A Structure-guided Active Site Affinity Ligand Unmasks a Stress-Sensitizing Role for BLVRB in the Endoplasmic Reticulum

and cellular nanoBRET assays demonstrate specific energy transfer and inform equilibrium binding affinities, target engagement, and residence time analyses for diverse panels of BLVRB inhibitors. NanoBRET demonstrates strong concordance with enzymatic inhibition and is validated by crystallographic structures confirming active site binding. Live-cell imaging using affinity ligands reveals predominant endoplasmic reticulum localization and transient suppression of the ER stress chaperone GRP78/BiP without eliciting a canonical unfolded protein response. These studies inform a redox-regulated mechanism whereby spatiotemporal BLVRB active site engagement functions as a stress sensitizer modulating ER proteostasis.