Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compounds
Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compounds ..more
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 CA087660-05
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: MDACYTOX_INH_LUMI_384_4XCC50
Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compounds
Glutathione transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide-variety of both exogenous and endogenous compounds for biotransformation and/or removal . Using activity-based proteomic methods, we discovered that glutathione S-tranferase omega (GSTO1) is over-expressed in human cancer cell lines that show enhanced aggressiveness , and other studies have implicated GSTO1 in chemotherapeutic resistance . Cancer remains one of the most life-threatening diseases for which effective treatments and cures are lacking. Historically, cancer has been treated with general chemotoxic agents; however, because these agents essentially kill all cells at a rate proportional to their proliferation, general chemotoxins offer only a modest therapeutic window. Recently, targeted therapeutics (i.e., selective inhibitors that block individual enzymes) have shown great promise for the treatment of cancer. Inhibiting GSTO1 may thus offer a new therapeutic strategy for cancer. Development of a selective inhibitor would also aid in the investigation into GSTO1 involvement in the dysregulated biochemical pathways that support tumorigenesis. GSTO1 has a catalytic cysteine residue and is consequently sensitive to broadly reactive thiol alkylating agents, including N-ethylmaleimide ; however, selective inhibitors for GSTO1 have not yet been identified. A limited number of substrate assays have been developed, but these are not well-suited for HTS due to poor turnover rates and/or reliance on UV absorbance at short wavelengths (305 nm), where many small-molecules exhibit intrinsic absorbance . As such, inhibitor discovery by FluoPol-ABPP  offers a unique opportunity to develop inhibitors for this important enzyme.
1. Hayes, J.D., J.U. Flanagan, and I.R. Jowsey, Glutathione transferases. Annu. Rev. Parmacol. Toxicol., 2005. 45: 51-88.
2. Adam, G., E.J. Sorensen, and B.F. Cravatt, Proteomic Profiling of mechanistically distinct enzyme classes using a common chemotype. Nature Biotechnology, 2002. 20: 805-809.
3. Yan, X.D., et al., Identifcation of platinum resistance-associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines. J. Proteome Res., 2007. 6: 772-780.
4. Whitbread, A.K., et al., Characterization of the Omega Class of Glutathione Transferases. Methods in Enzymology, 2005. 401: 78-99.
5. Board, P.G., et al., S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1. Analytical Biochemistry, 2008. 374: 25-30.
6. Bachovchin, D.A., Brown, S.J., Rosen, H., and Cravatt, B.F. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat Biotechnol, 2009. 27: 387-394.
Late stage, late stage AID, assay provider, powders, GSTO1, oxidoreductase, glutathione S-transferase omega 1, cytotoxicity, viability, luminescence, dose response, counterscreen, MDA-MB-435, inhibitor, cancer, chemotherapeutic resistance, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to determine cytotoxicity of inhibitor compounds belonging to alpha-chloroacetamide scaffold. In this assay, MDA-MB-435 cells in either serum-free media (Assay 1) or serum-free media (Assay 2) are incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 7-point 1:10 dilution series starting at a nominal test concentration of 125 uM.
This assay was started by dispensing MDA-MB-435 cells in RPMI media (15 uL, 8 x 10E3 cells/well) into a 384-well plate. Both serum-free media (Assay 1) and media supplemented with fetal calf serum (FCS) (Assay 2) were tested. Compound (5 uL of 0-500 uM in media containing 5% DMSO) was added to each well, giving final compound concentrations of 0-125 uM. Cells were incubated for 48hrs at 37 C in a humidified incubator and cell viability was determined by the CellTitre-Glo assay (Promega) according to manufacturer instructions.
The % cell viability for each well was then calculated as follows:
% Cell Viability = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100
Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).
For each test compound, percent cell viability was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using GraphPad Prism (GraphPad Software Inc). The software-generated CC50 values are reported. For data where only the highest (125 uM) concentration point showed >60% cell death, the curve was fit by assuming 100% cell death at 10X, 100X, and 1000X (1.25 mM , 12.5 mM, 125 mM) higher concentrations, giving a conservative estimate of the CC50.
PubChem Activity Score and Outcome:
Compounds with a CC50 value of less than 10 uM were considered active (cytotoxic). Compounds with a CC50 value greater than 10 uM were considered inactive (non-cytotoxic).
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
MDA-MB-435 cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
Cell Titer-Glo (Promega, part G7572)
384-well plates (Corning 3704)
This assay was performed by the assay provider with compounds ordered as powders. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDS listed in the Related Bioassays section of this AID.
* Activity Concentration. ** Test Concentration. § Panel component ID.