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
Name: 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. ..more
BioActive Compound: 1
Depositor Specified Assays
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
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: LYPLA1_INH_LCMS_ABPP_SILAC_INSITU
Name: 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.
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, selective inhibitors of LYPLA1 or LYPLA2 would greatly aid investigations into the biological function of these 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 high affinity inhibitors, capable of achieving temporal and more complete control over activity, is critical.
1. Dekker, F.J., et al., Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol., 2010. 6(6): p. 449-56.
2. Duncan, J.A. and A.G. Gilman, A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J. Biol. Chem., 1998. 273(25): p. 15830-7.
3. Sugimoto, H., H. Hayashi, and S. Yamashita, Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. J. Biol. Chem., 1996. 271(13): p. 7705-11.
4. Toyoda, T., H. Sugimoto, and S. Yamashita, Sequence, expression in Escherichia coli, and characterization of lysophospholipase II. Biochim. Biophys. Acta, 1999. 1437(2): p. 182-93.
5. Biel, M., et al., Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Chemistry, 2006. 12(15): p. 4121-43.
6. Deck, P., et al., Development and biological evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Angew. Chem. Int. Ed. Engl., 2005. 44(31): p. 4975-80.
7. Jessani, N., et al., Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. U. S. A., 2002. 99(16): p. 10335-40.
8. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol., 2003. 21(6): p. 687-91.
9. Bachovchin, D.A., et al., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat. Biotechnol., 2009. 27(4): p. 387-94.
10. Forner, F., et al., Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol. Cell. Proteomics, 2006. 5(4): p. 608-19.
11. Schubert, C., The genomic basis of the Williams-Beuren syndrome. Cell. Mol. Life Sci., 2009. 66(7): p. 1178-97.
late stage, late stage AID, assay provider, powders, LYPLA1, lysophospholipase 1, APT1, acyl-protein thioesterase 1, serine hydrolase, palmitoylation, protein palmitoylation, counterscreen, inhibitor, inhibition, selectivity, anti-targets, liquid chromatography, LC, tandem mass spectrometry, MS/MS, activity-based protein profiling, ABPP, stable isotope labeling with amino acids in cell culture, SILAC, ABPP-SILAC, inhibitor, inhibition, fluorophosphonate-peg-biotin, FP-PEG-biotin, BW5147-derived murine T-cell hybridoma cells, BW5147, murine T-cell hybridoma, in situ, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
§ Panel component ID.
The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using stable isotope labeling with amino acids in cell culture (SILAC) ABPP. In this assay, cultured HEK293T cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with test compound and DMSO, respectively, in situ, followed by the cell-permeable alkyne-functionalized triazole urea ABPP probe NHU5. NHU5 covalently reacts with a subset of serine hydrolases, including LYPLA1 and LYPLA2, and shows slower reaction kinetics than other cell-permeable serine hydrolase-specific ABPP probes (e.g., FP-alkyne), facilitating analysis of target inhibition by reversible compounds. Cells are harvested, proteome fractions isolated and reacted with a biotin-functionalized azide tag under copper(I)-catalyzed azide-alkyne cycloaddition ("click chemistry") reaction conditions for enrichment of probe-tagged proteins. Light and heavy samples are combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block NHU5 probe labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, resulting in a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio close to 1.
HEK293T cells were initially grown for 6 passages in either light or heavy SILAC DMEM medium supplemented with 10% dialyzed FCS and 1x PenStrep Glutamine in a humidified incubator (37 C, 5% CO2). Light medium was supplemented with 100 ug/mL L-arginine (Sigma) and 100 ug/mL L-lysine (Sigma). Heavy medium was supplemented with 100 ug/mL [(isotope-13)C6(isotope-15)N4]-L-Arginine (Isotek) and 100 ug/mL [(isotope-13)C6(isotope-15)N2]-L-Lysine (Isotek). Light cells were treated with test compound (5 uM) and heavy cells were treated with DMSO for 3 hours at 37 C. Activity-based alkyne probe NHU5 (50 uM) was added, and cells incubated for 1 hour at 37 C. After 1 hour, cells were washed (4x DPBS), harvested, isolated by centrifugation at 1,400 x g for 3 minutes, re-suspended in methanol/chloroform/dH2O (4:1:3 v/v), and vortexed. After centrifugation at 13,000 x g for 3 minutes, the upper layer was removed and 3 volumes of methanol were added and the solution vortexed. Protein was pelleted by centrifugation at 13,000 x g for 6 minutes. The supernatant was removed and the pellet was air-dried and re-suspended in PBS by sonication. Light and heavy proteomes were mixed in 1:1 (w/w) ratio to generate one membrane and one soluble sample. For affinity purification of probe-labeled proteins, click chemistry with a biotin-azide tag (biotin-N3; 100 uM) was carried out under standard conditions (1 mM TCEP, 100 uM TBTA ligand, 1 mM Cu(II)sulfate, 1 hour, 25 C), and excess reagents removed by washing the precipitated protein with cold methanol (3 x 750 uL). The protein pellets were resolubilized in 2.5% SDS/DPBS (750 uL) by sonication and heating (60 C, 5 minutes). The concentration of SDS was reduced to 0.5% with DPBS (3 mL), and biotinylated proteins enriched with streptavidin beads (50 uL, 1 hour, 25 C). The beads were washed with 1% SDS in DPBS (1x), 6 M urea (1x), and DPBS (2x), then resuspended in in 6 M urea (150 uL), reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25 C in the dark, and urea concentration was reduced to 2 M with 2x volume DPBS. On-bead digestions were performed for 12 hours at 37 C with sequence-grade modified trypsin (Promega; 2 ug) in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid and stored at -80 C prior to analysis.
Samples were analyzed by multidimensional liquid chromatography tandem mass spectrometry (MudPIT) using an Agilent 1100-series quaternary pump and Thermo Scientific LTQ Orbitrap ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (60 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 7 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.1; publicly available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the Sequest algorithm against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. Sequest searches allowed for static modification of cysteine residues (+57.02146 due to alkylation), methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, +8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the --modstat, --mass, and --trypstat options (applies different statistical models for the analysis of high resolution masses, peptide digestion state, and methionine oxidation state respectively). Ratios of light/heavy peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were >3 standard deviations from the median peptide value. Proteins with less than three peptides per protein ID were not included in the analysis.
Ratio = Average( AUC_light / AUC_heavy ) calculated for all unique peptides
AUC_light is the area-under-the-curve for the light peptide pair from cells treated with test compound.
AUC_heavy is the area-under-the-curve for the heavy peptide pair from cells treated with DMSO.
PubChem Activity Score and Outcome:
The following applies to each panel in this assay:
A compound was considered active for a particular target/anti-target with a light/heavy ratio of less than or equal to 0.5. A compound was considered inactive for a specified target/anti-target with a light/heavy ratio of greater than 0.5.
Overall Outcome and Score:
A compound was considered active if it was active for LYPLA1 and inactive for all other serine hydrolases tested, otherwise inactive.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
HEK293T cells (provided by Assay Provider)
SILAC DMEM Medium (Thermo 89985)
dialyzed FCS (Gemini 100-108)
1x PenStrep Glutamine (CellGro 30-002-CI)
L-Arginine (Sigma A6969)
L-Lysine (Sigma L9037)
[(isotope-13)C6(isotope-15)N4]-L-Arginine (Sigma 608033)
[(isotope-13)C6(isotope-15)N2]-L-Lysine (Sigma 608041)
DPBS (Cellgro 20-031-CV)
FP-PEG-biotin (provided by Assay Provider)
PD-10 desalting columns (GE Healthcare 17-0851-01)
SDS (Sigma L6026)
Urea (Fisher U15-3)
DTT (Sigma 43815)
Trypsin (Promega V5111)
CaCl2 (Sigma C1016)
streptavidin beads (Pierce 20349)
Fused-silica (Agilent 160-2635-10)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Formic acid (Fluka 06440)
Triton-X100 (Fisher AC21568-0010)
This assay was performed by the assay provider with powder samples of synthetic compounds.
** Test Concentration. § Panel component ID.