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1.
Int J Mol Sci ; 25(12)2024 Jun 16.
Article in English | MEDLINE | ID: mdl-38928339

ABSTRACT

Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (Kd = 1.28 × 10-8 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein-peptide interaction, which highly specifically interacts with the Tag7 protein (Kd = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide-peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases.


Subject(s)
Apoptosis , Receptors, Tumor Necrosis Factor, Type I , Humans , Receptors, Tumor Necrosis Factor, Type I/metabolism , Receptors, Tumor Necrosis Factor, Type I/chemistry , Apoptosis/drug effects , Protein Binding , Molecular Docking Simulation , Cell Line, Tumor , Peptides/chemistry , Peptides/pharmacology , Peptides/metabolism , Molecular Dynamics Simulation , Signal Transduction/drug effects , Necroptosis/drug effects , Oligopeptides/chemistry , Oligopeptides/pharmacology , Oligopeptides/metabolism , Cytokines
2.
Mol Pharm ; 20(4): 1884-1897, 2023 04 03.
Article in English | MEDLINE | ID: mdl-36897792

ABSTRACT

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.


Subject(s)
Receptors, Tumor Necrosis Factor, Type II , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Receptors, Tumor Necrosis Factor, Type II/chemistry , Receptors, Tumor Necrosis Factor, Type II/metabolism , Ligands , Tumor Necrosis Factor Inhibitors , Tumor Necrosis Factor-alpha/metabolism
3.
Nat Prod Res ; 36(22): 5817-5822, 2022 Nov.
Article in English | MEDLINE | ID: mdl-34937447

ABSTRACT

Tissue damage occurs in COVID-19 patients due to nsp3-induced Fas-FasL interaction/TNF-related apoptosis. Presently, possible therapeutic-drug, nigellidine against was screened by bioinformatics studies COVID-19. Atomic-Contact-Energy (ACE) and binding-blocking effects were explored of nigellidine (Nigella sativa L.) in the active/catalytic sites of viral-protein nsp3 and host inflammatory/apoptotic signaling-molecules Fas/TNF receptors TNFR1/TNFR2. A control binding/inhibition of Oseltamivir to influenza-virus neuraminidase was compared here. In AutoDock, Oseltamivir binding-energy (BE) and inhibition-constant (KI) was -4.12 kcal/mol and 959.02. The ACE values (PatchDock) were -167.02/-127.61/-124.91/-122.17/-54.81/-47.07. The nigellidine BE/KI with nsp3 was -7.61 and 2.66, respectively (ACE values were -221.40/-215.62/-113.28). Nigellidine blocked FAS dimer by binding with a BE value of -7.41 kcal/mol. Its strong affinities to TNFR1 (-6.81) and TNFR2 (-5.1) are demonstrated. Our present data suggest that nigellidine may significantly block the TNF-induced inflammatory/Fas-induced apoptotic death-signaling in comparison with a positive-control drug Oseltamivir. Further studies are necessary before proposing nigellidine as medical drug.


Subject(s)
COVID-19 Drug Treatment , Cuminum , Nigella sativa , Humans , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Receptors, Tumor Necrosis Factor, Type I/pharmacology , Receptors, Tumor Necrosis Factor, Type II/metabolism , Receptors, Tumor Necrosis Factor, Type II/pharmacology , Nigella sativa/metabolism , Cuminum/metabolism , SARS-CoV-2 , Oseltamivir/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Apoptosis , Seeds/metabolism , Virus Replication
4.
J Mol Recognit ; 34(10): e2922, 2021 10.
Article in English | MEDLINE | ID: mdl-34132435

ABSTRACT

Using a rat model of peritonitis, we herein report the inflammatory effect induced by the lectin isolated from Vatairea guianensis (VGL) seeds in the context of interactions between VGL and both toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1). Peritoneal macrophages were stimulated with VGL for dose-dependent gene expression and release of TNF-α. In vivo results showed that VGL (1 mg/kg; intraperitoneal) induced peritonitis in female Wistar rats. Leukocyte migration, macrophage activation, and protein leakage were measured 3 and 6 hours after induction. In vitro, peritoneal macrophages were stimulated with VGL for gene expression and TNF-α dosage (mean ± SEM (n = 6), analysis of variance, and Bonferroni's test (P < .05)). In silico, VGL structure was applied in molecular docking with representative glycans. It was found that (a) VGL increases vascular permeability and stimulates leukocyte migration, both rolling and adhesion; (b) lectin-induced neutrophil migration occurs via macrophage stimulation, both in vitro and in vivo; (c) lectin interacts with TLR4 and TNFR1; and (d) stimulates TNF-α gene expression (RT-PCR) and release from peritoneal macrophages. Thus, upon lectin-glycan binding on the cell surface, our results suggest that VGL induces an acute inflammatory response, in turn activating the release of peritoneal macrophages via TNF-α and TLR and/or TNFR receptor pathways.


Subject(s)
Fabaceae/chemistry , Glycoconjugates/metabolism , Macrophages, Peritoneal/drug effects , Plant Lectins/pharmacology , Animals , Cell Movement/drug effects , Cells, Cultured , Disease Models, Animal , Female , Gene Expression Regulation/drug effects , Glycoconjugates/chemistry , Leukocytes/drug effects , Macrophages, Peritoneal/metabolism , Peritonitis/chemically induced , Peritonitis/metabolism , Peritonitis/pathology , Plant Lectins/chemistry , Plant Lectins/metabolism , Rats, Wistar , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
5.
Sci Rep ; 11(1): 12256, 2021 06 10.
Article in English | MEDLINE | ID: mdl-34112868

ABSTRACT

Proteins are tiny players involved in the activation and deactivation of multiple signaling cascades through interactions in cells. The TNFR1 and MADD interact with each other and mediate downstream protein signaling pathways which cause neuronal cell death and Alzheimer's disease. In the current study, a molecular docking approach was employed to explore the interactive behavior of TNFR1 and MADD proteins and their role in the activation of downstream signaling pathways. The computational sequential and structural conformational results revealed that Asp400, Arg58, Arg59 were common residues of TNFR1 and MADD which are involved in the activation of downstream signaling pathways. Aspartic acid in negatively charged residues is involved in the biosynthesis of protein. However, arginine is a positively charged residue with the potential to interact with oppositely charged amino acids. Furthermore, our molecular dynamic simulation results also ensured the stability of the backbone of TNFR1 and MADD death domains (DDs) in binding interactions. This DDs interaction mediates some conformational changes in TNFR1 which leads to the activation of mediators proteins in the cellular signaling pathways. Taken together, a better understanding of TNFR1 and MADD receptors and their activated signaling cascade may help treat Alzheimer's disease. The death domains of TNFR1 and MADD could be used as a novel pharmacological target for the treatment of Alzheimer's disease by inhibiting the MAPK pathway.


Subject(s)
Death Domain Receptor Signaling Adaptor Proteins/chemistry , Guanine Nucleotide Exchange Factors/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Conformation , Protein Interaction Domains and Motifs , Receptors, Tumor Necrosis Factor, Type I/chemistry , Amino Acid Sequence , Binding Sites , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Humans , Models, Biological , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , Structure-Activity Relationship
6.
Nat Commun ; 12(1): 582, 2021 01 25.
Article in English | MEDLINE | ID: mdl-33495441

ABSTRACT

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.


Subject(s)
Protein Multimerization/drug effects , Receptors, Tumor Necrosis Factor, Type I/chemistry , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology , Tumor Necrosis Factor-alpha/chemistry , Algorithms , Animals , Binding, Competitive/drug effects , Humans , Models, Molecular , Protein Binding/drug effects , Protein Conformation/drug effects , Receptors, Tumor Necrosis Factor, Type I/metabolism , Small Molecule Libraries/chemistry , Tumor Necrosis Factor-alpha/metabolism
7.
Nat Commun ; 12(1): 583, 2021 01 25.
Article in English | MEDLINE | ID: mdl-33495445

ABSTRACT

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.


Subject(s)
Antibodies, Monoclonal/metabolism , Multiprotein Complexes/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Small Molecule Libraries/metabolism , Tumor Necrosis Factor-alpha/metabolism , Antibodies, Monoclonal/pharmacology , Cells, Cultured , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/metabolism , HEK293 Cells , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Protein Binding/drug effects , Protein Conformation/drug effects , Receptors, Tumor Necrosis Factor, Type I/chemistry , Signal Transduction/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Tumor Necrosis Factor-alpha/chemistry
8.
Mol Nutr Food Res ; 65(7): e2000425, 2021 04.
Article in English | MEDLINE | ID: mdl-33465830

ABSTRACT

SCOPE: Human milk oligosaccharides (hMOs) can attenuate inflammation by modulating intestinal epithelial cells, but the mechanisms of action are not well-understood. Here, the effects of hMOs on tumor necrosis factor-α (TNF-α) induced inflammatory events in gut epithelial cells are studied. METHODS AND RESULTS: The modulatory effects of 2'-fucosyllactose, 3-fucosyllactose (3-FL), 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), lactodifucotetraose (LDFT), and lacto-N-triaose (LNT2) on immature (FHs 74 Int) and adult (T84) intestinal epithelial cells with or without TNF-α are determined. Interleukin-8 (IL-8) secretion in FHs 74 Int and T84 are quantified to determine hMO induced attenuation of inflammatory events by ELISA. 3-FL, LNnT, and LDFT significantly attenuate TNF-α induced inflammation in FHs 74 Int, while LNT2 induces IL-8 secretion in T84. In addition, microscale thermophoresis assays and ELISA are used to study the possible mechanisms of interaction between effective hMOs and tumor necrosis factor receptor 1 (TNFR1). 3-FL, LNnT, and LDFT exert TNFR1 ectodomain shedding while LNnT also shows binding affinity to TNFR1 with a Kd of 900 ± 660 nM. CONCLUSION: The findings indicate that specific hMO types attenuate TNF-α induced inflammation in fetal gut epithelial cells through TNFR1 in a hMO structure-dependent fashion suggest possibilities to apply hMOs in management of TNF-α dependent diseases.


Subject(s)
Intestinal Mucosa/cytology , Milk, Human/chemistry , Oligosaccharides/pharmacology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Cell Line , Cell Survival , Gastroenteritis/drug therapy , Humans , Hydrolysis , Interleukin-8/metabolism , Intestinal Mucosa/drug effects , Intestinal Mucosa/embryology , Oligosaccharides/chemistry , Protein Domains , Receptors, Tumor Necrosis Factor, Type I/chemistry , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/adverse effects
9.
Angew Chem Int Ed Engl ; 60(12): 6733-6743, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33331089

ABSTRACT

Selective modulation of ligand-receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein-ligand with cell-identity recognition and receptor-mediated signaling in a cell-type-specific manner. Using embedded DNA-based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell-surface proteins via Boolean logic circuits. Therefore, it achieves cell-specific signal modulation by quickly unlocking the protein-ligand in proximity to the target cell-surface to activate its cognate receptor. As a proof of concept, we non-genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell-specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor-α (TNFα) to induce programmed cell death of target cell subpopulations through cell-specific modulation of TNFR1 signaling.


Subject(s)
DNA/metabolism , Hepatocyte Growth Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , DNA/chemistry , Hepatocyte Growth Factor/chemistry , Humans , Ligands , Models, Molecular , Receptors, Tumor Necrosis Factor, Type I/chemistry , Signal Transduction
10.
Methods Mol Biol ; 2248: 121-137, 2021.
Article in English | MEDLINE | ID: mdl-33185872

ABSTRACT

Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor-receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor-receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.


Subject(s)
Biosensing Techniques , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Receptors, Tumor Necrosis Factor, Type I/chemistry , Cell Line , Computational Biology/methods , Drug Discovery/methods , Fluorescence Resonance Energy Transfer , Fluorescent Antibody Technique , Gene Expression , Genes, Reporter , High-Throughput Nucleotide Sequencing , Humans , Ligands , Microscopy, Fluorescence , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Small Molecule Libraries , Software , Structure-Activity Relationship
11.
Sci Signal ; 13(614)2020 01 14.
Article in English | MEDLINE | ID: mdl-31937565

ABSTRACT

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.


Subject(s)
Cell Membrane/drug effects , Protein Multimerization , Receptors, Tumor Necrosis Factor, Type I/metabolism , Single Molecule Imaging/methods , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis/drug effects , Cell Membrane/metabolism , Cells, Cultured , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , HeLa Cells , Humans , Mice, Knockout , Mice, Transgenic , Models, Molecular , Mutation , NF-kappa B/metabolism , Protein Binding , Protein Transport/drug effects , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/genetics , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/metabolism
12.
Protein Sci ; 29(6): 1401-1415, 2020 06.
Article in English | MEDLINE | ID: mdl-31960514

ABSTRACT

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that plays a key role in the regulation of the inflammatory pathway. While inhibition of TNFR1 has been the focus of many studies for the treatment of autoimmune diseases such as rheumatoid arthritis, activation of the receptor is important for the treatment of immunodeficiency diseases such as HIV and neurodegenerative diseases such as Alzheimer's disease where a boost in immune signaling is required. In addition, activation of other TNF receptors such as death receptor 5 or FAS receptor is important for cancer therapy. Here, we used a previously established TNFR1 fluorescence resonance energy transfer (FRET) biosensor together with a fluorescence lifetime technology as a high-throughput screening platform to identify a novel small molecule that activates TNFR1 by increasing inter-monomeric spacing in a ligand-independent manner. This shows that the conformational rearrangement of pre-ligand assembled receptor dimers can determine the activity of the receptor. By probing the interaction between the receptor and its downstream signaling molecule (TRADD) our findings support a new model of TNFR1 activation in which varying conformational states of the receptor act as a molecular switch in determining receptor function.


Subject(s)
Receptors, Tumor Necrosis Factor, Type I/metabolism , Biosensing Techniques , Cells, Cultured , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Models, Molecular , Protein Conformation , Receptors, Tumor Necrosis Factor, Type I/chemistry
13.
Proteins ; 88(5): 698-709, 2020 05.
Article in English | MEDLINE | ID: mdl-31710744

ABSTRACT

The interactions between tumor necrosis factors (TNFs) and their corresponding receptors (TNFRs) play a pivotal role in inflammatory responses. Upon ligand binding, TNFR receptors were found to form oligomers on cell surfaces. However, the underlying mechanism of oligomerization is not fully understood. In order to tackle this problem, molecular dynamics (MD) simulations have been applied to the complex between TNF receptor-1 (TNFR1) and its ligand TNF-α as a specific test system. The simulations on both all-atom (AA) and coarse-grained (CG) levels achieved the similar results that the extracellular domains of TNFR1 can undergo large fluctuations on plasma membrane, while the dynamics of TNFα-TNFR1 complex is much more constrained. Using the CG model with the Martini force field, we are able to simulate the systems that contain multiple TNFα-TNFR1 complexes with the timescale of microseconds. We found that complexes can aggregate into oligomers on the plasma membrane through the lateral interactions between receptors at the end of the CG simulations. We suggest that this spatial organization is essential to the efficiency of signal transduction for ligands that belong to the TNF superfamily. We further show that the aggregation of two complexes is initiated by the association between the N-terminal domains of TNFR1 receptors. Interestingly, the cis-interfaces between N-terminal regions of two TNF receptors have been observed in the previous X-ray crystallographic experiment. Therefore, we provide supportive evidence that cis-interface is of functional importance in triggering the receptor oligomerization. Taken together, our study brings insights to understand the molecular mechanism of TNF signaling.


Subject(s)
Cell Membrane/chemistry , Molecular Dynamics Simulation , Receptors, Tumor Necrosis Factor, Type I/chemistry , Tumor Necrosis Factor-alpha/chemistry , Binding Sites , Cell Membrane/metabolism , Humans , Kinetics , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Receptors, Tumor Necrosis Factor, Type I/metabolism , Thermodynamics , Tumor Necrosis Factor-alpha/metabolism
14.
Sci Signal ; 12(592)2019 07 30.
Article in English | MEDLINE | ID: mdl-31363069

ABSTRACT

Tumor necrosis factor receptor 1 (TNFR1) is a central mediator of the inflammatory pathway and is associated with several autoimmune diseases such as rheumatoid arthritis. A revision to the canonical model of TNFR1 activation suggests that activation involves conformational rearrangements of preassembled receptor dimers. Here, we identified small-molecule allosteric inhibitors of TNFR1 activation and probed receptor dimerization and function. Specifically, we used a fluorescence lifetime-based high-throughput screen and biochemical, biophysical, and cellular assays to identify small molecules that noncompetitively inhibited the receptor without reducing ligand affinity or disrupting receptor dimerization. We also found that residues in the ligand-binding loop that are critical to the dynamic coupling between the extracellular and the transmembrane domains played a key gatekeeper role in the conformational dynamics associated with signal propagation. Last, using a simple structure-activity relationship analysis, we demonstrated that these newly found molecules could be further optimized for improved potency and specificity. Together, these data solidify and deepen the new model for TNFR1 activation.


Subject(s)
Protein Multimerization , Receptors, Tumor Necrosis Factor, Type I/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type I/chemistry , HEK293 Cells , Humans , Protein Domains , Protein Structure, Quaternary
15.
J Transl Med ; 17(1): 215, 2019 07 02.
Article in English | MEDLINE | ID: mdl-31266509

ABSTRACT

BACKGROUND: Tumor necrosis factor α (TNFα) is a multifunctional cytokine with a potent pro-inflammatory effect. It is a validated therapeutic target molecule for several disorders related to autoimmunity and inflammation. TNFα-TNF receptor-1 (TNFR1) signaling contributes to the pathological processes of these disorders. The current study is focused on finding novel small molecules that can directly bind to TNFα and/or TNFR1, preventing the interaction between TNFα or TNFR1, and regulating downstream signaling pathways. METHODS: Cheminformatics pipeline (pharmacophore modeling, virtual screening, molecular docking and in silico ADMET analysis) was used to screen for novel TNFα and TNFR1 inhibitors in the Zinc database. The pharmacophore-based models were generated to screen for the best drug like compounds in the Zinc database. RESULTS: The 39, 37 and 45 best hit molecules were mapped with the core pharmacophore features of TNFα, TNFR1, and the TNFα-TNFR1 complex respectively. They were further evaluated by molecular docking, protein-ligand interactions and in silico ADMET studies. The molecular docking analysis revealed the binding energies of TNFα, TNFR1 and the TNFα-TNFR1 complex, the basis of which was used to select the top five best binding energy compounds. Furthermore, in silico ADMET studies clearly revealed that all 15 compounds (ZINC09609430, ZINC49467549, ZINC13113075, ZINC39907639, ZINC25251930, ZINC02968981, ZINC09544246, ZINC58047088, ZINC72021182, ZINC08704414, ZINC05462670, ZINC35681945, ZINC23553920, ZINC05328058, and ZINC17206695) satisfied the Lipinski rule of five and had no toxicity. CONCLUSIONS: The new selective TNFα, TNFR1 and TNFα-TNFR1 complex inhibitors can serve as anti-inflammatory agents and are promising candidates for further research.


Subject(s)
Anti-Inflammatory Agents/isolation & purification , Computational Chemistry/methods , Drug Discovery/methods , Multiprotein Complexes/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type I/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Anti-Inflammatory Agents/analysis , Binding, Competitive , Catalytic Domain/drug effects , Computational Biology/methods , Computer Simulation , Drug Evaluation, Preclinical/methods , Humans , Ligands , Models, Molecular , Molecular Docking Simulation/methods , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/metabolism
16.
BMC Vet Res ; 15(1): 68, 2019 Feb 28.
Article in English | MEDLINE | ID: mdl-30819151

ABSTRACT

BACKGROUND: Refractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α. RESULTS: Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig's anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1ß and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α. CONCLUSIONS: Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application.


Subject(s)
Cell Death/drug effects , Cytokines/drug effects , Tumor Necrosis Factor Decoy Receptors/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis/drug effects , Cattle , Cell Line , Cells, Cultured , Cytokines/metabolism , Fibroblasts , Gene Expression , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/pharmacology , Inflammation/drug therapy , Inflammation/metabolism , Leukocytes, Mononuclear , Mice , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/pharmacology , Receptors, Tumor Necrosis Factor, Type II/chemistry , Receptors, Tumor Necrosis Factor, Type II/pharmacology
17.
EBioMedicine ; 41: 556-570, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30826358

ABSTRACT

BACKGROUND: Osteoarthritis (OA) is a common degenerative disease, and tumor necrosis factor (TNF-α) is known to play a critical role in OA. Cortistatin (CST) is a neuropeptide discovered over 20  years ago, which plays a vital role in inflammatory reactions. However, it is unknown whether CST is involved in cartilage degeneration and OA development. METHODS: The interaction between CST and TNF-α receptors was investigated through Coimmunoprecipitation and Biotin-based solid-phase binding assay. Western blot, Real-time PCR, ELISA, immunofluorescence staining, nitrite production assay and DMMB assay of GAG were performed for the primary chondrocyte experiments. Surgically induced and spontaneous OA models were established and western blot, flow cytometry, Real-time PCR, ELISA, immunohistochemistry and fluorescence in vivo imaging were performed for in vivo experiments. FINDINGS: CST competitively bound to TNFR1 as well as TNFR2. CST suppressed proinflammatory function of TNF-α. Both spontaneous and surgically induced OA models indicated that deficiency of CST led to an accelerated OA-like phenotype, while exogenous CST attenuated OA development in vivo. Additionally, TNFR1- and TNFR2-knockout mice were used for analysis and indicated that TNFRs might be involved in the protective role of CST in OA. CST inhibited activation of the NF-κB signaling pathway in OA. INTERPRETATION: This study provides new insight into the pathogenesis and therapeutic strategy of cartilage degenerative diseases, including OA. FUND: The National Natural Science Foundation of China, the Natural Science Foundation of Shandong Province, Key Research and Development Projects of Shandong Province and the Cross-disciplinary Fund of Shandong University.


Subject(s)
Neuropeptides/metabolism , Osteoarthritis/pathology , Receptors, Tumor Necrosis Factor, Type II/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Animals , Cartilage, Articular/cytology , Caspase 3/metabolism , Chondrocytes/cytology , Chondrocytes/drug effects , Chondrocytes/metabolism , Disease Models, Animal , Humans , Interleukin-1beta/blood , Knee Joint/pathology , Matrix Metalloproteinase 13 , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Neuropeptides/genetics , Neuropeptides/pharmacology , Neuropeptides/therapeutic use , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type II/chemistry , Receptors, Tumor Necrosis Factor, Type II/genetics , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology
18.
Nat Commun ; 10(1): 860, 2019 02 26.
Article in English | MEDLINE | ID: mdl-30808860

ABSTRACT

Target-centric drug development strategies prioritize single-target potency in vitro and do not account for connectivity and multi-target effects within a signal transduction network. Here, we present a systems biology approach that combines transcriptomic and structural analyses with live-cell imaging to predict small molecule inhibitors of TNF-induced NF-κB signaling and elucidate the network response. We identify two first-in-class small molecules that inhibit the NF-κB signaling pathway by preventing the maturation of a rate-limiting multiprotein complex necessary for IKK activation. Our findings suggest that a network-centric drug discovery approach is a promising strategy to evaluate the impact of pharmacologic intervention in signaling.


Subject(s)
NF-kappa B/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/metabolism , CRISPR-Cas Systems , Cell Line , Drug Development/methods , Gene Knock-In Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Models, Molecular , Protein Interaction Domains and Motifs/drug effects , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction/physiology , Systems Biology , TNF Receptor-Associated Factor 2/chemistry , TNF Receptor-Associated Factor 2/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors
19.
Protein Expr Purif ; 155: 21-26, 2019 03.
Article in English | MEDLINE | ID: mdl-30414969

ABSTRACT

The interaction between TNF-α with TNFR1 triggers important signaling pathways inducing diverse cellular phenomena including inflammation, apoptosis, etc., and is involved in the pathogenesis and progression of numerous autoimmune diseases. The extracellular domain (ECD) of TNFR has been successfully used to clinically treat such TNF-associated diseases. However, large-scale production of these biological material via eukaryotic cell expression systems is usually costly owing to the culture medium and complicated growth conditions. This study aimed to extract pure soluble human TNFR1-ECD and investigate its biological activity, using a prokaryotic expression system. Recombinant vector pMCSG7-TNFR1-ECD was constructed via ligation-independent cloning. The His-tag fusion protein was expressed in E. coli and localized in inclusion bodies. Recombinant TNFR1-ECD was refolded and purified via nickel-affinity chromatography, tag cleavage, followed by cation-exchange chromatography or size-exclusion chromatography. A purity of over 95% and a yield of 9.3 mg protein per liter of bacterial culture media was obtained. The purified protein showed significant affinity of 2.15 nM towards human TNF-α and inhibited TNF-α-mediated cytotoxicity in L929 cells, with an ED50 of 0.10 µg/ml. It formed a self-associated oligomer with a KD of 1.15 µM, detected via microscale thermophoresis. We thus established a highly efficient approach to construct, express, and purify the recombinant protein of human TNFR1-ECD from a prokaryotic system. The antagonistic bioactivities in vitro indicate this protein as a prospective molecules for drug research against autoimmune diseases characterized by TNF-α overexpression.


Subject(s)
Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Cell Line , Cloning, Molecular/methods , Escherichia coli/genetics , Fibroblasts/metabolism , Humans , Protein Domains , Protein Refolding , Receptors, Tumor Necrosis Factor, Type I/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolism
20.
Bull Exp Biol Med ; 165(3): 386-389, 2018 Jul.
Article in English | MEDLINE | ID: mdl-30003423

ABSTRACT

Death receptor 5 (DR5) is a promising target for antitumor therapy due to its high expression on different tumor cells. Resistance of various tumor cells against TRAIL, a natural ligand for the death receptors, reduces its therapeutic potential and prompts the search for novel agonists at these receptors. Previous screening across the combinatorial peptide library yielded a peptide sequence KVVLTHR that specifically binds DR5. Incorporation of this sequence into TNFα resulted in binding DR5 with mutant protein TNFα-mut and appearance of cytotoxicity against lymphoma cells.


Subject(s)
Apoptosis/drug effects , Gene Expression Regulation, Neoplastic , Lymphocytes/drug effects , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Tumor Necrosis Factor-alpha/genetics , Amino Acid Sequence , Apoptosis/genetics , Binding Sites , Cell Line, Tumor , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Lymphocytes/metabolism , Lymphocytes/pathology , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, TNF-Related Apoptosis-Inducing Ligand/antagonists & inhibitors , Receptors, TNF-Related Apoptosis-Inducing Ligand/chemistry , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand/chemistry , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology
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