Pinostrobin from plants and propolis against human coronavirus HCoV-OC43 by modulating host AHR/CYP1A1 pathway and lipid metabolism.

Coronaviruses, as enveloped positive-strand RNA viruses, manipulate host lipid compositions to enable robust viral replication. Temporal modulation of the host lipid metabolism is a potential novel strategy against coronaviruses. Here, the dihydroxyflavone pinostrobin (PSB) was identified through bioassay that inhibited the increment of human coronavirus OC43 (HCoV-OC43) in human ileocecal colorectal adenocarcinoma cells. Lipid metabolomic studies showed that PSB interfered with linoleic acid and arachidonic acid metabolism pathways. PSB significantly decreased the level of 12, 13- epoxyoctadecenoic (12, 13-EpOME) and increased the level of prostaglandin E2. Interestingly, exogenous supplement of 12, 13-EpOME in HCoV-OC43-infected cells significantly stimulated HCoV-OC43 virus replication. Transcriptomic analyses showed that PSB is a negative modulator of aryl hydrocarbon receptor (AHR)/cytochrome P450 (CYP) 1A1signaling pathway and its antiviral effects can be counteracted by supplement of FICZ, a well-known AHR agonist. Integrative analyses of metabolomic and transcriptomic indicated that PSB could affect linoleic acid and arachidonic acid metabolism axis through AHR/CYP1A1 pathway. These results highlight the importance of the AHR/CYP1A1 pathway and lipid metabolism in the anti-coronavirus activity of the bioflavonoid PSB.

Virtual screening and molecular dynamics simulation study of approved drugs as a binder to the linoleic acid binding site on spike protein of SARS-CoV-2 and double mutant (E484Q and L452R).

INTRODUCTION: Binding of linoleic acid (LA) to the spike trimer stabilizes it in closed conformation hindering its binding to angiotensin-converting enzyme-2, thus decreasing infectivity. In the current study, we tend to repurpose Food and Drug Administration-approved drugs as binder to the LA binding pocket in wild and double mutant spike protein. MATERIALS AND METHODS: Approved drugs from DrugBank database (n = 2456) were prepared using Ligprep module of Schrodinger. Crystal structure of LA bound to spike trimer was retrieved (PDB: 6ZB4) and prepared using protein preparation wizard and grid was generated. A virtual screening was performed. With the help of molecular dynamics (MD) studies interaction profile of screened drugs were further evaluated. The selected hits were further evaluated for binding to the double mutant form of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). RESULTS AND DISCUSSION: Following virtual screening, a total of 26 molecules were shortlisted, which were further evaluated using 1ns MD simulation study. Four ligands showing better root mean square deviation (RMSD), RMSD to LA with interaction profile similar to LA were further evaluated using 100 ns MD simulation studies. A total of 2 hits were identified, which performed better than LA (selexipag and pralatrexate). Both these ligands were also found to bind to LA binding site of the double mutant form (E484Q and L452R); however, the binding affinity of pralatrexate was found to be better. CONCLUSION: We have identified 2 ligands (selexipag and pralatrexate) as possible stable binders to the LA binding site in spike trimer (wild and mutant form). Among them, pralatrexate has shown in vitro activity against SARS-CoV-2, validating our study results.

Cryo-EM reveals binding of linoleic acid to SARS-CoV-2 spike glycoprotein, suggesting an antiviral treatment strategy.

The COVID-19 pandemic and concomitant lockdowns presented a global health challenge and triggered unprecedented research efforts to elucidate the molecular mechanisms and pathogenicity of SARS-CoV-2. The spike glycoprotein decorating the surface of SARS-CoV-2 virions is a prime target for vaccine development, antibody therapy and serology as it binds the host cell receptor and is central for viral cell entry. The electron cryo-microscopy structure of the spike protein revealed a hydrophobic pocket in the receptor-binding domain that is occupied by an essential fatty acid, linoleic acid (LA). The LA-bound spike protein adopts a non-infectious locked conformation which is more stable than the infectious form and shields important immunogenic epitopes. Here, the impact of LA binding on viral infectivity and replication, and the evolutionary conservation of the pocket in other highly pathogenic coronaviruses, including SARS-CoV-2 variants of concern (VOCs), are reviewed. The importance of LA metabolic products, the eicosanoids, in regulating the human immune response and inflammation is highlighted. Lipid and fatty-acid binding to a hydrophobic pocket in proteins on the virion surface appears to be a broader strategy employed by viruses, including picornaviruses and Zika virus. Ligand binding stabilizes their protein structure and assembly, and downregulates infectivity. In the case of rhinoviruses, this has been exploited to develop small-molecule antiviral drugs that bind to the hydrophobic pocket. The results suggest a COVID-19 antiviral treatment based on the LA-binding pocket.

Linoleic acid binds to SARS-CoV-2 RdRp and represses replication of seasonal human coronavirus OC43.

Fatty acids belong to a group of compounds already acknowledged for their broad antiviral efficacy. However, little is yet known about their effect on replication of human coronaviruses. To shed light on this subject, we first screened 15 fatty acids, three lipid-soluble vitamins, and cholesterol, on SARS-CoV-2 RdRp, and identified the four fatty acids with the highest RdRp inhibitory potential. Among them, linoleic acid was found to have the greatest interaction with SARS-CoV-2 RdRp, with its direct binding to the cavity formed by the RNA double helix and protein. Linoleic acid forms hydrophobic interactions with multiple residues, and at the same time forms electrostatic interactions including the hydrogen bond with Lys593 and Asp865. In line with these results, a dose-dependent inhibition of HCoV-OC43 replication in vitro was observed, additionally strengthened by data from in vivo study, which also confirmed anti-inflammatory potential of linoleic acid. Based on these results, we concluded that our study provides a new understanding of the antiviral properties of fatty acids against human coronaviruses including the SARS-CoV-2 strain. Particularly, they lays down a new prospect for linoleic acid's RdRp-inhibitory activity, as a candidate for further studies, which are warranted to corroborate the results presented here.

Saliva metabolomic profile of COVID-19 patients associates with disease severity.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is strongly linked to dysregulation of various molecular, cellular, and physiological processes that change abundance of different biomolecules including metabolites that may be ultimately used as biomarkers for disease progression and severity. It is important at early stage to readily distinguish those patients that are likely to progress to moderate and severe stages. OBJECTIVES: This study aimed to investigate the utility of saliva and plasma metabolomic profiles as a potential parameter for risk stratifying COVID-19 patients. METHOD: LC-MS/MS-based untargeted metabolomics were used to profile the changes in saliva and plasma metabolomic profiles of COVID-19 patients with different severities. RESULTS: Saliva and plasma metabolites were screened in 62 COVID-19 patients and 18 non-infected controls. The COVID-19 group included 16 severe, 15 moderate, 16 mild, and 15 asymptomatic cases. Thirty-six differential metabolites were detected in COVID-19 versus control comparisons. SARS-CoV-2 induced metabolic derangement differed with infection severity. The metabolic changes were identified in saliva and plasma, however, saliva showed higher intensity of metabolic changes. Levels of saliva metabolites such as sphingosine and kynurenine were significantly different between COVID-19 infected and non-infected individuals; while linoleic acid and Alpha-ketoisovaleric acid were specifically increased in severe compared to non-severe patients. As expected, the two prognostic biomarkers of C-reactive protein and D-dimer were negatively correlated with sphingosine and 5-Aminolevulinic acid, and positively correlated with L-Tryptophan and L-Kynurenine. CONCLUSION: Saliva disease-specific and severity-specific metabolite could be employed as potential COVID-19 diagnostic and prognostic biomarkers.

Systematic identification of chemical components in Fufang Shuanghua oral liquid and screening of potential active components against SARS-CoV-2 protease.

Coronavirus disease (COVID-19) caused by SARS-COV-2 infection has been widely prevalent in many countries and has become a common challenge facing mankind. Traditional Chinese medicine (TCM) has played a prominent role in this pandemic, and especially TCM with the function of "heat-clearing and detoxifying" has shown an excellent role in anti-virus. Fufang Shuanghua oral liquid (FFSH) has been used to treat the corresponding symptoms of influenza such as fever, nasal congestion, runny nose, sore throat, and upper respiratory tract infections in clinic, which are typical symptoms of COVID-19. The content of chlorogenic acid, andrographolide and dehydrated andrographolide as the quality control components of FFSH is not less than 1.0 mg/mL, 60 µg/mL and 60 µg/mL respectively. In this study, UPLC-Q-TOF-MS/MS was employed to describe the chemical profile of FFSH. Virtual screening and fluorescence resonance energy transfer (FRET) were used to screen the effective components of FFSH acting on SARS-CoV-2 main protease (Mpro). As a result, 214 compounds in FFSH were identified or preliminarily characterized by UPLC-Q-TOF-MS/MS, and 61 active ingredients with potential inhibitory effects on Mpro were selected through receptor-based and ligand-based virtual screening. In particular, quercetin, forsythoside A, and linoleic acid showed a good inhibitory effect on Mpro in FRET evaluation with IC50 values of 26.15 µM, 22.26 µM and 47.09 µM respectively, and had a strong binding affinity with the receptor Mpro (6LU7) in molecular docking. CYS145 and HIS41 were the main amino acid residues affected by small molecules in the protein binding domain. In brief, we characterized, for the first time, 214 chemical components in FFSH, and three of them, including quercetin, forsythoside A and linoleic acid, were screened out to exert beneficial anti-COVID-19 effects through CYS145 and HIS41 sites, which may provide a new research strategy for TCM to develop new therapeutic drugs against COVID-19.

sEH-derived metabolites of linoleic acid drive pathologic inflammation while impairing key innate immune cell function in burn injury.

Fatty acid composition in the Western diet has shifted from saturated to polyunsaturated fatty acids (PUFAs), and specifically to linoleic acid (LA, 18:2), which has gradually increased in the diet over the past 50 y to become the most abundant dietary fatty acid in human adipose tissue. PUFA-derived oxylipins regulate a variety of biological functions. The cytochrome P450 (CYP450)­formed epoxy fatty acid metabolites of LA (EpOMEs) are hydrolyzed by the soluble epoxide hydrolase enzyme (sEH) to dihydroxyoctadecenoic acids (DiHOMEs). DiHOMEs are considered cardioprotective at low concentrations but at higher levels have been implicated as vascular permeability and cytotoxic agents and are associated with acute respiratory distress syndrome in severe COVID-19 patients. High EpOME levels have also correlated with sepsis-related fatalities; however, those studies failed to monitor DiHOME levels. Considering the overlap of burn pathophysiology with these pathologies, the role of DiHOMEs in the immune response to burn injury was investigated. 12,13-DiHOME was found to facilitate the maturation and activation of stimulated neutrophils, while impeding monocyte and macrophage functionality and cytokine generation. In addition, DiHOME serum concentrations were significantly elevated in burn-injured mice and these increases were ablated by administration of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a sEH inhibitor. TPPU also reduced necrosis of innate and adaptive immune cells in burned mice, in a dose-dependent manner. The findings suggest DiHOMEs are a key driver of immune cell dysfunction in severe burn injury through hyperinflammatory neutrophilic and impaired monocytic actions, and inhibition of sEH might be a promising therapeutic strategy to mitigate deleterious outcomes in burn patients.

Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2.

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

The need for precision nutrition, genetic variation and resolution in Covid-19 patients.

The health of the individual and the population in general is the result of interaction between genetics and various environmental factors, of which diet/nutrition is the most important. The focus of this paper is on the association of high n-6 PUFA or low n-3 PUFA due to genetic variation and/or dietary intake, with changes in specialized pro-resolving mediators (SPMs), cytokine storm, inflammation-resolution and Covid-19. Human beings evolved on a diet that was balanced in the n-6 and n-3 essential fatty acids with a ratio of n-6/n-3 of 1-2/1 whereas today this ratio is 16/1. Such a high ratio due to high amounts of n-6 fatty acids leads to a prothrombotic and proinflammatory state and is associated with obesity, diabetes, cardiovascular disease, and some forms of cancer. In addition to the high intake of n-6 fatty acids that increases inflammation there is genetic variation in the biosynthesis of n-6 linoleic acid (LA) to arachidonic acid (ARA) and of linolenic (ALA) to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Present day humans have two common FADS haplotypes that differ dramatically in their ability to generate long-chain fatty acids. The more efficient, evolutionary derived haplotype increases the efficiency of synthesizing essential long-chain fatty acids from precursors and could have provided an advantage in environments with limited access to dietary long-chain fatty acids ARA, EPA and DHA. In the modern world this haplotype has been associated with lifestyle-related diseases, such as cardiovascular disease, obesity, diabetes, all of which are characterized by increased levels of inflammation. African Americans and Latino populations have increased susceptibility and higher death rates from SARS-CoV-2 than whites. These populations are characterized by increased numbers of persons (about 80%) that are fast metabolizers, leading to increased production of ARA, as well as poor intake of fruits and vegetables. The combinations of fast metabolism and high n-6 intake increases their inflammatory status and possibly susceptibility of SARS-CoV-2. In vitro and human studies indicate that the specialized pro-resolving mediators (SPM) produced from the n-3, EPA and DHA influence the resolution of inflammation, allowing the tissues to return to function and homeostasis. The SPMs each counter-regulate cytokine storms, as well as proinflammatory lipid mediators via NFκB and inflammasome down regulation and reduce the proinflammatory eicosanoids produced from ARA. The nutritional availability of dietary n-3 fatty acids from marine oils enriched with SPM intermediate precursors, along with increasing local biosynthesis of SPMs to functional concentrations may be an approach of value during SARS-CoV2 infections, as well as in prevention, and shortening their recovery from infections. It is evident that populations differ in their genetic variants and their frequencies and their interactions with the food they eat. Gene-nutrient interactions is a very important area of study that provides specific dietary advice for individuals and subgroups within a population in the form of Precision Nutrition. Nutritional science needs to focus on Precision Nutrition, genetic variants in the population and a food supply composed of Nutrients that have been part of our diet throughout evolution, which is the diet that our genes are programmed to respond.

Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.

Obesity-Driven Deficiencies of Specialized Pro-resolving Mediators May Drive Adverse Outcomes During SARS-CoV-2 Infection.

Obesity is a major independent risk factor for increased morbidity and mortality upon infection with Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), which is responsible for the current coronavirus disease pandemic (COVID-19). Therefore, there is a critical need to identify underlying metabolic factors associated with obesity that could be contributing toward increased susceptibility to SARS-CoV-2 in this vulnerable population. Here, we focus on the critical role of potent endogenous lipid metabolites known as specialized pro-resolving mediators (SPMs) that are synthesized from polyunsaturated fatty acids. SPMs are generated during the transition of inflammation to resolution and have a vital role in directing damaged tissues to homeostasis; furthermore, SPMs display anti-viral activity in the context of influenza infection without being immunosuppressive. We cover evidence from rodent and human studies to show that obesity, and its co-morbidities, induce a signature of SPM deficiency across immunometabolic tissues. We further discuss how the effects of obesity upon SARS-CoV-2 infection are likely exacerbated with environmental exposures that promote chronic pulmonary inflammation and augment SPM deficits. Finally, we highlight potential approaches to overcome the loss of SPMs using dietary and pharmacological interventions. Collectively, this mini-review underscores the need for mechanistic studies on how SPM deficiencies driven by obesity and environmental exposures may exacerbate the response to SARS-CoV-2.