The unfolded protein response (UPR) pathway plays an essential role in health and diseases from cancer to neurodegeneration. Of the three UPR pathway branches, the IRE1-XBP1 pathway is the most highly conserved in comparison with the PERK and ATF6 pathways. It has been shown that selective genetic activation of XBP1 in the IRE1 pathway is effective in multiple neurodegenerative disorders from Alzheimer’s disease (AD) to Parkinson’s disease (PD). Using an XBP1-driven luciferase reporter system, we screened a library of newly designed macrocycles called rapafucins that are modeled after the immunosuppressive and anticancer drug rapamycin and identified an activator of XBP1 named Rapireon. Further characterization of Rapireon revealed that it acts as a molecule glue by recruiting FKBP12 to form a ternary complex with the cytosolic domain of IRE1. Moreover, the FKBP12-Rapireon complex was found to selectively activate the nuclease activity of IRE1 without affecting its kinase activity. In this application, we propose to carry out in-depth characterization of the mode of action of Rapireon using both biochemical and structural biology approaches. In preliminary studies, we already obtained a low-resolution cryo-EM structural model of the FKBP12- Rapireon-IRE1 ternary complex. We will continue to collect more data and refine the the structure to better than 2.5 Å resolution. In parallel, we will build on our existing high- resolution (below 2 Å) FKBP12-Rapireon binary complex x-ray crystal structure and attempt to obtain the crystal structure of the ternary complex. To improve the potency and specificity of the existing Rapireon lead, we will design and synthesize a DNA- encoded library (DEL) of Rapireon analogs and develop a solution-phase proximity ligation/deep sequencing platform to identify new analogs of Rapireon from the DEL. Once a potent lead compound is identified, we will attempt to improve the pharmacokinetic property and bioavailability of Rapireon through structural optimization of Raprieon, giving rise to a Rapireon analog that can serve as a tool compound for exploring its potential in treating neurodegenerative diseases.