Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, D-89081 Ulm, Germany.
Anal Biochem. 2010 Jun 1;401(1):30-7. doi: 10.1016/j.ab.2010.02.020. Epub 2010 Feb 20.
Sesquiterpene lactones such as helenalin have generally been considered as highly promising compounds for the treatment of inflammatory disorders. Although sesquiterpene lactones are known to inhibit signaling through transcription factor nuclear factor-kappaB (NF-kappaB), the nature of their molecular targets remains controversial. To characterize the interactions of helenalin with putative target proteins, a surface plasmon resonance-based method was developed and validated to analyze the interactions of helenalin with the NF-kappaB protein p65/RelA, with recombinant IkappaB kinases (IKKs) alpha and beta, and with the intracellular antioxidant glutathione, all immobilized on sensor chips. At pH 7.4, helenalin is interacting with RelA (K(D)=4.8microM), yet it failed to bind either IKKalpha or IKKbeta. When DNA with NF-kappaB binding sites was immobilized on sensor chips, the binding of RelA was inhibited by helenalin with an IC(50) of 5.0microM. At pH 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a K(D) of 24microM, but no significant interaction was observed at pH 7.4. Thus, with this optimized method, we showed that the sesquiterpene lactone helenalin interacts with the NF-kappaB protein RelA but not with IKKalpha or IKKbeta. Moreover, at physiological pH, helenalin does not interact with glutathione to any significant extent.
倍半萜内酯类如白头翁素通常被认为是治疗炎症性疾病的极有前途的化合物。尽管倍半萜内酯类物质被认为可以抑制转录因子核因子-κB（NF-κB）的信号转导，但它们的分子靶点的性质仍存在争议。为了表征白头翁素与假定的靶蛋白的相互作用，开发并验证了一种基于表面等离子体共振的方法，用于分析白头翁素与 NF-κB 蛋白 p65/RelA、重组 IkappaB 激酶（IKK）α和β以及细胞内抗氧化剂谷胱甘肽的相互作用，所有这些都固定在传感器芯片上。在 pH 值为 7.4 时，白头翁素与 RelA 相互作用（K(D)=4.8μM），但它不能与 IKKalpha 或 IKKbeta 结合。当具有 NF-κB 结合位点的 DNA 固定在传感器芯片上时，白头翁素以 5.0μM 的 IC(50)抑制 RelA 的结合。在 pH 值为 8.0 时，白头翁素还可以与还原但不氧化的谷胱甘肽相互作用，K(D)为 24μM，但在 pH 值为 7.4 时没有观察到明显的相互作用。因此，使用这种优化的方法，我们表明倍半萜内酯白头翁素与 NF-κB 蛋白 RelA 相互作用，但不与 IKKalpha 或 IKKbeta 相互作用。此外，在生理 pH 值下，白头翁素与谷胱甘肽没有显著的相互作用。
