The aim of this work was to investigate how the photoaffinity labelling (PAL) approach can positively impact and complement current methods of small molecule target-based drug discovery. For this broad application, an operationally simple and versatile synthetic protocol was required. An Ugi multicomponent reaction protocol for the expedient one-step synthesis of PAL probes was developed. The reaction couples an amine affinity function (compound of interest) with commonly used photoreactive groups and a variety of handle functionalities. Each component can be independently changed, providing access to a wide variety of PAL probes. For proof-of-concept studies, a series of pan-BET selective bromodomain PAL probes were obtained by parallel synthesis. Subsequent studies on the effect of different photoreactive groups, linker lengths, and irradiation wavelengths on photocrosslinking efficiency provided valuable insights into photoaffinity probe design. The observed trends in labelling were interpreted with additional consideration of the protein topology surrounding the active site, guided by crystallography and LC-MSMS studies. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles. From the optimisation studies above, PAL probes with high labelling efficiencies of recombinant BRD4 BD1 and BD2 protein were identified. The most promising probe was used to develop a novel biochemical PAL displacement assay, where the affinities of other competitor compounds to both BD1 and BD2 could be determined in the same experiment. This dual-domain PAL displacement assay represents the exciting and unexplored potential for PAL probes in biochemical assays.Additionally, preliminary investigations into the impact of PAL on fragment-based biochemical screening were performed. A series of photoreactive BET-targeting fragments were prepared and their labelling efficiencies to recombinant BRD4 BD1 were found to be concentration-dependent and correlated well with the affinity of the fragment.Overall, this work demonstrates new and exciting ways that the PAL approach can assist small-molecule target-based drug discovery, such as photoreactive fragment hit finding and novel biochemical PAL screening methods with recombinant protein. Additionally, the synthetic aspect of PAL-based chemoproteomics is made more accessible through the described one-step Ugi protocol and the information obtained regarding optimal probe design.
| Date of Award | 13 Dec 2019 |
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| Original language | English |
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| Awarding Institution | - University Of Strathclyde
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| Sponsors | University of Strathclyde |
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| Supervisor | Nick Tomkinson (Supervisor) & Glenn Burley (Supervisor) |
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