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BioAssay: AID 504633

A cell-based HTS for delayed death inhibitors of the malarial parasite plastid Measured in Microorganism System Using Plate Reader - 2126-01_Inhibitor_Dose_DryPowder_Activity

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 more ..
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AID: 504633
Data Source: Broad Institute (2126-01_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: Assay Vendor
BioAssay Version:
Deposit Date: 2011-03-31
Hold-until Date: 2012-02-18
Modify Date: 2012-02-18

Data Table ( Complete ):           Active    All
BioActive Compounds: 9
Depositor Specified Assays
AIDNameTypeComment
488774Quantitative high throughput screen for delayed death inhibitors of the malarial parasite plastid: Summarysummary
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 3D7 P. falciparum strain against novel small molecules will be determined by HTS using SYBR green-based fluorescence assays. Parasites and 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 3D7 P. 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.EC50 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 '>'
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_0.0000245uM_(%) (2.45e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
9Activity_at_0.000049uM_(%) (4.9e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity_at_0.000098uM_(%) (9.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.000195uM_(%) (0.000195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.000391uM_(%) (0.000391μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.000781uM_(%) (0.000781μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.00156uM_(%) (0.00156μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.00244uM_(%) (0.00244μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.00312uM_(%) (0.00312μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.00488uM_(%) (0.00488μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.00625uM_(%) (0.00625μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.00977uM_(%) (0.00977μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.0125uM_(%) (0.0125μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.0195uM_(%) (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.025uM_(%) (0.025μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.0391uM_(%) (0.0391μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.05uM_(%) (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.0781uM_(%) (0.0781μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.156uM_(%) (0.156μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.312uM_(%) (0.312μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.625uM_(%) (0.625μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_1.25uM_(%) (1.25μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_2.5uM_(%) (2.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_5.0uM_(%) (5μM**)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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