Time- and spatially-resolved TRAP analysis identifies drug targets and deciphers signaling network in vitro and in vivo

Target identification reveals how drugs act, and the discovered targets represent a valuable repertoire for therapeutic target discovery. Current approaches mostly investigate the direct binding targets (directTs) for drugs and lack the ability to decipher the downstream effector proteins (defined as "indirectTs"). Previously, we developed a target-responsive accessibility profiling (TRAP) approach, which measures ligand-binding induced changes in accessibility for target identification by global labeling of reactive proteinaceous lysines via reductive demethylation (Nat Chem Biol., 2023, 19, 1480–1491). We proposed that when the directTs bind to its indirectTs, the chemical accessibility also changes and can thus be detected by TRAP. Since the engagement between the drug and its directTs precedes the engagement between the directTs and the indirectTs, we sought to perform time-resolved TRAP to simultaneously identify the direct -indirect target networks.

To meet the high throughput requirements of time-resolved TRAP, data-independent acquisition (DIA) (Nature, 2016, 537, 347–355) is incorporated into the TRAP workflow for label-free quantification of TRAP-labelled peptides. Using TEPP-46 as a model compound, we incubated it with cells and then performed TRAP labelling at 6 different time points after administration (0, 5 min, 15 min, 30 min, 1 hr, 2 hr). Subsequent DIA-TRAP analysis allowed us to identify its bona fide target, PKM2, which showed the most significant decrease in accessibility at the earliest time point (5 min). Interestingly, changes in the accessibility of glycolytic enzymes such as ALDOA and GAPDH were observed at 30 min post-administration. These proteins have been annotated as interacting proteins of PKM2. Surprisingly, at a later time point (2 h), we observed that proteins involved in the cellular response to stress and the organic cyclic compounds metabolic process, which have not been linked to PKM2, also showed altered accessibility. AlphaFold was used to assess the possibility of interactions and biochemical experiments are ongoing to elucidate the signaling pathways involved.

With the analysis power enabled by DIA-TRAP, we asked whether we can assess ligands-induced accessibility changes in vivo and identify targets in a spatially-resolved manner. As a proof-of-concept, we administered TEPP-46 to mice inoculated with transplanted tumors and performed TRAP analysis on divided tumor tissues (core and periphery). TRAP analysis showed that the target PKM2 most significantly responded among all quantified TRAP peptides in all assayed regions. Notably, TRAP ratio of PKM2 extracted from tumor periphery most markedly changed when compared to the core, agreeing with the in vivo distribution of TEPP-46.

Overall, we demonstrate that DIA-TRAP enables time and spatially-resolved TRAP analysis. It allows us to identify targets for bioactive ligands and profile the downstream effectors, and is applicable to in situ contexts.