The oxidoreductase Tpx: dissection of an unusual inhibitor binding mode and structural basis for its function in the hydroperoxide clearance cascade in trypanosomatids

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Parasites cause devastating diseases and threathen the health of both people and their livestock. Trypanosomatids, including the causative agents of African Sleeping Sickness (Trypanosoma brucei spp.), Chagas disease (T. cruzi) and visceral and cutaneous Leishmaniosis (Leishmania spp.) are unicellular eukaryotic protozoans that are characterized by a unique thiol redox metabolism centered on trypanothione as the global electron donor for DNA synthesis, protein repair and hydroperoxide detoxification. These reactions are carried out by peroxidases such as PxIII that are reduced via tryparedoxin (Tpx), an essential parasitic oxidoreductase. We recently determined the first structure of Tpx in complex with a small, covalent inhibitor that shows anti-trypanosomal potential in vitro and in vivo. Intriguingly, this inhibitor (CFT) induces Tpx dimerization through intricate protein-inhibitor, protein-protein and inhibitor-inhibitor interactions thus constituting a novel concept in the area of chemically induced dimerization. We further described multiple Tpx point mutations that affect oxidoreductase function and/or its interaction with CFT, trypanothione and peroxidases. Despite these advances, a detailed mechanistic understanding of Tpx-catalyzed redox reactions is currently hampered by the lack of reliable structural data for Tpx in complex with its upstream and downstream partners. Finally, the development of improved Tpx inhibitors requires a detailed molecular description of the relevant parameters dictating protein-inhibitor interactions and the observed CFT-induced protein dimerization. We thus aim to employ an integrated structural biology approach to elucidate the structural and dynamical basis of Tpx interaction with the inhibitor CFT, trypanothione and PxIII. Some of the Tpx-CFT interactions involve the protein backbone and can therefore not be investigated by mutagenesis. In these cases, we will chemically modify the inhibitor to probe specific determinants of the intermolecular CFT-Tpx interaction. Tpx is highly conserved across trypanosomatids. This includes the most widely distributed and the most medically relevant human pathogenic species T. cruzi, L. infantum, L. donovani and L. major. Thus, a detailed molecular characterization of small molecule inhibitor Tpx interactions has unprecedented potential for the development of novel drugs against these neglected tropical diseases. Consequently, we will investigate the interaction of our inhibitors with the tryparedoxin oxidoreductases from these organisms on a structural and functional level.