Computational protocol: Fragment-BasedApproaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors

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Protocol publication

[…] Untagged CYP121 protein and crystals were prepared as described previously with the following adaptations. Crystals were prepared by vapor diffusion in 1.5–2.1 M ammonium sulfate and 0.1 M sodium MES or Cacodylate from pH 5.5–6.15. Additional crystallization was also conducted on the nanolitre scale using a Mosquito pipetting robot (Molecular Dimensions, Newmarket, UK), and crystals were produced in the Morpheus crystallization screen (Molecular Dimensions) in 800 nL drops with protein-to-mother liquor at a ratio of 1:1. Ligand soaks were carried out either by directly dissolving solid ligand at saturation or by the addition of 2–5 mM ligand solution in DMSO to the mother liquor and soaking was carried out for 24 h. Crystals were cryoprotected and frozen as described previously except from Morpheus conditions that were frozen directly. Data were collected on beamline I24 (wavelength 0.9173/1.0118 Å) and I02 (wavelength 0.9795 Å) at the Diamond Light Source Facility (Oxfordshire, UK). Data were processed and refined as previously described. Data tables and statistics are provided in Table S5, Supporting Information. CYP121–ligand cocrystal structures are deposited in the Protein Data Bank ( under the accession codes: 6, 5IBJ, 7; 5EDT, 19a; 5IBF, 24a; 5IBD, 25a; 5IBE, 25b; 5IBG, 26a; 5IBI, and 26h; 5IBH. Images for presentation were rendered using an academic version of the PyMOL Molecular Graphics System, version 1.3, 2010, Schrödinger, LLC. [...] Lead compound 2, retrofragments 5 and 6, and all Ar1, Ar2, and Ar3 analogues were prepared using the LigPrep v3.2 and Epik v3.0 functions of Schrödinger suite software (Schrödinger LLC, NY), selecting to include metal binding states when generating ligand ionization states. Ligands were docking into the crystal structure of CYP121 bound to lead 2 (PDB 4KTL) or the previously reported ligand 4-(1H-1,2,4-triazol-1-yl)quinolin-6-amine (PDB 4G1X). Proteins were prepared using the internal Protein Preparation function of the Schrödinger suite software. Ionization states were generated to be compatible with metal-binding interactions, and the heme-iron was manually adjusted to the ferric (+3) oxidation state. All water molecules were removed from the structure PDB 4G1X. Duplicate energy minimized (OPLS 2005) structures of PDB 4KTL were prepared either with all water molecules removed or retaining the axial heme–water ligand only. Ar1 and Ar3 analogues were docked using core constraints to replicate the position of the aminopyrazole ring of lead 2. Ar2 analogues were docked using core constraints to replicate the heme binding interactions of the 4-(1H-1,2,4-triazol-1-yl)quinolin-6-amine ligand in PDB 4G1X. Images were generated using the PyMOL Molecular Graphics System, version 1.3, 2010 (Schrödinger, LLC). […]

Pipeline specifications

Software tools PyMOL, LigPrep
Applications Drug design, Small-angle scattering
Organisms Mycobacterium tuberculosis, Dipturus trachyderma, Homo sapiens
Diseases Tuberculosis