Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes.

Elucidation of the cyclization mechanism catalyzed by terpene synthases is important for the rational engineering of terpene cyclases. We developed a chemoenzymatic method for the synthesis of systematically deuterium-labeled geranylgeranyl diphosphate (GGPP), starting from site-specifically deuterium-labeled isopentenyl diphosphates (IPPs) using IPP isomerase and three prenyltransferases. We examined the cyclization mechanism of tetracyclic diterpene phomopsene with phomopsene synthase. A detailed EI-MS analysis of phomopsene labeled at various positions allowed us to propose the structures corresponding to the most intense peaks, and thus elucidate a cyclization mechanism involving double 1,2-alkyl shifts and a 1,2-hydride shift via a dolabelladien-15-yl cation. Our study demonstrated that this newly developed method is highly sensitive and provides sufficient information for a reliable assignment of the structures of fragmented ions.

Seed dormancy breaking diterpenoids from the liverwort Plagiochila sciophila and their differentiation inducing activity in human promyelocytic leukemia HL-60 cells.

To obtain the structural diversity of bioactive compounds similar to cotylenins and fusicoccins that modulate 14-3-3 protein-protein interactions in eukaryotes, screening tests were carried out using the lettuce seed dormancy breaking-assay. An acetone extract of the liverwort Plagiochila sciophila exhibited significant activity against the seeds in the presence of the plant hormone abscisic acid. Activity-guided fractionation of the extract afforded the isolation of seven novel fusicoccane-type diterpenoids, named fusicosciophins A-E (1-5), 8-deacetyl (6) and 9-deacetyl fusicosciophin E (7). Their structures were determined by spectroscopic methods and X-ray crystallographic analyses. All the pure isolated compounds (1-7) exhibited moderate lettuce seed dormancy breaking activity. In addition, the differentiation-inducing activity and cytotoxicity of these isolates, together with fusicoccin A (FC-A) and all-trans retinoic acid (ATRA), were evaluated in human promyelocytic leukemia HL-60 cells and human mouth epidermal carcinoma KB cells, respectively. Fusicosciophins (2 and 4) and FC-A exhibited moderate differentiation-inducing activity (EC50 31.2-59.1 microM) compared with ATRA (EC50 0.3 microM), while 2, 4 and ATRA exhibited higher selectivity indices (IC50/EC50 >3.38-667) than FC-A (IC50/EC50 1.05). This is the first report on the isolation of fusicoccane-type diterpenoids from liverworts having seed dormancy breaking activity and differentiation-inducing activity in mammal cells.

Seed dormancy breaking diterpenoids, including novel brassicicenes J and K, from fungus Alternaria brassicicola, and their necrotic/apoptotic activities in HL-60 cells.

To find new metabolites similar to cotylenins and fusicoccins from the fungus Alternaria brassicicola, screening tests were carried out using the lettuce seed dormancy breaking assay. Activity-guided fractionation of the EtOAc extract from the culture using the assay afforded the isolation of two novel fusicoccane diterpenoids named brassicicenes J (1) and K (2), along with three known brassicicenes A (3), B (4), and F (5). Their structures were elucidated from extensive NMR spectral data and by comparison of these with those reported in the literature. Brassicicenes (1-5) exhibited weak to moderate seed dormancy breaking activities against lettuce seeds in the presence of abscisic acid. In addition, the necrotic/apoptotic activities of the brassicicenes (1-5), fusicoccin A (6) and cotylenin A (7) were evaluated by determining their cytotoxicity, cell viability and caspase-3/7 activation on the HL-60 cell line. Brassicicene K (2) exhibited similar cytostatic profiles to that of cotylenin A (7), and brassicicenes J (1), A (3), B (4), and F (5) exhibited necrotic activity. This is the first report of the seed dormancy breaking activity of brassicicenes in plants, and of necrotic/apoptotic activity in mammalian cells.

Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers.

One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas. However, they paradoxically promote the growth of RAS mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in RAS mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth in vitro and in vivo. This defines a novel pharmacologic strategy for treatment of RAS mutant cancers.

Molecular breeding of a fungus producing a precursor diterpene suitable for semi-synthesis by dissection of the biosynthetic machinery.

Many clinically useful pharmaceuticals are semi-synthesized from natural products produced by actinobacteria and fungi. The synthetic protocols usually contain many complicated reaction steps and thereby result in low yields and high costs. It is therefore important to breed microorganisms that produce a compound most suitable for chemical synthesis. For a long time, desirable mutants have been obtained by random mutagenesis and mass screening. However, these mutants sometimes show unfavorable phenotypes such as low viability and low productivity of the desired compound. Fusicoccin (FC) A is a diterpene glucoside produced by the fungus Phomopsis amygdali. Both FC and the structurally-related cotylenin A (CN) have phytohormone-like activity. However, only CN exhibits anti-cancer activity. Since the CN producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out, and elimination of the hydroxyl group at position 12 of FC was essential to mimic the CN-like activity. Based on detailed dissection of the biosynthetic machinery, we constructed a mutant producing a compound without a hydroxyl group at position 12 by gene-disruption. The mutant produced this compound as a sole metabolite, which can be easily and efficiently converted into an anti-cancer drug, and its productivity was equivalent to the sum of FC-related compounds produced by the parental strain. Our strategy would be applicable to development of pharmaceuticals that are semi-synthesized from fungal metabolites.

An enzyme catalyzing O-prenylation of the glucose moiety of fusicoccin A, a diterpene glucoside produced by the fungus Phomopsis amygdali.

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane-bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant-pathogenic fungus Phomopsis amygdali and has a unique O-prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6'-hydroxy group of the glucose moiety of FC A to yield an O-prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K(m) values were calculated as 0.49±0.037 µM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 µM for DMAPP, with a k(cat) of 55.3±3.3×10⁻³ s. The enzyme did not act on representative substrates of the above-mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.

A novel fusicoccin derivative preferentially targets hypoxic tumor cells and inhibits tumor growth in xenografts.

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy. We report here that a novel fusicoccin derivative (ISIR-042), but not its parent or related compounds such as fusicoccin A and cotylenin A, is more cytotoxic to hypoxic cells than to normoxic cells. The hypoxia-induced accumulation of hypoxia-inducible factor (HIF)-1α and the phosphorylation of Akt were effectively inhibited by treatment with ISIR-042, suggesting that the preferential cytotoxicity toward hypoxic cells is associated with a reduction of HIF-1α and Akt activation. ISIR-042 inhibited the growth of human pancreatic cancer MIAPaCa-2 cells while sparing normal endothelial cells, and significantly inhibited the growth of MIAPaCa-2 cells as xenografts without apparent adverse effects. Pancreatic cancer cells expressing CD24 and CD44 exhibited characteristics of stem cells. Treatment with gemcitabine increased this stem cell-enriched population, and this effect was significantly inhibited by ISIR-042, suggesting that ISIR- 042 preferentially inhibits stem/progenitors in pancreatic cancer cell lines compared with chemotherapeutic agents. These results suggest that ISIR-042 may be a potential therapeutic agent for hypoxic tumors such as pancreatic cancer.

Dioxygenases, key enzymes to determine the aglycon structures of fusicoccin and brassicicene, diterpene compounds produced by fungi.

Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8ß,16-diol. In this study, we report that a α-ketoglutarate dependent dioxygenase, the gene coding for which was located in the cluster, catalyzed a hydroxylation at the 3-position of fusicocca-2,10(14)-diene-8ß,16-diol. On the other hand, a α-ketoglutarate-dependent dioxygenase, which had been identified in a fusicoccin A biosynthetic gene cluster, catalyzed the 16-oxidation of fusicocca-2,10(14)-diene-8ß,16-diol to yield an aldehyde (8ß-hydroxyfusicocca-1,10(14)-dien-16-al), although both dioxygenases had 51% amino acid sequence identity. These findings suggested that the dioxygenases played critical roles for the formation of the fusicoccin A-type and cotylenin A-/brassicicene C-type aglycons. Moreover, we showed that short-chain dehydrogenase/reductase located in the fusicoccin A biosynthetic gene cluster catalyzed the reduction of the aldehyde to yield fusicocca-1,10(14)-diene-8ß,16-diol.

Cotylenin A inhibits cell proliferation and induces apoptosis and PAX6 mRNA transcripts in retinoblastoma cell lines.

PURPOSE: Retinoblastoma, a childhood cancer of the retina, is caused by inactivation of the tumor suppressor gene retinoblastoma (RB). Cotylenin A (CN-A), a novel fusicoccane-diterpene glycoside, accelerates the differentiation of several types of myeloid cell lines and is a candidate for a new type of anticancer therapeutic agent with this effect. However, whether CN-A has the same effect on retinoblastoma cells is unknown. We studied the response of two retinoblastoma cell lines, Y-79 and WERI-Rb-1, to CN-A. METHODS: We studied the response of two retinoblastoma cell lines to CN-A with respect to cell growth, apoptosis, morphology, mRNA, protein expression analysis of specific genes (N-myc, cyclin-dependent kinase inhibitor 1A [P21], paired box gene 6 [PAX6], and rhodopsin [RHO]), and activity of three PAX6 promoters (P0, P1, and Palpha). RESULTS: CN-A inhibited cell proliferation and induced apoptosis via caspase activity in the two retinoblastoma cell lines. In addition, CN-A induced mRNA expression of P21, PAX6, and RHO and protein expression of P21. In Y-79 cells, PAX6 P1 promoter was activated by CN-A. In WERI-Rb-1 cells, PAX6 P0, P1, and Palpha promoter were activated by CN-A. CN-A decreased mRNA and protein expression of N-myc in two retinoblastoma cell lines. CONCLUSIONS: The responses of retinoblastoma cells to CN-A include inhibition of cell growth, induction of apoptosis, and the potential to change neuroblastoma characteristics of retinoblastoma cells.

Functional deficits in the extrastriate body area during observation of sports-related actions in schizophrenia.

Exercise and sports are increasingly being implemented in the management of schizophrenia. The process of action perception is as important as that of motor execution for learning and acquiring new skills. Recent studies have suggested that body-selective extrastriate body area (EBA) in the posterior temporal-occipital cortex is involved not only in static visual perception of body parts but also in the planning, imagination, and execution of actions. However, functional abnormality of the EBA in schizophrenia has yet to be investigated. Using functional magnetic resonance imaging (fMRI) with a task designed to activate the EBA by sports-related actions, we aimed to elucidate functional abnormality of the EBA during observation of sports-related actions in patients with schizophrenia. Twelve schizophrenia patients and 12 age-sex-matched control participants participated in the study. Using sports-related motions as visual stimuli, we examined brain activations during observation of context-congruent actions relative to context-incongruent actions by fMRI. Compared with controls, the patients with schizophrenia demonstrated diminished activation in the EBA during observation of sports-related context-congruent actions. Furthermore, the EBA activation in patients was negatively correlated with the severity of negative and general psychopathology symptoms measured by the Positive and Negative Syndrome Scale. Dysfunction of the EBA might reflect a difficulty in representing dynamic aspects of human actions and possibly lead to impairments of simulation, learning, and execution of actions in schizophrenia.

Functional analyses of cytochrome P450 genes responsible for the early steps of brassicicene C biosynthesis.

We previously revealed that Orf8 and Orf6, which were identified in the brassicicene C biosynthetic gene cluster in Alternaria brassicicola strain ATCC96836, were fusicoccadiene (FD) synthase and 16-O-methyltransferase, respectively. In the present Letter, the early biosynthetic steps after the formation of FD were investigated. Plasmids carrying the FD synthase gene, one (or two) of five cytochrome P450 genes (orf1, orf2, orf5, orf7, and orf11) identified in the cluster and a cytochrome P450 reductase gene cloned from strain ATCC96836 were constructed and introduced into Saccharomyces cerevisiae. Based on the structures of the compounds produced by the transformants, Orf1 is suggested to be an 8beta-hydroxylation enzyme that yields FD 8beta-ol (4), followed by 16-hydroxylation by Orf7 to produce FD 8beta16-diol (5).

Transannular proton transfer in the cyclization of geranylgeranyl diphosphate to fusicoccadiene, a biosynthetic intermediate of fusicoccins.

Enzymatic cyclization of geranylgeranyl diphosphate to fusicoccadiene involves a transannular proton transfer process. Label distribution in the cyclized products derived from deuterium-labeled GGDPs showed that a proton generated from C-10 migrates to C-6 in the intermediary dolabellane framework prior to the second ring formation. Although a direct 1,5-proton transfer would achieve this process, semiempirical MO calculations suggested an alternative pathway, which involves successive 1,4- and 1,5-proton transfers using C-2 as a springboard.

A structural rationale for selective stabilization of anti-tumor interactions of 14-3-3 proteins by cotylenin A.

Cotylenin A, a fungal metabolite originally described as a cytokinin-like bioactive substance against plants shows differentiation-inducing and anti-tumor activity in certain human cancers. Here, we present the crystal structure of cotylenin A acting on a 14-3-3 regulatory protein complex. By comparison with the closely related, but non-anticancer agent fusicoccin A, a rationale for the activity of cotylenin A in human cancers is presented. This class of fusicoccane diterpenoids are possible general modulators of 14-3-3 protein-protein interactions. In this regard, specificities for individual 14-3-3/target protein complexes might be achieved by varying the substituent pattern of the diterpene ring system. As the different activities of fusicoccin A and cotylenin A in human cancers suggest, hydroxylation of C12 might be a sufficient determinant of structural specificity.

Biosynthetic gene-based secondary metabolite screening: a new diterpene, methyl phomopsenonate, from the fungus Phomopsis amygdali.

The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.

Identification and functional analysis of brassicicene C biosynthetic gene cluster in Alternaria brassicicola.

The biosynthetic gene cluster of brassicicene C was identified in Alternaria brassicicola strain ATCC 96836 from genome database search. In vivo and in vitro study clearly revealed the function of Orf8 and Orf6 as a fusicoccadiene synthase and methyltransferase, respectively. The understanding toward the biosynthetic pathway promises construction of this type of diterpene compounds with genetic engineering.

Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali.

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.

Enhanced activation in the extrastriate body area by goal-directed actions.

AIM: Neuroimaging studies on biological motion have established the view that the posterior superior temporal sulcus (pSTS) is involved in detecting intention of others. Those studies have consistently reported other regions such as body-selective extrastriate body area (EBA) and motion-sensitive middle temporal, in close proximity to pSTS. Whether EBA responds only to static body parts or has a more extended role as part of a system for inferring intention of others has remained an elusive issue. The aim of the present study was to investigate the role of EBA in processing goal-directed actions. METHODS: Twelve healthy volunteers participated in the present study. Using sports-related motions as visual stimuli, brain activations were examined during observation of goal-directed actions and non-goal-directed actions on functional magnetic resonance imaging. RESULTS: Compared to non-goal-directed actions, goal-directed actions produced greater activations in EBA along with the mirror neuron system. CONCLUSIONS: EBA might contribute to understanding others' actions by representing the dynamic aspects of human motions.

Diterpene phytoalexins are biosynthesized in and exuded from the roots of rice seedlings.

Rice (Oryza sativa L.) produces a variety of diterpene phytoalexins, such as momilactones, phytocassanes, and oryzalexins. Momilactone B was previously identified as an allelopathic substance exuded from the roots of rice. We identified in this present study momilactone A and phytocassanes A-E in extracts of, and exudates from, the roots of rice seedlings. The concentration of each compound was of the same order of magnitude as that of momilactone B. Expression analyses of the diterpene cyclase genes responsible for the biosynthesis of momilactones and phytocassanes suggest that these phytoalexins found in roots are primarily biosynthesized in those roots. None of phytocassanes B-E exhibited allelopathic activity against dicot seedling growth, whereas momilactone A showed much weaker allelopathic activity than momilactone B. The exudation of diterpene phytoalexins from the roots might be part of a system for defense against root-infecting pathogens.

Comparison of the enzymatic properties of ent-copalyl diphosphate synthases in the biosynthesis of phytoalexins and gibberellins in rice.

The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 muM GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 muM Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1.

Dose-finding study of paliperidone ER based on striatal and extrastriatal dopamine D2 receptor occupancy in patients with schizophrenia.

RATIONALE: Paliperidone ER is a novel antipsychotic drug in an extended-release (ER) formulation. As with all antipsychotics, careful dose setting is necessary to avoid side effects. OBJECTIVES: In this study, we measured striatal and extrastriatal dopamine D2 receptor occupancy during paliperidone ER treatment in patients with schizophrenia using positron emission tomography (PET) to compare regional occupancy and to estimate the optimal dose. MATERIALS AND METHODS: Thirteen male patients with schizophrenia participated in this 6-week multiple-dose study. Six of them took 3 mg of paliperidone ER per day, four took 9 mg, and three took 15 mg. Two to 6 weeks after first drug intake, two PET scans, one with [11C]raclopride and one with [11C]FLB 457, were performed in each patient on the same day. The relationship between the dose or plasma concentration of paliperidone and dopamine D2 receptor occupancy was calculated. RESULTS: The dopamine D2 receptor occupancies in the striatum measured with [11C]raclopride and the temporal cortex measured with [11C]FLB 457 were 54.2-85.5% and 34.5-87.3%, respectively. ED50 values of the striatum and temporal cortex were 2.38 and 2.84 mg/day, respectively. There was no significant difference in dopamine D2 receptor occupancy between the striatum and the temporal cortex. CONCLUSIONS: The data from this study suggest that paliperidone ER at 6-9 mg provides an estimated level of dopamine D2 receptor occupancy between 70-80% and that the magnitude of dopamine D2 receptor occupancy is similar between the striatum and temporal cortex.