Tryptoline stereoprobe elaboration identifies inhibitors of the GRPEL1-HSPA9 complex.

Activity-based protein profiling has identified hundreds of human proteins from diverse classes that react at cysteine residues with stereochemically defined electrophilic compounds (stereoprobes). 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. Most stereoprobe liganding events occurred at nonorthosteric sites and evaded assignment by the machine learning-based cofolding model Boltz-2, which instead tended to misplace stereoprobes in orthosteric pockets (an outcome we term 'orthostery burnout'). We found that stereoprobes reacting with C124 in the nucleotide exchange factor GRPEL1 disrupt interactions with the mitochondrial HSP70 chaperone HSPA9/mortalin, leading to impairments in mitochondrial protein import and mitophagy induction. Our results highlight tryptoline acrylamides as a versatile source of covalent ligands targeting nonorthosteric sites on proteins, including tool compounds that perturb the mitochondrial HSP70 chaperone system.