Carrimycin inhibits coronavirus replication by decreasing the efficiency of programmed -1 ribosomal frameshifting through directly binding to the RNA pseudoknot of viral frameshift-stimulatory element.

The pandemic of SARS-CoV-2 worldwide with successive emerging variants urgently calls for small-molecule oral drugs with broad-spectrum antiviral activity. Here, we show that carrimycin, a new macrolide antibiotic in the clinic and an antiviral candidate for SARS-CoV-2 in phase III trials, decreases the efficiency of programmed -1 ribosomal frameshifting of coronaviruses and thus impedes viral replication in a broad-spectrum fashion. Carrimycin binds directly to the coronaviral frameshift-stimulatory element (FSE) RNA pseudoknot, interrupting the viral protein translation switch from ORF1a to ORF1b and thereby reducing the level of the core components of the viral replication and transcription complexes. Combined carrimycin with known viral replicase inhibitors yielded a synergistic inhibitory effect on coronaviruses. Because the FSE mechanism is essential in all coronaviruses, carrimycin could be a new broad-spectrum antiviral drug for human coronaviruses by directly targeting the conserved coronaviral FSE RNA. This finding may open a new direction in antiviral drug discovery for coronavirus variants.

Rogersonins C-F, 9H-Imidazo[2,1-i]purine-Incorporating Adenine-Polyketide Hybrids from an Ophiocordyceps-Associated Clonostachys rogersoniana.

Rogersonins C-F (1-4), four unprecedented adenine-polyketide hybrids featuring a rare 9H-imidazo[2,1-i]purine (1,N6-ethenoadenine) moiety, were isolated from an Ophiocordyceps-associated fungus, Clonostachys rogersoniana. Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-4 were assigned by a combination of the modified Mosher method, chemical degradation, electronic circular dichroism (ECD) calculations, and X-ray crystallography using Cu Kα radiation. Compound 3 downregulated the expression of PD-L1 protein in MDA-MB-231 and A549 cells, but did not show detectable effect on mRNA transcription of the PD-L1-encoding gene CD274.

Spirocitrinols A and B, citrinin derivatives with a spiro[chromane-2,3'-isochromane] skeleton from Penicillium citrinum.

Spirocitrinols A (1) and B (2), two new citrinin-derived metabolites possessing a spiro[chromane-2,3'-isochromane] skeleton, were isolated from cultures of Penicillium citrinum. Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned by electronic circular dichroism calculations. Compound 2 is the first naturally occurring trimeric citrinin derivative with a spiro[chromane-2,3'-isochromane] core. Compound 1 showed modest cytotoxicity against A549 human tumor cells.

Lachnochromonin, a fungal metabolite from Lachnum virgineum, inhibits cell growth and promotes apoptosis in tumor cells through JAK/STAT3 signaling.

Natural compounds that interfere with tumor cell growth have potential to be used as therapeutic agents to treat cancers. Lachnochromonin (p71) is a small molecule isolated from Lachnum virgineum. Here, we reported the effect of p71 on human tumor cells, especially on breast cancer MCF-7 cells. We found that p71 significantly suppresses cell growth and induces apoptosis. The luciferase results demonstrated that p71 specifically attenuates the activation of JAK/STAT3 signaling. Biochemical analysis revealed that p71 blocks the phosphorylation of STAT3 tyrosine 705 and serine 727, resulting in down-regulation of c-Myc and Cyclin D1 expression level. Importantly, p71 inhibited cell growth, colony-formation, and migration through affecting STAT3 activity. These results implied that p71 may be used as a therapeutic agent against breast cancer.

Altersteroids A-D, 9,11-Secosteroid-Derived γ-Lactones from an Alternaria sp.

Altersteroids A-D (1-4), four new 9,11-secosteroid-derived γ-lactones, were isolated from cultures of the ascomycete fungus Alternaria sp. Their structures were elucidated primarily by NMR experiments. The absolute configuration of 1 was established by X-ray crystallographic analysis of its di-p-nitrobenzenesulfonate 1a using Cu Kα radiation, whereas those for 2-4 were assigned by quantum-chemical calculations. Compounds 1-4 incorporate a γ-lactone moiety fused to the steroid D ring at C-13/C-14. Compound 3 showed moderate cytotoxicity toward four tumor cell lines and induced an apoptotic process in A549 cells. Notably, compound 3 showed equipotent activity against the cisplatin-sensitive MB49 and -resistant MB49 CisR cells, with an IC50 value of 12.7 µM.

Cladoxanthones A and B, Xanthone-Derived Metabolites with a Spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'-tetraone Skeleton from a Cladosporium sp.

Cladoxanthones A (1) and B (2), two xanthone-derived metabolites featuring a new spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'(4a'H)-tetraone skeleton, were isolated from cultures of the ascomycete fungus Cladosporium sp., together with the known mangrovamide J (3). Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned by X-ray crystallography using Cu Kα radiation. Compound 1 could be generated from the hypothetical precursors related to α-methylene ketone and dihydro-xanthone via a Diels-Alder reaction, while 2 could be an oxidative coupling product resulting from 1 and 3. Compounds 1 and 2 showed weakly cytotoxic effects.

Prebiotic-like cyclodextrin assisted silybin on NAFLD through restoring liver and gut homeostasis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with no currently approved treatment. The natural compound silybin (SLN) has versatile hepatoprotective efficacy with negligible adverse effects; however, poor absorption limits its clinical applications. Gut microbiota has been proposed to play a crucial role in the pathophysiology of NAFLD and targeted for disease control. Cyclodextrins, the cyclic oligosaccharides, were documented to have various health benefits with potential prebiotic properties. This study aimed to develop a silybin-2-hydroxypropyl-ß-cyclodextrin inclusion (SHßCD) to improve the therapeutic efficacy of SLN and elucidate the mechanisms of improvement. The results showed that SLN formed a 1:1 stoichiometric inclusion complex with HP-ß-CD. The solubility of SLN was increased by generating SHßCD, resulting in improved drug permeability and bioavailability. In high-fat diet (HFD)-fed hamsters, SHßCD modulated gut health by restoring the gut microbiota and intestinal integrity. SHßCD showed superior anti-lipid accumulation, antioxidant, and anti-inflammatory effects compared with SLN alone. Transcriptome analysis in the liver tissue implied that the improved inflammation and/or energy homeostasis was the potential mechanism. Therefore, SHßCD may be a promising alternative for the treatment of NAFLD, attributing to the dual functions of HßCD on drug absorption and gut microbial homeostasis.

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.

Neopestolides A-D, Diphenyl Ether Derivatives from the Plant Endophytic Fungus Neopestalotiopsis sp.

Four new diphenyl ether derivatives, neopestolides A-D (2-5), were isolated from cultures of the plant endophytic fungus Neopestalotiopsis sp., along with the known metabolite pestalotiollide A (1); their structures were elucidated primarily by NMR experiments. The absolute configurations of 2 and 3-5 were deduced by electronic circular dichroism calculations and via Snatzke's method, respectively. Compounds 2-4 incorporate tetrahydrofuran moieties attached to the dibenzo[b,g][1,5]dioxocin-5(7H)-one skeleton via C-C linkages. Compounds 1 and 2 showed modest cytotoxicity against HepG2 cells.

Bicyclol Alleviates Atherosclerosis by Manipulating Gut Microbiota.

Atherosclerosis (AS) is associated with high morbidity and mortality, thus imposing a growing burden on modern society. Herb-derived bicyclol (BIC) is a versatile bioactive compound that can be used to treat AS. However, its efficacy in AS is not yet described. Here, it is shown that BIC normalizes gut microflora dysbiosis induced by a high fat diet in Apoe(-/-) mice. Metagenome-wide association study analysis verifies that the modulation on carbohydrate-active enzymes and short-chain fatty acid generating genes in gut flora is among the mechanisms. The gut healthiness, especially the gut immunity and integrity, is restored by BIC intervention, leading to improved systemic immune cell dynamic and liver functions. Accordingly, the endothelial activation, macrophage infiltration, and cholesterol ester accumulation in the aortic arch are alleviated by BIC to lessen the plaque onset. Moreover, it is proved that the therapeutic effect of BIC on AS is transmissible by fecal microbiota transplantation. The current study, for the first time, demonstrates the antiatherosclerotic effects of BIC and shows that its therapeutic value can at least partially be attributed to its manipulation of gut microbiota.

Overproduction of medicinal ergot alkaloids based on a fungal platform.

Privileged ergot alkaloids (EAs) produced by the fungal genus Claviceps are used to treat a wide range of diseases. However, their use and research have been hampered by the challenging genetic engineering of Claviceps. Here we systematically refactored and rationally engineered the EA biosynthetic pathway in heterologous host Aspergillus nidulans by using a Fungal-Yeast-Shuttle-Vector protocol. The obtained strains allowed the production of diverse EAs and related intermediates, including prechanoclavine (PCC, 333.8 mg/L), chanoclavine (CC, 241.0 mg/L), agroclavine (AC, 78.7 mg/L), and festuclavine (FC, 99.2 mg/L), etc. This fungal platform also enabled the access to the methyl-oxidized EAs (MOEAs), including elymoclavine (EC), lysergic acid (LA), dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA), by overexpressing a P450 enzyme CloA. Furthermore, by optimizing the P450 electron transfer (ET) pathway and using multi-copy of cloA, the titers of EC and DHLG have been improved by 17.3- and 9.4-fold, respectively. Beyond our demonstration of A. nidulans as a robust platform for EA overproduction, our study offers a proof of concept for engineering the eukaryotic P450s-contained biosynthetic pathways in a filamentous fungal host.

Azvudine is a thymus-homing anti-SARS-CoV-2 drug effective in treating COVID-19 patients.

Azvudine (FNC) is a nucleoside analog that inhibits HIV-1 RNA-dependent RNA polymerase (RdRp). Recently, we discovered FNC an agent against SARS-CoV-2, and have taken it into Phase III trial for COVID-19 patients. FNC monophosphate analog inhibited SARS-CoV-2 and HCoV-OC43 coronavirus with an EC50 between 1.2 and 4.3 µM, depending on viruses or cells, and selective index (SI) in 15-83 range. Oral administration of FNC in rats revealed a substantial thymus-homing feature, with FNC triphosphate (the active form) concentrated in the thymus and peripheral blood mononuclear cells (PBMC). Treating SARS-CoV-2 infected rhesus macaques with FNC (0.07 mg/kg, qd, orally) reduced viral load, recuperated the thymus, improved lymphocyte profiles, alleviated inflammation and organ damage, and lessened ground-glass opacities in chest X-ray. Single-cell sequencing suggested the promotion of thymus function by FNC. A randomized, single-arm clinical trial of FNC on compassionate use (n = 31) showed that oral FNC (5 mg, qd) cured all COVID-19 patients, with 100% viral ribonucleic acid negative conversion in 3.29 ± 2.22 days (range: 1-9 days) and 100% hospital discharge rate in 9.00 ± 4.93 days (range: 2-25 days). The side-effect of FNC is minor and transient dizziness and nausea in 16.12% (5/31) patients. Thus, FNC might cure COVID-19 through its anti-SARS-CoV-2 activity concentrated in the thymus, followed by promoted immunity.

Repurposing carrimycin as an antiviral agent against human coronaviruses, including the currently pandemic SARS-CoV-2.

COVID-19 pandemic caused by SARS-CoV-2 infection severely threatens global health and economic development. No effective antiviral drug is currently available to treat COVID-19 and any other human coronavirus infections. We report herein that a macrolide antibiotic, carrimycin, potently inhibited the cytopathic effects (CPE) and reduced the levels of viral protein and RNA in multiple cell types infected by human coronavirus 229E, OC43, and SARS-CoV-2. Time-of-addition and pseudotype virus infection studies indicated that carrimycin inhibited one or multiple post-entry replication events of human coronavirus infection. In support of this notion, metabolic labelling studies showed that carrimycin significantly inhibited the synthesis of viral RNA. Our studies thus strongly suggest that carrimycin is an antiviral agent against a broad-spectrum of human coronaviruses and its therapeutic efficacy to COVID-19 is currently under clinical investigation.

Disulfide Cleavage in a Dimeric Epipolythiodioxopiperazine Natural Product Diminishes Its Apoptosis-Inducing Effect but Enhances Autophagy in Tumor Cells.

Gliocladicillin C (3) is a cytotoxic epipolythiodioxopiperazine (ETP) isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana. Although the disulfides/polysulfides in ETPs are believed to account for their cytotoxicity, and 11'-deoxyverticillin A was demonstrated to induce apoptosis and autophagy, how they mediate apoptosis and autophagy remained unknown. Here, we revealed that 3 activated caspase-dependent apoptosis and autophagy in human tumor cells, while the prepared disulfide-cleavage product failed to induce reactive oxygen species production and PARP cleavage, but further enhanced the autophagic flux compared to 3. Gliocladicillin C and its derivative also increased the phosphorylation of AMP-activated protein kinase and stimulated autophagy by affecting the glycolytic pathway. These results demonstrated that the disulfides played an essential role in inducing apoptosis, but not autophagy.

Sarcosenones A-C, highly oxygenated pimarane diterpenoids from an endolichenic fungus Sarcosomataceae sp.

Three new highly oxygenated pimarane diterpenoids, sarcosenones A-C (1-3), and the known 9α-hydroxy-1,8(14),15-isopimaratrien-3,7,11-trione (4), were isolated from cultures of an endolichenic fungus Sarcosomataceae sp. Their structures were elucidated based on NMR spectroscopic data and electronic circular dichroism (ECD) calculations. Compound 1 showed moderate cytotoxicity against a small panel of four human tumor cell lines, with IC50 values of 7.5-26.4 µM.

Enzymatic Intermolecular Hetero-Diels-Alder Reaction in the Biosynthesis of Tropolonic Sesquiterpenes.

Diels-Alder reactions are among the most powerful synthetic transformations to construct complex natural products. Despite that increasing of enzymatic intramolecular Diels-Alder reactions have been discovered, natural intermolecular Diels-Alderases are rarely described. Here, we report an intermolecular hetero-Diels-Alder reaction in the biosynthesis of tropolonic sesquiterpenes and functionally characterize EupfF as the first fungal intermolecular hetero-Diels-Alderase. We demonstrate that EupfF catalyzed the dehydration of a hydroxymethyl-containing tropolone (5) to generate a reactive tropolone o-quinone methide (6) and might further stereoselectively control the subsequent intermolecular hetero-Diels-Alder reaction with (1E,4E,8Z)-humulenol (8) to produce enantiomerically pure neosetophomone B (1). Our results reveal the biosynthetic pathway of 1 and expand the repertoire of activities of Diels-Alder cyclases.

The disruption of verM activates the production of gliocladiosin A and B in Clonostachys rogersoniana.

Gliocladiosin A (1) and B (2), two dipeptides conjugated with macrolides, were identified from a verM disruption mutant of the Cordycep-colonizing fungus Clonostachys rogersoniana. The structures and absolute configurations of 1 and 2 were determined on the basis of spectroscopic data analysis, including MS, NMR, CD and X-ray diffraction. A biogenetic pathway for 1 and 2 was proposed. These two compounds showed moderate antibacterial effects.

Grb2 binds to PTEN and regulates its nuclear translocation to maintain the genomic stability in DNA damage response.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein critical for signal transduction and endocytosis, but its role in DNA damage response (DDR) remains unknown. Here, we report that either knockdown of Grb2 or overexpression of the mutated Grb2 promotes micronuclei formation in response to oxidative stress. Furthermore, Grb2 was demonstrated to interact with phosphatase and tensin homologue (PTEN; a tumor suppressor essential for nuclear stability), and the loss of Grb2 reduced the nuclear-localized PTEN, which was further decreased upon stimulation with hydrogen peroxide (H2O2). Overexpression of the T398A-mutated, nuclear-localized PTEN reduced micronuclei frequency in the cells deficient of functional Grb2 via rescuing the H2O2-dependent expression of Rad51, a protein essential for the homologous recombination (HR) repair process. Moreover, depletion of Grb2 markedly decreased the expression of Rad51 and its interaction with PTEN. Notably, Rad51 showed a preference to immunoprecipation with the T398A-PTEN mutant, and silencing of Rad51 alone accumulated micronuclei concurring with decreased expression of both Grb2 and PTEN. Our findings indicate that Grb2 interacts with PTEN and Rad51 to regulate genomic stability in DDR by mediating the nuclear translocation of PTEN to affect the expression of Rad51.

Phomanolides C-F from a Phoma sp.: Meroterpenoids Generated via Hetero-Diels-Alder Reactions.

Phomanolides C-F (1-4), four new meroterpenoids, were isolated from a Phoma sp., together with the known phomanolides A (5) and B (6); their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-3 were assigned by electronic circular dichroism calculations, and that of 4 was established by X-ray diffraction analysis using Cu Kα radiation. Compounds 1-3 incorporate an unprecedented trioxa[4.4.3]propellane subunit in their skeletons. Compounds 2 and 4 were weakly cytotoxic.