Hsu C Y, Persons P E, Spada A P, Bednar R A, Levitzki A, Zilberstein A
Rhône-Poulenc Rorer Central Research, King of Prussia, Pennsylvania 19406.
J Biol Chem. 1991 Nov 5;266(31):21105-12.
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 less than or equal to 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 microM, whereas the overall dissociation constant is estimated to be less than or equal to 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.
据报道，拉文达斯汀 - A是一种有效的表皮生长因子（EGF）受体酪氨酸激酶抑制剂（小野田，T.，饭沼，H.，佐佐木，Y.，滨田，M.，石比，K.，长泽，H.，竹内，T.，辰田，K.，和梅泽，K.（1989年）《天然产物杂志》52卷，1252 - 1257页）。使用杆状病毒表达的表皮生长因子受体（EGFR - IC）重组细胞内结构域详细研究了其抑制动力学。拉文达斯汀 - A（RG 14355）是受体酪氨酸激酶的一种缓慢且紧密结合的抑制剂。稳态前动力学分析表明，这种抑制作用对应于一个两步机制，其中首先快速形成初始酶 - 抑制剂复合物（EI），随后是一个缓慢的异构化步骤，形成紧密复合物（EI*）。初始快速形成复合物的解离常数为370 nM，而总体解离常数估计小于或等于1 nM。这两个值之间的差异是由于抑制剂与EI*中酶的紧密结合特性。在存在一种底物的情况下，使用预孵育方案使酶与抑制剂预平衡进行的动力学分析表明，拉文达斯汀 - A对于ATP和肽底物都是双曲线混合型抑制剂，对两种底物的结合亲和力都有主要影响。拉文达斯汀 - A的一种类似物（RG 14467）显示出与拉文达斯汀 - A相似的抑制动力学。稳态前分析的结果也与所提出的两步机制一致。在这种情况下，初始快速形成复合物的解离常数为3.4 microM，而总体解离常数估计小于或等于30 nM。它对于ATP是部分（双曲线）竞争性抑制剂。当肽与抑制剂同时添加到酶中时，其抑制作用会因不同的肽底物而不同程度地降低。当用保护作用最小的肽K1（一种包含表皮生长因子受体主要自磷酸化位点的肽）进行研究时，RG 14467对于该肽是双曲线非竞争性抑制剂。
