Late 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)
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 MetRS of T. brucei (Round1). ..more
BioActive Compounds: 66
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: TBRUCEI-METRS_INH_RAD_0096_3X%INH MDCSRUN (Round1) (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 MetRS of T. brucei (Round1).
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.
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.
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Radioactivity-based, cell-based inhibition assays.
§ Panel component ID.
The purpose of this assay is to determine whether compounds identified as MetRS inhibitor probe candidates can inhibit the activity of MetRS of T. brucei. Aminoacylation assay run by Assay Provider.
Enzyme activity is quantified by the attachment of [3H]L-methionine to tRNA in the presence of T. brucei MetRS enzyme. Reactions are performed in 96-well filter plates with Durapore membranes (MSHVN4B10; Millipore) in volumes of 75 ul. The reaction is performed with 25 mM HEPES-KOH, pH 7.9, 10 mM MgCl2, 50 mM KCl, 0.2 mM spermine, 0.1 mg/ml bovine serum albumin, 2.5 mM dithiothreitol, 0.1 mM ATP, 240 nM [3H]L-methionine (83 Ci/mmol), and 0.1 U/ml pyrophosphatase (I1643; Sigma). Recombinant enzyme (10 nM) and compound inhibitors (10 uM and 1 uM) are mixed with the buffer and preincubated for 15 min. To start the reaction, 400 ug/ml bulk Escherichia coli tRNA (R4251; Sigma) is added. The plate is incubated without shaking at room temperature for 120 min. The reactions are stopped by the addition of 100 ul/well cold 10% trichloroacetic acid. The reaction components are separated from tRNA by filtration through a vacuum manifold and washed three times with 300 ul/well cold 10% trichloroacetic acid. The filter plates are dried, 25 ul/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 uM and 1 uM).
Prior to the start of the assay 66 ul/well of TbMetRS solution (0.23 mM Spermine, 0.11 mg/ml BSA, 28.41 mM HEPES-KOH pH 7.9, 11.36 mM MgCl2, 56.82 mM KCl, 2.84 mM DTT, 0.11 U/ml pyrophosphatase, 0.11 mM ATP, 272.73 nM [3H]L-methionine, 11.36 nM T. brucei MetRS) to all wells. Next, 1.5 ul/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 ul of 400 ug/ml bulk E. coli tRNA or ddH20 (low control) were distributed into the appropriate wells. The plates were then incubated for 2 hours at room temperature. After incubation, 100 ul/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 ul/well of cold 10% trichloroacetic acid. The filter plates were dried, the rubber backing removed, and 25 ul/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
Test_Compound is defined as wells containing test compound,
Low_Control is defined as wells containing DMSO without 400 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 100-51, and for inactive compounds 44-0.
List of Reagents:
T. brucei MetRS protein (supplied by Assay Provider)
Magnesium Chloride Hexahydrate (Fisher, part 7791-18-6)
1M HEPES (Lonza, part 17-737E)
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)
Microbeta2 plate reader (PerkinElmer)
Ultima Gold scintillation fluid (PerkinElmer, part 6013329)
MultiScreenHTS vacuum manifold (Millipore, part MSVMHTS00)
Excel 2013 (Microsoft)
This assay was performed by the assay provider. In this assay an Inactive assignment indicates that the compound does not inhibit the T. brucei MetRS enzyme, while an Active assignment indicates that a compound inhibits the T. brucei 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.
The NCBI protein and taxonomy identifiers referenced in this Assay represent the closest available in NCBI Entrez to the T. brucei bloodstream form strain 427 that was utilized in this Assay. Please refer to reference  for more information on the actual source strain.
** Test Concentration. § Panel component ID.