LBPO.CH01 · 化学 · Late-Breaking
一个化学蛋白质组学光亲和平台(PAfBPP)实现马兜铃酸诱导肾毒性的靶点鉴定和机制解析
A chemoproteomic photoaffinity platform(PAfBPP) enables target identification and mechanistic dissection of aristolochic acid-induced nephrotoxicity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
鉴定生物活性小分子的直接分子靶点仍是药物发现中的一大挑战，尤其对于源自表型筛选或作用机制不明确的化合物。为应对这一挑战，我们开发了一个化学蛋白质组学光亲和探针蛋白质谱分析(PAfBPP)平台，用于在天然生物系统中进行全蛋白质组范围的靶点鉴定和验证。以马兜铃酸(AA)——一类与严重肾损伤相关的肾毒性天然产物——作为模型系统，我们通过设计和合成一种基于AA的光亲和探针，在肾组织中构建了全面的AA结合蛋白图谱。严格的竞争性谱分析和内部开发的数据分析流程使高置信度AA结合蛋白得以优先排序，从而鉴定出HIGD1A为一个高亲和力AA靶点，这一结果经表面等离子体共振(KD = 59.6 nM)和等温滴定量热法(KD = 195 nM)验证，并伴有显著的AA诱导的热稳定性位移。在活细胞中使用AA光亲和探针进行位点分辨的定位鉴定出HIGD1A上一个直接的AA结合位点，该位点经突变分析确认，Met72的替换显著减弱了AA诱导的细胞毒性。在机制上，AA结合破坏了HIGD1A与TFAM之间的相互作用，导致TFAM通过自噬-溶酶体途径降解。总之，本研究建立了一个稳健的化学蛋白质组学PAfBPP平台，用于生物活性小分子的靶点解卷积，并揭示了一种此前未被认识的AA诱导肾毒性的分子机制，凸显了该方法在药物发现和化学毒理学中的价值。
查看英文原文 English abstract
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.
利益披露 Disclosure
N. Chen, None..
B. Liu, None..
W. Meng, None.