Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.

23Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid 24 arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of 25 reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated 26 auranofin resistant T. gondii lines through chemical mutagenesis in order to identify the 27 molecular target of this drug. Resistant clones were confirmed with a competition assay 28 using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference 29 strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of 30 our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal 31 a consensus resistance locus, although many have point mutations in genes encoding 32 redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide 33 reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient 34 to confer resistance when introduced into wild-type parasites. Resistant clones 35 accumulated less ROS than their wild type counterparts. Our results demonstrate that 36 resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through 37 diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin 38 anti-parasitic activity as disruption of redox homeostasis. 39 40 41 42 3 45While this is currently a critical therapeutic strategy, currently approved drugs cannot 46 eliminate latent parasites in cysts that are found in chronically infected individuals. 47Moreover, these drugs have significant bone marrow toxicity, are suspected teratogens 48 and the emergence of resistance remains a potential threat to treatment. Repurposing 49 FDA-approved drugs will accelerate drug discovery for neglected parasitic diseases. 50Most recently, auranofin, a reprofiled drug that is FDA-approved for treatment of 51 rheumatoid arthritis, has emerged as a promising anti-parasitic agent. It has 52 antiproliferative activity against Plasmodium falciparum (1) , Schistosoma mansoni (2), 53Leishmania infantum (3) and Entamoeba histolytica (4) as well as other parasites with 54 public health significance (5)(6)(7)(8)(9)(10)(11). Despite this promising broad spectrum of anti-parasitic 55 activity, the molecular target of auranofin has only been indirectly implicated as 56 thioredoxin reductase. 57Thioredoxin reductase enzymes are found in archaea, bacteria and diverse 58 eukaryotes and play a key role in reduction of disulfide bonds that is essential for cell 59 replication and survival. Inhibition of human thioredoxin reductase 1 induces expression 60 of heme oxygenase-1 to repress inflammation(12). We previously demonstrated that 61 auranofin reduces T. gondii infection of host cells in vitro and a single dose of auranofin 62 allows survival of chicken embryos infected with this parasite (13). It is hypothesized 63 that the anti-parasitic activity of auranofin comes from inhibition of the thioredoxin 64 reductase enzyme of thes...