Phage Display as a Tool for Probing Lipid A Biosynthesis.
dc.contributor.author | Jenkins, Ronald J. | en_US |
dc.date.accessioned | 2013-06-12T14:26:52Z | |
dc.date.available | 2013-06-12T14:26:52Z | |
dc.date.issued | 2013 | en_US |
dc.date.submitted | 2013 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/98051 | |
dc.description.abstract | The lipid A biosynthetic pathway is exclusive to gram-negative bacteria, thus making it an ideal target for antimicrobial drug discovery. Furthermore, two distinct acyltransferases, UDP-GlcNAc acyltransferase (LpxA) and UDP-3-O-(Acyl)-GlcN acyltransferase (LpxD), display structural and functional similarities within the pathway. Such similarities offer the potential to design inhibitors capable of targeting both active sites. This provides a unique paradigm to combating antimicrobial resistance by decreasing the likelihood that the bacteria would obtain resistance, through increasing the number of mutations necessary for the microbe to survive the therapeutic. Phage display was used to identify several LpxD-inhibitory peptides, one of which (RJPXD33) also inhibited LpxA (LpxD Kd = 7 uM; LpxA Kd = 22 uM) and one which was found to be selective for LpxD (RJPXD34; Kd = 31 uM). Both peptides displayed antimicrobial activity when expressed as N-terminal fusions to thioredoxin. A fluorescence polarization (FP) binding assay was developed for LpxD utilizing a fluorescein-labeled RJPXD33 (Kd = 600 nM) and for LpxA using a fluorescein-labeled Peptide 920 (Kd = 200 nM). With the FP binding assay, RJPXD33 was shown to bind competitively with acyl-ACP. RJPXD33 was co-crystallized with LpxA, in order to gain an understanding of how RJPXD33 binds to LpxA. The structural data suggested that RJPXD33 mimics the acyl-phosphopantetheine moiety of acyl-acyl carrier protein (ACP), the native substrate of LpxA. Biochemical characterization of truncated variations of RJPXD33 confirmed this model and showed that smaller peptides could be synthesized that could inhibit LpxA with similar potency. While RJPXD33 could not be crystallized with LpxD, a crosslinking strategy using photo-affinity derivatives of RJPXD33 was developed for mapping the peptide-protein interactions. Finally, the FP binding assay was employed to screen a small molecule library (~120,000 compounds) against LpxD. The hits were reconfirmed with a continuous, fluorescent enzyme assay developed for both LpxA and LpxD. Eleven compounds ranging in potency (IC50’s = 0.1 - 30 uM) were identified, three of which demonstrated in vivo toxicity in Escherichia coli lacking the multidrug efflux pump, TolC. These molecules provide a foundation for the future development of more potent small molecule inhibitors of LpxD. | en_US |
dc.language.iso | en_US | en_US |
dc.subject | Lipid A | en_US |
dc.subject | Phage Display | en_US |
dc.subject | LpxA | en_US |
dc.subject | LpxD | en_US |
dc.subject | Escherichia Coli | en_US |
dc.title | Phage Display as a Tool for Probing Lipid A Biosynthesis. | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Medicinal Chemistry | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.contributor.committeemember | Dotson, Garry Dean | en_US |
dc.contributor.committeemember | Fierke, Carol A. | en_US |
dc.contributor.committeemember | Woodard, Ronald W. | en_US |
dc.contributor.committeemember | Soellner, Matthew Bryan | en_US |
dc.subject.hlbsecondlevel | Biological Chemistry | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/98051/1/jenkinsr_1.pdf | |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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