Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): gel-based activity-based protein profiling (ABPP) selectivity assay with endogenous enzyme
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): gel-based activity-based protein profiling (ABPP) selectivity assay with endogenous enzyme. ..more
BioActive Compounds: 5
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_ENDOG_SEL
Name: Late stage assay provider results from the probe development effort to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1 (GSTO1): gel-based activity-based protein profiling (ABPP) selectivity assay with endogenous enzyme.
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, gel-based ABPP, ABPP, activity-based protein profiling, SE-Rh, rhodamine, sulfonate ester, CA-Rh, chloroacetamide, recombinant, inhibition, counterscreen, selectivity, anti-target, fold selectivity, 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 whether compounds identified as active in the assays "Fluorescence polarization-based counterscreen for RBBP9 inhibitors: primary biochemical high throughput screening assay to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1)" (AID 1974) and "Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1)" (AID 2176) can inhibit endogenous GSTO1 in a complex proteomic lysate and to assess the extent of their anti-target reactivity. In this activity-based protein profiling (ABPP) assay, MDA-MB-435 soluble proteome is incubated with test compound followed by reaction with a rhodamine-conjugated sulfonate ester (SE-Rh) or rhodamine-conjugated alpha-chloroacetamide (CA-Rh) activity-based probe. 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 (IOD) of the bands. As designed, 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 band in the gel.
MDA-MB-435 soluble proteome (1 mg/mL in DPBS) was incubated with 0.1, 1, or 10 uM test compound (1 uL of a 50X stock in DMSO) for 30 minutes at 25 C. The proteome was then labeled with 10 uM SE-Rh (1 uL of a 50X stock in DMSO) or 5 uM CA-Rh (1 uL of a 50X stock in DMSO) for 1 hour at 25 C. The reaction was 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 GSTO1 or anti-target band relative to a DMSO-only (no compound) control. Anti-target bands at 70 kDa, 55 kDa, 5 2kDa, 49 kDa, 38 kDa, 34 kDa, 32 kDa, and 15 kDa were assessed.
% Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Test_Compound is defined as GSTO1 or anti-target treated with test compound.
High_Control is defined as GSTO1 or anti-target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
Anti-targets with > 50% inhibition were given the designation "INH" in the data table. Anti-targets with > two-fold activation were given the designation "ACT" in the data table. For anti-targets with <= 50% inhibition and <= two-fold activation there is a blank entry in the data table.
Fold selectivity is reported as:
[Conc_<=50%_INH_Anti-target] / [Conc_>50%_INH_GSTO1]
Conc_<=50%_INH_Anti-target is the test compound concentration at which less than or equal to 50% inhibition of the anti-target is observed.
Conc_>50%_INH_GSTO1 is the test compound concentration at which greater than 50% inhibition of GSTO1 is observed.
If the concentrations are both 1 uM, fold selectivity is reported as < 100. If the value for [Conc_<=50%_INH_Anti-target] is unknown, fold selectivity is not determined.
PubChem Activity Outcome and Score:
Compounds with >= 80% inhibition at 0.1 uM and no anti-targets were considered active. Compounds with < 80% inhibition at 0.1 uM and/or one or more anti-targets were considered inactive.
Active compounds were given a score of 100 and inactive compounds were given a score of 0.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
MDA-MB-435 soluble proteome (provided by Assay Provider)
DPBS (Cellgro 20-031-CV)
SE-Rh (provided by the Assay Provider)
CA-Rh (provided by the Assay Provider)
This assay was performed in the laboratory of the Assay Provider with powder or liquid 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.
Data Table (Concise)