Computational protocol: Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13

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[…] In this study, 6- to 8-week-old female specific-pathogen-free immunocompetent BALB/c mice (Charles River, Wilmington, MA) were infected via a low-dose aerosol exposure to M. tuberculosis Erdman in a Middlebrook aerosol generation device (Glas-Col Inc., Terre Haute, IN). One day post aerosol infection, three mice were sacrificed to verify the uptake of an average of about 100 CFU of bacteria per mouse. Following infection, the mice were randomly divided into treatment groups. Negative control mice remained untreated. Positive control mice received INH (at 25 mg/kg of body weight). Each group consisted of five mice at each time point. Treatment was started day 27 post aerosol and continued for 4 weeks. Five infected mice were killed at the start of treatment as pretreatment controls. Drugs were administered in canola oil, 5 days per week by oral gavage. To determine drug efficacies, mice from each treatment group were sacrificed after 4 weeks of treatment. The mice were humanely euthanized by CO2 inhalation. The spleens and left lung lobes were aseptically removed and disrupted in a tissue homogenizer. The number of viable organisms was determined by serial dilution of the homogenates on nutrient Middlebrook 7H11 agar plates (GIBCO BRL, Gaithersburg, MD). The plates were incubated at 37°C in ambient air for 4 weeks prior to determine the total number of culturable mycobacteria per organ. For statistical analysis, the number of culturable mycobacteria were converted to logarithms, which were then evaluated by a one-way analysis of variance, followed by a multiple-comparison analysis of variance by a one-way Tukey test (SigmaPlot software program). Differences were considered significant at the 95% level of confidence. [...] For diffraction data collection the crystals were cryo-protected using Fomblin (Sigma) and flash frozen in liquid nitrogen. High resolution data was collected at wavelengths of 0.98 – 1.03 Å on the beamlines 19-ID and 23-ID at the Advanced Photon Source (APS) of the Argonne National Laboratory, Chicago, IL, USA. All the datasets were processed and scaled with HKL2000 (). Analysis of the integrated and scaled data by Xprep () indicated that Pks13-TE crystallized in P21212 space group. Solvent content analysis in CCP4 suite indicated the presence of two molecules (VM 2.16, VS 43.2%) in the asymmetric unit (). [...] The structure of the Pks13-TE domain was solved by molecular replacement method (MR) using E. coli EntF (PDB: 3tej) structure, as search model. A single MR solution was obtained using Phenix AutoMR () which was input into the AutoBuild wizard to generate the initial model for apo-Pks13-TE. The initial model was improved by further manual rebuilding in COOT (). The final model was obtained after iterative cycles of model building and Phenix refinement with simulated annealing yielding a 1.72 Å resolution apo-Pks13-TE model with Rcryst of 16.9% and an Rfree of 20.1% with good stereochemistry (). The final refined apo-model has two chains, designated A and B, a fragment of additive PPG P400 and 471 water molecules in the asymmetric unit. The crystal structures Pks13-TE-inhibitor complex structures, as well as the D1607N mutant structures were refined with simulated annealing (start temperature 5000 K, Phenix). Inspection of electron density maps showed clear |Fo-Fc| positive difference density for the ligands which were fit into the density using Ligandfit routine in Phenix (). The ligand model and geometry restraint files were created in ELBOW BUILDER of the Phenix suite (). Iterative cycles of model building and NCS-restrained maximum likelihood refinement with simulated annealing yielded high quality models for Pks13-TE-inhibitor complexes ( and ). In all of the structures > 98% of residues are placed in the favored region of the Ramachandran plot (MolProbity, ). Figures of the structures were made with UCSF Chimera package () and PyMOL Molecular Graphics System version 1.4.1 (Schrodinger, LLC). Structural analysis of Apo Pks13-TE for the identification of tunnels and channels was done using CAVER 3.0 PyMol plugin (). Electrostatic surface potentials were calculated using APBS () and displayed using the APBS plugin for PyMOL. Atomic coordinates and structure factors for the reported crystal structures ( and ) have been deposited with the Protein Data Bank under accession codes: PDB: 5V3W (Apo Pks13-TE), PDB: 5V3X (Pks13-TE:TAM1), PDB: 5V3Y (Pks13-TE:TAM16), PDB: 5V3Z (Pks13-TE(D1607N)), PDB: 5V40 (Pks13-TE:TAM6), PDB: 5V41 (Pks13-TE:TAM5), and PDB: 5V42 (Pks13-TE:TAM3). […]

Pipeline specifications

Software tools SigmaPlot, CCP4, PHENIX, Coot, MolProbity, UCSF Chimera, PyMOL, CAVER
Applications Miscellaneous, Protein structure analysis
Organisms Mus musculus, Mycobacterium tuberculosis
Chemicals Isoniazid, Rifampin, Tamoxifen