ABSTRACT
COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC50 < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔGbind) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro.
Subject(s)
COVID-19 , Coronavirus 3C Proteases , Peptidomimetics , Humans , SARS-CoV-2/metabolism , Peptidomimetics/pharmacology , Molecular Docking Simulation , Protease Inhibitors/chemistry , Amino Acids , Molecular Dynamics Simulation , Antiviral Agents/pharmacology , Antiviral Agents/chemistryABSTRACT
Aurones are a small subgroup of flavonoids in which the basic C6-C3-C6 skeleton is arranged as (Z)-2-benzylidenebenzofuran-3(2H)-one. These compounds are structural isomers of flavones and flavonols, natural products reported as potent inhibitors of SARS-CoV-2 replication. Herein, we report the design, synthesis, and anti-SARS-CoV-2 activity of a series of 25 aurones bearing different oxygenated groups (OH, OCH3, OCH2OCH3, OCH2O, OCF2H, and OCH2C6H4R) at the A- and/or B-rings using cell-based screening assays. We observed that 12 of the 25 compounds exhibit EC50 < 3 µM (8e, 8h, 8j, 8k, 8l, 8m, 8p, 8q, 8r, 8w, 8x, and 8y), of which five presented EC50 < 1 µM (8h, 8m, 8p, 8q, and 8w) without evident cytotoxic effect in Calu-3 cells. The substitution of the A- and/or B-ring with OCH3, OCH2OCH3, and OCF2H groups seems beneficial for the antiviral activity, while the corresponding phenolic derivatives showed a significant decrease in the anti-SARS-CoV-2 activity. The most potent compound of the series, aurone 8q (EC50 = 0.4 µM, SI = 2441.3), is 2 to 3 times more effective than the polyphenolic flavonoids myricetin (2) and baicalein (1), respectively. Investigation of the five more active compounds as inhibitors of SARS-CoV-2 3CLpro based on molecular dynamic calculations suggested that these aurones should detach from the active site of 3CLpro, and, probably, they could bind to another SARS-CoV-2 protein target (either receptor or enzyme).
Subject(s)
Benzofurans , COVID-19 , Humans , SARS-CoV-2 , Benzofurans/pharmacology , Flavonoids/pharmacology , Flavonoids/chemistry , Antiviral Agents/pharmacology , Protease Inhibitors/pharmacology , Molecular Docking SimulationABSTRACT
Annual vaccination against influenza is the best tool to prevent deaths and hospitalizations. Regular updates of trivalent inactivated influenza vaccines (TIV) are necessary due to high mutation rates in influenza viruses. TIV effectiveness is affected by antigenic mismatches, age, previous immunity, and other host factors. Studying TIV effectiveness annually in different populations is critical. The serological responses to Southern-Hemisphere TIV and circulating influenza strains were evaluated in 2018−2020 among Brazilian volunteers, using hemagglutination inhibition (HI) assays. Post-vaccination titers were corrected to account for pre-vaccination titers. Our population achieved >83% post-vaccination seroprotection levels, whereas seroconversion rates ranged from 10% to 46%. TIV significantly enhanced antibody titers and seroprotection against all prior and contemporary vaccine and circulating strains tested. Strong cross-reactive responses were detected, especially between H1N1 subtypes. A/Singapore/INFIMH-16-0019/2016, included in the 2018 TIV, induced the poorest response. Significant titer and seroprotection reductions were observed 6 and 12 months after vaccination. Age had a slight effect on TIV response, whereas previous vaccination was associated with lower seroconversion rates and titers. Despite this, TIV induced high seroprotection for all strains, in all groups. Regular TIV evaluations, based on regional influenza strain circulation, should be conducted and the factors affecting response studied.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza Vaccines , Influenza, Human , Adult , Antibodies, Viral , Brazil/epidemiology , Hemagglutination Inhibition Tests , Humans , Influenza A Virus, H1N1 Subtype/genetics , Seasons , Vaccines, InactivatedABSTRACT
Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.
Subject(s)
Herpes Simplex , Herpesviridae Infections , Herpesvirus 1, Human , Acyclovir/pharmacology , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Chlorocebus aethiops , Herpes Simplex/drug therapy , Herpesviridae Infections/drug therapy , Herpesvirus 1, Human/physiology , Pyrazoles , Pyridines/pharmacology , Pyridines/therapeutic use , Vero Cells , Virus ReplicationABSTRACT
The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.
Subject(s)
COVID-19/immunology , COVID-19/virology , Immunity, Cellular , Models, Biological , SARS-CoV-2/physiology , Adult , Alveolar Epithelial Cells/virology , COVID-19/blood , COVID-19/genetics , Cytokines/genetics , Cytokines/metabolism , Female , Gene Expression Regulation , Humans , Immunologic Memory/immunology , Killer Cells, Natural/immunology , Leukocytes, Mononuclear/virology , Male , Middle Aged , Phenotype , T-Lymphocytes/immunology , Virus Replication/physiology , Young AdultABSTRACT
Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.
Subject(s)
COVID-19/complications , Inflammation Mediators/metabolism , Inflammation/etiology , Lipid Droplets/pathology , SARS-CoV-2/isolation & purification , Animals , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Case-Control Studies , Chlorocebus aethiops , Humans , Inflammation/metabolism , Inflammation/pathology , Vero Cells , Virus ReplicationABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.
Subject(s)
Antiviral Agents/pharmacology , Atazanavir Sulfate/pharmacology , Betacoronavirus/drug effects , Cytokines/metabolism , Ritonavir/pharmacology , Animals , Atazanavir Sulfate/chemistry , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , COVID-19 , Cell Death/drug effects , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Infections/pathology , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Drug Therapy, Combination , Humans , Inflammation/metabolism , Inflammation/virology , Lopinavir/pharmacology , Molecular Docking Simulation , Monocytes/virology , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Pneumonia, Viral/pathology , Protease Inhibitors/pharmacology , SARS-CoV-2 , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , COVID-19 Drug TreatmentABSTRACT
Neuraminidase inhibitors (NAIs) are the main class of antivirals currently used for the treatment of influenza infections. As influenza viruses are constantly evolving, drug-resistance can emerge resulting in reduced effectiveness of treatment. This study evaluated the presence of molecular markers associated with NAI susceptibility in 724 influenza A(H1N1)pdm09 positive samples from Brazilian surveillance system from the 2014-2016 seasons, including 76 isolates tested for oseltamivir (OST) susceptibility and 23 isolates also tested for zanamivir, peramivir and laninamivir susceptibility. We identified the H275Y (nâ¯=â¯3) and I223K (nâ¯=â¯1) NA substitutions, associated with reduced inhibition (RI) by the NAIs. Noteworthy, no epidemiological links were identified among the patients infected with the mutant viruses. Phylogenetic analysis from NA and hemagglutinin genes showed that mutant viruses were not clustered. All mutant virus strains carried the permissive substitutions V241I and N369K, in addition to the N386K, which has been shown to destabilize the NA structure. Functional NA analysis of one virus containing the H275Y mutation confirmed its highly RI profile to OST and peramivir and demonstrated that it had decreased viral replication and NA thermostability compared to the wild type virus. The remaining tested isolates presented normal inhibition profile to the NAIs tested. In conclusion, the overall frequency of influenza A(H1N1)pdm09 viruses bearing mutations associated with NAI RI was 0.6%, similar to what has been observed in recent global studies.