Fragment-based photoaffinity probes for examining protein-small molecule and protein-protein interactions

Abstract

Proteins are main players in the execution and regulation of biological processes in all organisms. They are able to specifically bind with small molecules, nucleic acids and other proteins in cells, which is the basis of them performing diverse functions including catalysis, stimuli responding, supporting cellular structure, transporting biomolecules and regulating gene expression as well as protein synthesis. Such interactions, if dysregulated, will lead to compromised protein functions and eventually cause various diseases. To elucidate key disease-related pathways and to develop novel therapeutics, building platforms and techniques for investigating protein-protein interaction and protein-small molecule interaction is highly desirable. One classic method to study protein-small molecule interactions is affinity chromatography. Despite its decades of success, it has the limitation of not being performed in a cellular context and its preference towards more abundant or strongly bound proteins. To overcome these drawbacks, activity-based protein profiling (ABPP) and affinity-based protein profiling (AfBPP) have been applied to identify targets of natural products and to screen inhibitors for given proteins. Our group have put great effort on developing photoaffinity probes to investigate the binding partners of proteins-of-interest. The probes bear photo-crosslinkers that react with adjacent molecules upon UV irradiation and form covalent bonds, which is especially valuable when detecting dynamic, weak or transient interactions. By installing bio-orthogonal handles on the probe, the photo-crosslinked products are readily enriched, which facilitates the detection of low abundance proteins among complex cellular proteome. In Chapter 2, a small library of fragment-based photoaffinity probes for ligand and target discovery was synthesized. Each probe bears a diazirine group as the photo-crosslinker and an alkyne as the bio-orthogonal handle. Photoaffinity-based protein profiling in HeLa cells showed that the probes labelled diverse classes of proteins including those that are commonly considered “undruggable”. A case study of Probe 22 having preferential binding with the second bromodomain of Bromodomain-containing protein 4 (BRD4) was carried out with binding site identification result, preliminary binding affinity data and evidence from cellular experiments such as photo-crosslinking and pulldown and fluorescence recovery after photobleaching (FRAP). Various methods of investigating protein-protein interactions have been developed including affinity purification, co-immunoprecipitation (co-IP), crosslinkingassisted co-IP and label transfer protein interaction analysis. Previously, our group reported the design and synthesis of photo-lysine (pLys), a photo-crosslinkable lysine for metabolic labelling of cellular proteins which facilitated the identification of interacting proteins of histones that recognize histone lysine post-translational modifications. We would like to broaden the application of pLys by combining metabolic labeling with overexpression of tagged proteins that can be enriched after photo-crosslinking, which we termed “Photo-ID”. In Chapter 3, FLAG-His-tagged proteins were overexpressed in HEK293T cells followed by metabolic labeling of pLys. Preliminary mass spectrometry data of histone H3 interacting proteins showed that Photo-ID could identify large number of known H3 interactors. Performing Photo-ID in cells arrested at different stages of the cell cycle was also attempted in the hope of identifying cell-cycle dependent interactors of protein-of-interest.