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1.
Anticancer Res ; 40(9): 5211-5219, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878809

RESUMO

BACKGROUND/AIM: CBP is a transcriptional coactivator in the Wnt/ß-catenin pathway that is related to cell kinetics and differentiation. This study aimed to characterize ß-catenin-activated hepatocellular carcinoma (HCC) and evaluate the direct effects of PRI-724 (a selective inhibitor of Wnt/ß-catenin/CBP signaling) on HCC. MATERIALS AND METHODS: Immunohistochemistry for ß-catenin was performed in 199 HCC resected samples. Moreover, using cultured HCC cell lines, cell kinetics and its related proteins were analyzed after treatment of cells with C-82 (active form of PRI-724). RESULTS: Nuclear ß-catenin expression was found in 18% of HCC cases and the tumor sizes in these positive samples were larger. In HCC cell lines with a constitutively activated ß-catenin, C-82 inhibited cell proliferation. C-82 led to an increase in the percentage of cells in the G0/G1 phase of the cell cycle. The percentage of cells in the sub-G1 phase also increased. Moreover, C-82 treatment significantly decreased the expression of cell proliferating markers and increased the expression of apoptosis-related proteins. CONCLUSION: PRI-724(C-82) may be a novel drug for ß-catenin-activated HCC therapy.


Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Pirimidinonas/farmacologia , beta Catenina/metabolismo , Biomarcadores , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores
2.
PLoS One ; 15(9): e0238572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32898143

RESUMO

Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multi-kinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words).


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Rabdomiossarcoma/tratamento farmacológico , Tiazolidinas/farmacologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia
3.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
4.
PLoS One ; 15(7): e0234103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645016

RESUMO

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).


Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Humanos , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia
5.
Life Sci ; 259: 118150, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32726663

RESUMO

Conventional therapeutic methods against cancer, including chemotherapy, radiotherapy, surgery, and combination therapy, have exhibited different toxicity levels due to their unspecific mechanism of action. To overcome the challenges facing conventional cancer therapies, newly developed methods are being investigated. Significant levels of specificity, remarkable accumulation at the tumor site, limited side effects, and minimal off-target effects enable the newly synthesized nanoparticles (NPs) to become the preferred drug delivery method in anticancer therapeutic approaches. According to the literature, CD73 has a pivotal role in cancer progression and resistance to chemotherapy and radiotherapy. Therefore, CD73 has attracted considerable attention among scientists to target this molecule. Accordingly, FDA approved CDK inhibitors such as Dinaciclib that blocks CDK1, 2, 5, and 9, and exhibits significant anticancer activity. So in this study, we intended to simultaneously suppress CD73 and CDKs in cancer cells by using the folic acid (FA)-conjugated chitosan-lactate (CL) NPs loaded with anti-CD73 siRNA and Dinaciclib to control tumor progression and metastasis. The results showed that NPs could effectively transfect cancer cells in a FA receptor-dependent manner leading to suppression of proliferation, survival, migration, and metastatic potential. Moreover, the treatment of tumor-bearing mice with this combination strategy robustly inhibited tumor growth and enhanced survival time in mice. These findings imply the high potential of FA-CL NPs loaded with anti-CD73 siRNA and Dinaciclib for use in cancer treatment shortly.


Assuntos
5'-Nucleotidase/efeitos dos fármacos , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ácido Fólico , Nanopartículas , Compostos de Piridínio/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , 5'-Nucleotidase/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada , Quinases Ciclina-Dependentes/efeitos dos fármacos , Progressão da Doença , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Humanos , Camundongos , Metástase Neoplásica/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Ensaio Tumoral de Célula-Tronco
6.
Mol Pharmacol ; 98(4): 328-342, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690626

RESUMO

Epibatidine is a potent analgetic agent with very high affinity for brain nicotinic acetylcholine receptors (nAChR). We determined the activity profiles of three epibatidine derivatives, RTI-36, RTI-76, and RTI-102, which have affinity for brain nAChR equivalent to that of epibatidine but reduced analgetic activity. RNAs coding for nAChR monomeric subunits and/or concatamers were injected into Xenopus oocytes to obtain receptors of defined subunit composition and stoichiometry. The epibatidine analogs produced protracted activation of high sensitivity (HS) α4- and α2-containing receptors with the stoichiometry of 2alpha:3beta subunits but not low sensitivity (LS) receptors with the reverse ratio of alpha and beta subunits. Although not strongly activated by the epibatidine analogs, LS α4- and α2-containing receptors were potently desensitized by the epibatidine analogs. In general, the responses of α4(2)ß2(2)α5 and ß3α4ß2α6ß2 receptors were similar to those of the HS α4ß2 receptors. RTI-36, the analog closest in structure to epibatidine, was the most efficacious of the three compounds, also effectively activating α7 and α3ß4 receptors, albeit with lower potency and less desensitizing effect. Although not the most efficacious agonist, RTI-76 was the most potent desensitizer of α4- and α2-containing receptors. RTI-102, a strong partial agonist for HS α4ß2 receptors, was effectively an antagonist for LS α4ß2 receptors. Our results highlight the importance of subunit stoichiometry and the presence or absence of specific accessory subunits for determining the activity of these drugs on brain nAChR, affecting the interpretation of in vivo studies since in most cases these structural details are not known. SIGNIFICANCE STATEMENT: Epibatidine and related compounds are potent ligands for the high-affinity nicotine receptors of the brain, which are therapeutic targets and mediators of nicotine addiction. Far from being a homogeneous population, these receptors are diverse in subunit composition and vary in subunit stoichiometry. We show the importance of these structural details for drug activity profiles, which present a challenge for the interpretation of in vivo experiments since conventional methods, such as in situ hybridization and immunohistochemistry, cannot illuminate these details.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Agonistas Nicotínicos/farmacologia , Subunidades Proteicas/metabolismo , Piridinas/química , Receptores Nicotínicos/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Humanos , Estrutura Molecular , Complexos Multiproteicos/metabolismo , Agonistas Nicotínicos/química , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Tropanos/química , Tropanos/farmacologia , Xenopus/genética
7.
PLoS One ; 15(4): e0231265, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267872

RESUMO

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in the regulation of cell proliferation and differentiation. TAZ activity changes in response to the cellular environment such as mechanic and nutritional stimuli, osmolarity, and hypoxia. To understand the physiological roles of TAZ, chemical compounds that activate TAZ in cells are useful as experimental reagents. Kaempferol, TM-25659, and ethacridine are reported as TAZ activators. However, as each TAZ activator has a distinct property in cellular functions, additional TAZ activators are awaiting. We screened for TAZ activators and previously reported IB008738 as a TAZ activator that promotes myogenesis in C2C12 cells. In this study, we have characterized IBS004735 that was obtained in the same screening. IBS004735 also promotes myogenesis in C2C12 cells, but is not similar to IBS008738 in the structure. IBS004735 activates TAZ via Akt and has no effect on TAZ phosphorylation, which is the well-described key modification to regulate TAZ activity. Thus, we introduce IBS004735 as a novel TAZ activator that regulates TAZ in a yet unidentified mechanism.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Imidazóis/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologia , Transativadores/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Mioblastos Esqueléticos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Transativadores/genética , Transfecção
8.
Psychopharmacology (Berl) ; 237(6): 1681-1689, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32125484

RESUMO

RATIONALE: A variety of neural systems are involved in drug addiction, and some of these systems are shared across different addictive drugs. We have found several different types of drug treatments that successfully reduce nicotine self-administration. OBJECTIVES: The current set of studies is the first in a series to determine if drug treatments that have been found to significantly reduce nicotine self-administration would reduce opiate self-administration. METHODS: Amitifadine, a triple reuptake inhibitor of dopamine, norepinephrine, and serotonin, was assessed in female Sprague-Dawley rats to determine whether it significantly reduces remifentanil self-administration with either acute or chronic treatment. RESULTS: Acutely, amitifadine doses of 5, 10, and 20 mg/kg each significantly reduced remifentanil self-administration. In a chronic study, repeated treatment with 10 mg/kg of amitifadine continued to reduce remifentanil self-administration, even after the cessation of treatment. However, amitifadine was not found to attenuate the rise in remifentanil self-administration with continued access. This study and our earlier one showed that the 10 mg/kg amitifadine dose did not significantly affect food motivated responding. Amitifadine did not attenuate remifentanil-induced antinociception as measured on the hot plate test but extended and maintained antinociceptive effects. CONCLUSIONS: These studies show the promise of amitifadine as a treatment for countering opiate self-administration for adjunctive use with opioids for analgesia. Further studies are needed to determine the possible efficacy of amitifadine for combating opiate addiction or preventing it in humans during adjunctive use with opioids for chronic pain.


Assuntos
Analgésicos Opioides/administração & dosagem , Compostos Aza/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Inibidores da Captação de Dopamina/uso terapêutico , Norepinefrina/antagonistas & inibidores , Remifentanil/administração & dosagem , Inibidores de Captação de Serotonina/uso terapêutico , Animais , Compostos Aza/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Motivação/efeitos dos fármacos , Motivação/fisiologia , Nicotina/administração & dosagem , Norepinefrina/metabolismo , Dor/tratamento farmacológico , Dor/metabolismo , Ratos , Ratos Sprague-Dawley , Autoadministração , Serotonina/metabolismo , Inibidores de Captação de Serotonina/farmacologia , Estereoisomerismo
9.
Obesity (Silver Spring) ; 28(4): 724-732, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32202075

RESUMO

OBJECTIVE: This study aimed to evaluate ertugliflozin in patients with overweight and obesity with type 2 diabetes mellitus. METHODS: Data from three placebo-controlled, randomized, Phase 3 studies were pooled. Patients with baseline BMI ≥ 25 (1,377/1,544; 89%) were assessed with a stratification by BMI subgroup. RESULTS: At week 26, reductions from baseline in glycated hemoglobin A1c (HbA1c), fasting plasma glucose, body weight (BW), and systolic blood pressure (SBP) were greater with ertugliflozin versus placebo. For placebo, ertugliflozin 5 mg, and ertugliflozin 15 mg, respectively, least squares mean change was 0.1%, -0.8%, and -0.9% for HbA1c and -1.2 kg, -3.1 kg, and -3.2 kg for BW. HbA1c reductions were consistent across BMI subgroups. For ertugliflozin 5 mg and 15 mg, least squares mean change (placebo adjusted) in absolute BW was -1.4 kg and -1.2 kg for BMI 25 to < 30, -1.8 kg and -1.9 kg for BMI 30 to < 35, and -2.5 kg and -2.9 kg for BMI ≥ 35. Percent BW changes were similar across BMI subgroups. Incidence of adverse events was 52.5%, 44.6%, and 50.1% with placebo, ertugliflozin 5 mg, and ertugliflozin 15 mg, respectively. CONCLUSIONS: Meaningful reductions in HbA1c, fasting plasma glucose, BW, and SBP were observed with ertugliflozin in patients with overweight and obesity with type 2 diabetes mellitus. Ertugliflozin improved HbA1c and SBP and reduced BW across BMI subgroups. Ertugliflozin was generally well tolerated.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Obesidade/tratamento farmacológico , Sobrepeso/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
10.
Nat Commun ; 11(1): 1228, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144272

RESUMO

The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the 'primed' state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Complexo II de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Sulfonamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Resistencia a Medicamentos Antineoplásicos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Mutação , Oxirredução/efeitos dos fármacos , Seleção de Pacientes , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Tenoiltrifluoracetona/farmacologia , Translocação Genética
11.
Biochem Biophys Res Commun ; 525(3): 549-556, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32113682

RESUMO

As a proapoptotic death effect domain (DED)-containing protein, DED-containing DNA-binding protein (DEDD) has been demonstrated to inhibit tumor growth, invasion and metastasis in our previous studies. Here, we demonstrated that knockdown of DEDD in MCF-7 cells resulted in characteristic drug resistance to doxorubicin and paclitaxel, and overexpression of DEDD in MDA-MB-231 cells increased their sensitivity to doxorubicin and paclitaxel. The expression levels of DEDD were positively correlated with Bcl-2 in breast cancer cell lines as well as in human breast cancer tissue. Knockdown of DEDD downregulated the transcriptional activity of the bcl-2 gene and shortened the time for Bcl-2 degradation. DEDD interacts with and stabilizes Bcl-2, and breast cancer cells with low DEDD expression were more sensitive to treatment with a BH3 mimetic, ABT-199, than were those with high DEDD expression. In total, our findings highlight a new strategy for treating breast cancer with no/low DEDD expression by targeting Bcl-2 with the BH3 mimetic ABT-199.


Assuntos
Neoplasias da Mama/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Proteínas de Ligação a DNA/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfonamidas/uso terapêutico , Transcrição Genética/efeitos dos fármacos
12.
Nat Commun ; 11(1): 929, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066735

RESUMO

Current treatments for clear cell renal cell cancer (ccRCC) are insufficient because two-thirds of patients with metastases progress within two years. Here we report the identification and characterization of a cancer stem cell (CSC) population in ccRCC. CSCs are quantitatively correlated with tumor aggressiveness and metastasis. Transcriptional profiling and single cell sequencing reveal that these CSCs exhibit an activation of WNT and NOTCH signaling. A significant obstacle to the development of rational treatments has been the discrepancy between model systems and the in vivo situation of patients. To address this, we use CSCs to establish non-adherent sphere cultures, 3D tumor organoids, and xenografts. Treatment with WNT and NOTCH inhibitors blocks the proliferation and self-renewal of CSCs in sphere cultures and organoids, and impairs tumor growth in patient-derived xenografts in mice. These findings suggest that our approach is a promising route towards the development of personalized treatments for individual patients.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Proteínas Wnt/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Rim/patologia , Neoplasias Renais/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Pirimidinonas/farmacologia , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Esferoides Celulares , Proteínas Wnt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Med Chem ; 63(10): 5102-5118, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32083858

RESUMO

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase da Agamaglobulinemia/química , Animais , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cristalografia por Raios X/métodos , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Endogâmicos Lew , Ovinos
14.
Leukemia ; 34(9): 2342-2353, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32094466

RESUMO

Acute myeloid leukemia (AML) results from the enhanced proliferation and impaired differentiation of hematopoietic stem and progenitor cells. Using an ex vivo functional screening assay, we identified that the combination of the BTK inhibitor ibrutinib and BCL2 inhibitor venetoclax (IBR + VEN), currently in clinical trials for chronic lymphocytic leukemia (CLL), demonstrated enhanced efficacy on primary AML patient specimens, AML cell lines, and in a mouse xenograft model of AML. Expanded analyses among a large cohort of hematologic malignancies (n = 651 patients) revealed that IBR + VEN sensitivity associated with selected genetic and phenotypic features in both CLL and AML specimens. Among AML samples, 11q23 MLL rearrangements were highly sensitive to IBR + VEN. Analysis of differentially expressed genes with respect to IBR + VEN sensitivity indicated pathways preferentially enriched in patient samples with reduced ex vivo sensitivity, including IL-10 signaling. These findings suggest that IBR + VEN may represent an effective therapeutic option for patients with AML.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Camundongos , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Nat Biotechnol ; 38(4): 460-470, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32094658

RESUMO

Generation of pancreatic ß cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived ß (SC-ß) cells with improved in vitro and in vivo function. SC-ß cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.


Assuntos
Engenharia Celular/métodos , Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células-Tronco Pluripotentes/citologia , Actinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Diabetes Mellitus/terapia , Endoderma/citologia , Endoderma/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/transplante , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismo , Tiazolidinas/farmacologia , Transativadores/metabolismo , Moduladores de Tubulina/farmacologia
16.
Nat Commun ; 11(1): 465, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974362

RESUMO

The ability to quantitatively measure a small molecule's interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Chaperonas Moleculares/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Benzoquinonas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Imunofluorescência , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Hidroxibutiratos/metabolismo , Hidroxibutiratos/farmacologia , Lactamas Macrocíclicas/farmacologia , Espectrometria de Massas , Chaperonas Moleculares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Proteoma/análise , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptor ErbB-2/metabolismo
17.
PLoS One ; 15(1): e0227454, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914150

RESUMO

Cholangiocarcinoma (CCA), a malignant tumor originating in the biliary tract, is well known to be associated with adverse clinical outcomes and high mortality rates due to the lack of effective therapy. Evasion of apoptosis is considered a key contributor to therapeutic success and chemotherapy resistance in CCA, highlighting the need for novel therapeutic strategies. In this study, we demonstrated that the induction of necroptosis, a novel regulated form of necrosis, could potentially serve as a novel therapeutic approach for CCA patients. The RNA sequencing data in The Cancer Genome Atlas (TCGA) database were analyzed and revealed that both receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), two essential mediators of necroptosis, were upregulated in CCA tissues when compared with the levels in normal bile ducts. We demonstrated in a panel of CCA cell lines that RIPK3 was differentially expressed in CCA cell lines, while MLKL was more highly expressed in CCA cell lines than in nontumor cholangiocytes. We therefore showed that treatment with both tumor necrosis factor-α (TNF-α) and Smac mimetic, an inhibitor of apoptosis protein (IAP) antagonist, induced RIPK1/RIPK3/MLKL-dependent necroptosis in CCA cells when caspases were blocked. The necroptotic induction in a panel of CCA cells was correlated with RIPK3 expression. Intriguingly, we demonstrated that Smac mimetic sensitized CCA cells to a low dose of standard chemotherapy, gemcitabine, and induced necroptosis in an RIPK1/RIPK3/MLKL-dependent manner upon caspase inhibition but not in nontumor cholangiocytes. We further demonstrated that Smac mimetic and gemcitabine synergistically induced an increase in TNF-α mRNA levels and that Smac mimetic reversed gemcitabine-induced cell cycle arrest, leading to cell killing. Collectively, our present study demonstrated that TNF-α and gemcitabine induced RIPK1/RIPK3/MLKL-dependent necroptosis upon IAP depletion and caspase inhibition; therefore, our findings have pivotal implications for designing a novel necroptosis-based therapeutic strategy for CCA patients.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Desoxicitidina/análogos & derivados , Necroptose/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias do Sistema Biliar/metabolismo , Neoplasias do Sistema Biliar/patologia , Inibidores de Caspase/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Humanos , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
19.
Curr Opin Hematol ; 27(2): 76-80, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31895103

RESUMO

PURPOSE OF REVIEW: Since its approval in November 2018, venetoclax with a hypomethylating agent backbone has shown promising efficacy for older, newly diagnosed acute myeloid leukemia (AML) patients who are unfit for standard intensive induction chemotherapy. This regimen is well tolerated, allows for deep and durable responses and may be increasing the prevalence of the disease. Although there is justifiable excitement, it remains to be seen to what extent venetoclax-based regimens, as they are currently administered, will have a long-term impact on the treatment of AML. This review aims to evaluate the strengths of the regimen that deserve enthusiasm as well as its shortcomings, which should be viewed as opportunities for improvement. RECENT FINDINGS: The clinical efficacy as well as the novel mechanism of venetoclax with hypomethylating agents will be described here. SUMMARY: Venetoclax with hypomethylating agents do not represent the holy grail for AML, but this regimen is a promising step in the right direction, and proof of principle that a low-intensity therapy can have a major impact on this disease.


Assuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Sulfonamidas/uso terapêutico , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Humanos , Sulfonamidas/farmacologia
20.
Exp Hematol ; 81: 32-41, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31954171

RESUMO

Gemcitabine (Gem), busulfan (Bu), and melphalan (Mel) are used for hematopoietic stem cell transplantation. To further improve their efficacy, a preclinical study on their synergism with the histone deacetylase inhibitor panobinostat (Pano) and the BCL2 inhibitor venetoclax/ABT199 was performed. Multiple myeloma cell lines MM.1R and MC/CAR were exposed to ∼IC20 levels of the drugs. Synergistic cytotoxicity was observed in cells exposed to the five-drug combination as indicated by combination indexes <1, supported by ∼86% inhibition of proliferation and ∼84% annexin V positivity in MM.1R and ∼58% inhibition of proliferation and ∼46% annexin V positivity in MC/CAR cells. Activation of the DNA damage response and apoptosis were suggested by a modest increase in the phosphorylation of ATM and its substrates; significant cleavage of PARP1, caspase 3, and heat shock protein 90; DNA fragmentation; mitochondrial membrane depolarization; and reactive oxygen species production. The five-drug combination significantly decreased the levels of PI3K, AKT, mTOR, RAPTOR, P-P70S6K, and eIF2α, with concomitant increases in P-AMPK and its substrate Tuberin/TSC2, suggesting that the mTOR signaling pathway was compromised. Endoplasmic reticulum stress through activation of the unfolded protein response was also observed as suggested by increases in the levels of calnexin, BiP/GRP78, ERO1-Lα, and protein disulfide isomerase, which may relate to venetoclax-mediated inhibition of BCL2 in the endoplasmic reticulum. This is the first report on the effects of a venetoclax-containing regimen on the unfolded protein response. These results provide a rationale to propose a clinical trial on use of Gem + Bu + Mel + Pano + Venetoclax as part of a conditioning regimen for multiple myeloma patients undergoing autologous hematopoietic stem cell transplantation.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mieloma Múltiplo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bussulfano/farmacologia , Linhagem Celular Tumoral , Citotoxinas/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melfalan/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Panobinostat/farmacologia , Sulfonamidas/farmacologia
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