RESUMO
In drug discovery and development, the direct target identification of bioactive small molecules plays a significant role for understanding the mechanism of action, predicting the side effects, and rationally designing more potent compounds. However, due to the complicated regulatory processes in a cell together with thousands of biomacromolecules, target identification is always the major obstacle. New methods and technologies are continuously invented to tackle this problem. Nevertheless, the mainly used tools possess several disadvantages. High synthetic skills are typically required to laboriously synthesize a probe for protein enrichment. To detect the ligand-protein interaction by analyzing proteins' responses to proteolytic or thermal treatment, costly and precise instruments are always necessary. Therefore, convenient and practical techniques are urgently needed. Over the past decades, a strategy using native compounds without the requirement of chemical modification, also termed Native-compound-Coupled Affinity Matrix (NCAM), is developing continuously. Two practical tactics based on "label-free" compounds have been invented and used, that is Photo-cross-linked Small-molecule Affinity Matrix (PSAM) and Native-compound-Coupled CNBr-activated Beads (NCCB). Presently, we will elucidate the characteristics, coupling mechanism, advantages and disadvantages, and future prospect of NCAM in specific target identification and validation.
Assuntos
Descoberta de Drogas , Peptídeo Hidrolases , Proteólise , Moléculas de Adesão de Célula NervosaRESUMO
Agonists of the stimulator of interferon gene (STING) are considered as promising therapeutics for cancer immunotherapy. However, drug-delivery barriers and adverse effects limit the clinical application of STING agonists. Therefore, it is an urgent need to develop an ideal delivery system to deliver STING agonists and avoid side effects. Here, we discovered that STING agonists significantly stimulated type I interferon (IFN) secretion in Clec9a+ dendritic cells (DCs). Then, we designed an engineered peptide-expressed biomimetic cancer cell membrane (EPBM)-coated nanovaccine drug-delivery system (PLGA/STING@EPBM) to deliver STING agonists and tumor antigens to Clec9a+ DCs. The PLGA/STING@EPBM nanovaccine significantly enhanced IFN-stimulated expression of genes and antigen cross-presentation of Clec9a+ DCs, thus eliciting strong antitumor effects in both anti-PD-1-responsive and -resistant tumor models without obvious cytotoxicity. Moreover, the PLGA/STING@EPBM nanovaccine combined with radiotherapy exhibited remarkable synergistic antitumor effects. Our work highlights the great potential of a EPBM-coated nanovaccine for systemic STING agonist delivery as an attractive tool for cancer immunotherapy.
Assuntos
Vacinas Anticâncer , Células Dendríticas , Proteínas de Membrana , Neoplasias , Apresentação de Antígeno , Antígenos de Neoplasias/farmacologia , Humanos , Imunoterapia , Lectinas Tipo C/genética , Proteínas de Membrana/agonistas , Nanopartículas , Neoplasias/terapia , Receptores Mitogênicos/metabolismoRESUMO
Metformin, as the first-line drug for the treatment of type 2 diabetes mellitus, has been shown to possess a capability to activate or inhibit the production of reactive oxygen species (ROS) in different ways. However, the detailed mechanisms of the opposite effect are poorly understood. Here we provide evidence that metformin induces accumulation of ROS by inhibiting the expression of a core antioxidant transcription factor nuclear factor erythroid 2 like 1 (NFE2L1/Nrf1) in human hepatocellular carcinoma HepG2 cells. In the present study, we originally found that the increased ROS induced by metformin was blunted in NFE2L1 knockdown cell line. Furtherly by examining the effects of metformin on endogenous and exogenous NFE2L1, we also found metformin could not only inhibit the transcription of NFE2L1 gene, but also promote the degradation of NFE2L1 protein at the post-transcriptional level, whereas this effect can be reversed by high glucose. The inhibitory effect of metformin on NFE2L1 was investigated to occur through the N-terminal domain (NTD) of NFE2L1 protein, and its downregulation by metformin was in an AMP-activated protein kinase (AMPK)-independent manner. But the activation of AMPK signaling pathway by metformin in NFE2L1 knockdown HepG2 cells is reversed, indicating that NFE2L1 may be an important regulator of AMPK signal. Altogether, this work provides a better understanding of the relationship between metformin and oxidative stress, and hence contributes to translational study of metformin through its hypoglycemic and tumor suppressive effects.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Metformina/farmacologia , Fator 1 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fator 1 Relacionado a NF-E2/genética , Transdução de SinaisRESUMO
BACKGROUND: TIGIT, as a novel immune checkpoint molecule involved in T cell and NK cell anergy, could induce the immune tolerance and escape through binding with its ligand PVR. Blockade of TIGIT/PVR is considered as a promising strategy in cancer immunotherapy. However, to facilitate the design of inhibitors targeting TIGIT/PVR, the structural characteristics and binding mechanism still need to be further studied. METHODS: In this study, molecular dynamics (MD) simulations and in silico mutagenesis were used to analyze the interaction between TIGIT and its ligand PVR. Then, PVR mutants were designed and their activities were determined by using TIGIT overexpressed Jurkat cells. RESULTS: The results suggested that the loops of PVR (CC' loop, C'Câ³ loop, and FG loop) underwent a large intra-molecular rearrangement, and more hydrogen bond crosslinking between PVR and TIGIT were formed during MD simulations. The potential residues for PVR to interact with TIGIT were identified and utilized to predict high affinity PVR mutants. Through the biological activity evaluation, four PVR mutants (PVRS72W, PVRS72R, PVRG131V and PVRS132Q) with enhanced affinity to TIGIT were discovered, which could elicit more potent inhibitory effects compared with the wild type PVR. CONCLUSIONS: The MD simulations analysis provided new insights into the TIGIT/PVR interaction model, and the identified PVR mutants (PVRS72W, PVRS72R, PVRG131V and PVRS132Q) could serve as new candidates for immunotherapy to block TIGIT/PVR. Video Abstract.
Assuntos
Receptores Imunológicos/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Animais , Células CHO , Técnicas de Cocultura , Desenho Assistido por Computador , Cricetulus , Células HEK293 , Humanos , Células Jurkat , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Receptores Imunológicos/química , Receptores Virais/químicaRESUMO
BACKGROUND: Inhibitors targeting immune checkpoint were proved effective in cancer immunotherapy, such as PD-1/PD-L1 blockade. The novel immune checkpoint TIGIT/PVR plays critical roles in suppressing the anti-tumor effects of CD8+ T and NK cells, and dual blockade of TIGIT/PVR and PD-1/PD-L1 by antibody can elicit synergistic effects in tumor models and clinical trials. However, small molecules for TIGIT/PVR blockade have not been investigated. METHODS: The expression of PVR in tumors were analyzed by using TCGA, Oncomine and GEO database, and in cancer cell lines examined by flow cytometry. Natural product compounds were docked to PVR for virtual screening by using the software Molecular Operating Environment (MOE). Candidate compounds were further tested by biolayer interferometry-based binding assay, microscale thermophoresis assay and cell based blocking assay. The in vitro activity of the candidate compound was determined by MTT, peripheral blood mononuclear cells (PBMCs) activation assay and coculture assay. The anti-tumor effects and mechanism were also investigated by using MC38 tumor-bearing mice model and immune cell depletion tumor model. RESULTS: PVR was over-expressed in many tumor tissues and cancer cell lines, making it a promising therapeutic target. Through virtual screening, binding, and blocking assay, liothyronine was discovered to bind PVR and block the interaction of TIGIT/PVR. Liothyronine could enhance the function of CD4+ and CD8+ T cells in PBMCs. Besides, in the Jurkat-hTIGIT and CHOK1-hPVR coculture assay, liothyronine could reverse the IL-2 secretion inhibition resulted by TIGIT/PVR ligation. Although had no influence on the proliferation of tumor cells in vitro, liothyronine could significantly inhibit tumor growth when administrated in vivo, by enhancing CD8+ T cell infiltration and immune responses in the tumor bearing mice. The immune cell depletion model showed that the anti-tumor effects of liothyronine depends on CD4+ T cells, CD8+ T cells and NK cells. CONCLUSIONS: A small molecule liothyronine was discovered to serve as a potential candidate for cancer immunotherapy by blocking the immune checkpoint TIGIT/PVR. Video abstract.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/terapia , Receptores Imunológicos/antagonistas & inibidores , Receptores Virais/antagonistas & inibidores , Tri-Iodotironina/uso terapêutico , Animais , Linhagem Celular Tumoral , Reposicionamento de Medicamentos , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Receptores Imunológicos/imunologia , Receptores Virais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Tri-Iodotironina/farmacologiaRESUMO
The antioxidant transcription factor NFE2L1 (also called Nrf1) acts as a core regulator of redox signaling and metabolism homeostasis, and thus, its dysfunction results in multiple systemic metabolic diseases. However, the molecular mechanism(s) by which NFE2L1 regulates glycose and lipid metabolism remains elusive. Here, we found that loss of NFE2L1 in human HepG2 cells led to a lethal phenotype upon glucose deprivation and NFE2L1 deficiency could affect the uptake of glucose. Further experiments revealed that glycosylation of NFE2L1 enabled it to sense the energy state. These results indicated that NFE2L1 can serve as a dual sensor and regulator of glucose homeostasis. The transcriptome, metabolome, and seahorse data further revealed that disruption of NFE2L1 could reprogram glucose metabolism to aggravate the Warburg effect in NFE2L1-silenced hepatoma cells, concomitant with mitochondrial damage. Co-expression and Co-immunoprecipitation experiments demonstrated that NFE2L1 could directly interact and inhibit AMPK. Collectively, NFE2L1 functioned as an energy sensor and negatively regulated AMPK signaling through directly interacting with AMPK. The novel NFE2L1/AMPK signaling pathway delineate the mechanism underlying of NFE2L1-related metabolic diseases and highlight the crosstalk between redox homeostasis and metabolism homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP , Fator 1 Relacionado a NF-E2 , Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Glucose , Homeostase , Fator 1 Relacionado a NF-E2/metabolismo , Transdução de SinaisRESUMO
Dendritic cells (DCs) can process the antigens of cancer vaccine and thus stimulate the CD8+ T cells to recognize and kill the tumor cells that express these antigens. However, lack of promising carriers for presenting the antigens to DCs is one of the main barriers to the development of clinically effective cancer vaccines. Another limitation is the risk of inflammatory side effects induced by the adjuvants. It is still unclear how we can develop ideal adjuvant-free DC vaccine carriers without adjuvants. Methods: A 12-mer peptide carrier (CBP-12) with high affinity for Clec9a expressed on DCs was developed using an in silico rational optimization method. The therapeutic effects of the adjuvant-free vaccine comprising CBP-12 and exogenous or endogenous antigenic peptides were investigated in terms of antigen cross-presentation efficacy, specific cytotoxic T lymphocyte response, and antitumor activity. We also explored the mechanism involved in the antitumor effects of the adjuvant-free CBP-12 vaccine. Finally, we assessed the effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Results: Here, we developed CBP-12 as a vaccine carrier that enhanced the uptake and cross-presentation of the antigens, thus inducing strong CD8+ T cell responses and antitumor effects in both anti-PD-1-responsive (B16-OVA) and -resistant (B16) models, even in adjuvant-free conditions. CBP-12 bound to and activated Clec9a, thereby stimulating Clec9a+ DC to product IL-21, but not IL-12 by activating of Syk. The antitumor effects of the CBP-12 conjugated peptide vaccines could be blocked by an IL-21 neutralizing antibody. We also observed the synergistic antitumor effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Conclusions: CBP-12 could serve as an adjuvant-free peptide vaccine carrier for cancer immunotherapy.
Assuntos
Vacinas Anticâncer , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos , Interleucinas/imunologia , Lectinas Tipo C/imunologia , Melanoma Experimental/imunologia , Peptídeos , Receptores Imunológicos/imunologia , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/imunologia , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Feminino , Interleucinas/genética , Lectinas Tipo C/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Knockout , Peptídeos/imunologia , Peptídeos/farmacologia , Receptores Imunológicos/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Quinase Syk/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/farmacologiaRESUMO
Tryptophan 2,3-dioxygnease 2 (TDO2) is specific for metabolizing tryptophan to kynurenine (KYN), which plays a critical role in mediating immune escape of cancer. Although accumulating evidence demonstrates that TDO2 overexpression is implicated in the development and progression of multiple cancers, its tumor-promoting role in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we observed that TDO2 was overexpressed in ESCC tissues and correlated significantly with lymph node metastasis, advanced clinical stage, and unfavorable prognosis. Functional experiments showed that TDO2 promoted tumor cell proliferation, migration, and colony formation, which could be prevented by inhibition of TDO2 and aryl hydrocarbon receptor (AHR). Further experimentation demonstrated that TDO2 could promote the tumor growth of KYSE150 tumor-bearing model, tumor burden of C57BL/6 mice with ESCC induced by 4-NQO, enhance the expression of phosphorylated AKT, with subsequent phosphorylation of GSK3ß, and polarization of M2 macrophages by upregulating interleukin-8 (IL-8) to accelerate tumor progression in the tumor microenvironment (TME). Collectively, our results discovered that TDO2 could upregulate IL-8 through AKT/GSK3ß to direct the polarization of M2 macrophages in ESCC, and suggested that TDO2 could represent as an attractive therapeutic target and prognostic marker to ESCC.
RESUMO
BACKGROUND: Immunotherapy has achieved remarkable advances via a variety of strategies against tumor cells that evade immune surveillance. As important innate immune cells, macrophages play important roles in maintaining homeostasis, preventing pathogen invasion, resisting tumor cells and promoting adaptive immune response. CD47 is found to be overexpressed on tumor cells and act as a don't eat me' signal, which contributes to immune evasion. Macrophages mediated phagocytosis via blockade CD47/SIRPα (signal regulatory protein alpha) interaction was proved to induce effective antitumor immune response. METHODS: A novel peptide pep-20, specifically targeting CD47 and blocking CD47/SIRPα interaction, was identified via high-throughput phage display library bio-panning. The capability to enhance the macrophage-mediated phagocytosis activities and antitumor effects of pep-20 were investigated. The mechanism of pep-20 to induce T-cell response was explored by ex vivo analysis and confirmed via macrophage depleting strategy. The structure-activity relationship and D-amino acid substitution of pep-20 were also studied. The antitumor effects and mechanism of a proteolysis resistant D-amino acid derivate pep-20-D12 combined with irradiation (IR) were also investigated. RESULTS: Pep-20 showed remarkable enhancement of macrophage-mediated phagocytosis to both solid and hematologic tumor cells in vitro, and inhibited tumor growth in immune-competent tumor-bearing mice. Furthermore, pep-20 promoted macrophages to mobilize the antitumor T-cell response with minimal toxicity. Furthermore, systemic administration of the derivate pep-20-D12 showed robust synergistic antitumor efficacy in combination with IR. CONCLUSION: In summary, these results demonstrated that CD47/SIRPα blocking peptides, pep-20 and its derivate, could serve as promising candidates to promote macrophages-mediated phagocytosis and immune response in cancer immunotherapy.
Assuntos
Antígeno CD47/metabolismo , Imunoterapia/métodos , Neoplasias/imunologia , Peptídeos/imunologia , Animais , Humanos , CamundongosRESUMO
Immunotherapy emerges as an effective avenue for tumor elimination and has many advantages compared with traditional surgery, radiotherapy, and chemotherapy. Tumor vaccines play an important role in cancer immunotherapy, while the application of tumor vaccines in clinical usage is limited because only limited response can be induced by primary tumor antigens. Accordingly, it is a key point to activate T-cell response with some novel tumor vaccines. Here, we applied phage display biopanning and screened a peptide (TY) that could combine with bone-marrow-derived-dendritic-cells (BMDCs) specifically and spleenic DCs. Then we developed mesoporous silica nanoparticles (MSN-TY/OVA/CpG), with peptide TY and OVA/CpG to target and activate DCs, respectively. Our results showed that the nanoparticles (NPs) could be specifically absorbed by DC in vitro, which enhanced the maturation and activation of DCs in vitro and in vivo. The in vitro study demonstrated the efficiency of nanoparticles in antigen uptake by BMDCs and in the activation of antigen-specific cytotoxic CD8+ T cells. Moreover, MSN-TY/OVA/CpG could activate antigen-specific CD8+ T cells and elicited the cytotoxic T lymphocyte (CTL) priming in naive C57BL/6J mice. Therapeutic application of MSN-TY/OVA/CpG enhanced the activation of DCs and the introducing of CD8+ T cell-mediated immune response to promote tumor elimination, prolong survival of tumor-bearing mice, and cause less systemic toxicity. All these results showed that the targeted nanovaccines could deliver antigen into DCs and activate cancer immunotherapy.