A Novel Fluorogenic Substrate for the Measurement of Phospholipase A1 Activity

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as phospholipase A2 (PLA2) enzymes. In order to distinguish the PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants and other nonhomogeneous systems a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and Hepatic lipase (HL), and was cleaved with a much lower efficiency by lipases that exhibit primarily triglyceride (TG) lipase activity, such as lipoprotein lipase (LPL), or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent to identify modulators of PLA1 enzymes, such as EL, and aids in characterizing their mechanisms of action. 2 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom Endothelial lipase (EL), a phospholipase A1 (PLA1) lipase, has gained attention as a potential therapeutic target for raising high density lipoprotein (HDL) and protection against atherosclerotic plaque formation (1, 2). EL is a secreted enzyme that subsequently becomes associated with the surface of endothelial cells, where it retains its enzymatic activity (1, 2). Therefore, EL resides in proximity with the plasma compartment where it alters lipoproteins and regulates lipoprotein metabolism. Its primary function is the hydrolysis of HDL phospholipids, which increases HDL turnover and decreases HDL cholesterol (HDL-C) and apoA-I content (3-5), resulting in an overall decrease in plasma levels of HDL particles. In addition to reducing plasma HDL-C, EL elicits other proatherogenic effects including increased free fatty acid and lyso PC, monocyte adhesion to the vessel wall (6), and increased plasma small dense LDL levels (7). EL is expressed in mouse aorta (8) and its expression is upregulated in response to various inflammatory mediators to such as IL-1β, TNFα (9) and INFγ (10). EL expression is also upregulated in the aortas of apo E mice, a model of atherosclerosis (8). Thus, EL’s role in lipoprotein metabolism is highly responsive to the proinflammatory conditions found in atherosclerotic plaques. Inhibition of EL with an anti-EL antibody increases HDL in several mouse strains with no increase in TG, LDL, or VLDL (11). In addition, HDL particles were increased approximately 60% in EL knockout mice (12, 13). More significantly, atherosclerotic lesion area in EL/apoE double knockout mice was reduced by 70% (6) and was accompanied by increased plasma HDL and reduction in vessel macrophage content. However a more recent study of both EL/apoE and EL/LDLR double knockout mice did not show significant improvement in lesion area despite clear increases in HDL (14). 3 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom The reason for this discrepancy is not known but may be the result of differences in environmental factors or stage of development of the atherosclerotic lesions at the time of measurement. Five separate studies have reported human mutations in EL that are associated with increased plasma HDL-C (13, 15-18). In particular, two non-synonymous mutations in human subjects were shown to promote elevated HDL by either reduced EL secretion (19) or reduced EL catalytic activity (20). In addition, EL expression was evident in infiltrating cells, macrophages and smooth muscle cells within human atheromatous plaques (21). Intriguingly, high levels of plasma EL are correlated with low levels of HDL-C, high levels of small dense LDL, and metabolic syndrome (22). Elevated EL is also associated with increased visceral adiposity (23) and plasma inflammatory markers (24). Thus, increases in EL activity may contribute to the low HDL levels observed in patients with obesity and metabolic syndrome. Overall, the data indicate that EL plays an important role in HDL metabolism in humans and that inhibition of EL should increase plasma HDL, especially in patients with low levels of HDL-C. Given the association of low levels of HDL with increased risk of atherosclerotic cardiovascular events in humans (2, 25-27) EL represents a promising target for potential inhibition of atherosclerotic disease. To identify inhibitors of EL as potential cardiovascular disease therapeutics, we developed an assay that can be used to reliably detect cell-based EL enzymatic activity and that was also adaptable to a high-throughput compound screening format. Although EL is a secreted enzyme, it remains associated with the cell surface via interactions with proteoglycans through a conserved heparin binding site (28, 29) similar to the related 4 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom PLA1 enzymes, lipoprotein lipase (LPL) and hepatic lipase (HL). Thus, cells expressing EL are amenable to monitoring EL enzymatic activity with a suitable substrate. In order to distinguish the PLA1 activity of EL from non-specific PLA2 activity in cell-based assays, we designed and characterized a novel fluorogenic substrate with selectivity toward PLA1 specific hydrolysis. This unique substrate has a BODIPY fluor conjugated to the fatty acyl chain at the sn-1 position. In addition, it has an ether bond at the sn-2 position, to preclude hydrolysis by PLA2 enzymes. Finally, the PLA1 substrate has a dinitrophenyl group on the phospholipid head. This chemical moiety quenches the BODIPY fluor until hydrolysis of the substrate takes place, creating distance between the fluor and quencher allowing fluorescence to occur. The use of this substrate in PLA1 assays allows the continuous monitoring of enzymatic activity over time. 5 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom MATERIALS AND METHODS Materials and reagents We conceived the PLA1 specific substrate [N-((6-(2,4-dinitrophenyl) amino) hexanoyl)-1-(4,4-difluoro-5,7-dimethyl-4-bora-3a,-4a-diaza-s-indacene-3-pentanoyl)-2hexyl-sn-glycero-3-phosphoethanolamine] (mw = 879) (Fig. 1A) based on our need for a PLA1-sensitive substrate for a high-throughput screen for endothelial lipase inhibitors. Since this substrate was not commercially available at the time (2005) we contracted a custom synthesis by Molecular Probes. Following its custom synthesis for our internal use, Invitrogen made the substrate available for sale as PED-A1 in its 2007 catalogue. PED6 [N-((6-(2,4-dinitrophenyl) amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora3a,-4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3phosphoethanolamine] (mw = 1136) (Fig. 1B) and the cleavage product for both PED6 and the PLA1 substrate [4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3pentanoic acid (BODIPY FL C5)] (Fig. 2A) were purchased from Invitrogen’s Molecular Probes (Eugene, OR). Bovine lipoprotein lipase, ebelactone B, TNFα and IL-1β were purchased from Sigma (St. Louis, MO) while bee venom PLA2 was from Cayman Chemical Co. (Ann Arbor, Michigan). Expression and purification of mouse EL The cDNA for mouse EL was modified by changing most of the nucleotide bases at the wobble position of the amino acid codons. The resulting cDNA retains the ability to encode full length wild type mouse EL polypeptide, however, it is unable to hybridize with the human EL cDNA sequence under high stringency conditions. The modified mouse EL cDNA was synthesized by DNA2.0 (Menlo Park, CA) with both 6xHIS and 6 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom 3xFLAG tags at the C-terminus, and then subcloned using unique BamHI and XbaI sites into pVL1393. The final construct was sequence confirmed (Seqwright, Houston, TX). Spodoptera frugiperda, Sf9, insect cells were routinely maintained between 0.8 X 10 and 4.0 X 10 cells/ml in ESF921 medium (Expression Systems, Woodland, CA) at 27C. To generate recombinant EL baculovirus, the pVL1393/EL vector was transfected into Sf9 insect cells using BaculoGold transfection kit (BD Biosciences, San Diego, CA) according to the manufacturer’s protocol. Five days post-transfection, passage 0 (P0), virus was harvested and stored at 4°C. High-titer virus (P3) was obtained through two sequential rounds of amplification and used for large-scale production of EL. Trichoplusia ni, High Five, cells were used for large-scale production. During growth, cells were maintained between 0.3 X 10 and 4 X 10 cells/mL in ESF921 media at 27°C. The cells were infected with the recombinant P3 EL baculovirus at a density of ~1.6 X 10 cells/mL and an MOI of 1 at 27C. Following 72 h of infection, the media was cleared by centrifugation at 800 x g for 10 min at 4°C. Further clarification was achieved with an additional centrifugation at 6000 x g for 10 min at 4°C. Expression was validated by immunoblots using an anti-FLAG (M2) antibody (Sigma). Media (6 L) was concentrated to 0.7 L on a Kvick Lab system (GE Healthcare, Piscataway, NJ). Concentrated media was buffer exchanged by adding 4 L of 50 mM Tris pH 7.4, 150 mM NaCl (buffer A) and re-concentrated to 0.7 L. Buffer exchanged media was clarified by centrifugation at 6000 x g for 1 h. The cleared media was loaded on a 10 mL anti-FLAG M2 affinity gel (Sigma) at 1 mL/min and 4°C using the AKTA Explorer System (GE Healthcare, Piscataway, NJ). The column was washed with 15-20 column volumes of buffer A. Mouse EL was eluted with 5 column volumes of buffer A 7 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom containing 0.1 mg/mL 3X-FLAG peptide (Sigma). Fractions were analyzed by SDSPAGE and those containing mouse EL were pooled. Expression yields following purification were ~ 1 mg/L as determined by Bradford assay using the protein assay kit from BioRad (Hercules, CA) according to manufacturer’s instructions with BSA as a standard. Assays with purified lipases To assay for EL activity, using purified EL protein, 15 μL assay buffer [Hank’s Balanced Salt Solution without calcium, magnesium or phenol red with 25 mM HEPES (Mediatech, Manassas, VA)] was placed into a 384 well PCR plate (Abgene, Epsom, UK). Three μL of PLA1 or PED6 substrate (50 μM) dissolved in DMSO was added using a Multidrop reagent dispenser (Thermo Fisher, Waltham, MA) for a final substrate concentration of 5 μM, or as indicated in the figure. The plate was incubated for 10 min at 37°C to avoid the lag phase. Purified mouse EL protein (12 μL; for a final concentration of 0.4 μM) was added for a final assay volume of 30 μL. Fluorescence signal was monitored for 40 min at 37°C on a Safire II plate reader (Tecan, Raleigh, NC) in kinetic mode (80 cycles, kinetic interval: 30 sec) with an excitation wavelength of 490 nm and an emission wavelength of 515 nm. Linear regression of the fluorescence intensity collected from 400 to 1500 sec was used to calculate the reaction rate (the slope) and the slopes were used to calculate IC50 values where appropriate. Conversion of relative fluorescent unit (RFU) to moles of substrate turnover was accomplished by determining the specific activity (RFU x M) of C5-bodipy following linear regression of 0.05 to 200 nM (serial dilution) under the exact assay conditions used for each 8 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom experiment. This was completed at multiple substrate concentrations (each substrate concentration used in all experiments) and multiple instrument gain settings to account for interfilter effects from the substrate. This must be determined empirically for experiments as the RFU x M is dependent upon the instrument, instrument settings, assay conditions (buffer and other components), temperature, and the assay plate. To assay for purified bovine LPL (Sigma #L2254) and bee venom PLA2 (Cayman #765001) activities, 80 μL assay buffer [DMEM with no glutamine, no phenol red with 25 mM HEPES pH 7.2 (Mediatech)] was placed into a Corning #3340 96-well black plate with clear bottom (Corning, NY). PLA1 or PED6 substrate (100 μL diluted in assay buffer for a series of final concentrations of between 0.25-10 μM) was added using a Multidrop reagent dispenser. Purified bovine LPL, or bee venom PLA2 protein were added to a final concentration of 2.5 μg/mL in a 200 μl assay volume. Fluorescence signal was monitored for 90 min at 37°C on a Safire II plate reader (Tecan, Raleigh, NC) with an excitation wavelength of 490 nm and an emission wavelength of 515 nm. The relative fluorescent units were converted to nM x min of free BODIPY-C5 liberated over time from a BODIPY FL C5 (Molecular Probes) standard curve. Analysis of PLA1 substrate hydrolysis by EL using LCMS Purified mouse EL (200 nM) was incubated with the PLA1 substrate (10 μM) and reactions were quenched with MeOH at a final concentration of 25%. Samples were subjected to LCMS analysis (using negative ion mode) monitoring A220, A503, h/z 319 for selective ion monitoring (SIM) or scanning the h/z range 300-350 and 879 (SIM, scan 800-900) and Ex/Em 490/525 (8 nm bp) using an Agilent 1100 LC coupled to an Agilent 9 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom MSD system (Agilent Technologies, Santa Clara, CA). Stationary and mobile phases consisted of 10 mM NH4OAc (Sigma Ultra pH 7.5) and 10 mM NH4OAc + 95% MeOH (EMD LCMS grade), respectively. Chromatography was accomplished with a 20-95% MeOH gradient over 3 min, where the mobile phase was maintained at 100% for an additional 3 min, and the column equilibrated for 4 min at 20% MeOH at 0.5 ml x min using a Jupiter C5 50 x 3 mm column (Phenomenex, Torrance, CA). Quantitation of substrate hydrolysis used the following equations: for A505 data, [S] x AUCproduct/(AUCproduct + AUCsubstrate) and for LCMS SIM: AUCproduct/7.53 x 10 x uM, where 7.53 x 10 is the AUC units x uM of purified BODIPY FL C5, as determined empirically from standard curve analysis. Assays with HEK293 cells stably expressing lipases The murine EL (LIPG) cDNA [NM_010720] sequence was modified, optimized for codon preference and fused to an in-frame C-terminal 6xHIS 3xFLAG affinity epitope tag (as described above). The resulting cDNA was subcloned into pcDNA3.1(+) with neomycin resistance (Invitrogen, Carlsbad, CA). Human HL (LIPC) [NM_000236] and human LPL (LPL) [NM_000237] were obtained from the OriGene Technologies, TrueCloneTM collection [SC120025 and SC120026, respectively]. The full length HL cDNA, isolated as a Not I fragment, and the full length LPL cDNA, isolated as an Eco RI fragment, were subcloned into the mammalian expression vector pcDNA3Neo (Invitrogen, Carlsbad, CA). HEK293 cells were transfected with either the EL, HL or LPL cDNAs using SuperFect transfection reagent (Qiagen, Valencia, CA), as directed by the manufacturer and selected for stable expression of the desired plasmid with 1200 10 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom mg/mL G418 (Mediatech). The stable HEK293 cell lines were maintained in DMEM (Mediatech), 10% fetal bovine serum (HyClone, Logan, Utah), 2 mM L-glutamine (Mediatech), 50 U/mL penicillin and 50 μg/mL streptomycin (GIBCO/Invitrogen, Grand Island, NY), and 1200 mg/mL G418. Immunocytochemistry was performed using: 1) the FLAG M2 MoAb (Sigma), to identify clones that expressed EL, 2) anti-hHL MoAb (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to identify clones that expressed HL, and 3) anti-LPL MoAb (Abcam, Cambridge, MA) to identify clones that expressed LPL. PLA1 or PED6 substrate stock solution consisted of 5 mM substrate in DMSO, stored at -80°C. The stock was diluted 1:625 in HBSS with 25 mM HEPES to achieve an 8 μM working solution. To assay for cell surface lipase activity, cells were plated in CellBIND 384-well plates (Corning, Lowell, MA) in 25 μL serum free medium at a density of 2000 cells/well. After an 18-24 h incubation at 37°C, medium was removed and replaced with 15 μl of the working solution (containing either PLA1 or PED6 substrate) and 15 μl of HBSS with 25 mM HEPES (1:2 dilution) to achieve a final concentration of 4 μM (or the final concentrations indicated in the figure legends). Substrate was dispensed using a Multidrop reagent dispenser. Fluorescence signal was monitored for 30 min at 37°C on a Safire II plate reader in kinetic mode (60 cycles, kinetic interval: 30 seconds) with an excitation wavelength of 490 nm and an emission wavelength of 515 nm. Linear regression of the fluorescence intensity collected from 480 to 1500 sec was used to calculate the reaction rate (the slope) and the slopes were used to calculate IC50 values where appropriate. The amount of BODIPY-labeled product generated was calculated at the 30 min time point as determined from standard curve analysis of purified BODIPY FL C5. In all studies using the inhibitor ebelactone B, 11 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom consistent results were obtained when it was dissolved as a stock in DMSO immediately before use. HUVEC assay Pooled human umbilical vein endothelial cells, HUVEC (Cambrex/Lonza, Walkersville, MD), were cultured with EBM2 medium containing 5% fetal bovine serum and EGM supplements (Cambrex/Lonza) and plated for assays at a density of 2000 cells/well into BD Collagen coated 384 well plates (BD Biosciences, San Jose, CA) and incubated 24 h at 37°C. Cells were then washed with phosphate buffered saline and incubated with serum-free medium containing 10 ng/ml TNF-α and 1 ng/ml IL-1β for an additional 24 h at 37°C. Medium was removed and replaced with 15 μL assay buffer and assayed as described for the stable HEK293 cell lines. 12 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom RESULTS In order to develop a cell-based assay that would distinguish the activity of a PLA1 versus a PLA2 enzyme, we conceived and designed a phospholipid substrate with a BODIPY fluor affixed at the sn-1 position and an alkyl chain attached to the sn-2 position with an ether bond (Fig. 1A). Since this molecule was not commercially available at the time (2005), we contracted its custom synthesis at Invitrogen (Note: Subsequent to our custom synthesis, Invitrogen added the novel substrate to its regular catalog (2007). The potentially fluorogenic phospholipid, herein referred to as the PLA1 substrate, is similar in structure to PED6 (Fig. 1B), a commercially available substrate for PLA2 enzymes. Although PLA1 enzymes are able to hydrolyze PED6, they do so with much lower efficiency than PLA2 lipases. Therefore, the PED6 substrate is not favorable for the detection of PLA1 enzymatic activity in a cell-based assay. We engineered the PLA1 substrate to have an ether bond at the sn-2 position. Therefore, this substrate should not be readily cleaved by PLA2 enzymes and would be a preferred substrate for PLA1 lipases. In addition to the BODIPY fluor, the PLA1 substrate also contains a dinitrophenyl quencher on the phospholipid head group, allowing for the continuous measurement of fluorescence increase associated with PLA1 hydrolysis. Confirmation of activity and appropriate products using purified EL A depiction of how the PLA1 substrate is predicted to be hydrolyzed by a PLA1 enzyme along with its products is presented in Fig. 2A. The prescribed mechanism, an acid/base catalysis with an acyl-enzyme intermediate following the release of product 1 (P1), has been described previously (Fig. 2B) (7, 29-33). An oxyanion hole formed by 13 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom amide backbone nitrogen atoms within the active site, stabilizes the enzyme-product 2 (P2) complex. A catalytic water molecule then enters the active site and allows for completion of the cleavage reaction releasing P2 and the free enzyme is ready for another round of catalysis. LCMS analysis confirmed that the PLA1 substrate was cleaved appropriately as a function of time using 200 nM mouse EL enzyme. The data in Fig. 3A shows the mass of the substrate (inset 4) and the C5-BODIPY product (inset 3) are consistent with cleavage at the sn-1 position (Fig. 1). The production of the appropriate product accumulates as a function of time and is consistent with its predicted molecular weight throughout the time course of the experiment. The proportionate reduction in substrate signal and C5-BODIPY product accumulation as a function of time, as well as the absence of additional A505 peaks or peaks at A220 in the chromatogram (data not shown), indicate absence of substrate hydrolysis at alternative sites. As shown in Fig. 3B, the increase in fluorescence of the PLA1 substrate turnover monitored by A505, corresponded with its quantification by mass spectroscopy as a function of time. Thus, the cleavage of the PLA1 substrate by EL is occurring exclusively at the appropriate sn-1 position. An SDS PAGE examination of the purified murine EL used in our analysis indicates that our preparation is predominantly intact as shown in Fig. 3C. However, it also contained inactive cleavage products we confirmed by N-terminal sequence analysis, due to partial processing by proprotein convertases, as previously identified by others (34, 35). Using the PLA1 substrate, this purified mouse EL had an average Km of 10.0 ± 3.8 μM, and a Kcat/Km of 84 ± 4.5 M x sec as calculated from a total of seven independent determinations. 14 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom PLA1 enzymes demonstrate preferential cleavage of the PLA1 substrate We investigated the preference for cleavage of the PLA1 substrate initially using a standard PLA2 enzyme, bee venom PLA2. As indicated in Fig. 4, purified bee venom PLA2 displayed robust enzymatic activity when incubated with the PED6 substrate, but under identical conditions displayed negligible enzymatic activity when incubated with the PLA1 substrate. This reflected the inability of bee venom PLA2 to cleave the PLA1 substrate at the sn-1 position. Purified bovine LPL, which has been reported to more readily cleave triglyceride versus phospholipid substrates (3-5), demonstrated limited enzymatic activity on the PLA1 substrate. The PLA1 substrate was then directly compared to that of PED6 in a cell-based assay format using cells stably expressing murine EL, human HL or human LPL. As shown in Fig. 5A, under the assay conditions used, the PED6 substrate showed no significant cleavage in cells expressing EL, HL or LPL above non-transfected control cells at all substrate concentrations tested. In contrast to PED6, the PLA1 substrate demonstrated significant cleavage with these three lipases (Fig. 5B). The rank order of cleavage was observed to be EL > HL > LPL > untransfected control cells. Moreover, these stably expressing cell lines were determined to have comparable levels of lipase expression (data not shown). EL demonstrated the highest activity on the PLA1 substrate. This is consistent with its phospholipid preference and strong bias for the sn-1 acyl ester bond (5, 28, 29). 15 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom Inhibition of the PLA1 substrate cleavage with ebelactone B We next examined the potential of a small molecule inhibitor, ebelactone B, to block the hydrolytic activity of the PLA1 lipases in our cell-based assays. Ebelactone B is known to be a promiscuous, covalent inhibitor of lipases (36). Using the HEK293 cells stably expressing EL, HL, LPL or control non-transfected HEK293 cells, the PLA1 substrate was assayed in the absence or the presence of 10 μM ebelactone B. As shown in Fig. 6, with the exception of non-transfected HEK293 cells, PLA1 substrate hydrolysis was decreased significantly when ebelactone B was present. As determined by subsequent ebelactone B dose-response curves in these cell-based assays, the IC50 values for the three lipases were determined to be: 0.15 ± 0.06 μM [n=28] for murine EL; 0.05 ± 0.01 μM [n=24] for human HL; and 0.12 ± 0.07 μM [n=24] for human LPL. This indicates that use of the PLA1 substrate can help identify and characterize small molecule inhibitors of EL as well as determine selectivity in counter-screen assays of related lipases. Furthermore, the murine EL HEK293 cell line was used as a high-throughput screen (HTS) of 200,000 compounds in order to detect inhibitors of EL activity. Statistical parameters for this HTS were as follows: 12.9 ± 1.5 RFU/sec; CV% = 11.9 and z’ = 0.50. When used as an assay to screen compounds, generate dose-response curves and determine their IC50 values, the z’ factor per 384 well plate ranged between 0.5 and 0.75. To further explore how the PLA1 substrate can be used to measure EL activity in a cell-based assay, we used HUVEC cells in which EL expression is known to be upregulated by cytokines (9, 37). Following stimulation with TNF-α and IL-1β, HUVEC cells were used in a cell-based assay with the PLA1 substrate and increasing 16 by gest, on A uust 5, 2017 w w w .j.org D ow nladed fom concentrations of ebelactone B. As shown in Fig. 7 hydrolysis of the PLA1 substrate by cells endogenously expressing EL was inhibited in a dose-dependent manner by increasing concentrations of ebelactone B. Using this HUVEC cell assay we determined the IC50 for ebelactone B inhibition to be 0.15 ± 0.03 μM [n=18]. This value is in agreement with the IC50 value of ebelactone B using the EL expressing HEK293. When used as an assay to screen compounds, generate dose-response curves and determine their IC50 values, the statistical parameters of the assay were as follows: 125.6 ± 18.7 RFU/min; CV% = 14.9 and the z’ factor ranged between 0.46 and 0.70. DISCUSSION We conceived and hypothesized the specificity of a novel BODIPY-labeled, selfquenched PLA1 substrate that could be used in homogeneous high-throughput kinetic assays to measure PLA1-specific phospholipase activities in order to identify small molecular inhibitors of EL as potential therapeutic agents. Since it was not commercially available at the time we contracted the custom synthesis of this substrate at Invitrogen. EL showed robust phosopholipase activity using this substrate in both cell-free and cellbased assays to monitor its hydrolytic activity. We have routinely run the cell-based PLA1 substrate assay in a 384-well format. This novel PLA1 substrate was compared to a widely used PED6 substrate and found to have superior characteristics, making it a more favorable substrate to monitor EL activity. In addition, the PLA1 substrate can be used to assay the hydrolytic activity of other lipases in counter-screens for monitoring compound