HTS for Bacterial rRNA inhibitors Measured in Microorganism-Based System Using Plate Reader - 7056-01_Inhibitor_SinglePoint_HTS_Activity
Osteoporosis is a progressive metabolic disease of the skeletal system that leads to debilitating loss in bone mass and elevated risk for fracture. More than half of the population over the age of 55 in the United States is diagnosed to have or at risk to develop osteoporosis. With the dramatic expansion of the aged population and the increase in fracture incidence at earlier ages, the burden of more ..
BioActive Compounds: 5500
Depositor Specified Assays
Keywords: rRNA, antibiotics, protein synthesis inhibitor, 23S ribosome, resistance
Osteoporosis is a progressive metabolic disease of the skeletal system that leads to debilitating loss in bone mass and elevated risk for fracture. More than half of the population over the age of 55 in the United States is diagnosed to have or at risk to develop osteoporosis. With the dramatic expansion of the aged population and the increase in fracture incidence at earlier ages, the burden of illness associated with osteoporosis will only continue to increase. The most widely used therapeutic for the treatment of osteoporosis, bisphosphonates, has been linked to adverse events including osteonecrosis of the jaw, atypical fractures and esophageal cancer. These findings highlight a growing need to identify novel therapeutic agents that prevent bone loss associated with this disease. Based on our extensive in vitro and in vivo data demonstrating that reduced levels of the large zinc-finger protein Schnurri-3 (Shn3), also known as kappa recognition component (KRC), is one of the few genes that regulates mature osteoblast
formation. In addition to the role in osteoblasts, Shn3 binds transcription factor c-Jun and regulates IL-2 in T cells. However, Schn3 -/- knockout mice had increased bone mass, but had normal thymus development. The overall goal is to identify compounds that selectively reduce Shn3 protein levels through a post-transcriptional mechanism targeting the 3'UTR of this gene.
Assay Overview: Antibiotic hypersusceptible E. coli strains (one wild-type and six humanized
mutants including the control A2058G) are grown individually overnight (for 18h) at 37 degrees C in LB medium supplemented with 100 mug/mL ampicillin and 50 mug/mL spectinomycin. Cultures are then diluted to OD600 = 0.05 and are grown for ~3.5 h to reach OD600 ~ 0.5 (exponential growth phase). Cells are mixed (final OD600 = 0.02) in the proportion 50% wild-type/8.33% each of 6 mutants in LB medium supplemented with 100 mug/mL ampicillin and 1 mM IPTG, which is used to induce the expression of fluorescent proteins. 30 muL of the cell mixture is then dispensed to the wells of a 384-well "assay ready plate (ARP)". An ARP is a plate in which the library compounds and controls have been previously dispensed.The final concentration of library compounds is 25 muM per well. The microplates are incubated for 24 h at 37 degrees C. Each plate contains 16 wells of positive
control (erythromycin), 32 wells of negative control (DMSO), and 16 wells of chloramphenicol (background fluorescence). The OD (600 nm) and fluorescence values (GFP: lambdaExc = 485 nm, lambdaEm = 535 nm; RFP: lambdaExc = 550 nm, lambdaEm = 595 nm) are measured in an Envision microplate reader. Compounds exhibiting fluorescence RFP/GFPratios >3 and OD >0.2 will be selected for further investigation.
Expected Outcome: Compounds exhibiting fluorescence RFP/RFP+GFP ratios >3 and OD >0.2 will be selected for further investigation.
Bac rRNA Automation Protocol
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.
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.
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.
Steristore (37 C, 95% humiodity, 0% CO2,)
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
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
Data Table (Concise)