Chemoproteomics Reveals The Cellular Targets Of DDR1 Kinase Inhibitor

Activity-based protein profiling?ABPP?has been widely used in recent years to explore the cellular target of bioactive molecules in living systems,which is of great significance in drug research and development.Recently,a small molecule DR,exhibiting potent inhibitory activity against kinase DDR1 in vitro,whose derivatives also showed promising anti-inflammatory effect in vivo using mouse model,was reported by our group,while its cellular targets remain unknown.As a result,in this work,two photoaffinity probes DR-2 and DR-4 were synthesized using photoaffinity labeling techniques,which were further applied to identify and validate the cellular targets through chemoproteomics and bioimaging.These works mainly includes the following parts:1.The N-methylpiperazine of DR was found to be solvent-accessible by investigating the co-crystal structures of inhibitor DR and kinase DDR1,indicating that modification of this group of DR would not compromise the bioactivities.Thus,a minimalist photo-crosslinker was incorporated into DR to afford the probes,DR-2 and DR-4.2.The inhibitory activities against DDR1 of these probes were tested,and the parent inhibitor DR were tested concurrently as a positive control.The results showed that both two probes retain moderate inhibitory activities against DDR1 with IC₅₀0 258.6 nM and576.6 nM3.Concentration dependent,time dependent and competitive proteome labeling were then carried out and both two probes showed high labeling efficiency both in situ and in vitro settings.The known target DDR1 was successfully captured in native cellular environment via pull-down/western blot experiments.The cellular targets were found to mainly locate in cytoplasm and cell membrane by performing competitive bioimaging.Collectively,these results proved that both probes can efficiently capture the intended cellular targets of DR.4.Subsequently,Pull-down/LC-MS/MS experiments were performed and Cathepsin D?CTSD?was found to be the main cellular target of DR,which was further validated by western blot and CETSA experiments.Furthermore,the interaction between DR and CTSD was found to be enhanced through compound accumulation in acidic lysosomes.In addition,as DDR1 has been considered as a potential therapeutic target for the treatment of inflammation and other related diseases,we further synthesized two probes DR-1 and DR-3 with a bioorthogonal handle trans-cyclooctene.By performing kinase activity inhibition assay and cell imaging experimenst,probe DR-3 was expected to label DDR1 in living cells and monitor the subcellular localization and expression level of DDRl,providing potential application in disease diagnosis and prediction.