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BioAssay: AID 720542

qHTS for Inhibitors of AMA1-RON; Towards Development of Antimalarial Drug Lead: Primary Screen

Plasmodium, the causative agent of malaria, is one of the world's deadliest parasitic diseases. Currently there is no vaccine available and there is widespread resistance to common anti-malarial drugs. This warrants novel, alternative approaches for developing improved therapy. During infection, one such essential interaction is between two parasite proteins, the apical membrane antigen1 (AMA1) more ..
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 Tested Compounds
 Tested Compounds
All(364084)
 
 
Active(735)
 
 
Inactive(356209)
 
 
Inconclusive(7154)
 
 
 Tested Substances
 Tested Substances
All(364447)
 
 
Active(738)
 
 
Inactive(356551)
 
 
Inconclusive(7158)
 
 
 Related BioAssays
 Related BioAssays
AID: 720542
Data Source: NCGC (AMA1100)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-07-24
Modify Date: 2013-07-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 735
Related Experiments
AIDNameTypeComment
720515qHTS for Inhibitors of AMA1-RON; Towards Development of Antimalarial Drug Lead: SummarySummarydepositor-specified cross reference
Description:
Plasmodium, the causative agent of malaria, is one of the world's deadliest parasitic diseases. Currently there is no vaccine available and there is widespread resistance to common anti-malarial drugs. This warrants novel, alternative approaches for developing improved therapy. During infection, one such essential interaction is between two parasite proteins, the apical membrane antigen1 (AMA1) that is translocated onto the merozoite surface and the rhoptry neck protein 2 (RON2), which is transferred to the red blood cells (RBC) membrane during invasion (Besteiro et.al., 2009). Recently, the Miller lab has shown that the interaction between AMA1 and RON2 is essential for junction formation of the parasite with the host cell (Srinivasan et.al., 2011). The binding of the RON2 peptide to the hydrophobic pocket in AMA triggers the formation of the moving junction which provides a firm anchor for the parasite to pull itself into the RBC using its actin-myosin motor; an irreversible step that commits the parasite for invasion. Only one AMA1 and RON2 genes exists in Plasmodium and there are no alternate pathways. Moreover, the region corresponding to RON2 peptide is conserved in all P. falciparum isolates for which sequences are available. The key residues in the AMA1 hydrophobic pocket that binds RON2 are also conserved and indicate a functional constraint governing this essential step in invasion since a short RON2 peptide (RON2L) corresponding to the binding region on AMA1 is sufficient to compete with the native RON2 protein and inhibits invasion (Tonkin et.al, 2011). Therefore, developing small molecule inhibitors of this interaction offers a promising target opening a new arena for anti-malarial therapeutics drug design.

In collaboration with the Miller lab, a quantitative High Throughput Screen using the AlphScreen technology was developed using a recombinant his-tagged AMA1 and a biotinylated RON2 peptide. This fluorescent screen was assayed against the NIH Molecular Libraries Small Molecule Repository (MLSMR). Inhibition of this protein-protein interaction is the desired outcome of this assay. However, due the limitations of this AlphaScreen assay, a large number of artifacts were observed. An AlphaScreen counterscreen was run to identify the true inhibitors, triage compound activity, and weed out false positives.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH099752
Assay Submitter (PI): Louis Miller, National Institute of Allergy and Infectious Disease (NIAID)
Protocol
Two microliters of his-tagged AMA1 3D7 allele protein (final concentration 25 nM) was dispensed into a 1,536-well assay plate. Small molecules and positive control peptides were pin-transferred (23 nL or 46 nL) via Kalypsys pin-tool equipped with a 1,536-pin array, resulting in final compound and peptide concentration ranges of 91.5 nM - 57.2 muM, and 15.6 nM - 2.00 muM, respectively. Unlabeled RON2 peptide or R1 peptide (VFAEFLPLFSKFGSRMHILK) that specifically binds the 3D7 AMA1 was used as a positive control that inhibited the binding of RON2L to AMA1. After 15 minutes incubation, 1 uL of biotinylated RON2 peptide (final concentration 25 nM) or buffer were dispensed and the assay plate was incubated for an additional 30 minutes at room temperature and protected from light. This was followed by an addition of 1 uL mixture of donor and acceptor beads (10 ug/mL final concentrations for each). The plates were incubated for 30 min at room temperature and read using the EnVision(R) plate reader (PerkinElmer). Maximum inhibition response was normalized to the positive control signal while no inhibition response was normalized to the DMSO treated wells.
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Activity_ScoreActivity score.Integer
6Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0003270000 uM (0.000327μM**)% Activity at given concentration.Float%
16Activity at 0.0009544551 uM (0.000954455μM**)% Activity at given concentration.Float%
17Activity at 0.00288 uM (0.00288052μM**)% Activity at given concentration.Float%
18Activity at 0.00366 uM (0.00366μM**)% Activity at given concentration.Float%
19Activity at 0.00867 uM (0.0086659μM**)% Activity at given concentration.Float%
20Activity at 0.021 uM (0.0207733μM**)% Activity at given concentration.Float%
21Activity at 0.030 uM (0.0304393μM**)% Activity at given concentration.Float%
22Activity at 0.050 uM (0.0499056μM**)% Activity at given concentration.Float%
23Activity at 0.094 uM (0.0936549μM**)% Activity at given concentration.Float%
24Activity at 0.198 uM (0.197722μM**)% Activity at given concentration.Float%
25Activity at 0.251 uM (0.251189μM**)% Activity at given concentration.Float%
26Activity at 0.464 uM (0.463786μM**)% Activity at given concentration.Float%
27Activity at 0.897 uM (0.897245μM**)% Activity at given concentration.Float%
28Activity at 1.696 uM (1.69622μM**)% Activity at given concentration.Float%
29Activity at 2.322 uM (2.32247μM**)% Activity at given concentration.Float%
30Activity at 4.534 uM (4.53393μM**)% Activity at given concentration.Float%
31Activity at 7.297 uM (7.29707μM**)% Activity at given concentration.Float%
32Activity at 11.61 uM (11.6073μM**)% Activity at given concentration.Float%
33Activity at 22.22 uM (22.2187μM**)% Activity at given concentration.Float%
34Activity at 49.68 uM (49.6809μM**)% Activity at given concentration.Float%
35Activity at 58.24 uM (58.2424μM**)% Activity at given concentration.Float%
36Activity at 116.2 uM (116.175μM**)% Activity at given concentration.Float%
37Activity at 202.9 uM (202.927μM**)% Activity at given concentration.Float%
38Activity at 287.0 uM (287μM**)% Activity at given concentration.Float%
39Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH099752

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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