Antiviral miliusanes and isolation of an unprecedented miliusane dimer from Miliusa balansae.

In an extensive screening endeavor for anti-coronaviral compounds, we examined 824 tropical plant extracts from the Annonaceae and Rutaceae families. The screening identified an ethyl acetate extract from the aerial parts of Miliusa balansae for its potent inhibitory activity against Human coronavirus HCoV-229E. Subsequent bioassay-guided fractionation of this extract revealed two unreported miliusanes including a complex dimeric structure and seven known compounds, comprising miliusane XXXVI, (+)-miliusol, bistyryls, styryl-pyranones, and the flavonoid rhamnetin. The absolute configuration of the new dimeric miliusane was determined by X-ray crystallography and a putative biogenetic origin was proposed. Investigation of the antiviral effect of these nine phytochemicals within HCoV-229E-infected Huh-7 cells showed that (+)-miliusol and miliusane XXXVI exert antiviral activity at non-cytotoxic concentrations, with IC50 values of 1.15 µM and 19.20 µM, respectively. Furthermore, these compounds significantly inhibited SARS-CoV-2 infection in Vero cells, presenting IC50 values of 11.31 µM for (+)-miliusol and 17.92 µM for miliusane XXXVI. Additionally, both compounds exhibited a potent antiviral effect against the emergent mosquito-borne Zika virus, with IC50 values of 1.34 µM and 23.45 µM, respectively. Time-of-addition assays suggest that their mechanism of action might target later stages of the viral cycle, indicating potential modulation of specific cellular pathways. These findings reinforce the invaluable contribution of medicinal flora as reservoirs of natural antiviral agents and emphasize their prospective role in combatting viruses of medical interest.

New Phenolic Lipids from the Leaves of Clausena harmandiana Inhibit SARS-CoV-2 Entry into Host Cells.

Induced by the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic underlined the clear need for antivirals against coronaviruses. In an effort to identify new inhibitors of SARS-CoV-2, a screening of 824 extracts prepared from various parts of 400 plant species belonging to the Rutaceae and Annonaceae families was conducted using a cell-based HCoV-229E inhibition assay. Due to its significant activity, the ethyl acetate extract of the leaves of Clausena harmandiana was selected for further chemical and biological investigations. Mass spectrometry-guided fractionation afforded three undescribed phenolic lipids (1-3), whose structures were determined via spectroscopic analysis. The absolute configurations of 1 and 2 were determined by analyzing Mosher ester derivatives. The antiviral activity against SARS-CoV-2 was subsequently shown, with IC50 values of 0.20 and 0.05 µM for 2 and 3, respectively. The mechanism of action was further assessed, showing that both 2 and 3 are inhibitors of coronavirus entry by acting directly on the viral particle. Phenolic lipids from Clausena harmandiana might be a source of new antiviral agents against human coronaviruses.

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

In vitro evaluation of NA1-115-7-loaded nanoemulsions, an MCL-1-specific inhibitor of natural origin, intended to treat B-cell lymphoproliferative disorders after oral administration.

MCL-1, an anti-apoptotic member of the BCL-2 protein family, is overexpressed in many types of cancer and contributes to chemotherapy resistance. The drimane derivative NA1-115-7 is a natural compound isolated from Zygogynum pancheri that can be considered as a very promising lead for treating MCL-1-dependent hematological malignancies. As this drug suffers from low stability in acidic conditions and poor aqueous solubility, we evaluated the potential oral use of NA1-115-7 by encapsulating it in lipid nanoemulsions (NA-NEs) prepared by spontaneous emulsification. NA-NEs showed a particle size of 41.9 ± 2.2 nm, PDI of 0.131 ± 0.016, zeta potential of -5.8 ± 3.4 mV, encapsulation efficiency of approximately 100 % at a concentration of 24 mM. The stability of NA-1-115-7 was sixfold higher than that of the unencapsulated drug in simulated gastric fluid. NA-NEs significantly restored apoptosis and halved the effective doses of NA1-115-7 on BL2, a Burkitt lymphoma cell line, without toxicity in normal cells. Such a drug-delivery system appears to be particularly interesting for the oral administration of NA1-115-7, as it improves its solubility and stability, as well as efficacy, by reducing the therapeutic dose, making it possible to further consider in-vivo studies of this promising drug in BL2 xenografted mice.

Antiviral Effect of Stenocline ericoides DC. and Stenocline inuloides DC., Two Flavonoid-Rich Endemic Plants from Madagascar, against Dengue and Zika Viruses.

Dengue and Zika viruses are identified as the most medically important arthropod-borne viral pathogens. Over the past 20 years, the global dengue incidence has dramatically increased with epidemics of severe dengue where the case fatality rate can reach up to 20% in untreated patients. The association between Zika virus infection and severe congenital anomalies was first reported in 2015. Today no specific antiviral therapies are available for dengue and Zika virus infections, accentuating the need of adapted antiviral strategies based on medicinal plant drug discovery. Plants are a potential source of antiviral phytocompounds which act primarily by blocking virus entry in the host-cell. In the present study, we evaluated whether crude extracts from Stenocline ericoides DC. and Stenocline inuloides DC., two endemic plants from Madagascar, may have antiviral effects against dengue and Zika viruses. We showed that S. ericoides has virucidal action whereas S. inuloides inhibits the early steps of virus infection with a non-cytotoxic effect in human cells. The administration of S. ericoides and S. inuloides extracts in zebrafish had no effect on the behavior of animals at the active doses against dengue and Zika viruses, suggesting the absence of adverse effects at these doses. LC-HRMS2 and molecular networking analyses revealed the richness of these two plants in polyphenols and flavonoid with the presence of clusters of phytocompounds specific to each Stenocline species. Consequently, S. ericoides and S. inuloides represent potential sources for natural and safe antiviral phytocompounds against flaviviruses of medical concern.

Synthesis and Anti-Leishmanial Properties of Quinolones Derived from Zanthosimuline.

Quinoline derivatives and especially quinolones are considered as privileged structures in medicinal chemistry and are often associated with various biological properties. We recently isolated a series of original monoterpenyl quinolones from the bark of Codiaeum peltatum. As this extract was found to have a significant inhibitory activity against a Leishmania species, we decided to study the anti-leishmanial potential of this type of compound. Leishmaniasis is a serious health problem affecting more than 12 million people in the world. Available drugs cause harmful side effects and resistance for some of them. With the aim of finding anti-leishmanial compounds, we developed a synthetic strategy to access natural quinolones and analogues derived from zanthosimuline. We showed the versatility of this natural compound toward cyclization conditions, leading to various polycyclic quinolone-derived structures. The natural and synthetic compounds were evaluated against amastigote forms of Leishmania infantum. The results obtained confirmed the interest of this family of natural compounds but also revealed promising activities for some intermediates deriving from zanthosimuline. Following the same synthetic strategy, we then prepared 14 new analogues. In this work, we identified two promising molecules with good activities against intramacrophage L. infantum amastigotes without any cytotoxicity. We also showed that slight changes in amide functional groups affect drastically their anti-parasitic activity.

NA1-115-7, from Zygogynum pancheri, is a new selective MCL-1 inhibitor inducing the apoptosis of hematological cancer cells but non-toxic to normal blood cells or cardiomyocytes.

The overexpression of antiapoptotic members (BCL-2, BCL-xL, MCL-1, etc.) of the BCL-2 family contributes to tumor development and resistance to chemotherapy or radiotherapy. Synthetic inhibitors targeting these proteins have been developed, and some hematological malignancies are now widely treated with a BCL-2 inhibitor (venetoclax). However, acquired resistance to venetoclax or chemotherapy drugs due to an upregulation of MCL-1 has been observed, rendering MCL-1 an attractive new target for treatment. Six MCL-1 inhibitors (S64315, AZD-5991, AMG-176, AMG-397, ABBV-467 and PRT1419) have been evaluated in clinical trials since 2016, but some were affected by safety issues and none are currently used clinically. There is, therefore, still a need for alternative molecules. We previously described two drimane derivatives as the first covalent BH3 mimetics targeting MCL-1. Here, we described the characterization and biological efficacy of one of these compounds (NA1-115-7), isolated from Zygogynum pancheri, a plant belonging to the Winteraceae family. NA1-115-7 specifically induced the apoptosis of MCL-1-dependent tumor cells, with two hours of treatment sufficient to trigger cell death. The treatment of lymphoma cells with NA1-115-7 stabilized MCL-1, disrupted its interactions with BAK, and rapidly induced apoptosis through a BAK- and BAX-mediated process. Importantly, a similar treatment with NA1-115-7 was not toxic to erythrocytes, peripheral blood mononuclear cells, platelets, or cardiomyocytes. These results highlight the potential of natural products for use as specific BH3 mimetics non-toxic to normal cells, and they suggest that NA1-115-7 may be a promising tool for use in cancer treatment.

Alkyl-Resorcinol Derivatives as Inhibitors of GDP-Mannose Pyrophosphorylase with Antileishmanial Activities.

Leishmaniasis is a vector-borne disease caused by the protozoan parasite Leishmania found in tropical and sub-tropical areas, affecting 12 million people around the world. Only few treatments are available against this disease and all of them present issues of toxicity and/or resistance. In this context, the development of new antileishmanial drugs specifically directed against a therapeutic target appears to be a promising strategy. The GDP-Mannose Pyrophosphorylase (GDP-MP) has been previously shown to be an attractive therapeutic target in Leishmania. In this study, a chemical library of 5000 compounds was screened on both L. infantum (LiGDP-MP) and human (hGDP-MP) GDP-MPs. From this screening, oncostemonol D was found to be active on both GDP-MPs at the micromolar level. Ten alkyl-resorcinol derivatives, of which oncostemonols E and J (2 and 3) were described for the first time from nature, were then evaluated on both enzymes as well as on L. infantum axenic and intramacrophage amastigotes. From this evaluation, compounds 1 and 3 inhibited both GDP-MPs at the micromolar level, and compound 9 displayed a three-times lower IC50 on LiGDP-MP, at 11 µM, than on hGDP-MP. As they displayed mild activities on the parasite, these compounds need to be further pharmacomodulated in order to improve their affinity and specificity to the target as well as their antileishmanial activity.

Drimane Derivatives as the First Examples of Covalent BH3 Mimetics that Target MCL-1.

Drimane sesquiterpenoid dialdehydes are natural compounds with antiproliferative properties. Nevertheless, their mode of action has not yet been discovered. Herein, we demonstrate that various drimanes are potent inhibitors of MCL-1 and BCL-xL, two proteins of the BCL-2 family that are overexpressed in various cancers, including lymphoid malignancies. Subtle changes in their structure significantly modified their activity on the target proteins. The two most active compounds are MCL-1 selective and bind in the BH3 binding groove of the protein. Complementary studies by NMR spectroscopy and mass spectrometry analyses, but also synthesis, showed that they covalently inhibit MCL-1 though the formation of a pyrrole adduct. In addition, cytotoxic assays revealed that these two compounds show a cytotoxic selectivity for BL2, a MCL-1/BCL-xL-dependent cell line and induce apoptosis.

Isolation of Picrotoxanes from Austrobuxus carunculatus Using Taxonomy-Based Molecular Networking.

A unique collection of 292 extracts from 107 New Caledonian Euphorbiaceae species sensu lato was profiled by LC-MS2 and the metabolite content organized by molecular networking. Based on the assumption that taxon-specific molecules are more likely to be structurally novel, taxonomic data were mapped on spectral networks to detect genus-specific clusters. Using this approach, a group of compounds unique to the genus Austrobuxus was highlighted. The subsequent MS-guided purification of the fruit EtOAc extract of Austrobuxus carunculatus led to the isolation of 13 new monolactone and "norditerpene" picrotoxanes (2-14), along with the known tutin (1). The structures of the new compounds were elucidated by HRESIMS and NMR spectroscopic data analysis, and the absolute configurations of compounds 1, 3, 7, 11, 12, and 14 were determined by single-crystal X-ray diffraction analysis. The relative and absolute configurations of compounds 4 and 5 were ascertained by chemical transformation of compound 3. The absolute configurations of other members of the series have been proposed on the basis of biogenetic considerations and specific rotation values of similar sign and magnitude. Compounds 1-14 were evaluated for their antiproliferative activities against HCT116 colon, U87-MG glioblastoma, and A549 lung human cancer cell lines. Compounds bearing an acyl chain at C-2 (i.e., 2, 4, and 13) showed IC50 values in the micromolar range for the three cell lines used.

Anti-diabetic and lipid-lowering effects of drimane sesquiterpenoids isolated from Zygogynum pancheri.

Recently, it has been shown that drimane-type sesquiterpenoids isolated from Zygogynum pancheri, a species native to New Caledonia, possessed significant α-amylase inhibitory activities. To further explore their antidiabetic potential, we investigated the effect of 1ß-O-(E-cinnamoyl)-6α-hydroxy-9epi-polygodial (D) and 1ß-E-O-p-methoxycinnamoyl-bemadienolide (L), two of the most active compounds of the series, on diabetic model rats. Compounds D and L (2 mg kg/day) were daily and orally administrated for 30 days to streptozotocin (STZ) (150 mg/kg) induced male diabetic Wistar rats. Animals were allocated into five groups of six rats. Comparatively to diabetic rats, treatments with D and L compounds were able to significantly (P < 0.05) decrease Fasting Blood Glucose (FBG) (70.15%, 71.02%), serum total cholesterol (46.27% and 39.38%), triglycerides (56.60% and 58.15%), creatinine (37.31% and 36.49%) and uric acid levels (67.76% and 69.68%), respectively. Compounds D and L also restored the altered plasma enzyme (aspartate aminotransferase, AST (47.83% and 43.20%), alanine aminotransferase, ALT (49.76% and 48.35%, alkaline phosphatase, ALP (72.78% and 73.21%)) and lactate dehydrogenase, LDH (47.95% and 53.93%) levels to near normal, respectively. Administration of Glymepiride, significantly (p < 0.05) reduced FBG (73.94%) in STZ induced diabetic rats. Additionally, the compounds D and L exhibited inhibitory effects in vivo on lipase activity of diabetic rats (54.83% and 52.25%), respectively. The outcomes of this study suggested that these two drimanes could be considered as efficient hypoglycemic, hypolipidemic and antiobesity agents for diabetes management and its complications.

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.

N-myristoyltransferases inhibitory activity of ellagitannins from Terminalia bentzoë (L.) L. f. subsp. bentzoë.

N-myristoylation (Myr) is an eukaryotic N-terminal co- or post-translational protein modification in which the enzyme N-myristoyltransferase (NMT) transfers a fatty acid (C14:0) to the N-terminal glycine residues of several cellular key proteins. Depending on the cellular context, NMT may serve as a molecular target in anticancer or anti-infectious therapy, and drugs that inhibit this enzyme may be useful in the treatment of cancer or infectious diseases. As part of an on-going project to identify natural Homo sapiens N-myristoyltransferase 1 inhibitors (HsNMT1), two ellagitannins, punicalagin (1) and isoterchebulin (2), along with eschweilenol C (3) and ellagic acid (4) were isolated from the bark of Terminalia bentzoë (L.) L. f. subsp. bentzoë. Their structures were determined by means of spectroscopic analyses and comparison with literature data. Punicalagin (1) and isoterchebulin (2) showed significant inhibitory activity towards HsNMT1, and also against Plasmodium falciparum NMT (PfNMT) both in vitro and in cellulo, opening alternative paths for new NMT inhibitors development. This is the first report identifying natural products from a botanical source as inhibitors of HsNMT and PfNMT.

Bioactive Natural Products Prioritization Using Massive Multi-informational Molecular Networks.

Natural products represent an inexhaustible source of novel therapeutic agents. Their complex and constrained three-dimensional structures endow these molecules with exceptional biological properties, thereby giving them a major role in drug discovery programs. However, the search for new bioactive metabolites is hampered by the chemical complexity of the biological matrices in which they are found. The purification of single constituents from such matrices requires such a significant amount of work that it should be ideally performed only on molecules of high potential value (i.e., chemical novelty and biological activity). Recent bioinformatics approaches based on mass spectrometry metabolite profiling methods are beginning to address the complex task of compound identification within complex mixtures. However, in parallel to these developments, methods providing information on the bioactivity potential of natural products prior to their isolation are still lacking and are of key interest to target the isolation of valuable natural products only. In the present investigation, we propose an integrated analysis strategy for bioactive natural products prioritization. Our approach uses massive molecular networks embedding various informational layers (bioactivity and taxonomical data) to highlight potentially bioactive scaffolds within the chemical diversity of crude extracts collections. We exemplify this workflow by targeting the isolation of predicted active and nonactive metabolites from two botanical sources (Bocquillonia nervosa and Neoguillauminia cleopatra) against two biological targets (Wnt signaling pathway and chikungunya virus replication). Eventually, the detection and isolation processes of a daphnane diterpene orthoester and four 12-deoxyphorbols inhibiting the Wnt signaling pathway and exhibiting potent antiviral activities against the CHIKV virus are detailed. Combined with efficient metabolite annotation tools, this bioactive natural products prioritization pipeline proves to be efficient. Implementation of this approach in drug discovery programs based on natural extract screening should speed up and rationalize the isolation of bioactive natural products.

Endiandric acid analogues from Beilschmiedia ferruginea as dual inhibitors of Bcl-xL/Bak and Mcl-1/Bid interactions.

A rapid screening by (1)H and (1)H-(13)C HSQC NMR spectroscopy of EtOAc extracts of Endiandra and Beilschmiedia species allowed the selection of Beilschmiedia ferruginea leaves and flowers extract for a chemical investigation, leading to the isolation of 11 new tetracyclic endiandric acid analogues, named ferrugineic acids A-K (1-11). Their structures were determined by 1D and 2D NMR spectroscopic analysis in combination with HRMS data. These compounds were assayed for Bcl-xL and Mcl-1 binding affinities. Ferrugineic acids B, C, and J (2, 3, and 10) exhibited significant binding affinity for both antiapoptotic proteins Bcl-xL (Ki = 19.2, 12.6, and 19.4 µM, respectively) and Mcl-1 (Ki = 14.0, 13.0, and 5.2 µM, respectively), and ferrugineic acid D (4) showed only significant inhibiting activity for Mcl-1 (Ki = 5.9 µM).

Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/cyclin B and DYRK1A.

In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

Antiplasmodial, antitrypanosomal, and cytotoxic activities of prenylated flavonoids isolated from the stem bark of Artocarpus styracifolius.

In continuation of our efforts to find new antimalarial drugs, a systematic IN VITRO evaluation using a chloroquine resistant strain of PLASMODIUM FALCIPARUM (FcB1) was undertaken on extracts prepared from various parts of Vietnamese plants. The ethyl acetate extract obtained from the stem bark of ARTOCARPUS STYRACIFOLIUS (Moraceae) exhibited strong antiplasmodial activity (87 % at 10 µg/mL) whereas weak cytotoxicity was observed in a human fibroblast cell line (MRC-5). Phytochemical investigation of this extract led to isolation of two new prenylated flavonoids, styracifolins A and B ( 1 and 2), as well as the known artoheterophyllin A ( 3) and B ( 4), artonins A ( 5), B ( 6), and F ( 7), and heterophyllin ( 8). Structures of 1 and 2 were elucidated by spectroscopic methods and through comparison with data reported in the literature. Compounds 1- 8 exhibited antiplasmodial activities with IC (50) values ranging from 1.1 µM to 13.7 µM, and compounds 1, 2, 6, and 8 showed significant antitrypanosomal activities.

Cytotoxic sesquiterpenoids from Winteraceae of Caledonian rainforest.

One secobutanolide, two butanolides and six drimane sesquiterpenoids were isolated from the bark and leaves of Zygogynum pancheri and Zygogynum acsmithii (Winteraceae) along with six known drimanes, isodrimanial, 1beta-O-p-methoxy-E-cinnamoyl-bemadienolide, 7-ketoisodrimenin, drimenin, polygodial and 1beta-E-cinnamoyl-6alpha-hydroxypolygodial. Their structures were elucidated through analysis of spectroscopic data. Drimane sesquiterpenoids with a dialdehyde function exhibited significant inhibitory activities in the in vitro cytotoxic assays against KB, HL60 and HCT116 cancer cell lines.