Identification of a binding target of triptolide and related studies

Triptolide, a diterpene triepoxide extracted from traditional Chinese medicinal herb Tripterygium wilfordii Hook. F has been shown to have profound inhibitory effects against tumor progression, pathological angiogenesis and inflammation. However, the mechanisms by which triptolide exerts these effects remain unclear. To understand its cellular mode of action, biotinylated/desthiobiotinylated and fluorophore-labeled triptolide derivatives were used as probes to identify cellular proteins that bind to triptolide. By using two different approaches for screening drug-protein interactions, the most prominent cellular protein bound to triptolide was confirmed to be peroxiredoxin 1 (PRDX1). This result was validated by demonstrating the ability of triptolide or its conjugated probes to bind recombinant human PRDX1. Specificity of the drug-protein interaction was established by competitive inhibition of binding of fluorophore-labeled triptolide to PRDX1 by triptolide itself. Two binding sites of triptolide to PRDX1 were found, one of which being Cys173 as confirmed by orbitrap LC-MS/MS analysis. Further study by size exclusive chromatography revealed that triptolide altered the oligomeric state of PRDX1. The decameric form of PRDX1 was dissociated into lower molecular weight species in the presence of triptolide. This observation was responsible for attenuation of PRDX1’s chaperone activity upon triptolide treatment, which was supported by evidence from both light scattering and native mass spectrometry studies. Functionally, triptolide’s synergistic effect on stress-induced cell apoptosis may be mediated, at least in part, by the interaction of triptolide with PRDX1 and the consequent inhibition of its chaperone activity. Several natural products, Celastrol, Withaferin A and Radiciol were discovered as new PRDX1 inhibitors and confirmed to physically interact with PRDX1 and exert similar functional effects as triptolide. The interaction between PRDX1 and those natural products may shed light on the detailed mechanism of their biological actions and render PRDX1 a potential target for cancer therapy.published_or_final_versionChemistryDoctoralDoctor of Philosoph