<p>The human interactome presents a goldmine of potentially powerful therapeutic targets, yet very few small molecule modulators of protein-protein interactions (PPI) have been identified. PPI pose a particular challenge for drug discovery, and one of the major obstacles to fully exploiting these interactions is a lack of appropriate technologies to screen for modulating compounds. This thesis aims to address the need for function- based approaches that target PPI by using magnetic beads (MB) and mass spectrometry (MS) to develop efficient assays to monitor these interactions and their modulation by small molecules. The work begins with the validation of a novel magnetic “fishing” assay, which uses affinity-capture MB to isolate intact complexes of a “bait” protein from solution. By monitoring the recovery of the secondary binding partner, this assay was used to functionally screen a library of 1000 compounds for small molecule modulators of a calmodulin/melittin (CaM/Mel) model system. The versatility of magnetic “fishing” is clearly demonstrated during a study of a more relevant CaM-based system, which uncovered a novel mode of interaction for the CaM-binding domain of transcription factor SOX9. In addition to the MB-based approach, a simple MS-based competitive displacement assay is developed to identify minimal inhibitory fragments of a target complex as indicators of potential ‘hot-spots’. The assay was used to probe a DNA repair complex of XRCC4/ligaseIV, and identified a short helix that can be used as a more defined target surface for future high-throughput screening and rational drug design. The functional MS-based assays herein are highly adaptable tools to monitor PPI, and will facilitate the study of these and other important biomolecular interactions.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/13715 |
| Date | 04 1900 |
| Creators | McFadden, Meghan J. |
| Contributors | Brennan, John D., Junop, Murray S., Capretta, Alfredo, Chemistry and Chemical Biology |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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