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BioAssay: AID 720706

HTS for Bacterial rRNA inhibitors Measured in Microorganism-Based System Using Plate Reader - 7056-01_Inhibitor_SinglePoint_HTS_Activity

Successful treatment of bacterial infections has become increasingly difficult due to the rapid emergence of drug resistant strains. The human and economic cost of antibiotic resistance is enormous. Despite the urgent need to develop new antibiotics, only four classes have been launched since 1970. Among hundreds of essential enzymes in the bacterial cell, the ribosome is an evolutionary preferred antibiotic target. ..more
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Active(5500)
 
 
Inactive(342659)
 
 
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Active(5501)
 
 
Inactive(345743)
 
 
 Related BioAssays
 Related BioAssays
AID: 720706
Data Source: Broad Institute (7056-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-10-29
Modify Date: 2014-03-05

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 5500
Related Experiments
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AIDNameTypeComment
720694Inhibitors of Bacterial Protein Synthesis with Novel Modes of Action_7056_Inhibitor_ProjectSummarydepositor-specified cross reference: Summary
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743480HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_CherryPick_Activity_Set4Confirmatorysame project related to Summary assay
743484A549 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-06_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
977621Bacterial rRNA profiling, H69-RFP Measured in Cell-Based System Using Plate Reader - 7056-04_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977622Bacterial rRNA profiling, h38-RFP Measured in Cell-Based System Using Plate Reader - 7056-03_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977623Bacterial rRNA profiling, H69-GFP Measured in Cell-Based System Using Plate Reader - 7056-10_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977624Bacterial rRNA profiling, h34-RFP Measured in Cell-Based System Using Plate Reader - 7056-07_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977625Bacterial rRNA profiling, h34-GFP Measured in Cell-Based System Using Plate Reader - 7056-13_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977626Bacterial rRNA profiling, G2058-GFP Measured in Cell-Based System Using Plate Reader - 7056-08_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977627Bacterial rRNA profiling, H38-GFP Measured in Cell-Based System Using Plate Reader - 7056-12_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977628Bacterial rRNA profiling, h24-RFP Measured in Cell-Based System Using Plate Reader - 7056-05_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977629Bacterial rRNA profiling, h24-GFP Measured in Cell-Based System Using Plate Reader - 7056-11_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
977630Bacterial rRNA profiling, H38-RFP Measured in Cell-Based System Using Plate Reader - 7056-06_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
1035470Bacterial rRNA profiling, G2058-RFP Measured in Cell-Based System Using Plate Reader - 7056-02_Inhibitor_Dose_CherryPick_ActivityOthersame project related to Summary assay
Description:
Keywords: rRNA, antibiotics, protein synthesis inhibitor, 23S ribosome, resistance

Biological Relevance:
Successful treatment of bacterial infections has become increasingly difficult due to the rapid emergence of drug resistant strains. The human and economic cost of antibiotic resistance is enormous. Despite the urgent need to develop new antibiotics, only four classes have been launched since 1970. Among hundreds of essential enzymes in the bacterial cell, the ribosome is an evolutionary preferred antibiotic target.
Approximately half of all known natural antibiotics and a large fraction of clinical antibiotics inhibit bacterial growth by binding to the ribosome and interfering with translation. The ribosome is a 2.5-megadalton
ribonucleoprotein complex composed of a small and a large subunit. RNA is the main structural and functional component of the ribosome, and most of the clinical translation inhibitors interact directly with ribosomal RNA
(rRNA). The redundancy of rRNA genes in bacterial genomes makes it difficult for the bacterium to develop resistance by mutating the site of drug action. Therefore, clinical resistance arises mainly from acquisition of
natural resistance genes circulating in microbial communities. Many of these genes code for enzymes that target the ribosomal sites of action of natural antibiotics. For example, the Erm methyltransferases modify 23S
rRNA at a specific residue (A2058 in E. coli), rendering bacteria resistant to macrolides, lincosamides and the streptogramin B group of antibiotics by impairing their binding to the ribosome. One of the most recently
marketed ribosome inhibitors is linezolid (FDA-approved in 2000). Because of its synthetic nature, it held the promise of evading natural resistance mechanisms. However, it has been shown that linezolid acts upon the
peptidyl transferase center (PTC), a ribosomal site that is targeted by several classes of natural translation inhibitors. In 2007, we described the first clinical isolate of methicillin-resistant S. aureus (MRSA) to
develop linezolid resistance by acquisition of a natural resistance gene, cfr. This gene encodes an rRNA methylase that targets the nucleotide A2503 in the PTC. Cfr likely originated from a bacterial producer of
a natural PTC inhibitor to resist its action.
We engineered several bacterial strains that express a hybrid ribosome in which the site of interest was mutated to mimic the counterpart sequence of the human cytoplasmic ribosome. Because the humanized
strains express a fluorescent protein different from that of the wild-type bacterial strain, strains will be grown in a mixed culture. Compounds that selectively inhibit the growth of the wild-type strain, but not the humanized
strain, likely act on the target ribosomal site, because the resistant humanized strain contains mutations at that site. Because of the nature of selection, we expect the compounds identified here to be selective protein
synthesis inhibitors, which inhibit bacterial growth by acting upon a novel ribosomal site.

Assay Overview:
We engineered several bacterial strains that express a hybrid ribosome in which the site of interest was mutated to mimic the counterpart sequence of the human cytoplasmic ribosome. Because the humanized
strains express a fluorescent protein different from that of the wild-type bacterial strain, strains will be grown in a mixed culture. Compounds that selectively inhibit the growth of the wild-type strain, but not the humanized strain, likely act on the target ribosomal site, because the resistant humanized strain contains mutations at that site. Because of the nature of selection, we expect the compounds identified here to be selective protein synthesis inhibitors, which inhibit bacterial growth by acting upon a novel ribosomal site.

Expected Outcome: Compounds that reduce GFP (wild type ribosome-expressing GFP-labeled E. coli) significantly (2x) greater than RFP (at least one of five strains of mutant humanized ribosome-expressing RFP-labeled E. coli) and do not significantly reduce total OD600 (< 30% inhibition) will be considered selective inhibitors. The exact mutant RFP strain that is spared will be deconvoluted in subsequent assays.
Protocol
Bac rRNA Automation Protocol
Day 1:
1. Grow each E. coli strain (one wild type (GFP) and six humanized mutants (RFP) including the control G2058) individually. Pick each strain into a separate 50 mL conical tube with 10 mL of LB medium supplemented with 100 ug/mL amplicillin and 50 ug/mL spectinomycin.
2. Incubate strains overnight at 37 degrees with shaking at 190 RPM.
Day 2:
1. In the morning, check OD600 of the cultures.
2. Dilute the cultures to OD 600 = 0.05 in LB medium supplanted with 100 ug/mL ampicillin.
3. Return strains to 37 degrees with shaking at 190 RPM for about 3. 5 hours. Grow strains to reach OD 600 about 0.5 (exponential growth phase).
4. Mix strains (final OD600= 0.02) in the proportion 50% wild-type, 8.33% each of the six mutants in LB medium supplemented with 100 ug/mL ampicillin and 1 mM IPTG (which is used to induce expression of fluorescent proteins).
5. Dispense 30 uL/well of bacteria mixture to 384 well Assay Ready Plates (ARPs) containing positive controls (Erythromycin 15 mg/mL, 50 nL in column 1 and Choramphenicol 15 mg/mL, 50 nL in column 23).
6. Place breathable film on plate using Plate Loc.
7. Incubate plates in Steristore at 37 degrees, 0% CO2 for 24 hours, 95% humidity.
Day 3:
1. Remove assay plate from Steristore.
2. Remove breathable film with X-Peel.
3. Read GFP signal using EnVision plate reader (mirror: FITC #403, Ex: FITC 485 #102, Em: FITC 535 #206).
4. Read RFP signal using EnVision plate reader (mirror: BODIPY TMR #405, Ex: photometric 550 #312, Em: Cy3 595 #229).
5. Read OD600 signal using EnVision plate reader (Ex: Photometric 600 #319).
6. Store plate in Steristore incubator.
Equipment Required
Combi
Plate Loc
Steristore (37 C, 95% humiodity, 0% CO2,)
X-Peel
2 EnVisions
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control
wells (PC) were included on every plate. One positive control,
Chloramphenicol, was used to determine the baseline fluorescence and OD
as it was expected to non-selectively kill all 6 strains, GFP wild-type
and RFP mutant. The other positive control, Erythromycin, was expected
to selectively spare one RFP control strain (G2058) and was therefore
used as a guideline for calculating the performance of a selective GFP
inhibitor.
NORMALIZATION:
Each of three layers was independently normalized using the 'Neutral
Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to
a normalized activity value of 0.
The a raw signal of 0 was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Assay
Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the
normalized plate data for each layer.
AGGREGATE COMPOUND LAYER SCORING: In addition to the reported normalized
percent activities above, each replicate compound measurement was
converted to a z-score relative to the corresponding DMSO-control
distribution, as described for ChemBank (Seiler et al., 2008). ChemBank
composite Z scores were computed to combine replicates for each channel
(RFP, GFP, and OD). We sought compounds that affected the GFP-expressing
wild-type strain while not affecting at least one of the mutant RFP
strains and not affecting overall viability as measured by OD; i.e.,
they score with negative composite Z in the GFP channel with little or
no effect in the RFP or OD channels. To combine the results of the three
channels quantitatively, we defined a trigonometric scaling function to
express our preferences that (1) GFP composite Z scores be negative, and
(2) RFP and OD composite Z score amplitudes exceed GFP composite Z score
amplitudes; such compounds should fall left of the DMSO-control
distribution, but above the main diagonal in a plot of GFP composite Z
versus either OD or RFP composite Z. Using the two ratios of ChemBank
composite Z scores (GFP/RFP and GFP/OD), we defined q1 = arctan(GFP/RFP)
and q2 = arctan(GFP/OD) and expressed our scale factor as F =
cos(-q1)cos(2q1)cos(-q2)cos(2q2), with the four factors respectively
expressing our preferences. As a three-assay composite score, we take
-(ZGFP) x F, which clearly discriminates the compounds of interest when
plotted against RFP or OD.
PUBCHEM_ACTIVITY_SCORE: The above three-assay composite score was
normalized on a scale of 1-100, with 1 being those with the least
desirable observed score representing compounds increasing in GFP signal
and decreasing in RFP or OD, and 100 being the most ideal observed score
representing a loss in GFP with no change in RFP or OD.
PUBCHEM_ACTIVITY_OUTCOME: The above three-assay composite scores were
fit to a standard distribution and a corresponding Holm-Bonferroni
corrected p value significance was calculated. Compounds with a FDR
corrected p value less than 0.05, normalized OD percent score > -30,
negative GFP composite Z score, and the expression Abs(Cos(-q)Cos(2q)) >
(sqrt2)/2 for both q1 and q2 were assigned a score of 2 (active).
Compounds failing any of these criteria were assigned a score of 1
(inactive.)
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Organism-based
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1R_RepAThe normalized percent activity of RFP measurements for the indicated sample.Float%
2G_RepAThe normalized percent activity of GFP measurements for the indicated sample.Float%
3OD_RepAThe normalized percent activity of OD measurements for the indicated sample.Float%
4Rz_RepAThe calculated Z score of RFP measurements for the indicated sampleFloat
5Gz_RepAThe calculated Z score of GFP measurements for the indicated sampleFloat
6ODz_RepAThe calculated Z score of OD measurements for the indicated sampleFloat
7R_RepBThe normalized percent activity of RFP measurements for the indicated sample.Float%
8G_RepBThe normalized percent activity of GFP measurements for the indicated sample.Float%
9OD_RepBThe normalized percent activity of OD measurements for the indicated sample.Float%
10Rz_RepBThe calculated Z score of RFP measurements for the indicated sampleFloat
11Gz_RepBThe calculated Z score of GFP measurements for the indicated sampleFloat
12ODz_RepBThe calculated Z score of OD measurements for the indicated sampleFloat
13RczComposite Z score of the replicate RFP measurements for the indicated sampleFloat
14GczComposite Z score of the replicate GFP measurements for the indicated sampleFloat
15ODczComposite Z score of the replicate OD measurements for the indicated sampleFloat
16pvalCalculated P value for the indicated sampleString
Additional Information
Grant Number: 1 RO3 DA035191-01

Data Table (Concise)
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