Omicsynin B4 potently blocks coronavirus infection by inhibiting host proteases cathepsin L and TMPRSS2.

The emergence of SARS-CoV-2 variants represents a major threat to public health and requires identification of novel therapeutic agents to address the unmet medical needs. Small molecules impeding viral entry through inhibition of spike protein priming proteases could have potent antiviral effects against SARS-CoV-2 infection. Omicsynin B4, a pseudo-tetrapeptides identified from Streptomyces sp. 1647, has potent antiviral activity against influenza A viruses in our previous study. Here, we found omicsynin B4 exhibited broad-spectrum anti-coronavirus activity against HCoV-229E, HCoV-OC43 and SARS-CoV-2 prototype and its variants in multiple cell lines. Further investigations revealed omicsynin B4 blocked the viral entry and might be related to the inhibition of host proteases. SARS-CoV-2 spike protein mediated pseudovirus assay supported the inhibitory activity on viral entry of omicsynin B4 with a more potent inhibition of Omicron variant, especially when overexpression of human TMPRSS2. Moreover, omicsynin B4 exhibited superior inhibitory activity in the sub-nanomolar range against CTSL, and a sub-micromolar inhibition against TMPRSS2 in biochemical assays. The molecular docking analysis confirmed that omicsynin B4 fits well in the substrate binding sites and forms a covalent bond to Cys25 and Ser441 in CTSL and TMPRSS2, respectively. In conclusion, we found that omicsynin B4 may serve as a natural protease inhibitor for CTSL and TMPRSS2, blocking various coronavirus S protein-driven entry into cells. These results further highlight the potential of omicsynin B4 as an attractive candidate for broad-spectrum antiviral therapy that could rapidly respond to emerging variants of SARS-CoV-2.

Cytotoxic hexadepsipeptides and anti-coronaviral 4-hydroxy-2-pyridones from an endophytic Fusarium sp.

Three new hexadepsipeptides (1-3), along with beauvericin (4), beauvericin D (5), and four 4-hydroxy-2-pyridone derivatives (6-9) were isolated from the endophytic fungus Fusarium sp. CPCC 400857 that derived from the stem of tea plant. Their structures were determined by extensive 1D and 2D NMR, and HRESIMS analyses. The absolute configuration of hexadepsipeptides were elucidated by the advanced Marfey's method and chiral HPLC analysis. Compounds 4, and 7-9 displayed the cytotoxicity against human pancreatic cancer cell line, AsPC-1 with IC50 values ranging from 3.45 to 29.69 µM, and 7 and 8 also showed the antiviral activity against the coronavirus (HCoV-OC43) with IC50 values of 13.33 and 6.65 µM, respectively.

Ginkgolic acid and anacardic acid are reversible inhibitors of SARS-CoV-2 3-chymotrypsin-like protease.

Because of the emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different regions of the world, the battle with infectious coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been seesawing. Therefore, the identification of antiviral drugs is of particular importance. In order to rapidly identify inhibitors for SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), an enzyme essential for viral replication, we combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) and developed a novel sandwich-like FP screening assay. Through high-throughput screening, two hits of 3CLpro inhibitors, ginkgolic acid (GA) and anacardic acid (AA) were identified, which showed IC50 values of 11.29 ± 0.48 and 12.19 ± 0.50 µM, respectively. Their binding modes were evaluated by HPLC-Q-TOF-MS. There was no mass increase detected for SARS-CoV-2 3CLpro incubated with either GA or AA, indicating the absence of covalent adducts. The kinetic analysis clearly demonstrated that both GA and AA inhibit SARS-CoV-2 3CLpro via reversible and mixed-inhibition manner. Our results argue against conclusion that GA and AA act as irreversible and covalent inhibitors against SARS-CoV-2 3CLpro, which is based on the studies by Chen et al.

Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. RESULTS: In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. CONCLUSIONS: This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

Multi-Omics-Guided Discovery of Omicsynins Produced by Streptomyces sp. 1647: Pseudo-Tetrapeptides Active Against Influenza A Viruses and Coronavirus HCoV-229E.

Many microorganisms have mechanisms that protect cells against attack from viruses. The fermentation components of Streptomyces sp. 1647 exhibit potent anti-influenza A virus (IAV) activity. This strain was isolated from soil in southern China in the 1970s, but the chemical nature of its antiviral substance(s) has remained unknown until now. We used an integrated multi-omics strategy to identify the antiviral agents from this streptomycete. The antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) analysis of its genome sequence revealed 38 biosynthetic gene clusters (BGCs) for secondary metabolites, and the target BGCs possibly responsible for the production of antiviral components were narrowed down to three BGCs by bioactivity-guided comparative transcriptomics analysis. Through bioinformatics analysis and genetic manipulation of the regulators and a biosynthetic gene, cluster 36 was identified as the BGC responsible for the biosynthesis of the antiviral compounds. Bioactivity-based molecular networking analysis of mass spectrometric data from different recombinant strains illustrated that the antiviral compounds were a class of structural analogues. Finally, 18 pseudo-tetrapeptides with an internal ureido linkage, omicsynins A1-A6, B1-B6, and C1-C6, were identified and/or isolated from fermentation broth. Among them, 11 compounds (omicsynins A1, A2, A6, B1-B3, B5, B6, C1, C2, and C6) are new compounds. Omicsynins B1-B4 exhibited potent antiviral activity against IAV with the 50% inhibitory concentration (IC50) of approximately 1 µmol∙L-1 and a selectivity index (SI) ranging from 100 to 300. Omicsynins B1-B4 also showed significant antiviral activity against human coronavirus HCoV-229E. By integrating multi-omics data, we discovered a number of novel antiviral pseudo-tetrapeptides produced by Streptomyces sp. 1647, indicating that the secondary metabolites of microorganisms are a valuable source of novel antivirals.