An anti-mitotic compound, (+)-6-tuliposide a, isolated from the Canadian glacier lily, Erythronium grandiflorum.

The Canadian prairie ecosystem is subjected to abiotic and biotic conditions that induce plants to produce secondary metabolites that affect mammalian physiology. Extracts prepared from certain plant species native to Canadian prairie and montane cordillera ecosystems have previously been shown to have anti-mitotic activity on human cancer cell lines. In this study, we investigated the glacier lily, Erythronium grandiflorum (Liliaceae), in which the species was the most taxonomically distant from Asteraceae and had anti-mitotic activity. When added to cell lines, E. grandiflorum extracts induced rounded cell morphology and arrested cells in the G2/M phase of the cell cycle. Of the cells that displayed a rounded phenotype, all were positive for phospho-histone H3 and contained a distorted mitotic spindle. This anti-mitotic activity was distinct from that of the compound colchicine, which has been previously isolated from the Liliaceae family. By biology-guided fractionation, we isolated the natural product (+)-6-tuliposide A and are the first to report its anti-mitotic activity. These results reveal a chemical motif in secondary metabolites and expand the range of Canadian prairie plants with anti-mitotic activity that can become new scientific tools or used in the development of anti-proliferative medicines.

Tetrahydroxanthene-1,3(2H)-diones and Oxidized Hexadiene Derivatives from Uvaria leptopoda and Their Biological Activities.

The first phytochemical investigation of the twig extract of Uvaria leptopoda resulted in the isolation and identification of three new tetrahydroxanthene-1,3(2H)-diones, uvarialeptones A-C, two new oxidized hexadiene derivatives, uvarialeptols A and B, together with ten known compounds. Their structures were elucidated by spectroscopic techniques and mass spectrometry. Uvarialeptones A and B were unprecedented tetrahydroxanthene-1,3(2H)-dione dimers which exhibited a cyclobutane ring via [2 + 2] cycloaddition from uvarialeptone C and 9a-O-methyloxymitrone, respectively. The structure of uvarialeptone A was confirmed by X-ray diffraction analysis using Mo Kα radiation. Compound 3 inhibited NO production at an IC50 value of 6.7 ± 0.1 µM.

Highly oxygenated cyclohexenes from Uvaria dac Pierre ex Finet & Gagnep. and their α-glucosidase inhibitory activity.

Phytochemical investigations of the twig and leaf extracts of Uvaria dac Pierre ex Finet & Gagnep. resulted in the isolation and identification of five new highly oxygenated cyclohexenes, uvaridacols M - Q (1-3, 5, and 6), and six known compounds (4 and 7-11). All new structures were elucidated by spectroscopic methods and HRESITOFMS data. The absolute configuration of 1, 5, and 6 was confirmed by single X-ray diffraction analysis with Cu Kα radiation. In contrast, other compounds were established by comparing their specific rotation and ECD spectra with those of known compounds. Some of the isolated compounds with sufficient quantity were evaluated for their α-glucosidase inhibitory activity. Of these, (-)-1,6-desoxypipoxide (10) showed α-glucosidase inhibitory activity with an IC50 value of 28.6 µM. The in silico molecular docking of active compounds was also studied.

Piperazate-Guided Isolation of Caveamides A and B, Cyclohexenylalanine-Containing Nonribosomal Peptides from a Cave Actinomycete.

Hybrid genome-mining/15N-NMR was used to target compounds containing piperazate (Piz) residues, leading to the discovery of caveamides A (1) and B (2) from Streptomyces sp. strain BE230, isolated from New Rankin Cave (Missouri). Caveamides are highly dynamic molecules containing an unprecedented ß-ketoamide polyketide fragment, two Piz residues, and a new N-methyl-cyclohexenylalanine residue. Caveamide B (2) exhibited nanomolar cytotoxicity against several cancer cell lines and nanomolar antimicrobial activity against MRSA and E. coli.

Styryl lactone derivatives and aristolactam alkaloids from Goniothalamus tapis Miq. and their α-glucosidase inhibitory activity.

Two new styryl lactone derivatives, goniothapic acids A (1) and B (2), and 18 known compounds, were isolated from the twig and leaf extracts of Goniothalamus tapis Miq. The structures of new compounds were characterised by spectroscopic methods and HRESITOFMS. Their absolute configuration was established by comparing the experimental and calculated ECD spectra. Eleven compounds were evaluated for their α-glucosidase inhibitory activity. Of these, (-)-goniothalamin (5) and oldhamactam (16) showed the best α-glucosidase inhibitory activity with IC50 values of 54.8 and 57.9 µM, respectively.

Curcuphenol possesses an unusual histone deacetylase enhancing activity that counters immune escape in metastatic tumours.

Curcuphenol, a common component of the culinary spices, naturally found in marine invertebrates and plants, has been identified as a novel candidate for reversing immune escape by restoring expression of the antigen presentation machinery (APM) in invasive cancers, thereby resurrecting the immune recognition of metastatic tumours. Two synthetic curcuphenol analogues, were prepared by informed design that demonstrated consistent induction of APM expression in metastatic prostate and lung carcinoma cells. Both analogues were subsequently found to possess a previously undescribed histone deacetylase (HDAC)-enhancing activity. Remarkably, the H3K27ac ChIPseq analysis of curcuphenol-treated cells reveals that the induced epigenomic marks closely resemble the changes in genome-wide pattern observed with interferon-γ, a cytokine instrumental for orchestrating innate and adaptive immunity. These observations link dietary components to modifying epigenetic programs that modulate gene expression guiding poised immunity.

New norclerodane diterpenoids from bulbils of Dioscorea bulbifera L.

Investigation of extracts from bulbils of Dioscorea bulbifera L. yielded two new norclerodane diterpenoids, diosbulbin N acetate (1) and epi-diosbulbin B (3), together with eleven known compounds. Their structures were established based on spectroscopy. The absolute configurations of 1 and diosbulbin B (2) were determined by X-ray crystallographic analysis using Cu Kα radiation. The absolute configuration of 3 was determined by comparison of its ECD spectrum to that of 2. Isolated phenanthrenes 7, 9 and 10 exhibited moderate cytotoxicity against the HelaS3 cell line with IC50 values of 9.03 ± 0.04, 27.13 ± 6.86 and 10.88 ± 2.75 µM, respectively. In addition, 7-9 and 11 showed potent inhibition of NO production by LPS-induced RAW 264.7 macrophages.

Synthetic Analogs of the Sponge Sesterterpenoid Alotaketal C are Potent Inhibitors of SARS-CoV-2 Omicron BA.1 and BA.5 Infections of Human Lung Cells.

The protein kinase C-activating sponge natural product alotaketal C (1) potently inhibits the infection of human Calu-3 lung cells by SARS-CoV-2 Omicron BA.1 and BA.5 variants. Simplified analogs of 1 have been synthesized and tested for anti-SARS-CoV-2 activity providing SAR data for the antiviral pharmacophore of 1. Analogs 19 and 23, which are missing the C-11 substituents in 1 and have modified C-13 appendages, are ∼2- to 7-fold more potent than 1 and have equal or larger selectivity indices.

A novel cell-based screen identifies chemical entities that reverse the immune-escape phenotype of metastatic tumours.

Genetic and epigenetic events have been implicated in the downregulation of the cellular antigen processing and presentation machinery (APM), which in turn, has been associated with cancer evasion of the immune system. When these essential components are lacking, cancers develop the ability to subvert host immune surveillance allowing cancer cells to become invisible to the immune system and, in turn, promote cancer metastasis. Here we describe and validate the first high-throughput cell-based screening assay to identify chemical extracts and unique chemical entities that reverse the downregulation of APM components in cell lines derived from metastatic tumours. Through the screening of a library of 480 marine invertebrate extracts followed by bioassay-guided fractionation, curcuphenol, a common sesquiterpene phenol derived from turmeric, was identified as the active compound of one of the extracts. We demonstrate that curcuphenol induces the expression of the APM components, TAP-1 and MHC-I molecules, in cell lines derived from both metastatic prostate and lung carcinomas. Turmeric and curcumins that contain curcuphenol have long been utilized not only as a spice in the preparation of food, but also in traditional medicines for treating cancers. The remarkable discovery that a common component of spices can increase the expression of APM components in metastatic tumour cells and, therefore reverse immune-escape mechanisms, provides a rationale for the development of foods and advanced nutraceuticals as therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Phaeanthuslucidines A-D, dimeric aporphine alkaloid derivatives from Phaeanthus lucidus Oliv.

The first phytochemical investigation of the twigs of Phaeanthus lucidus Oliv. resulted in the isolation and identification of four undescribed alkaloids, including two aporphine dimers, phaeanthuslucidines A and B, a hybrid of aristolactam-aporphine, phaeanthuslucidine C, and a C-N linked aporphine dimer, phaeanthuslucidine D, together with two known compounds. Their structures were determined by extensive analysis of spectroscopic data, and by comparison of their spectroscopic and physical data with previous reports. Phaeanthuslucidines A-C and bidebiline E were analysed and resolved by chiral HPLC to yield the (Ra) and (Sa) atropisomers, whose absolute configurations were respectively determined by ECD calculations. Phaeanthuslucidines A and B, bidebiline E, and lanuginosine showed α-glucosidase inhibitory activities with IC50 values in the range of 6.7-29.2 µM. Moreover, molecular docking simulations of α-glucosidase inhibition of active compounds were studied.

Alkaloids and Styryl lactones from Goniothalamus ridleyi King and Their α-Glucosidase Inhibitory Activity.

Gonioridleylactam (1), a new compound, is a unique dimeric aristolactam isolated from the EtOAc extract of the twigs of Goniothalamus ridleyi King. The structure of gonioridleylactam (1) consists of two different aristolactams linked together with two methylenedioxy bridges at C-3/C-3' and C-4/C-4', generating a ten-membered ring of [1,3,6,8]tetraoxecine. A new natural product, gonioridleyindole (3-hydroxymethyl-1-methyl-1H-benz[f]indole-4,9-dione, 2), together with eight known compounds (3-10) were also isolated from this plant. Their structures were extensively characterized by spectroscopic methods and comparisons were made with the literature. Compounds 1-4, 7, and 9 were evaluated for their α-glucosidase inhibitory activity. Of these, 3,5-demethoxypiperolide (7) displayed the highest α-glucosidase inhibitory activity, with an IC50 value of 1.25 µM.

Dimeric aporphine alkaloids from the twigs of Trivalvaria costata (Hook.f. & Thomson) I.M.Turner.

A phytochemical investigation of the twig extract of Trivalvaria costata (Hook.f. & Thomson) I.M.Turner has identified ten undescribed dimeric aporphine alkaloids, trivalcostatines A-J, one undescribed isoquinoline alkaloid, trivalcostaisoquinoline, and four known aporphine alkaloids. Their structures were elucidated by detailed analysis of NMR and HRESITOFMS data. Three of the dimeric aporphine structures were confirmed by single crystal X-ray diffraction analysis. All of the dimeric aporphine alkaloids were isolated as mixtures of atropisomers. Several of them were resolved by chiral-phase HPLC and the absolute configurations of the pure atropisomers were assigned by calculated and experimental ECD analysis. Bidebilines A and B, heteropsine, and urabaine showed α-glucosidase inhibitory activities with IC50 values in the range of 4.1-11 µM.

Thorectidiol A Isolated from the Marine Sponge Dactylospongia elegans Disrupts Interactions of the SARS-CoV-2 Spike Receptor Binding Domain with the Host ACE2 Receptor.

Thorectidiols isolated from the marine sponge Dactylospongia elegans (family Thorectidae, order Dictyoceratida) collected in Papua New Guinea are a family of symmetrical and unsymmetrical dimeric biphenyl meroterpenoid stereoisomers presumed to be products of oxidative phenol coupling of a co-occurring racemic monomer, thorectidol (3). One member of the family, thorectidiol A (1), has been isolated in its natural form, and its structure has been elucidated by analysis of NMR, MS, and ECD data. Acetylation of the sponge extract facilitated isolation of additional thorectidiol diacetate stereoisomers and the isolation of the racemic monomer thorectidol acetate (6). Racemic thorectidiol A (1) showed selective inhibition of the SARS-CoV-2 spike receptor binding domain (RBD) interaction with the host ACE2 receptor with an IC50 = 1.0 ± 0.7 µM.

Discovery of lead natural products for developing pan-SARS-CoV-2 therapeutics.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC50) below 50 µM against mNG-SARS-CoV-2; 16 of these had EC50 values below 10 µM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants.

Metabolomics Reveals a 26-Membered Macrolactone Produced by Endophytic Colletotrichum spp. from Alcatrazes Island, Brazil.

Comparative metabolomics analysis of nonphytotoxic endophytic Colletotrichum spp. isolated from Anthurium alcatrazense endemic to Alcatrazes island (Brazil) and phytopathogenic Colletotrichum spp. isolated from the mainland of Brazil revealed significant differences in chemical composition. Examination of endophytic Colletotrichum spp. from Alcatrazes island led to the discovery of a 26-member macrolactone, colletotrichumolide (1), containing a phosphatidyl choline side chain. Further examination of the phytopathogenic strains from the mainland identified a family of phytopathogenic metabolites not present in the nonpathogenic island-derived strains, suggesting that geographical isolation could influence the secondary metabolism of fungal strains.

Rotenoids and isoflavones from the leaf and pod extracts of Millettia brandisiana Kurz.

Phytochemical investigations of the leaf and pod extracts of Millettia brandisiana Kurz led to the isolation and identification of four previously undescribed rotenoids, (-)-(6aS,12aS)-millettiabrandisins A-C and (-)-(6aS,12aS)-6-deoxyclitoriacetal, two previously undescribed isoflavones, millettiabrandisins D and E, and 20 known compounds. The structures of previously undescribed compounds were determined on the basis of NMR and MS data. The absolute configurations of (-)-(6aS,12aS)-millettiabrandisins A-C were determined from the comparison of their experimental and calculated ECD spectra. (-)-(6aR,12aR)-12a-Hydroxy-α-toxicarol was also confirmed by X-ray crystallographic data. Some isolated compounds were evaluated for their cytotoxicity against three cancer cell lines, including lung cancer (A549), colorectal cancer (SW480), and leukemic cells (K562). Of these, α-toxicarol displayed the best cytotoxicity against lung cancer (A549) and leukemic cells (K562) with the IC50 values of 104.4 and 67.5 µM, respectively. 6″,6″-Dimethylchromene-[2″,3″:7,8]-flavone showed the highest cytotoxicity against colorectal cancer (SW480) with an IC50 value of 97.2 µM.

Antidiabetic and Cytotoxic Activities of Rotenoids and Isoflavonoids Isolated from Millettia pachycarpa Benth.

A phytochemical investigation of the root and leaf extracts of Millettia pachycarpa Benth resulted in the isolation and identification of 16 compounds, including six rotenoids (1-6) and 10 prenylated isoflavonoids (7-16). Compound 4 was isolated as a scalemic mixture, which was resolved by chiral HPLC to afford (-)-(6aS,12aS)-12a-hydroxy-α-toxicarol (4) and (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol (4). (+)-(6aR,12aR)-Millettiapachycarpin (3) and (-)-(6aS,12aS)-12a-hydroxy-α-toxicarol (4) were isolated as new compounds. The absolute configuration of (-)-(6R)-pachycarotenoid (2), (+)-(6aR,12aR)-millettiapachycarpin (3), (-)-(6aS,12aS)-4 and (+)-(6aR,12aR)-12a-hydroxy-α-toxicarol (4), (+)-(6aS,12aS)-(5), and (-)-(6aS,12aS,2″R)-sumatrol (6) were identified by electronic circular dichroism (ECD) data. (-)-(6aS,12aS,2″R)-Sumatrol (6) was also confirmed by X-ray diffraction analysis using Cu-Kα radiation. Antidiabetic activities, including α-glucosidase and α-amylase inhibitory activities, and cytotoxicities against lung cancer A549, colorectal cancer SW480, and leukemic K562 cells of some isolated compounds were evaluated. Of these, isolupalbigenin (11) exhibited the highest α-glucosidase inhibitory activity, with an IC50 value of 11.3 ± 0.2 µM, whereas the scalemic mixture of 12a-hydroxy-α-toxicarol (4) displayed the best α-amylase inhibitory activity, with an IC50 value of 106.9 ± 0.2 µM. Euchrenone b10 (15) exhibited the highest cytotoxicity against lung cancer A549, colorectal cancer SW480, and leukemic K562 cells, with IC50 values of 40.3, 39.1, and 15.1 µM, respectively. In addition, molecular docking simulations of α-glucosidase inhibition of the active compounds were studied.

Free Piperazic Acid as a Precursor to Nonribosomal Peptides.

Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen-nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use in vitro reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.

Natural Products Produced in Culture by Biosynthetically Talented Salinispora arenicola Strains Isolated from Northeastern and South Pacific Marine Sediments.

Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.