ABSTRACT
The TG6002.03 trial is a dose-escalation phase 1 clinical trial of TG6002 infusion via the hepatic artery in patients with liver-dominant colorectal cancer metastases. TG6002 is an engineered Copenhagen strain oncolytic Vaccinia virus, deleted of thymidine kinase and ribonucleotide reductase to enhance tumor selective viral replication and expressing FCU1, an enzyme converting the non-cytotoxic prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). In this trial, patients with advanced unresectable liver-dominant metastatic colorectal cancer who had failed previous oxaliplatin and irinotecan-based chemotherapy were treated with up to 2 cycles of TG6002 infusion 6 weeks apart via the hepatic artery on day 1 combined with oral 5-FC on days 5 to 14 (where day 1 = TG6002 infusion). TG6002 infusion was performed over 30 minutes via selective catheterization of the hepatic artery proper. 5-FC oral dosing was 50mg/kg x4 daily. Blood was sampled for TG6002 pharmacokinetics and 5-FC and 5-FU measurements. Sampling of liver metastases was performed at screening and on day 4 or day 8 for virus detection and 5-FC and 5-FU quantification. In total, 15 patients (median age 61 years, range 37-78) were treated in 1 UK centre and 2 centres in France and received a dose of TG6002 of 1 x 106 (n=3), 1 x 107 (n=3), 1 x 108 (n=3), or 1 x 109 pfu (n=6). Fourteen of the 15 patients received a single cycle of treatment, including one patient who did not received 5-FC, and one patient received two cycles. TG6002 was transiently detected in plasma following administration, suggesting a strong tissue selectivity for viral replication. In the highest dose cohort, a virus rebound was observed on day 8, concordant with replication time of the virus. In serum samples, 5-FU was present on day 8 in all patients with a high variability ranging from 0.8 to 1072 ng/mL and was measurable over several days after initiation of therapy. Seven of the 9 patients evaluable showed the biodistribution of the virus in liver lesions by PCR testing on day 4 or day 8. Translational blood samples showed evidence for T-cell activation and immune checkpoint receptor-ligand expression. At 1 x 109 pfu, there was evidence for T-cell proliferation and activation against tumour-associated antigens by ELISpot and for immunogenic cell death. In terms of safety, a total of 34 TG6002-related adverse events were reported, of which 32 were grade 1-2 and 2 were grade 3. The maximum tolerated dose was not reached, and a single dose-limiting toxicity was observed consisting of a myocardial infarction in a context of recent Covid-19 infection in a 78-year-old patient. These results indicate that TG6002 infused via the hepatic artery in combination with oral 5-FC was well tolerated, effectively localized and replicated in the tumor tissues, expressed its therapeutic payload and showed anti-tumoral immunological activity.
ABSTRACT
It is well known that vitamin D's general immunomodulatory actions are helpful in viral infections and that a shortage is linked to a more serious prognosis for Covid-19. In this sistematic review, we examined the existing literature on evidence as to whether there is also link between vitamin D range levels in pediatric population and the outcome of the Covid-19 infection. We looked for studies that measured vitamin D blood concentrations and examined the effects of vitamin D supplementation in young infected patients. Vitamin D may decrease the risk of respiratory infections in a number of ways through its interactions with numerous cells, including by decreasing viral survival and replication, reducing the cytokine storm, raising angiotensin-converting enzyme 2 concentrations (ACE2) while not damaging the endothelial integrity. The incidence or severity of Covid-19 is linked with blood 25-hydroxyvitamin D concentrations, according to many observational studies. However experimental verification is still needed. Given their safety and broad therapeutic window, vitamin D supplements seem to be an effective way for individuals and doctors to prevent or treat Covid-19. Nonetheless, the outcomes of significant vitamin D randomized controlled trials are further needed.Copyright © 2022 Maria Nicolae et al., published by Sciendo.
ABSTRACT
Background: The emergence of Coronavirus disease (COVID-19) has been declared a pandemic and made a medical emergency worldwide. Various attempts have been made, including optimizing effective treatments against the disease or developing a vaccine. Since the SARS-CoV-2 protease crystal structure has been discovered, searching for its inhibitors by in silico technique becomes possible. Objective(s): This study aims to virtually screen the potential of phytoconstituents from the Begonia genus as 3Cl pro-SARS-CoV- 2 inhibitors, based on its crucial role in viral replication, hence making these proteases "promising" for the anti-SARS-CoV-2 target. Method(s): In silico screening was carried out by molecular docking on the web-based program DockThor and validated by a retrospective method. Predictive binding affinity (Dock Score) was used for scoring the compounds. Further molecular dynamics on Desmond was performed to assess the complex stability. Result(s): Virtual screening protocol was valid with the area under curve value 0.913. Molecular docking revealed only beta-sitosterol-3-O-beta-D-glucopyranoside with a lower docking score of -9.712 kcal/mol than positive control of indinavir. The molecular dynamic study showed that the compound was stable for the first 30 ns simulations time with Root Mean Square Deviation <3 A, despite minor fluctuations observed at the end of simulation times. Root Mean Square Fluctuation of catalytic sites HIS41 and CYS145 was 0.756 A and 0.773 A, respectively. Conclusion(s): This result suggests that beta-sitosterol-3-O-beta-Dglucopyranoside might be a prospective metabolite compound that can be developed as anti-SARS-CoV-2.Copyright © 2023, Page Press Publications. All rights reserved.
ABSTRACT
Background/Objectives: Rosmarinus Officinalis L.(Rosemary) extract Carnosic acid(CA) has been investigated for its antimicrobial and antioxidative properties(1). Only limited number of publications reported the utilization of this extract in SARSCoV-2 infection. Also, the mechanistic understanding of CA remains to be determined. Our goal was to elucidate the potential role of CA in COVID19. To obtain mechanistic insight of pharmacogenomic action of CA, comprehensive in silico analyses were performed. Further in vitro experiments were done to illustrate the cytotoxicity of CA and confirm in silico findings. Method(s): CA was extracted from Rosmarinus Officinalis L. by HPLC. Stimulation assays were performed using the COVID19 samples. In silico pharmacogenomic properties of CA were performed by using SwissADME. SwissTargetPrediction tool was utilized to define the possible targets. SARS-CoV-2-interacting proteins were evaluated using STRING(2). To verify in silico findings, gene expression levels were analyzed using qPCR. Result(s): Among the top 15 SwissTargetPrediction target molecules(out of 100), Prostaglandin E synthase(PTGES) had the highest probability for CA. Among 332 proteins identified using the STRING, PGES2 was found to be interacting with the nsp7, important molecule for viral replication. The stimulation assays and gene expression analyses confirmed the viral inhibitory role of CA through PTGES pathway. Conclusion(s): To our knowledge, our work is the first to reveal the inhibitory role of CA in COVID19 through PTGES pathway. Given the crucial role of PTGES in inflammation, it is noteworthy to examine CA as potential anti-SARS-CoV2 therapeutics.
ABSTRACT
Cumulative data regardingCOVID-19 infection during pregnancy have demonstrated the ability of SARS-CoV-2 to infect the placenta. However, the mechanisms of SARS-CoV-2 placental viral entry are yet to be defined. SARS-CoV-2 infects cells by binding to the ACE2 receptor. However, SARS-CoV-2 cell entry also requires co-localization of spike protein cleavage by the serine protease TMPRSS2. However, the co-expression of ACE2 and TMPRSS2 in placental cells is debated, raising the question of whether potential non-canonical molecular mechanismsmay be involved in SARS-CoV-2 placental cells' viral entry. Although published data regarding the ability of the SARS-CoV- 2 to infect the fetus are contradicting, the placenta appears to be an immunological barrier to active SARS-CoV-2 infection and vertical transmission;however, the mechanism is unclear. Our experiments demonstrated the ability of the SARS-CoV-2 virus to directly infect the placenta and induce transcriptomic responses in COVID-positive mothers. These transcriptomic responses were characterized by differential expression of specific mRNAs and miRNAs associated with SARS-CoV-2 infection, with induction of specific placental miRNAs that can inhibit viral replication. Failure in such mechanisms may be associated with vertical transmission. Since the start of the COVID-19 pandemic, the COVID-19 mRNA vaccines have been widely used to reduce the morbidity and mortality of SARS-CoV-2 infection. Historically, non-live vaccines have not caused any harm to pregnant mothers;however, it is unclear whether our current understanding of the effects of non-live vaccines serves as a reliable precedent owing to the novel technology used to create these mRNA vaccines. Since there are no definitive data on the possible biodistribution of mRNA vaccines to the placenta, the likelihood of vaccine mRNA reaching the fetus remains uncertain. Little has been reported on the tissue localization of the lipid nanoparticles (LNPs) after intramuscular (IM) administration of the mRNA vaccine. The biodistribution of LNPs containing the mRNA vaccine has been investigated in animal models but not humans. In the murine model, the vaccine LNPs were rapidly disseminated to several organs, including the heart, liver, kidney, lung, and spleen, following IM administration. However, no traditional pharmacokinetic or biodistribution studies have been performed with the mRNA vaccines, including possible biodistribution to breast milk or the placenta.
ABSTRACT
Within the conditions of the ongoing COVID-19 pandemic, when many questions regarding prevention and treatment strategies remain unsolved and the search for the best antiviral agents is underway, attention should be paid to the role of trace elements zinc and selenium in increasing the body's resistance to viral infections and their direct antiviral activity against SARS-CoV-2. Experimental data show that trace elements zinc and selenium not only actthrough regulating the immune response at all levels of humoral and cellular immunity, but also can play a significant role in adjuvant therapy for viral diseases. This is especially relevant in the case of COVID-19. Studies of the direct antiviral effect of these micro-elements testify to its 3 main ways to SARS-Cov-2: I - counteraction to virus replication and its transcription through: (i) their covalent binding to the SH-group of the cysteine of the main protease M(Pro) of the virus;(ii) inhibition of its RNA polymerase activity by zinc;II - preventing the penetration of the virus into cells due to blocking SH-groups of protein disulfide isomerase (RDI) of the protein of its spikes (peplomers);III - decreasing the adsorption capacity of the virus due to the blocking of the electrostatic interaction of SARS-CoV-2 peplomers and angiotensin-converting enzyme (ACE-2) in ultra-low, uncharacteristic oxidation states (Zn+1and Se-2). The intensity of the antiviral action of these trace elements may depend on their chemical form. It was found that zinc citrate (a five-membered complex of zinc with citric acid) and monoselenium citric acid obtained with the help of nanotechnology have a greater intensity of action and higher chemical purity. Taking into account the immunostimulating and direct antiviral effect of zinc and selenium, their use in the form of pharmaceuticals and dietary supplements should be considered as adjunctive therapy for SARS-CoV-2 in patients, or as a preventive strategy for uninfected people from risk groups during the spread of COVID-19.Copyright © Publisher PH <<Akademperiodyka>> of the NAS of Ukraine, 2023.
ABSTRACT
SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, is a highly contagious positive-sense RNA virus. Its explosive community spread and the emergence of new mutant strains have created palpable anxiety even in vaccinated people. The lack of effective anticoronavirus therapeutics continues to be a major global health concern, especially due to the high evolution rate of SARS-CoV-2. The nucleocapsid protein (N protein) of SARS-CoV-2 is highly conserved and involved in diverse processes of the virus replication cycle. Despite its critical role in coronavirus replication, N protein remains an unexplored target for anticoronavirus drug discovery. Here, we demonstrate that a novel compound, K31, binds to the N protein of SARS-CoV-2 and noncompetitively inhibits its binding to the 5' terminus of the viral genomic RNA. K31 is well tolerated by SARS-CoV-2-permissive Caco2 cells. Our results show that K31 inhibited SARS-CoV-2 replication in Caco2 cells with a selective index of ~58. These observations suggest that SARS-CoV-2 N protein is a druggable target for anticoronavirus drug discovery. K31 holds promise for further development as an anticoronavirus therapeutic. IMPORTANCE The lack of potent antiviral drugs for SARS-CoV-2 is a serious global health concern, especially with the explosive spread of the COVID-19 pandemic worldwide and the constant emergence of new mutant strains with improved human-to-human transmission. Although an effective coronavirus vaccine appears promising, the lengthy vaccine development processes in general and the emergence of new mutant viral strains with a potential to evade the vaccine always remain a serious concern. The antiviral drugs targeted to the highly conserved targets of viral or host origin remain the most viable and timely approach, easily accessible to the general population, in combating any new viral illness. The majority of anticoronavirus drug development efforts have focused on spike protein, envelope protein, 3CLpro, and Mpro. Our results show that virus-encoded N protein is a novel therapeutic target for anticoronavirus drug discovery. Due to its high conservation, the anti-N protein inhibitors will likely have broad-spectrum anticoronavirus activity.
Subject(s)
COVID-19 , Humans , SARS-CoV-2 , COVID-19 Vaccines , Pandemics/prevention & control , Caco-2 Cells , Drug Discovery , Antiviral Agents/therapeutic use , Nucleocapsid ProteinsABSTRACT
Background: Implementation of vaccination programmes has had a transformational impact on control of the SARS-CoV-2 pandemic, but the need for effective antiviral drugs remains. Molnupiravir (MPV) targets viral RNA polymerase inhibiting replication via lethal mutagenesis and nirmatrelvir (NTV) is a protease inhibitor boosted with ritonavir when given clinically. This study aimed to assess the virological efficacy of NTV and MPV individually and in combination against the SARS-CoV-2 BA.1 Omicron variant in a K18-hACE2 mouse model. Method(s): K18-hACE2 mice were inoculated intranasally with 103 PFU of SARSCoV-2 BA.1 Omicron (B.1.1.529). After 24 hours, mice were orally dosed q12H, as outlined in Figure 1. At 2, 3, and 4-days post infection mice were sacrificed, and lung samples harvested. Animals were weighed and monitored daily throughout. Subsequently, viral replication in the lung was quantified using qRT-PCR to measure total (N-gene) and sub-genomic (E-gene) viral RNA. Data were normalized to 18S for quantitation. Viral exposures expressed as Areas Under viral load Curves (AUCs) were calculated by the trapezoidal method using mean values at each timepoint. Separate studies in Syrian golden hamsters using individual drugs were also conducted, and total serum IgG was measured by ELISA at 4-days post infection. Result(s): Mice gained weight in all groups post-treatment, with no significant difference between groups. A reduction in lung viral exposure was evident in all treatment groups compared to the vehicle control dosed mice (Figure 1). Coadministration of NTV with MPV displayed a trend towards lower lung viral exposure compared to the vehicle control with ~40-and ~45-fold reduction in AUC for N-and SgE-gene assays, respectively. Dosed individually, NTV and MPV reduced viral exposure 5.7-and 7.7-fold for the N-gene assay, respectively. Differences in total serum IgG concentrations were evident between vehicle and NTV-(34-fold reduction, P=0.018), and MPV-(4.2-fold reduction, P=0.053) treated hamsters. Conclusion(s): These data show virological efficacy of NTV and MPV against the SARS-CoV-2 BA.1 Omicron variant. The combination of NTV and MPV demonstrated a lower viral RNA exposure in the lung than either drug alone, albeit not statistically significant. Initial data indicate potential immune alterations in NTV and MPV dosed hamsters. Studies to clarify the utility of NTV/ MPV combinations and further characterize the impact of antiviral therapy on IgG are warranted.
ABSTRACT
Background: The SARS-CoV-2 Omicron variant is highly immune evasive but is attenuated in cell and animal models of infection, which many reports attribute to spike mutations. However, the phenotype and contribution to viral fitness of Omicron non-spike mutations remain unknown. Method(s): To study mutations across the entire genome independent of spike, we developed a novel cloning and replicon system capable of generating mutants within 6 hours and obtaining phenotypic results within 3-4 days. Result(s): Using a series of Omicron replicons, we found that ORF1ab harbors critical mutations, especially in the nonstructural protein 6 (NSP6), which lower viral fitness and are currently evolving in Omicron subvariants. In addition, Omicron mutations in several NSPs epistatically interact and are critical for viral replication and polyprotein processing. Conclusion(s): Collectively, we describe a robust replicon technology to study mutations across the genome and our data highlight the need to vigilantly study and monitor non-spike mutations in emerging Omicron subvariants.
ABSTRACT
Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.
ABSTRACT
The re-emergence of SARS-CoV, known as SARS-CoV-2, has proven extremely infectious that has infected a huge population worldwide. SARS-CoV-2 genome is translated into polyproteins that is processed by virus-specific protease enzymes. 3CLprotease is named as the main protease (Mpro) enzyme that cleaves nsp4 to nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. Till date, there is no effective commercially available drug against COVID-19 and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. We have used a high-throughput virtual screening approach to examine FDA approved library, natural compound library, and LOPAC 1280 (Library of Pharmacologically Active Compounds, Sigma-Aldrich, St. Louis, MO) library against Mpro. Primary screening identified potential drug molecules for the target, among which ten molecules were studied further using biophysical and biochemical techniques. SPR was used to validate the binding of inhibitors to purified Mpro and using FRET-based biochemical protease assay these inhibitors were confirmed to have Mpro inhibitory activity. Based on the kinetic studies, the antiviral efficacy of these compounds was further analysed by cell-culture based antiviral assays. Four out of ten molecules inhibited SARS-CoV-2 replication in Vero cells at a concentration range of 12.5 to 50 muM. The antiviral activity was evaluated by RT-PCR assay and TCID50 experiments. The co-crystallization of Mpro in complex with inhibitor for determining their structures is being carried out. Collectively, this study will provide valuable mechanistic and structural insights for development of effective antiviral therapeutics against SARS-CoV-2.
ABSTRACT
Background: We previously screened 10 human lung and upper airway cell lines expressing variable levels of endogenous ACE2/TMPRSS2. We found that H522 human lung adenocarcinoma cells supported SARS-CoV-2 replication independent of ACE2, whereas the ACE2 positive cell lines were not permissive to infection. Type I/III interferons (IFNs) potently restrict SARS-CoV-2 replication through the actions of hundreds of interferon-stimulated genes (ISGs) that are upregulated upon IFN signaling. Here we report that a number of ACE2 positive airway cell lines are unable to support SARS-CoV-2 replication due to basal activation of the cGAS-STING DNA sensing pathway and subsequent upregulation of IFNs and ISGs which restrict SARS-CoV-2 replication. Method(s): SARS-CoV-2 WT strain 2019-nCoV/USA-WA1/2020 viral replication was detected through analysis of cell associated RNA. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK1/2 inhibitor. A siRNA-mediated screen was used to determine the molecular basis of basally high expression of ISGs in cell lines. CRISPR knockout of IFN-alpha receptor and cGAS-STING pathway components was conducted in parallel Results: Here we show that higher basal levels of IFN pathway activity underlie the inability of ACE2+ cell lines to support virus replication. Importantly, this IFN-induced block can be overcome by chemical inhibition and genetic disruption of the IFN signaling pathway or by ACE2 overexpression, suggesting that one or more saturable ISGs underlie the lack of permissivity of these cells. Ruxolitinib treatment increased SARS-CoV-2 RNA levels by nearly 3 logs in OE21 and SCC25. Furthermore, the baseline activation of the STING-cGAS pathway accounts for the high ISG levels and genetic disruption of the cGAS-STING pathway enhances levels by nearly 2 and 3 logs of virus replication in the two separate ACE2+ cell line models respectively. Conclusion(s): Our findings demonstrate that cGAS-STING-dependent activation of IFN-mediated innate immunity underlies the inability of ACE2+ airway cell lines to support SARS-CoV-2 replication. Our study highlights that in addition to ACE2, basal activation of cGAS-STING pathway, IFNs and ISGs may play a key role in defining SARS-CoV-2 cellular tropism and may explain the complex SARS-CoV- 2 pathogenesis in vivo.
ABSTRACT
Background: The role of myeloid cells in the pathogenesis of SARS-CoV-2 is well established, in particular as drivers of cytokine production and systemic inflammation characteristic of severe COVID-19. However, the potential for myeloid cells to act as bona fide targets of productive SARS-CoV-2 infection remains unclear. Method(s): Using anti-SARS-CoV-2 mAbs with a range of neutralisation potencies and binding specificities, we performed a detailed assessment of mAb-mediated infection of monocytes/macrophages. THP-1 cells were used as a model system, with results confirmed in primary macrophages. Result(s): Infection of THP-1 cells was seen via mAbs targeting the spike RBD, but not with those targeting the NTD or S2 subunit. mAbs with the most consistent potential to mediate infection targeted a conserved region of the RBD (group 1/class IV). No infection was seen with the same quantity of virus but in the absence of antibody, and pre-treating the cells with FcgammaRI and -II blocking antibodies inhibited infection. Thus, antibody-FcR interactions are able to expand the tropism of SARS-CoV-2. Time-course studies demonstrated high-level and productive infection. Studies performed in human iPSC-derived macrophages and primary monocyte-derived macrophages paralleled results seen in THP-1 cells but with lower infection levels. Up to 2% of macrophages were infected, with infected cells appearing multinucleated and syncytial. Addition of ruxolitinib, an inhibitor of JAK1/2 signalling, increased infection up to 10-fold, indicating limitation of infection through innate immune mechanisms. Sera from primary infections (n=80) mediated rare infection events, with a minority of samples (n=3) promoting significant infection. Competition assays confirmed results seen in sera, with the addition of neutralising mAbs diminishing the infection seen with infection-mediating mAbs. Thus, the presence of antibodies with potential to mediate infection is not sufficient to predict myeloid cell infection, rather, the context in which the antibodies are produced is key. Conclusion(s): We hypothesise that a nascent antibody response during peak viral replication in primary infection presents a window of opportunity for myeloid cells to become infected, while establishment of a robust polyclonal response via vaccination or prior infection reduces the likelihood of this occurring. Infection via antibody-FcR interactions could contribute to pathogenesis in primary infection, systemic virus spread or persistent infection.
ABSTRACT
Background: Different viruses employ similar pathways for replication, revealing key intracellular hotspots to target with host-directed therapies and achieve a broad-spectrum antiviral activity. Plitidepsin is a clinically approved antitumoral agent that blocks the elongation factor eEF1A required for protein translation. This drug counteracts SARS-CoV-2 replication and shows a favorable safety profile in COVID-19 patients. Yet, the precise antiviral mechanism of action of plitidepsin remains unknown. Method(s): Here we used a deep quantitative proteomic analysis to measure the impact of plitidepsin on the proteome of SARS-CoV-2-infected Vero E6 cells. This was complemented with transmission electron microscopy assays, which unraveled the subcellular and morphological changes associated to plitidepsin treatment. In addition, we performed functional in vitro assays to dissect the antiviral activity of plitidepsin against SARS-CoV-2 and other viruses. Result(s): We found that this drug inhibited the synthesis of all SARS-CoV-2 proteins in a dose-dependent manner. These included the R1AB polyproteins, which facilitate the synthesis of non-structural proteins involved in the formation of double membrane vesicles (DMV) required for viral replication. Plitidepsin reduced DMV formation and the morphogenesis of new viruses, having a greater impact on viral than on host proteins. Less than 14% of the cellular proteome was significantly affected by plitidepsin, inducing the up-regulation of key molecules associated with protein biosynthesis, such as the translation initiation factors eIF4A2 and eIF2S3. Therefore, plitidepsin induced a compensatory state that rescued protein translation. This proteostatic response explains how cells preserve the cellular proteome after treatment with a translation inhibitor such as plitidepsin. In addition, it suggests that plitidepsin could inhibit other RNA-dependent and non-integrated DNA viruses, as we confirmed in vitro using Zika virus, Hepatitis C virus replicon and Herpes simplex virus. However, the compensatory proteostasis induced by plitidespin also explains why this drug failed to inhibit the replication of integrated DNA proviruses such as HIV-1. Conclusion(s): Unraveling the mechanism of action of host-directed therapies like plitidepsin is imperative to define the indications and antiviral profile of these compounds. This knowledge will be key to develop broad-spectrum treatments and have them ready to deploy when future pandemic viruses break through.
ABSTRACT
RNA viruses are the major class of human pathogens responsible for many global health crises, including the COVID-19 pandemic. However, the current repertoire of U.S. Food and Drug Administration (FDA)-approved antivirals is limited to only nine out of the known 214 human-infecting RNAviruses, and almost all these antivirals target viral proteins. Traditional antiviral development generally proceeds in a virus-centric fashion, and successful therapies tend to be only marginally effective as monotherapies, due to dose-limiting toxicity and the rapid emergence of drug resistance. Host-based antivirals have potential to alleviate these shortcomings, but do not typically discriminate between infected and uninfected cells, thus eliciting unintended effects. In infected cells where host proteins are repurposed by a virus, normal host protein functions are compromised;a situation analogous to a loss-of-function mutation, and cells harboring the hypomorph have unique vulnerabilities. As well-established in model systems and in cancer therapeutics, these uniquely vulnerable cells can be selectively killed by a drug that inhibits a functionally redundant protein. This is the foundation of synthetic lethality (SL). To test if viral induced vulnerabilities can be exploited for viral therapeutics, we selectively targeted synthetic lethal partners of GBF1, a Golgi membrane protein and a critical host factor for many RNA viruses including poliovirus, Coxsackievirus, Dengue, Hepatitis C and E virus, and Ebola virus. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. A genome-wide chemogenomic CRISPR screen identified synthetic lethal partners of GBF1 and revealed ARF1 as the top hit. Disruption of ARF1, selectively killed cells that synthesize poliovirus 3A alone or in the context of a poliovirus replicon. Combining 3A expression with sub-lethal amounts of GCA - a specific inhibitor of GBF1 further exacerbated the GBF1-ARF1 SL effect. Together our data demonstrate proof of concept for host-based SL targeting of viral infection. We are currently testing all druggable synthetic lethal partners of GBF1 from our chemogenomic CRISPR-screen, in the context of dengue virus infection for their abilities to selectively kill infected cells and inhibit viral replication and infection. Importantly, these SL gene partners of viral-induced hypomorphs only become essential in infected cells and in principle, targeting them will have minimal effects on uninfected cells. Our strategy to target SL interactions of the viral-induced hypomorph has the potential to change the current paradigm for host-based therapeutics that can lead to broad-spectrum antivirals and can be applied to other intracellular pathogens. This work is supported by National Institutes of Health grants R01 GM112108 and P41 GM109824, R21 AI151344 and foundation grant FDN-167277 from the Canadian Institutes of Health Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Background: The CoV-2 envelope (E) protein plays an important role in virus assembly, budding, immunopathogenesis and disease severity. E protein has ion channel activity, is located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we report that BIT225, an investigational HIV clinical compound, inhibits E ion channel activity and prevents body weight loss and mortality and reduces inflammation in lethally infected K18-hACE2 transgenic mice. BIT225 efficacy was observed when dosing was initiated before or 24 h or 48 h after infection. Method(s): SARS-CoV-2 E protein ion channel activity and Xenopus TMEM16A were measured in Xenopus oocytes. K18-hACE2 transgenic mice were infected intranasally with 104 pfu SARS CoV 2 (US-WA1/2020) and dosed orally twice daily with BIT225 for up to 12 Days. Dosing was initiated 12 h pre-infection or 24 h or 48 h post-infection. Disease parameters measured were survival, body weight, viral RNA by qPCR and infectious virus titre (plaque assay) in lung tissue homogenates and serum. In addition, levels of pro-inflammatory cytokines (IL-6, IL-1alpha, IL-1beta, TNFalpha & TGFbeta, MCP-1) were measured in lung and serum samples. Result(s): BIT225 inhibited ion channel activity of E-protein, but not that of TMEM16A in Xenopus oocytes. BIT225 dosed at 300mg/kg BID for 12 days starting 12 h pre-infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). Figure 1 shows results from a time of addition study: When treatment with BIT225 started at 24 h post-infection, body weight loss and mortality was also prevented (100% survival, n=5). In the group of mice where treatment started at 48 h after infection, body weight loss and mortality were prevented in 4 of 5 mice. Treatment efficacy was associated with significant reduction in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. Conclusion(s): BIT225 is an inhibitor of SARS-CoV-2 E-protein viroporin activity. In the K18 model BIT225 protected mice from weight loss and death, inhibited virus replication and reduced inflammation. These effects were noted when treatment with BIT225 was initiated before or 24-48 hours after infection and validate viroporin E as a viable antiviral target and support the clinical study of BIT225 in treatment of SARS-CoV-2.
ABSTRACT
Background: Viral infections pose some of the most serious human health concerns worldwide. The infections caused by several viruses, including coronavirus, hepatitis virus, and human immunodeficiency virus, are difficult to treat. Method(s): This review details the findings of a literature search performed on the antiviral properties of luteolin. The keywords engaged in the search are "virus" along with "luteolin." Results: Luteolin possesses antiviral properties, which is the basis for the current review. It is an important natural flavonoid with numerous important biological properties, including anti-inflammatory, immune regulatory, and antitumor effects, and is found in vegetables, fruits, and several medicinal plants. Recent studies have revealed that many traditional Chinese medicines that contain luteolin inhibit the replication of coronaviruses. Conclusion(s): Luteolin effectively inhibits the replication of coronavirus, influenza virus, enterovirus, rotavirus, herpes virus, and respiratory syncytial virus, among others. In particular, it prevents viral infection by improving the body's nonspecific immunity and antioxidation capacity and inhibiting many pathways related to virus infection and replication, such as MAPK, PI3K-AKT, TLR4/8, NF-kappaB, Nrf-2/hemeoxygenase-1, and others. It also regulates the expression of some receptors and factors, including hepatocyte nuclear factor 4alpha, p53, NLRP3, TNF-alpha, and interleukins, thereby interfering with the replication of viruses in cells. Luteolin also promotes the repair of damaged cells induced by proinflammatory factors by regulating the expression of inflammatory molecules. The overall effect of these processes is the reduction in viral replication and, consequently, the viral load. This review summarizes the antiviral effect of luteolin and the mechanism underlying this property.Copyright © The Author(s) 2023.
ABSTRACT
Background: Infection with SARS-CoV-2 triggers reprogramming through global transcriptomic changes that drive the development of Coronavirus disease 2019 (COVID-19). Although the expression and functions of proteincoding transcripts have been widely studied in SARS-CoV-2 infection, most of the transcriptome consists of non-protein-coding RNAs (ncRNAs). Long noncoding RNAs (lncRNAs), which constitute a large proportion of the transcriptome, regulate immune responses and play prominent roles in health and disease. However, the impact of lncRNAs on SARS-CoV-2 infection is poorly understood. Our study will provide fundamental insights into the role of lncRNAs in SARS-CoV-2 infection. Method(s): We hypothesized that SARS-CoV-2-induced lncRNAs are critical regulators of viral replication and immune response. To test our hypothesis, we identified lncRNAs with significant differential expression in SARS-CoV-2 infected vs. uninfected cells across two cell types (A549-hACE2 and Calu) from published transcriptome data. We silenced the expression of the top lncRNA Bre- AS1 (BA) a human lung epithelial cell model (A549 cells stably expressing hACE2 and hTMPRSS2, A549AT) using lncRNA-specific ASO (lncsi) or negative control (NC) and compared viral replication in lncsi vs. NC cells. BA-silencing (BA-si) increased SARS-CoV-2 replication. and inhibited the expression of antiviral interferon-stimulated genes (ISG). (Tyr 705) pSTAT3 forms suppressor molecular complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) that inhibit ISG transcription. Using molecular methods such as gene-silencing, immunoprecipitation, western blot, and measuring promoter activity, we further show that Bre-AS1 inhibits the phosphorylation of STAT3 and enhances ISG transcription. Result(s): Our data show that cellular lncRNA, Bre-AS1 enhances antiviral interferon-stimulated genes (ISG) expression and inhibits replication of SARSCoV- 2. Our data show that Bre-AS1 inhibits the (Tyr705) phosphorylation of STAT3 that forms ISG repressor complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) and thus enhances ISG transcription. Conclusion(s): Cellular lncRNA Bre-AS1 enhances expression of antiviral interferon-stimulated genes and inhibits the replication of SARS-CoV-2. Our data show that cellular lncRNAs could play significant roles in immune response and viral propagation. Thus, unraveling the mechanisms of lncRNA-mediated regulation of virus replication and immune response may lead to identifying new, highly selective therapeutic targets Bre-AS1 inhibits STAT3 phosphorylation and enhances ISG transcription.