Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by lavendustin-A and its analogue

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be ≤1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 μM, whereas the overall dissociation constant is estimated to be ≤30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.