Structural Insights Into the Terpene Cyclization Domains of Two Fungal Sesterterpene Synthases and Enzymatic Engineering for Sesterterpene Diversification.

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg2+), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research.

Hyrtiosins F and G, two new scalarane sesterterpenes from the South China sea sponge Hyrtios erecta.

Two new scalarane sesterterpenes, hyrtiosins F and G (1 and 2), along with two known related compounds, hyrtiosin D and sesterstatin 6 (3 and 4), were isolated from the Hainan marine sponge Hyrtios erecta. The structures of new compounds 1 and 2 were determined by detailed analysis of 1D and 2D NMR spectra and by comparison of the spectroscopic data with those reported in the literatures.

Arthproliferins A-D, Four New Sesterterpenes from the Mangrove-Sediment-Derived Fungus Arthrinium sp. SCSIO41221.

Four new sesterterpenes, arthproliferins A-D (1-4), together with four known derivatives, were isolated and characterized from the mangrove-sediment-derived fungus Arthrinium sp. SCSIO41221. Their structures were determined using detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses. Some of the isolated compounds were evaluated for their cytotoxicity in vitro. The results revealed that terpestacin (6) exhibited significant activity with an IC50 value of 20.3 µM, and compounds 2 and 5 were found to show weak inhibitory effects against U87MG-derived GSCs.

Functional Prediction of trans-Prenyltransferases Reveals the Distribution of GFPPSs in Species beyond the Brassicaceae Clade.

Terpenoids are the most diverse class of plant primary and specialized metabolites, and trans-prenyltransferases (trans-PTs) are the first branch point to synthesize precursors of various chain lengths for further metabolism. Whereas the catalytic mechanism of the enzyme is known, there is no reliable method for precisely predicting the functions of trans-PTs. With the exponentially increasing number of available trans-PTs genes in public databases, an in silico functional prediction method for this gene family is urgently needed. Here, we present PTS-Pre, a web tool developed on the basis of the "three floors" model, which shows an overall 86% prediction accuracy for 141 experimentally determined trans-PTs. The method was further validated by in vitro enzyme assays for randomly selected trans-PTs. In addition, using this method, we identified nine new GFPPSs from different plants which are beyond the previously reported Brassicaceae clade, suggesting these genes may have occurred via convergent evolution and are more likely lineage-specific. The high accuracy of our blind prediction validated by enzymatic assays suggests that PTS-Pre provides a convenient and reliable method for genome-wide functional prediction of trans-PTs enzymes and will surely benefit the elucidation and metabolic engineering of terpenoid biosynthetic pathways.

Cytotoxic Scalarane Sesterterpenes from the Sponge Hyrtios erectus.

Twelve new sesterterpenes along with eight known sesterterpenes were isolated from the marine sponge Hyrtios erectus collected off the coast of Chuuk Island, the Federated State of Micronesia. Based upon a combination of spectroscopic and computational analyses, these compounds were determined to be eight glycine-bearing scalaranes (1-8), a 3-keto scalarane (9), two oxidized-furan-bearing scalaranes (10 and 11), and a salmahyrtisane (12). Several of these compounds exhibited weak antiproliferation against diverse cancer cell lines as well as moderate anti-angiogenesis activities. The antiproliferative activity of new compound 4 was found to be associated with G0/G1 arrest in the cell cycle.

Dactylospenes A-E, Sesterterpenes from the Marine Sponge Dactylospongia elegans.

Chemical investigation on a marine sponge, Dactylospongia elegans, yielded five new γ-oxygenated butenolide sesterterpene derivatives, dactylospenes A-E (1-5), as well as two known biosynthetically related compounds, luffariellolide (6) and furospinosulin B (7). The structures of these compounds were elucidated on the basis of their spectroscopic data, experimental and calculated electronic circular dichroism (ECD) analysis, as well as comparison of the NMR data with those of known analogs. These metabolites are the first γ-oxygenated butenolide sesterterpenes to be reported from this genus. These compounds were evaluated in antimicrobial, anti-inflammatory, and cytotoxic assays. Only compounds 1, 3, and 6 exhibited moderate cytotoxicity against DU145, SW1990, Huh7, and PANC-1 cancer cell lines with IC50 values in the range of 2.11-13.35 µM. Furthermore, compound 2, without cytotoxicity, exhibited significant inhibitory effects (inhibitory rate 77.5%) on nitric oxide production induced by lipopolysaccharide at 10 µM.

On the mass spectrometric fragmentations of the bacterial sesterterpenes sestermobaraenes A-C.

A 13C-labelling was introduced into each individual carbon of the recently discovered sestermobaraenes by the enzymatic conversion of the correspondingly 13C-labelled isoprenyl diphosphate precursors with the sestermobaraene synthase from Streptomyces mobaraensis. The main compounds sestermobaraenes A, B, and C were analysed by gas chromatography-mass spectrometry (GC-MS), allowing for a deep mechanistic investigation of the electron impact mass spectrometry (EIMS) fragmentation reactions of these sesterterpene hydrocarbons.

Chemistry and biology of ophiobolin A and its congeners.

Ophiobolin A is a fungal secondary metabolite that was found to have significant activity against apoptosis-resistant glioblastoma cells through the induction of a non-apoptotic cell death, offering an innovative strategy to combat this aggressive cancer. The current article aims to make the bridge between the anti-cancer effects of ophiobolin A and its unique reaction with primary amines and suggests that pyrrolylation of lysine residues on its intracellular target protein(s) and/or phosphatidylethanolamine lipid is responsible for its biological effects. The article also discusses chemical derivatization of ophiobolin A to establish first synthetically generated structure-activity relationship. Finally, the reported total synthesis efforts toward the ophiobolin class of sesterterpenes are discussed and identified as a fertile area for improvement in pursuit of these molecules as anticancer agents.

Bipolarolides†A-G: Ophiobolin-Derived Sesterterpenes with Three New Carbon Skeletons from Bipolaris sp.†TJ403-B1.

Bipolarolides A-G (1-7), seven novel ophiobolin-derived sesterterpenes with three new types of skeletons, were characterized from fungus Bipolaris sp. TJ403-B1. Their structures were determined via spectroscopic analyses, X-ray crystallography, and quantum chemical 13 C NMR and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 were uniquely defined by a multicyclic caged oxapentacyclo[9.3.0.01,6 .05,9 .18,12 ]pentadecane-bridged system. Compounds 3 and 4 featured an unprecedented 5-5-5-5-fused core skeleton, while 3 also contained an unexpected C-3-C-14 oxygen bridge to construct the caged architecture. Compounds 5-7 form a new class of highly modified pentacyclic oxaspiro[4.4]nonane-containing sesterterpene-alkaloid hybrids. Their biosynthetic pathways and potential HMG-CoA reductase inhibitory and antimicrobial activities are also discussed.

Scalalactams A⁻D, Scalarane Sesterterpenes with a γ-Lactam Moiety from a Korean Spongia Sp. Marine Sponge.

Intensive study on the chemical components of a Korean marine sponge, Spongia sp., has led to the isolation of four new scalarane sesterterpenes, scalalactams A⁻D (1⁻4). Their chemical structures were elucidated from the analysis of spectroscopic data including 1D-and 2D-NMR as well as MS data. Scalalactams A⁻D (1⁻4) possess a scalarane carbon skeleton with a rare structural feature of a γ-lactam moiety within the molecules. Scalalactams A and B (1 and 2) have an extended isopropanyl chain at the lactam ring, and scalalactams C and D (3 and 4) possess a phenethyl group at the lactam ring moiety. Scalalactams A⁻D (1⁻4) did not show FXR antagonistic activity nor cytotoxicity up to 100 µM.

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate.

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short-chain all-trans (E) prenyl diphosphates geranyl diphosphate (GDP, C10 ), farnesyl diphosphate (FDP, C15 ) or geranylgeranyl diphosphate (GGDP, C20 ). In the genome of Arabidopsis thaliana, 15 trans-product-forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC-MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C25 ) instead of GGDP as their major product in enzyme assays performed in vitro. Over-expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N-terminal to the first aspartate-rich motif is responsible for C25 versus C20 product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C20 ) as a product and a larger R group (Met) resulting in GFDP (C25 ).

The Role of Spongia sp. in the Discovery of Marine Lead Compounds.

A comprehensive review on the chemistry of Spongia sp. is here presented, together with the biological activity of the isolated compounds. The compounds are grouped in sesquiterpene quinones, diterpenes, C21 and other linear furanoterpenes, sesterterpenes, sterols (including secosterols), macrolides and miscellaneous compounds. Among other reports we include studies on the intraspecific diversity of a Mediterranean species, compounds isolated from associated sponge and nudibranch and compounds isolated from S. zimocca and the red seaweed Laurentia microcladia. Under biological activity a table of the reported biological activities of the various compounds and the biological screening of extracts are described. The present review covers the literature from 1971 to 2015.

Cytotoxic scalarane-type sesterterpenes from the Saudi Red Sea sponge Hyrtios erectus.

The CHCl3/MeOH extract of the marine sponge Hyrtios erectus showed cytotoxicity against three cancer cell lines HepG2, A549, and PC-3 with IC50 0.055, 0.044, and 0.023 µg/ml, respectively. The CH2Cl2 soluble fraction afforded three scalarane sesterterpenes (1-3) along with a cholestane derivative (4) and an indole alkaloid (5). Chemical structures were established by spectroscopic techniques and comparison with data reported in the literature. Scalarinol (1) was found as a new metabolite, while heteronemin (2) and 12-O-deacetyl-19-deoxyscalarin (3) are known compounds. 1-3 exhibited cytotoxic activity against the cancer cell lines with IC50 values ranging from 14 to 230 µM. The molecular affinity to the DNA was employed as marker to examine the proposed mechanism of cytotoxic activities. Compound 2, with IC50 28 µg/ml, displayed the highest affinity to the DNA.

A New Bioactive Metabolite Isolated from the Red Sea Marine Sponge Hyrtios erectus.

Chemical investigation of the lipophilic fraction of Hyrtios erectus, a Red Sea sponge, yielded a new pentacyclic nitrogen-containing scalarane; 24-methoxypetrosaspongia C (1), together with the previously reported scalaranes sesterstatin 3 (2), 12-deacetyl-12-epi-scalaradial (3) and 12-deacetyl-12,18-di-epi-scalaradial (4). The compounds were identified using HRESIMS, 1D and 2D NMR experiments. The isolated compounds showed growth inhibitory activity against hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116) and breast adenocarcinoma cells (MCF-7).

Research Progress on Fungal Sesterterpenoids Biosynthesis.

Sesterterpenes are 25-carbon terpenoids formed by the cyclization of dimethyl allyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) as structural units by sesterterpenes synthases. Some (not all) sesterterpenoids are modified by cytochrome P450s (CYP450s), resulting in more intricate structures. These compounds have significant physiological activities and pharmacological effects in anti-inflammatory, antibacterial, antitumour, and hypolipidemic communities. Despite being a rare class of terpenoids, sesterterpenoids derived from fungi show a wide range of structural variations. The discovered fungal sesterterpenoid synthases are composed of C-terminal prenyltransferase (PT) and N-terminal terpene synthase (TS) domains, which were given the name PTTSs. PTTSs have the capacities to catalyze chain lengthening and cyclization concurrently. This review summarizes all 52 fungal PTTSs synthases and their biosynthetic pathways involving 100 sesterterpenoids since the discovery of the first PTTSs synthase from fungi in 2013.

Molecular Docking and Dynamics Simulation Study of Hyrtios erectus Isolated Scalarane Sesterterpenes as Potential SARS-CoV-2 Dual Target Inhibitors.

Presently, the world is under the toll of pandemic coronavirus disease-2019 (COVID-19) outbreak caused by SARS-CoV-2. Lack of effective and safe therapeutics has stressed the scientific community for developing novel therapeutics capable of alleviating and stopping this pandemic. Within the presented study, molecular docking, ADME properties and all-atom molecular dynamic (MD) simulation, along with two standard antiviral agents (lopinavir and benzopurpurin-4B), were applied to investigate 15 scalaranes sesterterpenes natural compounds, purified from the Red Sea marine sponge Hyrtios erectus, as potential COVID-19 dual-target inhibitors. Following multi-step docking within COVID-19 main protease and Nsp15 endoribonuclease cavities, nine promising drug-like compounds exhibited higher docking scores as well as better interactions with the target's crucial residues than those of reference ligands. Compounds 2, 6, 11, and 15, were predicted to simultaneously subdue the activity of the two COVID-19 targets. Dynamics behavior of the best-docked molecules, compounds 15 and 6, within COVID-19 target pockets showed substantial stability of ligand-protein complexes as presented via several MD simulation parameters. Furthermore, calculated free-binding energies from MD simulation illustrated significant ligand's binding affinity towards respective target pockets. All provided findings supported the utility of scalarane-based sesterterpenes, particularly compounds 15 and 6, as promising lead candidates guiding the development of effective therapeutics against SARS-CoV-2.

Sesterterpenes and macrolide derivatives from the endophytic fungus Aplosporella javeedii.

Five sesterterpenes (1-5) including two new compounds (1 and 2), as well as a new (6) and a known macrolide (7) were isolated from the endophytic fungus Aplosporella javeedii. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and HRMS data as well as by comparison with the literature. Compound 4 and its acetyl derivatives 4a, 4b, 4c which were prepared by acetylation of 4 exhibited moderate cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 6.2 to 12.8 µM, respectively. Moreover, 4a and 4c exhibited also cytotoxicity against human leukemia (Jurkat J16) and lymphoma (Ramos) cell lines. Compound 7 showed strong cytotoxicity against the L5178Y cell line, as well as against human Jurkat J16 and Ramos cells with IC50 values of 0.4, 5.8, and 4.4 µM, respectively. Mechanistic studies indicated that 7 induces apoptotic cell death. In addition, compounds 3, 4 and 7 showed low antibacterial activities against Mycobacterium tuberculosis H37Rv and compound 6 against Staphylococcus aureus, respectively, with MICs of 100 µM. Preliminary structure-activity relationships are discussed.

Novel Antimicrobial Compounds as Ophiobolin-Type Sesterterpenes and Pimarane-Type Diterpene From Bipolaris Species TJ403-B1.

Six previously undescribed ophiobolin-type sesterterpenes, namely, bipolatoxins A-F (1-6); and one previously undescribed pimarane-type diterpene, namely, 1ß-hydroxy momilactone A (7); together with three known compounds, namely, 25-hydroxyophiobolin I (8), ophiobolin I (9), and ophiobolin A lactone (10); were isolated and identified from the endophytic fungus Bipolaris species TJ403-B1. Their structures with absolute configurations were elucidated on the basis of extensive spectroscopic analyses (including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy data), single-crystal X-ray diffraction analyses, and comparison of experimental circular dichroism data. All compounds (except for 5) were evaluated for antimicrobial potential, which indicated that bipolatoxin D (4) showed significant inhibitory activity against Enterococcus faecalis with a minimum inhibitory concentration (MIC) value of 8 µg/mL, and ophiobolin A lactone (10) showed significant inhibitory activity against Acinetobacter baumannii and E. faecalis with MIC values of 8 and 8 µg/mL, respectively.

Bipolarins A-H, eight new ophiobolin-type sesterterpenes with antimicrobial activity from fungus Bipolaris sp. TJ403-B1.

Bipolarins A-H (1-8), eight new tetracyclic ophiobolin-type sesterterpenes featuring a rare oxaspiro[4.4]nonane moiety, were isolated from cultures of fungus Bipolaris sp. TJ403-B1. Their structures and absolute configurations were elucidated by comprehensive spectroscopic analyses, single-crystal X-ray diffraction experiments, electronic circular dichroism and 13C NMR calculations. Additionally, compound 5 exhibited significant selective antimicrobial activity against Enterococcus faecalis with an MIC value 8 µg·mL-1.

Ophiobolins P-T, five new cytotoxic and antibacterial sesterterpenes from the endolichenic fungus Ulocladium sp.

Five ophiobolane sesterterpenes, ophiobolins P-T, and three known compounds, 6-epi-21,21-O-dihydroophiobolin G, 6-epi-ophiobolin G and 6-epi-ophiobolin K, were isolated from the acetone extract of the endolichenic fungus Ulocladium sp. by using OSMAC method. Their structures were elucidated on the basis of spectroscopic analysis. The absolute configuration of the 18,19-diol moieties in ophiobolin Q was assigned using the Frelek's method. The cytotoxic effects on KB and HepG2 cell lines, antibacterial activity against Bacillus subtilis, methicillin-resistant Staphylococcus aureus, and Bacille Calmette-Guerin were evaluated for all isolated compounds. Ophiobolin T and 6-epi-ophiobolin G exhibited the most potent cytotoxic activity against HepG2 with IC50 of 0.24 and 0.37 µM, respectively. In antibacterial assay, ophiobolins P and T showed moderate antibacterial activity against B. subtilis and meticillin-resistant S. aureus. Ophiobolin T also displayed moderate antibacterial activity against the Bacille Calmette-Guerin strain.