Luminescence-based biochemical primary high throughput screening assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS)
Name: Luminescence-based biochemical primary high throughput screening assay to identify inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS). ..more
BioActive Compounds: 1456
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_1X%INH PRUN
Name: Luminescence-based biochemical primary high throughput screening assay to identify 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.
primary, 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 identify compounds that 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 singlicate at a final nominal concentration of 11.9 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)
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of low control wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter 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 100-5, and for inactive compounds 11-5.
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)
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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)