Solvent-Induced partial Cellular Fixation Approach enables systematic identification of drug targets and temporal progression of downstream biochemical pathways in living cells

In this study, we introduce a novel approach, the Solvent-Induced Partial Cellular Fixation Approach (SICFA), to monitor drug targets and the temporal progression of downstream biochemical pathways in living cells. We observed that proteins in living cells undergo unfolding and aggregation when exposed to increasing concentrations of solvent-based fixatives, a process modifiable by molecular interactions and intracellular dynamics. Combined with quantitative proteomics, SICFA can precisely detect the effects of drugs on the stability of target and downstream effector proteins. As proof of concept, the intracellular targets and downstream effector proteins of several drugs were identified. Additionally, the IsoSolvent Dose-Response (ISDR)-SICFA workflow was developed to quantify drug-target binding potency within cells. To distinguish between direct drug targets and downstream biochemical pathways, time-dependent experiments were integrated with SICFA, systematically identifying the molecular initiation, intermediate, and outcome events triggered by 5-fluorouracil (5-FU). Notably, SICFA revealed that the early stages of 5-FU treatment primarily impact RNA post-transcriptional modification and ribosome biogenesis pathways. Unlike protein expression data, the stability data provided by SICFA offers higher sensitivity, filling the gap in understanding early biochemical events during drug treatment.

本研究提出一种全新方法——溶剂诱导部分细胞固定法（Solvent-Induced Partial Cellular Fixation Approach, SICFA），用于监测活细胞内的药物靶点及下游生化通路的时序动态变化。研究观察到，活细胞内的蛋白质在暴露于浓度递增的溶剂型固定剂时会发生解折叠与聚集，该过程可通过分子相互作用及细胞内动态过程进行调控。结合定量蛋白质组学技术，SICFA可精准检测药物对靶点及下游效应蛋白稳定性的影响。作为概念验证，本研究已鉴定出多种药物的细胞内靶点及下游效应蛋白。此外，本研究还开发了等溶剂剂量响应（IsoSolvent Dose-Response, ISDR）-SICFA工作流程，用于量化细胞内药物靶点的结合效力。为区分直接药物靶点与下游生化通路，本研究将时序依赖性实验与SICFA相结合，系统鉴定了氟尿嘧啶（5-fluorouracil, 5-FU）所触发的分子起始事件、中间事件与结局事件。值得注意的是，SICFA揭示5-FU处理的早期阶段主要影响RNA转录后修饰及核糖体生物发生通路。与蛋白质表达数据不同，SICFA所提供的稳定性数据具有更高的灵敏度，填补了人们对药物治疗早期生化事件认知的空白。