Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess inhibitor binding mode
Name: Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess inhibitor binding mode. ..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_GELBASEDABPP_BINDINGMODE
Name: Late stage assay provider results from the extended probe development effort to identify inhibitors of LYPLA1 and LYPLA2: Fluorescence-based biochemical gel-based ABPP gel filtration assay to assess inhibitor binding mode.
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 on the NIH Bookshelf at http://www.ncbi.nlm.nih.gov/books/NBK47352/). 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, activity-based protein profiling, cell lysate, proteome, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, gel filtration, reversible, irreversible, binding mode, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Assay with Multiple Targets/Outcomes
§ Panel component ID.
The purpose of this assay is to determine whether powder samples of test compounds inhibit LYPLA1 and LYPLA2 in a reversible or irreversible manner. In this assay, a complex proteome is incubated with test compound and a fraction of the assay mixture is passaged over a Sephadex G-25M column to remove small molecules before reaction with the serine-hydrolase-specific fluorophosphonate-rhodamine (FP-Rh) 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 relative to a DMSO (no compound) control. As designed, test compounds that act as irreversible inhibitors will prevent enzyme-probe interactions both before and after gel filtration, leading to low fluorescence intensity in the band in the gel. In contrast, compounds that act as reversible inhibitors will show recovery of probe labeling (and higher fluorescence intensity in the band in the gel) following gel filtration.
HeLa cell lysate (1 mL of 1 mg/ml in DPBS) was treated with test compound (0.2 uM [Assay 1] or 1 uM [Assay 2], from 50x stock in DMSO) or DMSO only (no compound control). Test compounds were incubated for 30 minutes at 37 degrees Celsius. An aliquot (50 uL) was removed from each reaction, and the remaining sample was passaged over a Sephadex G-25M desalting column. An aliquot (50 uL, 1 mg/mL protein, eluted in DPBS) was removed, and pre- and post- filtration aliquots were reacted with FP-Rh (50x stock in DMSO; 2 uM final concentration). 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 LYPLA1 and LYPLA2 bands (quantified together) relative to a DMSO-only (no compound) control. The percent recovery was calculated by comparing percent inhibition before and after gel filtration for each test compound concentration.
Assay calculations were performed as follows;
% 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.
% Recovery = %_Inhibition_PreFilt - %_Inhibition_PostFilt
%_Inhibition_PostFilt is defined as percent inhibition post gel-filtration.
%_Inhibition_PreFilt is defined as percent inhibition pre gel-filtration.
Compounds were designated as reversible inhibitors if they exhibited greater than or equal to 50% recovery of probe labeling following gel filtration. Compounds were designated as irreversible inhibitors if they exhibited less than 50% recovery of probe labeling following gel filtration.
Compounds identified as irreversible in the Binding Mode assay were considered active. Compounds identified as reversible in the Binding Mode assay were considered inactive.
PubChem Activity Outcome:
Compounds active in both assays were considered active. Compounds inactive both assays were considered inactive.
List of Reagents:
HeLa cell lysate (provided by the Assay Provider)
Desalting columns (GE Life Sciences #17-0851-01)
FP-Rh (Thermo #88318)
DPBS (Cellgro 20-031-CV)
This assay was performed by the assay provider with powder samples of synthetic test compounds.
** Test Concentration. § Panel component ID.