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BioAssay: AID 504661

Cell-based assay to measure Apicoplast Disruption using Imaging

Assay Overview: The parasite apicoplast is an organelle of cyanobacterial origin that employs a prokaryotic transcription and translation machinery. During asexual blood stage development, this organelle adopts an elongated and branched structure as the parasite matures into its trophozoite form, then segregates into individual organelles that parse with the individual daughter merozoites prior more ..
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 Related BioAssays
 Related BioAssays
AID: 504661
Data Source: Broad Institute (2126-04_Inhibitor_SinglePoint_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Screening Center Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-04-13
Hold-until Date: 2012-02-18
Modify Date: 2012-02-18

Data Table ( Complete ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeComment
488774Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: SummarySummarydepositor-specified cross reference: Project Summary
488745Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid, 96 hour incubationConfirmatorysame project related to Summary assay
488752Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid, 48 hour incubationConfirmatorysame project related to Summary assay
504599Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid, 96 hour incubation.Confirmatorysame project related to Summary assay
504602Quantitative high throughput screen to test cell viability of anti-malarial compounds targeting the delayed death phenotypeConfirmatorysame project related to Summary assay
504604Quantitative high throughput screen to test activity of anti-malarial compounds in the Dd2 strain of Plasmodium FalciparumConfirmatorysame project related to Summary assay
504608Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid, 48 hour incubation.Confirmatorysame project related to Summary assay
504631A cell-based HTS for delayed death inhibitors of the malarial parasite plastid Measured in Microorganism System Using Plate Reader - 2126-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504633A cell-based HTS for delayed death inhibitors of the malarial parasite plastid Measured in Microorganism System Using Plate Reader - 2126-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504658FACS-based assay to assess parasite growth inhibition 48 hr Measured in Cell-Based System Using Flow Cytometry - 2126-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504659FACS-based assay to assess parasite growth inhibition 96 hr Measured in Cell-Based System Using Flow Cytometry - 2126-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504832Primary qHTS for delayed death inhibitors of the malarial parasite plastid, 48 hour incubationConfirmatorysame project related to Summary assay
504834Primary qHTS for delayed death inhibitors of the malarial parasite plastid, 96 hour incubationConfirmatorysame project related to Summary assay
624206Quantitative high throughput screen to test cell viability of anti-malarial compounds targeting the delayed death phenotype.Confirmatorysame project related to Summary assay
624328Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Hit Validation at 48 hrConfirmatorysame project related to Summary assay
624329Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Hit Validation with 3D7 at 48 hr using Flow CytometryConfirmatorysame project related to Summary assay
624332Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Hit Validation at 96 hrConfirmatorysame project related to Summary assay
624335Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Hit Validation with 3D7 at 96 hr using Flow CytometryConfirmatorysame project related to Summary assay
624336Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Hit Validation with Dd2 at 48 hr using Flow CytometryConfirmatorysame project related to Summary assay
624337Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Hit Validation with Dd2 at 96hr using Flow CytometryConfirmatorysame project related to Summary assay
Description:
Keywords: malaria, apicoplast, gene expression, Plasmodium falciparum

Assay Overview: The parasite apicoplast is an organelle of cyanobacterial origin that employs a prokaryotic transcription and translation machinery. During asexual blood stage development, this organelle adopts an elongated and branched structure as the parasite matures into its trophozoite form, then segregates into individual organelles that parse with the individual daughter merozoites prior to their release from the infected erythrocyte. To verify if the lead compounds interfere with the apicoplast, we used a whole cell imaging screen with a recombinant parasite line that has GFP-labeled apicoplasts. Parasites were treated with the test compounds at 3 x their IC50 value for four days. The infected cells were then stained with a Hoechst DNA dye to allow the parasite developmental stage to be ascertained. The parasites were also stained with mito-tracker red for comparative purposes. Parasites treated with compounds that disrupt the apicoplast demonstrate diffuse GFP staining, but intact mitochondrial staining. Parasites treated with fast acting compounds generally display poor mito-tracker staining, but intact apicoplast structures.

Expected Outcome: Antibiotics known to interfere with the apicoplast, such as azithromycin and doxycycline, have been observed to disrupt the morphology of the apicoplast in this GFP expressing line. While GFP is still produced in parasites treated with these drugs it is distributed throughout the cytoplasm. This difference in GFP distribution can be measured by comparing the Standard Deviation of the GFP signal between treated and untreated parasites. Parasites with intact apicoplasts have condensed regions of bright GFP staining with almost no signal detectable in the cytoplasm, leading to a high standard deviation. Parasites with disrupted apicoplasts have uniform levels of GFP staining throughout the cytoplasm leading to low Standard Deviations.
Protocol
Parasites expressing GFP targeted to the apicoplast, 3D7 ACP(L)-GFP, were treated with the indicated compounds in 200 uL of culture media at a 1% hematocrit. Azithromycin was used as a positive control, while parasites treated with drug vehicle (medium containing 0.05 - 0.1% DMSO) served as a negative control. Assays were started with synchronized ring stages, and parasites were harvested on day four. At 48 hrs, 30ul of culture was harvested and stained with Hoechst dye (1ug/ml) and Mitotracker-red (20nM) (Invitrogen) for 30 min at 37 degrees C. Stained parasites were washed, deposited on poly-lysine coated slides and imaged on a Nikon Ti inverted microscope with a 100x objective.
Comment
Expected outcome: An active compound will lead to a decreasing numerical value.
NORMALIZATION:
Normalization was applied to the raw data signals such that an activity score of 100 is most active and a value of 0 is inactive. Data was compared to the positive control, azithromycin.
PATTERN CORRECTION: No plate pattern correction algorithm was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
For this imaging assay, compounds were not tested at all doses. Instead, apicoplast morphology was assessed at a concentration 2 to 10 fold higher than the IC50 values from previous screening assays. Data was determined with imaging analysis software to determine raw values.
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
b) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1REPLICATE_A_ACTIVITY_SCOREThe calculated percent activity for the indicated sampleFloat%
2REPLICATE_B_ACTIVITY_SCOREThe calculated percent activity for the indicated sampleFloat%
3TESTED_CONCENTRATION(nM)Concentration at which the compound was tested.FloatnM
Additional Information
Grant Number: R21 NS059500

Data Table (Concise)
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