Fragment-based Ligand Discovery in Proteomes by Reversible Covalent Chemistry
Final Report Abstract
Covalent inhibitors with their potential for increased affinity and selectivity have garnered a lot of interest over the recent years. Nevertheless, covalent, non-mechanism-based inhibitors have been almost exclusively designed to target cysteine residues in proteins. This excludes the large number of proteins that do not harbor suitable cysteines from the design of covalent ligands. Lysine is a very intriguing addition as lysine is a very common amino acid in human proteomes, as it frequently resides on the surface of proteins and in protein pockets making it accessible to covalent ligands and as diverse chemistry exists to modify amines. Nevertheless, only very few lysine-directed covalent protein ligands have been designed. In this project, we set out to develop a broadly applicable platform that can be used to monitor a large number of lysine residues in the proteome in parallel and in this way to find new ligandable sites in proteins that can be addressed with lysine-directed covalent compounds. Using an alkyne-containing activated ester as our general lysine reactive probe, we were able to adapt a chemoproteomics method termed isoTOP-ABPP in order to monitor more than 8000 lysines in the human proteome. Using this technology we were able to identify especially reactive lysines in the proteome and show that these are enriched in functional sites and can be utilized for gel-based identification of effectors. Furthermore, we screened a small library of lysine reactive fragment-based electrophiles on their ability to specifically ligand lysines in the human proteome using the isoTOP-ABPP method. We identified more than 100 proteins that are liganded with lysine-directed covalent compounds. Using a novel quantitative mass-spectrometric method we were further able to show for selected cases that these compounds directly and quantitatively label the detected lysines. For several protein examples we could show that liganding a lysine blocks the function of the proteins. We demonstrate that enzymes can in this way be inhibited through active-site directed as well as allosteric mechanisms and that protein-protein-interactions can be disrupted using lysine-directed electrophiles. Altogether, the established platform will guide the design of covalent ligands that target lysine residues. Especially, the design of more stable and progressable lysine-directed electrophiles will be an important prerequisite to eventually arrive at inhibitors that can be used in complex system. We are convinced that the established platform will be a very important tool to allow the development of these electrophiles and also to screen for specific targets as well as off-targets for the developed compounds. In this way, it will largely help to expand the scope of covalent, non-mechanism-based inhibitors.
