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Antibody-assisted target identification reveals afatinib, an EGFR covalent inhibitor, down-regulating ribonucleotide

Cheng-Han Yu1, Chi-Chi Chou2, Hsin-Fang Tu3

  • 1Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan.

Oncotarget
|May 17, 2018
PubMed

Insights

Afatinib, a non-small cell lung cancer drug, targets Ribonucleotide reductase (RNR), inhibiting its activity and reducing its protein levels. This reveals new therapeutic strategies for cancer treatment.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Oncology

Background:

  • Afatinib is a first-line treatment for EGFR-mutated non-small cell lung cancer (NSCLC).
  • Afatinib functions by irreversibly inhibiting the epidermal growth factor receptor (EGFR).
  • Identifying off-target effects is crucial for understanding drug mechanisms and potential toxicities.

Purpose of the Study:

  • To identify novel protein targets of afatinib in NSCLC cells.
  • To investigate the functional consequences of afatinib targeting on identified proteins.
  • To explore the potential of afatinib as a lead compound for RNR-targeted therapies.

Main Methods:

  • Development of an immunoprecipitation assay using anti-afatinib antiserum.
  • Mass spectrometry analysis to identify afatinib-binding proteins.
  • In vitro assays to measure intracellular Ribonucleotide reductase (RNR) activity.
  • Western blot analysis to assess RNR subunit protein levels.
  • Assessment of DNA damage markers in afatinib-treated cells.

Main Results:

  • Ribonucleotide reductase (RNR) was identified as a novel target of afatinib.
  • Afatinib potently inhibited intracellular RNR activity in permeabilized cancer cells.
  • Afatinib treatment led to increased DNA damage markers in NSCLC cells.
  • Long-term afatinib treatment reduced protein levels of RNR subunits M1 and M2.
  • Afatinib repressed gemcitabine-induced upregulation of RNR subunit M2.
  • Similar effects were observed in EGFR-null Chinese hamster ovary (CHO) cells, suggesting EGFR-independent mechanisms.

Conclusions:

  • Afatinib covalently modifies and inhibits RNR, contributing to its therapeutic and off-target effects.
  • RNR inhibition and downregulation by afatinib present a potential therapeutic strategy for cancer.
  • Afatinib serves as a lead compound for developing novel chemotherapeutic drugs targeting RNR.
  • The developed method is applicable for identifying targets of other covalent drugs.
Keywords:
afatinibcovalent druggemcitabineribonucleotide reductasetarget identification

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