Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Substrate-based fluorescence-based biochemical determination of kinetic parameters
Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Substrate-based fluorescence-based biochemical determination of kinetic parameters. ..more
BioActive Compound: 1
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: LYPLA2_INH_FLUO_SUBSTRATE_4XKINETICS
Name: Late stage assay provider results from the probe development effort to identify selective inhibitors of LYPLA2: Substrate-based fluorescence-based biochemical determination of kinetic parameters.
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, LYPLA2, lysophospholipase 2, APT2, acyl-protein thioesterase 2, serine hydrolase, palmitoylation, protein palmitoylation, counterscreen, inhibitor, inhibition, substrate, resorufin acetate, kinetic, kinetic parameter, Michealis-Menten, Michealis-Menten kinetics, Cheng-Prusoff, Cheng-Prusoff equation, Vmax, Km, Ki, IC50, dose response, fluorescence, fluorescence intensity, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
§ Panel component ID.
The purpose of this assay is to determine kinetic parameters Vmax and Km for LYPLA2, and kinetic parameters Ki and IC50 values for powder samples of test compounds that act as reversible inhibitors for LYPLA2 using a fluorogenic substrate (resorufin acetate)-based assay. To determine Vmax and Km values, LYPLA2 is incubated with varying concentrations of substrate and the rate of enzymatic hydrolysis (as indicated by fluorescence intensity) is monitored as a function of time. Initial velocities for each substrate concentration are determined and used to calculate Vmax and Km using standard Michaelis-Menten kinetics. To obtain IC50 values, LYPLA2 is incubated with varying concentrations of inhibitor at a fixed substrate concentration, and fluorescence intensity is monitored as a function of time. Initial velocities determined for each inhibitor concentration are used to calculate IC50 values. From these data, Ki values can be calculated using the Cheng-Prusoff equation. For all assays, enzyme activity is calculated relative to a catalytically-dead (LYPLA2-S112A) enzyme control.
Substrate resorufin acetate was dissolved in DMSO. Active or catalytically-dead LYPLA1 enzyme solutions (10 nM) were prepared in DPBS adjusted to pH 6.5 with sodium acetate and 0.2% pluronic F127. In a black-bottom half-area 96 well plate, 5 uL of substrate was aliquoted at varying concentrations. The assay was initiated upon addition of enzyme (95 uL of 10 nM) using a multi-channel pipette, and reactions quickly mixed by pipetting up and down several times. Fluorescence intensity was measured on a Tecan F500 plate reader at room temperature every 30 seconds using a 525/35 nM excitation filter, a 600/10 nM emission filter, and a 560 LP dichroic filter. Each concentration was performed as 4 separate replicates for both the active and dead enzymes. After subtracting background (average fluorescence intensity of catalytically-dead enzyme at each time point for each assay condition), fluorescence intensity was plotted vs. time and initial velocities were calculated using standard straight-line plots (with non-linear regression) in GraphPad Prism using the first ~6 minutes of the reaction. The initial velocities (+/- SEM, plotted vs. substrate concentration) were analyzed using standard Michaelis-Menten kinetics (GraphPad Prism) to derive the Vmax and Km values. Software-generated values and SEM are reported.
To calculate the Ki values, the same experimental setup was used. Active or catalytically-dead LYPLA2-S122A enzyme (10 nM) was incubated with varying inhibitor concentrations (11-point series from 10 uM to 0.05 uM, and 0 uM) for 15 minutes (95 uL total volume), and then aliquoted into 96-well plate wells containing a fixed concentration of resorufin acetate substrate (50 uM, 5 uL). Fluorescence intensity was monitored as described above. After background subtraction, the initial velocities were calculated as described above, plotted vs. inhibitor concentration, and analyzed to derive IC50 values (standard one phase decay, GraphPad Prism). Software-generated values and SEM are reported. The Ki values were calculated using the Cheng-Prusoff equation:
Ki = ( IC50 ) / ( 1 + [S] / Km )
IC50 = IC50
[S] = substrate concentration
Km = Michaelis constant
PubChem Activity Outcome and Score:
Compounds with an IC50 value of less than or equal to 10 uM were considered active. Compounds with an IC50 value of greater than 10 uM were considered inactive.
Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
Resorufin acetate (SigmaAldrich, 83636)
Recombinant human LYPLA1 (provided by the Assay Provider)
Catalytically-dead (S122A) recombinant human LYPLA2 (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
Sodium acetate (FisherScientific, BP333)
Pluronic F127 (Invitrogen, P6866)
This assay was performed by the assay provider with powder samples of synthetic compounds.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: = ( [Y0] - [Plateau] ) * exp( -[Rate Constant] * [Concentration] ) + [Plateau]
Assay: CurveFit : Equation: = [Vmax] * [Substrate Concentration] / ( [Km] + [Substrate Concentration] )
* Activity Concentration. ** Test Concentration. § Panel component ID.