Linzhuan Wu

Identification of 4,5-Dihydro-4-hydroxygeldanamycins As Shunt Products of Geldanamycin Biosynthesis

Two new geldanamycin (GDM) analogues, (4S)-4,5-dihydro-4-hydroxygeldanamycin (1) and (4R)-4,5-dihydro-4-hydroxygeldanamycin (2), were identified from Streptomyces hygroscopicus 17997. Compounds 1 and 2 were not normal intermediates of GDM biosynthesis but shunt products of C-4,5 oxidation catalyzed by GdmP, a cytochrome P450 oxidase acting as a desaturase in GDM biosynthesis. Preliminary assays implied that, compared with GDM, 1 and 2 exhibited decreased cytotoxicity.

A pair of sulfur-containing geldanamycin analogs, 19-S-methylgeldanamycin and 4,5-dihydro- 19-S-methylgeldanamycin, from Streptomyces hygroscopicus 17997

Geldanamycin (GDM, Figure 1) is a benzoquinone ansamycin produced by Streptomyces hygroscopicus.1, 2, 3 It is a specific inhibitor of human heat shock protein 90 (Hsp90), which is a molecular chaperone assisting in protein folding, cell signaling and tumor repression, and a potential cellular target for anti-tumor agent. GDM is only used as a promising lead compound for anti-cancer drug development because of its poor water solubility and severe liver toxicity.4, 5, 6

Methanethiol as a catabolite of methionine provides methylthio- group for chemical formation of 19- S -methylgeldanamycin and 17,19-dimethylthioherbimycin A

Methanethiol as a catabolite of methionine provides methylthio- group for chemical formation of 19- S -methylgeldanamycin and 17,19-dimethylthioherbimycin A

7-O-descarbamoyl-7-hydroxygeldanamycin, a minor component from the gdmN disruption mutant of Streptomyces hygroscopicus 17997.

7- O -descarbamoyl-7-hydroxygeldanamycin, a minor component from the gdmN disruption mutant of Streptomyces hygroscopicus 17997

Cytotoxic Dibohemamines D–F from a Streptomyces Species

Three dimeric analogues of bohemamines, dibohemamines D-F (1-3), together with dibohemamine A (4), were isolated from Streptomyces sp. CPCC 200497. Their structures were solved using a combination of mass spectrometry, 1D and 2D NMR spectroscopy, and CD. Dibohemamines D and E were new dimeric analogues of bohemamines, and dibohemamine F was a known compound obtained previously by semisynthesis. Dibohemamine F displayed potent cytotoxicity against cancer cell lines A549 and HepG2 with IC50 values of 1.1 and 0.3 μM, respectively. Dibohemamines D and E showed moderate cytotoxicity against cancer cell lines A549 and HepG2.

6-Deoxy-13-hydroxy-8,11-dione-dihydrogranaticin B, an intermediate in granaticin biosynthesis, from Streptomyces sp. CPCC 200532

A new granaticin analogue and its hydrolysis product were isolated from Streptomyces sp. CPCC 200532. Their structures were determined to be 6-deoxy-13-hydroxy-8,11-dione-dihydrogranaticins B (1) and A (2), respectively, by detailed analysis of spectroscopic data. Compound 1 was regarded as an intermediate in granaticin biosynthesis, as it was bioconvertable to granaticin B. Compared to granaticin B, 1 showed similar cytotoxicity against cancer cell line HCT116, but decreased cytotoxicity against cancer cell lines A549, HeLa, and HepG2. Compound 2 displayed lower cytotoxicity than 1 against all four cancer cell lines tested.

17- O -demethylreblastatin, a subnormal intermediate in geldanamycin biosynthesis

Geldanamycin (GDM), produced by Streptomyces hygroscopicus, is a 19-membered macrocyclic lactam related to benzoquinone ansamycins. Interest in GDM increased greatly upon the discovery of its remarkable antitumor properties, but severe hepatotoxicity made it unfitting for direct clinical uses.1 At present, GDM is a promising lead compound for antitumor drug development. A clear understanding of GDM biosynthesis is of great importance in creating novel GDM analogs by genetic manipulation or combinatorial biosynthesis. The biosynthesis of GDM involves the assembly of one starter unit (3-amino-5-hydroxybenzoic acid), with seven extender units (one acetate, four propionates and two methoxyacetates). The assembly process forms a polyketide backbone, which then undergoes a postPKS tailoring process that includes C-7 carbamoylation, C-17 hydroxylation, C-17 methylation, C-21 hydroxylation, C-4,5 oxidation and oxidation of hydroquinone to quinone, to form GDM finally.2,3 The genes required for GDM biosynthesis have been cloned, sequenced and analyzed from several strains of Streptomyces.3–6 Up to now, the order of post-PKS tailoring process in GDM biosynthesis is only defined partially.3,6,7 The C-7 carbamoylation must take place before C-4,5 oxidation. The C-4,5 oxidation takes place at a later time in the post-PKS tailoring process. The order of benzoquinone modifications of the post-PKS tailoring process, in particular, the order of C-17 hydroxylation and C-21 hydroxylation remains unclear. The isolation of reblastatin from S. hygroscopicus subsp. hygroscopicus SANK61995,8 a GDM producer, and the isolation of reblastatin-like ansamycins (17-O-demethylreblastatin and autolytimicin) from Streptomyces sp. S6699 and S. autolyticus JX-47,9,10 seem to suggest that C-17 hydroxylation (and C-17 methylation) take place before C-21 hydroxylation, but biological evidences are needed to prove this point. We are interested in understanding the biosynthetic details of GDM production. Recently, we reported the characterization of a minor component, 7-O-descarbamoyl-7-hydroxygeldanamycin, from the gdmN (encoding the enzyme for C-7 carbamoylation) disruption mutant of S. hygroscopicus 17997, and provided evidences that C-7 carbamoylation must take place before C-4,5 oxidation in GDM biosynthesis.7 Recently, in our monitoring of secondary metabolites of S. hygroscopicus 17997, a wild-type GDM (300–400 mg l 1) producer, we found a GDM analog from the mid-stage (40–60 h) fermentation broth(s) of S. hygroscopicus 17997 (Supplementary Figures S1 and S2). It was found to exist also in the gdmP (encoding the cytochrome P450 monooxygenase for C-4,5 oxidation) disruption mutant of S. hygroscopicus 17997 (Figures 1 and 2). The compound disappeared from the fermentation broths in older cultures of S. hygroscopicus 17997 or its gdmP disruption mutant, coinciding with the slowdown or stop of GDM or 4,5-dihydrogeldanamycin biosynthesis. Besides, we detected the descarbamoylated form of the compound from the gdmN disruption mutant of S. hygroscopicus 17997 (Supplementary Figure S3). The compound aroused our attention. We isolated the compound and determined its chemical structure, which is 17-O-demethylreblastatin (or 17-hydroxyautolytimicin, compound 3 in Figure 4), a non-benzoquinone GDM analog. In this paper, we reported the re-discovery of 17-O-demethylreblastatin and its verification as a subnormal intermediate of GDM biosynthesis in S. hygroscopicus 17997; besides, we proposed an order of benzoquinone modifications of the post-PKS tailoring process in GDM biosynthesis. Frozen stock spores of the gdmP disruption mutant of S. hygroscopicus 17997 were thawed and spread onto ISPII (0.4% yeast extract, 1.0% malt extract, 0.4% glucose and 1.5% agar power) plates, incubated at 28 1C for 8–10 days, for mycelium growth and sporulation, then slices of the plate culture were picked up as seed and inoculated into the fermentation medium (starch 2 1C, 0.5% cottonseed meal, 0.5% glucose, 1.0% corn steep liquor, 0.5% yeast