Luminescence-based biochemical high throughput dose response assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)
Name: Luminescence-based biochemical high throughput dose response assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS). ..more
BioActive Compounds: 137
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 DRUN
Name: Luminescence-based biochemical high throughput dose response assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS).
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.
DRUN, dose, 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.
The purpose of this assay is to determine dose response curves for compounds that confirmed MetRS inhibitor activity in a set of previous experiments entitled, "Luminescence-based biochemical high throughput confirmation assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)" (AID 624412). Compounds should inhibit the activity of the Trypanosoma brucei methionyl tRNA synthetase (MetRS).
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), or wells containing no methionine. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 119 uM.
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
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)
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. 119 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 119 uM.
PubChem Activity Outcome and Score:
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 100-90, and for inactive compounds 89-0.
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)
Distilled 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)
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
From BioAssay Depositor:
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
Assay: Dictionary: Version: 0.1
Assay Format: Biochemical
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)