Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: LC-MS/MS assay to assess binding of compounds to active site
Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: LC-MS/MS assay to assess binding of compounds to active site. ..more
BioActive Compounds: 3
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_LCMS
Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: LC-MS/MS assay to assess binding of compounds to active site.
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, liquid chromatography-tandem mass spectrometry, LC-MS/MS, active site, trypsin, 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 assess the binding mode of inhibitor compounds belonging to the alpha chloroacetamide scaffold and determine whether or not they covalently label the active site cysteine of GSTO1. In this assay, purified enzyme is reacted with inhibitor compounds, digested with trypsin, and the resulting peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting data are analyzed to identify sites of covalent labeling.
Four aliquots (25 uL) of 50 uM GSTO1 in DPBS were prepared. Three inhibitor compounds (1 uL of 250 mM in DMSO) were added to three separate aliquots, giving a final concentration of 10 uM. To the fourth (control) aliquot was added DMSO (1 uL). Reactions were gently vortexed and incubated at room temperature for 30 min. To each reaction was added solid urea (25 mg), followed by freshly prepared aqueous ammonium bicarbonate (5 uL of 250 mM), and Millipore water to bring to total volume to 50 uL, giving final concentrations of 8 M urea and 25 mM ammonium bicarbonate. The reactions were vortexed until the urea was dissolved. To each reaction was added freshly prepared DTT (0.5 uL of 1 M in water, 10 mM [final]), and the reactions were incubated at 65 C for 15 min. To each reaction was then added freshly prepared IAA (4 uL of 0.5 mM in water, giving 20 mM [final]), and the reactions were incubated for 30 min at room temperature in the dark. Aqueous ammonium bicarbonate (140 uL of 25 mM) was added to reduce the urea concentration to 2 M. To each reaction was added sequencing grade modified trypsin (1 ug), and reactions were incubated at 37 C for 4 hrs. The digests were acidified with formic acid (10 uL, giving 5% [final]). An Agilent 1200 series quaternary HPLC pump/autosampler system and Thermo Scientific Orbitrap Velos mass spectrometer were used for sample analysis. A fraction (15 uL) of the protein digest for each sample was autosampler-loaded onto a 100 micron fused-silica column (with a 5 micron in-house pulled tip) packed with 12 cm of Aqua C18 reversed-phase packing material. Chromatography was carried out using an increasing gradient of aqueous acetonitrile containing 0.1% formic acid over 125 minutes. Mass spectra were acquired in a data-dependent mode with dynamic exclusion and charge state screening (for rejection of unassigned and +1 charge states) enabled.
The MS/MS spectra generated for each run were searched against a human protein database concatenated to a reversed decoy database using Sequest. For cysteine, both a static modification of +57.021 (for IAA alkylation) and variable modifications to account for possible inhibitor labeling (SID 92709043: 310.148; SID 92709034: 355.134; SID 92709100: 274.057) were specified. The resulting peptide identifications were assembled into protein identifications using DTASelect, and filters were adjusted to maintain a false discovery rate (as determined by number of hits against the reversed database) of <1%. Any modified peptides identified in the DMSO-treated sample were discarded as spurious hits.
PubChem Activity Outcome and Score:
The PubChem Activity Score is assigned a value of 100 for active compounds.
Compounds observed to modify the GSTO1 active site cysteine are considered active. Compounds for which no covalent modification was observed are considered inactive.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
GSTO1 protein (provided by Assay Provider)
DPBS (CellGro 21-030-CV)
Urea (Fisher U15-3)
Ammonium bicarbonate (Acros, AC37093-0010)
DTT (dithiothreitol; Sigma 43815)
IAA (iodoacetamide; Sigma I1149)
Trypsin (Promega V5111)
TFA (trifluoroacetic acid; SigmaAldrich 302031)
Fused-silica (Agilent 160-2635-10)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Formic acid (Fluka 06440)
This assay was performed by the assay provider with compounds ordered as powders. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDS listed in the Related Bioassays section of this AID.
Data Table (Concise)