and mTOR, with most yielding >50% reduction in each protein activity and in cell viability when dosed at 1-10 µM. These results support the potential of generative modeling for polypharmacology.
Asunto(s)
Simulación del Acoplamiento Molecular , Humanos , Serina-Treonina Quinasas TOR/metabolismo , Polifarmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 1/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Unión Proteica , Descubrimiento de Drogas/métodos , Diseño de Fármacos , Supervivencia Celular/efectos de los fármacosAsunto(s)
Melanoma , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Desprendimiento de Retina , Humanos , Desprendimiento de Retina/diagnóstico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Masculino , Inhibidores de Proteínas Quinasas/efectos adversos , Femenino , Persona de Mediana Edad , Melanoma/tratamiento farmacológico , Ritmo Circadiano/fisiología , Anciano , Tomografía de Coherencia Óptica/métodos , MAP Quinasa Quinasa 1/antagonistas & inhibidoresRESUMEN
Melanomas frequently harbor activating NRAS mutations. However, limited advance has been made in developing targeted therapy options for patients with NRAS mutant melanoma. MEK inhibitors (MEKi) show modest efficacy in the clinic and their actions need to be optimized. In this study, we performed a genome-wide CRISPR-Cas9-based screen and demonstrated that loss of phosphoinositide-dependent kinase-1 (PDPK1) enhances the efficacy of MEKi. The synergistic effects of PDPK1 loss and MEKi was validated in NRAS mutant melanoma cell lines using pharmacologic and molecular approaches. Combined PDPK1 inhibitors (PDPK1i) with MEKi suppressed NRAS mutant xenograft growth and induced gasdermin E-associated pyroptosis. In an immune-competent allograft model, PDPK1i+MEKi increased the ratio of intratumoral CD8+ T cells, delayed tumor growth, and prolonged survival; the combination treatment was less effective against tumors in immune-deficient mice. These data suggest PDPK1i+MEKi as an efficient immunostimulatory strategy against NRAS mutant melanoma. SIGNIFICANCE: Targeting PDPK1 stimulates antitumor immunity and sensitizes NRAS mutant melanoma to MEK inhibition, providing rationale for the clinical development of a combinatorial approach for treating patients with melanoma.
Asunto(s)
GTP Fosfohidrolasas , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Melanoma , 1-Fosfatidilinositol 4-Quinasa/genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Línea Celular Tumoral , GTP Fosfohidrolasas/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
BACKGROUND: Alcohol is mainly catabolized by class I alcohol dehydrogenase (ADH1) in liver. ADH deficiency can aggravate ethanol-induced tissue injury. Extracellular signal-regulated kinases 1/2 (ERK1/2) is involved in alcohol metabolism. However, the relationship between ERK1/2 and ADH1 remains unclear. METHODS AND RESULTS: To inhibit ERK1/2, HepG2 and BNL cells were treated with mitogen-activated protein kinases 1/2 (MEK1/2) inhibitors (U0126 and PD98059), and C57BL/6J mice were fed U0126. After treatment, the protein and mRNA expression of ADH1 were determined by Western blot and quantitative real time-PCR. The activity of ADH1 promoter was detected using luciferase assay. The results showed MEK1/2 inhibitors significantly increased ADH1 protein expression by inducing its transcription activity. Then we demonstrated a farnesoid X receptor (FXR) response element (FXRE) in ADH1 promoter by ChIP assay. To test whether FXR mediates the induction of MEK1/2 inhibitors on ADH1, HepG2 cells were transfected with FXR siRNA or ADH1 promoters with FXRE mutation. We found both FXR siRNA and FXRE mutation in ADH1 promoter abolished MEK1/2 inhibitors-induced ADH1 expression, indicating the activation of MEK1/2 inhibitors on ADH1 depends on FXR. CONCLUSIONS: Our findings revealed inhibition of ERK1/2 can significantly increase ADH1 expression, indicating MEK1/2 inhibitors may possess potential application in alcohol-related diseases.
Asunto(s)
Alcohol Deshidrogenasa , Hepatocitos , Inhibidores de Proteínas Quinasas/farmacología , Alcohol Deshidrogenasa/genética , Animales , Hepatocitos/fisiología , Hígado , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , ARN Interferente PequeñoRESUMEN
The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.
Asunto(s)
Clorofila/análogos & derivados , Células Endoteliales/efectos de los fármacos , Eupatorium , Linfangiogénesis/efectos de los fármacos , Extractos Vegetales/farmacología , Butadienos/farmacología , Línea Celular , Clorofila/farmacología , Humanos , Vasos Linfáticos/efectos de los fármacos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Nitrilos/farmacologíaRESUMEN
Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Fenamatos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , SARS-CoV-2/efectos de los fármacos , Células A549 , Adulto , Animales , COVID-19/metabolismo , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Citocinas/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/metabolismo , SARS-CoV-2/fisiología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i-sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer-related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i-mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor-mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN-amplified neuroblastomas.
Asunto(s)
Apoptosis/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Proteína Proto-Oncogénica N-Myc/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Amplificación de Genes , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Pronóstico , Piridonas/farmacología , Pirimidinonas/farmacología , ARN Mensajero/genéticaRESUMEN
BACKGROUND AND AIMS: Patients with pancreatic ductal adenocarcinoma (PDA) have not yet benefitted from the revolution in cancer immunotherapy due in large part to a dominantly immunosuppressive tumor microenvironment. MEK inhibition combined with autophagy inhibition leads to transient tumor responses in some patients with PDA. We examined the functional effects of combined MEK and autophagy inhibition on the PDA immune microenvironment and the synergy of combined inhibition of MEK and autophagy with CD40 agonism (aCD40) against PDA using immunocompetent model systems. METHODS: We implanted immunologically "cold" murine PDA cells orthotopically in wide type C57BL/6J mice. We administered combinations of inhibitors of MEK1/2, inhibitors of autophagy, and aCD40 and measured anticancer efficacy and immune sequelae using mass cytometry and multiplexed immunofluorescence imaging analysis to characterize the tumor microenvironment. We also used human and mouse PDA cell lines and human macrophages in vitro to perform functional assays to elucidate the cellular effects induced by the treatments. RESULTS: We find that coinhibition of MEK (using cobimetinib) and autophagy (using mefloquine), but not either treatment alone, activates the STING/type I interferon pathway in tumor cells that in turn activates paracrine tumor associated macrophages toward an immunogenic M1-like phenotype. This switch is further augmented by aCD40. Triple therapy (cobimetinib + mefloquine + aCD40) achieved cytotoxic T-cell activation in an immunologically "cold" mouse PDA model, leading to enhanced antitumor immunity. CONCLUSIONS: MEK and autophagy coinhibition coupled with aCD40 invokes immune repolarization and is an attractive therapeutic approach for PDA immunotherapy development.
Asunto(s)
Autofagia/inmunología , Azetidinas/farmacología , Antígenos CD40/agonistas , Carcinoma Ductal Pancreático/inmunología , Mefloquina/farmacología , Neoplasias Pancreáticas/inmunología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunología , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Hidroxicloroquina/farmacología , Inmunoterapia , Interferón Tipo I/efectos de los fármacos , Interferón Tipo I/inmunología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Macrófagos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/inmunología , Ratones , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/inmunología , Escape del Tumor , Microambiente Tumoral/efectos de los fármacos , Macrófagos Asociados a Tumores/efectos de los fármacosRESUMEN
Crocetin is a main bioactive component with a carotenoid skeleton in Gardenia jasminoides, a typical traditional Chinese medicine with a long history in Southeast Asia. Crocetin is being commonly consumed as spices, dyes, and food colorants. Recent pharmacological studies had implied that crocetin may possess potent anti-inflammatory properties; however, the underlying molecular mechanism is not fully elucidated. In the present study, the regulatory effect of crocetin on redox balance was systematically investigated in lipopolysaccharide- (LPS-) stimulated RAW264.7 cells. The results showed that crocetin dose-dependently inhibited LPS-induced nitric oxide production and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells. Molecular data revealed that crocetin exerted its anti-inflammatory property by inhibiting the MEK1/JNK/NF-κB/iNOS pathway and activating the Nrf2/HO-1 pathway. The shRNA-knockdown (KD) of MEK1 and ERK1 confirmed that the activation of MEK1 and inhibition of JNK mediated the anti-inflammatory effect of crocetin. Moreover, the pull-down assay and computational molecule docking showed that crocetin could directly bind to MEK1 and JNK1/2. It is noticed that both KD and knockout (KO) of HO-1 gene blocked this action. More detailed data have shown that HO-1-KO blocked the inhibition of p-IκB-α by crocetin. These data indicated that crocetin exerted its anti-inflammatory property via modulating the crosstalk between the MEK1/JNK/NF-κB/iNOS pathway and the Nrf2/HO-1 pathway, highlighting HO-1 as a major player. Therefore, the present study reveals that crocetin can act as a potential candidate for redox-balancing modulation in charge of its anti-inflammatory and chemopreventive effect, which strengthens its potency in the subsequent clinic application in the near future.
Asunto(s)
Antiinflamatorios/farmacología , Carotenoides/farmacología , Transducción de Señal/efectos de los fármacos , Vitamina A/análogos & derivados , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Sitios de Unión , Carotenoides/química , Carotenoides/metabolismo , Hemo-Oxigenasa 1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Factor 2 Relacionado con NF-E2/metabolismo , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Células RAW 264.7 , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Vitamina A/química , Vitamina A/metabolismo , Vitamina A/farmacologíaRESUMEN
Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/análisis , Bencimidazoles/farmacología , Antígenos de Histocompatibilidad Clase I/inmunología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/inmunología , Presentación de Antígeno/efectos de los fármacos , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Células Tumorales CultivadasAsunto(s)
MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Síndrome de Noonan , Mutación Puntual , Proteínas Proto-Oncogénicas c-raf/genética , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Taquicardia , Femenino , Humanos , Recién Nacido , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/genética , Síndrome de Noonan/tratamiento farmacológico , Síndrome de Noonan/enzimología , Síndrome de Noonan/genética , Taquicardia/tratamiento farmacológico , Taquicardia/enzimología , Taquicardia/genéticaRESUMEN
There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF-V600E-mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo, we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor-resistant patient-derived xenograft models. In vitro, we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor-resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor-resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Aminopiridinas/administración & dosificación , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Piperazinas/administración & dosificación , Piridinas/administración & dosificación , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Pirroles/administración & dosificación , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The variety and complexity of drug targets are expanding rapidly. At the same time, there is significant interest in exploring a larger chemical space to identify new candidates. Fragment-based screening (FBS) has emerged as a popular alternative to traditional high-throughput screening campaigns to identify such drug candidates. FBS identifies hit fragments that exhibit weak interactions with the target of interest, thereby enabling the rational design of small-molecule compounds from the identified hit fragments, which serve as building blocks. This strategy reduces the number of molecules to screen while also allowing the exploration of a greater chemical space.Here we use temperature-related intensity change (TRIC) technology to perform FBS against the target MAPK/ERK kinase-1 (Mek1). TRIC describes the change in fluorescence intensity of a fluorescently labeled molecule upon a change in temperature. This intensity variation is dependent on the physicochemical environment in the vicinity of the dye and strongly affected by binding events. Thus, the detection of binding events is independent of mass, making TRIC an ideal tool for FBS.Using only 150 pmol of labeled Mek1, the authors screened 193 fragments from a prescreened library in less than 1 h of measurement time, leading to 66 hits. Among those hits, they identified more than 80% of the published top hits found using orthogonal techniques. Furthermore, TRIC allowed the identification of fragments that were of poor solubility but could be mistaken as false-positive hits in other methods.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Colorantes Fluorescentes , Humanos , TemperaturaAsunto(s)
Azetidinas/efectos adversos , Angiografía con Fluoresceína/métodos , Piperidinas/efectos adversos , Enfermedades de la Retina/diagnóstico , Tomografía de Coherencia Óptica/métodos , Vemurafenib/efectos adversos , Azetidinas/uso terapéutico , Craneofaringioma/tratamiento farmacológico , Femenino , Fondo de Ojo , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Persona de Mediana Edad , Piperidinas/uso terapéutico , Neoplasias Hipofisarias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , Enfermedades de la Retina/inducido químicamente , Vemurafenib/uso terapéuticoRESUMEN
Colorectal cancer, one of the most commonly diagnosed cancers worldwide, is often accompanied by uncontrolled proliferation of tumor cells. Dyskerin pseudouridine synthase 1 (DKC1), screened using the genome-wide RNAi strategy, is a previously unidentified key regulator that promotes colorectal cancer cell proliferation. Enforced expression of DKC1, but not its catalytically inactive mutant D125A, accelerates cell growth in vitro and in vivo. DKC1 knockdown or its inhibitor pyrazofurin attenuates cell proliferation. Proteomics, RNA immunoprecipitation (RIP)-seq, and RNA decay analyses reveal that DKC1 binds to and stabilizes the mRNA of several ribosomal proteins (RPs), including RPL10A, RPL22L1, RPL34, and RPS3. DKC1 depletion significantly accelerates mRNA decay of these RPs, which mediates the oncogenic function of DKC1. Interestingly, these DKC1-regulated RPs also interact with HRAS and suppress the RAS/RAF/MEK/ERK pathway. Pyrazofurin and trametinib combination synergistically restrains colorectal cancer cell growth in vitro and in vivo. Furthermore, DKC1 is markedly upregulated in colorectal cancer tissues compared to adjacent normal tissues. Colorectal cancer patients with higher DKC1 expression has consistently poorer overall survival and progression-free survival outcomes. Taken together, these data suggest that DKC1 is an essential gene and candidate therapeutic target for colorectal cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias Colorrectales/tratamiento farmacológico , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Amidas/administración & dosificación , Amidas/farmacología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Sinergismo Farmacológico , Femenino , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/administración & dosificación , Pirazoles/farmacología , Piridonas/administración & dosificación , Piridonas/farmacología , Pirimidinonas/administración & dosificación , Pirimidinonas/farmacología , Ribosa/administración & dosificación , Ribosa/farmacología , Proteínas Ribosómicas/metabolismo , Tasa de Supervivencia , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are located at the meeting-point of ERK and p38 MAPK signaling pathways, which can phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at the conserved serine 209 exclusively. MNKs modulate the translation of mRNA involved in tumor-associated signaling pathways. Consequently, selective inhibitors of MNK1/2 could reduce the level of phosphorylated eIF4E. Series of imidazopyrazines, imidazopyridazines and imidazopyridines derivatives were synthesized and evaluated as MNK1/2 inhibitors. Several compounds exhibited great inhibitory activity against MNK1/2 and selected compounds showed moderate to excellent anti-proliferative potency against diffuse large B-cell lymphoma (DLBCL) cell lines. In particular, compound II-5 (MNK1 IC50 = 2.3 nM; MNK2 IC50 = 3.4 nM) exhibited excellent enzymatic inhibitory potency and proved to be the most potent compound against TMD-8 and DOHH-2 cell lines with IC50 value of 0.3896 µM and 0.4092 µM respectively. These results demonstrated that compound II-5 could be considered as a potential MNK1/2 inhibitor for further investigation.
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Diseño de Fármacos , Imidazoles/farmacología , Isoquinolinas/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Isoquinolinas/química , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridazinas/síntesis química , Piridazinas/química , Relación Estructura-ActividadRESUMEN
Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer's disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.
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Membrana Celular/metabolismo , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/genética , Glicoproteínas de Membrana/genética , Microglía/metabolismo , Receptores Inmunológicos/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Bencimidazoles/farmacología , Benzotiazoles/farmacología , Membrana Celular/efectos de los fármacos , Colchicina/farmacología , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Interferón gamma/farmacología , Interleucinas/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglía/citología , Microglía/efectos de los fármacos , Nitrilos/farmacología , Cultivo Primario de Células , Piridonas/farmacología , Pirimidinonas/farmacología , Quinazolinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Células THP-1 , Factor de Crecimiento Transformador beta/farmacología , Zearalenona/análogos & derivados , Zearalenona/farmacologíaRESUMEN
Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.
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Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Neoplasias de la Mama Triple Negativas/metabolismo , Femenino , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 5/antagonistas & inhibidores , MAP Quinasa Quinasa 5/genética , Células MCF-7 , Proteínas Proto-Oncogénicas c-fos/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK's catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting.