Research Papers:

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

Cheng-Han Yu, Chi-Chi Chou, Hsin-Fang Tu, Wei-Chieh Huang, Ya-Yeh Ho, Kay-Hooi Khoo, Ming-Shyue Lee and Geen-Dong Chang _

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Oncotarget. 2018; 9:21512-21529. https://doi.org/10.18632/oncotarget.25177

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Cheng-Han Yu1, Chi-Chi Chou2, Hsin-Fang Tu3, Wei-Chieh Huang1, Ya-Yeh Ho1, Kay-Hooi Khoo1,2, Ming-Shyue Lee3 and Geen-Dong Chang1

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

2Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

3Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan

Correspondence to:

Geen-Dong Chang, email: [email protected]

Ming-Shyue Lee, email: [email protected]

Keywords: afatinib; target identification; ribonucleotide reductase; covalent drug; gemcitabine

Received: October 29, 2017     Accepted: April 05, 2018     Published: April 20, 2018


Afatinib, used for the first-line treatment of non-small-cell lung carcinoma (NSCLC) patients with distinct epidermal growth factor receptor (EGFR) mutations, inactivates EGFR by mimicking ATP structure and forming a covalent adduct with EGFR. We developed a method to unravel potential targets of afatinib in NSCLC cells through immunoprecipitation of afatinib-labeling proteins with anti-afatinib antiserum and mass spectrometry analysis. Ribonucleotide reductase (RNR) is one of target proteins of afatinib revealed by this method. Treatment of afatinib at 10-100 nM potently inhibited intracellular RNR activity in an in vitro assay using permeabilized PC-9 cells (formerly known as PC-14). PC-9 cells treated with 10 μM afatinib displayed elevated markers of DNA damage. Long-term treatment of therapeutic concentrations of afatinib in PC-9 cells caused significant decrease in protein levels of RNR subunit M2 at 1-10 nM and RNR subunit M1 at 100 nM. EGFR-null Chinese hamster ovary (CHO) cells treated with afatinib also showed similar effects. Afatinib repressed the upregulation of RNR subunit M2 induced by gemcitabine. Covalent modification with afatinib resulting in inhibition and protein downregulation of RNR underscores the therapeutic and off-target effects of afatinib. Afatinib may serve as a lead compound of chemotherapeutic drugs targeting RNR. This method can be widely used in the identification of potential targets of other covalent drugs.

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