Study of Drug-Protein Covalent Interactions by Mass Spectrometry. A case study: Epidermal Growth Factor Receptor

Study of Drug-Protein Covalent Interactions by Mass Spectrometry A case study: Epidermal Growth Factor Receptor Abstract The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein belonging to the human EGFR family, which is in turn composed of four members: EGFR (ErbB1), ErbB2, ErbB3 and ErbB4. It is characterized by the presence of an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmatic domain that is endowed with a tyrosine kinase (TK) activity. EGFR signaling pathway is an important mediator of cancer cell oncogenesis, proliferation, maintenance and survival. There is a compelling evidence for a direct link between EGFR and human cancer. It is overexpressed in 40-80% of non-small cell lung cancers (NSCLC) and in other kind of tumor such as breast and prostatic cancer. On the basis of this data EGFR has become a strategic target for drug development. Several therapeutic strategies are under investigation to inhibit EGFR and my attention was focused on small molecule tyrosine kinase inhibitors, that block EGFR activity by interacting with the adenosine triphosphate-binding site of the intracellular region of the receptor. The commercial fusion protein GST- EGFR (containing the cytoplasmatic domain of EGFR) was analysed employing both MALDI and ESI ionization techniques, different sample preparations and choice of proteolytic enzymes to characterize the precise site of interaction with the irreversible inhibitor PD168393. Very good coverages were obtained by MALDI MS analysis, with the proteolytic enzyme trypsin and chymotrypsin. Samples previously submitted to MALDI analysis were analyzed by ESI MS analysis, employing an ESI ion source coupled to different instruments representing the current state-of-the art in terms of mass analyzers. In different cases peptides without and with the covalent modification were found, and the cysteine residue modified was the one expected on the basis of x ray analysis and comparative data on conserved cysteine residue in the ATP binding region: Cys 797