New non-quinone geldanamycin analogs from genetically engineered Streptomyces hygroscopicus

Abstract

Heat shock protein 90 (Hsp90) is emerging as an important target in cancer therapeutics and several other diseases. 1,2 Hsp90 is an abundant and ubiquitously expressed molecular chaperone that is involved in the maturation of multiple client proteins, many of which are involved in regulating cell signaling, proliferation and survival. 3,4 Client proteins of Hsp90 include Her-2, Akt, Src, Abl, c-Met and Raf-1, which are currently being targeted for intervention within oncology drug discovery or in clinical development. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors display remarkable selectivity for cancer cells as compared with normal cells. 5,6 Hsp90 is effectively inhibited by benzoquinone ansamycins such as geldanamycin (GM, 1), herbimycin, macbecin and many of their derivatives, which bind to the ATP-binding site in the N-terminal domain. 7,8 However, the development of 1 as a clinical agent has so far been limited by its toxicity, especially liver toxicity. 9 However, the promising antitumor properties of 1 have spurred initiatives to develop biologically active derivatives. 10,11 17-Allylamino-17-demethoxyGM (17-AAG) has reduced toxicity compared with 1 and is the most advanced Hsp90 inhibitor in clinical development (Phase II/III). 12 17-AAG and its benzoquinone analogs conjugate with glutathione, leading to cellular depletion. This conjugation with sulfur-containing nucleophiles may contribute to the dose-limiting hepatotoxicity of these quinone-containing compounds. 13,14 For these reasons, new non-quinone 1 analogs with improved pharmacological profiles are needed. 15,16 Recently, we reported the development of non-quinone 1 analogs by a mutasynthetic approach and directed biosynthetic method. 16,17 Of these non-quinone 1 analogs, DHQ3 (3), a 15-hydroxyl-17-demethoxy non-quinone analog, was found to inhibit Hsp90 ATPase activity more than 1. 16 Moreover, during these studies, novel tricyclic 1 analogs were prepared from a genetically engineered strain (AC15) of Streptomyces hygroscopicus. 18 Presently, we describe the fermentation of mutant AC15, and the isolation, structural determination and bioactivity of new non-quinone 1 analogs produced by the mutant: DHQ7 (4) and DHQ8 (5).AC15 was constructed by a combinational mutation with site-directed mutagenesis of the first dehydratase domain of the geldanamycin polyketide synthase (PKS) gene (gelA) and a post-PKS modification gene (gel7) of S. hygroscopicus JCM4427, as previously reported. 16 The seed medium and production medium (YEME) consisted of sucrose 103.0 g, yeast extract 3.0 g, peptone (Difco, Sparks, MD, USA) 5.0 g, malt extract (Difco) 3.0 g, glucose 10.0 g and MgCl 2 Á6H 2 O 1.0 g in 1.0 l of distilled water. Spores that developed during growth on ISP4 medium were harvested and inoculated into 300 ml of YEME in a 1.0 l baffled flask and cultured for 3 days at 28 1C. Equal volumes of the seed culture were inoculated into 25 baffled flasks containing 300 ml of YEME medium. Fermentation was subsequently carried out for 7 days ...

Enzymatic Glycosylation of Nonbenzoquinone Geldanamycin Analogs via Bacillus UDP-Glycosyltransferase

“…As can be seen in Table 2, compound 7 exhibited weak antiproliferation activity against SK-Br3 cells, with a 50% inhibitory concentration (IC 50 ) of 19.0 M compared with that of the original substrates (9.4 M). In contrast, GM showed submillimolar antiproliferation activity against several human cancer cells, including SK-Br3 cells (34). Compound 6 did not demonstrate antiproliferation activity within the experimental concentration ranges used in this study.…”

“…17-Demethoxy-reblastatin (compound 4) was purified from the culture broths of S. hygroscopicus AC11, in which the benzoquinone ring oxidation gene (gel7) of the GM pathway was disrupted by the kanamycin resistance gene (15). 17-Demethoxy-15-hydroxylreblastatin (compound 5; DHQ3) was also purified from the culture broths of S. hygroscopicus AC15, a double mutation with the first dehydratase domain of the geldanamycin polyketide synthase (PKS) gene and the gel7 gene of S. hygroscopicus JCM4427, as previously reported (15,34).…”

“…For these reasons, new nonbenzoquinone GM analogs with improved pharmacological profiles are needed. Recently, we reported the development of nonbenzoquinone GM analogs using biosyntheic approaches (15,16,34). Moreover, a novel semisynthetic nonbenzoquinone GM showed improved efficacy (unpublished data).…”

Spotlight on the microbes that produce heat shock protein 90-targeting antibiotics

“…The benzoquinone, through redox cycling and the production of the unstable semiquinone radical, is able to generate appreciable oxidative stress in vivo [36]. For this reason S. hygroscopicus has recently been genetically engineered for the production of new non-quinone analogues of GdA, compounds with a better pharmacological profile than the natural antibiotics [37]. Without the unravelling of the details of GdA and herbimycin A biosynthesis [34], this could not have been achieved.…”

Non-benzoquinone geldanamycin analogs trigger various forms of death in human breast cancer cells

“…Therefore, new GA derivatives with improved pharmacological profiles are needed. DHQ3 and 17-demethoxy-reblastatin (17-DR), two non-quinone GA analogs, were obtained from a genetically modified strain of Streptomyces hygroscopicus JCM442 and their structures have been determined [ 8 , 9 ]. The phenolic structure effectively improved the water solubility as compared to the benzoquinone structure.…”