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) percent inhibition assay with recombinant 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) percent inhibition assay with recombinant enzyme. ..more
BioActive Compounds: 37
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_1XINH_RECOMB
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) percent inhibition assay with recombinant 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, recombinant, inhibition, inhibitor, counterscreen, recombinant enzyme, 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 liquid samples of cherry-picked 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 recombinant GSTO1 in an activity-based protein profiling (ABPP) assay. In this assay, recombinant GSTO1 is incubated with test compound followed by reaction with a rhodamine-conjugated sulfonate ester (SE-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.
Recombinant GSTO1 (250 nM in 50 uL of DPBS) was incubated with 1 uM test compound (1 uL of a 50X stock in DMSO) for 30 minutes at 25 C followed by reaction 10 uM SE-Rh (1 uL of 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 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 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.
PubChem Activity Outcome and Score:
Compounds with >= 75% inhibition were considered active. Compounds with inhibition < 75% inhibition were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-79, and for inactive compounds 78-0.
List of Reagents:
Purified GSTO1 (provided by Assay Provider)
DPBS (Cellgro 20-031-CV)
SE-Rh (provided by the Assay Provider)
This assay was performed in the laboratory of the Assay Provider with liquid samples of cherry-picked compounds. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.
** Test Concentration.
Data Table (Concise)