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Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis.

Mikuni S, Kodama K, Sasaki A, Kohira N, Maki H, Munetomo M, Maenaka K, Kinjo M - PLoS ONE (2015)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

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).

No MeSH data available.


Related in: MedlinePlus

Strategic judgment of “Hit” chemicals by FCCS.A. When a chemical has an inhibitory effect on FtsZ dimerization even in the presence of GTP, the cross-correlation amplitude remains low. These inhibitory chemicals are included in the subset in the red circle (B). C. After addition of GTP, cross-correlation amplitude is increased if the chemical does not affect FtsZ (D). A and C were evaluated by the “binding ratio” analyzed by FCCS. The magenta and green lines indicate the autocorrelation functions of FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry, respectively. The black line indicates the cross-correlation function. E. Fluorescent intensity (EGFP and/or mCherry) changed when FtsZ fragments were precipitated by addition of a chemical. Chemicals that induced intensity changes of more than 20% when DMSO was added are included in the subset in the brown circle (F). G. Diffusion of FtsZ fragments was slower by aggregation. Chemicals that increased the slow fraction by more than 20% in 2-component fitting are included in the subset shown in the purple circle (H). Finally, the chemicals included in the subset shown only in red, were determined to be “true Hit” chemicals.
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pone.0130933.g002: Strategic judgment of “Hit” chemicals by FCCS.A. When a chemical has an inhibitory effect on FtsZ dimerization even in the presence of GTP, the cross-correlation amplitude remains low. These inhibitory chemicals are included in the subset in the red circle (B). C. After addition of GTP, cross-correlation amplitude is increased if the chemical does not affect FtsZ (D). A and C were evaluated by the “binding ratio” analyzed by FCCS. The magenta and green lines indicate the autocorrelation functions of FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry, respectively. The black line indicates the cross-correlation function. E. Fluorescent intensity (EGFP and/or mCherry) changed when FtsZ fragments were precipitated by addition of a chemical. Chemicals that induced intensity changes of more than 20% when DMSO was added are included in the subset in the brown circle (F). G. Diffusion of FtsZ fragments was slower by aggregation. Chemicals that increased the slow fraction by more than 20% in 2-component fitting are included in the subset shown in the purple circle (H). Finally, the chemicals included in the subset shown only in red, were determined to be “true Hit” chemicals.

Mentions: In chemical screening, inhibition by chemicals was judged via reduction of cross-correlation amplitude (Gc(0)). As shown in Fig 2, if a chemical has an inhibitory effect on FtsZ dimerization, the cross-correlation amplitude should remain low even after addition of GTP (Fig 2A and 2B). In contrast, if the chemical has no effect on FtsZ dimerization, the cross-correlation amplitude should increase after addition of GTP (Fig 2C and 2D). The inhibitory effect was statistically evaluated as the “BR (binding ratio)” from 3 independent FCCS screenings. Chemicals with inhibitory effects on the binding ratio compared with the control (DMSO solution) were categorized into “Hit” as shown in the red circle in the Venn diagram in Fig 2. Moreover, to improve the precision of screening, inappropriate data such as unexpected aggregation and/or precipitation of the fragmented FtsZ were excluded from the subset “Hit” (Fig 2E and 2G). The exclusion criteria were a reduction in intensity of more than 20% from the average intensity of the control (DMSO solution) as precipitation (Fig 2E and 2F), and an increase of more than 20% of the slow fraction by 2-component fitting in diffusion analysis as aggregation (Fig 2G and 2H). Finally, the chemicals included in the subset of the area shown in red were determined to be “true Hit” chemicals.


Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis.

Mikuni S, Kodama K, Sasaki A, Kohira N, Maki H, Munetomo M, Maenaka K, Kinjo M - PLoS ONE (2015)

Strategic judgment of “Hit” chemicals by FCCS.A. When a chemical has an inhibitory effect on FtsZ dimerization even in the presence of GTP, the cross-correlation amplitude remains low. These inhibitory chemicals are included in the subset in the red circle (B). C. After addition of GTP, cross-correlation amplitude is increased if the chemical does not affect FtsZ (D). A and C were evaluated by the “binding ratio” analyzed by FCCS. The magenta and green lines indicate the autocorrelation functions of FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry, respectively. The black line indicates the cross-correlation function. E. Fluorescent intensity (EGFP and/or mCherry) changed when FtsZ fragments were precipitated by addition of a chemical. Chemicals that induced intensity changes of more than 20% when DMSO was added are included in the subset in the brown circle (F). G. Diffusion of FtsZ fragments was slower by aggregation. Chemicals that increased the slow fraction by more than 20% in 2-component fitting are included in the subset shown in the purple circle (H). Finally, the chemicals included in the subset shown only in red, were determined to be “true Hit” chemicals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496089&req=5

pone.0130933.g002: Strategic judgment of “Hit” chemicals by FCCS.A. When a chemical has an inhibitory effect on FtsZ dimerization even in the presence of GTP, the cross-correlation amplitude remains low. These inhibitory chemicals are included in the subset in the red circle (B). C. After addition of GTP, cross-correlation amplitude is increased if the chemical does not affect FtsZ (D). A and C were evaluated by the “binding ratio” analyzed by FCCS. The magenta and green lines indicate the autocorrelation functions of FtsZK175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry, respectively. The black line indicates the cross-correlation function. E. Fluorescent intensity (EGFP and/or mCherry) changed when FtsZ fragments were precipitated by addition of a chemical. Chemicals that induced intensity changes of more than 20% when DMSO was added are included in the subset in the brown circle (F). G. Diffusion of FtsZ fragments was slower by aggregation. Chemicals that increased the slow fraction by more than 20% in 2-component fitting are included in the subset shown in the purple circle (H). Finally, the chemicals included in the subset shown only in red, were determined to be “true Hit” chemicals.
Mentions: In chemical screening, inhibition by chemicals was judged via reduction of cross-correlation amplitude (Gc(0)). As shown in Fig 2, if a chemical has an inhibitory effect on FtsZ dimerization, the cross-correlation amplitude should remain low even after addition of GTP (Fig 2A and 2B). In contrast, if the chemical has no effect on FtsZ dimerization, the cross-correlation amplitude should increase after addition of GTP (Fig 2C and 2D). The inhibitory effect was statistically evaluated as the “BR (binding ratio)” from 3 independent FCCS screenings. Chemicals with inhibitory effects on the binding ratio compared with the control (DMSO solution) were categorized into “Hit” as shown in the red circle in the Venn diagram in Fig 2. Moreover, to improve the precision of screening, inappropriate data such as unexpected aggregation and/or precipitation of the fragmented FtsZ were excluded from the subset “Hit” (Fig 2E and 2G). The exclusion criteria were a reduction in intensity of more than 20% from the average intensity of the control (DMSO solution) as precipitation (Fig 2E and 2F), and an increase of more than 20% of the slow fraction by 2-component fitting in diffusion analysis as aggregation (Fig 2G and 2H). Finally, the chemicals included in the subset of the area shown in red were determined to be “true Hit” chemicals.

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

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).

No MeSH data available.


Related in: MedlinePlus