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

Antibody-assisted target identification reveals afatinib, an EGFR covalent inhibitor, down-regulating ribonucleotide reductase. Oncotarget. 2018 Apr 20;9(30):21512-21529 Authors: Yu CH, Chou CC, Tu HF, Huang WC, Ho YY, Khoo KH, Lee MS, Chang GD Abstract 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 ...
Source: Oncotarget - Category: Cancer & Oncology Tags: Oncotarget Source Type: research