Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity
Name: Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity. ..more
BioActive Compounds: 7
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_FLUO_GEL_1XINH_SEL
Name: Late stage assay provider results from the probe development effort to identify dual inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity.
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.
late stage, late stage AID, assay provider, powders, LYPLA1, lysophospholipase 1, LYPLA2, lysophospholipase 2, APT1, acyl-protein thioesterase 1, APT2, acyl-protein thioesterase 2, serine hydrolase, palmitoylation, counterscreen, inhibitor, selectivity, anti-targets, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Assays (see Protocol for details)
§ Panel component ID.
The purpose of this assay is to determine whether powder samples of test compounds can inhibit LYPLA1 and LYPLA2 in a complex proteomic lysate and to estimate compound selectivity in an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as LYPLA1 and/or LYPLA2 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.
Soluble proteome (1 mg/ml in DPBS) of BW5147-derived murine T-cells was treated with 30 nM, 200 nM, or 1.5 uM test compound (1 uL of a 50x stock in DMSO). Test compounds were incubated for 30 minutes at 25 C (50 uL reaction volume). FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM. The reaction was incubated for 30 minutes at 25 C, quenched with 2x SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the target (LYPLA1 and LYPLA2) and anti-target (alpha/beta hydrolase domain-containing protein 11 (ABHD11), esterase D/formylglutathione hydrolase (ESD), and N-acylaminoacyl-peptide hydrolase (APEH)) bands relative to a DMSO-only (no compound) control.
% Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Test_Compound is defined as target or anti-target treated with test compound.
High_Control is defined as target or anti-target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
Fold selectivity is > [Conc_<50%_INH_Anti-target] / [Conc_>=50%_INH_target]
[Conc_<50%_INH_Anti-target] is the test compound concentration at which less than 50% inhibition of the anti-target is observed.
[Conc_>=50%_INH_target] is the test compound concentration at which greater than or equal to 50% inhibition of LYPLA1 and LYPLA2 is observed.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with greater than or equal to 50% inhibition at 0.2 uM were considered active. Compounds with less than 50% inhibition at 0.2 uM were considered inactive.
The reported PubChem Activity Score has been normalized to 100% of the observed value at 0.2 uM.
LYPLA1 Score: The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 0-0.
LYPLA2 Score: The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 0-0.
ABDHD11 Score: The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 0-0.
ESD Score: The PubChem Activity Score range for active compounds is 100-66, and for inactive compounds 0-0.
APEH Score: The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 0-0.
Overall Outcome and Score:
Compounds that were active for inhibition of LYPLA1 and LYPLA2 and inactive for inhibition of ESD and APEH were assigned an overall outcome of active, regardless of the ADHD11 inhibition assay outcome. Compounds that were inactive for LYPLA1 and/or LYPLA2 were assigned a overall outcome of inactive. Compounds that were active for inhibition of LYPLA1 and LYPLA2 and active for inhibition of ESD and/or APEH were assigned a overall outcome of inactive.
The overall 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, and for inactive compounds 0-0.
List of Reagents:
Soluble proteome of BW5147-derived murine T-cells
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
This assay was performed by the assay provider with powder samples of synthetic compounds.
** Test Concentration. § Panel component ID.