Inhibition Of Replication Research Articles

The ortholog of human DNAJC9 promotes histone H3-H4 degradation and is counteracted by Asf1 in fission yeast.

Mammalian J-domain protein DNAJC9 interacts with histones H3-H4 and is important for cell proliferation. However, its exact function remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, loss of Djc9, the ortholog of DNAJC9, renders the histone chaperone Asf1 no longer essential for growth. Utilizing AlphaFold-based structural prediction, we identified a histone-binding surface on Djc9 that binds to helix α3 of H3 in a manner that precludes simultaneous helix α3-binding by Asf1. Djc9 and Asf1 indeed compete for binding to the H3-H4 dimer in vitro, and an H3-α3 mutation impeding Djc9 binding also renders Asf1 non-essential, indicating that the role of Asf1 needed for growth in fission yeast is to prevent histone binding by Djc9. In the absence of Asf1, cell growth is hindered due to unrestrained Djc9-mediated downregulation of H3 and H4. In the presence of Asf1, Djc9 confers resistance to the DNA replication inhibitor hydroxyurea and dominant negative disease-related histone mutants by promoting the degradation of superfluous or dysfunctional histones. Our findings provide new insights into the function and mechanism of this conserved histone-binding protein.

A genetically based computational drug repurposing framework for rapid identification of candidate compounds: application to COVID-19.

The development and approval of novel drugs are typically time-intensive and expensive. Leveraging a computational drug repurposing framework that integrates disease-relevant genetically regulated gene expression (GReX) and large longitudinal electronic medical record (EMR) databases can expedite the repositioning of existing medications. However, validating computational predictions of the drug repurposing framework remains a challenge. To benchmark the drug repurposing framework, we first performed a 5-method-rank-based computational drug prioritization pipeline by integrating multi-tissue GReX associated with COVID-19-related hospitalization, with drug transcriptional signature libraries from the Library of Integrated Network-Based Cellular Signatures. We prioritized FDA-approved medications from the 10 top-ranked compounds, and assessed their association with COVID-19 incidence within the Veterans Health Administration (VHA) cohort (~9 million individuals). In parallel, we evaluated in vitro SARS-CoV-2 replication inhibition in human lung epithelial cells for the selected candidates. Our in silico pipeline identified seven FDA-approved drugs among the top ten candidates. Six (imiquimod, nelfinavir and saquinavir, everolimus, azathioprine, and retinol) had sufficient prescribing rates or feasibility for further testing. In the VHA cohort, azathioprine (odds ratio [OR]=0.69, 95% CI 0.62-0.77) and retinol (OR=0.81, 95% CI 0.72-0.92) were significantly associated with reduced COVID-19 incidence. Conversely, nelfinavir and saquinavir demonstrated potent SARS-CoV-2 inhibition in vitro (~95% and ~65% viral load reduction, respectively). No single compound showed robust protection in both in vivo and in vitro settings. These findings underscore the power of GReX-based drug repurposing in rapidly identifying existing therapies with potential clinical relevance; four out of six compounds showed a protective effect in one of the two validation approaches. Crucially, our results highlight how a complementary evaluation-combining epidemiological data and in vitro assays-helps refine the most promising candidates for subsequent mechanistic studies and clinical trials. This integrated validation approach may prove vital for accelerating therapeutic development against current and future health challenges.

Mutations in chikungunya virus nsP4 decrease viral fitness and sensitivity to the broad-spectrum antiviral 4'-Fluorouridine.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that has re-emerged to cause large outbreaks of human infections worldwide. There are currently no approved antivirals for treatment of CHIKV infection. Recently, we reported that the ribonucleoside analog 4'-fluorouridine (4'-FlU) is a highly potent inhibitor of CHIKV replication, and targets the viral nsP4 RNA dependent RNA polymerase. In mouse models, oral therapy with 4'-FlU diminished viral tissue burdens and virus-induced disease signs. To provide critical evidence for the potential of 4'-FlU as a CHIKV antiviral, here we selected for CHIKV variants with decreased 4'-FlU sensitivity, identifying two pairs of mutations in nsP2 and nsP4. The nsP4 mutations Q192L and C483Y were predominantly responsible for reduced sensitivity. These variants were still inhibited by higher concentrations of 4'-FlU, and the mutations did not change nsP4 fidelity or provide a virus fitness advantage in vitro or in vivo. Pathogenesis studies in mice showed that the nsP4-C483Y variant caused similar disease and viral tissue burden as WT CHIKV, while the nsP4-Q192L variant was strongly attenuated. Together these results support the potential of 4'-FlU to be an important antiviral against CHIKV.

SARS-CoV-2 Treatment: Current Perceptions and Perspectives

Context: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China. From the outset, this highly contagious and dangerous illness posed significant risks and challenges for global healthcare systems and populations. Scientists and healthcare professionals worldwide have been striving to develop effective treatments for severe acute respiratory SARS-CoV-2 infection, employing both pre-existing and innovative approaches. However, a specific drug targeted explicitly against COVID-19 remains elusive. Despite the reduced prevalence of severe cases and the predominance of the SARS-CoV-2 Omicron variant, COVID-19 continues to pose significant risks, particularly for polymorbid patient groups. This review focuses on the potential antiviral effects of promising new drugs, including ensitrelvir, clazakizumab, upamostat, and others. Evidence Acquisition: An analytical review was conducted using the Scopus, PubMed, EMBASE, ScienceDirect, and Google Scholar databases for publications related to promising drugs against SARS-CoV-2, their mechanisms of action, and potential therapeutic effects up to November 2024. Selection criteria included: (1) Free-text availability; (2) English language; (3) relevance to the publication's theme a total of 104 articles were initially selected. Of these, 27 articles were excluded for not meeting the free-text criterion, 14 were excluded due to language incompatibility, and 28 were excluded for thematic irrelevance. Ultimately, data from 35 articles were analyzed and summarized for this review. Results: Thirty-five scientific studies were reviewed to describe current paradigms and emerging concepts in SARS-CoV-2 treatment. Current drugs with proven efficacy include: (1) Combined monoclonal antibodies targeting the S-protein (casirivimab/imdevimab); (2) monoclonal antibody preparations against the S-protein (pemivibart; vilobelimab); (3) viral replication inhibitors (molnupiravir); (4) protease inhibitors (nirmatrelvir/ritonavir); (5) immunosuppressants (dexamethasone; tocilizumab). The review also highlights drugs under development targeting traditional pathways [e.g., viral protease, interleukin-6 (IL-6)] and those aimed at novel mechanisms (e.g., antigalectin-3; adhesion blockers). Conclusions: Although numerous drug effects and potential drug combinations for COVID-19 have been described, limited clinical trials and research focus on identifying novel treatment approaches. Comprehensive investigations are needed to assess and evaluate the risks of different treatment strategies, minimizing potential short- and long-term complications for patients. Such advancements will pave the way for more optimal and effective medical treatment of COVID-19.

Predicting the Anti-SARS-CoV-2 Potential of Isoquinoline Alkaloids from Brazilian Siparunaceae Species Using Chemometric Tools.

The COVID-19 pandemic has caused over 7 million deaths globally in the past four years. Siparuna spp. (Siparunaceae), which is used in Brazilian folk medicine, is considered a genus with potential antiviral alternatives. This study explored the correlation between phytochemicals in Siparuna leaf extracts (S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa) and their potential against various SARS-CoV-2 targets. In vitro assays examined interactions between the spike protein and the ACE2 receptor, protease activity, and viral replication inhibition in Calu-3 cell models. UHPLC-MS/MS analysis, processed with MZmine and evaluated chemometrically, revealed isoquinoline alkaloids with bulbocapnine, showing promising therapeutic potential. Predictions regarding absorption, distribution, metabolism, excretion, and toxicity were conducted, along with molecular docking and dynamics simulations, to evaluate protein-ligand interaction stability. The results confirmed the antiviral activity of the Siparuna genus against SARS-CoV-2 targets, with 92% of the extracts maintaining over 70% cellular viability at 200 μg·mL-1 and 80% achieving more than 50% viral activity suppression at 50 μg·mL-1. These findings highlight the potential of isoquinoline alkaloids as novel anti-coronavirus agents and support the need for further exploration, isolation, and testing of Siparuna compounds in the fight against COVID-19.

MicroRNA-3145 as a potential therapeutic target for hepatitis B virus: inhibition of viral replication via downregulation of HBS and HBX.

Current treatments for hepatitis B virus (HBV), such as interferons and nucleic acid analogs, have limitations due to side effects like depression and the development of drug-resistant mutants, highlighting the need for new therapeutic approaches. In this study, we identified microRNA-3145 (miR-3145) as a host-derived miRNA with antiviral activity that is upregulated in primary hepatocytes during HBV infection. The expression of its precursor, pri-miR-3145, increased in response to the the virus infection, and miR-3145 downregulated the hepatitis B virus S (HBS) antigen and hepatitis B virus X (HBX), thereby inhibiting viral replication. The binding site for miR-3145 was located in the HBV polymerase (pol) region, as experimentally confirmed. Moreover, overexpression of HBS and HBX induced pri-miR-3145 expression through endoplasmic reticulum stress. The expression of pri-miR-3145 showed a strong correlation with the Nance-Horan syndrome-like 1 (NHSL1) gene, as it is encoded within an intron of NHSL1, and higher NHSL1 expression in hepatocellular carcinoma patients with HBV infection was associated with better prognosis. These findings suggest that miR-3145-3p, along with small molecules targeting its binding sites, holds promise as a potential therapeutic candidate for HBV treatment.

Development of molecular Trojan horses targeting New Delhi metallo-β-lactamase-1 for the restoration of meropenem susceptibility in drug-resistant bacteria.

The emergence of New Delhi metallo-β-lactamase-1 (NDM-1) poses a significant threat to the clinical application of antibiotics, as it possesses the ability to hydrolyze nearly all β-lactam antibiotics. Regrettably, there are currently no clinical drugs targeting NDM-1, making it imperative to develop highly potent and minimally toxic NDM-1 inhibitors. Herein, a series of molecular Trojan horses targeting NDM-1 were synthesized by introducing ebselen into 7-aminocephalosporanic acid derivatives via a C-Se bond. Representative compound 18b exhibited potent inhibitory activity against NDM-1, with an IC50 value of 7.03μM, and combining with meropenem (Mem) decreased the minimum inhibitory concentration (MIC) of Mem by 4-32-fold in NDM-1 expressing bacteria. Mechanistically, 18b released the ebselen moiety at the active site of NDM-1, forming a Se-S bond with Cys208 to achieve targeted drug delivery of ebselen. Importantly, 18b demonstrated potent inhibition of resistant bacterial growth and replication in mice when administered in combination with Mem. These results suggest that 18b is a promising candidate for treating infections caused by resistant bacteria expressing NDM-1.

Discovery of pentacyclic triterpene conjugates as HBV polymerase/NTCP dual-targeting inhibitors with potent anti-HBV activities.

The inhibition of HBV DNA and elimination of HBsAg has already been established as an indicator for HBV clinic cure, and a novel dual-targeting inhibitors of HBV polymerase/entry are designed and synthesized in this study. Pentacyclic triterpenes (PTs) scaffold of exhibiting a high affinity to NTCP, including glycyrrhitinic acid (GA), oleanolic acid (OA), ursolic acid (UA), and betulinic acid (BA) were linked neatly with the nucleoside drug zidovudine (AZT) through a molecular hybrid strategy to synthesize twenty of PTs-AZT conjugates for targeting HBV polymerase as well as sodium taurocholate cotransporting polypeptide (NTCP). The conjugates showed significant inhibitory effects on the secretion of HBsAg and HBeAg in HepG2.2.15 cells, and the activity on HBsAg were better. Moreover, HBV DNA replication was also notably suppressed after incubated with the conjugates. The IC50 value of BA-AZT1 on HBsAg inhibition was 0.65±0.07μM, and it was 284.2 times and 442.2 times higher comparing to corresponding parent compound BA and AZT. Additionally, the therapeutic index (TI) was also improved by 87.8 times than AZT. And the IC50 value of BA-AZT1 on inhibition of HBV DNA replication was 0.70±0.02μM, 10.4 times higher than that of AZT besides conspicuous TI. Molecular docking suggested that AZT skeleton of conjugate BA-AZT1 interacted with B region of HBV Polymerase reverse transcription region, and BA structure simultaneously targeted to C region of polymerase via hydrophobic chain, establishing strong binding interactions with the HBV Pol protein. In addition, docked with NTCP, BA-AZT1 with flat pentacyclic structure inserted into the interface and also formed hydrogen bonds, hydrophobic and van der Waals forces with the amino residue 157-165 of NTCP. Further SPR analysis demonstrated the binding affinity of BA-AZT1 to C region of polymerase was 19.55μM, stronger than 53.21μM of BA and 31.82μM of AZT. BA-AZT1 selectively bound to the 157-165 epitopes of NTCP receptors in host cell but not PreS1 of virus. As a result, we deduced that the designed conjugates targeted NTCP and HBV polymerase, not only prevented HBV from entering host cells via selective binding NTCP, but also inhibited HBV DNA replication through obstructing the function of HBV polymerase, and it could potentially serve as a promising dual-functional and dual-target inhibitor with both replication and entry inhibition to exert anti-HBV activity.

Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

Treatment options for viral infections are limited and viruses have proven adept at evolving resistance to many existing therapies, highlighting a significant vulnerability in our defenses. In response to this challenge, we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191-mediated reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 5342191-resistant variants of coronavirus or influenza. As with HIV-1, 5342191's inhibition of coronaviruses and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for the replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential of greater resilience in combating viral infections.

Replication stress increases de novo CNVs across the malaria parasite genome.

Changes in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes in many organisms. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs that are present in only a few genomes across a population are often overlooked but important; if beneficial under specific conditions, a de novo CNV that arises in a single genome can expand during selection to create a larger population of cells with novel characteristics. While the reach of single cell methods to study de novo CNVs is increasing, we continue to lack information about CNV dynamics in rapidly evolving microbial populations. Here, we investigated de novo CNVs in the genome of the Plasmodium parasite that causes human malaria. The highly AT-rich P. falciparum genome readily accumulates CNVs that facilitate rapid adaptation to new drugs and host environments. We employed a low-input genomics approach optimized for this unique genome as well as specialized computational tools to evaluate the de novo CNV rate both before and after the application of stress. We observed a significant increase in genome-wide de novo CNVs following treatment with a replication inhibitor. These stress-induced de novo CNVs encompassed genes that contribute to various cellular pathways and tended to be altered in clinical parasite genomes. This snapshot of CNV dynamics emphasizes the connection between replication stress, DNA repair, and CNV generation in this important microbial pathogen.

Advances and Challenges in Antiviral Development for Respiratory Viruses.

The development of antivirals for respiratory viruses has advanced markedly in response to the growing threat of pathogens such as Influenzavirus (IAV), respiratory syncytial virus (RSV), and SARS-CoV-2. This article reviews the advances and challenges in this field, highlighting therapeutic strategies that target critical stages of the viral replication cycle, including inhibitors of viral entry, replication, and assembly. In addition, innovative approaches such as inhibiting host cellular proteins to reduce viral resistance and repurposing existing drugs are explored, using advanced bioinformatics tools that optimize the identification of antiviral candidates. The analysis also covers emerging technologies such as nanomedicine and CRISPR gene editing, which promise to improve the stability and efficacy of treatments. While current antivirals offer valuable options, they face challenges such as viral evolution and the need for accessible treatments for vulnerable populations. This article underscores the importance of continued innovation in biotechnology to overcome these limitations and provide safe and effective treatments. Combining traditional and advanced approaches in developing antivirals is essential in order to address respiratory viral diseases that affect global health.

Design, synthesis, and biological activity evaluation of dihydromyricetin derivatives against SARS-CoV-2-Omicron virus

An oxidising and substituting one-pot reaction strategy has been developed to synthesise dihydromyricetin derivatives with the aim of enhancing the inhibitory activity of dihydromyricetin against SARS-CoV-2. Different ω-methoxy-ω-oxeylkyl was introduced in C7-OH site and yielded eight analogs, all of them showed good inhibitory activity against SARS-CoV-2 3CLpro with IC50 values ranging from 0.72 to 2.36 μM. In the Vero E6-cell, compound 3 has a good activity of anti-SARS-CoV-2 virus (Omicron virus BA.5) in the prevention model, with an EC50 of 15.84 μM, and so do compound 10 in the therapeutic model, with an EC50 of 11.52 μM. The results suggest that the introduction of long chain ω-oxeylkyl at C7-OH facilitate the inhibition of viral replication in the therapeutic model, which is consistent with the binding energies predicted from molecular docking conclusions. It implies that dihydromyricetin derivatives have the potential to become effective inhibitors of SARS-CoV-2 Omicron and other viruses.

A Model H5N2 Vaccine Strain for Dual Protection Against H5N1 and H9N2 Avian Influenza Viruses.

Background/Objective: Highly pathogenic (HP) H5Nx and low-pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose global threats to the poultry industry and public health, highlighting the critical need for a dual-protective vaccine. Methods: In this study, we generated a model PR8-derived recombinant H5N2 vaccine strain with hemagglutinin (HA) and neuraminidase (NA) genes from clade 2.3.2.1c H5N1 and Y439-like H9N2 viruses, respectively. To enhance the immunogenicity of the recombinant H5N2 vaccine strain, N-glycans of the HA2 subunit, NA, and M2e were modified. Additionally, we replaced M2e with avian M2e to enhance the antigenic homogeneity of AIVs for better protection. We also replaced PR8 PB2 with 01310 PB2, which is the PB2 gene derived from an LP H9N2 avian influenza virus, to eliminate pathogenicity in mammals. The productivity of the model vaccine strain (rvH5N2-aM2e-vPB2) in embryonated chicken eggs (ECEs), its potential risk of mammalian infection, and the immunogenicity associated with different inactivation methods (formaldehyde (F/A) vs. binary ethyleneimine (BEI)) were evaluated. Results: The rvH5N2-aM2e-vPB2 strain demonstrated high productivity in ECEs and exhibited complete inhibition of replication in mammalian cells. Furthermore, compared with using F/A inactivation, inactivation using BEI significantly enhanced the immune response, particularly against NA. This enhancement resulted in increased virus neutralization titers, supporting its efficacy for dual protection against H5Nx and H9N2 avian influenza viruses. Furthermore, we demonstrated that M2e-specific immune responses, difficult to induce with inactivated vaccines, can be effectively elicited with live vaccines, suggesting a strategy to enhance M2e immunogenicity in whole influenza virus vaccines. Conclusions: Finally, the successful development of the model rH5N2 vaccine strain is described; this strain provides dual protection, has potential applicability in regions where avian influenza is endemic, and can be used to promote the development of versatile H5N2 recombinant vaccines for effective avian influenza control.

CRISPR/Cas9 Eye Drop HSV-1 Treatment Reduces Brain Viral Load: A Novel Application to Prevent Neuronal Damage.

Herpes simplex virus-1 (HSV-1) can invade the central nervous system (CNS). However, antiviral drugs used to treat HSV-1 have significant toxicity and resistance. An alternative approach involves the use of the CRISPR/Cas9 complex as a viral replication inhibitor. Editing the UL39 gene with CRISPR/Cas9 results in >95% inhibition of HSV-1 replication in vitro; however, few studies have investigated alternative therapies in in vivo models. This study aimed to investigate the efficacy of CRISPR/Cas9 targeting the UL39 region, which was administered via the ocular route, to reduce the HSV-1 viral count in the CNS of BALB/c mice. Mice were inoculated with HSV-1 and treated using CRISPR/Cas9. The kinetics of CNS infection were assessed, and the effects of CRISPR/Cas9 were compared with those of topical acyclovir treatments. The brain viral load was analyzed, and histopathology and immunofluorescence of the nervous tissue were performed. The group treated with CRISPR/Cas9 showed a reduced viral load on the seventh day post-infection, and no brain inflammation or chromatin compaction was observed in animals that received CRISPR/Cas9 therapy. These findings suggest that CRISPR/Cas9 anti-UL39 therapy can reduce the HSV-1 viral load in brain tissue. Therefore, investigating viral detection and evaluating antiviral treatments in the brain is essential.

Identification of cepharanthine as an effective inhibitor of African swine fever virus replication

ABSTRACT African swine fever virus (ASFV) causes highly contagious swine disease, African swine fever (ASF), thereby posing a severe socioeconomic threat to the global pig industry and underscoring that effective antiviral therapies are urgently required. To identify safe and efficient anti-ASFV compounds, a natural compound library was screened by performing an established cell-based ELISA in an ASFV-infected porcine alveolar macrophage (PAM) model. In total, 6 effective anti-ASFV compounds with low cytotoxicity were identified. Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid, was the most potent inhibitor effect with an IC50 of 0.3223 μM. To further investigate the mechanism through which CEP inhibits ASFV replication, transcriptome profiles were generated in PAMs treated with CEP and/or infected with ASFV. ASFV infection dramatically altered immune response-associated gene expression. CEP treatment upregulated the expression of cholesterol biosynthesis-related genes, regardless of infection status. According to time-of-addition experiments, CEP primarily exerts its antiviral effect during the early stages of ASFV infection, specifically by inhibiting viral entry. Transcriptomic analysis suggested that CEP blocks ASFV entry through the clathrin-mediated endocytosis pathway by increasing EHD2 gene expression in macrophages. Disrupting EHD2 with small interfering RNA promoted ASFV entry into clathrin-positive vesicles. Finally, the protective effect of CEP in vivo was evaluated using ASFV-infected pigs. CEP could provide partial protection against ASFV infection, as indicated by an increase in survival time from 9.67 days to 16.67 days. Our findings imply that CEP exhibits potential antiviral activity against ASFV infection in PAMs, positioning it as a promising therapeutic strategy for ASF.

Identification of RdRp-NiRAN/JAK1 Dual-Target Drugs for COVID-19 Treatment.

Inhibition of virus replication and inflammatory response is important for the treatment of severe COVID-19 patients. RNA-dependent RNA polymerase (RdRp) is indispensable for SARS-CoV-2 replication, and Janus kinase (JAK) 1 inhibitors exert immunosuppressive effects. RdRp/JAK1 dual-target drugs are expected to ameliorate the severity of the COVID-19 disease. The N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain of RdRp is a pseudokinase, and it has structural similarities with JAK1. Herein, we evaluated the inhibitory effects of triphosphate forms of 31 nucleoside drugs in the DrugBank database on the NiRAN domain and JAK1 through a combination of theoretical and experimental methods. By analyzing the three properties of 31 nucleoside drugs (total hydrophobic surface area, number of hydrophobic atoms, and molecular weight), these drugs met the application rule of our developed molecular docking with conformer-dependent charges (MDCC). Based on the MDCC method combined with molecular dynamics simulations, Azvudine and Citicoline among these 31 drugs showed stronger predicted binding affinities with the NiRAN domain as well as JAK1 compared to the reference drug Remdesivir. Further experimental verification, including a thermal shift assay and homogeneous time-resolved fluorescence assay, demonstrated that Azvudine was an RdRp-NiRAN/JAK1 dual-target drug. This work provided a previously unexplored mechanism of Azvudine for COVID-19 treatment and proposed a design concept for RdRp-NiRAN/JAK1 dual-target nucleoside drugs.

4-Substituted-2-Thiazole Amides as Viral Replication Inhibitors of Alphaviruses.

2-(Methylthio)-N-(4-(naphthalen-2-yl)thiazol-2-yl)nicotinamide 1 was identified as an inhibitor against Chikungunya virus (CHIKV) with good antiviral activity [EC50 = 0.6 μM; EC90 = 0.93 μM and viral titer reduction (VTR) of 6.9 logs at 10 μM concentration] with no observed cytotoxicity (CC50 = 132 μM) in normal human dermal fibroblast (NHDF) cells. Structure-activity relationship (SAR) studies to further improve the potency, efficacy, and drug-like properties of 1 led to the discovery of a new potent inhibitor N-(4-(3-((4-cyanophenyl)amino)phenyl)thiazol-2-yl)-2-(methylthio)nicotinamide 26, which showed a VTR of 8.7 logs at 10 μM against CHIKV and an EC90 of 0.45 μM with considerably improved MLM stability (t1/2 = 74 min) as compared to 1. Mechanism of action studies show that 26 inhibits alphavirus replication by blocking subgenomic viral RNA translation and structural protein synthesis. The in vivo efficacy studies of compound 26 on CHIKV infection in mice are reported.

Long Noncoding RNA LINC02453 Inhibits HIV-1 Replication by Binding With SEC13 to Regulate the Viral Productive Cycle.

Emerging evidence underscores the pivotal role of long noncoding RNAs (lncRNAs) as crucial regulators within the HIV life cycle. However, the precise functions and detailed mechanisms by which lncRNAs operate in HIV-1 highly exposed but persistently seronegative (HESN) individuals remain currently unknown. Through RNA sequencing analysis of the HESN individual and the matched control, we identified potential lncRNAs. Then, we conducted validation experiments at the population level, while cellular models of HIV-1 infection were constructed for functional experimental investigations in vitro. Subcellular localization of the identified lncRNA was determined, followed by an exploration of the specific regulatory mechanism underlying HIV resistance through some experiments, such as RNA pull-down, western blot and Hirt assays. LncRNA LINC02453 is highly expressed in HESN. Moreover, LINC02453 is identified as a novel lncRNA associated with heightened resistance to HIV-1. LINC02453 is predominantly localized in the nucleus and binds to SEC13, a component of the nuclear pore complex, leading to the inhibition of HIV-1 replication by regulating key processes such as late reverse transcription, nuclear import, and DNA integration. Our findings suggest that LINC02453 may serve as a prospective target for the development of innovative anti-HIV therapeutics.

Berberine promotes K48-linked polyubiquitination of HNF4α, leading to the inhibition of HBV replication.

The current antiviral agents for the treatment of chronic infection with hepatitis B virus (HBV) do not completely remove covalently closed circular DNA (cccDNA) and integrated viral DNA fragments from patients. Berberine is an isoquinoline alkaloid extracted from various plants and has been reported to inhibit the replication of various types of DNA. In this study, we tested the effects of berberine and its derivatives on HBV infection. Berberine inhibited viral core promoter activity at the highest level among the compounds tested and suppressed HBV production and cccDNA synthesis in primary human hepatocytes and HBV-infected HepG2-NTCP cells at an EC50 value of 3.6 μM and a CC50 value of over 240.0 μM. Compared with other viral promoter activities, berberine treatment potently downregulated core promoter activity and reduced protein levels, but not RNA levels, of hepatic nuclear factor 4α (HNF4α), which primarily enhances enhancer II/core promoter activity. Furthermore, berberine treatment enhanced K48-linked, but not K63-linked, polyubiquitination and subsequent proteasome-dependent degradation of HNF4α. These results suggest that berberine enhances the polyubiquitination- and proteasome-dependent degradation of HNF4α and then inhibits HBV replication via the suppression of core promoter activity. The development of antiviral agents based on berberine may contribute to the amelioration of HBV-related disorders, regardless of the presence of residual cccDNA or integrated viral DNA fragments.

Comprehensive transcriptomic analysis identifies cholesterol transport pathway as a therapeutic target of porcine epidemic diarrhea coronavirus

Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that poses a serious threat to the global pig industry. Despite extensive efforts, the mechanism underlying virus entry for PEDV remains elusive. In this study, we first identified PEDV-susceptible and non-susceptible cell lines by using PEDV spike pseudotyped vesicular stomatitis virus. Subsequently, we conducted a comprehensive transcriptomic analysis on these cell lines. Through integrating differential expression gene analysis with weighted gene co-expression network analysis, we identified the key pathways that are correlated with the PEDV entry. Our analysis revealed a strong correlation between cholesterol, sterols, and lipid transport with PEDV entry, suggesting a potential role for cholesterol transport in the PEDV entry. For further investigation, we treated Huh7, Vero and LLC-PK1 cells with a cholesterol transport inhibitor, ezetimibe, and observed a significant inhibition of PEDV entry and subsequent viral replication in these cells. Interestingly, pre-treating Huh7 cells with ezetimibe resulted in an increase in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses. Moreover, we found that cholesterol could facilitate the entry of PEDV into Huh7 and Vero cells, and this promoting effect can be blocked by ezetimibe. These findings suggest that targeting cholesterol transport specifically inhibits PEDV entry into susceptible cells. Our study offers novel insights into the mechanism of PEDV entry and the development of new therapeutic strategies against this economically important virus.