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Living Cell-Target Responsive Accessibility Profiling Reveals Silibinin Targeting ACSL4 for Combating Ferroptosis.

Wenchao Yan1, Dexiang Wang2, Ning Wan2

  • 1School of Pharmacy, China Pharmaceutical University, Tongjiaxiang No. 24, Nanjing 210009, Jiangsu, China.

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Silibinin protects liver cells by inhibiting ACSL4, a key enzyme in ferroptosis. This study introduces a new method, LC-TRAP, for discovering natural product drug targets in living cells.

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Area of Science:

  • Biochemistry
  • Pharmacology
  • Cell Biology

Background:

  • Silibinin (SIL) is a natural product known for liver protection.
  • Identifying the specific protein targets of natural products is crucial for understanding their mechanisms of action.
  • HepG2 cells are a widely used model for studying liver function and disease.

Purpose of the Study:

  • To identify the protein targets of silibinin in HepG2 cells using a novel approach.
  • To investigate the role of ACSL4 in silibinin's hepatoprotective effects.
  • To demonstrate the utility of the LC-TRAP method for natural product target discovery.

Main Methods:

  • Living Cell-Target Responsive Accessibility Profiling (LC-TRAP) combined with quantitative proteomics.
  • Covalent lysine labeling to probe protein accessibility changes.
  • Biophysical assays, enzymatic assays, and chemical probes for target validation.

Main Results:

  • LC-TRAP identified a targetome for silibinin in HepG2 cells.
  • ACSL4 was identified as a key target, and silibinin was shown to protect against ACSL4-dependent ferroptosis.
  • Silibinin directly binds to and inhibits ACSL4 enzymatic activity.
  • LC-TRAP analysis revealed distinct binding patterns of silibinin and arachidonic acid to ACSL4.

Conclusions:

  • ACSL4 inhibition by silibinin is a potential hepatoprotective strategy.
  • The LC-TRAP method is a versatile tool for natural product target discovery without requiring ligand-derived probes.
  • This workflow is broadly applicable for identifying targets of natural products in living cells.