Development of a virus-based affinity ultrafiltration method for screening virus-surface-protein-targeted compounds from complex matrixes: Herbal medicines as a case study.

Herbal medicines (HMs) are one of the main sources for the development of lead antiviral compounds. However, due to the complex composition of HMs, the screening of active compounds within these is inefficient and requires a significant time investment. We report a novel and efficient virus-based screening method for antiviral active compounds in HMs. This method involves the centrifugal ultrafiltration of viruses, known as the virus-based affinity ultrafiltration method (VAUM). This method is suitable to identify virus specific active compounds from complex matrices such as HMs. The effectiveness of the VAUM was evaluated using influenza A virus (IAV) H1N1. Using this method, four compounds that bind to the surface protein of H1N1 were identified from dried fruits of Terminalia chebula (TC). Through competitive inhibition assays, the influenza surface protein, neuraminidase (NA), was identified as the target protein of these four TC-derived compounds. Three compounds were identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), and their anti-H1N1 activities were verified by examining the cytopathic effect (CPE) and by performing a virus yield reduction assay. Further mechanistic studies demonstrated that these three compounds directly bind to NA and inhibit its activity. In summary, we describe here a VAUM that we designed, one that can be used to accurately screen antiviral active compounds in HMs and also help improve the efficiency of screening antiviral drugs found in natural products.

Identification of the Imidazo[1,2-a]pyrazine Derivative A4 as a Potential Influenza Virus Nucleoprotein Inhibitor.

Influenza A viruses (IAVs) have gradually developed resistance to FDA-approved drugs, which increases the need to discover novel antivirals with new mechanisms of action. Here, we used a phenotypic screening strategy and discovered that the imidazo[1,2-a]pyrazine derivative A4 demonstrates potent and broad-spectrum anti-influenza activity, especially for the oseltamivir-resistant H1N1/pdm09 strain. Indirect immunofluorescence assays revealed that A4 induces clustering of the viral nucleoprotein (NP) and prevents its nuclear accumulation. Furthermore, upon conducting binding analyses between A4 and the influenza NP using surface plasmon resonance assays and molecular docking simulations, we were able to confirm that A4 binds directly to the viral NP. Additionally, A4 exhibits high human plasma metabolic stability (remaining120 min > 90%, T1/2 = 990 min) and moderate inhibitory effects on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 as well as low acute toxicity in Kunming mice. Overall, this study provides valuable insights and lays the groundwork for future efforts in medicinal chemistry to identify effective drugs against influenza.

Influenza virus cell entry and targeted antiviral development.

Influenza virus infection is currently one of the most prevalent and transmissible diseases in the world causing local outbreaks every year. It has the potential to cause devastating global pandemics as well. The development of anti-influenza drugs possessing novel mechanisms of action is urgently needed to control the spread of influenza infections; thus, drugs that inhibit influenza virus entry into target cells are emerging as a hot research topic. In addition to discussing the biological significance of hemagglutinin in viral replication, this article provides recent updates on the natural products, small molecules, proteins, peptides, and neutralizing antibody-like proteins that have anti-influenza potency.

Allopregnanolone targets nucleoprotein as a novel influenza virus inhibitor.

Influenza A virus (IAV) poses a global public health concern and remains an imminent threat to human health. Emerging antiviral resistance to the currently approved influenza drugs emphasizes the urgent need for new therapeutic entities against IAV. Allopregnanolone (ALLO) is a natural product that has been approved as an antidepressant drug. In the present study, we repurposed ALLO as a novel inhibitor against IAVs. Mechanistic studies demonstrated that ALLO inhibited virus replication by interfering with the nucleus translocation of viral nucleoprotein (NP). In addition, ALLO showed significant synergistic activity with compound 16, a hemagglutinin inhibitor of IAVs. In summary, we have identified ALLO as a novel influenza virus inhibitor targeting NP, providing a promising candidate that deserves further investigation as a useful anti-influenza strategy in the future.

Qingjin Huatan decoction protects mice against influenza a virus pneumonia via the chemokine signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingjin Huatan Decoction (QJHTT) consists of 11 herbal medicines: Scutellaria baicalensis Georgi, Gardenia jasminoides J.Ellis, Platycodon grandiflorus (Jacq.) A.DC., Ophiopogon japonicus (Thunb.) Ker Gawl., Morus alba L., Fritillaria thunbergii Miq., Anemarrhena asphodeloides Bunge, Trichosanthes kirilowii Maxim., Citrus reticulata Blanco, Poria cocos (Schw.) Wolf, and Glycyrrhiza uralensis Fisch. As a traditional compound Chinese medicinal formula, QJHTT has been used for more than 400 years in China. Historically, it was used to treat respiratory diseases and had shown beneficial clinical results for diseases related to lung inflammation. AIM OF THE STUDY: To investigate the therapeutic effect of QJHTT on influenza A virus (IAV) pneumonia in mice and explore its possible mechanism of action. MATERIALS AND METHODS: The components in QJHTT were analyzed by UPLC-Q-TOF-MS and some antiviral active components reported in the literature were determined and quantified by HPLC. The protective effects of QJHTT were investigated using lethal and sublethal doses (2 LD50 or 0.8 LD50 viral suspension, separately) of H1N1-infected mice. Mortality and lung lesions in H1N1-infected mice were used to evaluate the efficacy of QJHTT. The potential mechanism of QJHTT in the treatment of viral pneumonia was determined at the gene level by RNA sequencing and validated by qRT-PCR. Following this, the changes in protein levels of JAK2/STAT3 were analyzed since it is a key downstream target of the chemokine signaling pathways. Preliminary elucidation of the mechanism of QJHTT to protect mice against IAV pneumonia through this pathway was conducted. RESULTS: In this study, 12 antiviral active constituents including baicalin, geniposide, and mangiferin were identified from QJHTT. In vivo treatment of QJHTT reduced the virus titers of lung tissue significantly and improved the survival rate, lung index, and pulmonary histopathological changes; additionally, a reduction in the serum levels of TNF-α, IL-1ß, IL-6, and IFN-γ inflammatory factors in H1N1-infected mice was observed. RNA-seq analysis and qRT-PCR showed that QJHTT primarily reversed the activities CCL2, CCL7, CCR1, and other chemokines and their reception-related genes, suggesting that QJHTT may produce disease-resistant pneumonia by inhibiting the downstream JAK2/STAT3 pathway. Western blot analysis confirmed that QJHTT effectively reduced the protein levels of JAK2, STAT3, and related phosphorylated products in the lung tissue of H1N1-infected mice. CONCLUSIONS: Our results indicated that QJHTT alleviated IAV pneumonia in mice by regulating related chemokines and their receptor-related genes in lung tissue, thereby inhibiting JAK2/STAT3 pathway. This could pave way for the design of novel therapeutic strategies to treat viral pneumonia.

Targeting SARS-CoV-2 viral proteases as a therapeutic strategy to treat COVID-19.

The 21st century has witnessed three outbreaks of coronavirus (CoVs) infections caused by severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, spreads rapidly and since the discovery of the first COVID-19 infection in December 2019, has caused 1.2 million deaths worldwide and 226,777 deaths in the United States alone. The high amino acid similarity between SARS-CoV and SARS-CoV-2 viral proteins supports testing therapeutic molecules that were designed to treat SARS infections during the 2003 epidemic. In this review, we provide information on possible COVID-19 treatment strategies that act via inhibition of the two essential proteins of the virus, 3C-like protease (3CLpro ) or papain-like protease (PLpro ).

RNA-dependent RNA polymerase of SARS-CoV-2 as a therapeutic target.

The global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), named coronavirus disease 2019, has infected more than 8.9 million people worldwide. This calls for urgent effective therapeutic measures. RNA-dependent RNA polymerase (RdRp) activity in viral transcription and replication has been recognized as an attractive target to design novel antiviral strategies. Although SARS-CoV-2 shares less genetic similarity with SARS-CoV (~79%) and Middle East respiratory syndrome coronavirus (~50%), the respective RdRps of the three coronaviruses are highly conserved, suggesting that RdRp is a good broad-spectrum antiviral target for coronaviruses. In this review, we discuss the antiviral potential of RdRp inhibitors (mainly nucleoside analogs) with an aim to provide a comprehensive account of drug discovery on SARS-CoV-2.

Modulation of hepatitis B virus pregenomic RNA stability and splicing by histone deacetylase 5 enhances viral biosynthesis.

Hepatitis B virus (HBV) is a worldwide health problem without curative treatments. Investigation of the regulation of HBV biosynthesis by class I and II histone deacetylases (HDACs) demonstrated that catalytically active HDAC5 upregulates HBV biosynthesis. HDAC5 expression increased both the stability and splicing of the HBV 3.5 kb RNA without altering the translational efficiency of the viral pregenomic or spliced 2.2 kb RNAs. Together, these observations point to a broader role of HDAC5 in regulating RNA splicing and transcript stability while specifically identifying a potentially novel approach toward antiviral HBV therapeutic development.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.