Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in vivo
Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in vivo. ..more
BioActive Compounds: 2
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&2_INH_FLUO_ABPP_INVIVO
Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP evaluation of activity in vivo.
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, LYPLA2, lysophospholipase 2, APT1, acyl-protein thioesterase 1, APT2, acyl-protein thioesterase 2, serine hydrolase, palmitoylation, counterscreen, inhibitor, inhibition, selectivity, anti-targets, activity-based protein profiling, ABPP, gel-based ABPP, click chemistry, azide-alkyne cycloaddition, Rh-N3, rhodamine-azide tag, CC-ABPP, in vivo, C57 mice, mice, NHU5, inhibitor, inhibition, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit LYPLA1 and LYPLA2 in vivo. In this assay, test compounds are administered to mice followed by the in vivo active 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 in vivo active serine hydrolase-specific ABPP probes (e.g., FP-alkyne), facilitating analysis of target inhibition by reversible compounds. Mice are sacrificed, and their tissues harvested, homogenized, and the soluble fraction isolated and reacted with a rhodamine-functionalized azide tag under copper(I)-catalyzed azide-alkyne cycloaddition ("click chemistry") reaction conditions to allow visualization of probe-tagged proteins. 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 or LYPLA2 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound, fluorescent-tagged probe, giving lower fluorescence intensity in the band in the gel.
Test compounds were prepared as a homogeneous PEG solution by vortexing and sonicating neat compound directly into PEG300. Purpose-bred C57-black laboratory mice (< 6 months old, 20-28 g) were administered i.p. with test compound (50 mg/kg; 4uL/g) or vehicle only and, after 3 hours, with NHU5 probe (PEG solution; 100 mg/kg). After 1 hour, mice were sacrificed, and tissues were removed and Dounce-homogenized directly in methanol/chloroform/dH2O (4:1:3 v/v). After centrifugation at 13,000 x g for 3 minutes, the upper layer was removed and 3 volumes of methanol were added and 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. For visualization of probe-labeled proteins, click chemistry with a rhodamine-azide tag (Rh-N3; 100 uM) was carried out under standard conditions (1 mM TCEP, 100 uM TBTA ligand, 1 mM Cu(II)sulfate) to append the rhodamine fluorophore to the alkyne-functionalized NHU5 probe. Reactions were 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 LYPLA 1 and LYPLA2 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.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with greater than or equal to 50% inhibition in one or more tissues tested were considered active. Compounds with less than 50% inhibition in all tissue tested were considered inactive.
Overall Outcome and Score:
Compounds were considered active if they were active in either panel. Compounds were considered inactive if they were inactive in both assays.
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:
C57-black laboratory mice (provided by Assay Provider)
PEG300 (Fluka, 90878)
DPBS (Cellgro 20-031-CV)
NHU5 probe (provided by the Assay Provider)
Rh-N3 (Invitrogen T10182)
TCEP (Sigma-Aldrich 93284)
TBTA Ligand (Sigma-Aldrich 678937)
Copper (II) sulfate (Sigma-Aldrich 451657)
This assay was performed by the assay provider with powder samples of synthetic compounds.
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