A target and efficient synthetic strategy for structural and bioactivity optimization of a fungal natural product.

Drugs have been largely inspired from natural products, while enzymes underlying their biosynthesis have enabled complex structures and diverse bioactivities. Nevertheless, the high enzyme specificity and limited in vivo precursor types have restricted the natural product reservoir, but Nature has imprinted natural products with active sites, which can be readily modified by chemosynthesis with various functional groups for more favorable druggability. Here in the less exploited fungal natural products, we introduced CtvA, a polyketide synthase for a mycotoxin citreoviridin biosynthesis in Aspergillus, into an endophytic fungus Calcarisporium arbuscula to expand tetrahydrofuran (THF) into a dioxabicyclo-octane (DBO) ring moiety based on versatility and promiscuity of the aurovertin biosynthetic enzyme. Alternative acylations on the hydroxyl groups essential for cell toxicity by chemosynthesis produced compounds with improved anti-tumor activities and pharmacokinetics. Thus, we showed an effective strategic way to optimize the fungal natural product efficiently for more promising drug development.

An Aspergillus nidulans Platform for the Complete Cluster Refactoring and Total Biosynthesis of Fungal Natural Products.

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in Aspergillus nidulans. Our platform places our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted activation upon induction. We demonstrated the utility of our system by creating strains that can synthesize high-value NPs, citreoviridin (1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can be used not only for producing NPs of interests (i.e., total biosynthesis) but also for elucidating cryptic biosynthesis pathways.

Aurovertin B sensitizes colorectal cancer cells to NK cell recognition and lysis.

The natural killer group 2D (NKG2D) receptor on natural killer (NK) cells play an important role in immunosurveillance to cancer cells, which could mediate the eradication of tumor cells through specific interactions with NKG2D ligands on tumor cells. Here we report one natural compound aurovertin B from basidiomycete Albatrellus confluens significantly stimulates the expression of NKG2D ligands on tumor cells, which greatly sensitizes its recognition and lysis by NK cell. It is completely a novel role for aurovertin B to target tumor cells to death mediated by NK cells and our findings indicate aurovertin B may deserve further development as sensitizing agent in NK cell mediated cancer immunotherapy.

Synthesis and production of the antitumor polyketide aurovertins and structurally related compounds.

Aurovertins belong to a family of highly reducing polyketides sharing a polyene α-pyrone-type structure. These compounds comprise aurovertin, asteltoxin, avertoxin, citreoviridin, verrucosidin, and their derivatives, which exihibit potent antitumor, antiviral, and antibacterial activities. Until now, over 40 aurovertins and structurally related compounds have been found in the fungal kingdom. Due to the unique structural feature and interesting bioactivities, significant progresses have been achieved for the structural identification, chemical synthesis, and biosynthesis of the mentioned compounds. Understanding of aurovertin biosynthetic mechanism provides a solid basis for engineering the metabolic pathway of those compounds by rational design and realizing their production in the model fungal host.

Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans.

Aurovertins are the structurally diverse polyketides that distribute widely in different fungal species. They feature a 2,6-dioxabicyclo[3.2.1]-octane ring in structure and exhibit the potential antitumor activity against breast cancer as F1-ATPase ß subunit inhibitor. In this study, we constructed the biosynthetic pathway of aurovertin in an Aspergillus nidulans host and obtained seven aurovertin-type compounds. Surprisingly, three new aurovertin geometric isomers were characterized. By introducing an inducible promoter xylP(p) in the pathway gene acyltransferase aurG, we can control the product ratios among different aurovertin compounds by adding glucose and/or inducer xylose. The yields of aurovertins could be increased up to about 20 times by adding a constitutive promoter gpdA(p) to transcription factor AurF, which indicates AurF's positive role in the biosynthesis of aurovertin. Taken together, our results provided not only an efficient way to generate bioactive fungal natural products but also realized the rational controlling their yields with designed promoters.

Biosynthetic Pathway of the Reduced Polyketide Product Citreoviridin in Aspergillus terreus var. aureus Revealed by Heterologous Expression in Aspergillus nidulans.

Citreoviridin (1) belongs to a class of F1-ATPase ß-subunit inhibitors that are synthesized by highly reducing polyketide synthases. These potent mycotoxins share an α-pyrone polyene structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution using heterologous expression in Aspergillus nidulans are reported. Two intermediates were isolated that allowed the proposal of the biosynthetic pathway of citreoviridin.

Bioprospecting Chemical Diversity and Bioactivity in a Marine Derived Aspergillus terreus.

A comparative metabolomic study of a marine derived fungus (Aspergillus terreus) grown under various culture conditions is presented. The fungus was grown in eleven different culture conditions using solid agar, broth cultures, or grain based media (OSMAC). Multivariate analysis of LC/MS data from the organic extracts revealed drastic differences in the metabolic profiles and guided our subsequent isolation efforts. The compound 7-desmethylcitreoviridin was isolated and identified, and is fully described for the first time. In addition, 16 known fungal metabolites were also isolated and identified. All compounds were elucidated by detailed spectroscopic analysis and tested for antibacterial activities against five human pathogens and tested for cytotoxicity. This study demonstrates that LC/MS based multivariate analysis provides a simple yet powerful tool to analyze the metabolome of a single fungal strain grown under various conditions. This approach allows environmentally-induced changes in metabolite expression to be rapidly visualized, and uses these differences to guide the discovery of new bioactive molecules.

Efficient Biosynthesis of Fungal Polyketides Containing the Dioxabicyclo-octane Ring System.

Aurovertins are fungal polyketides that exhibit potent inhibition of adenosine triphosphate synthase. Aurovertins contain a 2,6-dioxabicyclo[3.2.1]octane ring that is proposed to be derived from a polyene precursor through regioselective oxidations and epoxide openings. In this study, we identified only four enzymes required to produce aurovertin E. The core polyketide synthase produces a polyene α-pyrone. Following pyrone O-methylation by a methyltransferase, a flavin-dependent mono-oxygenase and an epoxide hydrolase can iteratively transform the terminal triene portion of the precursor into the dioxabicyclo[3.2.1]octane scaffold. We demonstrate that a tetrahydrofuranyl polyene is the first stable intermediate in the transformation, which can undergo epoxidation and anti-Baldwin 6-endo-tet ring opening to yield the cyclic ether product. Our results further demonstrate the highly concise and efficient ways in which fungal biosynthetic pathways can generate complex natural product scaffolds.

Binding of citreoviridin to human serum albumin: multispectroscopic and molecular docking.

Citreoviridin (CIT), a mycotoxin produced by Penicillium citreonigrum, is a common contaminant of wide range of agriproducts and detrimental to human and animal health. In this study, the interaction of CIT with human serum albumin (HSA) is researched by steady-state fluorescence, ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD) methods, and molecular modeling. The association constants, binding site numbers, and corresponding thermodynamic parameters are used to investigate the quenching mechanism. The alternations of HSA secondary structure in the presence of CIT are demonstrated with UV-Vis, synchronous fluorescence, and CD spectra. The molecular modeling results reveal that CIT can bind with hydrophobic pocket of HSA with hydrophobic and hydrogen bond force. Moreover, an apparent distance of 3.25 nm between Trp214 and CIT is obtained via fluorescence resonance energy transfer method.

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Structures and biomimetic synthesis of novel α-pyrone polyketides of an endophytic Penicillium sp. in Catharanthus roseus.

Novel polyketides, citreoviripyrone A (1) and B (2), known citreomontanin (3), and (-)-citreoviridin (4) were isolated from the mycelium of the endophytic fungus. The endophytic fungus, which belongs to the genus Penicillium, was separated from surface-sterilized healthy leaves of Catharanthus roseus. The structures of 1 and 2 were determined on the basis of NMR data, and 1 was characterized as an α-pyrone polyketide featuring bicyclo[4.2.0]octadiene. The biomimetic synthesis of 1 and 2 from 3 elucidated a plausible biosynthetic pathway. Both Zn(II)-type and NAD(+)-dependent histone deacetylase inhibitors significantly enhanced the production of 1 and 3.

Nematodetoxic aurovertin-type metabolites from a root-knot nematode parasitic fungus Pochonia chlamydosporia.

Chemical investigation of one fungal strain P. chlamydosporia YMF 1.00613 isolated from root knots of tobacco infected by Meloidogyne incognita led to the isolation and identification of four aurovertin-type metabolites, which include a new compound, aurovertin I (A1), and three known metabolites, aurovertins E, F and D (A2-A4). Their structures were established by spectroscopic studies such as 1D- and 2D-NMR and MS analysis. Aurovertin I (A1) is the first natural product with an aurovertin skeleton with one less carbon. Compounds A3 and A4 showed the toxicity to the worms of the free-living nematode Panagrellus redivevus with the LC(50) values 88.6 and 41.7 microg/mL at 48 h, respectively. All four aurovertins did not show obvious inhibitory effects on egg hatch of root knot nematode Meloidogyne incognita. The results suggested that the aurovertin-type metabolites produced by P. chlamydosporia might be one of the pathogenic factors involved in the suppression of nematodes.

Aurovertins F-H from the entomopathogenic fungus Metarhizium anisopliae.

Analysis of fermentation broth of the entomopathogenic fungus Metarhizium anisopliae has led to isolation of aurovertin D ( 1) and three new aurovertin-type metabolites, aurovertin F ( 2), aurovertin G ( 3), and aurovertin H ( 4). Their structures were determined on the basis of spectroscopic analyses and chemical conversions.

Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein.

The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the inhibitor protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500-650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the inhibitor protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of inhibitor protein, indicating that the protein stabilizes the global structure of particulate F1.

Inhibition sites in F1-ATPase from bovine heart mitochondria.

High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 alpha3beta3gamma subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the epsilon-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia-reperfusion injury and some cancers.

Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity.

Mitochondrial F(1)F(0)-ATPase normally synthesizes ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal tissue but also inhibit ATP hydrolysis in ischemic myocardium. We characterized the profile of aurovertin and oligomycin in ischemic and nonischemic rat myocardium and compared this with the profile of BMS-199264, which only inhibits F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), at concentrations inhibiting ATPase activity, reduced ATP concentration and contractile function in the nonischemic heart but significantly reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent with nonselective F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) conserved ATP in rat hearts during ischemia while having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis was reduced by BMS-199264. ATP hydrolase activity ex vivo was selectively inhibited by BMS-199264. Therefore, excessive ATP hydrolysis by F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.

The structure of bovine F1-ATPase complexed with the antibiotic inhibitor aurovertin B.

In the structure of bovine mitochondrial F1-ATPase that was previously determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. Adenylyl-imidodiphosphate is bound to one beta-subunit (betaTP), ADP is bound to the second (betaDP), and no nucleotide is bound to the third (betaE). Here we show that the uncompetitive inhibitor aurovertin B binds to bovine F1 at two equivalent sites in betaTP and betaE, in a cleft between the nucleotide binding and C-terminal domains. In betaDP, the aurovertin B pocket is incomplete and is inaccessible to the inhibitor. The aurovertin B bound to betaTP interacts with alpha-Glu399 in the adjacent alphaTP subunit, whereas the aurovertin B bound to betaE is too distant from alphaE to make an equivalent interaction. Both sites encompass betaArg-412, which was shown by mutational studies to be involved in binding aurovertin. Except for minor changes around the aurovertin pockets, the structure of bovine F1-ATPase is the same as determined previously. Aurovertin B appears to act by preventing closure of the catalytic interfaces, which is essential for a catalytic mechanism involving cyclic interconversion of catalytic sites.