Summary of probe development efforts to identify cytotoxic compounds using the Jurkat human T-cell line
- Deposit:2009-04-09
- Modify:2009-04-09
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Peter Hodder, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Numbers: ML00112, ML00113, ML00114
Grant Proposal PI: Peter Hodder, TSRI
Name: Summary of probe development efforts to identify cytotoxic compounds using the Jurkat human T-cell line.
Description:
Compound cytotoxicity is an important parameter to measure when developing potential human therapeutics. Thus, it is necessary to utilize cell-based high throughput screening assays to determine cell viability in the presence of test compounds, while using a known cytotoxic compound as a control (1). Jurkat cells are 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.
Summary of Probe Development Effort:
This erves as a summary of the High Throughput Screening (HTS) campaign and probe development effort to identify compounds that are cytotoxic. This project encompassed an initial screening of the NIH starter set of 3316 compounds in singlicate and in titration assays (AID 364), Primary screening of the MLSCN collection in singlicate (AID 463), and titration assays in triplicate to determine potency (AID 464). This pilot project was proposed by the MLSCN project team for the SRIMSC to demonstrate its screening capabilities. The expected results of this effort were for the SRIMSC to be able to identify known cytotoxic compounds within the MLSMR screening library. The final dose response assay (AID 464) identified 331 active/706 tested compounds, with the majority of actives known to be cytotoxic compounds. This project was closed after completion of AID 464.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective , as detailed below.
References:
1. Fan, F and Wood, KV, Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol, 2007. 5(1): p. 127-36.
2. Schneider, U, Schwenk, HU and Bornkamm, G. Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer, 1977. 19(5): p. 621-6.
3. Riss T, Moravec R, Beck M, Hannah R, Wilson K and Swanson R. A Viable Solution for Cytotoxicity Screening. Promega Notes, 2002. Number 81: p. 2-5.
Keywords:
Summary AID, Jurkat, clone E6.1, T-cells, viability, cytotoxicity, proliferation, inhibition, ATP, metabolism, CellTiter-Glo, luminescence, luciferase, doxorubicin, primary, dose response, high throughput screening, HTS, 1536, Scripps, Scripps Florida, Molecular Library Probe Production Center Network, MLPCN.