Terpenoid bio-transformations and applications via cell/organ cultures: a systematic review.

Structurally diverse natural products are valued for their targeted biological activity. The challenge of working with such metabolites is their low natural abundance and complex structure, often with multiple stereocenters, precludes large-scale or unsophisticated chemical synthesis. Since select plants contain the enzymatic machinery necessary to produce specialized compounds, tissue cultures can be used to achieve key transformations for large-scale chemical and/or pharmaceutical applications. In this context, plant tissue-culture bio-transformations have demonstrated great promise in the preparation of pharmaceutical products. This review describes the capacity of cultured plant cells to transform terpenoid natural products and the specific application of such transformations over the past three decades (1988-2019).

Bioactive metabolites from Chaetomium aureum: structure elucidation and inhibition of the Hsp90 machine chaperoning activity.

Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48 h.

Protein kinase and HDAC inhibitors from the endophytic fungus Epicoccum nigrum.

A chemical investigation of the endophytic fungus Epicoccum nigrum isolated from leaves of Mentha suaveolens collected in Morocco resulted in the isolation of five new polyketides, epicocconigrones A and B (1 and 2), 3-methoxyepicoccone B (3), 3-methoxyepicoccone (4), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (5), together with five known compounds (6-10). The structures of the new compounds were unambiguously determined by extensive analysis of the 1D and 2D NMR and mass spectroscopic data. Compounds 1 and 10 showed potent inhibition of at least 15 protein kinases with IC50 values ranging from 0.07 to 9.00 µM. Moreover, compounds 1 and 10 inhibited histone deacetylase (HDAC) activities with IC50 values of 9.8 and 14.2 µM, respectively. A preliminary structure-activity relationship is discussed. Interestingly, compounds 1 and 10 exert mainly cytostatic effects in human lymphoma RAJI and U-937 cell lines.

Two trypanocidal dipeptides from the roots of Zapoteca portoricensis (Fabaceae).

Zapoteca portoricensis (Jacq) HM Hernández is used with remarkable efficacy in ethnomedicinal management of tonsillitis in the Eastern part of Nigeria. Previous pharmacological studies have validated the antiinflammatory and antimicrobial activities of the crude extract. In this study, two dipeptides, saropeptate (aurantiamide acetate) and anabellamide, were isolated from the methanol root extract of Zapoteca portoricensis and their chemical structures deduced by one dimensional and two dimensional NMR and mass spectrometry. These compounds were isolated for the first time from this plant, and no report has been found on their previous isolation from the genus Zapoteca. Evaluation of their trypanocidal activity showed that compound 1 exhibited potent activity against Trypanosoma brucei rhodesiense with an IC50 value of 3.63 µM and selectivity index of 25.3.

Pro-apoptotic and immunostimulatory tetrahydroxanthone dimers from the endophytic fungus Phomopsis longicolla.

Four tetrahydroxanthone dimers (1-4) and four biogenetically related monomers (5-8), including the new derivatives 4-6, were isolated from the endophyte Phomopsis longicolla. The absolute configurations of 2-4 were established for the first time by TDDFT electronic circular dichroism calculations, and that of phomoxanthone A (1) was revised by X-ray crystallography. Phomoxanthone A (1) showed the strongest pro-apoptotic activity when tested against a panel of human cancer cell lines, including cisplatin-resistant cells, whereas it was up to 100-fold less active against healthy blood cells. It was also the most potent activator of murine T lymphocytes, NK cells, and macrophages, suggesting an activation of the immune system in parallel to its pro-apoptotic activity. This dual effect in combating cancer cells could help in fighting resistance during chemotherapy. Preliminary structure-activity studies of isolated compounds and derivatives obtained by semisynthesis (9a-11) hinted at the location of the biaryl axis and the presence of acetyl groups as important structural elements for the biological activity of the studied tetrahydroxanthones.

Anticancer effect of altersolanol A, a metabolite produced by the endophytic fungus Stemphylium globuliferum, mediated by its pro-apoptotic and anti-invasive potential via the inhibition of NF-κB activity.

Altersolanol A, a natural product from the endophytic fungus Stemphylium globuliferum isolated from the medicinal plant Mentha pulegium (Lamiaceae) growing in Morocco, shows cytotoxic, cytostatic, anti-inflammatory and anti-migrative activity against human chronic myeloid K562 leukemia and A549 lung cancer cells in a dose dependent manner without affecting the viability of non cancerous cells. Altersolanol A induces cell death by apoptosis through the cleavage of caspase-3 and -9 and through the decrease of anti-apoptotic protein expression. Moreover, we report here the importance of the distinct structural features of altersolanol A by testing other related anthracene derivatives in order to identify preliminary structure-activity relationships. Acetylation of altersolanol A did not improve activity where other derivatives such as tetrahydroaltersolanol B and ampelanol that differ from altersolanol A by reduction of one of a carbonyl group and removal of hydroxyl substituents were inactive in comparison. Altogether our results suggest that altersolanol A may be considered as an interesting lead for further development of chemotherapeutic agents.

O-heterocyclic embeurekols from Embellisia eureka, an endophyte of Cladanthus arabicus.

Three new polyketides ((-)-1, (+)-1, and 2) were isolated from the EtOAc extract of the fungus Embellisia eureka, an endophyte of the Moroccan plant Cladanthus arabicus (Asteraceae). The structures of these new compounds were determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of (-)-1, (+)-1, and 2 were determined by TDDFT ECD calculations of solution conformers, online HPLC-ECD analysis, and the modified Mosher method.

Inhibition of inflammatory mediators and reactive oxygen and nitrogen species by some depsidones and diaryl ether derivatives isolated from Corynespora cassiicola, an endophytic fungus of Gongronema latifolium leaves.

In this study, some depsidones and diaryl ether derivatives isolated from Corynespora cassicola, a fungi endophyte of Gongronema latifolium, were assessed for their anti-inflammatory potentials. The isolated metabolites corynesidone A (1), corynesidone C (2), corynesidone D (3) and corynether A (4) were screened for their effects on tumour necrosis factor-α (TNF-α), inducible nitric oxide (iNO), and reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by stimulated RAW264.7 macrophages. Concentration of 1, 2, 3 and 4 up to 100 µM did not remarkably affect the viability of treated macrophages. The compounds were found to cause a concentration-dependent decrease in lipopolysaccharide-induced TNF-α and iNO in RAW264.7 cells. Pre-treatment with 100 µM of 1, 2, 3 and 4 suppressed iNO by as much as 96.28%, 95.71%, 78.14% and 73.28%; with IC(50) of 8.16, 9.49, 15.29 and 26.52 µM, respectively. Similarly, pre-treatment with 100 µM of 1, 2, 3 and 4 caused an inhibition of 99.17%, 99.59%, 95.02% and 74.07% in the formation of iNO production, respectively, with IC(50) of 1.88, 3.99, 7.48 and 37.22 µM. Treatment of with compounds 1-4 (10, 30 and 100 µM) followed by stimulation with phorbol 12-myristate 13-acetate (1 µM) caused significant (p < 0.05) suppression of ROS/RNS-evoked chemiluminescence of luminol by as much as 100.96 ± 1.88%, 98.59 ± 1.38%, 87.35 ± 1.41% and 79.22 ± 0.30%, respectively at 100 µM. The depsidone derivatives (1-4) showed more potent inhibition of TNF-α and NO production and better scavenging ROS/RNS than the diaryl ether derivative (4). These chemical scaffolds can serve as suitable lead molecules for further development into novel anti-inflammatory and/or anti-cancer agents.

Optimization of enniatin production by solid-phase fermentation of Fusarium tricinctum.

Enniatins are cyclic depsipeptides produced by fungi of the genus Fusarium that are of interest due to their pronounced biological activities; especially enniatins A, A1, B, and B1 possess anticarcinogenic and anti-HIV properties. In the present study, F. tricinctum was grown on seven solid media and in one liquid growth medium with or without addition of peptone or of amino acid precursors in order to identify favourable media with simple cultivation conditions for maximum enniatin production. Additionally, the optimal duration of growth was investigated for the highest yields of enniatins. From the different media analysed, white beans (Phaseolus vulgaris, solid medium) induced the highest accumulation of enniatins A, A1, B, and B1, that reached a maximum of 1,365 mg total enniatins in 1 L growth medium after 18 days of fermentation. Fermentation of F. tricinctum on white beans gave the highest yield of enniatins compared to all other media analysed in this study.

Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer.

Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.

NF kappa B inhibitors and antitrypanosomal metabolites from endophytic fungus Penicillium sp. isolated from Limonium tubiflorum.

Chemical investigation of the endophytic fungus Penicillium sp. isolated from Limonium tubiflorum growing in Egypt afforded four new compounds of polyketide origin, including two macrolides, penilactone (1) and 10,11-epoxycurvularin (2), a dianthrone, neobulgarone G (7), and a sulfinylcoumarin, sulfimarin (14), along with twelve known metabolites (3-6, 8-13, 15 and 16). The structures of all compounds were assigned by comprehensive spectral analysis (1D and 2D NMR) and mass spectrometry. Compounds 3, 4, 13 and 16 showed pronounced antitrypanosomal activity with mean MIC values ranging from 4.96 to 9.75µM. Moreover, when tested against a panel of three human tumor cell lines compounds 3, 4, 6 and 12 showed selective growth inhibition against Jurkat and U937 cell lines with IC(50) values ranging from 1.8 to 13.3µM. The latter compounds also inhibited TNFα-induced NF-κB activity in K562 cells with IC(50) values ranging from 1.6 to 10.1µM, respectively.

Fungal endophytes: unique plant inhabitants with great promises.

Fungal endophytes residing in the internal tissues of living plants occur in almost every plant on earth from the arctic to the tropics. The endophyte-host relationship is described as a balanced symbiotic continuum ranging from mutualism through commensalism to parasitism. This overview will highlight selected aspects of endophyte diversity, host specificity, endophyte-host interaction and communication as well as regulation of secondary metabolite production with emphasis on advanced genomic methods and their role in improving our current knowledge of endophytic associations. Furthermore, the chemical potential of endophytic fungi for drug discovery will be discussed with focus on the detection of pharmaceutically valuable plant constituents as products of fungal biosynthesis. In addition, selected examples of bioactive metabolites reported in recent years (2008-2010) from fungal endophytes residing in terrestrial plants are presented grouped according to their reported biological activities.

Bioactive metabolites from the endophytic fungus Stemphylium globuliferum isolated from Mentha pulegium.

The endophytic fungus Stemphylium globuliferum was isolated from stem tissues of the Moroccan medicinal plant Mentha pulegium. Extracts of the fungus, which was grown on solid rice medium, exhibited considerable cytotoxicity when tested in vitro against L5178Y cells. Chemical investigation yielded five new secondary metabolites, alterporriol G (4) and its atropisomer alterporriol H (5), altersolanol K (11), altersolanol L (12), stemphypyrone (13), and the known compounds 6-O-methylalaternin (1), macrosporin (2), altersolanol A (3), alterporriol E (6), alterporriol D (7), alterporriol A (8), alterporriol B (9), and altersolanol J (10). The structures were determined on the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry. Among the alterporriol-type anthranoid dimers, the mixture of alterporriols G and H (4/5) exhibited considerable cytotoxicity against L5178Y cells with an EC(50) value of 2.7 microg/mL, whereas the other congeners showed only modest activity. The compounds were also tested for kinase inhibitory activity in an assay involving 24 different kinases. Compounds 1, 2, 3, and the mixture of 4 and 5 were the most potent inhibitors, displaying EC(50) values between 0.64 and 1.4 microg/mL toward individual kinases.

A new tetrahydrofuran derivative from the endophytic fungus Chaetomium sp. isolated from Otanthus maritimus.

A hitherto unidentified endophytic strain of the genus Chaetomium, isolated from the medicinal plant Otanthus maritimus, yielded a new tetrahydrofuran derivative, aureonitolic acid (1), along with 5 known natural products, 2-6. The structure of 1 was determined by extensive spectroscopic analysis and comparison with reported data. Extracts of the fungus, grown either in liquid culture or on solid rice media, exhibited considerable cytotoxic activity when tested in vitro against L5178Y mouse lymphoma cells. Compounds 2 and 6 showed significant growth inhibition against L5178Y cells with EC50 values of 7.0 and 2.7 microg/mL, respectively, whereas 1 was inactive.

Flavonoid glycosides from Olax mannii: Structure elucidation and effect on the nuclear factor kappa B pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Olax mannii Oliv. (Olacaceae) is among the many medicinal plants used in Nigeria for the ethnomedicinal management of both cancer and inflammation. Such plants represent potential sources of innovative therapeutic agents for the treatment of cancer and other malignant disorders. While the majority of medicinal plants exert their anticancer effects by direct cytotoxicity on tumor cells, it is important that other mechanisms through which these plants can exhibit anticancer effects are investigated. Preliminary studies indicated that Olax mannii leaves are rich sources of novel flavonoid glycosides. The detailed chemistry as well the mechanisms through which these flavonoid constituents may exert their cancer chemo-preventive and therapeutic effects are, however, not yet investigated. AIM OF THE STUDY: The aim of this study is to carry out a detailed chemical investigation of Olax mannii leaves and the effects of the isolated constituents on the nuclear factor kappa B (NF-κB) pathway. MATERIALS AND METHODS: A methanol leaf extract was subjected to various chromatographic separations to achieve isolation of flavonoid glycosides and the structures of the isolated compounds were elucidated by a combination of 1D and 2D NMR and high resolution mass spectrometry. Biological activities were assessed by measurement of cellular viability and proliferation using quantitative IncuCyte videomicroscopy, trypan blue staining and by quantification of the number of metabolically active K562 cells based on quantitation of ATP. The effect of the compounds on the inhibition of the NF-κB pathway as well as toxicity towards peripheral blood mononuclear cells to evaluate differential toxicity was also assayed. RESULTS: Chemical investigation of the methanol leaf extract of the plant material led to the isolation of three new flavonoid triglycosides, kaempferol 3-O-[α-D-apiofuranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (1), kaempferol 3-O-[ß-D-glucopyranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (2), kaempferol 3-O-[ß-D-arabinopyranosyl-(1→4)-α-L-rhamnopyranoside]-7-O-α-L-rhamnopyranoside (3), in addition to fourteen known flavonoid glycosides (4-17). Of all the tested compounds, only compound 9 (kaempferol 3-O-α-L-rhamnopyranoside) exhibited promising and specific antiproliferative activity on human K562 chronic myelogenous leukemia cells and dose-dependently inhibited NF-κB transactivation. CONCLUSION: The presence of this flavonoid glycoside and derivatives may account for the reported efficacy of Olax mannii leaf extract in the ethnomedicinal management of cancer and inflammation.

Embellicines A and B: absolute configuration and NF-κB transcriptional inhibitory activity.

Two new metabolites, embellicines A and B (1 and 2), were isolated from the EtOAc extract of the fungus Embellisia eureka , an endophyte of the Moroccan plant Cladanthus arabicus (Asteraceae). The structures of these new compounds were determined on the basis of extensive one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configuration of embellicine A (1) was determined by TDDFT ECD calculations of solution conformers, whereas that of embellicine B (2) was deduced based on ROESY correlations and on biogenetic considerations in comparison to 1. Both embellicines (1 and 2) are cytostatic, cytotoxic, and inhibit NF-κB transcriptional activity, indicating that inhibition of NF-κB may be a possible mechanism of action of these compounds. Embellicine B (2) was the most active compound encountered in this study and acts at nanomolar concentrations without affecting tumor microenvironment.

Atropisomeric dihydroanthracenones as inhibitors of multiresistant Staphylococcus aureus.

Two bisdihydroanthracenone atropodiastereomeric pairs, including homodimeric flavomannin A (1) and the previously unreported flavomannin B (2), two new unsymmetrical dimers (3 and 4), and two new mixed dihydroanthracenone/anthraquinone dimers (5 and 6) were isolated from Talaromyces wortmannii , an endophyte of Aloe vera . The structures of 2-6 were elucidated by extensive NMR and mass spectrometric analyses. The axial chirality of the biaryls was determined using TDDFT ECD and VCD calculations, the combination of which however did not allow the assignment of the central chirality elements of 1. The compounds exhibited antibacterial activity against Staphylococcus aureus , including (multi)drug-resistant clinical isolates. Reporter gene analyses indicated induction of the SOS response for some of the derivatives, suggesting interference with DNA structure or metabolism. Fluorescence microscopy demonstrated defective segregation of the bacterial chromosome and DNA degradation. Notably, the compounds showed no cytotoxic activity, encouraging their further evaluation as potential starting points for antibacterial drug development.

Methods for isolation of marine-derived endophytic fungi and their bioactive secondary products.

Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some of them featuring new carbon frameworks hitherto unprecedented in nature. These compounds are of interest as new lead structures for medicine as well as for plant protection. The aim of this protocol is to give a detailed description of methods useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on rapid 'in house' screening systems. Some results exemplifying the positive outcomes of the protocol are given at the end. From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at least 3 months has to be scheduled.

Bioactive compounds from marine bacteria and fungi.

Marine bacteria and fungi are of considerable importance as new promising sources of a huge number of biologically active products. Some of these marine species live in a stressful habitat, under cold, lightless and high pressure conditions. Surprisingly, a large number of species with high diversity survive under such conditions and produce fascinating and structurally complex natural products. Up till now, only a small number of microorganisms have been investigated for bioactive metabolites, yet a huge number of active substances with some of them featuring unique structural skeletons have been isolated. This review covers new biologically active natural products published recently (2007-09) and highlights the chemical potential of marine microorganisms, with focus on bioactive products as well as on their mechanisms of action.

Enniatins A1, B and B1 from an endophytic strain of Fusarium tricinctum induce apoptotic cell death in H4IIE hepatoma cells accompanied by inhibition of ERK phosphorylation.

Enniatins are mycotoxins which have important impact on human health, e.g. as contaminants of cereals, but also are discussed as possible anticancer agents. We investigated toxic effects of enniatins A1, B and B1 isolated from Fusarium tricinctum on different cancer cell lines. The enniatins showed moderate activity in HepG2 and C6 cells (EC(50)-values approximately 10-25 microM), but were highly toxic in H4IIE cells (EC(50)-values approximately 1-2.5 microM). In H4IIE cells, all enniatins increased caspase 3/7 activity and nuclear fragmentation as markers for apoptotic cell death. Enniatin A1, enniatin B1, and, to a lesser extent, also enniatin B decreased the activation of extracellular regulated protein kinase (ERK) (p44/p42), a mitogen-activated protein kinase which is associated with cell proliferation. Furthermore, enniatins A1 and B1, but not enniatin B were able to inhibit moderately tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. Screening of 24 additional protein kinases involved in signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) showed no inhibitory activity of enniatins. We conclude that enniatins A1 and B1 and, to a lesser extent, enniatin B may possess anticarcinogenic properties by induction of apoptosis and disruption of ERK signalling pathway. Further analysis of these substances is necessary to analyse their usefulness for cancer therapy.