RESUMO
Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.
Assuntos
Dissuasores de Álcool/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Dissulfiram/farmacologia , Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Proteína com Valosina/metabolismoRESUMO
The non-small cell lung cancer (NSCLC) represents a malignant type of cancer worldwide. The atalantraflavone (AFL) is a natural product isolated from leaves of Atalantia monophylla (L.) DC. However, the function of atalantraflavone in NSCLC is still elusive. In present work, we have unraveled a novel function of AFL in NSCLC. AFL significantly inhibited NSCLC cell viability and colony formation. AFL increased sub-G1 fraction and apoptotic rates in a dose-dependent manner. Furthermore, Twist-related protein 1 (Twist1) was identified as the target of AFL. The association between AFL and Twist1 markedly decreased the stability of Twist1 via elevated ubiquitin mediated proteasomal degradation. AFL induced NSCLC suppression was mediated by Twist1 as Twist1 overexpression could partially reverse the inhibitory effect of AFL on migration and metastasis. Furthermore, AFL could also sensitize NSCLC cells to cisplatin treatment and consistently impair NSCLC proliferation and metastasis. Our current data have identified a tumor suppressive function for AFL in NSCLC by increasing Twist1 degradation. Therefore, the anti-tumor activity of AFL might provide critical insight into pharmaceutic lung cancer intervention to overcome cisplatin resistance.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Flavonas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Rutaceae/química , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Flavonas/isolamento & purificação , Humanos , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Folhas de Planta/química , Estabilidade ProteicaRESUMO
Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sistemas de Liberação de Medicamentos , Feminino , Via de Sinalização Hippo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição de Domínio TEA , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
The Virtual Screening (VS) study described herein aimed at detecting novel Bromodomain BRD4 binders and relied on knowledge from public databases (ChEMBL, REAXYS) to establish a battery of predictive models of BRD activity for in silico selection of putative ligands. Beyond the actual discovery of new BRD ligands, this represented an opportunity to practically estimate the actual usefulness of public domain "Big Data" for robust predictive model building. Obtained models were used to virtually screen a collection of 2 million compounds from the Enamine company collection. This industrial partner then experimentally screened a subset of 2992 molecules selected by the VS procedure for their high likelihood to be active. Twenty nine confirmed hits were detected after experimental testing, representing 1% of the selected candidates. As a general conclusion, this study emphasizes once more that public structure-activity databases are nowadays key assets in drug discovery. Their usefulness is however limited by the state-of-the-art knowledge harvested so far by published studies. Target-specific structure-activity information is rarely rich enough, and its heterogeneity makes it extremely difficult to exploit in rational drug design. Furthermore, published affinity measures serving to build models selecting compounds to be experimentally screened may not be well correlated with the experimental hit selection criterion (in practice, often imposed by equipment constraints). Nevertheless, a robust 2.6-fold increase in hit rate with respect to an equivalent, random screening campaign showed that machine learning is able to extract some real knowledge in spite of all the noise in structure-activity data.
Assuntos
Mineração de Dados/métodos , Descoberta de Drogas , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Ligantes , Aprendizado de Máquina , Relação Estrutura-AtividadeRESUMO
Preventing hepatic stellate cell (HSC) activation represents a promising approach to resolve liver fibrosis. Several drugs have been reported to delay/prevent HSCs activation, however with limited clinical benefits. The latter may be in part attributed to the limited ability of such drugs in targeting more than one pathway of HSC activation. Added to that, is their inability of reaching their target cell in sufficient amounts to induce a therapeutic effect. In this work, chitosan NPs were loaded with JQ1 and atorvastatin, two drugs that have been reported to prevent HSCs activation, however via different mechanisms. NPs were then modified with different densities of retinol (Rt) for active targeting of HSCs. The NP HSCs targeting ability as a function of Rt density was assessed in vitro on primary HSCs and in vivo in carbon tetrachloride (CCl4) induced fibrotic mouse models. In vitro NPs modified with a low Rt density (LRt-NPs) showed ≈2 folds enhanced HSCs uptake in comparison to unmodified NPs, whereas NPs modified with a high Rt density (HRt-NPs) showed ≈0.8 folds change in uptake relative to unmodified NPs. Similarly, in vivo LRt-NPs showed higher accumulation in fibrotic livers in comparison to healthy livers whereas HRt-NPs and unmodified NPs showed lower accumulation in fibrotic livers relative to healthy controls respectively. Finally, the ability of drug-loaded NPs in preventing HSCs activation was assessed by monitoring the reduction in α-smooth muscle actin (α-SMA) expression by Western blot. NPs loaded with both JQ1 and atorvastatin showed reduction in α-SMA expression. In addition, a synergistic reduction in α-SMA was observed when cells were co-treated with JQ1 and atorvastatin loaded NPs.
Assuntos
Atorvastatina/administração & dosagem , Azepinas/administração & dosagem , Células Estreladas do Fígado/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Cirrose Hepática/tratamento farmacológico , Triazóis/administração & dosagem , Actinas/metabolismo , Animais , Tetracloreto de Carbono/toxicidade , Quitosana/química , Modelos Animais de Doenças , Portadores de Fármacos/química , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Células HEK293 , Células Estreladas do Fígado/imunologia , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Masculino , Camundongos , Nanopartículas/química , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Resultado do Tratamento , Vitamina A/químicaRESUMO
The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Linfoma Extranodal de Células T-NK/tratamento farmacológico , Linfoma de Células T/tratamento farmacológico , Proteínas Nucleares/genética , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Animais , Proteínas de Ciclo Celular , Depsipeptídeos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Fator de Transcrição Ikaros/antagonistas & inibidores , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Imidazolinas/farmacologia , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Linfoma Extranodal de Células T-NK/genética , Linfoma Extranodal de Células T-NK/metabolismo , Linfoma Extranodal de Células T-NK/patologia , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Indução de Remissão , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Novel conformationally constrained BET bromodomain inhibitors have been developed. These inhibitors were optimized in two similar, yet distinct chemical series, the 6-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (A) and the 1-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (B). Each series demonstrated excellent activity in binding and cellular assays, and lead compounds from each series demonstrated significant efficacy in in vivo tumor xenograft models.
Assuntos
Proteínas Nucleares/antagonistas & inibidores , Piridonas/química , Fatores de Transcrição/antagonistas & inibidores , Animais , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Camundongos , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Piridonas/farmacocinética , Piridonas/farmacologia , Piridonas/uso terapêutico , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Transplante HeterólogoRESUMO
BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration-resistant prostate cancer (CRPC). Several BET inhibitors (BETi) that displace BRD4 from chromatin are being evaluated in clinical trials for CRPC. Here, we describe mechanisms of acquired resistance to BETi that are amenable to targeted therapies in CRPC. BETi-resistant CRPC cells displayed cross-resistance to a variety of BETi in the absence of gatekeeper mutations, exhibited reduced chromatin-bound BRD4, and were less sensitive to BRD4 degraders/knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR signaling due to CDK9-mediated phosphorylation of AR, resulting in sensitivity to CDK9 inhibitors and enzalutamide. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DDR genes was observed, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy; however, our BETi resistance data suggest unique opportunities for combination therapies in treating CRPC.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Dano ao DNA/genética , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential 'hits' against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.
Assuntos
Antifúngicos/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Subunidades Proteicas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Candida albicans , Avaliação Pré-Clínica de Medicamentos , Proteínas Fúngicas/química , Ligação de Hidrogênio , Ligantes , Proteínas Associadas aos Microtúbulos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Subunidades Proteicas/químicaRESUMO
A growing body of studies has documented the pathological influence of impaired alternative splicing (AS) events on numerous diseases, including cancer. In addition, the generation of alternatively spliced isoforms is frequently noted to result in drug resistance in many cancer therapies. To gain comprehensive insights into the impacts of AS events on cancer biology and therapeutic developments, this paper highlights recent findings regarding the therapeutic routes of targeting alternative-spliced isoforms and splicing regulators to treatment strategies for distinct cancers.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/terapia , Fatores de Processamento de RNA/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Caspase 9/genética , Caspase 9/metabolismo , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Cicloexilaminas/uso terapêutico , Compostos de Epóxi/uso terapêutico , Humanos , Macrolídeos/uso terapêutico , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos/uso terapêutico , Piranos/uso terapêutico , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Compostos de Espiro/uso terapêutico , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Bromodomain and extra-terminal (BET) proteins, a class of epigenetic reader domains has emerged as a promising new target class for small molecule drug discovery for the treatment of cancer, inflammatory, and autoimmune diseases. Starting from in silico screening campaign, herein we report the discovery of novel BET inhibitors based on [1,2,4]triazolo[4,3-a]quinoxaline scaffold and their biological evaluation. The hit compound was optimized using the medicinal chemistry approach to the lead compound with excellent inhibitory activities against BRD4 in the binding assay. The substantial antiproliferative activities in human cancer cell lines, promising drug-like properties, and the selectivity for the BET family make the lead compound (13) as a novel BRD4 inhibitor motif for anti-cancer drug discovery.
Assuntos
Antineoplásicos/química , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Quinoxalinas/química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinoxalinas/farmacocinética , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Ratos , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
G1-S checkpoint loss contributes to carcinogenesis and increases reliance upon the G2-M checkpoint for adaptation to stress and DNA repair, making G2-M checkpoint inhibition a target for novel therapeutic development. AZD1775, an inhibitor against the critical G2-M checkpoint protein WEE1, is currently in clinical trials across a number of tumor types. AZD1775 and DNA-damaging agents have displayed favorable activity in several preclinical tumor models, often in the molecular context of TP53 loss. Whether AZD1775 efficacy is modulated by other molecular contexts remains poorly understood. The tumor suppressor serine/threonine kinase 11 (LKB1/STK11) is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC) and is commonly comutated with oncogenic KRAS mutations. We investigated the preclinical effects of AZD1775 in the context of KRAS/LKB1 in NSCLC. Using NSCLC cell lines, we found that AZD1775 alone and in combination with DNA-damaging agents (e.g., cisplatin and radiation) decreased tumor cell viability in LKB1-deficient NSCLC cells. In vitro, LKB1 deficiency enhanced DNA damage and apoptosis in response to AZD1775 exposure compared with wild-type LKB1 cells. In a genetically engineered mouse model of mutant Kras with concomitant loss of Lkb1, combined AZD1775 and cisplatin extended overall survival compared with cisplatin alone. Our data suggest that lack of phosphorylation of LKB1 by ATM was involved in AZD1775-mediated cytotoxicity. Collectively, these findings provide a clinical application for AZD1775 with DNA-damaging agents in KRAS/LKB1 NSCLC. Cancer Res; 77(17); 4663-72. ©2017 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Quinases Ativadas por AMP , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Pirimidinonas , Células Tumorais CultivadasRESUMO
Bromodomain-containing protein 4 (BRD4) is implicated in the pathogenesis of a number of different cancers, inflammatory diseases and heart failure. Much effort has been dedicated toward discovering novel scaffold BRD4 inhibitors (BRD4is) with different selectivity profiles and potential antiresistance properties. Structure-based drug design (SBDD) and virtual screening (VS) are the most frequently used approaches. Here, we demonstrate a novel, structure-based VS approach that uses machine-learning algorithms trained on the priori structure and activity knowledge to predict the likelihood that a compound is a BRD4i based on its binding pattern with BRD4. In addition to positive experimental data, such as X-ray structures of BRD4-ligand complexes and BRD4 inhibitory potencies, negative data such as false positives (FPs) identified from our earlier ligand screening results were incorporated into our knowledge base. We used the resulting data to train a machine-learning model named BRD4LGR to predict the BRD4i-likeness of a compound. BRD4LGR achieved a 20-30% higher AUC-ROC than that of Glide using the same test set. When conducting in vitro experiments against a library of previously untested, commercially available organic compounds, the second round of VS using BRD4LGR generated 15 new BRD4is. Moreover, inverting the machine-learning model provided easy access to structure-activity relationship (SAR) interpretation for hit-to-lead optimization.
Assuntos
Descoberta de Drogas/métodos , Aprendizado de Máquina , Terapia de Alvo Molecular , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
In this issue of Molecular Cell, Barrow et al. (2016) use two complementary approaches-one an assessment of a chemical library, and the other a genome-wide CRISPR screen-that both identify bromodomain-containing protein 4 (Brd4) as a therapeutic target for mtDNA diseases affecting complex I.
Assuntos
Benzodiazepinas/farmacologia , Grupo dos Citocromos c/genética , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular , Fusão Celular , Linhagem Celular , Grupo dos Citocromos c/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Complexo IV da Cadeia de Transporte de Elétrons , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders that cause failures in energetic and metabolic function. Boosting residual oxidative phosphorylation (OXPHOS) activity can partially correct these failures. Herein, using a high-throughput chemical screen, we identified the bromodomain inhibitor I-BET 525762A as one of the top hits that increases COX5a protein levels in complex I (CI) mutant cybrid cells. In parallel, bromodomain-containing protein 4 (BRD4), a target of I-BET 525762A, was identified using a genome-wide CRISPR screen to search for genes whose loss of function rescues death of CI-impaired cybrids grown under conditions requiring OXPHOS activity for survival. We show that I-BET525762A or loss of BRD4 remodeled the mitochondrial proteome to increase the levels and activity of OXPHOS protein complexes, leading to rescue of the bioenergetic defects and cell death caused by mutations or chemical inhibition of CI. These studies show that BRD4 inhibition may have therapeutic implications for the treatment of mitochondrial diseases.
Assuntos
Benzodiazepinas/farmacologia , Grupo dos Citocromos c/genética , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular , Fusão Celular , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Grupo dos Citocromos c/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Complexo IV da Cadeia de Transporte de Elétrons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Metaboloma , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.
Assuntos
Adenocarcinoma de Células Claras/genética , Antineoplásicos/farmacologia , Dasatinibe/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Inibidores de Proteínas Quinases/farmacologia , Mutações Sintéticas Letais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Terapia de Alvo Molecular , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bromodomains (BRDs) are protein interaction modules that selectively recognize ε -N-lysine residues, serving as key epigenetic readers and play a key role in epigenetic regulation of gene transcription. Bromodomain-containing protein 4 (BRD4), a protein containing two BRDs termed BD1 and BD2, has emerged as an attractive candidate for the development of inhibitors targeting gene transcription in several types of cancers. In this study, we made structural modifications of previously reported BRD4 inhibitors, to develop new chemical scaffold 3,4-dihydroquinoxalin-2(1H)-one. Four series of compounds (compounds 7-10) were synthesized, and the BRD4-inhibitory activity and anti-proliferative effect of these compounds were evaluated. We found compound 10d has remarkable anti-proliferative activities toward leukemia cells and could induce apoptosis by mitochondrial pathways. Notably, the analysis of molecular docking suggested that hydrophobic interaction was essential for compound 10d to bind to BD1. In conclusion, these results demonstrate the potential of compound 10d to be utilized as a BRD4 inhibitor with apoptosis inducing effect in future leukemia therapy.
Assuntos
Benzodiazepinonas/química , Benzodiazepinonas/farmacologia , Desenho de Fármacos , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Benzodiazepinonas/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Human testis-specific and bromodomain-containing protein (hBRDT) is essential for chromatin remodeling during spermatogenesis and is therefore an attractive target for the discovery of male contraceptive drugs. In this study, pharmacophore modeling was carried out based on the crystal structure of hBRDT in complex with the inhibitor, JQ1. The established pharmacophore model was used as a 3D search query to identify potent hBRDT inhibitors from an in-house chemical database. A molecular docking analysis was carried out to filter the obtained hit compounds. A total of 125 compounds was finally selected based on the ranking order and visual examination. These compounds were further evaluated by a protein-based in vitro assay. Four compounds with new chemical scaffolds were identified to be hBRDT inhibitors. The most active of these compounds, T480, had a half maximal inhibitory concentration (IC50) of 9.02 µM. The detailed analysis of the binding mode of compound T480 provides important information for the further development of novel BRDT inhibitors.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Interface Usuário-Computador , Domínio Catalítico , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Domínios ProteicosRESUMO
The ATPase p97 is a ubiquitin targeted segregase that uses the energy of ATP binding and hydrolysis to extract ubiquitylated substrates from biological membranes, from other proteins, or from protein complexes to carry out myriad tasks in eukaryotes. Increased p97 activity has been linked to a poor prognosis in cancer patients, making p97 an anti-neoplastic target. In the present study, we show that dehydrocurvularin (DHC) and its chlorinated variants are covalent inhibitors of p97, interfering with its ATPase activity. Interestingly, cellular studies revealed both DHC and its monochloro analogue interfere with both the proteasome and p97, whereas its dichloro analogue showed p97 specificity.