On the other hand, if the small molecules are specifically inhibitory for TS, the addition of BSA should not impact their potency. pregnant. Under these conditions, the parasite can form cysts in the brain that can eventually result in depression, anxiety, and schizophrenia2 in addition to fatal toxoplasma encephalitis and birth defects. To combat the infection, molecular targets are needed for drug therapy. One suitable target is the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR), responsible for nucleotide synthesis. Thymidylate synthase catalyzes the transfer of a methylene group from methylene-tetrahydrofolate to dUMP to create dTMP necessary for DNA replication.3 Conserved arginines facilitate substrate binding by transversing the dimer interface and contacting the dUMP molecule in the adjacent monomer.4 Proper orientation of the TS monomers is therefore required for catalysis. Peptides targeting the dimer interface in the human TS have been recently reported as well as the crystal structure of human TS in the apo-active site form with the peptide bound at a cavity in the TS/TS interface (PDB ID: 3N5E).5 The structure of bifunctional TgTS-DHFR in the presence of dUMP and the folate inhibitor PDDF has also been solved (PDB ID: 4EIL).6 There is no obvious interface cavity in the apo-active site human TS without the peptide bound nor in the liganded Tg or human TS structures.4a, 6C7 This structural information suggests that the peptide in human TS causes the domains to move apart from one another, creating a pocket in which the peptide is able to bind. This conformational change upon nucleotide binding is significant in part due to its pharmacological relevance. While human and TgTS share a large degree of sequence and structural conservation, several differences in the primary sequence of TS/TS interface residues the two enzymes exist (Supplementary Figure 1). Given that one amino acid substitution is sufficient to significantly alter conformational changes in Timapiprant sodium human TS, these sequence differences could cause unique molecular motions for each version of TS, allowing for the design of selective, allosteric inhibitors.7C8 Peptides that bind to the interface between the apo-dUMP TS domains of both Tg and human TS disrupt the organization of the TS/TS interface and thus reduce TS activity.5, 9 Recent results suggest that the conformational changes that take place in unliganded human TS to allow for peptide binding could also occur TgTS.9 We therefore reasoned that small drug-like molecules could bind at the TS/TS interface in TS/TS interface binding site. (A) Superimposed model created by removing the DHFR domains from the TS-DHFR crystal structure and superimposing the TS structure on the peptide-bound human TS structure. Dashed lines indicate that the coordinates of the DHFR domains were removed to facilitate the superposition. (B) Homology model of TS created using the amino acid sequence of TgTS and the peptide-bound human TS structure. The second strategy Timapiprant sodium created a homology model using the amino acid sequence of TgTS (GenBank accession code: “type”:”entrez-protein”,”attrs”:”text”:”AAB00163″,”term_id”:”295357″,”term_text”:”AAB00163″AAB00163) and the structure of peptide-bound human TS (Figure 1b). The program SWISS-MODEL was used to generate the homology model.10 This strategy modeled the shift in monomers relative to each other as well as specific loop movements that take place upon peptide binding. Superimposing the homology model on the TgTS crystal structure provided an RMSD of 0.86 ?, indicating that most of the model matched the solved structure. The portions of the model that differed most significantly from the structure were near the predicted peptide-binding site at the TS/TS interface. For the superimposed model, we used the SiteMap function of the Schrodinger suite Glide software to locate a large continuous hydrophobic patch in the TS/TS interface pocket (Figure 2).11 This region was explored computationally using CASTp12 and LIGSITE.13 This analysis revealed that the cavity between the two TS subunits in the superimposed model had a volume of 104.3 ?3 compared to the 160 ?3 peptide-binding pocket in human TS. The cavity in the superimposed model was used for docking 14,400 compounds in the screening library Maybridge Hitfinder, a subset of the ZINC database containing drug-like screening compounds.14 This approach has been used to successfully target allosteric pockets in bifunctional TS-DHFR from other species.15 A selection criteria was used where the top hundred hits from the initial run were then screened against the TS active site rather than the allosteric site. The purpose of the selection criteria was to find compounds with the highest Glide XP score.SiteMap located a hydrophobic pocket at the TS/TS interface of the homology model at a similar location as the superimposed model, but with a different shape for the target area selected for molecular docking (Figure 3).11 After performing a virtual screen using Glide and the Maybridge Hitfinder library, the five compounds with the highest Glide score were selected for inhibition studies (Supplementary Table II). organism.1 Infection can occur when an individual is immunocompromised during HIV infection, in those having received organ transplants, and in women who are pregnant. Under these conditions, the parasite can form cysts in the brain that can eventually result in major depression, panic, and schizophrenia2 in addition to fatal toxoplasma encephalitis and birth defects. To combat the infection, molecular focuses on are needed for drug therapy. One appropriate target is the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR), responsible for nucleotide synthesis. Thymidylate synthase catalyzes the transfer of a methylene group from methylene-tetrahydrofolate to dUMP to produce dTMP necessary for DNA replication.3 Conserved arginines facilitate substrate binding by transversing the dimer interface and contacting the dUMP molecule in the adjacent monomer.4 Proper orientation of the TS monomers is therefore required for catalysis. Peptides focusing on the dimer interface in the human being TS have been recently reported as well as the crystal structure of human being TS in the apo-active site form with the peptide bound at a cavity in the TS/TS interface (PDB ID: 3N5E).5 The structure of bifunctional TgTS-DHFR in the presence of dUMP and the folate inhibitor PDDF has also been solved (PDB ID: 4EIL).6 There is no obvious interface cavity in the apo-active site human being TS without the peptide bound nor in the liganded Tg or human being TS constructions.4a, 6C7 This structural info suggests that the peptide in human being TS causes the domains to move apart from one another, developing a pocket in which the peptide is able to bind. This conformational switch upon nucleotide binding is definitely significant in part due to its pharmacological relevance. While human being and TgTS share a large degree of sequence and structural conservation, several differences in the primary sequence of TS/TS interface residues the two enzymes exist (Supplementary Number 1). Given that one amino acid substitution is sufficient to significantly alter conformational changes in human being TS, these sequence differences could cause unique molecular motions for each version of TS, allowing for the design of selective, allosteric inhibitors.7C8 Peptides that bind to the interface between the apo-dUMP TS domains of both Tg and human being TS disrupt the organization of the TS/TS interface and thus reduce TS activity.5, 9 Recent results suggest that the conformational changes that take place in unliganded human TS to allow for peptide binding could also occur TgTS.9 We therefore reasoned that small drug-like molecules could bind in the TS/TS interface in TS/TS interface binding site. (A) Superimposed model produced by removing the DHFR domains from your TS-DHFR crystal structure and superimposing the TS structure within MGC34923 the peptide-bound human being TS structure. Dashed lines show the coordinates of the DHFR domains were eliminated to facilitate the superposition. (B) Homology model of TS created using the amino acid sequence of TgTS and the peptide-bound human being TS structure. The second strategy produced a homology model using the amino acid sequence of TgTS (GenBank accession code: “type”:”entrez-protein”,”attrs”:”text”:”AAB00163″,”term_id”:”295357″,”term_text”:”AAB00163″AAbdominal00163) and the structure of peptide-bound human being TS (Number 1b). The program SWISS-MODEL was used to generate the homology model.10 This strategy modeled the shift in monomers relative to each other as well as specific loop movements that take place Timapiprant sodium upon peptide binding. Superimposing the homology model within the TgTS crystal structure offered an RMSD of 0.86 ?, indicating that most of the model matched the solved structure. The portions of the model that differed most significantly from the structure were near the expected peptide-binding site in the TS/TS interface. For the superimposed model, we used the SiteMap function of the Schrodinger suite Glide software to locate a large continuous hydrophobic patch in the TS/TS interface pocket (Number 2).11 This region was explored computationally using CASTp12 and LIGSITE.13 This analysis revealed the cavity between the two TS subunits in the superimposed magic size had a volume of 104.3 ?3 compared to the 160 ?3 peptide-binding pocket in human being TS. The cavity in the superimposed model was utilized for docking 14,400 compounds.