Screening method for antagonists that inhibit the binding of calmodulin to a target peptide using surface plasmon resonance
Screening method for antagonists that inhibit the binding of calmodulin to a target peptide using surface plasmon resonance
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DOI：
10.1016/s0003-2670(01)01290-9
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发表时间：
2001-11
影响因子：
6.2
通讯作者：
K. Sasaki;T. Ozawa;Y. Umezawa
中科院分区：
文献类型：
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作者：
K. Sasaki;T. Ozawa;Y. Umezawa
建立了一种新的筛选方法来评价钙调素(CaM)拮抗剂在钙信号通路中对CaM与其靶蛋白相互作用的影响。用表面等离子体共振(SPR)技术研究了肌球蛋白轻链激酶(MLCK)的靶肽M13与钙调素-CaM的结合。当将含有钙调素的样品溶液注入固定在SPR传感器表面的M13的流动池中时，SPR信号在3min内显著增加并趋于平稳。在样品溶液中加入钙调素拮抗剂W-7后，平衡态的SPR信号减弱。这种SPR信号的下降是由于W-7与钙-CaM结合，从而抑制了钙-CaM与M13之间的特异性相互作用。在其他拮抗剂如三氟拉嗪、戊二胺和苯丙地尔的情况下，随着这些拮抗剂浓度的增加，SPR信号的初始增加速率降低，在平衡状态下信号达到相同的值。用本方法测得的IC50值与先前由MLCK活性本身测得的IC50值一致。因此，本方法能够找到抑制钙-钙调素与MLCK相互作用的拮抗剂。本方法的适用性也被检验和讨论，以评估内分泌干扰化学物质对钙信号通路的影响。
A new screening method was developed for evaluating effects of calmodulin (CaM) antagonists on the interaction between CaM and its target protein in the Ca2+-signaling pathway. A binding of Ca2+-CaM to the target peptide, M13, derived from myosin light-chain kinase (MLCK) is monitored by surface plasmon resonance (SPR) technique. When a sample solution containing Ca2+-CaM was injected into a flow cell with M13 immobilized on the SPR sensor surface, the SPR signal largely increased and leveled-off within 3min. By adding W-7, a CaM antagonist, into the sample solution, the SPR signal at the equilibrium state decreased. This decrease in the SPR signal is due to the binding of W-7 to Ca2+-CaM, thereby inhibiting the specific interaction between Ca2+-CaM and M13. In the case of other antagonists such as trifluoperazine, prenylamine and bepridil, upon increasing the concentration of these antagonists, the initial rate of the increase in the SPR signals decreased, and the signals reached the same value under the equilibrium state. These IC50values obtained by the present method were consistent with ones obtained earlier by MLCK activity itself. The present method was thus capable of finding antagonists inhibiting the interaction between Ca2+-CaM and MLCK. The applicability of the present method for evaluating the effect of endocrine disrupting chemicals toward the Ca2+-signaling pathway was also examined and discussed.