Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPP in situ
Name: Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPP in situ. ..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
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: LYPLA1&2_INH_FLUO_3XIC50_IN_SITU_GEL_BASED_ABPP
Name: Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical dose-response gel-based ABPP 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 . 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) . 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 , 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 . Initially classified as lysophospholipase 1 (LYPLA1) , 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 . 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 and/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. We recently reported optimization of a covalent dual LYPLA1/LYPLA2 inhibitor ML211 (SID 99445338), and identification of reversible compounds ML348 (SID 160654487) and ML349 (SID 160654496) that act as selective LYPLA1 and LYPLA2 inhibitors, respectively (see Probe Reports). These compounds represent an important advance in chemical tools for investigating the biological function(s) of LYPLA1 and LYPLA2. However, ML211 (SID 99445338), owing to its modest solubility, does not have demonstrated in vivo activity. Irreversible chemical probes have distinct advantages vs. reversible compounds for use in living systems, as confirming target engagement is a comparatively straightforward process, and near-complete and sustained target inhibition can be achieved without extensive optimization of physicochemical and pharmacokinetic properties. The goal of this current investigation is to develop an irreversible, in vivo-active dual LYPLA1/LYPLA2 inhibitor to add to the arsenal of available chemical tools for identification of LYPLA1 and LYPLA2 substrates and evaluating the role of these enzymes in dynamic de-palmitoylation and tumorigenesis.
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, dose response, IC50, HeLa, cell-based, 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
The purpose of this assay is to determine IC50 values of powder samples of test compounds for LYPLA1 (Assay 1) and LYPLA2 (Assay 2) in situ using an activity-based proteomic profiling (ABPP) assay. In this assay, cultured cells are treated with test compound, harvested, lysed, and the soluble fraction is reacted with a serine hydrolase-specific 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. Compounds are tested in triplicate in a 6-point dilution series starting at a nominal concentration of 20000 nM.
To cultured HeLa cells (90% confluent) was added fresh DMEM (5 mL total volume; supplemented with 10% FCS) pre-mixed with DMSO or test compound (5 uL of a 1000x stock in DMSO; 20000 nM, 1000 nM, 250 nM, 50 nM, 1 nM, or 0.1 nM final concentration). After 2 hours at 37 degrees Celsius, cells were harvested by scraping, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 16 uL 4x SDS-PAGE loading buffer (reducing), 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 bands relative to a DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration.
%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Test_Compound is defined as target treated with test compound.
High_Control is defined as target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
For each test compound, percent inhibition was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported. In the event that the highest test concentration did not result in at least 50% inhibition, the IC50 value is reported as being greater than the highest test concentration.
Compounds with an IC50 less than or equal to 1 uM were considered active. Compounds with an IC50 greater than 1 uM were considered inactive.
PubChem Activity Outcome:
Compounds were considered active if they were active in both assays. Compounds were considered inactive if they were inactive in one or both assays.
List of Reagents:
HeLa cells (provided by the Assay Provider)
DMEM Medium (CellGro 10-017-CV)
FCS (Omega Scientific, FB-01)
1x PenStrep Glutamine (CellGro 30-002-CI)
FP-Rh (Thermo #88318)
DPBS (Cellgro 20-031-CV)
This assay was performed by the assay provider with powder samples of synthetic test compounds
* Activity Concentration. ** Test Concentration. § Panel component ID.