Two-way pharmacodynamic modeling of drug combinations and its application to pairs of repurposed Ebola and SARS-CoV-2 agents.

Existing pharmacodynamic (PD) mathematical models for drug combinations discriminate antagonistic, additive, multiplicative, and synergistic effects, but fail to consider how concentration-dependent drug interaction effects may vary across an entire dose-response matrix. We developed a two-way pharmacodynamic (TWPD) model to capture the PD of two-drug combinations. TWPD captures interactions between upstream and downstream drugs that act on different stages of viral replication, by quantifying upstream drug efficacy and concentration-dependent effects on downstream drug pharmacodynamic parameters. We applied TWPD to previously published in vitro drug matrixes for repurposed potential anti-Ebola and anti-SARS-CoV-2 drug pairs. Depending on the drug pairing, the model recapitulated combined efficacies as or more accurately than existing models and can be used to infer efficacy at untested drug concentrations. TWPD fits the data slightly better in one direction for all drug pairs, meaning that we can tentatively infer the upstream drug. Based on its high accuracy, TWPD could be used in concert with PK models to estimate the therapeutic effects of drug pairs in vivo.

Discovery of host-directed modulators of virus infection by probing the SARS-CoV-2-host protein-protein interaction network.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based method that expands the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19. We experimentally tested seven chemical inhibitors of the identified host proteins for modulation of SARS-CoV-2 infection in human cells that express ACE2 and TMPRSS2. Inhibition of the epigenetic regulators bromodomain-containing protein 4 (BRD4) and histone deacetylase 2 (HDAC2), along with ubiquitin-specific peptidase (USP10), enhanced SARS-CoV-2 infection. Such proviral effect was observed upon treatment with compounds JQ1, vorinostat, romidepsin and spautin-1, when measured by cytopathic effect and validated by viral RNA assays, suggesting that the host proteins HDAC2, BRD4 and USP10 have antiviral functions. We observed marked differences in antiviral effects across cell lines, which may have consequences for identification of selective modulators of viral infection or potential antiviral therapeutics. While network-based approaches enable systematic identification of host targets and selective compounds that may modulate the SARS-CoV-2 interactome, further developments are warranted to increase their accuracy and cell-context specificity.

Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Human Immunodeficiency Virus Is Associated With Higher Levels of Systemic Inflammation Among Kenyan Adults Despite Viral Suppression.

BACKGROUND: Systemic inflammation independently predicts future cardiovascular events and is associated with a 2-fold increase in cardiovascular disease (CVD) risk among persons living with human immunodeficiency virus (PLHIV). We examined the association between inflammatory markers, HIV status, and traditional CVD risk factors. METHODS: We conducted a cross-sectional study of Kenyan adults with and without HIV seeking care at Kisumu County Hospital. Using a multiplex immunoassay, we measured interleukin (IL) 1ß, IL-6, tumor necrosis factor α (TNF-α), and high-sensitivity C-reactive protein (hsCRP) concentrations. We compared inflammatory marker concentrations by HIV status using the Wilcoxon rank-sum test. Multivariable linear regression was used to evaluate associations between inflammatory biomarkers and HIV status, adjusting for CVD risk factors. RESULTS: We enrolled 286 PLHIV and 277 HIV-negative participants. Median duration of antiretroviral therapy for PLHIV was 8 years (interquartile range, 4-10) and 96% were virally suppressed. PLHIV had a 51% higher mean IL-6 concentration (P < .001), 39% higher mean IL-1ß (P = .005), 40% higher mean TNF-α (P < .001), and 27% higher mean hsCRP (P = .008) compared with HIV-negative participants, independent of CVD risk factors. Male sex, older age, and obesity were associated with higher concentrations of inflammatory markers. Restricting to PLHIV, viral load of ≥1000 copies/mL was associated with higher TNF-α levels (P = .013). CONCLUSIONS: We found higher levels of systemic inflammatory biomarkers among PLHIV who were virally suppressed, and this was independent of traditional CVD risk factors. Further longitudinal analyses to determine whether these inflammatory markers predict future CVD events, and are possible therapeutic targets among PLHIV, are warranted.

Inhibition of Arenaviruses by Combinations of Orally Available Approved Drugs.

Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.

Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus genome replication.

Arbidol (ARB, also known as umifenovir) is used clinically in several countries as an anti-influenza virus drug. ARB inhibits multiple enveloped viruses in vitro and the primary mode of action is inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. ARB is also an effective inhibitor of non-enveloped poliovirus types 1 and 3. In the current report, we evaluate the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen. ARB inhibits the replication of FMDV RNA sub-genomic replicons. ARB inhibition of FMDV RNA replication is not a result of generalized inhibition of cellular uptake of cargo, such as transfected DNA, and ARB can be added to cells up to 3 h post-transfection of FMDV RNA replicons and still inhibit FMDV replication. ARB prevents the recovery of FMDV replication upon withdrawal of the replication inhibitor guanidine hydrochloride (GuHCl). Although restoration of FMDV replication is known to require de novo protein synthesis upon GuHCl removal, ARB does not suppress cellular translation or FMDV internal ribosome entry site (IRES)-driven translation. ARB also inhibits infection with the related Aphthovirus, equine rhinitis A virus (ERAV). Collectively, the data demonstrate that ARB can inhibit some non-enveloped picornaviruses. The data are consistent with inhibition of picornavirus genome replication, possibly via the disruption of intracellular membranes on which replication complexes are located.

1,4-Benzodioxane Lignans: An Efficient, Asymmetric Synthesis of Flavonolignans and Study of Neolignan Cytotoxicity and Antiviral Profiles.

1,4-Benzodioxane lignans are a class of bioactive compounds that have received much attention through the years. Herein research pertaining to both 1,4-benzodioxane flavonolignans and 1,4-benzodioxane neolignans is presented. A novel synthesis of both traditional 1,4-benzodioxane flavonolignans and 3-deoxyflavonolignans is described. The antiviral and cytotoxic activities of 1,4-benzodioxane neolignans were then investigated; eusiderins A, B, G, and M, deallyl eusiderin A, and nitidanin, which contain the 1,4-benzodioxane motif but lack the chromanone motif found in the known antiviral flavonolignans, were tested. Notably, it was found that all eusiderin 1,4-benzodioxane neolignans exhibited greater antiviral activity than the potent and well-known silybin flavonolignans. While most modifications of the C-1' side chain did not significantly alter the cytotoxicity or antiviral activity, eusiderin M and nitidanin, which contain an allylic alcohol side chain, had lower cytotoxicity. All the eusiderins had similar antiviral activities, with eusiderin B having the best selectivity index. These results show that the chromanone moiety of the flavonolignans is not essential for bioactivity.

The Synthetic Antiviral Drug Arbidol Inhibits Globally Prevalent Pathogenic Viruses.

UNLABELLED: Arbidol (ARB) is a synthetic antiviral originally developed to combat influenza viruses. ARB is currently used clinically in several countries but not in North America. We have previously shown that ARB inhibits in vitro hepatitis C virus (HCV) by blocking HCV entry and replication. In this report, we expand the list of viruses that are inhibited by ARB and demonstrate that ARB suppresses in vitro infection of mammalian cells with Ebola virus (EBOV), Tacaribe arenavirus, and human herpesvirus 8 (HHV-8). We also confirm suppression of hepatitis B virus and poliovirus by ARB. ARB inhibited EBOV Zaire Kikwit infection when added before or at the same time as virus infection and was less effective when added 24 h after EBOV infection. Experiments with recombinant vesicular stomatitis virus (VSV) expressing the EBOV Zaire glycoprotein showed that infection was inhibited by ARB at early stages, most likely at the level of viral entry into host cells. ARB inhibited HHV-8 replication to a similar degree as cidofovir. Our data broaden the spectrum of antiviral efficacy of ARB to include globally prevalent viruses that cause significant morbidity and mortality. IMPORTANCE: There are many globally prevalent viruses for which there are no licensed vaccines or antiviral medicines. Some of these viruses, such as Ebola virus or members of the arenavirus family, rapidly cause severe hemorrhagic diseases that can be fatal. Other viruses, such as hepatitis B virus or human herpesvirus 8 (HHV-8), establish persistent infections that cause chronic illnesses, including cancer. Thus, finding an affordable, effective, and safe drug that blocks many viruses remains an unmet medical need. The antiviral drug arbidol (ARB), already in clinical use in several countries as an anti-influenza treatment, has been previously shown to suppress the growth of many viruses. In this report, we expand the list of viruses that are blocked by ARB in a laboratory setting to include Ebola virus, Tacaribe arenavirus, and HHV-8, and we propose ARB as a broad-spectrum antiviral drug that may be useful against hemorrhagic viruses.

Chemoselective fluorination and chemoinformatic analysis of griseofulvin: Natural vs fluorinated fungal metabolites.

Griseofulvin is a fungal metabolite and antifungal drug used for the treatment of dermatophytosis in both humans and animals. Recently, griseofulvin and its analogues have attracted renewed attention due to reports of their potential anticancer effects. In this study griseofulvin (1) and related analogues (2-6, with 4 being new to literature) were isolated from Xylaria cubensis. Six fluorinated analogues (7-12) were synthesized, each in a single step using the isolated natural products and Selectflour, so as to examine the effects of fluorine incorporation on the bioactivities of this structural class. The isolated and synthesized compounds were screened for activity against a panel of cancer cell lines (MDA-MB-435, MDA-MB-231, OVCAR3, and Huh7.5.1) and for antifungal activity against Microsporum gypseum. A comparison of the chemical space occupied by the natural and fluorinated analogues was carried out by using principal component analysis, documenting that the isolated and fluorinated analogues occupy complementary regions of chemical space. However, the most active compounds, including two fluorinated derivatives, were centered around the chemical space that was occupied by the parent compound, griseofulvin, suggesting that modifications must preserve certain attributes of griseofulvin to conserve its activity.

Human Cytokinome Analysis for Interferon Response.

UNLABELLED: Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-γ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN response-betacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-ß and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-ß response through the NF-κB pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-α against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities. IMPORTANCE: Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry.

Understanding how biological molecules are generated, metabolized and eliminated in living systems is important for interpreting processes such as immune response and disease pathology. While genomic and proteomic studies have provided vast amounts of information over the last several decades, interest in lipidomics has also grown due to improved analytical technologies revealing altered lipid metabolism in type 2 diabetes, cancer, and lipid storage disease. Mass spectrometry (MS) measurements are currently the dominant approach for characterizing the lipidome by providing detailed information on the spatial and temporal composition of lipids. However, interpreting lipids' biological roles is challenging due to the existence of numerous structural and stereoisomers (i.e. distinct acyl chain and double-bond positions), which are often unresolvable using present approaches. Here we show that combining liquid chromatography (LC) and structurally-based ion mobility spectrometry (IMS) measurement with MS analyses distinguishes lipid isomers and allows insight into biological and disease processes.

Direct, interferon-independent activation of the CXCL10 promoter by NF-κB and interferon regulatory factor 3 during hepatitis C virus infection.

Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein ß (C/EBP-ß) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-ß negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.

Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling.

Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed silymarin suppression of glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism. Anti-inflammatory effects arose with prolonged (i.e., 24 h) silymarin exposure, with suppression of multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB) and forkhead box O (FOXO). Studies with murine knock out cells revealed that silymarin inhibition of both mTOR and NF-κB was partially AMPK dependent, whereas silymarin inhibition of mTOR required DDIT4. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Thus, natural products activate stress and repair responses that culminate in an anti-inflammatory cellular phenotype. Natural products like silymarin may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B.

UNLABELLED: Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. CONCLUSION: Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.

Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin-dependent trafficking.

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti-HCV action of silymarin-derived compounds at the molecular level. By using live-cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV-infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin-coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin-coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well-known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin-mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad-spectrum antiviral therapy.

A unifying model to explain high nirmatrelvir therapeutic efficacy against SARS-CoV-2, despite low post-exposure prophylaxis efficacy and frequent viral rebound.

In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We developed a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulated viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly.

A unifying model to explain frequent SARS-CoV-2 rebound after nirmatrelvir treatment and limited prophylactic efficacy.

In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We develop a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulates viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly.