RESUMO
Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.
Assuntos
Proteínas de Ciclo Celular , Oxindóis , Proteólise , Ubiquitina-Proteína Ligases , Humanos , Animais , Proteólise/efeitos dos fármacos , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Oxindóis/farmacologia , Oxindóis/metabolismo , Oxindóis/química , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Células HEK293 , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Azepinas/farmacologia , Azepinas/química , Azepinas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Feminino , Proteínas que Contêm Bromodomínio , Receptores de Interleucina-17RESUMO
IGF1R plays an important role in regulating cellular metabolism and growth. As a single transmembrane protein, its structure is flexible. Although previous studies revealed some structures of IGF1R, the cryo-EM apo structures of the receptor have never been reported. Herein, we reported four distinct cryo-EM structures that reveal the apo states of IGF1R. These conformations were classified as "Resting states" and "Active states", according to the orientation of α-CT helices and structural symmetry. In addition, a "Ligand-pocket" was formed in the active conformations, which presented a new view of conformational changes of apo-IGF1R. These results suggest a new dynamic change model to show the details of why and how ligands can bind to IGF1R.
Assuntos
Receptor IGF Tipo 1 , Microscopia Crioeletrônica/métodos , Humanos , Ligantes , Conformação Proteica , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/ultraestruturaRESUMO
Sorafenib, a multikinase inhibitor, has been widely used as a first-line anticancer drug for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance to sorafenib is frequently observed in clinical applications. Potential nonkinase targets of sorafenib have not been well documented and may provide insights into reversing drug resistance and enhancing drug efficacy. Herein, we report that sorafenib exerts its anticancer effects by activating metallothionein 1 G (MT1G) expression. MT1G is a novel marker in HCC that correlates well with patient survival. MT1G overexpression suppressed the cellular proliferation, migration, invasion, and tumour formation of HCC and sensitised cells to sorafenib treatment. However, the disruption of MT1G attenuated the anticancer effects of sorafenib. Mechanistically, sorafenib upregulated MT1G expression via hypomethylation of its promoter region by binding and inhibiting DNA methyltransferase 1 (DNMT1) and increasing its promoter accessibility in HCC cells. Activation of MT1G also inhibited CA9 transcription through the suppression of HIF1A as mediated by KLF4. Our collective data revealed that sorafenib exerts its anticancer effects through epigenetic regulation of the DNMT1/MT1G/KLF4/CA9 axis in HCC and the activation of MT1G might constitute a strategy for enhancing the effect of sorafenib to suppress HCC cells.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Sorafenibe , Antígenos de Neoplasias , Antineoplásicos/farmacologia , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Humanos , Fator 4 Semelhante a Kruppel/genética , Fator 4 Semelhante a Kruppel/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Metalotioneína/genética , Metalotioneína/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologiaRESUMO
Cisplatin (DDP) is commonly used in the treatment of non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), and the primary cause for its clinical inefficacy is chemoresistance. Here, we aimed to investigate a novel mechanism of chemoresistance in LUAD cells, focusing on the calcium-sensing receptor (CaSR). In this study, high CaSR expression was detected in DDP-resistant LUAD cells, and elevated CaSR expression is strongly correlated with poor prognosis in LUAD patients receiving chemotherapy. LUAD cells with high CaSR expression exhibited decreased sensitivity to cisplatin, and the growth of DDP-resistant LUAD cells was inhibited by cisplatin treatment in combination with CaSR suppression, accompanied by changes in BRCA1 and cyclin B1 protein expression both in vitro and in vivo. Additionally, an interaction between CaSR and KIF11 was identified. Importantly, suppressing KIF11 resulted in decreased protein levels of BRCA1 and cyclin B1, enhancing the sensitivity of DDP-resistant LUAD cells to cisplatin with no obvious decrease in CaSR. Here, our findings established the critical role of CaSR in promoting cisplatin resistance in LUAD cells by modulating cyclin B1 and BRCA1 and identified KIF11 as a mediator, highlighting the potential therapeutic value of targeting CaSR to overcome chemoresistance in LUAD.
Assuntos
Adenocarcinoma de Pulmão , Proteína BRCA1 , Cisplatino , Ciclina B1 , Resistencia a Medicamentos Antineoplásicos , Cinesinas , Neoplasias Pulmonares , Receptores de Detecção de Cálcio , Humanos , Cisplatino/uso terapêutico , Cisplatino/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Cinesinas/metabolismo , Cinesinas/genética , Animais , Camundongos , Camundongos Nus , Feminino , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Masculino , Camundongos Endogâmicos BALB CRESUMO
This study comprehensively addresses the involvement of the protein CKLF-like Marvel transmembrane domain-containing family member 5 (CMTM5) in the context of demyelination and cytodegenerative autoimmune diseases, particularly multiple Sclerosis (MS). An observed reduction in CMTM5 expression in post-mortem MS lesions prompted further investigations in both in vitro and in vivo animal models. In the cuprizone animal model, we detected a decrease in CMTM5 expression in oligodendrocytes that is absent in other members of the CMTM protein family. Our findings also confirm these results in the experimental autoimmune encephalomyelitis (EAE) model with decreased CMTM5 expression in both cerebellum and spinal cord white matter. We also examined the effects of a Cmtm5 knockdown in vitro in the oligodendroglial Oli-neu mouse cell line using the CRISPR interference technique. Interestingly, we found no effects on cell response to thapsigargin-induced endoplasmic reticulum (ER) stress as determined by Atf4 activity, an indicator of cellular stress responses. Overall, these results substantiate previous findings suggesting that CMTM5, rather than contributing to myelin biogenesis, is involved in maintaining axonal integrity. Our study further demonstrates that the knockdown of Cmtm5 in vitro does not modulate oligodendroglial responses to ER stress. These results warrant further investigation into the functional role of CMTM5 during axonal degeneration in the context of demyelinating conditions.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Camundongos , Esclerose Múltipla/genética , Proteínas da Mielina/genética , Encefalomielite Autoimune Experimental/genética , Autopsia , OligodendrogliaRESUMO
Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in regulating cellular metabolism and cell growth and has been identified as an anticancer drug target. Although previous studies have revealed some structures of IGF1R with different ligands, the continuous dynamic conformation change remains unclear. Here, we report 10 distinct structures (7.9-3.6 Å) of IGF1R bound to IGF1 or insulin to reveal the polymorphic conformations of ligand-bound IGF1R. These results showed that the α-CT2, disulfide bond (C670-C670'), and FnIII-2 domains had the most flexible orientations for the conformational change that occurs when ligands bind to the receptor. In addition, we found one special conformation (tentatively named the diverter-switch state) in both complexes, which may be one of the apo-IGF1R forms under ligand-treatment conditions. Hence, these results illustrated the mechanism of how different ligands could bind to human IGF1R and provided a rational template for drug design.
Assuntos
Insulina , Receptor IGF Tipo 1 , Proliferação de Células , Microscopia Crioeletrônica , Humanos , Insulina/metabolismo , Ligantes , Domínios Proteicos , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismoRESUMO
Trace levels of antibiotics are increasingly being detected in aquatic environment and their potential toxicity to aquatic organisms is concerning. Sulfamethoxazole (SMX), a veterinary sulfonamide widely used across the globe, exists ubiquitously in aquatic environment with concentrations up to micrograms per liter. This study aims to investigate the effects of environmentally relevant levels (0.1, 1, 10, 100⯵g/L) of SMX on the health of zebrafish during early development. Our results show that SMX delays the hatchment of embryos and reduces the body length. A dose-response relationship of oxidative stress indicators including total-antioxidant capacity (T-AOC), inducible nitric oxide synthase (iNOS) and total nitric oxide synthase (TNOS), catalase (CAT) has been observed. Additionally, SMX up-regulates the gene expression of several key proinflammatory cytokines and their corresponding proteins including interleukin-1ß (IL-1ß), interferon-γ (IFN-γ) and interleukin-11 (IL-11) and the expression of genes including interleukin-6 (il-6), tumor necrosis factor-α (tnf-α). This indicates that early exposure of SMX may evoke inflammation response in healthy fish. Inhibition of lysozyme and recombination-activating genes (rags) suggests that SMX suppresses the ability of zebrafish to resist pathogen. The reduction of the expression of Toll-like receptors (TLRs) related genes and significant correlations between TLRs and other immune-related genes reveal that TLRs might be an immunoregulator of SMX for zebrafish embryos and larvae. The novelty of this study lies in that early exposure to environmental levels of SMX not only affects the growth and development of zebrafish larvae, but also triggers oxidative stress and inflammation, resulting in a reduction in host immune defense via TLRs in healthy fish.
Assuntos
Peixe-Zebra , Animais , Embrião não Mamífero , Larva , Sulfametoxazol , Poluentes Químicos da ÁguaRESUMO
BACKGROUND: Miscanthus × giganteus is widely recognized as a promising lignocellulosic biomass crop due to its advantages of high biomass production, low environmental impacts, and the potential to be cultivated on marginal land. However, the high costs of bioethanol production still limit the current commercialization of lignocellulosic bioethanol. The lignin in the cell wall and its by-products released in the pretreatment step is the main component inhibiting the enzymatic reactions in the saccharification and fermentation processes. Hence, genetic modification of the genes involved in lignin biosynthesis could be a feasible strategy to overcome this barrier by manipulating the lignin content and composition of M. × giganteus. For this purpose, the essential knowledge of these genes and understanding the underlying regulatory mechanisms in M. × giganteus is required. RESULTS: In this study, MgPAL1, MgPAL5, Mg4CL1, Mg4CL3, MgHCT1, MgHCT2, MgC3'H1, MgCCoAOMT1, MgCCoAOMT3, MgCCR1, MgCCR2, MgF5H, MgCOMT, and MgCAD were identified as the major monolignol biosynthetic genes in M. × giganteus based on genetic and transcriptional evidence. Among them, 12 genes were cloned and sequenced. By combining transcription factor binding site prediction and expression correlation analysis, MYB46, MYB61, MYB63, WRKY24, WRKY35, WRKY12, ERF021, ERF058, and ERF017 were inferred to regulate the expression of these genes directly. On the basis of these results, an integrated model was summarized to depict the monolignol biosynthesis pathway and the underlying regulatory mechanism in M. × giganteus. CONCLUSIONS: This study provides a list of potential gene targets for genetic improvement of lignocellulosic biomass quality of M. × giganteus, and reveals the genetic, transcriptional, and regulatory landscape of the monolignol biosynthesis pathway in M. × giganteus.
RESUMO
OBJECTIVE: To select potential molecules that can target viral spike proteins, which may potentially interrupt the interaction between the human angiotension-converting enzyme 2 (ACE2) receptor and viral spike protein by virtual screening. METHODS: The three-dimensional (3D)-coordinate file of the receptor-binding domain (RBD)-ACE2 complex for searching a suitable docking pocket was firstly downloaded and prepared. Secondly, approximately 15,000 molecular candidates were prepared, including US Food and Drug Administration (FDA)-approved drugs from DrugBank and natural compounds from Traditional Chinese Medicine Systems Pharmacology (TCMSP), for the docking process. Then, virtual screening was performed and the binding energy in Autodock Vina was calculated. Finally, the top 20 molecules with high binding energy and their Chinese medicine (CM) herb sources were listed in this paper. RESULTS: It was found that digitoxin, a cardiac glycoside in DrugBank and bisindigotin in TCMSP had the highest docking scores. Interestingly, two of the CM herbs containing the natural compounds that had relatively high binding scores, Forsythiae fructus and Isatidis radix, are components of Lianhua Qingwen (), a CM formula reportedly exerting activity against severe acute respiratory syndrome (SARS)-Cov-2. Moreover, raltegravir, an HIV integrase inhibitor, was found to have a relatively high binding score. CONCLUSIONS: A class of compounds, which are from FDA-approved drugs and CM natural compounds, that had high binding energy with RBD of the viral spike protein. Our work provides potential candidates for other researchers to identify inhibitors to prevent SARS-CoV-2 infection, and highlights the importance of CM and integrative application of CM and Western medicine on treating COVID-19.