Structural biology methods have determined 3D structures of many proteins describing their mechanism of action and providing insights into their biological function. In recent years, the link between structure and function has changed with the identification of intrinsically disordered proteins (IDPs) and regions (IDRs). These proteins do not have a defined structure but can specifically interact with their partners. Such interactions are being identified as important regulators in mammalian biology and there is great interest in their druggability. However, the identification of ligands that bind to IDPs or IDRs has been challenging.
Fragment-based lead discovery (FBLD) offers numerous advantages over conventional compound screening techniques. As well as sampling a potentially larger chemical space, fragments are more likely to bind to a protein, though with lower affinity. This requires biophysical methods to detect and characterise binding and this is the focus of this thesis.
Two IDPs were studied with their literature-reported compounds:
1. Aggregation-prone tau K18 was screened against literature ligands by NMR spectroscopy. One ligand was identified as binding and its analogues were further examined using NMR, MST and SPR. Electrophilic fragment screening by MS and NMR yielded several covalent hits, whereas an SPR-based screen yielded no hits.
2. One literature ligand was confirmed to bind to commercial cMyc peptide by NMR. Oncogenic cMyc peptides could not be produced for further studies due to degradation and purity problems.
Several fragment screens were also performed against IDP and IDR:
1. Hydrophilic acylated surface protein (HASPA), thought to be involved in leishmaniasis, was expressed and screened against a fragment library using NMR. The screening yielded no confirmed hits.
2. Protein Tyrosine Phosphatase 1B (PTP1B)(1-301) and (1-393), containing an IDR, was produced and investigated using NMR and SPR. 19F NMR fragment screening did not yield any hits binding to the IDR, whereas SPR identified several potentially IDR-directed fragments.
This study highlights the importance of orthogonal assays and direct observation of IDPs during the screening process in order to avoid potential artefacts.
