Surface plasmon resonance analysis of nuclear factor-κB protein interactions with the sesquiterpene lactone helenalin

摘要

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-κB (NF-κB), 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-κB protein p65/RelA, with recombinant IκB kinases (IKKs) α and β, and with the intracellular antioxidant glutathione, all immobilized on sensor chips. At pH 7.4, helenalin is interacting with RelA (K_D=4.8μM), yet it failed to bind either IKKα or IKKβ. When DNA with NF-κB binding sites was immobilized on sensor chips, the binding of RelA was inhibited by helenalin with an IC_(50) of 5.0μM. At pH 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a K_D of 24μM, 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-κB protein RelA but not with IKKα or IKKβ. Moreover, at physiological pH, helenalin does not interact with glutathione to any significant extent.