Integration of Wnt-inhibitory activity and structural novelty scoring results to uncover novel bioactive natural products: new Bicyclo[3.3.1]non-3-ene-2,9-diones from the leaves of Hymenocardia punctata.

In natural products (NPs) research, methods for the efficient prioritization of natural extracts (NEs) are key for discovering novel bioactive NPs. In this study a biodiverse collection of 1,600 NEs, previously analyzed by UHPLC-HRMS2 metabolite profiling was screened for Wnt pathway regulation. The results of the biological screening drove the selection of a subset of 30 non-toxic NEs with an inhibitory IC50 ≤ 5 µg/mL. To increase the chance of finding structurally novel bioactive NPs, Inventa, a computational tool for automated scoring of NEs based on structural novelty was used to mine the HRMS2 analysis and dereplication results. After this, four out of the 30 bioactive NEs were shortlisted by this approach. The most promising sample was the ethyl acetate extract of the leaves of Hymenocardia punctata (Phyllanthaceae). Further phytochemical investigations of this species resulted in the isolation of three known prenylated flavones (3, 5, 7) and ten novel bicyclo[3.3.1]non-3-ene-2,9-diones (1, 2, 4, 6, 8-13), named Hymenotamayonins. Assessment of the Wnt inhibitory activity of these compounds revealed that two prenylated flavones and three novel bicyclic compounds showed interesting activity without apparent cytotoxicity. This study highlights the potential of combining Inventa's structural novelty scores with biological screening results to effectively discover novel bioactive NPs in large NE collections.

Comparative metabolomic study of fungal foliar endophytes and their long-lived host Astrocaryum sciophilum: a model for exploring the chemodiversity of host-microbe interactions.

Introduction: In contrast to the dynamics observed in plant/pathogen interactions, endophytic fungi have the capacity to establish enduring associations within their hosts, leading to the development of a mutually beneficial relationship that relies on specialized chemical interactions. Research indicates that the presence of endophytic fungi has the ability to significantly modify the chemical makeup of the host organism. Our hypothesis proposes the existence of a reciprocal exchange of chemical signals between plants and fungi, facilitated by specialized chemical processes that could potentially manifest within the tissues of the host. This research aimed to precisely quantify the portion of the cumulative fungal endophytic community's metabolome detectable within host leaves, and tentatively evaluate its relevance to the host-endophyte interplay. The understory palm Astrocaryum sciophilum (Miq.) Pulle was used as a interesting host plant because of its notable resilience and prolonged life cycle, in a tropical ecosystem. Method: Using advanced metabolome characterization, including UHPLC-HRMS/MS and molecular networking, the study explored enriched metabolomes of both host leaves and 15 endophytic fungi. The intention was to capture a metabolomic "snapshot" of both host and endophytic community, to achieve a thorough and detailed analysis. Results and discussion: This approach yielded an extended MS-based molecular network, integrating diverse metadata for identifying host- and endophyte-derived metabolites. The exploration of such data (>24000 features in positive ionization mode) enabled effective metabolome comparison, yielding insights into cultivable endophyte chemodiversity and occurrence of common metabolites between the holobiont and its fungal communities. Surprisingly, a minor subset of features overlapped between host leaf and fungal samples despite significant plant metabolome enrichment. This indicated that fungal metabolic signatures produced in vitro remain sparingly detectable in the leaf. Several classes of primary metabolites were possibly shared. Specific fungal metabolites and/or compounds of their chemical classes were only occasionally discernible in the leaf, highlighting endophytes partial contribution to the overall holobiont metabolome. To our knowledge, the metabolomic study of a plant host and its microbiome has rarely been performed in such a comprehensive manner. The general analytical strategy proposed in this paper seems well-adapted for any study in the field of microbial- or microbiome-related MS and can be applied to most host-microbe interactions.

Chiral Separation of Stilbene Dimers Generated by Biotransformation for Absolute Configuration Determination and Antibacterial Evaluation.

A series of complex stilbene dimers have been generated through biotransformation of resveratrol, pterostilbene, and the mixture of both using the enzymatic secretome of Botrytis cinerea Pers. The process starts with achiral molecules and results in the generation of complex molecules with multiple chiral carbons. So far, we have been studying these compounds in the form of enantiomeric mixtures. In the present study, we isolated the enantiomers to determine their absolute configuration and assess if the stereochemistry could impact their biological properties. Eight compounds were selected for this study, corresponding to the main scaffolds generated (pallidol, leachianol, restrytisol and acyclic dimers) and the most active compounds (trans-δ-viniferin derivatives) against a methicillin-resistant strain of Staphylococcus aureus (MRSA). To isolate these enantiomers and determine their absolute configuration, a chiral HPLC-PDA analysis was performed. The analysis was achieved on a high-performance liquid chromatography system equipped with a chiral column. For each compound, the corresponding enantiomeric pair was obtained with high purity. The absolute configuration of each enantiomer was determined by comparison of experimental and calculated electronic circular dichroism (ECD). The antibacterial activities of the four trans-δ-viniferin derivatives against two S. aureus strains were evaluated.

Chemoenzymatic Synthesis of Original Stilbene Dimers Possessing Wnt Inhibition Activity in Triple-Negative Breast Cancer Cells Using the Enzymatic Secretome of Botrytis cinerea Pers.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancer and triple-negative breast cancer (TNBC) in particular depend upon Wnt pathway overactivation. Despite this importance, no Wnt pathway-targeting drugs are currently available, which necessitates novel approaches to search for therapeutically relevant compounds targeting this oncogenic pathway. Stilbene analogs represent an under-explored field of therapeutic natural products research. In the present work, a library of complex stilbene derivatives was obtained through biotransformation of a mixture of resveratrol and pterostilbene using the enzymatic secretome of Botrytis cinerea. To improve the chemodiversity, the reactions were performed using i-PrOH, n-BuOH, i-BuOH, EtOH, or MeOH as cosolvents. Using this strategy, a series of 73 unusual derivatives was generated distributed among 6 scaffolds; 55 derivatives represent novel compounds. The structure of each compound isolated was determined by nuclear magnetic resonance and high-resolution mass spectrometry. The inhibitory activity of the isolated compounds against the oncogenic Wnt pathway was comprehensively quantified and correlated with their capacity to inhibit the growth of the cancer cells, leading to insights into structure-activity relationships of the derivatives. Finally, we have dissected mechanistic details of the stilbene derivatives activity within the pathway.

Metabolomics in Ecology and Bioactive Natural Products Discovery: Challenges and Prospects for a Comprehensive Study of the Specialised Metabolome.

Metabolomics is playing an increasingly prominent role in chemical ecology and in the discovery of bioactive natural products (NPs). The identification of metabolites is a common/central objective in both research fields. NPs have significant biological properties and play roles in multiple chemical-ecological interactions. Classically, in pharmacognosy, their chemical structure is determined after a complex process of isolating and interpreting spectroscopic data. With the advent of powerful analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) the annotation process of the specialised metabolome of plants and microorganisms has improved considerably. In this article, we summarise the possibilities opened by these advances and illustrate how we harnessed them in our own research to automate annotations of NPs and target the isolation of key compounds. In addition, we are also discussing the analytical and computational challenges associated with these emerging approaches and their perspective.

Characterization of Pseudomonas aeruginosa Quorum Sensing Inhibitors from the Endophyte Lasiodiplodia venezuelensis and Evaluation of Their Antivirulence Effects by Metabolomics.

The opportunistic pathogen Pseudomonas aeruginosa is one of the "critical priority pathogens" due to its multidrug resistance to a wide range of antibiotics. Its ability to invade and damage host tissues is due to the use of quorum sensing (QS) to collectively produce a plethora of virulence factors. Inhibition of QS is an attractive strategy for new antimicrobial agents because it disrupts the initial events of infection without killing the pathogen. Highly diverse microorganisms as endophytes represent an under-explored source of bioactive natural products, offering opportunities for the discovery of novel QS inhibitors (QSI). In the present work, the objective was to explore selective QSIs within a unique collection of fungal endophytes isolated from the tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted, and screened for their antibacterial and specific anti-QS activities against P. aeruginosa. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation for its selective activity, leading to the isolation of eight compounds in a single step. Among them, two pyran-derivatives were found to be responsible for the QSI activity, with an effect on some QS-regulated virulence factors. Additional non-targeted metabolomic studies on P. aeruginosa documented their effects on the production of various virulence-related metabolites.

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.

Hibiscus sabdariffa, a Treatment for Uncontrolled Hypertension. Pilot Comparative Intervention.

In Iraq, in 2019, there were about 1.4 million Internally Displaced Persons (IDP); medical treatments were often interrupted. The feasibility of using Hibiscus sabdariffa (HS) decoction to curb hypertension was evaluated. A multicentric comparative pilot intervention for 121 participants with high blood pressure (BP) (≥140/90 mmHg) was conducted. Participants of the intervention group (with or without conventional medication) received HS decoction on a dose regimen starting from 10 grams per day. BP was measured five times over six weeks. The major active substances were chemically quantified. Results: After 6 weeks, 61.8% of participants from the intervention group (n = 76) reached the target BP < 140/90 mmHg, compared to 6.7% in the control group (n = 45). In the intervention group, a mean (±SD) reduction of 23.1 (±11.8) mmHg and 12.0 (±11.2) for systolic and diastolic BP, respectively, was observed, while in the control group the reduction was 4.4 (±10.2)/3.6 (±8.7). The chemical analysis of the starting dose indicated a content of 36 mg of total anthocyanins and 2.13 g of hibiscus acid. The study shows the feasibility of using HS decoction in IDP's problematic framework, as hibiscus is a safe, local, affordable, and culturally accepted food product.

In Vitro Anti-Inflammatory Activity in Arthritic Synoviocytes of A. brachypoda Root Extracts and Its Unusual Dimeric Flavonoids.

Arrabidaea brachypoda is a plant commonly used for the treatment of kidney stones, arthritis and pain in traditional Brazilian medicine. Different in vitro and in vivo activities, ranging from antinociceptive to anti-Trypanosoma cruzi, have been reported for the dichloromethane root extract of Arrabidaea brachypoda (DCMAB) and isolated compounds. This work aimed to assess the in vitro anti-inflammatory activity in arthritic synoviocytes of the DCMAB, the hydroethanolic extract (HEAB) and three dimeric flavonoids isolated from the DCMAB. These compounds, brachydin A (1), B (2) and C (3), were isolated both by medium pressure liquid and high-speed counter current chromatography. Their quantification was performed by mass spectrometry on both DCMAB and HEAB. IL-1ß activated human fibroblast-like synoviocytes were incubated with both extracts and isolated compounds to determine the levels of pro-inflammatory cytokine IL-6 by enzyme-linked immunosorbent assay (ELISA). DCMAB inhibited 30% of IL-6 release at 25 µg/mL, when compared with controls while HEAB was inactive. IC50 values determined for 2 and 3 were 3-fold higher than 1. The DCMAB activity seems to be linked to higher proportions of compounds 2 and 3 in this extract. These observations could thus explain the traditional use of A. brachypoda roots in the treatment of osteoarthritis.

Phosphatidylcholines from Pieris brassicae eggs activate an immune response in Arabidopsis.

Recognition of conserved microbial molecules activates immune responses in plants, a process termed pattern-triggered immunity (PTI). Similarly, insect eggs trigger defenses that impede egg development or attract predators, but information on the nature of egg-associated elicitors is scarce. We performed an unbiased bioactivity-guided fractionation of eggs of the butterfly Pieris brassicae. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry of active fractions led to the identification of phosphatidylcholines (PCs). PCs are released from insect eggs, and they induce salicylic acid and H2O2 accumulation, defense gene expression and cell death in Arabidopsis, all of which constitute a hallmark of PTI. Active PCs contain primarily C16 to C18-fatty acyl chains with various levels of desaturation, suggesting a relatively broad ligand specificity of cell-surface receptor(s). The finding of PCs as egg-associated molecular patterns (EAMPs) illustrates the acute ability of plants to detect conserved immunogenic patterns from their enemies, even from seemingly passive structures such as eggs.

Phytochemical and Biological Investigation of Helianthemum nummularium, a High-Altitude Growing Alpine Plant Overrepresented in Ungulates Diets.

Helianthemum nummularium is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that H. nummularium from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: p-hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulate's preference for this plant and possible use of it for self-medication.

A Cytotoxic Porphyrin from North Pacific Brittle Star Ophiura sarsii.

Triple-negative breast cancer (TNBC) represents the deadliest form of gynecological tumors currently lacking targeted therapies. The ethanol extract of the North Pacific brittle star Ophiura sarsii presented promising anti-TNBC activities. After elimination of the inert material, the active extract was submitted to a bioguided isolation approach using high-resolution semipreparative HPLC-UV, resulting in one-step isolation of an unusual porphyrin derivative possessing strong cytotoxic activity. HRMS and 2D NMR resulted in the structure elucidation of the compound as (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid. Never identified before in Ophiuroidea, porphyrins have found broad applications as photosensitizers in the anticancer photodynamic therapy. The simple isolation of a cytotoxic porphyrin from an abundant brittle star species we describe here may pave the way for novel natural-based developments of targeted anti-cancer therapies.

Chemical Constituents of Anacardium occidentale as Inhibitors of Trypanosoma cruzi Sirtuins.

Benznidazole and nifurtimox, the only drugs available for the treatment of Chagas disease, have limited efficacy and have been associated with severe adverse side effects. Thus, there is an urgent need to find new biotargets for the identification of novel bioactive compounds against the parasite and with low toxicity. Silent information regulator 2 (Sir2) enzymes, or sirtuins, have emerged as attractive targets for the development of novel antitrypanosomatid agents. In the present work, we evaluated the inhibitory effect of natural compounds isolated from cashew nut (Anacardium occidentale, L. Anacardiaceae) against the target enzymes TcSir2rp1 and TcSir2rp3 as well as the parasite. Two derivates of cardol (1, 2), cardanol (3, 4), and anacardic acid (5, 6) were investigated. The two anacardic acids (5, 6) inhibited both TcSir2rp1 and TcSir2rp3, while the cardol compound (2) inhibited only TcSir2rp1. The most potent sirtuin inhibitor active against the parasite was the cardol compound (2), with an EC50 value of 12.25 µM, similar to that of benznidazole. Additionally, compounds (1, 4), which were inactive against the sirtuin targets, presented anti-T. cruzi effects. In conclusion, our results showed the potential of Anacardium occidentale compounds for the development of potential sirtuin inhibitors and anti-Trypanosoma cruzi agents.

Antileishmanial Activity of Dimeric Flavonoids Isolated from Arrabidaea brachypoda.

Leishmaniasis are diseases caused by parasites belonging to Leishmania genus. The treatment with pentavalent antimonials present high toxicity. Secondary line drugs, such as amphotericin B and miltefosine also have a narrow therapeutic index. Therefore, there is an urgent need to develop new drugs to treat leishmaniasis. Here, we present the in vitro anti-leishmanial activity of unusual dimeric flavonoids purified from Arrabidaea brachypoda. Three compounds were tested against Leishmana sp. Compound 2 was the most active against promastigotes. Quantifying the in vitro infected macrophages revealed that compound 2 was also the most active against intracellular amastigotes of L. amazonensis, without displaying host cell toxicity. Drug combinations presented an additive effect, suggesting the absence of interaction between amphotericin B and compound 2. Amastigotes treated with compound 2 demonstrated alterations in the Golgi and accumulation of vesicles inside the flagellar pocket. Compound 2-treated amastigotes presented a high accumulation of cytoplasmic vesicles and a myelin-like structure. When administered in L. amazonensis-infected mice, neither the oral nor the topical treatments were effective against the parasite. Based on the high in vitro activity, dimeric flavonoids can be used as a lead structure for the development of new molecules that could be useful for structure-active studies against Leishmania.

The plant pathogen Pseudomonas aeruginosa triggers a DELLA-dependent seed germination arrest in Arabidopsis.

To anticipate potential seedling damage, plants block seed germination under unfavorable conditions. Previous studies investigated how seed germination is controlled in response to abiotic stresses through gibberellic and abscisic acid signaling. However, little is known about whether seeds respond to rhizosphere bacterial pathogens. We found that Arabidopsis seed germination is blocked in the vicinity of the plant pathogen Pseudomonas aeruginosa. We identified L-2-amino-4-methoxy-trans-3-butenoic acid (AMB), released by P. aeruginosa, as a biotic compound triggering germination arrest. We provide genetic evidence that in AMB-treated seeds DELLA factors promote the accumulation of the germination repressor ABI5 in a GA-independent manner. AMB production is controlled by the quorum sensing system IQS. In vitro experiments show that the AMB-dependent germination arrest protects seedlings from damage induced by AMB. We discuss the possibility that this could serve as a protective response to avoid severe seedling damage induced by AMB and exposure to a pathogen.

Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC) Bureau.

Arrabidaea brachypoda (DC) Bureau is a medicinal plant found in Brazil. Known as "cipó-una", it is popularly used as a natural therapeutic agent against pain and inflammation. This study evaluated the chemical composition and antinociceptive activity of the dichloromethane fraction from the roots of A. brachypoda (DEAB) and its mechanism of action. The chemical composition was characterized by high-performance liquid chromatography, and this fraction is composed only of dimeric flavonoids. The antinociceptive effect was evaluated in formalin and hot plate tests after oral administration (10-100 mg/kg) in male Swiss mice. We also investigated the involvement of TRPV1 (transient receptor potential vanilloid 1), TRPA1 (transient receptor potential ankyrin 1), TRPM8 (transient receptor potential melastatin 8), and ASIC (acid-sensing ion channel), as well as the opioidergic, glutamatergic, and supraspinal pathways. Moreover, the nociceptive response was reduced (30 mg/kg) in the early and late phase of the formalin test. DEAB activity appears to involve the opioid system, TRPM8, and ASIC receptors, clearly showing that the DEAB alleviates acute pain in mice and suggesting the involvement of the TRPM8 and ASIC receptors and the opioid system in acute pain relief.

Cell-based bioreporter assay coupled to HPLC micro-fractionation in the evaluation of antimicrobial properties of the basidiomycete fungus Pycnoporus cinnabarinus.

CONTEXT: Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns. OBJECTIVE: This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling. MATERIALS AND METHODS: A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation. RESULTS: Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone. DISCUSSION AND CONCLUSION: HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.

Biomimetic synthesis of Tramadol.

Tramadol has recently been isolated from the roots and bark of Nauclea latifolia. A plausible biosynthetic pathway has been proposed and the product-precursor relationship has been probed by (13)C position-specific isotope analysis. By further exploring this pathway, we demonstrate that a key step of the proposed pathway can be achieved using mild conditions that mimic in vivo catalysis.