Year of Publication

2011

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Dr. Jürgen Rohr

Abstract

Gilvocarcin V (GV), the principal product of Streptomyces griseoflavus Gö 3592 and other Streptomyces spp., is the most prominent member of a distinct class of antitumor antibiotics that share a polyketide derived coumarin-based aromatic core. GV and other members of this class including polycarcin V from Streptomyces polyformus, often referred to as gilvocarcin-like aryl C-glycosides, are particularly interesting because of their potent bactericidal, virucidal and antitumor activities at low concentrations while maintaining low in vivo toxicity. Although the precise molecular mechanism of GV bioactivity is unknown, gilvocarcin V has been shown to undergo a photoactivated [2+2] cycloaddition of its vinyl side chain with thymine residues of DNA in near-UV or visible blue light. In addition, GV was shown to selectively crosslink histone H3 with DNA, thereby effectively disrupting normal cellular processes such as transcription. Furthermore, GVs ability to inhibit topoisomerase II has also been attributed as a mechanism of action for gilvocarcin V activity. The excellent antitumor activity, as well as an unprecedented structural architecture, has made GV an ideal candidate for biosynthetic studies toward the development of novel analogues with improved pharmacological properties. Previous biosynthetic research has identified several candidate genes responsible for key steps during the biosynthesis of gilvocarcin V including an oxygenase cascade leading to C-C bond cleavage, methylations, lactone formation, C-glycosylation and vinyl side chain formation.

In this study, we further examined two critical biosynthetic transformations essential for the bioactivity of gilvocarcin V, namely starter unit incorporation and C-glycosylation, through the following specific aims: 1) creation of functional chimeric C-glycosyltransferases through domain swapping of gilvocarcin-like glycosyltransferases and identification and evaluation of the donor substrate flexibility of PlcGT, the polycarcin V pathway specific C-glycosyltransferase; 2) creation of a library of O-methylated-L-rhamnose analogues of polycarcin V for structure activity relationship studies; 3) identification of the role of GilP and GilQ in starter unit specificity during gilvocarcin V biosynthesis; and 4) creation of a plasmid based approach in which selective gilvocarcin biosynthetic genes were utilized to produce important gilvocarcin intermediates for further in vivo and in vitro experimentation.

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