Late 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)
Name: Late 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). ..more
BioActive Compounds: 50
Depositor Specified Assays
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_FP_1536_3XIC50 MDCSRUN (orthogonal)
Name: Late 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).
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.
MDSCRUN, orthogonal, counterscreen, triplicate, powders, SAR, enzyme, T. brucei, parasite, MetRS, methionyl tRNA synthetase, ligase, Aminoacyl-tRNA synthetase, aaRS, tRNA, methionine, methionyl, kinetic, biochemical, enzymatic, inhibit, inhibitor, inhibition, fluorescent, fluorescence polarization, FP, Transcreener, tracer, 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 available powder samples of compounds identified as active in a set of previous experiments entitled, "Fluorescent Polarization-based biochemical high throughput orthogonal assay for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)" (AID 651607). This assay employs an orthogonal assay detection (FP). Compounds should inhibit the activity of the Trypanosoma brucei methionyl tRNA synthetase (MetRS).
In this biochemical assay, methionine, bulk E. coli tRNA, and ATP are incubated with MetRS enzyme in the presence of test compounds. Following the incubation, AMP levels are measured as a function of the displacement of a fluorescent AMP analog initially bound to an antibody. Specifically this assay employs fluorescence polarization to monitor the AMP formation. Polarization is a measure of the change in molecular movement of a labeled species and is defined as the ratio of the difference between the vertical and horizontal components of emitted light over their sum. Because polarization is a dimensionless value, it is independent of the emitted light or concentration of fluorophore. 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). Free AMP, product of the reaction, displaces the fluorescent tracer from the antibody, leading to a decreased fluorescence polarization. Reductions in MetRS activity and ATP hydrolysis decrease the relative amount of AMP produced, preventing the displacement of the tracer resulting in no decrease in fluorescence polarization. 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.
Prior to the start of the assay 1.5 ul of a MetRS solution (0.4 mM Spermine, 0.2 mg/ml BSA, 45 mM HEPES, 18 mM 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 120 minutes at 25 C. After incubation, 3 ul of Transcreener reagent (16 nM AMP2/GMP2 AlexaFluor Tracer, 3 ug/ml AMP2/GMP2 Antibody, 25 mM HEPES, 10 mM MgCl2, 50 mM KCl ) were added to all 48 columns and plates were centrifuged and incubated for another 90 minutes at 25 C. Fluorescence polarization was read on an EnVision microplate reader (PerkinElmer, Turku, Finland) using a Cy5 FP filter set and a Cy5 dichroic mirror (excitation = 620 nm, emission = 688 nm). Fluorescence polarization was read for 50 seconds for each polarization plane (parallel and perpendicular).
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 (CID1312)
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. 125.1 uM) 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 100-74, and for inactive compounds 62-1.
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)
Transcreener AMP2/GMP2 (BellBrooks Lab, part 2102, 2101)
DMSO (Acros Organics, part 127790025)
1536-well plates (Corning, part 7254)
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.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)