Rational scaffold design mitigates mitochondrial complex I off-target inhibition in bifunctional degraders

Chemical inducers of proximity have transformed small-molecule pharmacology, but the large, bifunctional architectures they often employ introduce new and poorly understood off-target risks. During a targeted protein degrader synthesis project, we identified a subset of compounds that cause rapid and unexpected ATP depletion in cells. Mechanistic studies traced this effect to inhibition of mitochondrial complex I, a central component of oxidative phosphorylation. This inhibition does not stem from off-target binding by either of the two target ligands, but from the global molecular shape of the bifunctional molecules. Systematic truncation and modification revealed that long, linear molecular architectures enable effective occupancy of the hydrophobic ubiquinone-binding tunnel of mitochondrial complex I. Strikingly, this liability extends to structurally unrelated bifunctional molecules, including clinical candidates such as the androgen receptor degrader ARV-110, which inhibits mitochondrial complex I with nanomolar potency. In a proof-of-concept study, we redesigned the ARV-110 scaffold to mitigate mitochondrial liability. Disruption of linear molecular geometry through the introduction of structural "bumps" or "kinks" abolished mitochondrial complex I inhibition while preserving degrader potency, revealing a generalizable strategy for targeted protein degraders. These findings uncover a previously underappreciated structural determinant of off-target mitochondrial toxicity and establish new design principles for safer proximity-inducing therapeutics.