Enzyme Inhibition

Chemical compounds were tested as potential inhibitors of EGFR-TK activity in a substrate phosphorylation assay with a synthetic peptide substrate and a plasma membrane preparation derived from human A431 cells, which overexpress the EGFR. This assay first identified the anilinoquinazoline class of EGFR-TK inhibitors (Ward et al. 1994; Wakeling et al. 1996), and subsequently the candidate drug EGFR-TK inhibitor, ZD1839 (Gibson et al. 1997; Barker et al. 2001; Wakeling et al. 2002), designated Iressa. Iressa potency and kinase selectivity was demonstrated by comparing its activity against EGFR-TK with that against several other tyrosine and serine-threonine ki-nases (Wakeling et al. 2002). Enzyme kinetic methods described by Ward et al. (1994) showed that Iressa is competitive with ATP (Ki=2.1±0.2 nM) and non-competitive with substrate (Ki=15.0±1.0 nM). Thus, Iressa inhibits EGFR-TK activity by competing directly with ATP at the enzyme active site. EGF binding to EGFR stabilises a kinase active dimeric form of EGFR and stimulates intermolecular autophosphorylation of receptor homo- or hetero-(erbB)-dimers. Iressa inhibits EGF-stimulated EGFR autophosphorylation in several human tumour cell lines cells (Wakeling et al. 2002). Iressa blocked EGF-stimulated EGFR autophosphorylation in a dose-dependent and complete manner; complete inhibition was achieved at 0.16 mM in Du145 (prostate) and A549 (lung) cells, and at 0.8 mM Iressa in KB (oral squamous) and HT29 (colon) cells. Drug wash-out studies showed that this inhibition was sustained for at least 24 h following a 2-h drug treatment.

Investigation of TK inhibitory selectivity in cell lines expressing EGFR or ErbB2 showed that Iressa, at concentrations up to 5 mM, does not directly inhibit ErbB2 tyrosine phosphorylation (Christensen et al. 2001; Moulder et al. 2001; Normanno et al. 2002).

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