The Natural Stilbenoid (-)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9.

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (-)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.

Evaluation of natural resveratrol multimers as marine antifoulants.

In the current study we investigate the antifouling potential of three polyphenolic resveratrol multimers (-)-hopeaphenol, vaticanol B and vatalbinoside A, isolated from two species of Anisoptera found in the Papua New Guinean rainforest. The compounds were evaluated against the growth and settlement of eight marine microfoulers and against the settlement and metamorphosis of Amphibalanus improvisus barnacle cyprids. The two isomeric compounds (-)-hopeaphenol and vaticanol B displayed a high inhibitory potential against the cyprid larvae metamorphosis at 2.8 and 1.1 µM. (-)-Hopeaphenol was also shown to be a strong inhibitor of both microalgal and bacterial adhesion at submicromolar concentrations with low toxicity. Resveratrol displayed a lower antifouling activity compared to the multimers and had higher off target toxicity against MCR-5 fibroblasts. This study illustrates the potential of natural products as a valuable source for the discovery of novel antifouling leads with low toxicity.

The Natural Stilbenoid (-)-Hopeaphenol Inhibits Cellular Entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7, and B.1.351 Variants.

Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host angiotensin-converting enzyme II (ACE2) receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here, we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (-)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (-)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 µM, in contrast to an IC50 of 28.3 µM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index, 257.3). When assessed against the USA-WA1/2020 variant, (-)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect and yield reduction assays (50% effective concentrations [EC50s], 10.2 to 23.4 µM) without cytotoxicity and approaching the activities of the control antiviral remdesivir (EC50s, 1.0 to 7.3 µM). Notably, (-)-hopeaphenol also inhibited two emerging variants of concern, B.1.1.7/Alpha and B.1.351/Beta in both viral and spike-containing pseudovirus assays with similar or improved activities over the USA-WA1/2020 variant. These results identify (-)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants of concern.

Identification of Fromiamycalin and Halaminol A from Australian Marine Sponge Extracts with Anthelmintic Activity against Haemonchus contortus.

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus-a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC50) = 26.6 ± 0.74 µM) and L4 motility (IC50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.

Synthesis and antimalarial evaluation of amide and urea derivatives based on the thiaplakortone A natural product scaffold.

A series of amide (8­32, 40­45) and urea (33, 34, 36­39) analogues based on the thiaplakortone A natural product scaffold were synthesised and screened for in vitro antimalarial activity against chloroquine-sensitive (3D7) and chloroquine- and mefloquine-resistant (Dd2) Plasmodium falciparum parasite lines. Several analogues displayed potent inhibition of P. falciparum growth (IC50 <500 nM) and good selectivity for P. falciparum versus human neonatal foreskin fibroblast cells (selectivity index >100). Two of these compounds, 8 and 33, exhibited good aqueous solubility and metabolic stability, and when administered subcutaneously to mice (32 mg kg(-1)), plasma concentrations remained above 0.2 µM for at least 8 h. Both 8 and 33 were well tolerated in mice after subcutaneous administration of 32 mg kg(-1) twice daily for 4 days. Using this regimen blood stage P. berghei was suppressed by 52% for 8 and 26% for 33, relative to the vehicle control.

Isolation, structure determination and cytotoxicity studies of tryptophan alkaloids from an Australian marine sponge Hyrtios sp.

Mass-guided fractionation of the MeOH extract from a specimen of the Australian marine sponge Hyrtios sp. resulted in the isolation of two new tryptophan alkaloids, 6-oxofascaplysin (2), and secofascaplysic acid (3), in addition to the known metabolites fascaplysin (1) and reticulatate (4). The structures of all molecules were determined following NMR and MS data analysis. Structural ambiguities in 2 were addressed through comparison of experimental and DFT-generated theoretical NMR spectral values. Compounds 1-4 were evaluated for their cytotoxicity against a prostate cancer cell line (LNCaP) and were shown to display IC50 values ranging from 0.54 to 44.9 µM.

Solving the supply of resveratrol tetramers from Papua New Guinean rainforest anisoptera species that inhibit bacterial type III secretion systems.

The supply of (-)-hopeaphenol (1) was achieved via enzymatic biotransformation in order to provide material for preclinical investigation. High-throughput screening of a prefractionated natural product library aimed to identify compounds that inhibit the bacterial virulence type III secretion system (T3SS) identified several fractions derived from two Papua New Guinean Anisoptera species, showing activity against Yersinia pseudotuberculosis outer proteins E and H (YopE and YopH). Bioassay-directed isolation from the leaves of A. thurifera, and similarly A. polyandra, resulted in three known resveratrol tetramers, (-)-hopeaphenol (1), vatalbinoside A (2), and vaticanol B (3). Compounds 1-3 displayed IC50 values of 8.8, 12.5, and 9.9 µM in a luminescent reporter-gene assay (YopE) and IC50 values of 2.9, 4.5, and 3.3 µM in an enzyme-based YopH assay, respectively, which suggested that they could potentially act against the T3SS in Yersinia. The structures of 1-3 were confirmed through a combination of spectrometric, chemical methods, and single-crystal X-ray structure determinations of the natural product 1 and the permethyl ether analogue of 3. The enzymatic hydrolysis of the ß-glycoside 2 to the aglycone 1 was achieved through biotransformation using the endogenous leaf enzymes. This significantly enhanced the yield of the target bioactive natural product from 0.08% to 1.3% and facilitates ADMET studies of (-)-hopeaphenol (1).

(3R,4S,5S,8S,10R,13R)-3-Hy-droxy-kaura-9(11),16-dien-18-oic acid.

The title compound, C(20)H(28)O(3), was isolated during our investigation into the chemical composition and pharmacological activity of Centipeda cunninghamii (DC.) A. Braun & Asch. (Asteraceae). The enanti-opure compound, a diterpene with a carbon skeleton, is composed of three six- and one five-membered rings in chair, twist-boat, half-chair and envelope conformations, respectively. Each mol-ecule makes one intra- and one inter-molecular O-H⋯O hydrogen bond in the crystal lattice, forming hydrogen-bonded chains along [010]. The absolute configuration of the compound was assigned on the basis of optical rotation measurements.

(2S,3S)-sulfated pterosin C, a cytotoxic sesquiterpene from the Bangladeshi mangrove fern Acrostichum aureum.

Two new sesquiterpenes, (2R,3S)-sulfated pterosin C (1) and (2S,3S)-sulfated pterosin C (2), along with two known derivatives, (2S,3S)-pterosin C and (2R)-pterosin P, were isolated from a methanolic extract of the aerial parts of Acrostichum aureum. The structures of 1 and 2 were determined by the interpretation of their spectroscopic data. The isolated pterosins were evaluated for their cytotoxic activity against the AGS, HT-29, MDA-MB-231, and MCF-7 human cancer cell lines and the NIH3T3 normal mouse fibroblast cell line, using the MTT assay. Compound 2 showed IC50 values in the range 23.9-68.8 µM. The lowest IC50 value (23.9 µM) was recorded against AGS gastric adenocarcinoma cells. Compound 2 was found to exert an apoptotic effect on AGS cells within 24 h of treatment, which increased with time and was greater than the positive control, cycloheximide. The cytotoxicity of 2 seems to be due in part to the sulfate group on C-14 and the configuration at C-2.

Ethanolic and aqueous extracts derived from Australian fungi inhibit cancer cell growth in vitro.

Fifteen Australian macrofungi were investigated for cytotoxic activity. Ethanol, cold and hot water extracts of each species were screened for cytotoxic activity against normal mouse fibroblast cells (NIH/3T3), healthy human epithelial kidney cells (HEK-293), four cancer cell lines, gastric adenocarcinoma cells (AGS), two mammary gland adenocarcinoma cells (MDA-MB-231, MCF7) and colorectal adenocarcinoma cells (HT-29) with a validated MTT assay. Most extracts derived from Omphalotus nidiformis, Cordyceps cranstounii and Cordyceps gunnii demonstrated significant cytotoxic activity toward a variety of cancer cell lines. In contrast only some extracts from Coprinus comatus, Cordyceps hawkesii, Hypholoma fasciculare, Lepista nuda, Leratiomyces ceres and Ophiocordyceps robertsii displayed significant cytotoxic activity, which was usually selective for only one or two cancer cell lines tested. The least cytotoxic species evaluated in this study were Agaricus bitorquis, Coprinopsis atrametaria, Psathyrella asperospora, Russula clelandii, Tricholoma sp. AU2 and Xerula mundroola.

Triangulation of methods using insect cell lines to investigate insecticidal mode-of-action.

BACKGROUND: This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. RESULTS: We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. CONCLUSION: This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

Type III secretion inhibitors for the management of bacterial plant diseases.

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds-including molecules effective against human pathogens-for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down-regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases.

Medicinal Plants of the Australian Aboriginal Dharawal People Exhibiting Anti-Inflammatory Activity.

Chronic inflammation contributes to multiple ageing-related musculoskeletal and neurodegenerative diseases, cardiovascular diseases, asthma, rheumatoid arthritis, and inflammatory bowel disease. More recently, chronic neuroinflammation has been attributed to Parkinson's and Alzheimer's disease and autism-spectrum and obsessive-compulsive disorders. To date, pharmacotherapy of inflammatory conditions is based mainly on nonsteroidal anti-inflammatory drugs which in contrast to cytokine-suppressive anti-inflammatory drugs do not influence the production of cytokines such as tumour necrosis factor-α or nitric oxide. However, their prolonged use can cause gastrointestinal toxicity and promote adverse events such as high blood pressure, congestive heart failure, and thrombosis. Hence, there is a critical need to develop novel and safer nonsteroidal anti-inflammatory drugs possessing alternate mechanism of action. In this study, plants used by the Dharawal Aboriginal people in Australia for the treatment of inflammatory conditions, for example, asthma, arthritis, rheumatism, fever, oedema, eye inflammation, and inflammation of bladder and related inflammatory diseases, were evaluated for their anti-inflammatory activity in vitro. Ethanolic extracts from 17 Eucalyptus spp. (Myrtaceae) were assessed for their capacity to inhibit nitric oxide and tumor necrosis factor-α production in RAW 264.7 macrophages. Eucalyptus benthamii showed the most potent nitric oxide inhibitory effect (IC50 5.57 ± 1.4 µg/mL), whilst E. bosistoana, E. botryoides, E. saligna, E. smithii, E. umbra, and E. viminalis exhibited nitric oxide inhibition values between 7.58 and 19.77 µg/mL.

Antibacterial and antifungal screening of natural products sourced from Australian fungi and characterisation of pestalactams D-F.

Eighteen natural products sourced from Australian micro- or macro-fungi were screened for antibacterial and antifungal activity. This focused library was comprised of caprolactams, polyamines, quinones, and polyketides, with additional large-scale isolation studies undertaken in order to resupply previously identified compounds. Chemical investigations of the re-fermented culture from the endophytic fungus Pestalotiopsis sp. yielded three caprolactam analogues, pestalactams D-F, along with larger quantities of the known metabolite pestalactam A, which was methylated using diazomethane to yield 4-O-methylpestalactam A. The chemical structures of the previously undescribed fungal metabolites were determined by analysis of 1D/2D NMR and MS data. The structure of 4-O-methylpestalactam A was confirmed following single crystal X-ray diffraction analysis. The antibacterial and antifungal activity of all compounds was assessed, which identified three compounds, (1S,3R)-austrocortirubin, (1S,3S)-austrocortirubin, and 1-deoxyaustrocortirubin with mild activity (100 µM) against Gram-positive isolates and one compound, 2-hydroxy-6-methyl-8-methoxy-9-oxo-9H-xanthene-1-carboxylic acid, with activity against Cryptococcus neoformans and Cryptococcus gattii at 50 µM.

Entonalactams A-C: Isoindolinone derivatives from an Australian rainforest fungus belonging to the genus Entonaema.

Bioassay-guided fractionation of an antimalarial DCM/MeOH extract derived from the Australian rainforest fungus Entonaema sp. resulted in the isolation of three new isoindolinone derivatives, entonalactams A-C (1-3), along with the known natural products 3-methoxy-5-methylbenzene-1,2-diol (4), daldinal B (5), and ergosta-4,6,8(14),22-tetraen-3-one (6). The chemical structures of the new secondary metabolites were determined following extensive 1D/2D NMR and MS data analysis. A single crystal X-ray structure for entonalactam A (1) confirmed the NMR-based structure assignment. Entonalactams A-C (1-3) were all determined to be racemic based on chiro-optical data. All secondary metabolites were tested in vitro against Plasmodium falciparum malaria parasites, and ergosta-4,6,8(14),22-tetraen-3-one (6) was identified as the most active compound with 66% inhibition at 50 µM.

Pyridocoumarin, aristolactam and aporphine alkaloids from the Australian rainforest plant Goniothalamus australis.

Chemical investigation of the CH(2)Cl(2)/CH(3)OH extracts from aerial parts of the Australian plant Goniothalamus australis has resulted in the isolation of two pyridocoumarin alkaloids, goniothalines A (1) and B (2) as well as eight known natural products, aristolactam AII (3), enterocarpam II (4), caldensine (5), sauristolactam (6), (-)-anonaine (7), asimilobine (8), altholactone (9) and (+)-goniofufurone (10). The chemical structures of all compounds were determined by extensive spectroscopic and spectrometric analysis. Methylation of 2 using TMS-diazomethane afforded 1, which unequivocally established that both 1 and 2 possessed a 10-methyl-2H-pyrano[2,3-f]quinolin-2-one skeleton. These pyridocoumarin alkaloids are putatively proposed to arise biosynthetically from an aporphinoid precursor. Compounds 1-10 were evaluated for in vitro antimalarial activity against a chloroquine-sensitive Plasmodium falciparum line (3D7). Sauristolactam (6) and (-)-anonaine (7) exhibited the most potent antiparasitic activity with IC(50) values of 9 and 7 µM, respectively.

Chemical composition and cytotoxicity of oils and eremophilanes derived from various parts of Eremophila mitchellii Benth. (Myoporaceae).

A detailed investigation of the wood, leaf, branch and root oil of Eremophila mitchellii (Benth.) was carried out by a combination of GC-FID, GC-MS and NMR. The wood oil was composed predominantly of eremophilanes, a rare class of biologically active, bicyclic sesquiterpenoids. The root oil was also found to contain the eremophilanes together with the zizaene sesquiterpene, sesquithuriferone. 9-Hydroxy-1,7(11),9-eremophilatrien-8-one and the known 1(10),11-eremophiladien-9-one (eremophilone), 9-hydroxy-7(11),9-eremophiladien-8-one (2-hydroxyeremophilone), 8-hydroxy-11-eremophilen-9-one (santalcamphor), 8-hydroxy-10,11-eremophiladien-9-one, sesquithuriferone and 8-hydroxy-1,11-eremophiladien-9-one were purified and elucidated by NMR. Three approaches to the purification of the major eremophilanes from the wood oil are described. (+) Spathulenol, α-pinene, globulol, viridiflorene were the major constituents of the leaf oil. All of the essential oils and the eremophilanes exhibited cytotoxicity against P388D(1) mouse lymphoblast cells in-vitro.

Antibacterial metabolites from Australian macrofungi from the genus Cortinarius.

In this study, ethyl acetate and aqueous fractions from 117 collections of Australian macrofungi belonging to the mushroom genus Cortinarius were screened for antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Overall, the lipophilic fractions were more active than the aqueous fractions. The ethyl acetate fractions of most or all collections of 13 species, namely Cortinarius ardesiacus, C. archeri, C. austrosaginus, C. austrovenetus, C. austroviolaceus, C. coelopus, C. [Dermocybe canaria](2), C. clelandii, C. [D. kula], C. memoria-annae, C. persplendidus, C. sinapicolor, C. vinosipes and forty seven collections of un-described Cortinarius species exhibited IC(50) values of 0.09 mg/mL against S. aureus. In contrast, most or all collections of only four species, namely C. abnormis, C. austroalbidus, C. [D. kula], C. persplendidus, and eleven un-described Cortinarius collections exhibited similar effects against P. aeruginosa (IC(50)