Computational protocol: Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy

Similar protocols

Protocol publication

[…] Data was collected at SSRL beamlines 7-1 and 9-1 as summarized in Table . Complete, high quality datasets to 1.9 Å and 2.3 Å resolution were obtained for the BAU-bound and ligand-free crystal forms, respectively. Collected data was processed and reduced by the HKL2000 package with Denzo and Scalepack []. The higher resolution crystal is of the orthogonal space group P212121 with low mosaicity. The other crystal form belongs to the face-centered cubic space group F4132. Molecular replacement phasing of the data obtained on hUPP1 with BAU was successful using Molrep [] with dimeric homology models of hUPP1, based on the BAU-bound E. coli UPP structure (PDB ID: 1U1C) [], modified by Swiss-Model []. Solution phases were sufficient to resolve density for the unmodelled BAU ligand and other unmodelled residues. The initial model was rebuilt after phases were obtained using ARP/wARP []. Rounds of model building and refinement were performed using Coot [] and Refmac []. Due to a lack of electron-density, the first 15 residues of hUPP1 and the N-terminal cloning artifact residues 'MRGSHHHHHHGSPGLQEF' were not built. Additionally, for the same reason the final two C-terminal residues could not be modelled in the BAU-bound structure. Medium non-crystallographic symmetry restraints (between 4 chains) were retained for the loop residues 79–84 in the BAU-bound structure due to the low quality of the electron-density map in this region of the protein. The BAU-bound model includes both a BAU molecule and a phosphate ion per protein chain as ligands that could be clearly built into the electron density. The native hUPP1 structure was completed with a sulfate ion (coordinated by Arg64) and a cation (modelled as magnesium based on interatomic distances) positioned at a crystal contact point between the side chain oxygen atoms of Asp212 and Ser116 of a symmetry-related chain. The final structures were refined with Refmac to an Rfactor/Rfree of 20.5%/25.1% respectively for the BAU-bound structure, and 20.4%/22.1% for the ligand-free structure of the enzyme, with approximately 92% of residues in most favorable regions of the Ramachandran plot as analyzed by Procheck []. The models were further validated using Molprobity [], scoring in the 91st and 97th percentile, respectively. Figures were rendered using ICM Browser-Pro (Molsoft). The atomic coordinates and structure factors have been deposited in the Protein Data Bank (3EUF and 3EUE). […]

Pipeline specifications

Software tools Molrep, ARP/wARP, Coot, PROCHECK, MolProbity, ICM-Browser
Applications Drug design, Protein structure analysis
Organisms Homo sapiens
Diseases Neoplasms, Drug-Related Side Effects and Adverse Reactions
Chemicals Fluorouracil, Uracil, Uridine