Development of chemical probes to profile functional tyrosines in live cells using sulfur triazole exchange chemistry (SuTEx)

The recent advancement of chemoproteomic technologies has led to discoveries of electrophilic small molecule warheads targeting lysine and cysteine residues that have enormously expanded our understanding of biology by enabling the identification of druggable protein targets and their associated biological functions. Still, chemical ligands have been reported for only a small fraction of the human proteome. Approximately one in three human proteins fall into the “dark genome”, of which almost nothing is known regarding their structure and function. To expand the scope of ligandable proteins, chemical probes targeting residues beyond cysteine and lysine are needed.

This work introduces Sulfur-triazole exchange chemistry (SuTEx) as a platform for developing covalent probes for tunable targeted reactivity toward more than 10,000 unique tyrosine sites in ~3700 proteins in cell lysates and live cells. Modifications to the triazole leaving group furnished sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids. Approximately 70% of the proteins bound by the probes are not in the DrugBank database and include proteins involved in RNA-recognition and protein-protein interactions, which have historically been challenging to target with small molecules. Additionally, we discovered ~30% of the tyrosine sites labeled by the probes are annotated as phosphotyrosine sites. As a proof of concept, we applied SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites.

To expand the utility of the SuTEx platform, we designed small molecule fragments based on the tested probes to target proteins with no effective inhibitors. The small molecule fragments exhibited high selectivity and potency in live cells against two target proteins, Acetyl-CoA acetyltransferase 2 (ACAT2, IC50 = 5 µM) and prostaglandin reductase 2 (PTGR2, IC50 = 1 µM). PTGR2 is found to be expressed in pancreatic cancer tissues but absent in normal pancreatic tissue, and knockdown of its expression was found to reduce tumor growth and induce apoptosis. ACAT2 is involved in regulating lipid metabolism by catalyzing the synthesis of acetoacetyl-CoA from two acetyl-CoA molecules, which is later converted to cholesterol. ACAT2 deficiency has been shown to reduce amounts of atherosclerosis.

In summary, we demonstrated that sulfur triazole exchange chemistry is a powerful platform that can provide new biological insights and novel chemical probes for drug discovery.