X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action

Abstract

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution x-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys140-Cys143) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys286 was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with E. coli TrxR and Trx). We note that the –CXXC– catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

Phase I Clinical Trial Results of Auranofin, a Novel Antiparasitic Agent

“…We hypothesized that the monovalent gold from auranofin would bind the redox-active dithiol group in the active site of thioredoxin; however, this was not the case in crystal structural studies of EhTrxR and EhTrx, so the exact mechanism of action remains to be determined (21). The results from the phase I trial described here support the idea of the safety of auranofin in short-term, anti-infective therapy.…”

Structural characterization of Giardia duodenalis thioredoxin reductase ( g TrxR) and computational analysis of its interaction with NBDHEX

“…In Other L-TrxRs have been crystallized in the canonical FO CC conformation, in the absence of NADPH [21,30,58] but none in the conformation displayed by gTrxR monomer B. However, higher mobility has been observed for the NADPH-binding domain in the absence of the pyridine nucleotide cofactor in the 4CCR structure of Entamoeba hystolitica TrxR [21], and the TrxR of barley (PDB code 2WHD) was crystallized without NADPH in an intermediate conformation between FO and FR (with the NADPH and FAD domains rotated to each other of only 49.8°) [30].…”

“…Although the structure of EhTrxR in presence of auranofin has been solved, the mechanism of TrxR inhibition is far to be clarified. In fact, no Au(I) was found to bind in the active site to eventually prevent electron transfer M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 from TrxR and its substrate [21]. Overexpression of gTrxR in G. duodenalis indeed increases the susceptibility to 5-nitrocompounds (i.e.…”

“…The structures of several L-TrxRs have been solved [21,[27][28][29][30][31] showing that the catalytic cycle of L-TrxR requires a large conformational change of one domain. L-TrxRs display two different conformations.…”

Recent advances in Entamoeba biology: RNA interference, drug discovery, and gut microbiome

“…Transcriptional profiling identified E. histolytica thioredoxin reductase (EhTrxR) protein as a target of auranofin. It is proposed that a monovalent gold atom, Au, is released from auranofin, subsequently inactivating the EhTrxR protein, which interferes with the redox homeostasis in the parasite and subsequently makes the parasites more sensitive to reactive oxygen-mediated cell killing 84 . Jeelani and Nozaki recently highlighted this unique thiol-based redox metabolism system as a drug target against amoebiasis 85 .…”