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1.
Int J Mol Sci ; 23(3)2022 Feb 08.
Article in English | MEDLINE | ID: covidwho-1674674

ABSTRACT

Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor a (TNFα), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6, IL-1ß, and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management.


Subject(s)
Aldo-Keto Reductases/physiology , COVID-19/genetics , Cytokine Release Syndrome/genetics , Respiratory Distress Syndrome/genetics , Aldo-Keto Reductases/antagonists & inhibitors , Aldo-Keto Reductases/genetics , Animals , COVID-19/complications , COVID-19/metabolism , COVID-19/pathology , Case-Control Studies , Cells, Cultured , Cytokine Release Syndrome/metabolism , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/virology , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Humans , Macrophages/drug effects , Macrophages/metabolism , Mice , Patient Acuity , RAW 264.7 Cells , Respiratory Distress Syndrome/metabolism , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/virology , SARS-CoV-2/physiology , Transcriptome
2.
Chem Commun (Camb) ; 58(13): 2120-2123, 2022 Feb 10.
Article in English | MEDLINE | ID: covidwho-1639577

ABSTRACT

The coronavirus 2019 (COVID-19) pandemic is causing serious impacts in the world, and safe and effective vaccines and medicines are the best methods to combat the disease. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in interacting with the angiotensin-converting enzyme 2 (ACE2) receptor, and is regarded as an important target of vaccines. Herein, we constructed the adjuvant-protein conjugate Pam3CSK4-RBD as a vaccine candidate, in which the N-terminal of the RBD was site-selectively oxidized by transamination and conjugated with the TLR1/2 agonist Pam3CSK4. This demonstrated that the conjugation of Pam3CSK4 significantly enhanced the anti-RBD antibody response and cellular response. In addition, sera from the Pam3CSK4-RBD immunized group efficiently inhibited the binding of the RBD to ACE2 and protected cells from SARS-CoV-2 and four variants of concern (alpha, beta, gamma and delta), indicating that this adjuvant strategy could be one of the effective means for protein vaccine development.


Subject(s)
COVID-19/prevention & control , Lipopeptides/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Vaccines, Conjugate/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibody Formation , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , COVID-19/virology , Female , HEK293 Cells , Humans , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Protein Binding , Protein Domains/immunology , RAW 264.7 Cells , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/chemistry
3.
Pharmacol Res ; 176: 106083, 2022 02.
Article in English | MEDLINE | ID: covidwho-1638968

ABSTRACT

The pathogenic hyper-inflammatory response has been revealed as the major cause of the severity and death of the Corona Virus Disease 2019 (COVID-19). Xuanfei Baidu Decoction (XFBD) as one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, shows unique advantages in the control of symptomatic transition from moderate to severe disease states. However, the roles of XFBD to against hyper-inflammatory response and its mechanism remain unclear. Here, we established acute lung injury (ALI) model induced by lipopolysaccharide (LPS), presenting a hyperinflammatory process to explore the pharmacodynamic effect and molecular mechanism of XFBD on ALI. The in vitro experiments demonstrated that XFBD inhibited the secretion of IL-6 and TNF-α and iNOS activity in LPS-stimulated RAW264.7 macrophages. In vivo, we confirmed that XFBD improved pulmonary injury via down-regulating the expression of proinflammatory cytokines such as IL-6, TNF-α and IL1-ß as well as macrophages and neutrophils infiltration in LPS-induced ALI mice. Mechanically, we revealed that XFBD treated LPS-induced acute lung injury through PD-1/IL17A pathway which regulates the infiltration of neutrophils and macrophages. Additionally, one major compound from XFBD, i.e. glycyrrhizic acid, shows a high binding affinity with IL17A. In conclusion, we demonstrated the therapeutic effects of XFBD, which provides the immune foundations of XFBD and fatherly support its clinical applications.


Subject(s)
Acute Lung Injury/drug therapy , Drugs, Chinese Herbal/pharmacology , Interleukin-17/metabolism , Macrophages/drug effects , Neutrophils/drug effects , Programmed Cell Death 1 Receptor/metabolism , Signal Transduction/drug effects , Acute Lung Injury/metabolism , Animals , COVID-19/drug therapy , COVID-19/metabolism , Cell Line , China , Cytokines/metabolism , Leukocyte Count/methods , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Neutrophils/metabolism , RAW 264.7 Cells
4.
Cell Mol Immunol ; 19(2): 210-221, 2022 02.
Article in English | MEDLINE | ID: covidwho-1608557

ABSTRACT

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.


Subject(s)
Angiotensin-Converting Enzyme 2/administration & dosage , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , COVID-19/therapy , Cell- and Tissue-Based Therapy/methods , Cell-Derived Microparticles/metabolism , Cholesterol/metabolism , Endosomes/chemistry , Macrophages, Alveolar/metabolism , SARS-CoV-2/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Female , Humans , Hydrogen-Ion Concentration , Lysosomes/chemistry , Mice , Mice, Inbred ICR , Mice, Transgenic , Oxidation-Reduction , RAW 264.7 Cells , Treatment Outcome , Vero Cells
5.
Nutrients ; 13(12)2021 Dec 16.
Article in English | MEDLINE | ID: covidwho-1580557

ABSTRACT

The excessive synthesis of interleukin-6 (IL-6) is related to cytokine storm in COVID-19 patients. Moreover, blocking IL-6 has been suggested as a treatment strategy for inflammatory diseases such as sepsis. Sepsis is a severe systemic inflammatory response syndrome with high mortality. In the present study, we investigated the anti-inflammatory and anti-septic effects and the underlying mechanisms of Dracocephalum moldavica ethanol extract (DMEE) on lipopolysaccharide (LPS)-induced inflammatory stimulation in RAW 264.7 macrophages along with septic mouse models. We found that DMEE suppressed the release of inflammatory mediators NO and PGE2 and inhibited both the mRNA and protein expression levels of iNOS and COX-2, respectively. In addition, DMEE reduced the release of proinflammatory cytokines, mainly IL-6 and IL-1ß, in RAW 264.7 cells by inhibiting the phosphorylation of JNK, ERK and p65. Furthermore, treatment with DMEE increased the survival rate and decreased the level of IL-6 in plasma in LPS-induced septic shock mice. Our findings suggest that DMEE elicits an anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophages and an anti-septic effect on septic mouse model through the inhibition of the ERK/JNK/NF-κB signaling cascades and production of IL-6.


Subject(s)
Interleukin-6/metabolism , Lamiaceae/chemistry , Lipopolysaccharides/toxicity , MAP Kinase Signaling System/drug effects , Plant Extracts/pharmacology , Transcription Factor RelA/metabolism , Animals , Ethanol/chemistry , Extracellular Signal-Regulated MAP Kinases/metabolism , Inflammation/chemically induced , Inflammation/drug therapy , Inflammation/metabolism , MAP Kinase Kinase 4/metabolism , Male , Mice , Plant Extracts/chemistry , RAW 264.7 Cells
6.
J Ethnopharmacol ; 283: 114738, 2022 Jan 30.
Article in English | MEDLINE | ID: covidwho-1466608

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal importance and potential activity of Siddha herbal formulations have proved over several centuries against a wide range of causative agents as Influenza, Dengue, Chikungunya, and Tuberculosis. The traditional medicine system of Siddha is a valuable therapeutic approach for treating viral respiratory infections like Coronavirus disease 2019 (COVID-19) and can be effectively employed to target the host response and preventive care to boost the immune system. Kaba Sura Kudineer (KSK), an official polyherbal formulation has been used in Siddha traditional medicine for centuries. However, the role of KSK in regulating inflammation and the underlying molecular mechanisms has remained elusive. AIM OF THE STUDY: The goal of this study was to evaluate the anti-inflammatory effect of KSK using lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophage cells. MATERIALS AND METHODS: Raw 264.7 murine macrophage cells were used for this study. The Inflammatory mediators and cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The NF-κB nulcear translocation and protein expression of iNOS, COX-2 was analyzed with westernblot. RESULTS: KSK supplementation decreased LPS mediated TLR-4 production and secretion of pro-inflammatory mediators and cytokines including IL-6, TNF-α, COX-2 and PGE-2. Moreover, it inhibited the production of nitric oxide (NO) and thereby inhibited the expression of iNOS in the cell. The Western blot analysis further confirmed that KSK strongly prevented the LPS-induced degradation of IκB which is normally required for the activation of NF-κB and hereby suppressed nuclear translocation of NF-κB. The protein expression of iNOS, COX-2 was significantly decreased with the presence of KSK treatment. Results suggested that KSK manipulates its anti-inflammatory effects mainly through blocking the TLR mediated NF-κB signal transduction pathways. CONCLUSIONS: Together, this study has proven that KSK could be a potential therapeutic drug for alleviating excessive inflammation in many inflammation-associated diseases like COVID-19.


Subject(s)
COVID-19/drug therapy , Inflammation/drug therapy , Lipopolysaccharides/toxicity , Macrophages/drug effects , Medicine, Ayurvedic , Plant Preparations/therapeutic use , Animals , Anti-Inflammatory Agents/pharmacology , Dietary Supplements , Mice , Pharmaceutical Preparations , Phytotherapy , Plant Preparations/pharmacology , RAW 264.7 Cells , SARS-CoV-2
7.
mBio ; 12(5): e0254221, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1462902

ABSTRACT

Damage in COVID-19 results from both the SARS-CoV-2 virus and its triggered overactive host immune responses. Therapeutic agents that focus solely on reducing viral load or hyperinflammation fail to provide satisfying outcomes in all cases. Although viral and cellular factors have been extensively profiled to identify potential anti-COVID-19 targets, new drugs with significant efficacy remain to be developed. Here, we report the potent preclinical efficacy of ALD-R491, a vimentin-targeting small molecule compound, in treating COVID-19 through its host-directed antiviral and anti-inflammatory actions. We found that by altering the physical properties of vimentin filaments, ALD-491 affected general cellular processes as well as specific cellular functions relevant to SARS-CoV-2 infection. Specifically, ALD-R491 reduced endocytosis, endosomal trafficking, and exosomal release, thus impeding the entry and egress of the virus; increased the microcidal capacity of macrophages, thus facilitating the pathogen clearance; and enhanced the activity of regulatory T cells, therefore suppressing the overactive immune responses. In cultured cells, ALD-R491 potently inhibited the SARS-CoV-2 spike protein and human ACE2-mediated pseudoviral infection. In aged mice with ongoing, productive SARS-CoV-2 infection, ALD-R491 reduced disease symptoms as well as lung damage. In rats, ALD-R491 also reduced bleomycin-induced lung injury and fibrosis. Our results indicate a unique mechanism and significant therapeutic potential for ALD-R491 against COVID-19. We anticipate that ALD-R491, an oral, fast-acting, and non-cytotoxic agent targeting the cellular protein with multipart actions, will be convenient, safe, and broadly effective, regardless of viral mutations, for patients with early- or late-stage disease, post-COVID-19 complications, and other related diseases. IMPORTANCE With the Delta variant currently fueling a resurgence of new infections in the fully vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanisms of action that address both the viral infection and the overactive immune system in the pathogenesis of the disease. Unlike virus-directed therapeutics that may lose efficacy due to viral mutations, and immunosuppressants that require ideal timing to be effective, this agent, with its unique host-directed antiviral and anti-inflammatory actions, can work against all variants of the virus, be effective during all stages of the disease, and even resolve post-disease damage and complications. Further development of the compound will provide an important tool in the fight against COVID-19 and its complications, as well as future outbreaks of new viruses.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/metabolism , Organic Chemicals/therapeutic use , Spike Glycoprotein, Coronavirus/metabolism , Vimentin/metabolism , Animals , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Exosomes/drug effects , Exosomes/metabolism , HEK293 Cells , Humans , Mice , RAW 264.7 Cells
8.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Article in English | MEDLINE | ID: covidwho-1450313

ABSTRACT

Cancer therapy reduces tumor burden via tumor cell death ("debris"), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E2 receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.


Subject(s)
Eicosanoids/metabolism , Epoxide Hydrolases/biosynthesis , Macrophages/immunology , Neoplasm Metastasis/pathology , Receptors, Prostaglandin E, EP4 Subtype/biosynthesis , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular/pathology , Cell Death/drug effects , Cell Line, Tumor , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/prevention & control , Cytokines/metabolism , Hep G2 Cells , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Neoplasm Metastasis/prevention & control , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Phagocytosis/immunology , RAW 264.7 Cells
9.
J Ethnopharmacol ; 283: 114701, 2022 Jan 30.
Article in English | MEDLINE | ID: covidwho-1446835

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu Decoction (XFBD), one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, plays an important role in the treatment of mild and/or common patients with dampness-toxin obstructing lung syndrome. AIM OF THE STUDY: The present work aims to elucidate the protective effects and the possible mechanism of XFBD against the acute inflammation and pulmonary fibrosis. METHODS: We use TGF-ß1 induced fibroblast activation model and LPS/IL-4 induced macrophage inflammation model as in vitro cell models. The mice model of lung fibrosis was induced by BLM via endotracheal drip, and then XFBD (4.6 g/kg, 9.2 g/kg) were administered orally respectively. The efficacy and molecular mechanisms in the presence or absence of XFBD were investigated. RESULTS: The results proved that XFBD can effectively inhibit fibroblast collagen deposition, down-regulate the level of α-SMA and inhibit the migration of fibroblasts. IL-4 induced macrophage polarization was also inhibited and the secretions of the inflammatory factors including IL6, iNOS were down-regulated. In vivo experiments, the results proved that XFBD improved the weight loss and survival rate of the mice. The XFBD high-dose administration group had a significant effect in inhibiting collagen deposition and the expression of α-SMA in the lungs of mice. XFBD can reduce bleomycin-induced pulmonary fibrosis by inhibiting IL-6/STAT3 activation and related macrophage infiltration. CONCLUSIONS: Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.


Subject(s)
COVID-19/drug therapy , Drugs, Chinese Herbal , Inflammation/drug therapy , Macrophages/drug effects , SARS-CoV-2 , Signal Transduction/drug effects , Animals , Cell Survival/drug effects , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Gene Regulatory Networks , Humans , Interleukin-6/antagonists & inhibitors , Interleukin-6/genetics , Interleukin-6/metabolism , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Phytotherapy , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/prevention & control , RAW 264.7 Cells , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism
10.
Mar Drugs ; 19(8)2021 Jul 29.
Article in English | MEDLINE | ID: covidwho-1376892

ABSTRACT

Seaweed of Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO3 residues and (1→6)-ß-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKß) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Aquatic Organisms/chemistry , Fucose/pharmacology , Galactose/pharmacology , Seaweed/chemistry , Animals , Inhibitory Concentration 50 , Mice , RAW 264.7 Cells/drug effects , Zebrafish
11.
Drug Des Devel Ther ; 15: 3255-3276, 2021.
Article in English | MEDLINE | ID: covidwho-1360673

ABSTRACT

BACKGROUND: Huai Hua San (HHS), a famous Traditional Chinese Medicine (TCM) formula, has been widely applied in treating ulcerative colitis (UC). However, the interaction of bioactives from HHS with the targets involved in UC has not been elucidated yet. AIM: A network pharmacology-based approach combined with molecular docking and in vitro validation was performed to determine the bioactives, key targets, and potential pharmacological mechanism of HHS against UC. MATERIALS AND METHODS: Bioactives and potential targets of HHS, as well as UC-related targets, were retrieved from public databases. Crucial bioactive ingredients, potential targets, and signaling pathways were acquired through bioinformatics analysis, including protein-protein interaction (PPI), as well as the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Subsequently, molecular docking was carried out to predict the combination of active compounds with core targets. Lastly, in vitro experiments were conducted to further verify the findings. RESULTS: A total of 28 bioactive ingredients of HHS and 421 HHS-UC-related targets were screened. Bioinformatics analysis revealed that quercetin, luteolin, and nobiletin may be potential candidate agents. JUN, TP53, and ESR1 could become potential therapeutic targets. PI3K-AKT signaling pathway might play an important role in HHS against UC. Moreover, molecular docking suggested that quercetin, luteolin, and nobiletin combined well with JUN, TP53, and ESR1, respectively. Cell experiments showed that the most important ingredient of HHS, quercetin, could inhibit the levels of inflammatory factors and phosphorylated c-Jun, as well as PI3K-Akt signaling pathway in LPS-induced RAW264.7 cells, which further confirmed the prediction by network pharmacology strategy and molecular docking. CONCLUSION: Our results comprehensively illustrated the bioactives, potential targets, and molecular mechanism of HHS against UC. It also provided a promising strategy to uncover the scientific basis and therapeutic mechanism of TCM formulae in treating diseases.


Subject(s)
Colitis, Ulcerative/drug therapy , Drugs, Chinese Herbal/pharmacology , Medicine, Chinese Traditional , Molecular Docking Simulation , Animals , Mice , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins c-akt/physiology , Quercetin/pharmacology , RAW 264.7 Cells , Signal Transduction/drug effects
12.
Int J Mol Sci ; 22(9)2021 Apr 28.
Article in English | MEDLINE | ID: covidwho-1359279

ABSTRACT

Deeply understanding the virus-host interaction is a prerequisite for developing effective anti-viral strategies. Traditionally, the transporter associated with antigen processing type 1 (TAP1) is critical for antigen presentation to regulate adaptive immunity. However, its role in controlling viral infections through modulating innate immune signaling is not yet fully understood. In the present study, we reported that TAP1, as a product of interferon-stimulated genes (ISGs), had broadly antiviral activity against various viruses such as herpes simplex virus 1 (HSV-1), adenoviruses (AdV), vesicular stomatitis virus (VSV), dengue virus (DENV), Zika virus (ZIKV), and influenza virus (PR8) etc. This antiviral activity by TAP1 was further confirmed by series of loss-of-function and gain-of-function experiments. Our further investigation revealed that TAP1 significantly promoted the interferon (IFN)-ß production through activating the TANK binding kinase-1 (TBK1) and the interferon regulatory factor 3 (IRF3) signaling transduction. Our work highlighted the broadly anti-viral function of TAP1 by modulating innate immunity, which is independent of its well-known function of antigen presentation. This study will provide insights into developing novel vaccination and immunotherapy strategies against emerging infectious diseases.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 2/immunology , Antiviral Agents/immunology , Host Microbial Interactions/immunology , Interferon Type I/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 2/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 2/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 2/genetics , Animals , Gene Knockout Techniques , HEK293 Cells , Humans , Immunity, Innate , Interferon Regulatory Factor-3/immunology , Mice , Models, Immunological , RAW 264.7 Cells , Toll-Like Receptors/agonists , Virus Diseases/immunology
13.
Bioorg Chem ; 115: 105265, 2021 10.
Article in English | MEDLINE | ID: covidwho-1356144

ABSTRACT

In spite of possessing a wide range of pharmacological properties the anti-inflammatory activities of isoquinolin-1(2H)-ones were rarely known or explored earlier. PDE4 inhibitors on the other hand in addition to their usefulness in treating inflammatory diseases have been suggested to attenuate the cytokine storm in COVID-19 especially TNF-α. In our effort, a new class of isoquinolin-1(2H)-ones derivatives containing an aminosulfonyl moiety were designed and explored as potential inhibitors of PDE4. Accordingly, for the first time a CuCl2-catalyzed inexpensive, faster and ligand/additive free approach has been developed for the synthesis of these predesigned isoquinolin-1(2H)-one derivatives via the coupling-cyclization strategy. Thus, the CuCl2-catalyzed reaction of 2-iodobenzamides with appropriate terminal alkynes proceeded with high chemo and regioselectivity affording the desired compounds in 77-84% yield within 1-1.5 h. The methodology also afforded simpler isoquinolin-1(2H)-ones devoid of aminosulfonyl moiety showing a broader generality and scope of this approach. Several of the synthesized compounds especially 3c, 3k and 3s showed impressive inhibition (83-90%) of PDE4B when tested at 10 µM in vitro whereas compounds devoid of aminosulfonyl moiety was found to be less active. In spite of high inhibition showed at 10 µM these compounds did not show proper concertation dependent inhibition below 1 µM that was reflected in their IC50 values e.g. 2.43 ± 0.32, 3.26 ± 0.24 and 3.63 ± 0.80 µM for 3k, 3o and 3s respectively. The anti-inflammatory potential of these compounds was indicated by their TNF-α inhibition (60-50% at 10 µM). The in silico docking studies of these molecules suggested good interactions with PDE4B and selective inhibition of PDE4B by 3k over PDE4D that was supported by in vitro assay results. These observations together with the favorable ADME and safety predicted for 3kin silico not only suggested 3k as an interesting hit molecule for further studies but also reveal the first example of isoquinolin-1(2H)-one based inhibitor of PDE4B.


Subject(s)
Anti-Inflammatory Agents/chemistry , Copper/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 4/chemistry , Isoquinolines/chemistry , Phosphodiesterase 4 Inhibitors/chemistry , Animals , Anti-Inflammatory Agents/chemical synthesis , Catalysis , Cyclization , Enzyme Assays , Humans , Isoquinolines/chemical synthesis , Mice , Molecular Structure , Phosphodiesterase 4 Inhibitors/chemical synthesis , RAW 264.7 Cells , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/antagonists & inhibitors
14.
Mol Syst Biol ; 17(9): e10426, 2021 09.
Article in English | MEDLINE | ID: covidwho-1355289

ABSTRACT

Although 15-20% of COVID-19 patients experience hyper-inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N-terminal domain (NTD) of the SARS-CoV-2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning-based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD-induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA-approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD-mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS-CoV-2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide-mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS-CoV-2-mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Cytokines/metabolism , Host-Pathogen Interactions/physiology , Animals , Azetidines/pharmacology , Host-Pathogen Interactions/drug effects , Humans , Imidazoles/pharmacology , Interleukin-1 Receptor-Associated Kinases/metabolism , Janus Kinase 1/metabolism , Lipopolysaccharides/toxicity , Machine Learning , Male , Mice , Mice, Inbred C57BL , Neutrophils/virology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrazoles/pharmacology , Pyridazines/pharmacology , RAW 264.7 Cells , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Sulfonamides/pharmacology
15.
FASEB J ; 35(9): e21801, 2021 09.
Article in English | MEDLINE | ID: covidwho-1345745

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in mediating viral entry into host cells. However, whether it contributes to pulmonary hyperinflammation in patients with coronavirus disease 2019 is not well known. In this study, we developed a spike protein-pseudotyped (Spp) lentivirus with the proper tropism of the SARS-CoV-2 spike protein on the surface and determined the distribution of the Spp lentivirus in wild-type C57BL/6J male mice that received an intravenous injection of the virus. Lentiviruses with vesicular stomatitis virus glycoprotein (VSV-G) or with a deletion of the receptor-binding domain (RBD) in the spike protein [Spp (∆RBD)] were used as controls. Two hours postinfection (hpi), there were 27-75 times more viral burden from Spp lentivirus in the lungs than in other organs; there were also about 3-5 times more viral burden from Spp lentivirus than from VSV-G lentivirus in the lungs, liver, kidney, and spleen. Deletion of RBD diminished viral loads in the lungs but not in the heart. Acute pneumonia was observed in animals 24 hpi. Spp lentivirus was mainly found in SPC+ and LDLR+ pneumocytes and macrophages in the lungs. IL6, IL10, CD80, and PPAR-γ were quickly upregulated in response to infection in the lungs as well as in macrophage-like RAW264.7 cells. Furthermore, forced expression of the spike protein in RAW264.7 cells significantly increased the mRNA levels of the same panel of inflammatory factors. Our results demonstrated that the spike protein of SARS-CoV-2 confers the main point of viral entry into the lungs and can induce cellular pathology. Our data also indicate that an alternative ACE2-independent viral entry pathway may be recruited in the heart and aorta.


Subject(s)
Macrophages/immunology , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Spike Glycoprotein, Coronavirus/immunology , Acute Disease , Alveolar Epithelial Cells/virology , Animals , B7-1 Antigen , Cell Line , Inflammation Mediators , Interleukin-10 , Interleukin-6 , Lentivirus/genetics , Lentivirus/isolation & purification , Lentivirus/metabolism , Lung/immunology , Lung/pathology , Lung/virology , Macrophages/virology , Male , Membrane Glycoproteins , Mice , Mice, Inbred C57BL , PPAR gamma , RAW 264.7 Cells , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins
16.
Theranostics ; 11(15): 7379-7390, 2021.
Article in English | MEDLINE | ID: covidwho-1266907

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel strain of highly contagious coronaviruses that infects humans. Prolonged fever, particularly that above 39.5 °C, is associated with SARS-CoV-2 infection. However, little is known about the pathological effects of fever caused by SARS-CoV-2. Methods: Primary bovine alveolar macrophages (PBAMs), RAW264.7 mouse macrophages, and THP-1 human cells were transfected with plasmids carrying the genes encoding the SARS-CoV-2 spike (S) protein or receptor-binding domain (RBD). Proteins in the macrophages interacting with S-RBD at 39.5 °C or 37 °C were identified by immunoprecipitation-mass spectrometry. Glutathione S-transferase pulldown, surface plasmon resonance, and immunofluorescence were performed to evaluate the transient receptor potential vanilloid 2 (TRPV2) interaction with SARS-CoV-2-S-RBD at 39.5 °C. Using an RNA sequencing-based approach, cytokine gene expression induced by SARS-CoV-2 S transfection at 39.5 °C and 37.5 °C in primary alveolar macrophages was measured. Fluo-4 staining and enzyme-linked immunosorbent assays were used to assess the regulatory function of TRPV2 in intracellular Ca 2+ and cytokines under SARS-CoV-2-S-RBD at 39.5 °C. Additionally, cytokine release was examined after TRPV2 knockdown with shRNA oligonucleotides or inhibition using the SKF-96365 antagonist. Results: We identified an interaction between the primary alveolar macrophage receptor TRPV2 and S-RBD under febrile conditions. Febrile temperature promotes Ca2+ influx through SARS-CoV-2 infection in PBAMs, further activates the NF-κB p65 signaling pathway, and enhances the secretion of cytokines. Furthermore, knockdown or antagonist (with SKF-96365) of TRPV2 significantly decreased the release of cytokines that drive the inflammatory response. Conclusion: Collectively, our findings identified TRPV2 as a receptor of SARS-CoV-2 in conditions of febrile temperature, providing insight into critical interactions of SARS-CoV-2 with macrophages, as well as a useful resource and potential drug target for coronavirus disease 2019.


Subject(s)
COVID-19/virology , Fever/virology , Macrophages/metabolism , Macrophages/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , TRPV Cation Channels/metabolism , Virus Internalization , Animals , Calcium/metabolism , Cattle , Cells, Cultured , Cytokines/metabolism , Humans , Imidazoles/pharmacology , Kinetics , Macrophages/drug effects , Mice , NF-kappa B/metabolism , Protein Binding/drug effects , RAW 264.7 Cells , SARS-CoV-2/drug effects , Signal Transduction/drug effects , THP-1 Cells , Temperature , Virus Internalization/drug effects
17.
Nat Commun ; 12(1): 3431, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1262001

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.


Subject(s)
Reverse Genetics , SARS-CoV-2/genetics , Amino Acid Sequence , Animals , Base Sequence , Chlorocebus aethiops , Culicidae/virology , Furin/metabolism , Genome, Viral , HEK293 Cells , Humans , Mice , Mutation/genetics , NIH 3T3 Cells , Polymerase Chain Reaction , RAW 264.7 Cells , Receptors, Virus/metabolism , Vero Cells , Viral Proteins/chemistry , Virus Replication
18.
J Extracell Vesicles ; 10(8): e12110, 2021 06.
Article in English | MEDLINE | ID: covidwho-1258076

ABSTRACT

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.


Subject(s)
Extracellular Vesicles/chemistry , Immunologic Techniques/methods , Animals , Bodily Secretions/chemistry , COVID-19/blood , COVID-19/physiopathology , Chlorocebus aethiops , Circulating MicroRNA , High-Throughput Nucleotide Sequencing , Humans , MCF-7 Cells , Mice , RAW 264.7 Cells , Severity of Illness Index , Vero Cells
19.
Eur J Med Chem ; 221: 113514, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1228023

ABSTRACT

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 = 0.43 ± 0.11 and 0.54 ± 0.19 µM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/drug therapy , Isocoumarins/therapeutic use , Phosphodiesterase 4 Inhibitors/therapeutic use , Sulfonamides/therapeutic use , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/toxicity , Arthritis, Experimental/pathology , Catalysis , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Embryo, Nonmammalian/drug effects , Female , Isocoumarins/chemical synthesis , Isocoumarins/metabolism , Isocoumarins/toxicity , Knee Joint/drug effects , Knee Joint/pathology , Male , Mice , Molecular Docking Simulation , Molecular Structure , Palladium/chemistry , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/metabolism , Phosphodiesterase 4 Inhibitors/toxicity , Protein Binding , RAW 264.7 Cells , Rats, Wistar , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/metabolism , Sulfonamides/toxicity , Zebrafish
20.
ACS Appl Mater Interfaces ; 13(18): 20995-21006, 2021 May 12.
Article in English | MEDLINE | ID: covidwho-1209173

ABSTRACT

COVID-19 has been diffusely pandemic around the world, characterized by massive morbidity and mortality. One of the remarkable threats associated with mortality may be the uncontrolled inflammatory processes, which were induced by SARS-CoV-2 in infected patients. As there are no specific drugs, exploiting safe and effective treatment strategies is an instant requirement to dwindle viral damage and relieve extreme inflammation simultaneously. Here, highly biocompatible glycyrrhizic acid (GA) nanoparticles (GANPs) were synthesized based on GA. In vitro investigations revealed that GANPs inhibit the proliferation of the murine coronavirus MHV-A59 and reduce proinflammatory cytokine production caused by MHV-A59 or the N protein of SARS-CoV-2. In an MHV-A59-induced surrogate mouse model of COVID-19, GANPs specifically target areas with severe inflammation, such as the lungs, which appeared to improve the accumulation of GANPs and enhance the effectiveness of the treatment. Further, GANPs also exert antiviral and anti-inflammatory effects, relieving organ damage and conferring a significant survival advantage to infected mice. Such a novel therapeutic agent can be readily manufactured into feasible treatment for COVID-19.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Antiviral Agents/therapeutic use , Glycyrrhizic Acid/therapeutic use , Inflammation/drug therapy , Nanoparticles/therapeutic use , Virus Diseases/drug therapy , Animals , Anti-Inflammatory Agents/chemistry , Antioxidants/chemistry , Antioxidants/therapeutic use , Antiviral Agents/chemistry , COVID-19/drug therapy , Coronavirus Nucleocapsid Proteins/pharmacology , Cytokines/metabolism , Female , Glycyrrhizic Acid/chemistry , Humans , Liver/pathology , Lung/pathology , Mice , Mice, Inbred BALB C , Murine hepatitis virus/drug effects , Nanoparticles/chemistry , Phosphoproteins/pharmacology , RAW 264.7 Cells , SARS-CoV-2/chemistry , THP-1 Cells , Viral Load/drug effects , Virus Diseases/pathology , Virus Replication/drug effects
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