Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: 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 dual inhibitors of LYPLA1 and LYPLA2: LC-MS/MS assay to assess binding of compounds to active site. ..more
BioActive Compound: 1
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: LYPLA1_INH_LCMS
Name: Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: LC-MS/MS assay to assess binding of compounds to active site.
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.
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.
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The purpose of this assay is to assess the covalent nature of an inhibitor compound belonging to the urea triazole scaffold and determine whether or not it labels the active site serine of LYPLA1. In this assay, purified enzyme is reacted with inhibitor compound, 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.
Two aliquots (25 uL) of 50 uM LYPLA1 were prepared. To one aliquot was added inhibitor (0.5 uL of 10 mM in DMSO), giving a final concentration of 200 uM. To the second (control) aliquot was added DMSO (0.5 uL). Reactions were gently vortexed and incubated at room temperature for 30 minutes. To each reaction was added solid urea (50 mg), followed by freshly prepared aqueous ammonium bicarbonate (75 uL of 25 mM). The reactions were vortexed until the urea was dissolved. Final urea concentration was approximately 8 M. To each reaction was added freshly prepared TCEP (5 uL of 100 mM in water), and the reactions were incubated at 30 C for 30 minutes. To each reaction was then added freshly prepared IAA (10 uL of 100 mM in water), and the reactions were incubated for 30 min at room temperature in the dark. Aqueous ammonium bicarbonate (375 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 12 hours. Formic acid was added to 5% (v/v) final.
An Agilent 1200 series quaternary HPLC pump and Thermo Scientific LTQ-Orbitrap mass spectrometer were used for sample analysis. A fraction (10 uL) of the protein digest for each sample was pressure-loaded onto a 100 micron fused-silica column (with a 5 micron in-house pulled tip) packed with 10 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 enabled.
The MS/MS spectra generated for each run were searched against a human protein database concatenated to a reversed decoy database using Sequest. A static modification of +57.021 was specified cysteine, and a variable modification of +167.131 was specified for serine to account for possible probe labeling by AA64-2. 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:
Compounds observed to covalently modify the active site serine of LYPLA1 were considered active. Compounds for which no covalent modification was observed were considered inactive.
The PubChem Activity Score is assigned a value of 100 for active compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
LYPLA1 protein (provided by Assay Provider)
DPBS (CellGro 21-030-CV)
Urea (Fisher U15-3)
Ammonium bicarbonate (Acros, AC37093-0010)
TCEP (Tris[2-carboxyethyl]phosphine hydrochloride; Sigma C4706)
IAA (iodoacetamide; Sigma I1149)
Trypsin (Promega V5111)
Fused-silica (Agilent 160-2635-10)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Formic acid (Fluka 06440)
This assay was performed in the laboratory of the Assay Provider with powder samples of compounds.
Data Table (Concise)