Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): absorbance-based cell-based assay to identify compounds that inhibit growth of T. brucei.
Name: Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): absorbance-based cell-based assay to identify compounds that inhibit growth of T. brucei. ..more
BioActive Compounds: 12
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-GROWTH_INH_ABS_0096_3XIC50 MDCSRUN ROUND 0 (run by assay provider)
Name: Late stage assay provider counterscreen for inhibitors of Trypanosoma brucei methionyl tRNA synthetase (MetRS): absorbance-based cell-based assay to identify compounds that inhibit growth of T. brucei.
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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§ Panel component ID.
The purpose of this assay is to determine an EC50 against T.brucei by testing compounds against the parasites at 4 different concentrations Activity Cutoff: >10 uM on blood stream form T. brucei. Cell growth assay run by the assay provider.
T. brucei (bloodstream form strain 427 from K. Stuart, Seattle Biomedical Research Institute, Seattle, WA) is cultured in HMI-9 medium containing 10% fetal bovine serum, penicillin, and streptomycin at 37 degrees C with 5% CO2 (14). Drug sensitivity of the T. brucei strain is determined in 96-well microtiter plates in triplicate with an initial inoculum of 1x 104 trypomastigotes per well. Compound stock solutions are prepared in DMSO at 20 mM and/or 10 mM and added in serial dilutions for a final volume of 200 microl/well. Parasite growth was quantified at 48 h by the addition of AlamarBlue (Alamar Biosciences, Sacramento, CA) (25). Pentamidine isethionate (Aventis, Dagenham, United Kingdom) was included in each assay as a positive control. Compounds were tested in triplicate using a 4-point serial three dilution system starting at a test concentration of 10 uM.
Prior to the start of the assay 200 uL of sterile water is added around the perimeter of the plates. Initial dilutions of 20 uM (2X) were made in HMI-9 in sterile deep well plates. 150 ul of this 20 uM drug dilution in HMI-9 were added in triplicate to the plate, and 3 sets of serial 3 dilutions were made (for a total of 4 concentrations in triplicate per drug) by mixing 50 ul into 100 ul HMI-9. In this manner, 4 drugs were tested per plate. Each plate also contained 6 wells of a no drug control, and 6 wells containing only media to establish no growth and 100% growth. Plates were read using a flourescence reader with 530 nM excitation and 590 nM emmision filters.
The inhibition at the various concentrations and the EC50 value were calculated by the online service CDD (http://www.collaborativedrug.com) using an equation to describe the sigmoidal dose-response curve.
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 100-1, and for inactive compounds 0-0.
List of Reagents:
5mL and 10mL pipets
Sterile reagent reservoirs (Corning Costar 4870)
96-well flat bottom plate with lid (Corning Costar 3596)
96-deep well plate (Corning Costar 3960)
10, 250, and 1000 uL pipet tips (Rainin Brand GPS-L series)
P10, P20, P200, and P1000 pipet (Rainin Pipet-Lite series)
P200 and P1200 Multi-channel pipet with 12 channels (Rainin Pipet-Lite series)
Microfuge tube rack
Complete HMI-9 Medium (1 L IMDM (Gibco #12440-046), 28.2 mg bathocuprionedisulfonic acid disodium salt (Sigma #B1125), 182 mg L-Cysteine (Sigma #C7352), 10 mls 100X HT-Supplement (Gibco 11067-030), 10 mls Pen/Strep (Lonza 17-602E), 100 mls fetal bovine serum (Atlanta Biologicals #S11550) Heat deactivated, 14 ul 2-mercaptoethanol (Biorad #161-0710); filter sterilized over a 0.22 Millipore Steritop Filter (Millipore #SCGP S05 RE)
Alamar Blue (Invitrogen #DAL1100)
T. brucei BSF 427 parasites (gift from K. Stuart, Seattle Biomedical Research Institute, Seattle, WA)
Collaborative Drug Discovery Inc. (CDD)- web based drug discovery software (www.collaborativedrug.com)
Pentamidine isethionate (Aventis, Dagenham, United Kingdom)
FLx800 plate reader (BioTek)
This assay was performed by the assay provider. In this assay an Inactive assignment indicates that the compound is not cidal against T. brucei cells, while an Active assignment indicates that a compound is cidal against T. brucei cells. 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.
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.
* Activity Concentration. ** Test Concentration. § Panel component ID.