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  • Our current understanding of nucleotide metabolism in has ma

    2019-10-08

    Our current understanding of nucleotide metabolism in has made it possible to identify numerous proteins which serve essential roles in the life cycle of the parasite. One such protein is thymidylate synthase-dihydrofolate reductase (TS-DHFR), a bifunctional enzyme which is necessary for the production of thymidine monophosphate and folate in . TS-DHFR has been extensively characterized and shown to be a promising target for the development of inhibitors., , , Whereas TS and DHFR exist as monofunctional enzymes in humans and in bacteria, in the two enzymes are linked together on the same polypeptide chain and form a dimer via a TS-TS interface., , Interestingly, the DHFR domains of TS-DHFR also form dimer interactions in a manner which is unique to this class of enzymes. As revealed by the crystal structure of TS-DHFR (TS-DHFR), the linker region extends from each DHFR domain of the dimer, forms mostly hydrophobic interactions by way of the “crossover helix” motif with the opposite DHFR domain, and crosses back to the original monomer to complete the DHFR and TS domains (A). Recognizing that these interactions are unique to TS-DHFR led us to investigate whether such features could be explored to develop parasite-specific inhibitors. Contacts formed between the crossover helix and the DHFR domain have been shown by mutational analyses to be necessary for full catalysis and domain stability of DHFR., Earlier studies showed a decrease in catalytic activity when mutating phenylalanine 207 at the Caspase-1, human recombinant proteinase mass of the crossover helix to alanine. The resulting crystal structure of the TS-DHFR mutant revealed a small but significant shift in the position of the crossover helix which appears to weaken its interactions with the Helix B of the DHFR catalytic domain, explaining the observed decrease in catalytic activity for this mutant. With this information in mind, it is conceivable that disrupting crossover helix interactions in TS-DHFR with small molecules may lead to allosteric inhibition of the enzyme. In all available crystal structures of TS-DHFR, a non-active site pocket which may accommodate small molecules is formed just above the crossover helix and adjacent to the DHFR domain of the bifunctional protein (B). Residues from both the crossover helix and Helix B of the DHFR catalytic domain form a significant part of the pocket (C). The pocket was originally identified using the web-based application Q-SiteFinder and confirmed as a potential small molecule binding pocket using the SiteMap tool in Schrödinger (SiteScore ≥ 0.800). Visual inspection of the pocket reveals a concave surface of about 59 Å with multiple amino acid residues in position to form hydrogen-bonding interactions. The pocket also includes a phenylalanine at position 111 which is inaccessible to solvent, but capable of forming pi-stacking interactions with an aromatic group from an incoming ligand. We turned to an approach to search for compounds which can potentially maximize interactions within the proposed pocket. Computational virtual screening of large chemical libraries Caspase-1, human recombinant proteinase mass provides an inexpensive way to search for new leads targeting novel binding pockets. To aid in our efforts, the program Glide from the 2014-2 release of the Schrödinger Suite was accessed through the Structural Biology Grid and used to perform virtual screening of 14,400 commercially-available, drug-like small molecules from the Maybridge Hitfinder library. Input protein and ligand structures used for docking were prepared using Schrödinger’s Protein Preparation Wizard and LigPrep tools, respectively. Ligands were allowed to sample different binding orientations while the proposed binding pocket in TS-DHFR (PDB ID: , chain B from the A/B complex) was kept rigid. Docking was performed in successive standard precision (SP) and extra precision (XP) modes. Structures from the SP screen were ranked according to their Glide Score, and the top 15% (∼3000 structures) were subject to a second virtual screen using the XP mode in Glide. The results were ranked according to their Glide XP Scores, and the top 100 poses were inspected. Filtering was done manually to remove poses which did not contain at least two hydrogen bonds between the docked ligand and the crossover and/or catalytic Helix B of the DHFR domain. From 44 matching compounds, 15 were purchased for testing.