Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): fluorescence-based click chemistry assay
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): fluorescence-based click chemistry assay. ..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: GSTO1_INH_FLUO_ABPP_CLICKCHEMISTRY
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): fluorescence-based click chemistry assay.
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.
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The purpose of this assay is to compare inhibition of GSTO1 by an inhibitor compound in a competitive ABPP assay (Assay 1) compared to inhibition by the same compound in a direct ABPP assay using click chemistry (Assay 2). In Assay 1, cultured MDA-MB-435 cells were incubated with test compound. Cells were harvested and the soluble fraction was isolated and reacted with a rhodamine-conjugated sulfonate ester (SE-Rh) activity-based probe. In Assay 2, labeling of GSTO1 in proteome aliquots was assessed directly (rather than by competition) by appending an azide-conjugated rhodamine tag (Rh-N3) by click chemistry. For both assays, the reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining was determined by measuring the integrated optical density (IOD) of the bands. For Assay 1, the percentage activity remaining was determined by measuring the integrated optical density (IOD) of the bands; test compounds that act as GSTO1 inhibitors will prevent GSTO1-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the GSTO1 band in the gel. For Assay 2, the percent labeling was determined by measuring the IOD of the bands; compounds that act as GSTO1 inhibitors will bind GSTO1, and, following conjugation with the rhodamine tag, will cause an increase in the fluorescence intensity in the GSTO1 band in the gel.
MDA-MB-435 cells in media (5 mL total volume; supplemented with FCS) are treated with 1 uM, 0.5 uM, and 0.25 uM test compound (1 uL of 1000X stock (1 mM, 500 uM, or 250 uM, respectively) in DMSO) for 1 hour at 37 C. Cells are harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction is isolated by centrifugation (100K x g, 45 min) and the protein concentration is adjusted to 1 mg/mL with DPBS. For Assay 1, SE-Rh (1 uL of 50X stock in DMSO) is added to a final concentration of 10 uM in 50 ul total reaction volume. For Assay 2, proteome sample aliquots were reacted with a rhodamine-azide tag (Rh-N3; 50 uM) under click chemistry (CC) reaction conditions (1 mM TCEP, 100 uM TBTA ligand, 1 mM Cu(II)sulfate). For both assays, after addition of reagents, the reaction is incubated for 1 hr at 25 C, quenched with 2X SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning.
For Assay 1, the percentage activity remaining is determined by measuring the integrated optical density of the GSTO1 band relative to a DMSO-only (no compound) control.
% Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Test_Compound is defined as GSTO1 in cells treated with test compound.
High_Control is defined as GSTO1 in cells treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
For Assay 2, the IOD of the GSTO1 band was calculated relative to background.
Relative Band Intensity = IOD_GSTO1 / IOD_Low_Control
GSTO1 in the target band.
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
Assay 1 Outcome: Compounds with >= 90% inhibition were considered active. Compounds with inhibition < 90% inhibition were considered inactive.
Assay 2 Outcome: A GSTO1 signal of > 5-fold above background was considered active. A GSTO1 signal of < 5-fold above background was considered inactive.
The PubChem Activity Score is assigned a value of 100 for active compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
MDA-MB-435 cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
DPBS (Cellgro 20-031-CV)
SE-Rh (provided by the Assay Provider)
Rh-N3 (provided by Assay Provider)
TCEP (Sigma-Aldrich 93284)
TBTA Ligand (Sigma-Aldrich 678937)
Copper (II) sulfate (Sigma-Aldrich 451657)
This assay was performed in the laboratory of the Assay Provider with powder samples of compounds. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.
Categorized Comment - additional comments and annotations
** Test Concentration. § Panel component ID.