Multiple effects of silymarin on the hepatitis C virus lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell

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.Peer reviewe

Therapeutic Vaccine for Genital Herpes Simplex Virus-2 Infection: Findings from a Randomized Trial

Background. Genital herpes simplex virus type 2 (HSV-2) infection causes recurrent lesions and frequent viral shedding. GEN-003 is a candidate therapeutic vaccine containing HSV-2 gD2∆TMR and ICP4.2, and Matrix-M2 adjuvant. Methods. Persons with genital herpes were randomized into 3 dose cohorts to receive 3 intramuscular doses 21 days apart of 10 µg, 30 µg, or 100 µg of GEN-003, antigens without adjuvant, or placebo. Participants obtained genital swab specimens twice daily for HSV-2 detection and monitored genital lesions for 28-day periods at baseline and at intervals after the last dose. Results. One hundred and thirty-four persons received all 3 doses. Reactogenicity was associated with adjuvant but not with antigen dose or dose number. No serious adverse events were attributed to GEN-003. Compared with baseline, genital HSV-2 shedding rates immediately after dosing were reduced with GEN-003 (from 13.4% to 6.4% for 30 μg [P < .001] and from 15.0% to 10.3% for 100 µg [P < .001]). Lesion rates were also significantly (P < .01) reduced immediately following immunization with 30 µg or 100 µg of GEN-003. GEN-003 elicited increases in antigen binding, virus neutralizing antibody, and T-cell responses. Conclusions. GEN-003 had an acceptable safety profile and stimulated humoral and cellular immune responses. GEN-003 at doses of 30 µg and 100 µg reduced genital HSV shedding and lesion rates

Combinations of Host- and Virus-Targeting Antiviral Drugs Confer Synergistic Suppression of SARS-CoV-2

Three directly acting antivirals (DAAs) demonstrated substantial reduction in COVID-19 hospitalizations and deaths in clinical trials. However, these agents did not completely prevent severe illness and are associated with cases of rebound illness and viral shedding. Combination regimens can enhance antiviral potency, reduce the emergence of drug-resistant variants, and lower the dose of each component in the combination. Concurrently targeting virus entry and virus replication offers opportunities to discover synergistic drug combinations. While combination antiviral drug treatments are standard for chronic RNA virus infections, no antiviral combination therapy has been approved for SARS-CoV-2. Here, we demonstrate that combining host-targeting antivirals (HTAs) that target TMPRSS2 and hence SARS-CoV-2 entry, with the DAA molnupiravir, which targets SARS-CoV-2 replication, synergistically suppresses SARS-CoV-2 infection in Calu-3 lung epithelial cells. Strong synergy was observed when molnupiravir, an oral drug, was combined with three TMPRSS2 (HTA) oral or inhaled inhibitors: camostat, avoralstat, or nafamostat. The combination of camostat plus molnupiravir was also effective against the beta and delta variants of concern. The pyrimidine biosynthesis inhibitor brequinar combined with molnupiravir also conferred robust synergistic inhibition. These HTA+DAA combinations had similar potency to the synergistic all-DAA combination of molnupiravir plus nirmatrelvir, the protease inhibitor found in paxlovid. Pharmacodynamic modeling allowed estimates of antiviral potency at all possible concentrations of each agent within plausible therapeutic ranges, suggesting possible in vivo efficacy. The triple combination of camostat, brequinar, and molnupiravir further increased antiviral potency. These findings support the development of HTA+DAA combinations for pandemic response and preparedness. IMPORTANCE Imagine a future viral pandemic where if you test positive for the new virus, you can quickly take some medicines at home for a few days so that you do not get too sick. To date, only single drugs have been approved for outpatient use against SARS-CoV-2, and we are learning that these have some limitations and may succumb to drug resistance. Here, we show that combinations of two oral drugs are better than the single ones in blocking SARS-CoV-2, and we use mathematical modeling to show that these drug combinations are likely to work in people. We also show that a combination of three oral drugs works even better at eradicating the virus. Our findings therefore bode well for the development of oral drug cocktails for at home use at the first sign of an infection by a coronavirus or other emerging viral pathogens.Peer reviewe

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Studies On Trans-Acting Factors Involved In Chloroplast Rna Editing In Arabidopsis Thaliana

RNA editing is a post-transcriptional process that changes specific cytidines to uridines in the organelles of most land plants. Although members of the editosome have begun to be elucidated, the identity of the deaminase is still unknown. Members of the pentatricopeptide repeat (PPR) protein family are known to be the site-recognition factors in RNA editing. Most PPR editing factors also carry extra C-terminal motifs called the E domain and the DYW domain, where the DYW domain features amino acids conserved with the active site in other cytidine deaminases. I examined the DYW motifs of two chloroplast PPR-DYW editing factors: REQUIRED FOR ACCD RNA EDITING 1 (RARE1), which is responsible for editing a single site in the accD transcript, and QUINTUPLE EDITING FACTOR 1 (QED1), which is responsible for the editing of five plastid sites. Truncation of the DYW motifs greatly decreased the editing efficiency of RARE1 and QED1 when transfected into protoplasts. Using sitedirected mutagenesis, the conserved deaminase residues were targeted along with a highly conserved potential second zinc binding site. The mutant proteins could not efficiently complement the RNA editing phenotype when expressed in mutant protoplasts, indicating that these deaminase residues may be important for RNA editing efficiency for certain PPR editing factors. Since QED1 affects five different chloroplast sites including sites that changes the coded protein sequences of matK, ndhB, and rpoB, I further examined the functional consequences of these sites not being edited in qed1 mutants. qed1 mutants feature a stunted growth phenotype, the presence of yellow patches that increases with age, a lack of NA(P)H dehydrogenase (NDH) activity, and an abnormal chloroplast morphology as well as a smaller average cell size which can be observed in mutant protoplasts. These phenotypes were found to be at least partially complemented using qed1 mutant lines which have a stably integrated QED1 with a 3xFLAG-StrepII tag attached to its N-terminus. The line carrying one stably integrated allele, though not able to complement the mutant phenotypes to wild-type levels, was able to partially complement the mutant phenotypes, likely due to the presence of only one allele rather than two