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