Protein binding modulates inhibition of the epidermal growth factor receptor kinase and DNA synthesis by tyrphostins

Inhibition of growth factor-stimulated DNA synthesis carried out in defined medium is often compared with inhibition of serum-stimulated DNA synthesis so as to assess the selectivity of growth-factor-receptor tyrosine kinase inhibitors such as tyrphostins. We investigated whether protein binding may influence the interpretation of these experiments. Protein binding of tyrphostins was determined by ultrafiltration, equilibrium dialysis or spectrophotometer, and was quantitated by high-performance liquid chromatography (HPLC). For growth factor-stimulated DNA synthesis, we used the non-small-cell lung cancer cell line L23/P stimulated by transforming growth factor alpha (TGF alpha). The epidermal growth factor (EGF)-receptor kinase was assayed by phosphorylation of a peptide substrate or by receptor autophosphorylation. Protein binding of a number of tyrphostins ranged from 64% to 98%. There was a positive correlation (r = 0.995) between the degree of protein binding and the hydrophobicity. Inhibition of the EGF-receptor tyrosine kinase activity by the highly protein-bound tyrphostin B56 [N-(4-phenylbutyl)-3,4-dihydroxybenzylidene cyanoacet-amide] was reduced by bovine serum albumin (BSA), but BSA had less of an effect on inhibition of the EGF-receptor kinase by the weakly protein-bound tyrphostin A47 (RG 50864: 3,4-dihydroxybenzylidene cyanothioacetamide). Tyrphostins B46 [N-(3-phenylpropyl)-3,4-dihydroxybenzylidene cyanoacetamide] and B56 (both highly protein-bound) inhibited DNA synthesis of L23/P cells with approximately 3-fold greater potency in 0.5% serum than in 10% serum, but the inhibition of DNA synthesis in 0.5% serum was reduced by the addition of BSA. Tyrphostins B46 and B56 inhibited DNA synthesis stimulated by TGF alpha in defined medium to a greater extent than DNA synthesis stimulated by serum. However, this apparent selectivity for inhibition of TGF alpha-stimulated DNA synthesis was lost when the protein concentration in the defined medium was made equivalent to that in the serum-containing medium. By contrast, BSA enhanced the selective inhibition of TGF alpha-stimulated DNA synthesis by tyrphostin A47. These results demonstrate that protein binding accounts for the apparent selectivity of some highly protein-bound tyrphostins for TGF alpha-stimulated DNA synthesis of L23/P cells. Therefore, protein binding should be taken into consideration in assessments of the selectivity of tyrphostins.