Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue.
Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue.
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Lavendustin-A及其类似物抑制表皮生长因子受体酪氨酸激酶的动力学分析。
DOI：
10.1016/s0021-9258(18)54826-2
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Asher Zilberstein
中科院分区：
文献类型：
--
作者：
C. Y. Hsu;P. Persons;A. Spada;R. Bednar;A. Levitzki;Asher Zilberstein
据报道，Lavendustin-A是表皮生长因子(EGF)受体(Onoda，T.，Iinuma，H.，Sasaki，Y.，Hamada，M.，Isshibi，K.，Naganawa，H.，Takeuchi，T.，Tatsuta，K.和Umezawa，K.(1989)J.NAT)的有效酪氨酸激酶抑制剂。戳。52、1252-1257)。利用杆状病毒表达的重组EGF受体胞内区(EGFR-IC)对其抑制动力学进行了详细的研究。Lavendustin-A(RG 14355)是一种缓慢而紧密结合的受体酪氨酸激酶抑制剂。稳态前动力学分析表明，抑制作用符合两步反应机制，即快速形成酶-抑制剂复合体(EI)，然后缓慢异构化形成致密复合体(EI*)。初始快速形成的络合物的解离常数为370 nm，而总的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*中的酶具有紧密结合的性质。动力学分析表明，Lavendustin-A对ATP和多肽底物均为双曲线混合型抑制物，对两种底物的结合亲和力都有很大影响。Lavendustin-A的类似物(RG 14467)显示出与Lavendustin-A相似的抑制动力学。预稳态分析的结果也与所提出的两步机制相一致。在这种情况下，初始快速形成的络合物的解离常数为3.4微米，而总解离常数估计小于或等于30 nm。它是一种相对于ATP的部分(双曲)竞争性抑制物。当多肽与抑制剂同时加入到酶中时，不同的多肽底物会不同程度地降低其抑制作用。当用保护性最弱的多肽K1(一种含有表皮生长因子受体的主要自磷酸化位点的多肽)进行研究时，RG 14467对该多肽起到了双曲线非竞争性抑制作用。
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.