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 de fined medium is often compared with inhibition of serum-stimulated DNA synthesis so as to assess the selectivity of growth-factor-receptor t yrosine 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, eq uilibrium dialysis or spectrophotometer, and was quantitated by high-p erformance 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 epider mal growth factor (EGF)-receptor kinase was assayed by phosphorylation of a peptide substrate or by receptor autophosphorylation. Protein bi nding of a number of tyrphostins ranged from 64% to 98%. There was a p ositive 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 cyanoacetamide] 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-p henylpropyl)-3,4-dihydroxybenzylidene cyanoacetamide] and B56 (both hi ghly protein-bound) inhibited DNA synthesis of L23/P cells with approx imately 3-fold greater potency in 0.5% serum than in 10% serum, but th e inhibition of DNA synthesis in 0.5% serum was reduced by the additio n of BSA. Tyrphostins B46 and B56 inhibited DNA synthesis stimulated b y TGF alpha in defined medium to a greater extent than DNA synthesis s timulated by serum. However, this apparent selectivity for inhibition of TGF alpha-stimulated DNA synthesis was lost when the protein concen tration 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 result s demonstrate that protein binding accounts for the apparent selectivi ty of some highly protein-bound tyrphostins for TGF alpha-stimulated D NA synthesis of L23/P cells. Therefore, protein binding should be take n into consideration in assessments of the selectivity of tyrphostins.