A chemoproteomic photoaffinity platform(PAfBPP) enables target identification and mechanistic dissection of aristolochic acid-induced nephrotoxicity

Identifying the direct molecular targets of bioactive small molecules remains a major challenge in drug discovery, particularly for compounds emerging from phenotypic screens or with poorly defined mechanisms of action. To address this challenge, we developed a chemoproteomic photoaffinity-based protein profiling (PAfBPP) platform for proteome-wide target identification and validation in native biological systems. Using aristolochic acids (AA), a class of nephrotoxic natural products associated with severe kidney injury, as a model system, we constructed a comprehensive AA-binding protein atlas in kidney tissues through the design and synthesis of an AA-based photoaffinity probe. Rigorous competitive profiling and an internally developed data analysis pipeline enabled prioritization of high-confidence AA-binding proteins, leading to the identification of HIGD1A as a high-affinity AA target, as validated by surface plasmon resonance (KD = 59.6 nM) and isothermal titration calorimetry (KD = 195 nM), together with pronounced AA-induced thermal stability shifts. Site-resolved mapping using the AA photoaffinity probe in living cells identified a direct AA-binding site on HIGD1A, which was confirmed by mutagenesis, with substitution of Met72 markedly attenuating AA-induced cellular toxicity. Mechanistically, AA binding disrupted the interaction between HIGD1A and TFAM, resulting in TFAM degradation through the autophagy-lysosome pathway. Collectively, this study establishes a robust chemoproteomic PAfBPP platform for target deconvolution of bioactive small molecules and uncovers a previously unrecognized molecular mechanism underlying AA-induced nephrotoxicity, highlighting the value of this approach for drug discovery and chemical toxicology.