<p> Lipopolysaccharide (LPS) a major non-protein component of the outer membrane of Gram-negative bacteria, defines many of the host-bacterium interactions and provides an effective barrier by inhibiting diffusion of bile salts, detergents, and lipophilic antibiotics. The heptose molecule is an essential component of the bacterial LPS inner core and the synthesis of this subunit is an attractive antimicrobial target. A major challenge to exploit this pathway for antibiotic discoveries has been the unavailability of substrates. </p> <p> TktA, GmhA, HidE and GmhB proteins involved in the biosynthesis of the LPS precursor ADP-D-glycero-β-manno-heptose have been cloned and overexpressed in Escherichia coli to develop a simultaneous in vitro assay suitable for high-throughput screening (HTS) of small molecules. Readily accessible ribose-5-phosphate and fructose-6-phosphate were used as substrates for the heptose pathway and to circumvent the obstacles of substrate availability and stability. We have optimized an in-vitro pathway assay and report its use to screen a small molecule library that identifies the first reported inhibitor of heptose synthesis. The inhibitor, 2-Methyl-6-methylamino-7-oxo-78naphthol [ 1 ,2,3-de]quinoline-4-sulfonic acid was determined to be a competitive inhibitor of the HidE kinase with Ki of 63 ± 8 f..LM. This assay enables probing an important biochemical pathway that otherwise would be highly challenging and allows for efficient detection of novel LPS inhibitors that could potentially lead into antimicrobial treatment. </p> <p> This study also exploited structure-based drug design via protein crystallization. The crystal apoprotein structure of GmhA was determined to a 1.95 A resolution. The GmhA with the substrate bound was determined to a 2.79 A resolution which is the first reported structure of this protein bound with the substrate. By applying knowledge and techniques earned from a combination of advances in assay development, compound library development, robotic instrumentation and protein crystallization, identification and modification of novel inhibitors is possible and has the potential to advance antibiotic research. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21363 |
| Date | 11 1900 |
| Creators | de Leon, Gladys |
| Contributors | Wright, G. D., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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