Diphenyl Diselenide and SARS-CoV-2: in silico Exploration of the Mechanisms of Inhibition of Main Protease (Mpro) and Papain-like Protease (PLpro).

The SARS-CoV-2 pandemic has prompted global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro) and the papain-like protease (PLpro) are essential for viral replication and are key targets for therapeutic development. In this work, we investigate the mechanisms of SARS-CoV-2 inhibition by diphenyl diselenide (PhSe)2 which is an archetypal model of diselenides and a renowned potential therapeutic agent. The in vitro inhibitory concentration of (PhSe)2 against SARS-CoV-2 in Vero E6 cells falls in the low micromolar range. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations [level of theory: SMD-B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ] are used to inspect non-covalent inhibition modes of both proteases via π-stacking and the mechanism of covalent (PhSe)2 + Mpro product formation involving the catalytic residue C145, respectively. The in vitro CC50 (24.61 µM) and EC50 (2.39 µM) data indicate that (PhSe)2 is a good inhibitor of the SARS-CoV-2 virus replication in a cell culture model. The in silico findings indicate potential mechanisms of proteases' inhibition by (PhSe)2; in particular, the results of the covalent inhibition here discussed for Mpro, whose thermodynamics is approximatively isoergonic, prompt further investigation in the design of antiviral organodiselenides.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

Aurones: A Promising Scaffold to Inhibit SARS-CoV-2 Replication.

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).

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case.

BACKGROUND: According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE: To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS: We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS: We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS: Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

Anti-SARS-CoV-2 Activity of Ampelozizyphus amazonicus (Saracura-Mirá): Focus on the Modulation of the Spike-ACE2 Interaction by Chemically Characterized Bark Extracts by LC-DAD-APCI-MS/MS.

Traditional medicine shows several treatment protocols for COVID-19 based on natural products, revealing its potential as a possible source of anti-SARS-CoV-2 agents. Ampelozizyphus amazonicus is popularly used in the Brazilian Amazon as a fortifier and tonic, and recently, it has been reported to relieve COVID-19 symptoms. This work aimed to investigate the antiviral potential of A. amazonicus, focusing on the inhibition of spike and ACE2 receptor interaction, a key step in successful infection. Although saponins are the major compounds of this plant and often reported as its active principles, a polyphenol-rich extract was the best inhibitor of the spike and ACE2 interaction. Chemical characterization of A. amazonicus bark extracts by LC-DAD-APCI-MS/MS before and after clean-up steps for polyphenol removal showed that the latter play an essential role in maintaining this activity. The effects of the extracts on viral replication were also assessed, and all samples (aqueous and ethanol extracts) demonstrated in vitro activity, inhibiting viral titers in the supernatant of Calu-3 cells after 24 hpi. By acting both in the SARS-CoV-2 cell entry process and its replication, A. amazonicus bark extracts stand out as a multitarget agent, highlighting the species as a promising candidate in the development of anti-SARS-CoV-2 drugs.

Antiviral Effect of 5'-Arylchalcogeno-3-aminothymidine Derivatives in SARS-CoV-2 Infection.

The understanding that zidovudine (ZDV or azidothymidine, AZT) inhibits the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and that chalcogen atoms can increase the bioactivity and reduce the toxicity of AZT has directed our search for the discovery of novel potential anti-coronavirus compounds. Here, the antiviral activity of selenium and tellurium containing AZT derivatives in human type II pneumocytes cell model (Calu-3) and monkey kidney cells (Vero E6) infected with SARS-CoV-2, and their toxic effects on these cells, was evaluated. Cell viability analysis revealed that organoselenium (R3a-R3e) showed lower cytotoxicity than organotellurium (R3f, R3n-R3q), with CC50 ≥ 100 µM. The R3b and R3e were particularly noteworthy for inhibiting viral replication in both cell models and showed better selectivity index. In Vero E6, the EC50 values for R3b and R3e were 2.97 ± 0.62 µM and 1.99 ± 0.42 µM, respectively, while in Calu-3, concentrations of 3.82 ± 1.42 µM and 1.92 ± 0.43 µM (24 h treatment) and 1.33 ± 0.35 µM and 2.31 ± 0.54 µM (48 h) were observed, respectively. The molecular docking calculations were carried out to main protease (Mpro), papain-like protease (PLpro), and RdRp following non-competitive, competitive, and allosteric inhibitory approaches. The in silico results suggested that the organoselenium is a potential non-competitive inhibitor of RdRp, interacting in the allosteric cavity located in the palm region. Overall, the cell-based results indicated that the chalcogen-zidovudine derivatives were more potent than AZT in inhibiting SARS-CoV-2 replication and that the compounds R3b and R3e play an important inhibitory role, expanding the knowledge about the promising therapeutic capacity of organoselenium against COVID-19.

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators.

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.

The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle.

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.

Bioassay-Guided Fractionation of Siparuna glycycarpa n-Butanol Extract with Inhibitory Activity against Influenza A(H1N1)pdm09 Virus by Centrifugal Partition Chromatography (CPC).

Siparuna glycycarpa occurs in the Amazon region, and some species of this genus are used in Brazilian folk medicine. A recent study showed the inhibitory effect of this species against influenza A(H1N1)pdm09 virus, and in order to acquire active fractions, a polar solvent system n-butanol-methanol-water (9:1:10, v/v) was selected and used for bioassay-guided fractionation of n-butanol extract by centrifugal partition chromatography (CPC). The upper phase was used as stationary phase and the lower phase as mobile (descending mode). Among the collected fractions, the ones coded SGA, SGC, SGD, and SGO showed the highest antiviral inhibition levels (above 74%) at 100 µg·mL-1 after 24 h of infection. The bioactive fractions chemical profiles were investigated by LC-HRMS/MS data in positive and negative ionization modes exploring the Global Natural Products Social Molecular Networking (GNPS) platform to build a molecular network. Benzylisoquinoline alkaloids were annotated in the fractions coded SGA, SGC, and SGD collected during elution step. Aporphine alkaloids, O-glycosylated flavonoids, and dihydrochalcones in SGO were acquired with the change of mobile phase from lower aqueous to upper organic. Benzylisoquinolinic and aporphine alkaloids as well as glycosylated flavonoids were annotated in the most bioactive fractions suggesting this group of compounds as responsible for antiviral activity.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells.

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Atazanavir, Alone or in Combination with Ritonavir, Inhibits SARS-CoV-2 Replication and Proinflammatory Cytokine Production.

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.

Identification of SARS-CoV-2 and additional respiratory pathogens cases under the investigation of COVID-19 initial phase in a Brazilian reference laboratory.

Coronavirus disease 2019 (COVID-19) surveillance, in Brazil, initiated shortly after its description, in China. Our aim was to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and additional pathogens in samples from the initial phase of the outbreak in Brazil, from late February to late March. From 707 samples analysed, 29 (4.1%) were SARS-CoV-2 positive. Fever and cough were their most prevalent symptoms. Co-detection of rhinovirus was observed in 2 (6.9%) cases. Additional pathogens were identified in 66.1% of the SARS-CoV-2 negative cases, mainly rhinovirus and influenza A(H1N1)pdm09. Thus, we emphasise the importance of differential diagnosis in COVID-19 suspected cases.

Influenza virus RNA polymerase may be activated inside the virion.

Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.

An observational clinical case of Zika virus-associated neurological disease is associated with primary IgG response and enhanced TNF levels.

Descriptive clinical data help to reveal factors that may provoke Zika virus (ZIKV) neuropathology. The case of a 24-year-old female with a ZIKV-associated severe acute neurological disorder was studied. The levels of ZIKV in the cerebrospinal fluid (CSF) were 50 times higher than the levels in other compartments. An acute anti-flavivirus IgG, together with enhanced TNF-alpha levels, may have contributed to ZIKV invasion in the CSF, whereas the unbiased genome sequencing [obtained by next-generation sequencing (NGS)] of the CSF revealed that no virus mutations were associated with the anatomic compartments (CSF, serum, saliva and urine).

Insights from genome of Clostridium butyricum INCQS635 reveal mechanisms to convert complex sugars for biofuel production.

Clostridium butyricum is widely used to produce organic solvents such as ethanol, butanol and acetone. We sequenced the entire genome of C. butyricum INCQS635 by using Ion Torrent technology. We found a high contribution of sequences assigned for carbohydrate subsystems (15-20 % of known sequences). Annotation based on protein-conserved domains revealed a higher diversity of glycoside hydrolases than previously found in C. acetobutylicum ATCC824 strain. More than 30 glycoside hydrolases (GH) families were found; families of GH involved in degradation of galactan, cellulose, starch and chitin were identified as most abundant (close to 50 % of all sequences assigned as GH) in C. butyricum INCQS635. KEGG metabolic pathways reconstruction allowed us to verify possible routes in the C. butyricum INCQS635 and C. acetobutylicum ATCC824 genomes. Metabolic pathways for ethanol synthesis are similar for both species, but alcohol dehydrogenase of C. butyricum INCQS635 and C. acetobutylicum ATCC824 was different. The genomic repertoire of C. butyricum is an important resource to underpin future studies towards improved solvents production.

Whole-genome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season.

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.

Cholesterol depletion induces transcriptional changes during skeletal muscle differentiation.

BACKGROUND: Myoblasts undergo major changes in their plasma membrane during the initial steps of skeletal muscle differentiation, including major alterations in the distribution of cholesterol. Cholesterol is involved in crucial membrane functions, such as fluidity, and permeability, and in the organization of specialized membrane microdomains (or lipid rafts). We have previously shown that alterations in cholesterol levels in myoblasts induce changes in proliferation and differentiation, which involves activation of Wnt/beta-catenin signaling pathway. In this study we used methyl-ß-cyclodextrin (MbCD) to extract cholesterol from the membrane of chick skeletal muscle cells grown in culture. Using Ion Torrent-based sequencing, we compared the transcriptome of untreated and MbCD treated cells. Our aim was to define the genes that are expressed in these two conditions and relate their expression to cellular functions. RESULTS: Over 5.7 million sequences were obtained, representing 671.38 Mb of information. mRNA transcriptome profiling of myogenic cells after cholesterol depletion revealed alterations in transcripts involved in the regulation of apoptosis, focal adhesion, phagosome, tight junction, cell cycle, lysosome, adherens junctions, gap junctions, p53 signaling pathway, endocytosis, autophagy and actin cytoskeleton. Lim domain only protein 7 mRNA was found to be the highest up-regulated feature after cholesterol depletion. CONCLUSIONS: This is the first study on the effects of membrane cholesterol depletion in mRNA expression in myogenic cells. Our data shows that alterations in the availability of plasma membrane cholesterol lead to transcriptional changes in myogenic cells. The knowledge of the genes involved in the cellular response to cholesterol depletion could contribute to our understanding of skeletal muscle differentiation.

Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV­2 Mpro).

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.

Myrtucommulones and Related Acylphloroglucinols from Myrtaceae as a Promising Source of Multitarget SARS-CoV-2 Cycle Inhibitors.

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causative agent of the COVID-19 pandemic, a global public health problem. Despite the numerous studies for drug repurposing, there are only two FDA-approved antiviral agents (Remdesivir and Nirmatrelvir) for non-hospitalized patients with mild-to-moderate COVID-19 symptoms. Consequently, it is pivotal to search for new molecules with anti-SARS-CoV-2 activity and to study their effects in the human immune system. Ebselen (Eb) is an organoselenium compound that is safe for humans and has antioxidant, anti-inflammatory, and antimicrobial properties. Diphenyl diselenide ((PhSe)2) shares several pharmacological properties with Eb and is of low toxicity to mammals. Herein, we investigated Eb and (PhSe)2 anti-SARS-CoV-2 activity in a human pneumocytes cell model (Calu-3) and analyzed their toxic effects on human peripheral blood mononuclear cells (PBMCs). Both compounds significantly inhibited the SARS-CoV-2 replication in Calu-3 cells. The EC50 values for Eb and (PhSe)2 after 24 h post-infection (hpi) were 3.8 µM and 3.9 µM, respectively, and after 48 hpi were 2.6 µM and 3.4 µM. These concentrations are safe for non-infected cells, since the CC50 values found for Eb and (PhSe)2 on Calu-3 were greater than 200 µM. Importantly, the concentration rates tested on viral replication were not toxic to human PBMCs. Therefore, our findings reinforce the efficacy of Eb and demonstrate (PhSe)2 as a new candidate to be tested in future trials against SARS-CoV-2 infection/inflammation conditions.