Tackling redox pathways in eukaryotic parasites by a structural biology approach

All aerobic organisms are exposed to reactive oxygen species (ROS) generated as byproducts of their metabolism during their lifetime. To prevent and repair ROS-derived damage and maintain cell homeostasis, both prokaryotic and eukaryotic organisms have evolved enzymatic and non-enzymatic antioxidant systems. Among them, thioredoxin reductase (TrxR) and glutathione reductase (GR), belonging to the pyridine nucleotide-disulfide oxidoreductase family, play key roles in the regulation of redox pathways across all organisms. In particular, parasites are repeatedly exposed to the host defense machinery in their invasive stages and have to cope with oxidative stress generated both by their metabolic reactions and by the host immune system. On the global scale of public health impact, infectious diseases still deserve attention, and new therapies are urgently needed to combat devastating parasitic infections worldwide. Given their involvement in parasite survival, redox pathways represent an outstanding source of druggable target for novel antiparasitic drug discovery. The present thesis work undertakes the investigation by a structural biology approach of key thiol-dependent enzymes from two human eucaryotic parasites: Thioredoxin Glutathione Reductase from Schistosoma mansoni (SmTGR) and Thioredoxin Reductase from Cryptosporidium parvum (CpTrxR). The two studies are at different stages of research but are both grounded in a thorough characterization of the structure-function relationship of the identified drug targets, with the goal of exploiting them in a structure-based drug design approach. SmTGR is a validated and well-characterized drug target against Schistosomiasis, a neglected tropical disease affecting 280 million people and resulting in 200 000 deaths annually. To date, only praziquantel is available for the treatment of schistosomiasis and, due to massive drug administration, less sensitive parasite strains are emerging, making the identification of new therapies urgent. However, selective drug development for this class of enzyme is challenging, mainly due to the reliance on irreversible and/or covalent inhibition strategies, which are associated with unacceptable off-targets effects. This challenge was further exacerbated, until a few years ago, by the lack of structural data. Recently, a breakthrough by means of an X-ray crystallography fragment screening allowed us to identify the so-called "doorstop pocket", a novel regulatory and druggable site of TGR. The initial molecular fragments identified were ligated and partially optimized, but further attempts to determine the binding mode of these inhibitors by X-ray crystallography were unsuccessful. Thus, we settle on an integrative structural biology approach by switching to the cutting-edge cryo-EM technique that enables us to solve the first structure of TGR-inhibitor complex by this method, validating the doorstop pocket as a druggable site. Since this allosteric site is present in the whole protein family, this breakthrough offers opportunities for selectively inhibiting other pyridine nucleotide-disulfide oxidoreductases essential for various pathogens and holds implications for combating cancer. The experimental work carried out on this project has been supported by grants of the National Institute of Allergy and Infectious diseases (NIH/NIAID). On the other hand, TrxR from the apicomplexan Cryptosporidium parvum has attracted increasing attention as a promising drug target against cryptosporidiosis, the leading cause of diarrheal disease worldwide. Despite the established association with pediatric morbidity and mortality in low- and middle-income countries and with a chronic and life-threatening enteric disease in HIV-AIDS patients, there are no vaccines and treatments options are severely limited. Drug development is further hindered by the lack of many conventional target exploited for other parasitic diseases and limited ex vivo Cryptosporidium models. Compared to the related and most known malaria parasite Plasmodium falciparum, too little is known about antioxidant defense systems in Cryptosporidium. While P. falciparum relies on both thioredoxin (Trx) and glutathione (GSH) systems, no GR is encoded in the genome of C. parvum, and TrxR is the cornerstone of the antioxidant defense, supplying electrons to both the Trx and the GSH pathways. Given its role in parasite's redox homeostasis, we focus on functional and structural characterization of CpTrxR, illustrating the unique C-terminal -CGGGKCG motif, exclusive to apicomplexan parasites. Our study reports crystal structures of the enzyme with the C-terminal tail captured in different competent catalytic position, unveiling new aspects of the apicomplexan TrxR's mechanism of action. Moreover, has been shown that Auranofin (AF), a gold-containing compound and FDA-orphan drug, kills parasites in culture and here we validate the inhibition mechanism by enzymatic assays and providing the crystal structure of CpTrxR in complex with AF. These results offer crucial insights for the design of selective inhibitors, being the apicomplexan C-terminal motif notably distinct from the -GCU/CG one found in mammalian and insect TrxRs and emphasize CpTrxR as a critical target for anti-parasitic drug development.