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

Area of Science:

• Chemical Biology
• Molecular Cell Biology

Background:

• Activity-based protein profiling (ABPP) uses electrophilic compounds (stereoprobes) to identify protein targets.
• Understanding structure-activity relationships of stereoprobe-protein interactions is crucial but poorly understood.

Purpose of the Study:

• Investigate how structural modifications of tryptoline acrylamide stereoprobes affect their protein interactions.
• Characterize the binding sites and functional consequences of these interactions.

Main Methods:

• Synthesized and utilized tryptoline acrylamide stereoprobes with varying structures.
• Employed activity-based protein profiling to identify protein targets.
• Used a machine learning model (Boltz-2) for predicting binding sites.
• Assessed functional impacts on mitochondrial protein import and mitophagy.

Main Results:

• Stereoprobe interactions were sensitive to modifications distal to the reactive group.
• Most liganding occurred at non-orthosteric sites, often missed by the Boltz-2 model ('orthostery burnout').
• Stereoprobes targeting C124 in GRPEL1 disrupted HSPA9/mortalin interaction, impairing mitochondrial protein import and inducing mitophagy.

Conclusions:

• Tryptoline acrylamides are versatile covalent ligands for non-orthosteric protein sites.
• These probes can be used as tool compounds to perturb chaperone systems, such as mitochondrial HSP70.
• Understanding distal modifications is key to designing specific covalent inhibitors.