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  • Book
    Tracy Curtis Holmes, II.
    Digital2012
    Developing novel therapies for gram-negative bacterial infections and glioblastoma multiforme I. cloning and characterization of the guadinomine biosynthetic gene cluster II. developing a novel chemo-sensitizing agent to treat glioblastoma. This thesis explores the development of novel therapies for the treatment of two complicated problems: Gram-negative bacterial infections and glioblastoma multiforme, the most aggressive form of brain cancer. Part I of the thesis summarizes the current body of knowledge regarding guadinomines, their biosynthesis and implications for developing novel anti-infective agents. Part II of the thesis summarizes the development of the small molecule, ERW1227B, and its ability to sensitize glioblastoma cells to standard therapies. Part I. Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509. They are the first microbial metabolites shown to inhibit the Type III Secretion System (TTSS) of Gram-negative bacteria. The TTSS is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli, Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. Inhibition of the TTSS can mitigate virulence which is important considering that Gram-negative bacteria infect millions each year, leading to considerable morbidity and mortality. The guadinomine (gdn) biosynthetic gene cluster has been sequenced, and encodes a chimeric multimodular polyketide synthase -- nonribosomal peptide synthetase spanning 26 open reading frames and 51.2 kb. It also encodes enzymes responsible for the biosynthesis of the unusual aminomalonyl-ACP extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by genetic inactivation of the cluster, as well as heterologous expression and analysis of enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these biologically important compounds. Part II. Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that both promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. Previously, we described the ability of the small molecule, KCC009, a tissue transglutaminase inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with a nucleophilic cysteine thiol residue in one (or more) target protein(s) that have affinity for the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesions and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.