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: GSTO1_INH_FLUO_ABPP_3XIC50_SET2

Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50 Set 2.

Description:

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 [1]. 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 [2], and other studies have implicated GSTO1 in chemotherapeutic resistance [3]. 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 [4]; 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 [5]. As such, inhibitor discovery by FluoPol-ABPP [6] offers a unique opportunity to develop inhibitors for this important enzyme.

References:

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.

Keywords:

Late stage, late stage AID, assay provider, powders, GSTO1, oxidoreductase, glutathione S-transferase omega 1, inhibitor, counterscreen, fluorescence, gel-based ABPP, activity-based protein profiling, SE-Rh, sulfonate ester, cancer, chemotherapeutic resistance, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Assay Overview:

The purpose of this assay is to determine the IC50 values for powder samples of synthesized compounds. In this assay, a rhodamine-conjugated sulfonate ester (SE-Rh) activity-based probe is used to label GSTO1 in the presence of test compounds. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as GSTO1 inhibitors will prevent GSTO1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe, giving lower fluorescence intensity in the band in the gel.

Protocol Summary:

Recombinant GSTO1 (250 nM) in assay buffer (DPBS with 0.01% pluronic detergent) was incubated with DMSO or compound for 30 minutes at 25 C before the addition of PS-Rh at a final concentration of 10 uM in 50 ul total reaction volume. The reaction was incubated for 1 hour at 25 C, quenched with 2X SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the bands. IC50 values for inhibition of GSTO1 were determined from dose-response curves from three trials at each inhibitor concentration (7-point series from 3 uM to 3 nM).

The % inhibtion was then calculated as follows:

% Inhibition = ( IOD_Test_Compound - MedianIOD_Low_Control ) / ( MedianIOD_High_Control - MedianIOD_Low_Control ) * 100

Where:

Test_Compound is defined as the GSTO1 treated with test compound.
High_Control is defined as GSTO1 treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.

For each test compound, percent inhibition was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported.

PubChem Activity Outcome and Score:

Compounds with an IC50 value of less than 1 uM were considered active. Compounds with an IC50 value greater than 1 uM were considered inactive.

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. There are no inactive compounds.

List of Reagents:

Recombinant GSTO1 (provided by Assay Provider)
SE-Rh (provided by Assay Provider)
DPBS (Cellgro 20-031-CV)
Pluronic (Calbiochem 540025)

This assay was performed by the assay provider with compounds synthesized as powders. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.

Grant Number: 1 R01 CA087660-05