Zong-Qiang Tian

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation

ABSTRACT Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.

Potent cytotoxic C-11 modified geldanamycin analogues.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the activity of Hsp90, an important target for treatment of cancers. In an effort to identify analogues of geldanamycin (GDM) with properties superior to those of 17-AAG, we synthesized C-11 modified derivatives of GDM including ethers, esters, carbazates, ketones, and oximes and measured their affinity for Hsp90 and their ability to inhibit growth of human cancer cells. In accordance with crystal structures reported for complexes of GDMs with Hsp90, bulky groups attached to C-11 interfered with Hsp90 binding while smaller groups such as 11-O-methyl allowed Hsp90 binding. In addition, these analogues also showed in vitro cytotoxicity against human cancer cell lines. Esterification of the 11-OH of 17-AAG eliminated Hsp90 binding in vitro. The readily hydrolyzed esters acted as prodrugs during the measurement of cytotoxicity. Thus, during these experiments, the esters were hydrolyzed, releasing 17-AAG. Several 11-O-methyl-17-alkylaminogeldanamycin analogues were identified with improved potency relative to 17-AAG.

Efficacy of Ambruticin Analogs in a Murine Model of Invasive Pulmonary Aspergillosis

ABSTRACT Ambruticins are a family of polyketides. The antifungal activity of an ambruticin, KOSN-2079, was tested in the mouse model of invasive aspergillosis. KOSN-2079 significantly reduced pulmonary fungal burdens and improved survival over that with the vehicle control. These results support the continued development of ambruticins as antifungal agents.

Investigating Amine Derivatives of Ambruticin VS‐5 and VS‐4

A structure–activity relationship around the amine group of the ambruticin VS series has been developed for antifungal activity. It was shown that the amine can be alkylated through reductive amination without loss of potency. However, if it is converted into either an amide, carbamate, or urea, a significant loss of potency is observed. Of the alkyl amines, small nonpolar groups are optimal for both potency and oral bioavailability. As a result of this study, one compound (KOS‐2079) was taken into an animal efficacy model with success.