Yamamoto Mizuki, Horie Ryouichi, Takeiri Masatoshi, Kozawa Ikuko, Umezawa Kazuo
Center for Chemical Biology, School of Fundamental Science and Technology, Keio University, Yokohama 223-0061, Japan.
J Med Chem. 2008 Sep 25;51(18):5780-8. doi: 10.1021/jm8006245. Epub 2008 Aug 26.
Previously, we designed and synthesized a potent NF-kappaB inhibitor, DHMEQ. Although DHMEQ showed potent anti-inflammatory and anticancer activities in animals, its molecular target has not been elucidated. In the present study, its target protein was found to be p65 and other Rel homology proteins. We found that (-)-DHMEQ bound to p65 covalently with a 1:1 stoichiometry by conducting SPR and MALDI-TOF MS analyses. MS analysis of the chymotrypsin-digested peptide suggested the binding of (-)-DHMEQ to a Cys residue. Formation of Cys/(-)-DHMEQ adduct in the protein was supported by chemical synthesis of the adduct. Substitution of specific Cys in p65 and other Rel homology proteins resulted in the loss of (-)-DHMEQ binding. (-)-DHMEQ is the first NF-kappaB inhibitor that was proven to bind to the specific Cys by chemical methodology. These findings may explain the highly selective inhibition of NF-kappaB and the low toxic effect of (-)-DHMEQ in cells and animals.
此前，我们设计并合成了一种强效的核因子-κB（NF-κB）抑制剂DHMEQ。尽管DHMEQ在动物实验中表现出强效的抗炎和抗癌活性，但其分子靶点尚未阐明。在本研究中，发现其靶蛋白为p65和其他Rel同源蛋白。通过表面等离子体共振（SPR）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析，我们发现（-）-DHMEQ以1:1的化学计量比与p65共价结合。胰凝乳蛋白酶消化肽段的质谱分析表明（-）-DHMEQ与一个半胱氨酸（Cys）残基结合。通过该加合物的化学合成，证实了蛋白质中Cys/（-）-DHMEQ加合物的形成。p65和其他Rel同源蛋白中特定Cys的取代导致（-）-DHMEQ结合丧失。（-）-DHMEQ是首个通过化学方法被证明与特定Cys结合的NF-κB抑制剂。这些发现可能解释了（-）-DHMEQ在细胞和动物中对NF-κB的高度选择性抑制以及低毒效应。
