Counterscreen 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 cells
Name: Counterscreen 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 cells ..more
BioActive Compounds: 161
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: JURKAT_INH_LUMI_1536_3X%INH CSRUN (MetRS)
Name: Counterscreen 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 cells
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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The purpose of this assay is to determine whether compounds identified as active in a set of previous experiments entitled, "Luminescence-based biochemical primary high throughput screening assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)" (AID 624268) are nonselective or cytotoxic.
This assay employs Jurkat cells, a human T-cell line originally isolated from an adolescent male with T cell leukemia (2). The cells are grown in suspension. The end point assays presented here employed the CellTiter-Glo luminescent reagent (3), which contains luciferase enzyme to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of Jurkat cell ATP. Since metabolically active cells produce ATP, an increase in the number of dead or dying cells will correlate with a reduction in ATP levels. As designed, compounds that inhibit cell viability and reduce intracellular ATP will reduce the catalytic conversion of luciferin into oxyluciferin, resulting in decreased luciferase activity and well luminescence. This assay included as a positive control doxorubicin, an antibiotic used as an anti-cancer drug. Compounds are tested in triplicate at a final nominal concentration of 8.3 uM.
Jurkat cells (clone E6.1) were routinely cultured in suspension in Roller bottles at 37 C in 95% relative humidity (RH) at 5% CO2 in growth media. Growth media consisted of RPMI-1640 containing 10% dialyzed fetal bovine serum, 0.1 mM NEAA, 1mM Sodium Pyruvate, 25mM HEPES, 5mM L Glutamine, and 1x antibiotic.
Prior to the start of the assay, cells were suspended to a concentration of 100,000 cells/ml in assay media consisting of RPMI-1640 containing 10% dialyzed fetal bovine serum, 0.1 mM NEAA, 1mM Sodium Pyruvate, 25 mM HEPES, 5mM L Glutamine, and 1x antibiotic. To start the assay, 5 uL of assay media was dispensed into the first two columns of a 1536 well plate and 5 ul of cell suspension to the remaining wells (500 cells per well). The assay was started immediately by dispensing 25 nL of test compound in DMSO, Doxurubicin (8 uM final concentration) or DMSO alone (0.6% final concentration) to the appropriate wells. The plates were then incubated for 48 hours at 37 C, 5% CO2 and 95% RH).
Following the two day incubation, plates were equilibrated to room temperature for 10 minutes and 5 ul of CellTiter-Glo reagent was added to each well. Plates were centrifuged and incubated at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader. The percent inhibition for each compound was calculated as follows:
%_Inhibition = ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing 0.6% DMSO. (0% inhibition)
High_Control is defined as wells containing 8 uM doxorubicin in 0.6% DMSO (100% inhibition).
PubChem Activity Outcome and Score:
The average percent inhibition and standard deviation of each compound tested were calculated. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-15, and for inactive compounds 15-0.
List of Reagents:
Jurkat cells (clone E6.1; ATCC Cat# TIB-152, Invitrogen Cat# K1045)
Cell Titer Glo (Promega, part G7573)
Doxorubicin (Fisher, part BP251610)
RPMI (Invitrogen, part 11875)
Fetal Bovine Serum (Fisher, part SH3008803)
NEAA (Invitrogen, part 11140-050)
Hepes (Invitrogen, part 15630-080)
Anti-Anti (Invitrogen, part 15240)
L-Glutamine (Invitrogen, part 25030-081)
Sodium Pyruvate (Invitrogen, part 11360-070)
Roller Bottle (BD, part 353154)
1536-well plates (Corning, part 7254)
Due to the increasing size of the MLPCN compound library, 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)