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BioAssay: AID 504631

A cell-based HTS for delayed death inhibitors of the malarial parasite plastid Measured in Microorganism System Using Plate Reader - 2126-02_Inhibitor_Dose_DryPowder_Activity

Assay Overview:We have developed an assay that would allow us to identify compounds that inhibit Plasmodium falciparum by acting through a delayed death mechanism. Our assay will enrich for molecules targeting the apicoplast by identifying compounds that inhibit growth in the second generation of drug exposure. We will measure parasite growth inhibition using SYBR Green I, which becomes highly more ..
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Active(9)
 
 
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 Related BioAssays
 Related BioAssays
AID: 504631
Data Source: Broad Institute (2126-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-31
Hold-until Date: 2012-02-18
Modify Date: 2012-02-18

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 9
Related Experiments
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AIDNameTypeComment
488774Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: SummarySummarydepositor-specified cross reference
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
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
504661Cell-based assay to measure Apicoplast Disruption using ImagingOthersame 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, Plasmodium falciparum, apicoplast


Assay Overview:We have developed an assay that would allow us to identify compounds that inhibit Plasmodium falciparum by acting through a delayed death mechanism. Our assay will enrich for molecules targeting the apicoplast by identifying compounds that inhibit growth in the second generation of drug exposure. We will measure parasite growth inhibition using SYBR Green I, which becomes highly fluorescent when bound to double stranded DNA. This assay takes advantage of the fact that human erythrocytes lack nucleic acids, therefore only the infected erythrocytes provide a fluorescent signal. Parasite cultures will be incubated for 72 hours with compounds, allowing for growth inhibition of later generations.

The susceptibility of the Dd2 P. falciparum strain against novel small molecules will be determined using SYBR green-based fluorescence assays. Parasites and human erythrocytes will be cultured in the presence of serial dilutions of test compounds. Parasites are synchronized at ring stage using the sorbitol method before performing the assay. Assay plates incubate at 37 C for 72 hours in a low oxygen environment. After this incubation, SYBR lysis buffer is added. Plates are incubated 20-36 hrs at room temperature before reading the SYBR green fluorescence intensity. Assays are performed with six technical replicates per drug concentration. EC50 values are determined by regression analysis performed in the Collaborative Drug Discovery database system.


Expected Outcome: Loss of Signal
The goal of this project is to identify compounds that cause delayed death in the P. falciparum organism. Therefore, a successful compound will kill the organism causing a decrease in double stranded DNA and a subsequent loss of SYBR green signal.
Protocol
The susceptibility of the Dd2 Plasmodium falciparum line against novel small molecules will be determined using a SYBR green-based fluorescence assay in 384-well plates.

1.Parasites are cultured in the presence of serial dilutions of test compounds in 40 ul of RPMI containing 4.16 mg/ml Albumax. Parasites are synchronized at ring stage using the sorbitol method before performing the assay.

2.Two-fold serial drug dilutions are prepared in RPMI/Albumax culture medium in a 96-well plate, and 10 ul of each drug solution per well is transferred into a black Greiner GNF clear-bottom 384-well plate (Greiner Bio-One Ltd.) using the Bravo Liquid Handling Platform (Velocity 11, CA) to give final 1x concentrations in 40 ul of assay volume. Assays are performed with six technical replicates per drug concentration.

3.30 ul of parasite culture at 1.0% parasitemia and 1% hematocrit (a.k.a. packed cell volume of erythrocytes) is transferred into each well of 384-well plate with sterilized cartridges using a Matrix WellMate (Thermo Scientific).

4.Assay plates are transferred to a Thermo three-gas incubator for a further 72 hours incubation at 37 degrees Celsius in a low oxygen gas environment (1% O2, 4.1% CO2, balance N2).

5.10 ul of SYBR lysis buffer (Saponin 0.16%, Tris-HCl (pH 7.5) 20 mM, EDTA (disodium salt) 5 mM, Triton X-100 6% v/v, SYBR Green I (Invitrogen) 1000X) is added to the each well of an assay plate and briefly centrifuged at 1000 rpm for 1 minute to settle the contents within the plate wells.

6.Plates are incubated 20-36 hrs at room temperature before reading the SYBR green fluorescence intensity (EX 480 nm, EM 530 nM) using a Molecular Devices SpectraMax M5 plate reader.

7.AC50 values are determined by regression analysis performed in the Collaborative Drug Discovery database system.
Comment
- Curves were fit using the algorithms at Collaborative Drug Discovery, Inc.

- The curve fit is performed using the standard Hill equation, or the four parameter logistic curve:

response = S0 + ((Sinf-S0)/(1+10;((logAC50-X)*HillSlope)))

where
Response is the measured response
S0 is the minimum response
Sinf is the maximum response
AC50 is the concentration (uM) at 50% response
pAC50 is the (-1)(log10) of concentration (M)
X is the log of concentration
HillSlope is the Hill coefficient


- The minimum required number of doses is 3, but there is no upper limit. Hill slope will automatically be restricted to 1, if only 3 doses are provided.

- For the response values that are imported into CDD with a numeric modifier (<, >, <=, >=, etc), the modifier is disregarded during curve fitting.

- Replicates of a molecule are plotted on a single curve, and one fit and one EC50 value are calculated per molecule/run

- The curve fit is done by the Marquardt-Levenberg algorithm (minimizing root mean squared error). See the wikipedia article for more information: http://en.wikipedia.org/wiki/Levenberg-Marquardt_algorithm

- PUBCHEM_ACTIVITY_SCORE was calculated to be 10*pAC50;
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M

- PUBCHEM_ACTIVITY_OUTCOME was set to '2' (active) if the AC50 calculation resulted in an AC50_Qualifier of '=', and to '1' (inactive) if the AC50 calculation resulted in an AC50_Qualifier of '>'
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based/Organism-based
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: NULL
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Activity_at_5.0uM_(%) (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
9Activity_at_2.5uM_(%) (2.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity_at_1.25uM_(%) (1.25μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.625uM_(%) (0.625μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.312uM_(%) (0.312μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.156uM_(%) (0.156μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.0781uM_(%) (0.0781μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.0391uM_(%) (0.0391μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.0195uM_(%) (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.00977uM_(%) (0.00977μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.00488uM_(%) (0.00488μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.00244uM_(%) (0.00244μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.05uM_(%) (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.025uM_(%) (0.025μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.0125uM_(%) (0.0125μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.00625uM_(%) (0.00625μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.00312uM_(%) (0.00312μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.00156uM_(%) (0.00156μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.000781uM_(%) (0.000781μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.000391uM_(%) (0.000391μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.000195uM_(%) (0.000195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_0.000098uM_(%) (9.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_0.000049uM_(%) (4.9e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_0.0000244uM_(%) (2.44e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R21 NS059500

Data Table (Concise)
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