Primary uHTS assay to identify LYPLA2 inhibitors (AID 2177)

Assay Overview: The purpose of this assay was to identify compounds that act as LYPLA2 inhibitors. This assay also serves as a counterscreen for a set of previous experiments entitled, “Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1)" (AID 2130). This competitive activity-based protein profiling (ABPP) assay uses fluorescence polarization to investigate enzyme-substrate functional interactions based on active site-directed molecular probes [3, 15]. A serine hydrolase-specific fluorophosphonate-rhodamine (FP-Rh) probe, which broadly targets enzymes from the serine hydrolase family [14] was used to label LYPLA2 in the presence of test compounds. The reaction was excited with linear polarized light and the intensity of the emitted light was measured as the polarization value (mP). As designed, test compounds that act as LYPLA2 inhibitors will prevent LYPLA2-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Omission of enzyme (which gives the same result as use of a catalytically-dead enzyme) serves as a positive control. Compounds were tested at a nominal concentration of 5.9 µM.

Protocol Summary: Prior to the start of the assay, Assay Buffer (4.0 µL; 0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1mM DTT) containing LYPLA2 protein (9.38 nM) was dispensed into 1536-well microtiter plates. Next, test compound (30 nL in DMSO) or DMSO alone (0.59% final concentration) was added to the appropriate wells and incubated for 30 minutes at 25 °C. The assay was started by dispensing FP-Rh probe (1.0 µL of 375 nM in Assay Buffer) to all wells. Plates were centrifuged and, after 10 minutes of incubation at 25 °C, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software. Assay Cutoff: Compounds that inhibited LYPLA2 greater than 25.78% (mean + 3 x standard deviation) were considered active.

Confirmation uHTS assay to identify LYPLA2 inhibitors (AID 2232)

Assay Overview: The purpose of this assay was to confirm activity of compounds identified as active in the primary uHTS screen (AID 2177). In this assay, the FP-Rh probe was used to label LYPLA2 in the presence of test compounds and analyzed as described above (AID 2177). Compounds were tested in triplicate at a nominal concentration of 5.9 µM.

Protocol Summary: The assay was performed as described above (AID 2177), except that compounds were tested in triplicate. Assay Cutoff: Compounds that inhibited LYPLA2 greater than 25.78% were considered active.

Inhibition of LYPLA2 by top HTS hits (AID 652029)

Assay Overview: The purpose of this assay is to determine whether test compounds can inhibit LYPLA2 in a complex proteomic lysate using a competitive activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome, containing spiked-in recombinant form of LYPLA2 is incubated with test compound followed by reaction 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 of the bands. As designed, test compounds that act as 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.

Protocol Summary: To soluble proteome prepared from mouse brain (1 mg/ml in DPBS) is added 20 nM purified recombinant human (rh) LYPLA2. Proteome was treated with 20 µM test compound (1 µL of a 50x stock in DMSO) for 30 minutes at 25 ºC (50 µL reaction volume). FP-Rh (1 µL of 50x stock in DMSO) was added to a final concentration of 2 µM. The reaction was incubated for 30 minutes at 25 ºC, 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 target rhLYPLA2 band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition were considered active.

Inhibition of LYPLA2 and selectivity vs. LYPLA1 of top HTS hits (AID 652030)

Assay Overview: The purpose of this assay is to determine whether test compounds can inhibit LYPLA2 in a complex proteomic lysate and assess selectivity vs. LYPLA1 using a competitive activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome, containing spiked-in recombinant form of LYPLA2 is incubated with test compound followed by reaction 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 of the bands. As designed, test compounds that act as 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.

Protocol Summary: To membrane proteome prepared from mouse brain (1 mg/ml in DPBS) is added 20 nM purified recombinant human (rh) LYPLA2. Proteome was treated with 10 µM test compound (1 µL of a 50x stock in DMSO) for 30 minutes at 25 ºC (50 µL reaction volume). FP-Rh (1 µL of 50x stock in DMSO) was added to a final concentration of 2 µM. The reaction was incubated for 30 minutes at 25 ºC, 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 target rhLYPLA2 band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 30% inhibition were considered active.

Gel-based competitive ABPP analysis of reversible (AID 651988) and triazole urea (AID 652018) SAR libraries

Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds can inhibit LYPLA2 and LYPLA1 in a complex proteomic lysate and to estimate compound selectivity in a competitive activity-based protein 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 [12]. 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 of the bands. As designed, test compounds that act as LYPLA2 and/or LYPLA1 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.

Protocol Summary: Soluble proteome (50 µL of 1 mg/ml in DPBS) of HEK293T cells was treated with 300 nM, 1 µM, or 10 µM test compound (1 µL of a 50x stock in DMSO). Test compounds were incubated for 30 minutes at 37 °C. FP-PEG-Rh (1 µL of 50x stock in DMSO) was added to a final concentration of 5 µM. 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 LYPLA2 and LYPLA1 bands relative to a DMSO-only (no compound) control. From among the 20+ other distinct bands visible, anti-targets are only listed if greater than or equal to 50% inhibition is observed at any concentration tested. Assay Cutoff: Compounds with greater than or equal to 50% inhibition at 300 nM were considered active.

Determination of IC50 values for LYPLA2 and LYPLA1 by competitive ABPP (AID 651990)

Assay Overview: The purpose of this assay is to determine IC50 values of powder samples of test compounds for LYPLA2 and LYPLA1 in a complex proteomic lysate using a competitive activity-based protein 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 [12]. 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 of the bands. As designed, test compounds that act as LYPLA2 and/or LYPLA1 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.

Protocol Summary: Soluble proteome (50 µL of 1 mg/ml in DPBS) of HEK293T cells was treated with varying concentrations of test compound (1 µL of a 50x stock in DMSO) for 30 minutes at 37 °C. FP-PEG-Rh (1 µL of 50x stock in DMSO) was added to a final concentration of 5 µM. 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 LYPLA2 and LYPLA1 bands relative to a DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration: 3000 nM to 1000 nM, 300 nM to 100 nM, 30 nM, and 1 nM. Assay Cutoff: Compounds with an IC50 less than or equal to 500 nM were considered active.

Kinetic analysis of inhibition of LYPLA2 (AID 652001) and LYPLA1 (AID 652003) by substrate assay

Assay Overview: The purpose of this assay is to determine kinetic parameters (Vmax, Km, Ki, and IC50 values) for powder samples of test compounds that act as reversible inhibitors for LYPLA2 and LYPLA1 using a fluorogenic substrate (resorufin acetate)-based assay. To determine Vmax and Km values, LYPLA2 or LYPLA1 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 or LYPLA1 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- S122A or LYPLA1-S119A) enzyme control.

Protocol Summary: Substrate resorufin acetate was dissolved in DMSO. Active or catalytically-dead 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 µL of substrate was aliquoted at varying concentrations (LYPLA2: 200 µM, 180 µM, 160 µM, 140 µM, 100 µM, 80 µM, 40 µM, 30 µM, 20 µM, 17.5 µM, 15 µM, 12.5 µM, 10 µM, 7.5 µM, 5 µM, 2.5 µM, 0 µM ; LYPLA1: 180 µM, 140 µM, 100 µM, 70 µM, 60 µM, 50 µM, 35 µM, 25 µM, 15 µM, 7.5 µM, 3.75 µM, 0 µM). The assay was initiated upon addition of enzyme (95 µL 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 enzyme (10 nM) was incubated with varying inhibitor concentrations (LYPLA2: 10 µM, 6 µM, 4 µM, 2 µM, 1 µM, 0.8 µM, 0.6 µM, 0.4 µM, 0.2 µM, 0.1 µM, 0.05 µM, 0 µM ; LYPLA1: 11-point 2-fold dilution series from 25 µM to 24 nM, 0 nM) for 15 minutes (95 µL total volume), and then aliquoted into 96-well plate wells containing a fixed concentration of resorufin acetate substrate (LYPLA2: 30 µM, 5 µL; LYPLA1: 50 µM, 5 µL). 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. Assay Cutoff: Compounds with an IC50 value of less than or equal to 10 µM were considered active.

Analysis of Cytotoxicity (AID 651991)

Assay Overview: The purpose of this assay is to determine cytotoxicity of powder samples of test compounds. In this assay, HEK293T cells in either serum-free medium or medium containing fetal calf serum (FCS) are incubated with test compounds, followed by determination of cell viability. The assay utilizes the WST-1 substrate which is converted into colorimetric formazan dye by the metabolic activity of viable cells. The amount of formed formazan directly correlates to the number of metabolically active cells in the culture. As designed, compounds that reduce cell viability will result in decreased absorbance of the dye.

Protocol Summary: This assay was started by dispensing HEK293T in DMEM medium supplemented with 10% FCS (100 µL, 15,000 cells/well) into a 96-well plate. Cells were incubated for 24 hours at 37 °C in a humidified incubator, medium was removed, and 100 µL of fresh, serum-free medium or medium supplemented with 10% FCS was added. Compound (10 µL of 11x stocks in medium containing 10% DMSO) or an equal volume medium containing 10% DMSO only was added to each well. Cells were incubated for 48 hours at 37 °C in a humidified incubator and cell viability was determined by the WST-1 assay (Roche) according to manufacturer instructions. CC50 values were determined from dose-response curves from six replicates at each inhibitor concentration (50000, 10000, 2000, 400, 80, 16, and 3.2 nM). Assay Cutoff: Compounds with CC50 values less than or equal to 10 µM were considered active (cytotoxic).

Gel filtration to assess mode of action (AID 651987)

Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds inhibit LYPLA2 and LYPLA1 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 before reaction with the serine-hydrolase-specific fluorophosphonate-PEG-rhodamine (FP-PEG-Rh) probe. FP-PEG-Rh is similar to FP-Rh, but shows slower reaction kinetics, facilitating analysis of target inhibition by reversible compounds [12]. 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 to remove small molecules from the sample.

Protocol Summary: Soluble proteome (2 mL of 1 mg/ml in DPBS) of HEK293T cells was treated with 10 µM or 20 µM test compound (50x stock in DMSO) or DMSO (control). Test compounds were incubated for 30 minutes at 37 °C. An aliquot (50 µL) was removed from each reaction, and the remaining sample was passaged over a Sephadex G-25M column 1, 2, or 3x, with an aliquot (50 µL) set aside after each passage. Aliquot volumes were adjusted to equalize protein concentrations across all samples and reacted with FP-PEG-Rh (50x stock in DMSO; 5 µM final concentration). 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 LYPLA2 and LYPLA1 bands relative to a DMSO-only (no compound) control. Assay Cutoff: 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.

In situ inhibition of LYPLA2 and LYPLA1 by competitive ABPP (AID 651986)

Assay Overview: The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit LYPLA2 and LYPLA1 in situ. In this assay, cultured HEK293T cells or BW5147-derived murine T-cells are incubated with test compound followed by the cell-permeable alkyne-functionalized triazole urea ABPP probe NHU5 [12]. NHU5 covalently reacts with a subset of serine hydrolases, including LYPLA2 and LYPLA1, and shows slower reaction kinetics than other cell-permeable serine hydrolase-specific ABPP probes (e.g., FP-alkyne), facilitating analysis of target inhibition by reversible compounds. Cells are harvested, homogenized, and reacted with a rhodamine-functionalized azide tag under copper(I)-catalyzed azide-alkyne cycloaddition (“click chemistry”) reaction conditions to allow visualization of probe-tagged proteins. 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 of the bands. As designed, test compounds that act as LYPLA2 and/or LYPLA1 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound, fluorescent-tagged probe, giving lower fluorescence intensity in the band in the gel.

Protocol Summary: HEK293T cells were grown in DMEM medium (supplemented with 10% FCS and 1x Penicillin/Streptomycin/Glutamine solution) and BW5147-derived murine T-cell hybridoma cells were grown in RPMI-1640 medium (supplemented with 10% FCS and 1x Penicillin/Streptomycin/Glutamine solution) at 37 °C in a humidified incubator (5% CO₂ atmosphere). Test compounds (5 µM final concentration) or DMSO only were directly added to the cell culture medium. After 3 hours, the cell-permeable, alkyne-functionalized probe NHU5 (50 µM final concentration) was added. After 1 hour, cells were washed (4x DPBS), harvested, isolated by centrifugation at 1,400 x g for 3 minutes, re-suspended in methanol/chloroform/dH₂O (4:1:3 v/v), and vortexed. After centrifugation at 13,000 x g for 3 minutes, the upper layer was removed and 3 volumes of methanol were added and the solution vortexed. Protein was pelleted by centrifugation at 13,000 x g for 6 minutes. The supernatant was removed and the pellet was air-dried and re-suspended in PBS by sonication. For visualization of probe-labeled proteins, click chemistry with a rhodamine-azide tag (Rh-N3; 100 µM) was carried out under standard conditions (1 mM TCEP, 100 µM TBTA ligand, 1 mM Cu(II)sulfate, 1 hour, 25 °C) to append the rhodamine fluorophore to the alkyne-functionalized NHU5 probe. Reactions were 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 bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition were considered active.

In vivo inhibition of LYPLA2 and LYPLA1 by competitive ABPP (AID 651985)

Assay Overview: The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit LYPLA2 and LYPLA1 in vivo. In this assay, test compounds are administered to mice followed by the in vivo active alkyne-functionalized triazole urea ABPP probe NHU5 [12]. NHU5 covalently reacts with a subset of serine hydrolases, including LYPLA2 and LYPLA1, and shows slower reaction kinetics than other in vivo active serine hydrolase-specific ABPP probes (e.g., FP-alkyne), facilitating analysis of target inhibition by reversible compounds. Mice are sacrificed, and their tissues harvested, homogenized, and the soluble fraction isolated and reacted with a rhodamine-functionalized azide tag under copper(I)-catalyzed azide-alkyne cycloaddition (“click chemistry”) reaction conditions to allow visualization of probe-tagged proteins. 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 of the bands. As designed, test compounds that act as inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound, fluorescent-tagged probe, giving lower fluorescence intensity in the band in the gel.

Protocol Summary: Test compounds were prepared as a homogeneous PEG solution by vortexing and sonicating neat compound directly into PEG300. Purpose-bred C57-black laboratory mice (<6 months old, 20–28 g) were i.p. administered with test compound (50 mg/kg; 4µL/g) or vehicle only and, after 3 hours, with NHU5 probe (PEG solution; 100 mg/kg). After 1 hour, mice were sacrificed, and tissues were removed and Dounce-homogenized directly in methanol /chloroform/dH₂O (4:1:3 v/v). After centrifugation at 13,000 x g for 3 minutes, the upper layer was removed and 3 volumes of methanol were added and solution vortexed. Protein was pelleted by centrifugation at 13,000 x g for 6 minutes. The supernatant was removed and the pellet was air-dried and re-suspended in PBS by sonication. For visualization of probe-labeled proteins, click chemistry with a rhodamine-azide tag (Rh-N3; 100 µM) was carried out under standard conditions (1 mM TCEP, 100 µM TBTA ligand, 1 mM Cu(II)sulfate) to append the rhodamine fluorophore to the alkyne-functionalized NHU5 probe. Reactions were 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 bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition in one or more tissues tested were considered active.

Selectivity analysis by ABPP-SILAC in vitro (AID 651981)

Assay Overview: The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using stable isotope labeling with amino acids in cell culture (SILAC) activity-based protein profiling (ABPP) in vitro. In this assay, cultured BW5147-derived murine T-cells are metabolically labeled with light or heavy amino acids. Cells are harvested, homogenized, and proteome fractions isolated. Light and heavy samples are treated with inhibitor and DMSO, respectively, followed by the serine-hydrolase-specific ABPP affinity probe fluorophosphonate-PEG-biotin (FP-PEG-biotin). FP-PEG-biotin is similar to FP-biotin, but shows slower reaction kinetics, facilitating analysis of target inhibition by reversible compounds. Light and heavy samples are combined in a 1:1 (w/w) ratio, and biotinylated proteins are enriched, trypsinized, and analyzed by LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block FP-PEG-biotin labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, giving a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio close to 1.

Sample Preparation. BW5147-derived murine T-cell hybridoma cells were initially grown for 6 passages in either light or heavy SILAC RPMI 1640 medium supplemented with 10% dialyzed FCS and 1x PenStrep Glutamine. Light medium was supplemented with 100 µg/mL L-arginine (Sigma) and 100 µg/mL L-lysine (Sigma). Heavy medium was supplemented with 100 µg/mL [¹³C₆¹⁵N₄]-L-Arginine (Isotek) and 100 µg/mL [¹³C₆¹⁵N₂]-L-Lysine (Isotek). Cells were harvested and homogenized by sonication in DPBS. The soluble and membrane fractions were isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS. Membrane and soluble fractions of light cells were treated with test compound (1 µM) and membrane and soluble fractions of heavy cells were treated with DMSO for 30 minutes at 37 °C. All samples were reacted with the activity-based affinity probe FP-PEG-Biotin (5 µM) for 30 minutes at 25 °C. Light and heavy proteomes were mixed in 1:1 (w/w) ratio to generate one membrane and one soluble sample, which were desalted over PD-10 columns (GE Healthcare). SDS was added to a final concentration of 0.5% in 3 mL total reaction volume and biotinylated proteins were enriched on streptavidin beads (50 µL, 1 hour, 25 °C). The beads were washed with 1% SDS in DPBS (1x), 6M urea (1x), and DPBS (2x), then resuspended in 6 M urea (150 µL), reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25 °C in the dark, and urea concentration was reduced to 2 M with 2x volume DPBS. On-bead digestions were performed for 12 hours at 37 °C with sequence-grade modified trypsin (Promega; 2 µg) in the presence of 2 mM CaCl₂. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid and stored at -80 °C prior to analysis.

LC-MS/MS analysis. Samples were analyzed by multidimensional liquid chromatography tandem mass spectrometry (MudPIT) using an Agilent 1100-series quaternary pump and Thermo Scientific LTQ Orbitrap ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (60 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 7 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.1; publicly available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the Sequest algorithm against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. Sequest searches allowed for static modification of cysteine residues (+57.02146, alkylation), variable methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, +8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the --modstat, --mass, and --trypstat options (applies different statistical models for the analysis of methionine oxidation, high resolution masses, and peptide digestion state, respectively). Ratios of light/heavy peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were >3 standard deviations from the median peptide value. Proteins with fewer than three peptides per protein ID were not included in the analysis. Assay Cutoff: A compound was considered active for a particular target/anti-target with a light/heavy ratio of ≤0.5.

Selectivity analysis by ABPP-SILAC in situ (AID 651980)

Assay Overview: The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using stable isotope labeling with amino acids in cell culture (SILAC) ABPP. In this assay, cultured HEK293T cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with inhibitor and DMSO, respectively,in situ, followed by the cell-permeable alkyne-functionalized triazole urea ABPP probe NHU5 [12]. NHU5 covalently reacts with a subset of serine hydrolases, including LYPLA2 and LYPLA1, and shows slower reaction kinetics than other cell-permeable serine hydrolase-specific ABPP probes (e.g., FP-alkyne), facilitating analysis of target inhibition by reversible compounds. Cells are harvested, proteome fractions isolated and reacted with a biotin-functionalized azide tag under copper(I)-catalyzed azide-alkyne cycloaddition (“click chemistry”) reaction conditions for enrichment of probe-tagged proteins. Light and heavy samples are combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block NHU5 probe labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, giving a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio close to 1.

Sample Preparation. HEK293T cells were initially grown for 6 passages in either light or heavy SILAC DMEM medium supplemented with 10% dialyzed FCS and 1x PenStrep Glutamine in a humidified incubator (37 °C, 5% CO₂). Light medium was supplemented with 100 µg/mL L-arginine (Sigma) and 100 µg/mL L-lysine (Sigma). Heavy medium was supplemented with 100 µg/mL [¹³C₆¹⁵N₄]-L-Arginine (Isotek) and 100 µg/mL [¹³C₆¹⁵N₂]-L-Lysine (Isotek). Light cells were treated with test compound (5 µM) and heavy cells were treated with DMSO for 3 hours at 37 °C. Activity-based alkyne probe NHU5 (50 µM) was added, and cells incubated for 1 hour at 37 °C. After 1 hour, cells were washed (4x DPBS), harvested, isolated by centrifugation at 1,400 x g for 3 minutes, re-suspended in methanol/chloroform/dH₂O (4:1:3 v/v), and vortexed. After centrifugation at 13,000 x g for 3 minutes, the upper layer was removed and 3 volumes of methanol were added and the solution vortexed. Protein was pelleted by centrifugation at 13,000 x g for 6 minutes. The supernatant was removed and the pellet was air-dried and re-suspended in PBS by sonication. Light and heavy proteomes were mixed in 1:1 (w/w) ratio to generate one membrane and one soluble sample. For affinity purification of probe-labeled proteins, click chemistry with a biotin-azide tag (100 µM) was carried out under standard conditions (1 mM TCEP, 100 µM TBTA ligand, 1 mM Cu(II)sulfate, 1 hour, 25 °C), and excess reagents removed by washing the precipitated protein with cold methanol (3 × 750 µL). The protein pellets were resolubilized in 2.5% SDS/DPBS (750 µL) by sonication and heating (60 degrees C, 5 minutes). The concentration of SDS was reduced to 0.5% with DPBS (3 mL), and biotinylated proteins enriched with streptavidin beads (50 µL, 1 hour, 25 °C). The beads were washed with 1% SDS in DPBS (1x), 6 M urea (1x), and DPBS (2x), then resuspended in in 6 M urea (150 µL), reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25 °C in the dark, and the urea concentration was reduced to 2 M with 2x volume DPBS. On-bead digestions were performed for 12 hours at 37 °C with sequence-grade modified trypsin (Promega; 2 µg) in the presence of 2 mM CaCl₂. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid and stored at -80 °C prior to analysis.

LC-MS/MS analysis. Samples were analyzed by multidimensional liquid chromatography tandem mass spectrometry (MudPIT) using an Agilent 1100-series quaternary pump and Thermo Scientific LTQ Orbitrap ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (60 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 7 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.1; publicly available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the Sequest algorithm against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. Sequest searches allowed for static modification of cysteine residues (+57.02146 due to alkylation), variable methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, +8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the --modstat, --mass, and --trypstat options (applies different statistical models for the analysis of methionine oxidation state, high resolution masses, and peptide digestion state, respectively). Ratios of light/heavy peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were >3 standard deviations from the median peptide value. Proteins with fewer than three peptides per protein ID were not included in the analysis. Assay Cutoff A compound was considered active for a particular target/anti-target with a light/heavy ratio of ≤0.5.