This fellowship has three Aims.
The first Aim i s to use the create analogs of the guadinomines through a combined synthetic and biosynthetic approach. Hypothesis-driven analogs will be synthesized, each addressing a question about which structural features are necessary for Type III Secretion System (TTSS) inhibition. Analogs will also be made that should serve as substrates for precursor-directed biosynthesis, which should allow the rapid formation of guadinomine analogs.
The second Aim i s to devise an improved isolation protocol for guadinomines. Natural guadinomines A and B are present at levels of ~1 mg/L of culture media, and require tedious purification procedures. Since guadinomines A and B both contain a vicinal diamine, an uncommon structural subunit, we will exploit its reactivity to enhance its isolation. We have shown that diamines react cleanly with fluorine-containing aldehydes to make aminals at room temperature without added catalysts (May, Khosla - unpublished results). This will allow for solid phase fluorous extraction from the remaining material in the culture media.
The third Aim i s to determine the scope of TTSS inhibition of the guadinomines, and determine how they affect their targets. We have shown that the plant pathogen Xanthomonas is inhibited by guadinomine A (May, Mudgett - unpublished results) and will continue to study plant pathogens with natural guadinomines and their analogs. We will use a Yersinia enterolitica assay to determine which effector proteins, structural proteins, and chaperones are affected by guadinomines. This knowledge will enable a second-generation design of new and more potent analogs.
Pathogenic Gram-negative bacteria use a Type III Secretion System (TTSS) to infect hosts. The goals of the proposal are to study how the guadinomines inhibit TTSS, and to make new and more potent analogs.
|Holmes, Tracy C; May, Aaron E; Zaleta-Rivera, Kathia et al. (2012) Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor. J Am Chem Soc 134:17797-806|