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 2
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 2. ..more
BioActive Compounds: 2
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_ASSAY2
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 2.
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, fluorescence, SE-Rh, rhodamine, sulfonate ester, Rh-N3, azide, alkyne, click chemistry, soluble proteome, 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 compare inhibition of GSTO1 by an inhibitor compound in a competitive ABPP assay (Assay 1) to inhibition by the same compound in a direct ABPP assay using click chemistry (Assay 2). In Assay 1, MDA-MB-435 soluble proteome was incubated with test compound and then 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 to the alkyne analog test compounds. For both assays, the reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. 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 GSTO1 relative to the highest test concentration; 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 soluble proteome (1 mg/mL in DPBS) is treated with 1 uM, 250 nM, 63 nM or 16 nM test compound (1 uL of 50X stock in DMSO) for 30 minutes at 25 C. 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, separate proteome sample aliquots are reacted with a rhodamine-azide tag (Rh-N3; 5 0uM) under click chemistry reaction conditions (1 mM TCEP, 100 uM TBTA ligand, 1mM Cu(II)sulfate). Compound N03 was not tested in Assay 2 because Assay 2 is not applicable to non-alkyne compounds. For both assays, after addition of reagents, the reaction is incubated for 1hr 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 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 Assay 2, the IOD of the GSTO1 band for the lower concentrations (250 nM, 63 nM and 16 nM) was calculated relative to IOD of GSTO1 for the highest concentration (1 uM) of test compound.
Relative Band Intensity = ( IOD_GSTO1 - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control )
GSTO1 is the target band.
High_Control is defined as corresponding signal at 1 uM.
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with value >= 80% at 63 nM were considered active. Compounds with a value < 80% at 63 nM were considered inactive.
The reported PubChem Activity Score has been normalized to 100% of the observed value at 63 nM.
Assay 1 Score: The PubChem Activity Score range for active compounds is 100-89, and for inactive compounds 22-22.
Assay 2 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 42-42.
Overall Outcome and Score:
The overall outcome was active if the compound was active in at least one panel, inactive otherwise.
The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.
The PubChem Activity Score range for active compounds is 100-50, 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)
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.