Abstract:
The work described in this Thesis has focused on the application of varies biophysical techniques to discover novel inhibitors and fragments to clinically significant protein targets. The combined use of high-throughput virtual screening, thermal shift assaying and protein nuclear magnetic resonance spectroscopy (NMR) to discover enzyme inhibitors was investigated. Heat shock protein 90 (HSP90), which plays an essential role in protein folding and stabilisation, was used as a model system. The combined screening strategy has led to the discovery of several HSP90 inhibitors. Notably, four of those contained the resorcinol scaffold, which was also found in HSP90 inhibitors that are currently in clinical trial. The results there validated this combined computational and biophysical approach to discover enzyme inhibitors. The use of ligand-observe NMR spectroscopy, thermal shift assaying and X-ray crystallography to conduct fragment-based screening for fibroblast growth factor receptor 1 (FGFR1) was investigated. A significant differences in hit rate were obtained by these different methods. Competition-based experiments were conducted to confirm the results although it was not conclusive, presumably due to significant non-specific interactions between the fragments and FGFR1. Future work, including the optimisation of screening conditions, may enable more conclusive fragment-screening of FGFR1 inhibitors. A literature review was conducted to investigate the use of protein-directed dynamic combinatorial chemistry for inhibitor discovery. Using literature examples, the set-up of the dynamic combinatorial library, the types of reversible reactions and the different biophysical methods that have be applied were reviewed.