State Key Laboratory of Quality Research in Chinese Medicine, Biotechnology Laboratory of Chinese Medicine, Macau University of Science and Technology, Taipa, Macau.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jan 15;881-882:55-62. doi: 10.1016/j.jchromb.2011.11.041. Epub 2011 Dec 6.
The compound 4,4'-diphenylmethane-bis(methyl) carbamate (CM1) has a protective activity on AGEs-induced endothelial dysfunction on human umbilical vein endothelial cell (HUVEC) in our previous study. It suggested that CM1 which may act as a competitive antagonist to the blockade of AGEs to receptor of AGEs (RAGE) and attenuate the HUVEC damage. In order to testify that hypothesis, the cell membrane chromatography (CMC) combined with high performance liquid chromatography (HPLC) was developed for analyzing the competitive binding properties on RAGE of HUVEC between CM1 and MG-H1, the agonist of RAGE. The results from saturation binding of CM1 and MG-H1 on cells demonstrated that dissociation equilibrium constants (K(d)) of CM1 and MG-H1 were 3.653 nM and 4.12 nM, respectively; while maximum binding capacity (B(max)) of CM1 and MG-H1 were 30.08 and 18.72 fmol/mg protein, respectively. In competition experiments, IC₅₀ of CM1 with pre-incubation 10⁻¹⁰ M and 10⁻⁹ M MG-H1 were 1.37 × 10⁻⁹ M and 4.56 × 10⁻⁸ M, respectively. The present findings indicated that CM1 conjugated competitively to cells with RAGE ligand MG-H1. The primary study illustrated that CMC combined with HPLC analysis method could be an alternative, rapid and efficient approach for the interaction of drug molecule and receptor, and that CM1 intervene the AGEs inducing HUVEC damage may via the competitively block the AGEs-RAGE path way.
在我们之前的研究中，化合物 4,4'-二苯甲烷双（甲基）氨基甲酸酯（CM1）对人脐静脉内皮细胞（HUVEC）中 AGEs 诱导的内皮功能障碍具有保护作用。这表明 CM1 可能作为 AGEs 受体（RAGE）的竞争性拮抗剂发挥作用，从而减轻 HUVEC 的损伤。为了验证这一假设，采用细胞膜色谱（CMC）结合高效液相色谱（HPLC）法分析 CM1 与 RAGE 的激动剂 MG-H1 对 HUVEC 上 RAGE 的竞争性结合特性。CM1 和 MG-H1 在细胞上的饱和结合结果表明，CM1 和 MG-H1 的解离平衡常数（K(d)）分别为 3.653 nM 和 4.12 nM；而 CM1 和 MG-H1 的最大结合容量（B(max)）分别为 30.08 和 18.72 fmol/mg 蛋白。在竞争实验中，预孵育 10⁻¹⁰ M 和 10⁻⁹ M MG-H1 时，CM1 的 IC₅₀ 分别为 1.37 × 10⁻⁹ M 和 4.56 × 10⁻⁸ M。这些发现表明，CM1 与 RAGE 配体 MG-H1 以竞争方式结合到细胞上。初步研究表明，CMC 结合 HPLC 分析方法可能是一种替代的、快速有效的药物分子与受体相互作用的方法，而 CM1 通过竞争性阻断 AGEs-RAGE 通路来干预 AGEs 诱导的 HUVEC 损伤。
