Reactive fragment screening for expansion of the liganded proteome

Abstract

Advances in genomic analyses enable the identification of proteins that are associated with disease. In order to validate these targets for prioritisation in drug discovery programmes, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space, however, traditional techniques often lack the sensitivity / throughput to profile weak-binding fragments. The use of electrophilic fragment libraries has emerged within chemical biology as a powerful approach for the identification of fragment–protein interactions, particularly in a cellular context.

The Chemical Biology group at GSK have developed reactive fragment platforms for identification of ligands through screening with purified protein and by chemoproteomic profiling in cells. Work with purified protein has employed cysteine- and photo-reactive libraries for the identification of tools for protein targets of interest. More recently, sulfonyl fluoride libraries have enabled identification of ‘beyond-cysteine’ covalent ligands that react with lysine, tyrosine and histidine residues, greatly expanding the portion of the proteome that can be accessed with covalent ligands. Proof of concept studies have enabled identification of tool molecules for multiple protein classes including E3-ligases, bromodomains and oncology targets.1,2,3

The application of reactive fragment screening in cells by chemoproteomics offers potential for expansion of the liganded proteome. A protocol has been developed employing data independent acquisition (DIA) to enable rapid and robust profiling of fragment-proteins interactions across the proteome. Additionally, a more focussed platform has been developed for the study of the ‘DUBome’, which has been employed for the identification of covalent inhibitors for selected deubiquitinases. These platforms highlight the opportunity for systems chemical biology approaches, where covalent ligands are employed directly in live cell experiments, circumventing the requirement for protein purification and providing more physiologically relevant data.