Primary uHTS assay to identify DAGL-β inhibitors (AID 504411)

Assay Overview: The purpose of this assay is to identify compounds that inhibit the activity of human diacylglycerol lipase-beta (DAGL-β). In this assay, compounds are preincubated with membranes derived from mouse DAGL-β-transfected HEK293T cells (Open Biosystems Accession BC016105), followed by incubation with the fluorogenic lipase substrate EnzChek. Fluorescence is determined at a specific time point. As designed, compounds that act as DAGL-β inhibitors will slow the rate of substrate hydrolysis, resulting in a decrease in well fluorescence. Compounds are tested in singlicate at a final nominal concentration of 5.17 μM.
Protocol Summary: Prior to the start of the assay, DAGL-β membrane lysate was sonicated three times for 5 seconds, 1 minute between each pulse using a microtip. Then, 4.0 μL of Assay Buffer (50 mM HEPES pH7.2, 100 mM NaCl, 5 mM CaCl2, 0.1% Triton X-100, 10% DMSO and 0.5 mM DTT) containing 0.375 mg/mL of DAGL-β membrane lysate were dispensed into 1536 microtiter plates. Next, 26 nL of test compound in DMSO or DMSO alone (0.52% final concentration) were added to the appropriate wells and incubated for 60 minutes at 25°C. The assay was started by dispensing 1.0 μL of 5 μM EnzChek lipase substrate in Assay Buffer to all wells. Plates were centrifuged and incubated for 60 minutes at 25°C. Fluorescence was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a FITC filter set (excitation = 480 nm, emission = 540 nm) and a FITC dichroic mirror for 1 second. Assay Cutoff: Compounds that inhibited DAGL-β ≥34.1% were considered active.

Confirmation uHTS assay to identify DAGL-β inhibitors (AID 504445)

Assay Overview: The purpose of this assay is to confirm activity of compounds identified as active in the primary uHTS pPAFAH screen (AID 504411). In this assay, the fluorogenic lipase substrate EnzChek is used to assess inhibition of DAGL-β in a complex membrane preparation as described above (AID 504411). Compounds were tested in singlicate at a final nominal concentration of 5.17 μM.
Protocol Summary: The assay was performed as described above (AID 504411), except that compounds were tested in triplicate. Assay Cutoff: Compounds that inhibited DAGL-β ≥34.1% were considered active.

Gel-based competitive ABPP inhibition of overexpressed DAGL-β in vitro: triazole urea library (AID 602303)

Assay Overview: The purpose of this assay is to determine whether test compounds belonging to the triazole urea scaffold can inhibit DAGL-β in a complex proteome using a gel-based competitive activity-based proteomic profiling (ABPP) assay. In this assay, the target enzyme DAGL-β is incubated with test compound followed by reaction with a fluorescently-tagged serine-hydrolase specific activity-based probe, fluorophosphonate-rhodamine (FP-Rh, Thermo #88318). For this assay and all other gel-based ABPP assays described below, 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 DAGL-β inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. See also refs. [8, 9].
Protocol Summary: Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse DAGL-β (25 μL of 0.3 mg/mL) in Dulbecco’s PBS (DPBS) was treated with test compound (0.5 μL of a 50x stock in DMSO; 500 nM final concentration) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe FP-Rh (0.5 μL of a 50x stock in DMSO; 5 μM final concentration) was added, and the reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage of DAGL-β activity remaining was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition were considered active.

Gel-based competitive ABPP inhibition of ABHD6 in vitro (AID 602319)

Assay Overview: The purpose of this assay is to determine whether test compounds can inhibit endogenous ABHD6 using a gel-based ABPP assay. In this assay, a complex proteome natively expressing ABHD6 is incubated with test compound followed by reaction with a fluorescently-labeled activity-based probe, HT-01 [9], which selectively labels several serine hydrolases including ABHD6.The reaction products are analyzed as described for AID 602303.
Protocol Summary: Membrane proteome of Neuro-2A murine neuroblastoma cells (25 μL of 1 mg/mL) in Dulbecco’s PBS (DPBS) was treated with test compound (0.5 μL of a 50x stock in DMSO; 1 nM or 10 nM final concentration) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe HT-01 (0.5 μL of a 50x stock in DMSO; 1 μM final concentration) was added, and the reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage of ABHD6 activity remaining was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition at 10 nM compound concentration were considered active.

Gel-based competitive ABPP inhibition of overexpressed anti-target DAGL-β in vitro (AIDs 602299 and 602300)

Assay Overview: The purpose of this assay is to determine whether test compounds can inhibit anti-target DAGL-β in a complex proteome using a gel-based ABPP assay. In this assay, DAGL-β is incubated with test compound followed by reaction with a fluorescently-labeled activity-based probe, HT-01 [9], which selectively labels several serine hydrolases including DAGL-β. The reaction products are analyzed as described for AID 602303.
Protocol Summary: Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse DAGL-β (25 μL of 0.3 mg/mL) in Dulbecco’s PBS (DPBS) was treated with test compound (0.5 μL of a 50x stock in DMSO; 10 μM [AID 602299] or 1 μM [AID 602300] final concentration) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe HT-01 (0.5 μL of a 50x stock in DMSO; 1 μM final concentration) was added, and the reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage of DAGL-β activity remaining was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition were considered active.

Gel-based competitive ABPP inhibition of overexpressed ABHD11 in vitro (AID 602312)

Assay Overview: The purpose of this assay is to determine whether test compounds can inhibit anti-target ABHD11 in a complex proteome using a gel-based competitive ABPP assay. In this assay, ABHD11 is spiked into a complex proteome and incubated with test compound followed by reaction with the ABPP probe FP-Rh. The reaction products are analyzed as described for AID 602303.
Protocol Summary: Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse DAGL-β (25 μL of 0.3 mg/mL; Open Biosystems Accession BC016105) with added recombinant mouse ABHD11 (0.050 μM, Open Biosystems Accession BC069866) in Dulbecco’s PBS (DPBS) was treated with test compound (0.5 μL of a 50x stock in DMSO; 10 μM, 3.3 μM, 1 μM, or 0.3 μM final concentration) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe FP-Rh (0.5 μL of a 50x stock in DMSO; 5 μM final concentration) was added, and the reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage of ABHD11 activity remaining was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control.Assay Cutoff: Compounds with ≥50% inhibition were considered active.

Gel-based competitive ABPP inhibition of SHs in mouse brain membrane with FP-Rh (AIDs 602311 and 602321)

Assay Overview: The purpose of this assay is to assay test compound inhibition of serine hydrolases (SHs) using a gel-based competitive ABPP assay. In this assay, a complex proteome is incubated with test compound followed by reaction with the ABPP probe FP-Rh. The reaction products are analyzed as described for AID 602303.
Protocol Summary: Mouse brain membrane proteome (25 μL of 1 mg/mL in Dulbecco’s PBS [DPBS]) was treated with test compound (0.5 μL of a 50x stock in DMSO; 10 μM final concentration for AID 602311, 1 μM and 10 μM final concentration for AID 602321) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe FP-Rh (0.5 μL of a 50x stock in DMSO; 1 μM final concentration) or HT-01 (0.5 μL of a 50x stock in DMSO; 1 μM final concentration) was added, and the reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage activity remaining for each SH was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control. SHs with at least 50% inhibition by one or more compounds (FP-Rh: ABHD6, APEH, FAAH, KIAA1363, ABHD12, MAGL, LYPLA1, and LYPLA2; HT-01: DAGL-β, ABHD6, PLA2G7, and a SH with MW of 80kDa) are reported. Assay Cutoff: Compounds with greater than or equal to 50% inhibition for a given SH were considered active.

Gel-based competitive ABPP inhibition of ABHD6 and DAGL-β for IC50 determination in vitro (AID 602322)

Assay Overview: The purpose of this assay is to determine the IC50 values of powder samples of test compounds for ABHD6 and anti-target DAGL-β inhibition in a complex proteome lysate using a competitive ABPP assay. In this assay, the ABPP probe HT-01 is used to label a complex proteome natively expressing ABHD6 and anti-target DAGL-β in the presence of test compounds. The reaction products are analyzed as described for AID 602303.
Protocol Summary: Membrane proteome of Neuro-2A murine neuroblastoma cells (25 μL of 1 mg/mL) in Dulbecco’s PBS (DPBS) was treated with test compound (0.5 μL of a 50x stock in DMSO) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe HT-01 (0.5 μL of a 50x stock in DMSO; 1 μM final concentration) was added, and the reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage activity remaining for ABHD6 and anti-target DAGL-β was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration (7-point 1:3 dilution series from either 3333 nM to 3.3 nM, 100 nM to 0.1 nM, or 100 nM to 0.033 nM). Note: inhibition of DAGL-β at each test concentration is also reported. Assay Cutoff: Compounds with an IC50 for ABHD6 less than or equal to 50 nM were considered active.

Gel-based competitive ABPP inhibition of SH anti-targets PNPLA6 and PLA2G15 in vitro (AID 602355)

Assay Overview: The purpose of this assay is to assay test compound inhibition of SHs using a gel-based competitive ABPP assay. In this assay, a complex proteome is incubated with test compound followed by reaction with the ABPP probe FP-Rh. The reaction products are analyzed as described for AID 602303.
Protocol Summary: The following proteome sources, prepared in Dulbecco’s PBS (DPBS), were used:PLA2G15: Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse PLA2G15 (0.3 mg/mL). PNPLA6: Mouse brain membrane proteome (1 mg/mL). Proteome (25 μL) was treated with test compound (0.5 μL of a 50x stock in DMSO, 10, 2, 0.4, 0.08, 0.016, or 0.003 μM final concentration) or DMSO (0.5 μL) for 30 minutes at 37°C. The activity-based probe FP-Rh (0.5 μL of a 50x stock in DMSO; 1 μM final concentration) was added. The reaction was incubated for 30 minutes at 37°C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage activity remaining for each SH was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control.Assay Cutoff: Compounds with greater than or equal to 50% inhibition for a given SH at 2 μM test compound concentration were considered active.

Gel-based competitive ABPP inhibition of ABHD6 and DAGL-β for IC50 determination in situ (AID 602335)

Assay Overview: The purpose of this assay is to determine the IC50 values of powder samples of test compounds for ABHD6 and anti-target DAGL-β inhibition in situ using a competitive ABPP assay. In this assay, the ABPP probe HT-01 is used to label ABHD6 and DAGL-β in the presence of test compounds. The reaction products are analyzed as described for AID 602303.
Protocol Summary: Cultured Neuro-2A murine neuroblastoma cells (5 mL DMEM medium with 10% FCS) were treated with test compound (1 μL of 5000x stock in DMSO) or DMSO only (1 μL) and incubated for 4 hours at 37°C. Cells were washed with DPBS (4x), harvested, and homogenized by sonication. The protein concentration was adjusted to 2 mg/mL with Dulbecco’s PBS (DPBS), and an aliquot (50 μL) was reacted with HT-01 (1 μL of a 50x stock in DMSO; 1 μM final concentration) for 30 minutes at 37°C. Samples were quenched with an equal volume of 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 ABHD6 and DAGL-β bands relative to the DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration (8-point 1:3 dilution series from 100 nM to 0.033 nM). Assay Cutoff: Compounds with an IC50 less than or equal to 50 nM were considered active.

Analysis of triazole urea cytotoxicity (AID 602337)

Assay Overview: The purpose of this assay is to determine cytotoxicity of powder samples of synthetic inhibitor compounds belonging to the triazole urea scaffold. In this assay, Neuro-2A murine neuroblastoma cells in either serum-free medium (Assay 1) or medium containing fetal calf serum (FCS) (Assay 2) 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 Neuro-2A murine neuroblastoma cells 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 (Assay 1) or medium supplemented with 10% FCS (Assay 2) 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 (100, 50, 10, 1, 0.1, and 0.01 μM). Assay Cutoff: Compounds with CC50 values less than or equal to 1 μM were considered active (cytotoxic).

LC-MS/MS-based ABPP-SILAC analysis of selectivity in situ for ML295 (AID 602341)

Assay Overview: The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using ABPP in combination with stable isotope labeling with amino acids in cell culture (SILAC) [10] as described [7, 9]. In this assay, cultured Neuro-2A cells are metabolically labeled with heavy or light amino acids. Heavy and light cells are treated with test compound and DMSO, respectively, in situ. Cells are lysed, proteomes are treated with the serine-hydrolase-specific activity-based fluorophosphonate-biotin (FP-biotin, [11]) affinity probe, and combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by multi-dimensional liquid chromatography tandem mass spectrometery LC/LC-MS/MS (MudPIT) [12, 13]. 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-biotin labeling, reducing enrichment in the inhibitor-treated (heavy) sample relative to the DMSO-treated (light) sample, giving a smaller heavy/light ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio of 1.
Protocol Summary: Sample Preparation. Neuro-2A murine neuroblastoma cells were initially grown for 10 passages in either light or heavy SILAC DMEM medium supplemented with 10% dialyzed FCS and 2 mM L-glutamine. Light medium was supplemented with 100 ug/mL L-arginine and 100 ug/mL L-lysine. Heavy medium was supplemented with 100 ug/mL [¹³C₆¹⁵N₄]-L-Arginine and 100 ug/mL [¹³C₆¹⁵N₂]-L-Lysine. Heavy cells (in 10 mL medium) were treated with test compound (10 μL of a 1000x stock in DMSO; 25 nM final concentration) and light cells were treated with DMSO (10 μL) for 4 hours at 37°C. Cells were washed with DPBS (4x), 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 for each fraction was adjusted to 2 mg/mL with DPBS. The light and heavy proteomes were labeled with the activity-based affinity probe FP-biotin (500 μL total reaction volume, 10 μM final concentration) for 2 hours at 25°C. After incubation, light and heavy proteomes were mixed in 1:1 ratio, and the membrane proteomes were additionally solubilized with 1% Triton-X100. Samples were desalted over PD10 columns (GE Healthcare) in DPBS, and biotinylated proteins enriched with streptavidin beads (50 μL beads; conditions: 1 hour, 25°C 0.5% SDS in DPBS). 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. 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 1200-series quaternary pump and Thermo Scientific LTQ-Orbitrap Velos 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 (20 seconds, repeat of 1). Specifically, one full MS (MS1) scan (400–1800 m/z) was followed by 30 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.9.2; publicly available athttp://fields.scripps.edu/researchtools.php). MS2 spectra data were searched using the ProLuCID algorithm (publicly available at http://fields.scripps.edu/downloads.php) against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. ProLucid searches allowed for static modification of cysteine residues (+57.02146 due to alkylation), 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 2.0.41, http://fields.scripps.edu/researchtools.php) using the –modstat, –mass, and –trypstat options (applies different statistical models for the analysis of high resolution masses, peptide digestion state, and methionine oxidation state respectively). Ratios of heavy/light (test compound/DMSO) 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 less 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 less than or equal to 0.5.

Gel-based competitive ABPP inhibition of ABHD6 in vivo (AID 602349, AID 624041, AID 602343, and AID 624077)

Assay Overview: The purpose of this assay is to determine whether or not powder samples of test compounds are active against ABHD6 in vivo using a gel-based competitive ABPP assay. In this assay, test compounds are administered to mice. Mice are sacrificed, and their brain and/or liver tissue is harvested, homogenized, and the membrane fraction isolated and reacted with either the ABPP probe FP-Rh or HT-01. The reaction products are analyzed as described for AID 602303.
Protocol Summary: Purpose-bred C57-black laboratory mice were administered test compound by either intraperitonial injection (i.p.) (1, 0.5, and 0.1 mg/kg for AID 602349; 20 and 5 mg/kg for AID 624041) or oral administration (p.o.) (10, 5, or 1 mg/kg for AID 602343 and AID 624077) or vehicle only (18:1:1 saline:PEG300:EtOH); n = 1 per group. After 4 hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and tissue (brain or liver) was removed and snap frozen in liquid nitrogen. Tissues were homogenized and the membrane fraction isolated by centrifugation (45 minutes, 100K x g) and adjusted to 1 mg/mL in DPBS. Aliquots (50 μL) were reacted with the activity-based probes HT-01 or FP-Rh (1 μL of a 50x stock in DMSO, 1 μM final concentration) for 30 minutes at 37°C. The reactions were quenched with an equal volume of 2x 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 test compound bands relative to vehicle bands. Assay Cutoff: Compounds with greater than or equal to 50% inhibition at 1 mg/kg compound (AID 602349), at 5 mg/kg compound (AID 624041), or 10 mg/kg compound (AIDs 602343 and 624077) were considered active.

LC-MS/MS-based ABPP-MudPIT analysis of selectivity in vivo for ML295 (AID 602351)

Assay Overview: The purpose of this assay is to determine the selectivity profile of powder samples of test compounds in vivo by ABPP in combination with the multidimensional protein identification technology (MudPIT) liquid chromatography tandem mass spectrometry (LC-M/MS) analysis platform [9, 14]. In this assay, mice are administered test compound or vehicle only. Mice are sacrificed, and primary macrophages removed, lysed, and reacted with the serine hydrolase specific activity-based fluorophosphonate-biotin (FP-biotin) affinity probe. Biotinylated proteins are enriched, trypsinized, and analyzed by MudPIT. Inhibition of target and anti-target activity is quantified by comparing spectra counts between the test compound- and DMSO-treated samples. As designed, compounds that act as inhibitors will block FP-biotin labeling, reducing enrichment in the inhibitor-treated sample relative to the DMSO-treated sample, thus giving a lower number of smaller spectra counts in the inhibitor treated sample. Proteins not targeted by inhibitors would be expected to have a spectra count ratio similar to DMSO control.
Protocol Summary: Sample Preparation. Purpose-bred C57-black laboratory mice, injected 4 days prior with thioglycollate, were administered test compound (5 mg/kg in 18:1:1 saline:PEG300:EtOH vehicle solution, i.p.) or vehicle only (n=3 per group). After 4 hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and primary macrophages were removed and snap frozen in liquid nitrogen. Macrophages were homogenized and the protein concentration for each lysate was adjusted to 2 mg/mL with DPBS. The macrophage proteomes were labeled with the activity-based affinity probe FP-biotin (500 μL total reaction volume, 10 μM final concentration) for 2 hours at 25°C. After incubation, the proteomes were additionally solubilized with 1% Triton-X100. Samples were desalted over PD10 columns (GE Healthcare) in DPBS, and biotinylated proteins enriched with streptavidin beads (50 μL beads; conditions: 1 hour, 25°C 0.5% SDS in DPBS). The beads were washed with 1% SDS in DPBS (1x), 6 M 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. 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 ug) 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. Digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (MudPIT) using an Agilent 1200-series quaternary pump and Thermo Scientific LTQ 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, with chromatographic elution following each salt pulse accomplished using an increasing gradient of aqueous acetonitrile containing 0.1% formic acid over 125 minutes. Data were collected in data-dependent acquisition mode with dynamic exclusion turned on (90 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; publically available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the PROLUCID algorithm (publically available athttp://fields.scripps.edu/downloads.php) against the latest version of the mouse UniProt database concatenated with the reversed database for assessment of false-discovery rates. PROLUCID searches allowed for variable oxidation of methionine (+16), static modification of cysteine (+57 due to alkylation), and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0.41) using the –trypstat and –modstat options, which apply different statistical models for the analysis of tryptic, half-tryptic, non-tryptic, and modified peptides. Spectra count abundance ratios (test compound/DMSO) were calculated for all serine hydrolases with an average of 10 or more spectra counts in the DMSO control samples, as well as for anti-target ABHD6, which had fewer spectra counts than the 10-count cutoff. A Student’s T-test (unpaired, equal variances) was utilized to determine statistical significance between the average spectra counts in compound vs. DMSO samples. Assay Cutoff: A compound was considered active for a particular target/anti-target if there was a statistical difference between the average spectra counts in the test compound- and DMSO-treated samples (P < 0.01).

Figure 1

IC50 curves for probes ML295 (SID 125269138) and ML296 (SID 125269096) and compound 12 (SID 125269091). Determined by gel-based competitive-ABPP with the activity-based probe HT-01 against endogenous ABHD6 in a complex proteome lysate (AID 602322, left)(more…)

Figure 2

Structures of prior-art ABHD6 carbamate inhibitors. WWL70 has only been characterized in vitro and in situ, and WWL123 is only active at high (20 mg/kg) dose in vivo. As such, the triazole urea probes ML295 and ML296 are both more potent and better characterized (more…)