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BioAssay: AID 720620

Late 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.

Name: Late 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. ..more
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 Tested Compounds
 Tested Compounds
All(54)
 
 
Active(1)
 
 
Inactive(53)
 
 
 Tested Substances
 Tested Substances
All(54)
 
 
Active(1)
 
 
Inactive(53)
 
 
AID: 720620
Data Source: The Scripps Research Institute Molecular Screening Center (HUMAN-METRS_INH_RAD_0096_3X%INH MDCSRUN ROUND 0 (run by assa..)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-09-06
Hold-until Date: 2014-08-23
Modify Date: 2014-08-26

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
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)
686967Late stage luminescence-based biochemical dose response assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)Confirmatorydepositor-specified cross reference: Late Stage Dose Response (MetRS inhibitors in triplicate)
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 cellsConfirmatorydepositor-specified cross reference: Late Stage Dose Response (Jurkat in triplicate)
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)Confirmatorydepositor-specified cross reference: Late Stage Dose Response (MetRS orthogonal in triplicate)
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
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.Confirmatorysame project related to Summary assay
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.Screeningsame project related to Summary assay
1053198On Hold
1053199On Hold
1053200On Hold
1053201On Hold
1053203On Hold
1053204On Hold
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: HUMAN-METRS_INH_RAD_0096_3X%INH MDCSRUN ROUND 0 (run by assay provider)

Name: Late 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.

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, assay provider, 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 whether compounds identified as MetRS inhibitor probe candidates can inhibit the activity of MetRS in human mitochondria. This assay is performed by the assay provider.
Enzyme activity is quantified by the attachment of [3H]L-methionine to tRNA in the presence of Human mitochondrial MetRS enzyme. Reactions are performed in 96-well filter plates with Durapore membranes (MSHVN4B10; Millipore) in volumes of 75 microl. The reaction is performed with 50 mM Tris-HCl pH 8.0, 6 mM MgCl2, 2.5 mM KCl, 0.2 mM spermine, 0.2 mg/ml bovine serum albumin, 2.5 mM dithiothreitol, 2.5 mM ATP, 240 nM [3H]L-methionine (83 Ci/mmol), and 0.1 U/ml pyrophosphatase (I1643; Sigma). Recombinant enzyme (13 nM) and compound inhibitors (10 microM and 1 microM) are mixed with the buffer and preincubated for 15 min. To start the reaction, 200 microg/ml bulk Escherichia coli tRNA (R4251; Sigma) is added. The plate is incubated without shaking at room temperature for 60 min. The reactions are stopped by the addition of 100 microl/well cold 10% trichloroacetic acid. The reaction components are separated from tRNA by filtration through a vacuum manifold and washed three times with 300 microl/well cold 10% trichloroacetic acid. The filter plates are dried, 25 microl/well scintillation fluid added, and the counts on the plates determined in a scintillation plate counter. Samples are run in triplicate, and the average activity of inhibitors are compared to that in control wells without inhibitors. Compounds are tested in triplicate at two concentrations final (10 microM and 1 microM).
Protocol Summary:
Prior to the start of the assay 66 ul/well of HmMetRS solution (0.23 mM Spermine, 0.23 mg/ml BSA, 56.82 mM Tris-HCl pH 8.0, 6.82 mM MgCl2, 2.84 mM KCl, 2.84 mM DTT, 0.11 U/ml pyrophosphatase, 2.84 mM ATP, 272.73 nM [3H]L-methionine, 14.77 nM human mitochondrial MetRS) to all wells. Next, 1.5 microl/well of 50X test compounds diluted in 100% DMSO or DMSO alone (2% final concentration) were distributed into the appropriate wells. The plates were then incubated for 15 minutes at room temperature. The assay was started by the addition of 7.5 microl of 200 microg/ml bulk E. coli tRNA or ddH20 (low control) were distributed into the appropriate wells. The plates were then incubated for 1 hour at room temperature. After incubation, 100 microl/well of cold 10% trichloroacetic acid was added and the plates were placed in the -20o C for 12 minutes to stop the reaction. The reaction components were seperated from tRNA by filtration through a vacuum manifold and washed three times with 300 microl/well of cold 10% trichloroacetic acid. The filter plates were dried, the rubber backing removed, and 25 microl/well of scintillation fluid was added. Plates were read on the MicroBeta2 scintillation plate reader.
The percent inhibition for each compound was calculated using the following mathematical expression:
%_Inhibition = ( (Average_High_Control - Test_Compound ) / ( Average_High_Control - Average_Low_Control ) ) * 100
Where:
Test_Compound is defined as wells containing test compound,
Low_Control is defined as wells containing DMSO without 200 ug/mL of bulk E. coli tRNA
High_Control is defined as wells containing DMSO
PubChem Activity Outcome and Score:
Each test compound was tested at 10 uM and 1 uM and the percent inhibition was determined.
Compounds with a percent inhibtion less than 50% at 10 uM were considered inactive. Compounds with a percent inhibition greater than or equal to 50% at 10 uM were considered active.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 54-0.
List of Reagents:
Human Mitochondrial MetRS protein (supplied by Assay Provider)
Magnesium Chloride Hexahydrate (Fisher, part 7791-18-6)
Tris(Hydroxymethyl) Aminomethane (Sigma-Aldrich/Aldrich, part 154563)
Potassium Chloride (J.T. Baker, part 3040-01)
Distiller Water (Baxter, part 2F7114)
Spermine (Fluka, part 85588)
Bovine serum albumin (New England BioLabs, part B9001S)
Pyrophosphatase (Sigma, part I1643)
Dithiothreitol (Acros, part1 6568-0250)
Bulk E. coli tRNA (Sigma, part R4251)
ATP (Sigma, part A7699)
L-[Methyl-3H]-Methionine (PerkinElmer, part NET061X005MC)
Trichloroacetic Acid (Fisher Scientific, part UN1839)
DMSO (Sigma, part D8418)
96-well filter plates with Durapore membranes (Millipore, part MSHVN4B10)
Ultima Gold scintillation fluid (PerkinElmer, part 6013329)
MultiScreenHTS vacuum manifold (Millipore, part MSVMHTS00)
Comment
This assay was performed by the assay provider. In this assay an Inactive assignment indicates that the compound does not inhibit the human mitochondrial MetRS enzyme, while an Active assignment indicates that a compound inhibits the human mitochondrial MetRS enzyme. 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-assay basis.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 10 uM (10μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 10.0 uM.Float%
2Standard Deviation [Inhibition at 10 uM] (10μM**)Standard deviation derived from the normalized percent inhibition of the triplicate data for each compound.Float
3Inhibition at 10 uM [1] (10μM**)Percent inhibition of the confirmation screen at a compound concentration of 10.0 uM; replicate 1.Float%
4Inhibition at 10 uM [2] (10μM**)Percent inhibition of the confirmation screen at a compound concentration of 10.0 uM; replicate 2.Float%
5Inhibition at 10 uM [3] (10μM**)Percent inhibition of the confirmation screen at a compound concentration of 10.0 uM; replicate 3.Float%
6Average Inhibition at 1 uM (1μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 1.0 uM.Float%
7Standard Deviation [Inhibition at 1 uM] (1μM**)Standard deviation derived from the normalized percent inhibition of the triplicate data for each compound.Float
8Inhibition at 1 uM [1] (1μM**)Percent inhibition of the confirmation screen at a compound concentration of 1.0 uM; replicate 1.Float%
9Inhibition at 1 uM [2] (1μM**)Percent inhibition of the confirmation screen at a compound concentration of 1.0 uM; replicate 2.Float%
10Inhibition at 1 uM [3] (1μM**)Percent inhibition of the confirmation screen at a compound concentration of 1.0 uM; replicate 3.Float%

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

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