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 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.
Highlights
The epidermal growth factor receptor (EGFR) is one of four members of the ErbB family along with HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4)
As EGFR is the known target of afatinib, we attempted to confirm this notion by immunoprecipitation with antiEGFR antibody followed by immunoblotting with antiafatinib antiserum using detergent extract from HeLa cells treated with or without 10 μM afatinib for 1 h in culture
Treatment of afatinib in cellulo caused degradation of Ribonucleotide reductase (RNR) proteins resulting in DNA damage in PC-9 cells independent of the EGFR signal pathway since the similar effects can be observed in the EGFR-null Chinese hamster ovary (CHO) cells (Figure 5B and 5C)
Summary
The epidermal growth factor receptor (EGFR) is one of four members of the ErbB family along with HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Erlotinib and gefitinib are the firstgeneration EGFR tyrosine kinase inhibitors (TKIs) with high specificity to EGFR [4]. These two drugs bind reversibly to the ATP binding pocket of the catalytic domain and effectively block the downstream signaling initiated from EGFR ligand binding. Resistance to these drugs occurs frequently in NSCLC patients due to de novo EGFR mutations, especially deletions in exon 19 (EGFRdel19) and the exon 21 L858R mutation (EGFR L858R) [5]. Afatinib developed under Boehringer Ingelheim is a covalent inhibitor of ErbB family with www.oncotarget.com
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