Computational prediction and experimental validation of low-affinity target of triptolide and its analogues

Abstract

Triptolide, triptonide and triptriolide are active ingredients of traditional Chinese herbal medicine Tripterygium wilfordii Hook.f. Although these compounds are found to have significant anti-inflammatory, immune-suppressive or anti-tumour effects, the molecular mechanisms of actions, especially their binding to proteins remain unclear. Since the chemical structures of triptolide, triptonide and triptriolide are similar to steroid hormones, we try to identify potential target proteins from the steroid hormone receptors (or “nuclear receptors”). In this study, using the reverse docking strategy, 12 nuclear receptors are reversely docked to triptolide and ranked by the binding energy scores. Based on this, human estrogen receptor alpha (ERα) was selected as a potential interaction protein for triptolide and the binding mode of three compounds to ERα-LBD (ligand binding domain) was further assessed by Docking and Molecular Dynamics (MD) simulation. To further analyze the docking results, Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Reporter Gene assays were used to validate the interactions of ERα-LBD with the three compounds. SPR studies together with ITC measurements indicated that the three compounds could bind to ERα-LBD with weak affinity. Triptonide showed the highest affinity and triptriolide exhibited the weakest affinity. Furthermore, the binding of triptonide or triptolide to ERα significantly increased the reporter gene activity in human cervical cancer cell lines HeLa. This study not only further defines the binding proteins of triptolide and its analogues but also provides useful information for application of these compounds.