Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex

Activity-based protein profiling has identified hundreds of proteins from diverse classes that react site-specifically with stereo-defined electrophilic compounds (stereoprobes) in human cells. The structure-activity relationships underlying these stereoprobe-protein interactions, however, remain poorly understood. Here we show that the protein interaction landscape of tryptoline acrylamide stereoprobes can be profoundly altered by structural modifications distal to the acrylamide reactive group. Stereoprobe liganding events, many of which occurred at non-orthosteric sites, mostly evaded assignment by affinity prediction models (e.g., Boltz-2), which instead tended to redirect the ligands to orthosteric pockets (an outcome we refer to as “orthostery burnout”). We discovered that stereoprobes reacting with C124 in the nucleotide exchange factor GRPEL1 disrupt interactions with the mitochondrial Hsp70 chaperone (mortalin/HSPA9), leading to impairments in mitochondrial protein import and induction of mitophagy. Our results highlight tryptoline acrylamides as a versatile source of first-in-class covalent ligands targeting non-orthosteric sites on proteins, including tool compounds that perturb the mitochondrial HSP70 chaperone system.