Computational protocol: Pharmacophore Modeling for Anti-Chagas Drug Design Using the Fragment Molecular Orbital Method

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Protocol publication

[…] Recombinant TcDHODH expression, purification, and crystallization in complex with 43 orotate derivatives were performed essentially as previously reported []. Briefly, TcDHODH was purified to homogeneity from BL21(DE3)pET3a/TcDHODH using DEAE Sepharose Fast Flow (GE Healthcare) followed by Phenyl Sepharose H.P. (GE Healthcare) and TSK G3000SW (Tosoh). By this method, a total of 10–15 mg of TcDHODH with specific activity ranging from 12 to 18 μmol/min/mg could be purified from 10 L of culture. TcDHODH crystals formed in the presence of oxonate [] were used to soak overnight into crystallization buffer containing 1 mM of orotate derivatives that are listed in . X-ray diffraction data were collected at SPring-8 (beam lines BL32XU, BL41XU and BL44XU) or Photon Factory (beam lines AR-NW12A, AR-NE3A, BL-5A and BL-17A). All the data were processed and scaled with HKL2000 []. The co-crystal structures of TcDHODH in complex with orotate derivatives were initially solved by molecular replacement using coordinates from TcDHODH-oxonate complexed structure (3W1Q) and later by one of the open form structures (e.g., 3W1R). Manual building and crystallographic refinement were performed with the programs COOT [] and REFMAC5 [], respectively. The PDB IDs, resolution, R-value, R-Free, average of the occupancy of the ligands and average of the B-factors are listed in . The detailed synthetic process of the 43 orotate derivatives and the X-ray analysis performed in this study will be described elsewhere (Inaoka et al., manuscript in preparation). […]

Pipeline specifications

Software tools Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Trypanosoma cruzi, Homo sapiens
Diseases Chagas Disease
Chemicals Flavin Mononucleotide, Hydrogen