Yinggang Luo

Brevianamide J, A New Indole Alkaloid Dimer from Fungus Aspergillus versicolor

Brevianamide J (1), a new indole alkaloid dimer, was isolated together with four new diketopiperazine alkaloids (brevianamide K-N, 2-5) from the solid-state fermented culture of Aspergillus versicolor. Their structures were elucidated on the basis of spectral data. X-ray crystallographic analysis confirmed the structures of 1 and 4.

Tautomycetin and tautomycin suppress the growth of medullary thyroid cancer cells via inhibition of glycogen synthase kinase-3β

Medullary thyroid cancer (MTC) is a relatively uncommon neuroendocrine tumor that arises from the calcitonin-secreting parafollicular cells of the thyroid gland. Unfortunately, MTC frequently metastasizes, precluding curative surgical resection and causing significant morbidity. Thus, there is an urgent need for new treatment modalities. Tautomycin and tautomycetin are antifungal antibiotics isolated from Streptomyces spiroverticillatus and Streptomyces griseochromogens, respectively. Glycogen synthase kinase-3β is a serine/threonine protein kinase that regulates multiple cellular processes and is important in various cancers, including MTC. Treatment with tautomycin and tautomycetin decreased neuroendocrine markers, suppressed hormonal secretion, and inhibited growth through apoptosis in MTC cells. Importantly, we describe a novel action of these compounds: inhibition of glycogen synthase kinase-3β.[Mol Cancer Ther 2009;8(4):914–20]

Identification of Fredericamycin E from Streptomyces griseus: Insights into Fredericamycin A Biosynthesis Highlighting Carbaspirocycle Formation⊥

Fredericamycin (FDM) A ( 1), a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique asymmetric carbaspiro center, exhibits potent cytotoxicity and represents a novel anticancer drug lead. We have localized previously the fdm gene cluster to a 33 kb DNA segment of Streptomyces griseus ATCC49344, the involvement of which in the biosynthesis of 1 was confirmed by gene inactivation, complementation, and heterologous expression experiments. We now report the isolation and characterization of FDM E ( 5), a heretofore undetected intermediate for 1 biosynthesis from S. griseus, shedding new insight into the mechanism of carbaspirocycle formation. The structure of 5 was elucidated through the combination of spectroscopic methods and isotope-labeling experiments. The core spiro[4.5]decane scaffold of 5 is characterized by a unique cyclohexa-1,2,4-triketone moiety. Transformation of the spiro[4.5]decane 5 into the spiro[4.4]nonane 1 can be rationalized by a biosynthetic benzilic acid-like rearrangement. This unusual rearrangement can be mimicked in vitro by proceeding under aerobic conditions in the absence of enzyme. FDM E displays cytotoxic activity on par with 1 against a selected set of cancer cells, a finding that further supports the unique molecular topology, resulting from the unprecedented carbaspirocycle as exemplified by 1 and 5, as a novel pharmacophore for this family of anticancer agents.

Novel ceramides and a new glucoceramide from the roots of Incarvillea arguta

Novel ceramides, rel-(3S,4S,5S)-3-[(2R)-2-hydroxycosanoyl-hexacosanoylamino]-4-hydroxy-5-[(4Z)-tetradecane-4-ene]-2,3,4,5-tetrahydrofuran (1a-g), and a new glucoceramide, 1-O-β-d-glucopyranosyl-(2S,3S,4R,8E)-2-[(2R)-2-hydroxytetracosanoylamino]-1,3,4-octodecanetriol-8-ene (2) were isolated from the aqueous ethanolic extract of the roots of Incarvillea arguta, together with eight known compounds: β-sitosterol (3), oleanolic acid (4), ursolic acid (5), piperin (6), maslinic acid (7), β-sitosterol 6′-O-acyl-β-d-glucopyranoside (8), 8-epideoxyloganic acid (9), and plantarenaloside (10). Their structures were elucidated on the basis of spectral data including IR, MS, NMR [1H NMR, 13C NMR (distortionless enhancement by polarization transfer), 1H−1H COSY, heteronuclear multiplequantum coherence, and heteronuclear multiple-bond coherence correlations]. The relative configurations were established by nuclear Overhauser effect spectroscopy experiments and by comparison of the NMR spectral data and coupling constants with those already reported in the literature.

Dihydroagarofuran Derivatives from the Dried Roots of Tripterygium wilfordii

Five new sesquiterpene derivatives, including dihydroagarofuran pyridine macrolides 1-4 and dihydroagarofuran ester 18, and 13 known dihydroagarofuran derivatives were isolated from the aqueous EtOH extract of the dried roots of Tripterygium wilfordii. An in vitro antiherpetic activity assay indicated that compounds 11 and 17 displayed weak and moderate inhibition against herpes simplex virus type II, respectively.

Characterization of the tautomycetin biosynthetic gene cluster from Streptomyces griseochromogenes provides new insight into dialkylmaleic anhydride biosynthesis.

Tautomycetin (TTN) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces griseochromogenes. The biological activity of TTN makes it an important lead for drug discovery, whereas its rare dialkylmaleic anhydride moiety and structural similarity to tautomycin (TTM), another potent phosphatase inhibitor with tremendous medicinal potential, draws attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with TTN production, the ttn biosynthetic gene cluster from S. griseochromogenes was isolated and characterized, and its involvement in TTN biosynthesis confirmed by gene inactivation and complementation experiments. The ttn cluster was localized to a 79 kb DNA region, consisting of 19 open reading frames that encode two modular type I polyketide synthases (TtnAB), one type II thioesterase (TtnH), eight proteins for dialkylmaleic anhydride biosynthesis (TtnKLMNOPRS), four tailoring enzymes (TtnCDFI), two regulatory proteins (TtnGQ), and one resistance protein (TtnJ). A model for TTN biosynthesis is proposed on the basis of functional assignments from sequence analysis, which agrees well with previous feeding experiments, has been supported by in vivo gene inactivation experiments, and is supported by analogy to the recently reported ttm cluster. These findings set the stage to fully investigate TTN biosynthesis and to biosynthetically engineer new TTN analogues.

Enediyne Antitumor Antibiotic Maduropeptin Biosynthesis Featuring a C-Methyltransferase That Acts on a CoA-Tethered Aromatic Substrate

The enediyne antitumor antibiotic maduropeptin (MDP) is produced by Actinomadura madurae ATCC 39144. The biosynthetic pathway for the 3,6-dimethylsalicylic acid moiety of the MDP chromophore is proposed to be comprised of four enzymes: MdpB, MdpB1, MdpB2, and MdpB3. Based on the previously characterized biosynthesis of the naphthoic acid moiety of neocarzinostatin (NCS), we expected a biosynthetic pathway featuring carboxylic acid activation by the MdpB2 CoA ligase immediately before its coupling to an enediyne core intermediate. Surprisingly, the MDP aromatic acid biosynthetic pathway employs an unusual logic in which MdpB2-catalyzed CoA activation occurs before MdpB1-catalyzed C-methylation, demonstrating that MdpB1 is apparently unique in its ability to C-methylate a CoA-tethered aromatic acid. MdpB2 is a promiscuous CoA ligase capable of activating a variety of salicylic acid analogues, a property that could be potentially exploited to engineer MDP analogues.

Regiospecific O-Methylation of Naphthoic Acids Catalyzed by NcsB1, an O-Methyltransferase Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

Neocarzinostatin, a clinical anticancer drug, is the archetypal member of the chromoprotein family of enediyne antitumor antibiotics that are composed of a nonprotein chromophore and an apoprotein. The neocarzinostatin chromophore consists of a nine-membered enediyne core, a deoxyaminosugar, and a naphthoic acid moiety. We have previously cloned and sequenced the neocarzinostatin biosynthetic gene cluster and proposed that the biosynthesis of the naphthoic acid moiety and its incorporation into the neocarzinostatin chromophore are catalyzed by five enzymes NcsB, NcsB1, NcsB2, NcsB3, and NcsB4. Here we report the biochemical characterization of NcsB1, unveiling that: (i) NcsB1 is an S-adenosyl-l-methionine-dependent O-methyltransferase; (ii) NcsB1 catalyzes regiospecific methylation at the 7-hydroxy group of its native substrate, 2,7-dihydroxy-5-methyl-1-naphthoic acid; (iii) NcsB1 also recognizes other dihydroxynaphthoic acids as substrates and catalyzes regiospecific O-methylation; and (iv) the carboxylate and its ortho-hydroxy groups of the substrate appear to be crucial for NcsB1 substrate recognition and binding, and O-methylation takes place only at the free hydroxy group of these dihydroxynaphthoic acids. These findings establish that NcsB1 catalyzes the third step in the biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore and further support the early proposal for the biosynthesis of the naphthoic acid and its incorporation into the neocarzinostatin chromophore with free naphthoic acids serving as intermediates. NcsB1 represents another opportunity that can now be exploited to produce novel neocarzinostatin analogs by engineering neocarzinostatin biosynthesis or applying directed biosynthesis strategies.

Nitrogen-Containing Dihydro-β-agarofuran Derivatives from Tripterygium wilfordii

Thunder god vine, the dried roots of Tripterygium wilfordii, is a widely used traditional Chinese medicine. More than 200 bioactive complex natural products have been isolated from this herb. Inspired by the diversity of chemical structures and bioactivities of the components of this herb, the investigation to mine new chemical entities as potential drug leads led to the identification of 36 nitrogen-containing compounds. Among them, 18 new dihydro-β-agarofuran alkaloids (tripterygiumines A-L (1-12), M-Q (22-26), and R (33)) were identified from the spectroscopic data and chemical degradation studies. Tripterygiumine Q (26) exhibited immunosuppressive activity against human peripheral mononuclear cells with an IC50 value of 8.67 μM and showed no cytotoxicity, even at 100 μM, indicating that 26 may represent a novel scaffold for the development of new immunosuppressants.

Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.