Rational Biosynthetic Engineering for Optimization of Geldanamycin Analogues

Abstract

Tailor made: We report the rational biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound. A rational biosynthetic engineering approach was applied to the optimization of the pharmacological properties of the benzoquinone ansamycin, geldanamycin. Geldanamycin and its natural or semisynthetic derivatives have the potential to serve as anticancer chemotherapeutic agents. However, these first-generation Hsp90 inhibitors share an unfavorable structural feature that causes both reduced efficacy and toxicity during clinical evaluation. We report the rationally designed biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. A 15-hydroxyl-17-demethoxy non-quinone analogue, DHQ3, exhibited stronger inhibition of Hsp90 ATPase activity (4.6-fold) than geldanamycin. Taken together, the results of the present study indicate that rational biosynthetic engineering allows the generation of derivatives of geldanamycin with superior pharmacological properties.

New non-quinone geldanamycin analogs from genetically engineered Streptomyces hygroscopicus

“…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.…”

“…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.…”

“…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.…”

“…16 13 C NMR and HMQC spectra revealed the presence of 28 carbon signals comprising two carbonyl, six aromatic or olefinic quaternary, six aromatic or olefinic methine, four oxymethine, two aliphatic methine, two aliphatic methylene, two O-methyl, and four C-methyl carbons. Additionally, four exchangeable proton signals were observed in DMSO-d 6 : two hydroxyl protons (d 5.10 and 4.76), one phenolic hydroxyl proton (d 9.13) and one amide proton (d 9.33).…”

“…But, the 3 showed stronger ATPase inhibition activity (0.68 mM) compared with the original Hsp90 inhibitor 1 (3.19 mM), as previously demonstrated. 16 To further confirm that 4 and 5 bind and interact with the N-terminal nucleotide binding site of Hsp90 as an ATP mimetic, we examined whether these compounds could compete with a bead-immobilized derivative of ATP for Hsp90 binding. It is noteworthy that the compounds demonstrated significant activity compared with the DMSO control.…”