Kelly, Gilbert Thomson (2009-08). Probing the Biosynthesis and Mode of Action of Azinomycin B. Doctoral Dissertation. Thesis uri icon

abstract

  • Since the isolation of azinomycins A and B in 1954 from the soil bacterium, Streptomyces sahachiroi, these natural products have been synthetic targets. Both compounds exhibit in vitro cytotoxic activity at submicromolar levels and demonstrate anti-tumor activities comparable to that of mitomycin C in vivo. Unique to this class of natural products is the presence of an aziridine [1,2-a] pyrrolidine ring system. Coupled with an epoxide moiety, these structural functionalities impart the ability to form interstrand cross-links with DNA via the electrophilic C10 and C21 carbons of azinomycin and the N7 positions of suitably disposed purine bases. This dissertation investigates the global impact of azinomycin B treatment in a yeast model with special emphasis on DNA damage response, the resulting cell cycle effects, and cellular localization of the compound. The results provide the first demonstration of the in vivo actions of azinomycin B and are consistent with the proposed role of the drug as a DNA crosslinking agent. Biosynthesis of azinomycin B was investigated and appears to have polyketide, non-ribosomal peptide synthetase and alkaloid origins. In pursuit of elucidating the biosynthetic origin we developed both a cell culturing system and a cell-free extract procedure capable of supporting azinomycin synthesis; we used these. These were employed with labeled metabolites to probe the biosynthetic origins of the molecule. Investigations with this enzyme preparation imparted important information regarding the substrate and cofactor requirements of the pathway. These results supported the premise of a mixed origin for the biosynthesis of the molecule and paved the way for expansive stable isotope labeling studies, which largely revealed the biosynthetic precursors and probable construction of the azinomycins. Some of these studies corroborate while other results conflict with initial proposed biosynthetic routes based upon the azinomycin biosynthetic gene cluster sequence. Future azinomycin biosynthetic gene cluster enzyme characterization, mechanistic investigations, and genetic modifications will ultimately provide definitive proof for the intermediacy of proposed biosynthetic precursors and the involvement of specific cofactors. Better understanding of how nature constructs unique molecule may provide insight into eventual chemoenzymatic/gene thearapy based approaches toward cancer therapy.
  • Since the isolation of azinomycins A and B in 1954 from the soil bacterium,

    Streptomyces sahachiroi, these natural products have been synthetic targets. Both compounds

    exhibit in vitro cytotoxic activity at submicromolar levels and demonstrate anti-tumor activities

    comparable to that of mitomycin C in vivo. Unique to this class of natural products is the

    presence of an aziridine [1,2-a] pyrrolidine ring system. Coupled with an epoxide moiety, these

    structural functionalities impart the ability to form interstrand cross-links with DNA via the

    electrophilic C10 and C21 carbons of azinomycin and the N7 positions of suitably disposed

    purine bases.

    This dissertation investigates the global impact of azinomycin B treatment in a yeast

    model with special emphasis on DNA damage response, the resulting cell cycle effects, and

    cellular localization of the compound. The results provide the first demonstration of the in vivo

    actions of azinomycin B and are consistent with the proposed role of the drug as a DNA crosslinking

    agent. Biosynthesis of azinomycin B was investigated and appears to have polyketide,

    non-ribosomal peptide synthetase and alkaloid origins. In pursuit of elucidating the biosynthetic

    origin we developed both a cell culturing system and a cell-free extract procedure capable of

    supporting azinomycin synthesis; we used these. These were employed with labeled metabolites

    to probe the biosynthetic origins of the molecule. Investigations with this enzyme preparation

    imparted important information regarding the substrate and cofactor requirements of the

    pathway. These results supported the premise of a mixed origin for the biosynthesis of the

    molecule and paved the way for expansive stable isotope labeling studies, which largely revealed

    the biosynthetic precursors and probable construction of the azinomycins. Some of these studies

    corroborate while other results conflict with initial proposed biosynthetic routes based upon the

    azinomycin biosynthetic gene cluster sequence. Future azinomycin biosynthetic gene cluster enzyme characterization, mechanistic

    investigations, and genetic modifications will ultimately provide definitive proof for the

    intermediacy of proposed biosynthetic precursors and the involvement of specific cofactors.

    Better understanding of how nature constructs unique molecule may provide insight into eventual

    chemoenzymatic/gene thearapy based approaches toward cancer therapy.

publication date

  • August 2009