Synthesis, Structure-Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.

Drugs against SARS-CoV-2: What do we know about their mode of action?

The health emergency caused by the recent Covid-19 pandemic highlights the need to identify effective treatments against the virus causing this disease (SARS-CoV-2). The first clinical trials have been testing repurposed drugs that show promising anti-SARS-CoV-2 effects in cultured cells. Although more than 2400 clinical trials are already under way, the actual number of tested compounds is still limited to approximately 20, alone or in combination. In addition, knowledge on their mode of action (MoA) is currently insufficient. Their first results reveal some inconsistencies and contradictory results and suggest that cohort size and quality of the control arm are two key issues for obtaining rigorous and conclusive results. Moreover, the observed discrepancies might also result from differences in the clinical inclusion criteria, including the possibility of early treatment that may be essential for therapy efficacy in patients with Covid-19. Importantly, efforts should also be made to test new compounds with a documented MoA against SARS-CoV-2 in clinical trials. Successful treatment will probably be based on multitherapies with antiviral compounds that target different steps of the virus life cycle. Moreover, a multidisciplinary approach that combines artificial intelligence, compound docking, and robust in vitro and in vivo assays will accelerate the development of new antiviral molecules. Finally, large retrospective studies on hospitalized patients are needed to evaluate the different treatments with robust statistical tools and to identify the best treatment for each Covid-19 stage. This review describes different candidate antiviral strategies for Covid-19, by focusing on their mechanism of action.

Incretin combination therapy for the treatment of non-alcoholic steatohepatitis.

AIMS: To test specific mono-agonists to the glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic peptide receptor (GIPR), individually and in combination, in a mouse model of diet-induced non-alcoholic steatohepatitis (NASH) and fibrosis in order to decipher the contribution of their activities and potential additive effects to improving systemic and hepatic metabolism. MATERIALS AND METHODS: We induced NASH by pre-feeding C57BL/6J mice a diet rich in fat, fructose and cholesterol for 36 weeks. This was followed by 8 weeks of treatment with the receptor-specific agonists 1-GCG (20 µg/kg twice daily), 2-GLP1 (3 µg/kg twice daily) or 3-GIP (30 µg/kg twice daily), or the dual (1 + 2) or triple (1 + 2 + 3) combinations thereof. A dual GLP-1R/GCGR agonistic peptide, 4-dual-GLP1/GCGR (30 µg/kg twice daily), and liraglutide (100 µg/kg twice daily) were included as references. RESULTS: Whereas low-dose 1-GCG or 3-GIP alone did not influence body weight, liver lipids and histology, their combination with 2-GLP1 provided additional weight loss, reduction in liver triglycerides and improvement in histological disease activity score. Notably, 4-dual-GLP-1R/GCGR and the triple combination of selective mono-agonists led to a significantly stronger reduction in the histological non-alcoholic fatty liver disease activity score compared to high-dose liraglutide, at the same extent of body weight loss. CONCLUSIONS: GCGR and GIPR agonism provide additional, body weight-independent improvements on top of GLP-1R agonism in a murine model of manifest NASH with fibrosis.

Carneic Acids from an Endophytic Phomopsis sp. as Dengue Virus Polymerase Inhibitors.

Thirteen carneic acids were isolated from the fungal endophyte Phomopsis sp. SNB-LAP1-7-32. Their structures were identified by mass spectrometry and extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. Compounds 1-13 were investigated for their antipolymerase activities against DENV polymerase and Zika NS5. Five of them exhibited significant inhibition of dengue polymerase with IC50 values in the 10 to 20 µM range without cytotoxicity. None inhibited Zika virus NS5 protein.

Type 2/Th2-driven inflammation impairs olfactory sensory neurogenesis in mouse chronic rhinosinusitis model.

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear. METHODS: In order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)-mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro-olfactograms (EOGs). RESULTS: After 22 weeks, a typical type 2/Th2-mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)-4, IL-5, and IL-13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired. CONCLUSION: Our mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP-associated anosmia.

Antiviral Compounds from Codiaeum peltatum Targeted by a Multi-informative Molecular Networks Approach.

From a set of 292 Euphorbiaceae extracts, the use of a molecular networking (MN)-based prioritization approach highlighted three clusters (MN1-3) depicting ions from the bark extract of Codiaeum peltatum. Based on their putative antiviral potential and structural novelty, the MS-guided purification of compounds present in MN1 and MN2 afforded two new daphnane-type diterpenoid orthoesters (DDO), codiapeltines A (1) and B (2), the new actephilols B (3) and C (4), and four known 1,4-dioxane-fused phenanthrene dimers (5-8). The structures of the new compounds were elucidated by NMR spectroscopic data analysis, and the absolute configurations of compounds 1 and 2 were deduced by comparison of experimental and calculated ECD spectra. Codiapeltine B (2) is the first daphnane bearing a 9,11,13-orthoester moiety, establishing a new major structural class of DDO. Compounds 1-8 and four recently reported monoterpenyl quinolones (9-12) detected in MN3 were investigated for their selective activities against chikungunya virus replication and their antipolymerase activities against the NS5 proteins of dengue and zika viruses. Compounds 3-8 exhibited strong inhibitory activities on both dengue and zika NS5 in primary assays, but extensive biological analyses indicated that only actephilol B (3) displayed a specific interaction with the NS5 targets.

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives.

The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2'-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2'-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-l-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the αX α-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs.IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns, and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.

CD36 Is a Marker of Human Adipocyte Progenitors with Pronounced Adipogenic and Triglyceride Accumulation Potential.

White adipose tissue (WAT) expands in part through adipogenesis, a process involving fat cell generation and fatty acid (FA) storage into triglycerides (TGs). Several findings suggest that inter-individual and regional variations in adipogenesis are linked to metabolic complications. We aimed to identify cellular markers that define human adipocyte progenitors (APs) with pronounced adipogenic/TG storage ability. Using an unbiased single cell screen of passaged human adipose-derived stromal cells (hADSCs), we identified cell clones with similar proliferation rates but discordant capabilities to undergo adipogenic differentiation. Transcriptomic analyses prior to induction of differentiation showed that adipogenic clones displayed a significantly higher expression of CD36, encoding the scavenger receptor CD36. CD36+ hADSCs, in comparison with CD36-cells, displayed almost complete adipogenic differentiation while CD36 RNAi attenuated lipid accumulation. Similar findings were observed in primary CD45-/CD34+/CD31-APs isolated from human WAT where the subpopulation of MSCA1+/CD36+ cells displayed a significantly higher differentiation degree/TG storage capacity than MSCA1+/CD36-cells. Functional analyses in vitro and ex vivo confirmed that CD36 conferred APs an increased capacity to take up FAs thereby facilitating terminal differentiation. Among primary APs from subcutaneous femoral, abdominal and visceral human WAT, the fraction of CD36+ cells was significantly higher in depots associated with higher adipogenesis and reduced metabolic risk (i.e., femoral WAT). We conclude that CD36 marks APs with pronounced adipogenic potential, most probably by facilitating lipid uptake. This may be of value in developing human adipocyte cell clones and possibly in linking regional variations in adipogenesis to metabolic phenotype. Stem Cells 2017;35:1799-1814.

NF-L in cerebrospinal fluid and serum is a biomarker of neuronal damage in an inducible mouse model of neurodegeneration.

Accumulation of neurofilaments (NFs), the major constituents of the neuronal cytoskeleton, is a distinctive feature of neurological diseases and several studies have shown that soluble NFs can be detected in the cerebrospinal fluid (CSF) of patients with neurological diseases, such as multiple sclerosis and frontotemporal dementia. Here we have used an inducible transgenic mouse model of neurodegeneration, CamKII-TetOp25 mice, to evaluate whether NF-L levels in CSF or blood can be used as a biochemical biomarker of neurodegeneration. Induction of p25 transgene brain expression led to increase in CSF and serum NF-L levels that correlated with ongoing neurodegeneration. Switching off p25 prevented further increases in both CSF and serum NF-L levels and concomitantly stopped the progression of neurodegeneration. The levels of CSF NF-L detected in p25 mice are about 4-fold higher than the CSF levels detected in patients with chronic neurodegenerative diseases, such as symptomatic FTD (bvFTD). In addition, our data indicate that the NF-L detected in CSF is most likely a cleaved form of NF-L. These results suggest that CSF and serum NF-L are of interest to be further explored as potential translational dynamic biomarkers of neurodegeneration or as pharmacodynamics biomarkers at least in preclinical animal studies.

Betulinic Acid, The First Lupane-Type Triterpenoid Isolated from Both a Phomopsis sp. and Its Host Plant Diospyros carbonaria Benoist.

Following a biological screening using a dengue replicon virus-cell-based assay, Diospyros carbonaria AcOEt extract was investigated, affording six known lupane-type triterpenoids endowed with anti-DENV-2 NS5 polymerase activity. The study of the associated microbial community of this species permitted us to identify 38 endophytes belonging to five different orders. Nine out of these 38 strains showed significant activity on the dengue replicon assay. The chemical investigation of the most active one, Phomopsis sp. SNB-LAP1-7-32, led to the isolation of betulinic acid, an anti-viral secondary metabolite isolated previously from the host plant. This result is the first example of a lupane-type triterpenoid isolated from both an endophyte and its host plant. Its presence in the Phomopsis strain may result from gene transfer and/or specific niche selection.

Targeted identification of sialoglycoproteins in hypoxic endothelial cells and validation in zebrafish reveal roles for proteins in angiogenesis.

The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis.

Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.

Structure-activity relationship study of biflavonoids on the Dengue virus polymerase DENV-NS5 RdRp.

Dengue virus is the world's most prevalent human pathogenic arbovirus. There is currently no treatment or vaccine, and solutions are urgently needed. We previously demonstrated that biflavonoids from Dacrydium balansae, an endemic gymnosperm from New Caledonia, are potent inhibitors of the Dengue virus NS5 RNA-dependent RNA polymerase. Herein we describe the structure-activity relationship study of 23 compounds: biflavonoids from D. balansae (1-4) and from D. araucarioides (5-10), hexamethyl-amentoflavone (11), cupressuflavone (12), and apigenin derivatives (13-23). We conclude that 1) over the four different biflavonoid skeletons tested, amentoflavone (1) and robustaflavone (5) are the most promising ones for antidengue drug development, 2) the number and position of methyl groups on the biflavonoid moiety modulate their inhibition of Dengue virus NS5 RNA-dependent RNA polymerase, and 3) the degree of oxygenation of flavonoid monomers influences their antidengue potential. Sotetsuflavone (8), with an IC50 = 0.16 µM, is the most active compound of this series and is the strongest inhibitor of the Dengue virus NS5 RNA-dependent RNA polymerase described in the literature.

Notch activation shifts the fate decision of senescent progenitors toward myofibrogenesis in human adipose tissue.

Senescence is a key event in the impairment of adipose tissue (AT) function with obesity and aging but the underlying molecular and cellular players remain to be fully defined, particularly with respect to the human AT progenitors. We have found distinct profiles of senescent progenitors based on AT location between stroma from visceral versus subcutaneous AT. In addition to flow cytometry, we characterized the location differences with transcriptomic and proteomic approaches, uncovering the genes and developmental pathways that are underlying replicative senescence. We identified key components to include INBHA as well as SFRP4 and GREM1, antagonists for the WNT and BMP pathways, in the senescence-associated secretory phenotype and NOTCH3 in the senescence-associated intrinsic phenotype. Notch activation in AT progenitors inhibits adipogenesis and promotes myofibrogenesis independently of TGFß. In addition, we demonstrate that NOTCH3 is enriched in the premyofibroblast progenitor subset, which preferentially accumulates in the visceral AT of patients with an early obesity trajectory. Herein, we reveal that NOTCH3 plays a role in the balance of progenitor fate determination preferring myofibrogenesis at the expense of adipogenesis. Progenitor NOTCH3 may constitute a tool to monitor replicative senescence and to limit AT dysfunction in obesity and aging.

TEAD Inhibitors Sensitize KRASG12C Inhibitors via Dual Cell Cycle Arrest in KRASG12C-Mutant NSCLC.

KRASG12C is one of the most common mutations detected in non-small cell lung cancer (NSCLC) patients, and it is a marker of poor prognosis. The first FDA-approved KRASG12C inhibitors, sotorasib and adagrasib, have been an enormous breakthrough for patients with KRASG12C mutant NSCLC; however, resistance to therapy is emerging. The transcriptional coactivators YAP1/TAZ and the family of transcription factors TEAD1-4 are the downstream effectors of the Hippo pathway and regulate essential cellular processes such as cell proliferation and cell survival. YAP1/TAZ-TEAD activity has further been implicated as a mechanism of resistance to targeted therapies. Here, we investigate the effect of combining TEAD inhibitors with KRASG12C inhibitors in KRASG12C mutant NSCLC tumor models. We show that TEAD inhibitors, while being inactive as single agents in KRASG12C-driven NSCLC cells, enhance KRASG12C inhibitor-mediated anti-tumor efficacy in vitro and in vivo. Mechanistically, the dual inhibition of KRASG12C and TEAD results in the downregulation of MYC and E2F signatures and in the alteration of the G2/M checkpoint, converging in an increase in G1 and a decrease in G2/M cell cycle phases. Our data suggest that the co-inhibition of KRASG12C and TEAD leads to a specific dual cell cycle arrest in KRASG12C NSCLC cells.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Single-cell RNA-sequencing of PBMCs from SAVI patients reveals disease-associated monocytes with elevated integrated stress response.

Gain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-ß. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response.

Flacourtosides A-F, phenolic glycosides isolated from Flacourtia ramontchi.

In an effort to identify novel inhibitors of chikungunya (CHIKV) and dengue (DENV) virus replication, a systematic study with 820 ethyl acetate extracts of madagascan plants was performed in a virus-cell-based assay for CHIKV, and a DENV NS5 RNA-dependent RNA polymerase (RdRp) assay. The extract obtained from the stem bark of Flacourtia ramontchi was selected for its significant activity in both assays. Six new phenolic glycosides, named flacourtosides A-F (1-6), phenolic glycosides itoside H, xylosmin, scolochinenoside D, and poliothrysoside, and betulinic acid 3ß-caffeate were obtained using the bioassay-guided isolation process. Their structures were elucidated by comprehensive analyses of NMR spectroscopic and mass spectrometric data. Even though several extracts and fractions showed significant selective antiviral activity in the CHIKV virus-cell-based assay, none of the purified compounds did. However, in the DENV RNA polymerase assay, significant inhibition was observed with betulinic acid 3ß-caffeate (IC(50) = 0.85 ± 0.1 µM) and to a lesser extent for the flacourtosides A and E (1 and 5, respectively), and scolochinenoside D (IC(50) values ~10 µM).

Biflavonoids of Dacrydium balansae with potent inhibitory activity on dengue 2 NS5 polymerase.

In order to find new molecules for antiviral drug design, we screened 102 ethyl acetate extracts from New-Caledonian flora for antiviral activity against the dengue 2 virus RNA-dependant RNA polymerase (DV-NS5 RdRp). The leaf extract of Dacrydium balansae, which strongly inhibited the DV-NS5, was submitted to bioguided fractionation. Four biflavonoids ( 1- 4), three sterols ( 5- 7), and two stilbene derivatives ( 8- 9) were identified and evaluated for their antiviral potential on the DV-NS5 RdRp. Biflavonoids appeared to be potent inhibitors of DV-NS5 RdRp with IC (50)s between 0.26 and 3.12 µM. Inhibitory activity evaluations against the RNA polymerase from other Flaviviridae viruses allowed us to conclude that these compounds are specific inhibitors of the DV RNA polymerase. The strongest inhibitions were observed with hinokiflavone ( 4), but podocarpusflavone A ( 2) is the strongest noncytotoxic inhibitor of the DV-NS5 and it also displayed polymerase inhibitory activity in a DV replicon. A preliminary structure-activity relationship study (SARs) revealed the necessity of the biflavonoid skeleton, the influence of number and position of methoxylations, and the importance of a free rotation of the linkage between the two apigenin monomers of the biflavonoids. To the best of our knowledge, podocarpusflavone A ( 2) is the strongest noncytotoxic non-nucleotide molecule exhibiting a specific inhibitory activity against the RNA polymerase domain of DV-NS5 and thus is promising for chemotherapy development against dengue fever.

A Versatile Class of 1,4,4-Trisubstituted Piperidines Block Coronavirus Replication In Vitro.

There is a clear need for novel antiviral concepts to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of 1,4,4-trisubstituted piperidines, we here conducted a detailed analysis of the structure-activity relationship of these structurally unique inhibitors. Despite the presence of five points of diversity, the synthesis of an extensive series of analogues was readily achieved by Ugi four-component reaction from commercially available reagents. After evaluating 63 analogues against human coronavirus 229E, four of the best molecules were selected and shown to have micromolar activity against SARS-CoV-2. Since the action point was situated post virus entry and lying at the stage of viral polyprotein processing and the start of RNA synthesis, enzymatic assays were performed with CoV proteins involved in these processes. While no inhibition was observed for SARS-CoV-2 nsp12-nsp7-nsp8 polymerase, nsp14 N7-methyltransferase and nsp16/nsp10 2'-O-methyltransferase, nor the nsp3 papain-like protease, the compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was quite modest, the plausibility of binding to the catalytic site of Mpro was established by in silico studies. Therefore, the 1,4,4-trisubstituted piperidines appear to represent a novel class of non-covalent CoV Mpro inhibitors that warrants further optimization and development.