Late stage assay provider results from the probe development effort to identify selective 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 selective inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity. ..more
BioActive Compounds: 3
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_1X%INH_SEL
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 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, 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, protein palmitoylation, counterscreen, inhibitor, inhibition, selectivity, anti-targets, activity-based protein profiling, ABPP, HEK293T cells, gel-based ABPP, fluorophosphonate peg rhodamine, FP-PEG-Rh, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
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 a competitive activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with the serine-hydrolase-specific rhodamine-conjugated fluorophosphonate (FP-PEG-Rh) activity-based probe. FP-PEG-Rh is similar to FP-Rh, but shows slower reaction kinetics, facilitating analysis of target inhibition by reversible compounds. 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 (50 uL of 1 mg/ml in DPBS) of HEK293T cells was treated with 300 nM, 1 uM, or 10 uM test compound (1 uL of a 50x stock in DMSO). Test compounds were incubated for 30 minutes at 37 C. FP-PEG-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 5 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 LYPLA1 and LYPLA2 bands relative to a DMSO-only (no compound) control. From among the 20+ other distinct bands visible, no anti-targets with greater than or equal to 50% inhibition were observed at any concentration tested.
%_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 reported as:
[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 target is observed.
If [Conc_>=50%_INH_target] is not determined, then Fold Selectivity is not determined.
If [Conc_<50%_INH_Anti-target] < [Conc_>=50%_INH_target], Fold Selectivity is 0.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with greater than or equal to 50% inhibition at 300 nM were considered active. Compounds with less than 50% inhibition at 300 nM were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition at 300 nM. Negative % inhibition values are reported as activity score zero.
LYPLA1: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
LYPLA2: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 25-0.
Overall Outcome and Score:
Compounds were considered active if they were active in only one of the panels. Compounds were considered inactive if they were inactive in both panels, or active in both panels.
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, and for inactive compounds 0-0.
List of Reagents:
Soluble proteome of HEK293T cells (provided by the Assay Provider)
FP-PEG-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.
Categorized Comment - additional comments and annotations
** Test Concentration. § Panel component ID.