Telescoping a Prenyltransferase and a Diterpene Synthase to Transform Unnatural FPP Derivatives to Diterpenoids.

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.

Discovery of a terpene synthase synthesizing a nearly non-flexible eunicellane reveals the basis of flexibility.

Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely present in marine corals, but rarely found in bacteria and plants. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the mechanisms controlling flexibility remain unknown. The discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a nearly non-flexible eunicellane skeleton, enable us to propose a feasible theory about the flexibility in eunicellane structures. Parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-geranylgeranyl diphosphate incubations and density functional theory-based Boltzmann population computations, reveale that a trans-fused bicycle with a 2Z-configuration alkene restricts conformational flexibility resulting in a nearly non-flexible eunicellane skeleton. The catalytic route and the enzymatic mechanism of MicA are also elucidated by labeling experiments, density functional theory calculations, structural analysis of the artificial intelligence-based MicA model, and mutational studies.

Blocking smoothened cholesterylation: A strategy for overcoming drug resistance in cancer.

In this issue of Cell Chemical Biology, Liu et al.1 report the identification of Q29, a synthetic diterpenoid that blocks covalent cholesterol modification of smoothened (SMO) and inhibits hedgehog signaling. Q29 is capable of suppressing tumor cell growth, both in vitro and in vivo, and overcoming resistance to SMO inhibitors.

Total synthesis and structure-antifouling activity relationship of scabrolide F.

An efficient synthetic strategy for scabrolide F (7), a norcembranolide diterpene that was isolated from the Taiwanese soft coral Sinularia scabra, has only recently been reported by our group. Herein, we report details of the first total synthesis of 7. The tetrahydrofuran domain of 7 was stereoselectively constructed via the 5-endo-tet cyclization of a hydroxy vinyl epoxide. The reaction of alkyl iodide 30 with dithiane 38, followed by the introduction of an alkene moiety, afforded allylation precursor 41. The coupling of alkyl iodide 42 and allylic stannane 43 was examined as a model experiment of allylation. Because the desired allylated product 44 was not obtained, an alternative synthetic route toward 7 was investigated instead. In the second synthetic approach, fragment-coupling between alkyl iodide 56 and aldehyde 58, macrolactonization, and transannular ring-closing metathesis were used as the key steps to achieve the first total synthesis of 7. We hope that this synthetic strategy provides access to the total synthesis of other macrocyclic norcembranolides. We also evaluated the antifouling activity and toxicity of 7 and its synthetic intermediates toward the cypris larvae of the barnacle Amphibalanus amphitrite. This study is the first to report the antifouling activity of norcembranolides as well as the biological activity of 7.

Kallopterolides A-I, a New Subclass of seco-Diterpenes Isolated from the Southwestern Caribbean Sea Plume Antillogorgia kallos.

Kallopterolides A-I (1-9), a family of nine diterpenoids possessing either a cleaved pseudopterane or a severed cembrane skeleton, along with several known compounds were isolated from the Caribbean Sea plume Antillogorgia kallos. The structures and relative configurations of 1-9 were characterized by analysis of HR-MS, IR, UV, and NMR spectroscopic data in addition to computational methods and side-by-side comparisons with published NMR data of related congeners. An investigation was conducted as to the potential of the kallopterolides as plausible in vitro anti-inflammatory, antiprotozoal, and antituberculosis agents.

Combining the Elicitor Up-Regulated Production of Unusual Linear Diterpene-Derived Variants for an In-Depth Assessment of the Application Value and Risk of the Medicinal and Edible Basidiomycete Schizophyllum commune.

To better assess the practical value and avoid potential risks of the traditionally medicinal and edible basidiomycete Schizophyllum commune, which may arise from undescribed metabolites, a combination of elicitors was introduced for the first time to discover products from cryptic and low-expressed gene clusters under laboratory cultivation. Treating S. commune NJFU21 with the combination of five elicitors led to the upregulated production of a class of unusual linear diterpene-derived variants, including eleven new ones (1-11), along with three known ones (12-14). The structures and stereochemistry were determined by 1D and 2D NMR, HRESIMS, ECD, OR and VCD calculations. Notably, the elongation terminus of all the diterpenes was decorated by an unusual butenedioic acid moiety. Compound 1 was a rare monocyclic diterpene, while 2-6 possessed a tetrahydrofuran moiety. The truncated metabolites 4, 5 and 13 belong to the trinorditerpenes. All the diterpenes displayed approximately 70% scavenging of hydroxyl radicals at 50 µM and null cytotoxic activity at 10 µM. In addition, compound 1 exhibited potent antifungal activity against the plant pathogenic fungi Colletotrichum camelliae, with MIC values of 8 µg/mL. Our findings indicated that this class of diterpenes could provide valuable protectants for cosmetic ingredients and the lead compounds for agricultural fungicide development.

Bletilla striata polysaccharide-coated andrographolide nanomicelles for targeted drug delivery to enhance anti-colon cancer efficacy.

Background: Vitamin E, which is also known as tocopherol, is a compound with a polyphenol structure. Its esterified derivative, Vitamin E succinate (VES), exhibits unique anticancer and healthcare functions as well as immunomodulatory effects. Natural polysaccharides are proved to be a promising material for nano-drug delivery systems, which show excellent biodegradability and biocompatibility. In this study, we employed a novel bletilla striata polysaccharide-vitamin E succinate polymer (BSP-VES) micelles to enhance the tumor targeting and anti-colon cancer effect of andrographolide (AG). Methods: BSP-VES polymer was synthesized through esterification and its structure was confirmed using 1H NMR. AG@BSP-VES was prepared via the dialysis method and the drug loading, entrapment efficiency, stability, and safety were assessed. Furthermore, the tumor targeting ability of AG@BSP-VES was evaluated through targeted cell uptake and in vivo imaging. The antitumor activity of AG@BSP-VES was measured in vitro using MTT assay, Live&Dead cell staining, and cell scratch test. Results: In this study, we successfully loaded AG into BSP-VES micelles (AG@BSP-VES), which exhibited good stability, biosafety and sustained release effect. In addition, AG@BSP-VES also showed excellent internalization capability into CT26 cells compared with NCM460 cells in vitro. Meanwhile, the specific delivery of AG@BSP-VES micelles into subcutaneous and in-situ colon tumors was observed compared with normal colon tissues in vivo during the whole experiment process (1-24 h). What's more, AG@BSP-VES micelles exhibited significant antitumor activities than BSP-VES micelles and free AG. Conclusion: The study provides a meaningful new idea and method for application in drug delivery system and targeted treatment of colon cancer based on natural polysaccharides.

Anti-Leishmania effect of icetexanes from Salvia procurrens.

BACKGROUND AND PURPOSE: Leishmaniasis is a globally prevalent vector-borne disease caused by parasites of the genus Leishmania. The available chemotherapeutic drugs present problems related to efficacy, emergence of parasite resistance, toxicity and high cost, justifying the search for new drugs. Several classes of compounds have demonstrated activity against Leishmania, including icetexane-type diterpenes, previously isolated from Salvia and other Lamiaceae genera. Thus, in this study, compounds of Salvia procurrens were investigated for their leishmanicidal and immunomodulatory activities. METHODS: The exudate of S. procurrens was obtained by rapidly dipping the aerial parts in dichloromethane. The compounds were isolated by column and centrifugal planar chromatography over silica gel. The effects on L. amazonensis growth, survival, membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential and cytotoxicity of the compounds towards human erythrocytes, peripheral blood mononuclear cells and macrophages were evaluated. The effects on intracellular amastigote forms, nitric oxide (NO) and TNF-α production were also investigated. RESULTS: The exudate from the leaves afforded the novel icetexane 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2), fruticulin A (3) and demethylfruticulin A (4). The compounds (1-4) were tested against promastigotes of L. amazonensis and showed an effective inhibition of the parasite survival (IC50 = 4.08-16.26 µM). In addition, they also induced mitochondrial ROS production, plasma membrane permeability and mitochondrial dysfunction in treated parasites, and presented low cytotoxicity against macrophages. Furthermore, all diterpenes tested reduced the number of parasites inside macrophages, by mechanisms involving TNF-α, NO and ROS. CONCLUSION: The results suggest the potential of 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2),fruticulin A (3) and demethylfruticulin A (4) as candidates for use in further studies on the design of anti-leishmanial drugs.

Mechanistic characterisation of the diterpene synthase for clitopilene and identification of isopentalenene synthase from the fungus Clitopilus passeckerianus.

Two terpene synthases from the pleuromutilin producing fungus Clitopilus passeckerianus were functionally characterised. The first enzyme CpTS1 produces the new diterpene clitopilene with a novel 6-6-5-5 tetracyclic skeleton, while the second enzyme CpTS2 makes the new sesquiterpene isopentalenene. The CpTS1 reaction mechanism was studied in depth using experimental and theoretical approaches.

New hemisynthetic derivatives of sphaeropsidin phytotoxins triggering severe endoplasmic reticulum swelling in cancer cells.

Sphaeropsidins are iso-pimarane diterpenes produced by phytopathogenic fungi that display promising anticancer activities. Sphaeropsidin A, in particular, has been shown to counteract regulatory volume increase, a process used by cancer cells to avoid apoptosis. This study reports the hemi-synthesis of new lipophilic derivatives obtained by modifications of the C15,C16-alkene moiety. Several of these compounds triggered severe ER swelling associated with strong proteasomal inhibition and consequently cell death, a feature that was not observed with respect to mode of action of the natural product. Significantly, an analysis from the National Cancer Institute sixty cell line testing did not reveal any correlations between the most potent derivative and any other compound in the database, except at high concentrations (LC50). This study led to the discovery of a new set of sphaeropsidin derivatives that may be exploited as potential anti-cancer agents, notably due to their maintained activity towards multidrug resistant models.

Talaroacids A-D and Talaromarane A, Diterpenoids with Anti-Inflammatory Activities from Mangrove Endophytic Fungus Talaromyces sp. JNQQJ-4.

Five new diterpenes including four diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A-D (1-4) and an isopimarane diterpenoid talaromarane A (5) were isolated from the mangrove endophytic fungus Talaromyces sp. JNQQJ-4. Their structures and absolute configurations were determined by analysis of high-resolution electrospray ionization mass spectroscopy (HRESIMS), 1D/2D Nuclear Magnetic Resonance (NMR) spectra, single-crystal X-ray diffraction, quantum chemical calculation, and electronic circular dichroism (ECD). Talaromarane A (5) contains a rare 2-oxabicyclo [3.2.1] octan moiety in isopimarane diterpenoids. In bioassays, compounds 1, 2, 4, and 5 displayed significant anti-inflammatory activities with the IC50 value from 4.59 to 21.60 µM.

Diterpenoids with Antibacterial Activities from the Fungus Trichoderma harzianum.

A new fusicoccane diterpenoid, harziaderma A (1), two novel harziane diterpenoids, harzianones G and H (2 and 3), one revised harziane diterpenoid (4), and two known diterpenoids (5 and 6) were isolated from the fungus Trichoderma harzianum and established via NMR, HRESIMS, Mo2(OAc)4-induced circular dichroism (ICD) and electronic circular dichroism (ECD) calculations. It is worth noting that compound 1 represents the first instance of a fusicoccane-type diterpenoid derived from T. harzianum. The structure of furanharzianone B was revised to 4 via careful spectroscopic analyses. Additionally, compounds 2 and 5 could suppress the overall growth of the foodborne bacterial pathogen Bacillus cereus. Compound 4 showed a moderate suppressive impact on NO generation in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The discoveries from the current study not only expanded the structural variety of diterpenoids isolated from T. harzianum but also laid a robust foundation for the development of harziane diterpenoids as anti-foodborne pathogen agents.

On the Cholesterol Raising Effect of Coffee Diterpenes Cafestol and 16-O-Methylcafestol: Interaction with Farnesoid X Receptor.

The diterpene cafestol represents the most potent cholesterol-elevating compound known in the human diet, being responsible for more than 80% of the effect of coffee on serum lipids, with a mechanism still not fully clarified. In the present study, the interaction of cafestol and 16-O-methylcafestol with the stabilized ligand-binding domain (LBD) of the Farnesoid X Receptor was evaluated by fluorescence and circular dichroism. Fluorescence quenching was observed with both cafestol and 16-O-methylcafestol due to an interaction occurring in the close environment of the tryptophan W454 residue of the protein, as confirmed by docking and molecular dynamics. A conformational change of the protein was also observed by circular dichroism, particularly for cafestol. These results provide evidence at the molecular level of the interactions of FXR with the coffee diterpenes, confirming that cafestol can act as an agonist of FXR, causing an enhancement of the cholesterol level in blood serum.

Synthetic Modifications of Andrographolide Targeting New Potential Anticancer Drug Candidates: A Comprehensive Overview.

This review collects the synthetic modifications performed on andrographolide, a natural molecule derived from Andrographis paniculata, for oncology applications. Various pharmacomodulations were carried out, and the products were tested on different cancer cell lines. The impact of these modifications was analyzed with the aim of mapping the positions essential for activity to facilitate future research in this field. However, this study makes it clear that, in addition to structural modifications of the molecule, which can result in varying degrees of effectiveness in targeting interactions, the lipophilic capacity of the structures obtained through hemisynthesis is of significant importance.

Methods for the preparation and analysis of a bifunctional class II diterpene synthase, copalyl diphosphate synthase from Penicillium fellutanum.

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Homotypic cell membrane-camouflaged biomimetic PLGA nanoparticle loading triptolide for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated death worldwide. Beside early detection, early diagnosis, and early surgery, it is urgent to try new strategies for the treatment of HCC. Triptolide (TPL) has been employed to treat HCC. However, its clinical applications were restricted by the narrow therapeutic window, severe toxicity, and poor water-solubility. In this study, we developed cancer cell membrane-camouflaged biomimetic PLGA nanoparticles loading TPL (TPL@mPLGA) with the homologous targeting property for the treatment of HCC. The TPL@mPLGA was successfully prepared with particle size of 195.5 ± 7.5 nm and zeta potential at -21.5 ± 0.2 mV with good stability. The drug loading (DL) of TPL@mPLGA was 2.94%. After Huh-7 cell membrane coating, the natural Huh-7 cell membrane proteins were found to be retained on TPL@mPLGA, thus endowing the TPL@mPLGA with enhanced accumulation at tumor site, and better anti-tumor activity in vitro and in vivo when compared with TPL or TPL@PLGA. The TPL@mPLGA showed enhanced anti-tumor effects and reduced toxicity of TPL, which could be adopted for the treatment of HCC.

Albiflorenes A-L, polyoxygenated cyclohex(a/e)ne diterpene esters from Kaempferia albiflora.

Twelve polyoxygenated cyclohex(a/e)ne diterpene esters, named albiflorenes A-L (1-12), were isolated from the whole plants of Kaempferia albiflora, known as "Prao Mang Mum." Their structures and relative stereochemistry were determined by extensive spectroscopic analysis. Furthermore, the comparison of experimental electronic circular dichroism (ECD) curves with the curves predicted by TDDFT was used to determine the absolute configurations. Albiflorenes contain polyoxygenated cyclohexane (or cyclohexene) derivatives, which are linked to either isopimarane or abietane diterpene acid units. The discovery marks the first occurrence of a conjugate between polyoxygenated cyclohexane (or cyclohexene) rings and diterpenoids. Among the isolates, albiflorene C specifically exhibited antibacterial activity against Bacillus cereus with MIC and MBC values of 3.13 and 6.25 µg/mL, respectively.

A green and simple method for enrichment of major diterpenoids from the buds of Wikstroemia chamaedaphne with macroporous resins and their activation of latent human immunodeficiency virus activity.

In this study, a green and efficient enrichment method for the four majors active diterpenoid components: pimelotide C, pimelotide A, simplexin, and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate in the buds of Wikstroemia chamaedaphne was established using macroporous resin chromatography. The adsorption and desorption rates of seven macroporous resins were compared using static tests. The D101 macroporous resin exhibited the best performance. Static and dynamic adsorption tests were performed to determine the enrichment and purification of important bioactive diterpenoids in the buds of W. chamaedaphne. Diterpenoid extracts were obtained by using D101 macroporous resin from the crude extracts of W. chamaedaphne. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that most of the diterpenoids were enriched in diterpenoid extracts. These results confirmed that diterpenoids in the buds of W. chamaedaphne could be enriched using macroporous resin technology, and the enriched diterpenoid extracts showed more efficient activation of the latent human immunodeficiency virus. This study provides a novel strategy for discovering efficient and low-toxicity latency-reversing agents and a potential basis for the comprehensive development and clinical application of the buds of W. chamaedaphne.

Gibberellin-biosynthetic ent-kaurene synthases in higher plants do not require their non-catalytic domains for the catalysis.

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αßγ domains. Active sites of ent-CPS exist at the interface of ß and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or ßγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its ßγ domains. Domain-swapping experiments showed that replacing ßγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of ßγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without ßγ domains.