Harnessing Covalent Chemistry for Targeted Protein Labelling: A BTK Story

Abstract

Labelling of proteins in live cells is of great importance to study protein function, dynamics, and interactions in complex biological systems. Ligand-directed chemistry techniques enable the chemical modification of proteins in live cells without compromising enzymatic activity. Bruton’s tyrosine kinase (BTK) is a validated oncology target that plays a central role in B-cell proliferation and activation. Many existing covalent probes irreversibly inhibit BTK, limiting their utility for functional studies in living cells. This thesis aimed to develop activity-preserving covalent probes for BTK and to investigate covalent chemistry more broadly in the context of warhead reactivity and assay development. Ligand-directed covalent probes were designed and assessed using BTK as a model system. Fluorescent probes incorporating a methacrylamide warhead enabled labelling of BTK in cells without impairing the enzymatic activity and were applied to study BTK’s dynamic signalling pathway with confocal microscopy. To facilitate tuning of covalent warhead reactivity, an investigation of aromatic sulfoxide and sulfone warheads capable of undergoing nucleophilic aromatic substitution (SNAr) with nucleophiles was conducted. This included variation of aromatic scaffolds, leaving-groupelectronics and sulfur oxidation state. Kinetic assays showed pronounced chemoselectivity towards N-acetyl cysteine, spanning a range of reactivity profiles. Selected warheads were incorporated into BTK-probes, validating predictable labelling behaviour from reactivity in solution to protein assays. Computational studies aided with the design of a selective probe for endogenous BTK labelling which preserved enzymatic activity in cells, while binding assays validated the ligand-release mechanism. This thesis also explores how warhead orientation can influence labelling efficiency by incorporating chirality in the design of an enantiomeric pair of BTK-probes. The otherwise physicochemically equivalent probe pair displayed pronounced differences in activity and their capacity to form a covalent bond with recombinant BTK. The results highlighted the importance of stereoselective protein-ligand interactions, while also underscoring the complexity of translating these effects to cellular systems. To address limitations associated with conventional reactivity profiling methods, a fluorescence based assay was developed to assess intrinsic warhead reactivity. A FRET-based sensor incorporating an SNAr electrophile enabled quantitative kinetic analysis towards different nucleophiles in a high-throughput manner, providing a complementary and scalable approach to traditional analytical techniques. Overall, these findings address key opportunities and challenges in protein labelling and provide useful chemical tools for the design of covalent modalities. Beyond BTK, these tools may be applicable to other protein targets, making an important addition in the chemical biology toolbox.