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BioAssay: AID 686967

Late stage luminescence-based biochemical dose response assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)

Name: Late stage luminescence-based biochemical dose response assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS). ..more
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 Tested Compounds
 Tested Compounds
All(54)
 
 
Active(52)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(54)
 
 
Active(52)
 
 
Inactive(2)
 
 
AID: 686967
Data Source: The Scripps Research Institute Molecular Screening Center (METRS_INH_LUMI_1536_3XIC50 MDRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-05-03
Hold-until Date: 2014-05-02
Modify Date: 2014-05-02

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 52
Related Experiments
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AIDNameTypeComment
624268Luminescence-based biochemical primary high throughput screening assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Screeningdepositor-specified cross reference: Primary screen (MetRS inhibitors in singlicate)
624282Summary of the probe development efforts to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Summarydepositor-specified cross reference: Summary (MetRS inhibitors)
624412Luminescence-based biochemical high throughput confirmation assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Screeningdepositor-specified cross reference: Confirmation screen (MetRS inhibition in triplicate)
624413Counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): Luminescence-based cell-based high throughput assay to identify compounds that are cytotoxic to Jurkat human T lymphocyte cellsScreeningdepositor-specified cross reference: Counterscreen (Jurkat human T lymphocyte cells in triplicate)
651607Fluorescent Polarization-based biochemical high throughput orthogonal assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Screeningdepositor-specified cross reference: Counterscreen (MetRS orthogonal assay in triplicate)
651971Luminescence-based biochemical high throughput dose response assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Confirmatorydepositor-specified cross reference: Dose Response (MetRS inhibitors in triplicate)
651972Counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): Luminescence-based cell-based high throughput dose response assay to identify compounds that are cytotoxic to Jurkat human T lymphocyte cellsConfirmatorydepositor-specified cross reference: Counterscreen Dose Response(Cytotoxicity in triplicate)
651989Counterscreen Fluorescent Polarization-based biochemical high throughput orthogonal dose response assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Confirmatorydepositor-specified cross reference: Counterscreen Dose Response (Orthogonal in triplicate)
720620Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): radioactivity-based cell-based assay to identify compounds that inhibit human mitochondrial MetRS.Screeningdepositor-specified cross reference
720622Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): absorbance-based cell-based assay to identify compounds that inhibit growth of T. brucei.Confirmatorydepositor-specified cross reference
720623Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): radioactivity-based cell-based assay to identify compounds that inhibit MetRS of T. brucei.Screeningdepositor-specified cross reference
743060Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): radioactivity-based cell-based assay to identify compounds that inhibit MetRS of T. brucei (Round 1)Screeningdepositor-specified cross reference
743061Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): radioactivity-based cell-based assay to identify compounds that inhibit human mitochondrial MetRS (Round1)Screeningdepositor-specified cross reference
743068Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): absorbance-based cell-based assay to identify compounds that inhibit growth of T. brucei (Round 1)Confirmatorydepositor-specified cross reference
743153Late stage luminescence-based biochemical dose response assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS) (Round001)Confirmatorydepositor-specified cross reference
743154Late stage counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): Fluorescent Polarization-based biochemical dose response orthogonal assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)(Round001)Confirmatorydepositor-specified cross reference
743155Late stage counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): Luminescence-based cell-based high throughput dose response assay to identify compounds that are cytotoxic to Jurkat human T lymphocyte cells (Round001)Confirmatorydepositor-specified cross reference
743299On Hold
743300On Hold
743301On Hold
1053130On Hold
1053132On Hold
1053134On Hold
1053198On Hold
1053199On Hold
1053200On Hold
1053201On Hold
1053203On Hold
1053204On Hold
686968Late stage counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): Luminescence-based cell-based high throughput dose response assay to identify compounds that are cytotoxic to Jurkat human T lymphocyte cellsConfirmatorysame project related to Summary assay
686969Late stage counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): Fluorescent Polarization-based biochemical dose response orthogonal assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Confirmatorysame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: University of Washington
Assay Provider: Wilhelmus Hol, University of Washington
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 AI084004-01A1
Grant Proposal PI: Wilhelmus Hol, University of Washington
External Assay ID: METRS_INH_LUMI_1536_3XIC50 MDRUN

Name: Late stage luminescence-based biochemical dose response assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS).

Description:

Human African trypanosomiasis (HAT; also called sleeping sickness) is a neglected tropical disease that is caused by the protozoan Trypanosoma brucei, which employs the tsetse fly as its insect vector. Related tropical diseases include Chagas disease (caused by Trypanosoma cruzi) and leishmaniasis (caused by Leishmania species). Each of these diseases has a major impact on human health around the world and they lack adequate chemotherapeutic treatment options (1), as current therapies suffer from poor efficacy, oral bioavailability (2), toxicity, and difficult treatment regimens (3). As a result there is a great need to develop novel, more selective, and effective treatments (4). The aminoacyl-tRNA synthetases (aaRS) play essential roles in protein synthesis and cell survival and thus are attractive targets for the design of novel chemotherapeutic agents for these diseases (3). aaRS enzymes are essential to translating nucleotide-encoded gene sequences into proteins. Thus, inhibitors that interfere with these enzymes will inhibit formation of properly charged tRNA, leading to accumulation of uncharged tRNA on the ribosome, and disruption of normal protein chain elongation during translation, which are detrimental to cell viability. In particular, genomic studies have revealed sequence differences between the T. brucei trypanosome and mammalian methionyl-tRNA synthetases (MetRSs: which are members of the aaRS family), suggesting that selective inhibition of this enzyme and protozoan death can be achieved using drug-like molecules (2). Using RNA interference, T. brucei MetRS has been shown to be essential for parasite survival (3). In addition, since the MetRS enzymes from Trypanosomatid organisms are highly homologous (particularly in the methionine-ATP binding pocket) it is possible that compounds active against T. brucei MetRS will exhibit activity against the MetRS enzymes from T. cruzi and Leishmania.

References:

1. Gonzalez, M. and H. Cerecetto, Novel compounds to combat trypanosomatid infections: a medicinal chemical perspective. Expert Opin Ther Pat, 2011. 21(5): p. 699-715
2. Finn, J., M. Stidham, M. Hilgers, and C.K. G, Identification of novel inhibitors of methionyl-tRNA synthetase (MetRS) by virtual screening. Bioorg Med Chem Lett, 2008. 18(14): p. 3932-7.
3. Shibata, S., J.R. Gillespie, A.M. Kelley, A.J. Napuli, Z. Zhang, K.V. Kovzun, R.M. Pefley, J. Lam, F.H. Zucker, W.C. Van Voorhis, E.A. Merritt, W.G. Hol, C.L. Verlinde, E. Fan, and F.S. Buckner, Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice. Antimicrob Agents Chemother, 2011. 55(5): p. 1982-9.
4. Ding, D., Q. Meng, G. Gao, Y. Zhao, Q. Wang, B. Nare, R. Jacobs, F. Rock, M.R. Alley, J.J. Plattner, G. Chen, D. Li, and H. Zhou, Design, synthesis, and structure-activity relationship of Trypanosoma brucei leucyl-tRNA synthetase inhibitors as antitrypanosomal agents. J Med Chem, 2011. 54(5): p. 1276-87.

Keywords:

Late Stage, SAR, dose response, triplicate, enzyme, T. brucei, parasite, MetRS, methionyl tRNA synthetase, ligase, Aminoacyl-tRNA synthetase, aaRS, tRNA, methionine, methionyl, kinetic, biochemical, enzymatic, luciferase, luc, lumi, ATP depletion, luciferin, ATP, methionine, Luminescence, Lumi, Kinase Glo, RLU, inhibit, inhibitor, inhibition, Trypanosoma brucei., protozoa, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to determine dose response curves for available powder samples of compounds identified as active in a set of previous experiments entitled, "Luminescence-based biochemical high throughput dose response assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS) - BioAssay Summary" (AID 651971).
In this biochemical ATP depletion assay, methionine, bulk E. coli tRNA, and ATP are incubated with MetRS enzyme in the presence of test compounds. Following the incubation, remaining ATP levels are monitored using well chemiluminescence by addition of the luciferase-luciferin-based Kinase-Glo reagent to each well. As designed, a compound that inhibits MetRS activity will reduce the hydrolysis of ATP that normally occurs as the MetRS enzyme converts L-methionine to L-methionyl-tRNA(Met). Reductions in MetRS activity and ATP hydrolysis increase the relative amount of ATP available for luciferase-mediated cleavage of the luciferin substrate in the Kinase-Glo system, resulting in increased well luminescence. Negative controls include wells that do not contain ATP in the reaction. Positive controls include wells containing 1.5 uM of control compound (SID 136913750). Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 125.1 uM.
Protocol Summary:
Prior to the start of the assay 1.5 ul of a MetRS solution (0.4 mM Spermine, 0.2 mg/ml BSA, 45 nM HEPES, 18 nM MgCl2, 90 mM KCl, 5 mM DTT, 0.2 U/ml pyrophosphatase, 70 nM MetRS) to all wells. Plates were centrifuged. Next, 36 nL of test compounds or DMSO alone (0.9% final concentration) were distributed into the appropriate wells. The plates were then incubated for 15 minutes at 25 C. The assay was started by the addition of 1.5 ul of a mixture containing Met and tRNA (64 uM Met, 400 ug/ml tRNA) to column 1, 1.5 ul of a mixture containing ATP and tRNA (200 nM ATP, 400 ug/ml tRNA) to column 2 and 1.5 ul of a mixture containing ATP, t-RNA and Methionine (200 nM ATP, 400 ug/ml tRNA, 64 uM Met) to columns 3-48. The plates were then incubated for 2 hour at 25 C. After incubation, 3 ul of Kinase Glo reagent were added to all 48 columns and plates were incubated for another 10 minutes at 25 C. Plates were centrifuged and luminescence was measured by the ViewLux microplate reader.
The percent inhibition for each compound was calculated using the following mathematical expression:
%_Inhibition = ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) ) * 100
Where:
Test_Compound is defined as wells containing test compound,
Low_Control is defined as wells containing DMSO
High_Control is defined as wells containing 1.5 uM of control compound (SID 136913750)
PubChem Activity Outcome and Score:
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 125.1uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 125.1 uM.
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-61, and for inactive compounds 44-1.
List of Reagents:
MetRS protein (supplied by Assay Provider)
Magnesium Chloride Hexahydrate (Fisher, part 7791-18-6)
1M HEPES (Lonza, part 17-737)
Potassium Chloride (Fisher, part BP366)
Distiller Water (Gibco, part 15230)
Spermine (Fluka, part 85588)
Bovine serum albumin (Sigma, part A7906)
Pytophosphatase (Sigma, part I1643)
Dithiothreitol (Acros, part1 6568-0250)
E. coli tRNA (Sigma, part R4251)
ATP (Sigma, part A7699)
L-Methionine (Sigma, part M9625)
Kinase-Glo (Promega, part V6714)
DMSO (Acros Organics, part 127790025)
1536-well plates (Corning, part 7254)
Comment
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well Luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
2IC50*The concentration at which 50 percent of the inhibition in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in uM concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
7Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
8Response RangeThe range of Y.Float
9Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
10RsquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
11Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
12Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
13Inhibition at 0.006 uM [1] (0.00635574μM**)Value of percent Inhibition at 0.0064 uM compound concentration; replicate [1]Float%
14Inhibition at 0.006 uM [2] (0.00635574μM**)Value of percent Inhibition at 0.0064 uM compound concentration; replicate [2]Float%
15Inhibition at 0.006 uM [3] (0.00635574μM**)Value of percent Inhibition at 0.0064 uM compound concentration; replicate [3]Float%
16Inhibition at 0.019 uM [1] (0.0190672μM**)Value of percent Inhibition at 0.0191 uM compound concentration; replicate [1]Float%
17Inhibition at 0.019 uM [2] (0.0190672μM**)Value of percent Inhibition at 0.0191 uM compound concentration; replicate [2]Float%
18Inhibition at 0.019 uM [3] (0.0190672μM**)Value of percent Inhibition at 0.0191 uM compound concentration; replicate [3]Float%
19Inhibition at 0.057 uM [1] (0.0572016μM**)Value of percent Inhibition at 0.0572 uM compound concentration; replicate [1]Float%
20Inhibition at 0.057 uM [2] (0.0572016μM**)Value of percent Inhibition at 0.0572 uM compound concentration; replicate [2]Float%
21Inhibition at 0.057 uM [3] (0.0572016μM**)Value of percent Inhibition at 0.0572 uM compound concentration; replicate [3]Float%
22Inhibition at 0.172 uM [1] (0.171605μM**)Value of percent Inhibition at 0.1716 uM compound concentration; replicate [1]Float%
23Inhibition at 0.172 uM [2] (0.171605μM**)Value of percent Inhibition at 0.1716 uM compound concentration; replicate [2]Float%
24Inhibition at 0.172 uM [3] (0.171605μM**)Value of percent Inhibition at 0.1716 uM compound concentration; replicate [3]Float%
25Inhibition at 0.515 uM [1] (0.514815μM**)Value of percent Inhibition at 0.5147 uM compound concentration; replicate [1]Float%
26Inhibition at 0.515 uM [2] (0.514815μM**)Value of percent Inhibition at 0.5147 uM compound concentration; replicate [2]Float%
27Inhibition at 0.515 uM [3] (0.514815μM**)Value of percent Inhibition at 0.5147 uM compound concentration; replicate [3]Float%
28Inhibition at 1.5 uM [1] (1.54444μM**)Value of percent Inhibition at 1.5 uM compound concentration; replicate [1]Float%
29Inhibition at 1.5 uM [2] (1.54444μM**)Value of percent Inhibition at 1.5 uM compound concentration; replicate [2]Float%
30Inhibition at 1.5 uM [3] (1.54444μM**)Value of percent Inhibition at 1.5 uM compound concentration; replicate [3]Float%
31Inhibition at 4.6 uM [1] (4.63333μM**)Value of percent Inhibition at 4.6 uM compound concentration; replicate [1]Float%
32Inhibition at 4.6 uM [2] (4.63333μM**)Value of percent Inhibition at 4.6 uM compound concentration; replicate [2]Float%
33Inhibition at 4.6 uM [3] (4.63333μM**)Value of percent Inhibition at 4.6 uM compound concentration; replicate [3]Float%
34Inhibition at 13.9 uM [1] (13.9μM**)Value of percent Inhibition at 13.9 uM compound concentration; replicate [1]Float%
35Inhibition at 13.9 uM [2] (13.9μM**)Value of percent Inhibition at 13.9 uM compound concentration; replicate [2]Float%
36Inhibition at 13.9 uM [3] (13.9μM**)Value of percent Inhibition at 13.9 uM compound concentration; replicate [3]Float%
37Inhibition at 41.7 uM [1] (41.7μM**)Value of percent Inhibition at 41.7 uM compound concentration; replicate [1]Float%
38Inhibition at 41.7 uM [2] (41.7μM**)Value of percent Inhibition at 41.7 uM compound concentration; replicate [2]Float%
39Inhibition at 41.7 uM [3] (41.7μM**)Value of percent Inhibition at 41.7 uM compound concentration; replicate [3]Float%
40Inhibition at 125.1 uM [1] (125.1μM**)Value of percent Inhibition at 125.1 uM compound concentration; replicate [1]Float%
41Inhibition at 125.1 uM [2] (125.1μM**)Value of percent Inhibition at 125.1 uM compound concentration; replicate [2]Float%
42Inhibition at 125.1 uM [3] (125.1μM**)Value of percent Inhibition at 125.1 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R01 AI084004-01A1

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