RESUMEN
BACKGROUND AND OBJECTIVES: Smoothened (SMO), a key component of the hedgehog signaling pathway, represents a therapeutic target for triple negative breast cancer (TNBC), yet the chemotherapy response rate in TNBC patients is only 40-50%, underscoring the urgent need for the development of novel drugs to effectively treat this condition. The novel compound TPB15, an SMO inhibitor derived from [1,2,4] triazolo [4,3-α] pyridines, demonstrated superior anti-TNBC activity and lower toxicity compared to the first SMO inhibitor vismodegib in both in vitro and in vivo. However, the compound's pharmacokinetic properties remain unclear. The present work aims to develop a simple HPLC-MS/MS method to profile the pharmacokinetics and bioavailability of TPB15 in rats as a ground work for further clinical research. METHODS: Separation was performed on an Agilent ZORBAX StableBond C18 column by gradient elution using acetonitrile and 0.1% formic acid as mobile phase at a flow rate of 0.3 mL/min. Multiple reaction monitoring(MRM) in positive mode with the transitions of m/z 454.2 â 100.0, 248.1 â 121.1 was employed to determine TPB15 and internal standard tinidazole, respectively. The specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover of the method was validated. The pharmacokinetics and bioavailability study of TPB15 were carried out on rats through intravenous injection at the dose of 5 mg/kg and oral gavage at the dose of 25 mg/kg, and the pharmacokinetics parameters were calculated by the non-compartment analysis using the pharmacokinetics software DAS 2.1.1. RESULTS: The values of specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover satisfied the acceptable limits. The lower limit of quantification of this method was 10 ng/mL with a linear range of 10-2000 ng/mL. The validated method was then applied to pharmacokinetics and bioavailability studies in rat by dosing with gavage (25 mg/kg) and intravenous injection(5 mg/kg), and the oral bioavailability of TBP15 in rat was calculated as 16.4 ± 3.5%. The pharmacokinetic parameters were calculated as following: maximum of plasma concentration (Cmax) (PO: 2787.17 ± 279.45 µg/L), Time to maximum plasma concentration (Tmax) (PO: 4.20 ± 0.90 h), the area under the concentration-time curve 0 to time (AUC0-t) (PO: 17,373.03 ± 2585.18 ng/mL·h, IV: 21,129.79 ± 3360.84 ng/mL·h), the area under the concentration-time curve 0 to infinity (AUC0-∞) (PO: 17,443.85 ± 2597.63 ng/mL·h, IV: 17,443.85 ± 2597.63 ng/mL·h), terminal elimination half-life (t1/2) (PO: 7.26 ± 2.16 h, IV: 4.78 ± 1.09 h). CONCLUSIONS: TPB15, a promising candidate for treating TNBC, has demonstrated outstanding efficacy and safety in vitro and in vivo. This study established a simple, sensitive, and rapid HPLC-MS/MS bioanalytical method, developed and validated in accordance with FDA and EMA guidelines, for conducting pharmacokinetic and bioavailability studies of TPB15. The results revealed a favorable pharmacokinetic profile owing to its long t1/2. Nevertheless, the next phase of research should include formulation screening to enhance bioavailability, as well as clinical trials, metabolism pathway analysis, and assessment of potential drug-drug interactions.
Asunto(s)
Disponibilidad Biológica , Piridinas , Ratas Sprague-Dawley , Receptor Smoothened , Espectrometría de Masas en Tándem , Neoplasias de la Mama Triple Negativas , Animales , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ratas , Femenino , Cromatografía Líquida de Alta Presión/métodos , Receptor Smoothened/antagonistas & inhibidores , Espectrometría de Masas en Tándem/métodos , Piridinas/farmacocinética , Piridinas/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/administración & dosificación , Administración Oral , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.
Asunto(s)
Antineoplásicos , Neoplasias Pancreáticas , Purinas , Receptor Smoothened , Humanos , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Purinas/química , Purinas/farmacología , Purinas/síntesis química , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Ligandos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Animales , Ratones , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Línea Celular Tumoral , Células 3T3 NIH , Simulación del Acoplamiento Molecular , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidoresRESUMEN
In this issue of Cell Chemical Biology, Liu et al.1 report the identification of Q29, a synthetic diterpenoid that blocks covalent cholesterol modification of smoothened (SMO) and inhibits hedgehog signaling. Q29 is capable of suppressing tumor cell growth, both in vitro and in vivo, and overcoming resistance to SMO inhibitors.
Asunto(s)
Colesterol , Diterpenos , Resistencia a Antineoplásicos , Proteínas Hedgehog , Receptor Smoothened , Humanos , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Colesterol/metabolismo , Colesterol/química , Diterpenos/farmacología , Diterpenos/química , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Animales , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/química , Transducción de Señal/efectos de los fármacosRESUMEN
Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina , Resistencia a Antineoplásicos , Proteínas Hedgehog , Transducción de Señal , Receptor Smoothened , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/antagonistas & inhibidores , Animales , Humanos , Ratones , Resistencia a Antineoplásicos/genética , Transducción de Señal/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Elementos de Facilitación Genéticos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Sistemas CRISPR-CasRESUMEN
The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.
Asunto(s)
Proliferación Celular , Proteínas Hedgehog , Meduloblastoma , Transducción de Señal , Receptor Smoothened , Esteroles , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Transducción de Señal/efectos de los fármacos , Animales , Ratones , Proliferación Celular/efectos de los fármacos , Esteroles/química , Esteroles/farmacología , Esteroles/metabolismo , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/metabolismo , Meduloblastoma/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Estructura Molecular , Colesterol/metabolismoRESUMEN
Although basal cell carcinomas arise from ectopic Hedgehog pathway activation and can be treated with pathway inhibitors, sporadic basal cell carcinomas display high resistance rates, whereas tumors arising in patients with Gorlin syndrome with germline Patched (PTCH1) alterations are uniformly suppressed by inhibitor therapy. In rare cases, patients with Gorlin syndrome on long-term inhibitor therapy will develop individual resistant tumor clones that rapidly progress, but the basis of this resistance remains unstudied. In this study, we report a case of an SMO inhibitor-resistant tumor arising in a patient with Gorlin syndrome on suppressive SMO inhibitor for nearly a decade. Using a combination of multiomics and spatial transcriptomics, we define the tumor populations at the cellular and tissue level to conclude that Gorlin tumors can develop resistance to SMO inhibitors through the previously described basal to squamous cell carcinoma transition. Intriguingly, through spatial whole-exome genomic analysis, we nominate PCYT2, ETNK1, and the phosphatidylethanolamine biosynthetic pathway as genetic suppressors of basal to squamous cell carcinoma transition resistance. These observations provide a general framework for studying tumor evolution and provide important clinical insight into mechanisms of resistance to SMO inhibitors for not only Gorlin syndrome but also sporadic basal cell carcinomas.
Asunto(s)
Síndrome del Nevo Basocelular , Carcinoma Basocelular , Carcinoma de Células Escamosas , Resistencia a Antineoplásicos , Neoplasias Cutáneas , Receptor Smoothened , Humanos , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/patología , Síndrome del Nevo Basocelular/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Receptor Smoothened/genética , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma Basocelular/genética , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/patología , Masculino , Anilidas/uso terapéutico , Femenino , Transducción de Señal/efectos de los fármacos , Piridinas/uso terapéuticoRESUMEN
Epilepsy is one of the common encephalopathies caused by sudden abnormal discharges of neurons in the brain. About 30% of patients with epilepsy are insensitive and refractory to existing antiseizure medications. The sonic hedgehog signaling pathway is essential to the development and homeostasis of brain. Aberrant sonic hedgehog signaling is increased in refractory epileptic lesions and may involve the etiology of epilepsy. Thus, new inhibitors of Smoothened, a key signal transducer of this signaling pathway are urgently need for refractory epilepsy. We have established a high-throughput screening platform and discovered several active small molecules targeting Smoothened including TT22. Here we show that the novel Smoothened inhibitor TT22 could block the translocation of ßarrestin2-GFP to Smoothened, reduce the accumulation of Smoothened on primary cilia, displace Bodipy-cyclopamine binding to Smoothened, and inhibit the expression of downstream Gli transcription factor. Moreover, TT22 inhibits the abnormal seizure-like activity in neurons. Furthermore, we demonstrated that FDA-approved Smoothened inhibitor GDC-0449 and LDE-225 are able to inhibit abnormal seizure-like activity in neurons. Thus, our study suggests that targeting the sonic hedgehog signaling with new small-molecule Smoothened inhibitors might provide a potential new therapeutic avenue for refractory epilepsy.
Asunto(s)
Epilepsia Refractaria , Proteínas Hedgehog , Receptor Smoothened , Humanos , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Convulsiones , Transducción de Señal/fisiología , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismoRESUMEN
Basal cell carcinoma (BCC) is one of the most common neoplasms in the population. A good prognosis and mainly non-aggressive development have made it underdiagnosed and excluded from the statistics. Due to the availability of efficient surgical therapy, BCC is sometimes overlooked in the search for novel therapies. Most clinicians are unaware of its complicated pathogenesis or the availability of effective targeted therapy based on Hedgehog inhibitors (HHI) used in advanced or metastatic cases. Nevertheless, the concomitance and esthetic burden of this neoplasm are severe. As with other cancers, its pathogenesis is multifactorial and complicated with a network of dependencies. Although the tumour microenvironment (TME), genetic aberrations, and risk factors seem crucial in all skin cancers, in BCC they all have become accessible as therapeutic or prevention targets. The results of this review indicate that a central role in the development of BCC is played by the Hedgehog (Hh) signalling pathway. Two signalling molecules have been identified as the main culprits, namely Patched homologue 1 (PTCH1) and, less often, Smoothened homologue (SMO). Considering effective immunotherapy for other neoplastic growths being introduced, implementing immunotherapy in advanced BCC is pivotal and beneficial. Up to now, the US Food and Drug Administration (FDA) has approved two inhibitors of SMO for the treatment of advanced BCC. Sonidegib and vismodegib are registered based on their efficacy in clinical trials. However, despite this success, limitations might occur during the therapy, as some patients show resistance to these molecules. This review aims to summarize novel options of targeted therapies in BCC and debate the mechanisms and clinical implications of tumor resistance.
Asunto(s)
Antineoplásicos , Carcinoma Basocelular , Proteínas Hedgehog , Neoplasias Cutáneas , Receptor Smoothened , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Microambiente Tumoral , Estados Unidos , Receptor Patched-1/metabolismo , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismoRESUMEN
Allergic asthma is a common inflammatory airway disease in which Th2 immune response and inflammation are thought to be triggered by inhalation of environmental allergens. Many studies using mouse models and human tissues and genome-wide association have indicated that Sonic Hedgehog (Shh) and the Hedgehog (Hh) signaling pathway are involved in allergic asthma and that Shh is upregulated in the lung on disease induction. We used a papain-induced mouse model of allergic airway inflammation to investigate the impact of systemic pharmacological inhibition of the Hh signal transduction molecule smoothened on allergic airway disease induction and severity. Smoothened-inhibitor treatment reduced the induction of Shh, IL-4, and IL-13 in the lung and decreased serum IgE, as well as the expression of Smo, Il4, Il13, and the mucin gene Muc5ac in lung tissue. Smoothened inhibitor treatment reduced cellular infiltration of eosinophils, mast cells, basophils, and CD4+ T-cells to the lung, and eosinophils and CD4+ T-cells in the bronchoalveolar lavage. In the mediastinal lymph nodes, smoothened inhibitor treatment reduced the number of CD4+ T-cells, and the cell surface expression of Th2 markers ST2 and IL-4rα and expression of Th2 cytokines. Thus, overall pharmacological smoothened inhibition attenuated T-cell infiltration to the lung and Th2 function and reduced disease severity and inflammation in the airway.
Asunto(s)
Antiasmáticos/administración & dosificación , Antiinflamatorios/administración & dosificación , Asma/tratamiento farmacológico , Bencimidazoles/administración & dosificación , Quimiotaxis de Leucocito/efectos de los fármacos , Citocinas/metabolismo , Pulmón/efectos de los fármacos , Compuestos de Fenilurea/administración & dosificación , Neumonía/tratamiento farmacológico , Receptor Smoothened/antagonistas & inhibidores , Células Th2/efectos de los fármacos , Animales , Asma/inmunología , Asma/metabolismo , Modelos Animales de Enfermedad , Femenino , Inyecciones Intraperitoneales , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Neumonía/inmunología , Neumonía/metabolismo , Transducción de Señal , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Class F G protein-coupled receptors are characterized by a large extracellular domain (ECD) in addition to the common transmembrane domain (TMD) with seven α-helixes. For smoothened receptor (SMO), structural studies revealed dissected ECD and TMD, and their integrated assemblies. However, distinct assemblies were reported under different circumstances. Using an unbiased approach based on four series of cross-conjugated bitopic ligands, we explore the relationship between the active status and receptor assembly. Different activity dependency on the linker length for these bitopic ligands corroborates the various occurrences of SMO assembly. These results reveal a rigid "near" assembly for active SMO, which is in contrast to previous results. Conversely, inactive SMO adopts a free ECD, which would be remotely captured at "far" assembly by cholesterol. Altogether, we propose a mechanism of cholesterol flow-caused SMO activation involving an erection of ECD from far to near assembly.
Asunto(s)
Hidroxicolesteroles/metabolismo , Receptor Smoothened/metabolismo , Anilidas/síntesis química , Anilidas/metabolismo , Animales , Sitios de Unión , Células HEK293 , Humanos , Hidroxicolesteroles/síntesis química , Ligandos , Ratones , Células 3T3 NIH , Polietilenglicoles/síntesis química , Polietilenglicoles/metabolismo , Dominios Proteicos , Piridinas/síntesis química , Piridinas/metabolismo , Receptor Smoothened/agonistas , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/químicaRESUMEN
The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Purinas/farmacología , Receptor Smoothened/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HT29 , Proteínas Hedgehog/metabolismo , Humanos , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Purinas/química , Purinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/metabolismoRESUMEN
OBJECTIVE: Hypomethylating agents (HMAs) have been reported to target the Sonic Hedgehog (Shh) signaling pathway in myelodysplastic syndrome (MDS). However, the synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine in MUTZ-1 cell lines remains lacking. METHODS: We used a CCK-8 assay to detect the in-vitro proliferation rate of MUTZ-1 cell lines. Besides, the Annexin V-FITC/PI double staining flow cytometry was utilized to detect the apoptosis rate and cell cycle changes. The expression levels of mRNA were quantified by using qRT-PCR, and the western blot was employed to detect the expression of proteins. RESULTS: We found that the single-agent jervine or decitabine can significantly inhibit the proliferation rate of MUTZ-1 cell lines, and this inhibitory effect is time-dependent and concentration-dependent. The combined intervention of the jervine and decitabine can more significantly inhibit cell proliferation, induce cell apoptosis, and block the G1 phase of the cell cycle. The combined intervention of the two drugs significantly reduced Smo and G1i-1 mRNA expression in MUTZ-1 cells. Furthermore, after combining both of the drug treatments, the proteins levels of Smo, G1i-1, PI3K, p-AKT, Bcl2, and Cyclin Dl were significantly downregulated, and Caspase-3 is upregulated, indicating that jervine with its combination of decitabine might be effective for controlling the proliferation, apoptosis, and cell cycle. CONCLUSION: The Smo inhibitor jervine and its combination with decitabine have a synergistic effect on the proliferation, cell cycle, and apoptosis of MUTZ-1 cells, and its mechanism may be achieved by interfering with the Shh signaling pathway.
Asunto(s)
Decitabina/farmacología , Proteínas Hedgehog/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/antagonistas & inhibidores , Alcaloides de Veratrum/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Dipéptidos/farmacología , Guanidinas/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Compuestos Heterocíclicos con 3 Anillos/farmacología , Melanoma/tratamiento farmacológico , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dipéptidos/administración & dosificación , Sinergismo Farmacológico , Femenino , Guanidinas/administración & dosificación , Proteínas Hedgehog/metabolismo , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Humanos , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/antagonistas & inhibidores , Esferoides Celulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, â¿5 (20R, 24R) 23-oxo-24-methylsolacongetidine, â¿5 (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 â 2)-ß-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and ß1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC50 values of 0.72-14.31 µM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while ß1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC50 value of 5.35 µM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.
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Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Proliferación Celular/efectos de los fármacos , Meduloblastoma/patología , Receptor Smoothened/antagonistas & inhibidores , Esteroides/química , Veratrum/química , Alcaloides/química , Línea Celular Tumoral , Células HEK293 , Humanos , Estructura Molecular , Análisis Espectral/métodosRESUMEN
Hedgehog signaling is aberrantly activated in hematologic malignancies and solid tumors, and targeting it is a promising therapeutic strategy against these cancers. Resistance to clinically available hedgehog-targeted Smoothened inhibitor (SMOi) drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies identified inhibition of BET and CDK7 as two epigenetic/transcriptional-targeted therapeutic strategies for overcoming SMOi resistance, providing a promising direction for anti-hedgehog drug development. To uncover additional strategies for inhibiting aberrant hedgehog activity, here we performed CRISPR-Cas9 screening with an single-guide RNA library targeting epigenetic and transcriptional modulators in hedgehog-driven medulloblastoma cells, combined with tumor dataset analyses. Structure specific recognition protein 1 (SSRP1), a subunit of facilitates chromatin transcription (FACT) complex, was identified as a hedgehog-induced essential oncogene and therapeutic target in hedgehog-driven cancer. The FACT inhibitor CBL0137, which has entered clinical trials for cancer, effectively suppressed in vitro and in vivo growth of multiple SMOi-responsive and SMOi-resistant hedgehog-driven cancer models. Mechanistically, CBL0137 exerted anti-hedgehog activity by targeting transcription of GLI1 and GLI2, which are core transcription factors of the hedgehog pathway. SSRP1 bound the promoter regions of GLI1 and GLI2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 synergized with BET or CDK7 inhibitors to antagonize aberrant hedgehog pathway and growth of hedgehog-driven cancer models. Taken together, these results identify FACT inhibition as a promising epigenetic/transcriptional-targeted therapeutic strategy for treating hedgehog-driven cancers and overcoming SMOi resistance. SIGNIFICANCE: This study identifies FACT inhibition as an anti-hedgehog therapeutic strategy for overcoming resistance to Smoothened inhibitors and provides preclinical support for initiating clinical trials of FACT-targeted drug CBL0137 against hedgehog-driven cancers.
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Carbazoles/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Meduloblastoma/tratamiento farmacológico , Receptor Smoothened/antagonistas & inhibidores , Factores de Elongación Transcripcional/antagonistas & inhibidores , Animales , Apoptosis , Proliferación Celular , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Femenino , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Introduction: Hedgehog inhibitors are an alternative treatment option for patients with advanced BCCs not eligible for standard therapies due to lack of efficacy, high recurrence risk, and high-rate morbidity. Sonidegib, an oral smoothened antagonist, has been approved for the treatment of adult patients with locally advanced basal cell carcinoma. Several studies and randomized controlled trials have been conducted in order to evaluate the efficacy, safety, and tolerability of this new molecule.Areas covered: The aim of this article is to provide a complete overview on the use of sonidegib for the treatment of advanced BCCs describing the efficacy, safety, and drug tolerability of this drug.Expert opinion: Sonidegib, with a different pharmacokinetics profile from that of the other SMO-inhibitor vismodegib, demonstrated to be an efficacious and well-tolerated treatment in patients with locally advanced BCC. Although several drug-related adverse events have already been described, different strategies should be taken into account to better manage this small molecule while avoiding treatment discontinuation. The use of sonidegib as neoadjuvant therapy or combined with other hedgehog pathway inhibitors targeting different sites and to date, only available for pre-clinical studies, should also be considered.
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Compuestos de Bifenilo/administración & dosificación , Carcinoma Basocelular/tratamiento farmacológico , Piridinas/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Anilidas/administración & dosificación , Anilidas/efectos adversos , Anilidas/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Compuestos de Bifenilo/efectos adversos , Compuestos de Bifenilo/farmacocinética , Carcinoma Basocelular/patología , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Piridinas/efectos adversos , Piridinas/farmacocinética , Ensayos Clínicos Controlados Aleatorios como Asunto , Neoplasias Cutáneas/patología , Receptor Smoothened/antagonistas & inhibidoresRESUMEN
Advancements in the understanding of the pathogenesis of acute myeloid leukemia (AML) have led to the introduction and approval of a number of novel drugs in AML. Glasdegib, an oral hedgehog pathway inhibitor, was approved in 2018 in combination with low-dose cytarabine for the treatment of newly diagnosed AML in patients unfit for intensive chemotherapy. In this review, we discuss the preclinical rationale for glasdegib, important clinical trials that led to glasdegib's approval, and future trials of glasdegib in AML and other myeloid diseases. Notably, 2 large randomized, placebo-controlled phase 3 trials (AML BRIGHT 1019) are currently recruiting patients with newly diagnosed AML to evaluate glasdegib in combination with intensive chemotherapy or azacitidine, depending on the patient's ability to tolerate induction chemotherapy. While glasdegib and low-dose cytarabine have been eclipsed by venetoclax and hypomethylating agent combinations for newly diagnosed AML in the United States, we discuss other areas where glasdegib may still have an opportunity to improve outcomes in this devastating disease.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencimidazoles/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Compuestos de Fenilurea/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azacitidina/uso terapéutico , Bencimidazoles/farmacología , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Citarabina/uso terapéutico , Aprobación de Drogas , Evaluación Preclínica de Medicamentos , Humanos , Quimioterapia de Inducción/efectos adversos , Quimioterapia de Inducción/métodos , Ratones , Compuestos de Fenilurea/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Estados UnidosRESUMEN
Aberrantly activated Hedgehog (Hh) pathway is critical for driving the initiation and progression of multiple types of cancers, including medulloblastoma (MB) and basal cellular carcinoma (BCC). The majority of current Hh antagonist function by targeting the transmembrane domain of the oncoprotein Smoothened (Smo), a G-protein-coupled receptor-like receptor of Hh pathway. However, the primary and acquired resistance to current Smo inhibitors raise a critical need to develop next-generation of Smo inhibitors to improve their clinical efficacy. In this study, we identify that FDA approved drug ABT-199 significantly and selectively inhibits the Hh pathway. Mechanistically, ABT-199 acts as a competitive inhibitor of oxysterol by potentially targeting the cysteine rich domain (CRD) of Smo, rather as a BH3 mimetic. ABT-199 obviously inhibits the growth of Hh-driven tumors and possesses capacity of combating the primary and acquired resistance to Smo inhibitors caused by Smo mutations. Our data reposition ABT-199 as a Smo inhibitor for treating Hh-driven tumors, especially for those bearing Smo mutations and resistant to current Smo inhibitors. Meanwhile, our findings strengthen the argument that the CRD of Smo is a promising target for developing novel Smo inhibitors with capacity of combating the resistance to Smo inhibitors.
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Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Animales , Antineoplásicos/metabolismo , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Humanos , Hidroxicolesteroles/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células 3T3 NIH , Neoplasias/metabolismo , Unión Proteica , Receptor Smoothened/química , Receptor Smoothened/metabolismo , Sulfonamidas/metabolismoRESUMEN
Glasdegib is a potent, selective oral inhibitor of the Hedgehog signaling pathway. This phase 1 double-blind thorough QT study (NCT03162900) evaluated the effects of glasdegib on QTc interval. The study enrolled 36 healthy volunteers to receive a single dose of 150 mg glasdegib (representing a therapeutic dose), 300 mg glasdegib (representing a supratherapeutic dose), 400 mg moxifloxacin (positive control), or placebo under fasted conditions. The study demonstrated that therapeutic and supratherapeutic doses of glasdegib had no significant effect on QTc interval; the upper bound of the 2-sided 90% confidence intervals (CIs) for all time-matched least-squares mean differences in QT interval corrected using Fridericia's formula (QTcF) between glasdegib and placebo was below the prespecified criterion of 20 milliseconds (Food and Drug Administration correspondence reviewed and accepted). Based on an exposure-response analysis, glasdegib was determined not to have a meaningful effect on heart rate (change in RR interval). The mean (90%CI) model-derived baseline and placebo-adjusted QTcF at the average maximum observed concentration values corresponding to therapeutic and supratherapeutic glasdegib doses was 7.3 milliseconds (6.5-8.2 milliseconds) and 13.7 milliseconds (12.0-15.5 milliseconds), respectively. Together these results demonstrated that following therapeutic and supratherapeutic glasdegib dosing, the change in QTc from baseline was well below the 20-millisecond threshold of clinical concern in oncology.
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Bencimidazoles/farmacocinética , Corazón/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Compuestos de Fenilurea/farmacocinética , Receptor Smoothened/antagonistas & inhibidores , Adulto , Bencimidazoles/farmacología , Estudios de Casos y Controles , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Electrocardiografía/efectos de los fármacos , Electrocardiografía/métodos , Ayuno , Voluntarios Sanos/estadística & datos numéricos , Corazón/fisiología , Humanos , Masculino , Persona de Mediana Edad , Moxifloxacino/administración & dosificación , Compuestos de Fenilurea/farmacología , Placebos/administración & dosificación , Inhibidores de Topoisomerasa II/administración & dosificaciónRESUMEN
Primary cilia function as critical signaling hubs whose absence leads to severe disorders collectively known as ciliopathies; our knowledge of ciliogenesis remains limited. We show that Smo induces ciliogenesis through two distinct yet essential noncanonical Hh pathways in several cell types, including neurons. Surprisingly, ligand activation of Smo induces autophagy via an LKB1-AMPK axis to remove the satellite pool of OFD1. This is required, but not sufficient, for ciliogenesis. Additionally, Smo activates the Gαi-LGN-NuMA-dynein axis, causing accumulation of a portion of OFD1 at centrioles in early ciliogenesis. Both pathways are critical for redistribution of BBS4 from satellites to centrioles, which is also mediated by OFD1 centriolar translocation. Notably, different Smo agonists, which activate Smo distinctly, activate one or the other of these pathways; only in combination they recapitulate the activity of Hh ligand. These studies provide new insight into physiological stimuli (Hh) that activate autophagy and promote ciliogenesis and introduce a novel role for the Gαi-LGN-NuMA-dynein complex in this process.