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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Ting Yu  

LAB HEAD: Mingliang Ye

PROVIDER: PXD057157 | Pride | 2025-10-15

REPOSITORIES: Pride