Summary of probe development efforts to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).
Name: Summary of probe development efforts to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1). ..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 Fast Track
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: GST01_INH_SUMMARY
Name: Summary of probe development efforts to identify inhibitors of the oxidoreductase glutathione S-transferase omega 1(GSTO1).
Glutathione S-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 (1). GSTO1-1 was initially identified as a putative glutathione transferase by primary sequence alignment to known GSTs, although relatively distantly related with only ~20% sequence identity (2). The biochemical characterization and crystal structure of GSTO1-1 revealed that this enzyme was the founding member of a seventh family of glutathione transferases (humans have two omega-class enzymes) (2, 3). Unlike other GSTs, which possess a catalytic tyrosine or serine residue, GSTO1-1 has a catalytic cysteine residue in this conserved position. Consistent with this unusual feature, GSTO1-1 has unique activities in addition to glutathione transferase activity, including both dehydroascorbate and monomethylarsonate reductase activity not usually ascribed to glutathione transferases.
GSTO1-1 overexpression has been linked to cancer (4-6). High levels of GSTO1-1 were discovered in a drug resistant murine cancer leukemia cell (4) and in cells resistant to platinum-based chemotherapeutic agents (6). Using activity-based protein profiling (ABPP), it was discovered that activity of GSTO1-1 was correlated to invasiveness of human breast cancer cell lines (5). In addition, GSTO1-1 has been linked to the age at onset of both Alzheimer's and Parkinson's diseases (7). As GSTO1-1 has a reactive catalytic cysteine, it is sensitive to thiol alkylating agents, including N-ethylmaleimide (3). However, no selective chemical probe exists that would aid biological investigation of this cancer-related enzyme. Such a probe would be useful to evaluate the endogenous substrates of GSTO1-1 as well as its potential as an anticancer target, and therefore GSTO1-1 would benefit from a high-throughput screen for chemical inhibitors. In addition, our lab has determined that GSTO1-1 has an unusually reactive cysteine. A screen would also be a useful tool to survey the biologically reactive electrophilic species in the screening library.
Summary of Probe Development Effort:
This probe development effort is focused on the identification of GSTO1 inhibitors (AID 1974). This target also serves as a counterscreen for RBBP9 inhibitors (AID 1515). In the primary uHTS assay, compounds were screened by FluoPol-ABPP with a rhodamine (Rh)-conjugated sulfonate ester (SE) activity-based probe (SE-Rh) using fluorescence polarization readout (8). For the confirmation uHTS screen, 2,374 active compounds were retested in triplicate, and 1,286 compounds were confirmed as active. The first secondary assay assessed whether or not test compounds could inhibit recombinant GSTO1 using a gel-based competitive activity-based protein profiling (ABPP) assay with the SE-Rh activity-based probe. The next gel-based competitive-ABPP secondary assay was designed to assess whether or not compounds could inhibit endogenous GSTO1 in the context of a complex proteome. A third round of gel-based competitive-ABPP secondary screening involved assessment of potency in the complex proteome and an assessment of anti-target selectivity in the complex proteome using both SE-Rh and rhodamine-conjugated alpha-chloroacetamide (CA-Rh) activity-based probes. Because the two probes have different target enzymes, the search for anti-targets (> 30 ABPP probe-reactive enzymes) outside the standard profile of SE-Rh was expanded by using both. A final round of gel-based competitive-ABPP secondary screening aimed to determine whether or not compounds could inhibit GSTO1 in situ. Of the original 126 compounds subjected to secondary screening, compound SID 92709100 was identified as a highly potent and selective covalent inhibitor of GSTO1. It has an IC50 of 28 nM, and greater than 350-fold selectivity against potential anti-targets as assessed by competitive ABPP profiling. It has also been shown to be active in live cells, completely inhibiting GSTO1 at 250 uM concentration.
All AIDs that contain results associated with this project can be found in the "Related Bioassays" section of this Summary AID. A probe report has been submitted. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports. A probe report for SID 92709100 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML175.
1. Hayes, J.D., Flanagan, J.U., and Jowsey, I.R., Glutathione transferases. Annu. Rev. Parmacol. Toxicol., 2005. 45: p. 51-88.
2. Board, P.G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L.S., Schulte, G.K., Danley, D.E., Hoth, L.R., Griffor, M.C., Kamath, A.V., Rosner, M.H., Chrunyk, B.A., Perregaux, D.E., Gabel, C.A., Geoghegan, K.F., and Pandit, J., Identification, Characterization, and Crystal Structure of the Omega Class Glutathione Transferases. The Journal of Biological Chemistry, 2000. 275(32): p. 24798-24806.
3. Whitbread, A.K., Masoumi, A., Tetlow, N., Schmuck, E., Coggan, M., and Board, P.G., Characterization of the Omega Class of Glutathione Transferases. Methods in Enzymology, 2005. 401: p. 78-99.
4. Kodym, R., Calkins, P., and Story, M., The cloning and characterization of a new stress response protein: A mammalian member of a family of theta class glutathione S-transferase-like proteins. Journal of Biological Chemistry, 1999. 274: p. 5131-5137.
5. Adam, G., Sorensen, E.J., and Cravatt, B.F., Proteomic Profiling of mechanistically distinct enzyme classes using a common chemotype. Nature Biotechnology, 2002. 20: p. 805-809.
6. Giri, U., Terry, N.H., Kala, S.V., Lieberman, M.W., and Story, M., Elimination of the differential chemoresistance between the murine B-cell lymphoma LY-ar and LY-as cel lines after arsenic (As2O3) exposure via the overexpression of GSTO1 (p28). Cancer Chemother. Pharmacol., 2005. 55: p. 511-521.
7. Li, Y.J., Oliveira, S.A., u, P., Martin, E.R., Stenger, J.E., Scherzer, C.R., Hauser, M.A., Scott, W.K., Small, G.W., and Pericak, V., M. A., Glutathione S-transferase omega-1 modifies age-at-onset of Alzheimer disease and Parkinson disease. Hum. Mol. Genet., 2003. 12: p. 3259-3267.
8. 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: p. 387-394.
Summary, Summary AID, GSTO1, oxidoreductase, glutathione S-transferase omega 1, RBBP9, retinoblastoma binding protein 9, Fam 108B, BOG, cell cycle, cancer, fluorescence polarization, sulfonate ester, antagonist, inhibitor, primary, counterscreen, confirmation, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs.
Data Table (Concise)