title: Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis.
abstract: FtsZ is an attractive target for antibiotic research because it is an essential bacterial cell division protein that polymerizes in a GTP-dependent manner. To find the seed chemical structure, we established a high-throughput, quantitative screening method combining fluorescence cross-correlation spectroscopy (FCCS) and surface plasmon resonance (SPR). As a new concept for the application of FCCS to polymerization-prone protein, Staphylococcus aureus FtsZ was fragmented into the N-terminal and C-terminal, which were fused with GFP and mCherry (red fluorescent protein), respectively. By this fragmentation, the GTP-dependent head-to-tail dimerization of each fluorescent labeled fragment of FtsZ could be observed, and the inhibitory processes of chemicals could be monitored by FCCS. In the first round of screening by FCCS, 28 candidates were quantitatively and statistically selected from 495 chemicals determined by in silico screening. Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening. Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR. Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Mikuni
authors.affiliation: Creative Research Institution, Hokkaido University, Sapporo, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Kodama
authors.affiliation: Bio-Analytical Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sasaki
authors.affiliation: Discovery Research Laboratory for Core Therapeutic Areas, Shionogi & Co., Ltd., Toyonaka, Osaka, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kohira
authors.affiliation: Discovery Research Laboratory for Core Therapeutic Areas, Shionogi & Co., Ltd., Toyonaka, Osaka, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Maki
authors.affiliation: Information Initiative Center and Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Munetomo
authors.affiliation: Laboratory of Biomolecular Science, Faculty of Pharmaceutical Science, Hokkaido University, Sapporo, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
mesh_terms: D000465:Algorithms; D000900:Anti-Bacterial Agents / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D048749:Cytokinesis; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics; D008826:Microbial Sensitivity Tests; D025941:Protein Interaction Mapping; D055503:Protein Multimerization / Q000187:drug effects*; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000235:genetics*; D020349:Surface Plasmon Resonance
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D049452:Green Fluorescent Proteins