Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds set 2
Name: Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds set 2. ..more
Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds
Late stage dose response counterscreen (T-cell cytotoxicity in quadruplicate)
Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in vitro, Set 2
Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess binding mode
Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11
Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds
Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situ
Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis in situ
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 CA132630-01 Grant Proposal PI: Benjamin Cravatt, TSRI External Assay ID: TCELLCYTOX_INH_ABSORB_4XCC50_SET2
Name: Late stage assay provider results from the probe development effort to identify inhibitors of LYPLA1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds set 2.
Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Numerous proteins have been identified as targets of palmitoylation, including cytoskeletal proteins, kinases, receptors, and other proteins involved in various aspects of cellular signaling and homeostasis (1). Using a global chemo-proteomic method for the metabolic incorporation and identification of palmitoylated proteins, we were able to identify hundreds of palmitoylated proteins, revealing palmitoylation as a widespread post-translational modification (PTM) (2). Palmitoylation involves an acyl-thioester linkage to specific cysteines (3,4). Given the labile properties of thioesters, palmitoylation is potentially reversible and may be regulated in a manner analogous to other PTMs (e.g., phosphorylation). As such, identification of proteins responsible for the dynamic modulation of palmitoylation is paramount to understanding its patho/physiological roles. For example, multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation (5), suggesting protein palmitoyl thioesterases may have tumor suppressor activity required to repress aberrant growth signaling. More than a decade ago, the cytosolic serine hydrolase acyl-protein thioesterase 1 (APT1) was identified as an in vitro HRAS palmitoyl thioesterase (6). Initially classified as lysophospholipase 1 (LYPLA1) (7), the enzyme has since been demonstrated to have several hundred-fold higher activity as a protein thioesterase. While the in vitro data (6,8) provided an intriguing clue to its possible role in vivo, prior to our studies, little was known about the in vivo thioesterase activity of LYPLA1. Upon retroviral shRNA knockdown of LYPLA1, we found that HRAS was robustly hyper-palmitoylated, providing the first evidence that the endogenous enzyme is a functional protein palmitoyl thioesterase capable of regulating HRAS palmitoylation in mammalian cells. However, shRNA resulted in only an 80% reduction in LYPLA1 expression (unpublished). LYPLA2 (a.k.a. APT2) is 65% identical to LYPLA1, and also exhibits lysophospholipase activity in vitro, but its potential role as a thioesterase is unknown (9). shRNA knockdown studies of LYPLA2 revealed only partial knockdown of the enzyme, making substrate identification inconclusive (unpublished). A principle goal of post-genomic research is the determination of the molecular and cellular role of uncharacterized enzymes like LYPLA1 and LYPLA2. As such, a dual inhibitor selective for both LYPLA1 and LYPLA2 would greatly aid investigations into the biological function of these related enzymes. Several inhibitors of LYPLA1 have been described (10,11), but none of these agents have proven capable of inhibiting LYPLA1 activity in cells, and no selective inhibitors of LYPLA2 have been reported to date. To comprehensively identify LYPLA1 and LYPLA2 substrates and functionally test the role of these enzymes in dynamic de-palmitoylation and tumorigenesis, development of a high affinity inhibitor, capable of achieving temporal and more complete control over activity, is critical. Alpha/beta hydrolase domain-containing protein 11 (ABHD11) is a poorly characterized serine hydrolase; all that is known about its biology is that it is a mitochondrial enzyme (12) with broad tissue distribution, has little sequence homology to other proteins, and its gene is located in a region of chromosome 7 that is hemizygously deleted in Williams-Beuren syndrome, a rare genetic disease with symptoms that include vascular stenosis, mental retardation, and excessive sociability (13).
1. Smotrys, J.E. and Linder, M.E. PALMITOYLATION OF INTRACELLULAR SIGNALING PROTEINS: Regulation and Function. Annual Review of Biochemistry, 2004. 73: 559-587. 2. Martin, B.R. and Cravatt, B.F. Large-scale profiling of protein palmitoylation in mammalian cells. Nat Methods, 2009. 6: 135-138. 3. Magee, A.I., Koyama, A.H., Malfer, C., Wen, D. and Schlesinger, M. J. Release of fatty acids from virus glycoproteins by hydroxylamine. Biochimica et Biophysica Acta (BBA) - General Subjects, 1984. 798: 156-166. 4. Rose, J.K., Adams, G.A. and Gallione, C.J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci USA, 1984. 81: 2050-2054. 5. Willumsen, B.M., Cox, A.D., Solski, P.A., Der, C.J. and Buss, J.E. Novel determinants of H-Ras plasma membrane localization and transformation. Oncogene, 1996. 13: 1901-1909. 6. Duncan, J.A. and Gilman, A.G. A Cytoplasmic Acyl-Protein Thioesterase That Removes Palmitate from G Protein alpha Subunits and p21RAS. J Biol Chem, 1998. 273: 15830-15837. 7. Sugimoto, H., Hayashi, H. & Yamashita, S. Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. J Biol Chem, 1996. 271: 7705-7711. 8. Hirano, T. et al. Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2009. 1791: 797-805. 9. Toyoda, T., Sugimoto, H. and Yamashita, S. Sequence, expression in Escherichia coli, and characterization of lysophospholipase II. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1999. 1437: 182-193. 10. Biel, M., Deck, P., Giannis, A. and Waldmann, H. Synthesis and Evaluation of Acyl Protein Thioesterase 1 (APT1) Inhibitors. Chemistry - A European Journal, 2006. 12: 4121-4143. 11. Deck, P. et al. Development and Biological Evaluation of Acyl Protein Thioesterase 1 (APT1) Inhibitors. Angewandte Chemie International Edition, 2005. 44: 4975-4980. 12. Forner, F., et al., Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol. Cell. Proteomics, 2006. 5(4): p. 608-19. 13. Schubert, C., The genomic basis of the Williams-Beuren syndrome. Cell. Mol. Life Sci., 2009. 66(7): p. 1178-97.
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The purpose of this assay is to determine cytotoxicity of inhibitor compounds belonging to the urea triazole scaffold. In this assay, BW5147-derived murine T-cells in either serum-free media (Assay 1) or media containing FCS (Assay 2) are incubated with test compounds, followed by determination of cell viability. The assay utilizes the WST-1 substrate that is converted into colorimetric formazan dye by the metabolic activity of viable cells. The amount of formed formazan directly correlates to the number of metabolically active cells in the culture. As designed, compounds that reduce cell viability will result in decreased absorbance of the dye. Compounds were tested in quadruplicate in a 7-point 1:5 dilution series starting at a nominal test concentration of 50 uM.
This assay was started by dispensing BW5147-derived murine T cells in RPMI media (100uL, 10E4 cells/well) into a 96-well plate. Both serum-free media (Assay 1) and media supplemented with fetal calf serum (FCS) (Assay 2) were tested. Compound (5 uL of 0-1000 uM in media containing 5% DMSO) was added to each well, giving final compound concentrations of 0-50 uM. Cells were incubated for 48 hours at 37 C in a humidified incubator and cell viability was determined by the WST-1 assay (Roche) according to manufacturer instructions.
The % cell viability for each well was then calculated as follows:
Test_Compound is defined as wells containing cells in the presence of test compound. High_Control is defined as wells containing cells treated with media only (no compound). Low_Control is defined as wells containing no cells (media only).
For each test compound, percent cell viability was plotted against the log of the compound concentration. The CC50 is reported as ">X uM" (where X = the highest concentration tested for which > 50% cell survival was observed).
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with a CC50 value of less than 5 uM were considered active (cytotoxic). Compounds with a CC50 value greater than or equal to 5 uM were considered inactive (non-cytotoxic).
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
Assay 1 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
Assay 2 Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
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 inactive compounds is 0-0. There are no active compounds.
List of Reagents:
BW5147-derived murine T cells (provided by Assay Provider) RPMI Media (CellGro 10-040-CV) FCS (Omega Scientific, FB-01) WST-1 reagent (Roche) 96-well plates (Corning)