The Scripps Research Institute Molecular Screening Center (SRIMSC) recently completed a fluorescence-polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify inhibitors of protein phosphatase methylesterase-1 (PME-1). This campaign unveiled a phenomenal class of potent and selective inhibitors, the aza-beta lactams (ABLs), one of which, ML174, showed exceptional in situ and in vivo potency and selectivity for PME-1. During medicinal chemistry investigation of the ABLs for PME-1, we observed that one of the common anti-targets was the uncharacterized serine hydrolase abhydrolase domain containing protein 10 (ABHD10). This fortuitous discovery of inhibitor leads was of particular interest to us, as we had recently uncovered some exciting evidence that ABHD10 functions as a lipase in situ. A principle goal of post-genomic research is to elucidate the molecular and cellular roles of uncharacterized enzymes like ABHD10, an investigation which profits significantly from chemical tools for precise regulation of enzyme activity. Given that no selective inhibitors of ABHD10 have yet been reported in the literature, we completed a medicinal chemistry campaign to optimize an ABL probe for ABHD10, which is presented herein as ML257. This probe is highly potent against ABHD10 in vitro (IC50 = 17 nM), in situ (IC50 = 28 nM), and in vivo (active at 25 mg/kg, i.p., in mice), and exhibits remarkable selectivity among 40+ other serine hydrolases. Importantly, this probe demonstrates the potential for exploiting fortuitous inhibitor leads for orthogonal, “anti-target” enzymes, thus maximizing the benefits of a single HTS campaign, and highlights the “privileged” nature of the ABL scaffold for serine hydrolase inhibitor development.
Assigned Assay Grant #: 1 R01 CA132630-01
Screening Center Name & PI: The Scripps Research Institute Molecular Screening Center (SRIMSC), H Rosen
Chemistry Center Name & PI: SRIMSC, H Rosen
Assay Submitter & Institution: BF Cravatt, TSRI, La Jolla
PubChem Summary Bioassay Identifier (AID): 2143
Probe Structure & Characteristics
Recommendations for Scientific Use of the Probe
ABHD10 is one of many unannotated proteins that exist in the mammalian genome. We have preliminary evidence that suggests ABHD10 may function as a lipase in situ, but its role(s) in lipid metabolism and substrate profile are entirely unknown. This inhibitor is intended for primary research studies into the biochemical characterization of ABHD10, both in vitro and in cultured cells.
Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for more than 90% of all serine/threonine phosphatase activity . Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications (PTMs) rather than number. Mutations in several PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor . Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated [3, 4]. PME-1 is specifically responsible for demethylation of the carboxyl terminus , where the PTM is thought to control the binding of different subunits to PP2A; however, little is known about physiological significance of this post-translational modification in vivo. Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas .
As a serine hydrolase (SH), catalytically active PME-1 is readily labeled by fluorescent activity-based protein profiling (ABPP) probes bearing a fluorophosphonate (FP) reactive group . This reactivity can be exploited for inhibitor discovery using a competitive ABPP platform, whereby small molecule enzyme inhibition is assessed by the ability to out-compete ABPP probe labeling . Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, fluopol-ABPP . A fluopol-ABPP HTS assay for PME-1 inhibitor discovery unveiled a phenomenal class of potent and selective inhibitors, the aza-beta lactams (ABLs), which included a highly potent and selective and in vivo-active PME1 inhibitor, ML174 (see also reference ).
During our medicinal chemistry campaign to refine ABL inhibitors for PME-1 (See Probe Report for ML174), we observed that one of the common anti-targets of several ABL members was the uncharacterized SH abhydrolase domain containing protein 10 (ABHD10). This fortuitous discovery of inhibitor leads was of particular interest to us, as we had recently uncovered some exciting evidence, while conducting a global profile of dynamic protein palmitoylation , that ABHD10 functions as a lipase in situ.
Protein palmitoylation is a ubiquitous PTM that governs protein activity, trafficking, and membrane localization [13, 14]. Because palmitoylation involves appending palmitic acid to cysteine via a reversible thioester bond, this modification is potentially enzymatically reversible. Candidate protein palmitoyl thioesterases belonging to the SH superfamily, LYPLA1  and PPT1 , have been identified, but their relevance to dynamic regulation of palmitoylation in living systems has yet to be fully explored. To investigate the scope of SH involvement in regulation of palmitoylation, we designed a platform for identification of SHs with lipase activity in situ. The activity-based probe FP-Rh is a near-universal affinity label for the SH superfamily ; to restrict probe reactivity to SHs involved in depalmitoylation, we incorporated the SH-directed FP reactive moiety into palmitoyl-mimetic scaffold to generate a hexadecylfluorophosphonate (HDFP) activity-based probe . Reactivity profiling using the quantitative LC-MS/MS-based platform ABPP-SILAC , which combines competitive ABPP with stable isotope labeling of cells (SILAC) , revealed that enzymes susceptible to HDFP labeling included nearly all annotated lipases but few proteases, peptidases, or the proteasome. Interestingly, a handful of unannotated SHs, including ABHD10, also reacted strongly with HDFP, suggesting that they may function as lipases in situ. However, further investigation of their involvement in palmitoylation and lipid metabolism requires means to selectivity control activity, e.g., through the application of specific chemical inhibitors. As no known, selective inhibitors of ABHD10 have been reported, we completed a medicinal chemistry campaign to optimize an ABL probe for ABHD10, ML257.
As described herein and by Zuhl et al., , ML257 is highly potent against ABHD10 both in vitro (IC50 = 17 nM), in situ (IC50 = 28 nM), and in mice in vivo, and exhibits remarkable selectivity among dozens of SHs as assessed by both gel-based competitive ABPP and in-depth ABPP-SILAC profiling. ML257 is the first reported selective chemical inhibitor for ABHD10, which should greatly facilitate the metabolic and functional characterization of this intriguing enzyme. Importantly, this probe also demonstrates the potential for exploiting fortuitous inhibitor leads for orthogonal, “anti-target” enzymes, thus maximizing the benefits of a single time- and resource-intensive HTS campaign, and highlights the “privileged” nature of the ABL scaffold for SH inhibitor development.
2. Materials and Methods
All reagents were obtained from ThermoFisher or SigmaAldrich, unless otherwise noted. See AID protocol details for further details.
Probe Characterization Assays
Solubility in PBS. The solubility of compounds are tested in triplicate in phosphate buffered saline (PBS), pH 7.4. per well, 198 μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well filter plate: pore size: 0.45um (MSRLN0450). Test compounds are introduced from 10 mM DMSO stock solutions (2 μl). The final concentration of DMSO was 1 percent. Samples are allowed to incubate at 22 °C for 18 hours. In the morning the plate is centrifuged where the soluble portion passes through the filter and is collected in a capture plate. Clotrimazole is included as a control to assure the assay is working properly. The samples are analyzed by HPLC. Peak area is compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS-MS analysis is used.
Solubility in Media. The solubility of compounds are tested in triplicate in complete media (MDEM + 10% FBS). Per well, 198 μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well filter plate: pore size: 0.45um (MSRLN0450). Test compounds are introduced from 10 mM DMSO stock solutions (2 μl). The final concentration of DMSO was 1 percent. Samples are allowed to incubate at 22°C for 18 hours. In the morning the plate is centrifuged where the soluble portion passes through the filter and is collected in a capture plate. The samples are analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area is compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS-MS analysis is used.
Stability in PBS. Demonstration of stability in PBS was conducted by addition of 0.2 μM compound from a DMSO stock to PBS in HPLC autosampler vials. Samples are held in the HPLC autosampler at ambient temperature. At approximately 0, 1, 2, 4, 8, 24, and 48 hours the samples are injected on the HPLC. Peak area and retention time are compared between injections. Data is log transformed and represented as half life. DMSO is added as a co-solvent as needed for solubility.
Determination of Glutathione reactivity. Compound (10 μM) is incubated at 37°C for 6 hours in the presence of 50 μM freshly prepared reduced glutathione. At 0 and 6 hours the samples are injected on the HPLC. Peak area and retention time are compared between injections. Samples are evaluated for a glutathione dependent decrease in compound concentration. DMSO is added as a co-solvent as needed for solubility.
Primary uHTS assay to identify PME-1 inhibitors (AID 2130)
Assay Overview: The purpose of this assay was to identify compounds that act as PME-1 inhibitors. This competitive ABPP assay uses fluorescence polarization to investigate enzyme-substrate functional interactions based on active site-directed molecular probes. The SH-specific FP-Rh probe (now commercially available through Thermo Scientific, #88318) was used to label PME-1 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 PME-1 inhibitors will prevent PME-1-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 detergent, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1mM DTT) containing purified PME-1 protein (1.25 μM) 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 45 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 mP value was obtained via the PerkinElmer Viewlux software. Assay Cutoff: compounds that inhibited PME-1 greater than 26.13% (mean + 3x standard deviation) were considered active.
Confirmation uHTS assay to identify PME-1 inhibitors (AID 2171)
Assay Overview: The purpose of this assay was to confirm activity of compounds identified as active in the primary uHTS screen (AID 2130). In this assay, the FP-Rh probe was used to label PME-1 in the presence of test compounds and analyzed as described above (AID 2130). Compounds were tested in triplicate at a nominal concentration of 5.9 μM.
Protocol Summary: The assay was performed as described above (AID 2130), except that compounds were tested in triplicate. Assay Cutoff: compounds that inhibited PME-1 greater than 26.13% were considered active.
All secondary assays described below were conducted with power samples of synthetic compounds.
Inhibition and selectivity of ABLs for ABHD10 (AID 588807)
Assay Overview: The purpose of this assay is to assess the potency and selectivity of ABHD10-directed inhibition of test compounds in a complex proteomic lysate using an ABPP assay. In this assay, a complex proteome is incubated with test compound followed by reaction with the FP-Rh activity-based probe (now commercially available through Thermo Scientific, #88318). 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 ABHD10 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.
Protocol Summary: Mouse brain membrane proteome (1 mg/mL in Dulbecco’s PBS [DPBS]; 50 μL reaction volume) was treated with 10 μM, 1 μM or 0.1 μM test compound (1 μL of a 50× stock in DMSO) for 30 minutes at 37°C, and FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 1 μM. The reaction was incubated for 30 minutes at 25°C, quenched with 4× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the IOD of the target (ABHD10) and anti-target (APEH, PREP, KIAA1363, MAGL, and ABHD6) bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition of ABHD10 at 0.1 μM test compound concentration and less than 50% inhibition of all anti-targets at 1 μM concentration were considered active.
Inhibition of ABHD10 in situ (AID 588806)
Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds can inhibit ABHD10 in situ. In this assay, cultured cells are incubated with test compound. Cells are harvested, homogenized, and reacted with the FP-Rh activity-based probe. The reaction products are separated by SDS-PAGE and analyzed as described for AID 588807.
Protocol Summary: Cultured BW5147-derived murine T cells in serum-free RPMI medium (15 mL total volume) were treated with DMSO or test compound (250 nM, 100 nM, or 50 nM; 75 μL of a 200× stock in DMSO) for 4 hours at 37°C. Cells were washed with DPBS, resuspended in DPBS (400 μL), and homogenized by sonication. The protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 1 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25°C, quenched with 4× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the IOD of the ABHD10 band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with greater than or equal to 50% inhibition of ABHD10 at 250 nM were considered active.
Determination of IC50 values for ABHD10 inhibition in vitro (AID 588802 and AID 588796)
Assay Overview: The purpose of this assay is to determine the IC50 values of powder samples of test compounds for ABHD10 inhibition in a complex proteome lysate. In this assay, the FP-Rh activity-based probe is used to label ABHD10 in the presence of test compounds. The reaction products are separated by SDS-PAGE and analyzed as described for AID 588807.
Protocol Summary: Mouse brain membrane proteome (1 mg/mL in DPBS, 50 μL reaction volume) was incubated with DMSO or test compound (1 μL of a 50× stock in DMSO) for 30 minutes at 37°C before the addition of FP-Rh (1 μL of a 50× stock in DMSO, 1 μM final concentration). The reaction was incubated for 30 minutes at 25°C, quenched with 4× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the IOD of the bands relative to DMSO only (no compound) control. IC50 values for inhibition of ABHD10 were determined from dose-response curves from three replicates at each inhibitor concentration (AID 588802: 20 μM, 10 μM, 1 μM, 0.25 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM; AID 588796: 8-point 4-fold dilution series from 50 μM to 3 nM). Assay Cutoff: Compounds with IC50 less than or equal to 500 nM were considered active.
Determination of IC50 values for ABHD10 and anti-target inhibition in situ (AID 588801 and AID 588835)
Assay Overview: The purpose of this assay is to determine the IC50 values of powder samples of test compounds for in situ inhibition of ABHD10 (AID 588801) and anti-targets PREP and ABHD6 (AID 588835). In this assay, cultured cells are incubated with test compound. Cells are harvested, homogenized, and reacted with the FP-Rh activity-based probe. The reaction products are separated by SDS-PAGE and analyzed as described for AID 588807.
Protocol Summary: Cultured Neuro-2A murine neuroblastoma cells in serum-free DMEM medium (1 mL total volume) were treated with DMSO or test compound (10 μL of a 100× stock in DMSO) for 2 hours at 37°C. Cells were harvested, washed with DPBS (1 mL), and resuspended in DPBS (1 mL). Centrifugation (1000 × g, 5 minutes) provided a cell pellet which was resuspended in DPBS (200 μL) and homogenized by sonication. The protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 1 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25°C, quenched with 4× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the IOD of the ABHD10 or anti-target (ABHD6 and PREP) bands relative to a DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration (10 μM, 1 μM, 250 nM, 50 nM, 10 nM, 5 nM, and 1 nM). Assay Cutoff: Compounds with IC50 less than or equal to 500 nM were considered active.
Analysis of ABL cytotoxicity in Neuro-2A cells (AID 588804)
Assay Overview: The purpose of this assay is to determine cytotoxicity of test compounds belonging to the ABL scaffold. In this assay, cells cultured in either serum-free medium or medium supplemented with 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 seeding Neuro-2A murine neuroblastoma cells in DMEM medium (100 μL, 15,000 cells per well) into a 96-well plate. After 36 hours, the medium was removed and inhibitors were added in both serum-free medium and medium supplemented with 10% FCS final compound concentration (100 μL total volume, 1% DMSO). Cells were incubated for 48 hours at 37°C in a humidified incubator and cell viability was determined using the WST-1 assay (Roche) according to manufacturer instructions. CC50 values were determined from dose-response curves from six replicates at each inhibitor concentration (7-point 1:5 dilution series from 10 μM to 0.64 nM). Assay Cutoff: Compounds with CC50 ≤5 μM in either the serum-free assay or serum-supplemented assay were considered active (cytotoxic).
ABPP-SILAC analysis of ABHD10 and PME-1 inhibitor selectivity (AID 588805 and AID 588803)
Assay Overview: The purpose of this assay is to determine the selectivity profile of test compounds using competitive ABPP  in combination with stable isotope labeling with amino acids in cell culture (SILAC)  as described . In this assay, cultured Neuro-2A cells are metabolically labeled with light or heavy amino acids. Heavy and light cells are treated with test compound and DMSO, respectively, in situ. Cells are lysed, and proteomes are treated with the serine-hydrolase-specific activity-based fluorophosphonate-biotin (FP-biotin) 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) [20, 21]. Inhibition of target and anti-target activity is quantified by comparing intensities of heavy and light 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.
Stable isotope labeling with amino acids in cell culture (SILAC). 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 μg/mL L-arginine and 100 μg/mL L-lysine. Heavy medium was supplemented with 100 μg/mL [13C615N4]-L-Arginine and 100 μg/mL [13C615N2]-L-Lysine. Heavy cells (in 10 mL medium) were treated with 100 nM test compound (50 μL of a 200× stock in DMSO) and light cells were treated with DMSO (50 μL) for 2 hours at 37°C. Cells were washed 2 times with DPBS, harvested, and homogenized by sonication in DPBS (1 mL). The soluble and membrane fractions were isolated by centrifugation (100K × g, 45 minutes) and the protein concentration was adjusted to 2 mg/mL with DPBS in each fraction.
Sample preparation for ABPP-SILAC. The light and heavy proteomes were labeled with 10 μM of FP-biotin (500 μL total reaction volume) 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. The proteomes were desalted over PD-10 desalting columns (GE Healthcare) and biotinylated proteins were enriched with streptavidin beads. The beads were washed with 1% SDS in DPBS (1x), 6M urea (1x), and DPBS (2x), then resuspended in 6 M urea, reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25°C in the dark. On-bead digestions were performed for 12 hours at 37°C with sequence-grade modified trypsin (Promega; 2 μg) in 2M urea in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid, pressure-loaded on to a biphasic (strong cation exchange/reversed phase) capillary column and analyzed as described below.
LC-MS/MS analysis. Digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography (2D-LC) separation in combination with tandem mass spectrometry 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 enabled (20 s, 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 18.104.22.168; publicly available at http://fields.scripps.edu/downloads.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) 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 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: Compounds that were active (heavy/light ratio less than or equal to 0.5) for ABHD10 and inactive (heavy/light ratio greater than 0.5) for all anti-targets were considered active.
Inhibition and selectivity of ML257 for ABHD10 (AID 602468)
Assay Overview: The purpose of this assay is to assess selectivity among cysteine-containing proteins of powder samples of test compounds both in vitro (complex proteomic lysates) and in situ (cultured cells) using an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with a rhodamine-conjugated chloroacetamide (CA-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and analyzed as described for AID 588807.
In vitro MBM Assay: Mouse brain membrane (MBM) and soluble proteomes (1 mg/mL in DPBS; 50 μL reaction volume) was treated with 20, 10, 1, or 0.1 μM test compound (1 μL of a 50× stock in DMSO). Test compounds were incubated for 30 minutes at 37 degrees, and CA-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 5 μM. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 4× 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 anti-target bands relative to a DMSO-only (no compound) control. Only proteins for which at least 50% inhibition is observed at any test compound concentration are counted as anti-targets. Assay Cutoff: Compounds with anti-targets at any test compound concentration were considered active.
In situ Neuro-2A Assay: Cultured Neuro-2A murine neuroblastoma cells in serum-free DMEM medium (1 mL total volume) were treated with DMSO or test compound (5 μL of a 200× stock in DMSO, 100 nM or 250 nM final concentration) for 2 hours at 37 degrees Celsius. Cells were washed with DPBS, harvested, and resuspended in DPBS (1 mL). Centrifugation (1000 × g, 5 minutes) provided a cell pellet that was resuspended in DPBS (200 μL) and homogenized by sonication. The membrane and soluble fractions were separated by centrifugation (100,000 × g, 45 minutes), and the protein concentrations were adjusted to 1 mg/mL with DPBS. CA-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 5 μM. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 4× 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 anti-target bands relative to a DMSO-only (no compound) control. Only proteins for which at least 50% inhibition is observed at any test compound concentration are counted as anti-targets. Assay Cutoff: Compounds with anti-targets at any test compound concentration were considered active.
In vivo activity of ML257 for ABHD10 (AID 602485)
Assay Overview: The purpose of this assay is to determine whether or not test compounds can inhibit ABHD10 in vivo and to assess selectivity using a gel-based activity-based protein profiling (ABPP) assay. In this assay, test compounds are administered to mice. Mice are sacrificed, and their heart tissue harvested, homogenized, and the soluble fraction isolated and reacted with the serine-hydrolase-specific activity-based probe FP-Rh. The reaction products are separated by SDS-PAGE and analyzed as described for AID 588807.
Protocol Summary: Purpose-bred C57BL/6 laboratory mice were administered test compound (6, 13, or 25 mg/kg in vehicle solution, i.p.) or vehicle only (n=1 per group). After six hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and heart tissues removed and snap frozen in liquid nitrogen. Tissues were homogenized and the soluble fraction isolated by centrifugation (45 min, 100K × g) and adjusted to 1 mg/mL in 50 mM Dulbecco’s PBS (DPBS). For control, one aliquot (50 μL) of vehicle-treated proteome was reacted with test compound (1 μL of a 50× stock in DMSO, 1 μM final concentration) for 30 minutes at 25 degrees Celsius. All aliquots (50 μL) were treated with FP-Rh (1 μL of 50× stock in DMSO, 1 μM final concentration). The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 4× SDS-PAGE loading buffer (reducing), separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining of ABHD10 and anti-target bands was determined by measuring the integrated optical density of test compound bands relative to vehicle bands. Protein bands were counted as anti-targets if greater than or equal to 50% inhibition was observed at any test compound concentration. Assay Cutoff: Compounds with greater than or equal to 50% inhibition at 25 mg/kg and no anti-targets at any test compound concentration were considered active.
2.2. Probe Chemical Characterization
The probe structure was verified by 1H and 13C NMR, LCMS, and HRMS; both purity and ee were assessed to be greater than 99% by gas chromatographic analysis and chiral HPLC, respectively (Section 2.3; see also Figure S1 for chiral traces of the latter). Solubility (room temperature) was determined to be 3.3 μM in PBS. This low solubility is not expected to be a problem for in vitro and in situ use of the probe for ABHD10 biochemical investigation, as the IC50 of ML257 is significantly lower (17 nM in vitro, 28 nM in situ) than its solubility threshold. However, interpretation of some profiling assays used for characterization (gel-based anti-target selectivity, cytotoxicity) may require some caution in light of this low solubility. Assessment of probe stability gave a half-life of 11 hours in PBS. However, this modest stability has not compromised use of the probe for in vitro, in situ, and in vivo studies (see Sections 3.5 and 3.6).
2.3. Probe Preparation
Analytical data and procedures for the ABLs in the initial (PME-1) screening library are reported in our previous work . Nucleophilic catalyst PPY* was prepared by literature methods . p-Tolylacetic acid (Alfa Aesar), iodoethane (Aldrich), n-butyllithium solution (Aldrich), thionyl chloride (Aldrich), anhydrous CH2Cl2 (Aldrich), N,N-dimethylethylamine (Aldrich or Alfa Aesar), and diisopropylazodicarboxylate (Aldrich) were purchased and used as received. Tetrahydrofuran was dried by passage through a column of activated alumina under an argon atmosphere.
Ethyl p-tolyl ketene : The following procedure is adapted from our previously reported method . To a 0°C solution of p-tolylacetic acid (10.00 g, 66.59 mmol, 1.0 equiv) in THF (100 mL) was added a solution of n-BuLi (59.67 mL, 2.5 M in hexanes, 149.16 mmol, 2.24 equiv) dropwise over 20 minutes. Some bubbling was observed, and a precipitate formed during the addition. When the base addition was complete, the resulting orange-brown slurry was stirred for 2 hours. Neat iodoethane (6.42 mL, 79.91 mmol, 1.2 equiv) was added dropwise over 5 minutes. The mixture was warmed gradually to ambient temperature and stirred overnight. The reaction was quenched by addition of H2O (4 mL) and the volatiles were removed by rotary evaporation. The resulting paste was dissolved with Et2O (50 mL) and H2O (15 mL). The aqueous phase was brought to pH 1 by dropwise addition of concentrated HCl. The phases were separated and the aqueous phase was extracted with Et2O (3 × 20 mL). The organic phases were combined, washed with brine (1 × 7 mL), and dried over MgSO4. After filtration and concentration the crude acid  was obtained as an off-white solid and used directly in the next step.
1H NMR (500 MHz, CDCl3) δ 7.20 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 3.42 (app. t, J = 7.7 Hz, 1H), 2.33 (s, 3H), 2.09 (ddq, J = 14.9, 7.4, 7.4 Hz, 1H), 1.79 (ddq, J = 14.9, 7.5, 7.5 Hz, 1H), 0.90 (app. t, J = 7.4 Hz, 3H)
The crude butanoic acid from above was dissolved in CH2Cl2 (10 mL) and the flask was immersed in a room temperature water bath. Neat SOCl2 (14.49 mL, 199.77 mmol, 3.0 equiv) was added dropwise via syringe over 10 min. Some gas and heat evolution was observed. The homogenous solution was stirred for 15 hours. The mixture was concentrated to an oil by rotary evaporation. The residue was vacuum distilled through a short path distillation head to yield the acid chloride as a colorless oil (10.42 g, 80% yield for 2 steps).
bp 59–60 °C (400 mTorr), 80 °C oil bath temperature (lit. 118–120 °C (12 Torr)) 
1H NMR (500 MHz, C6D6) δ 6.93 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 3.49 (app. t, J = 7.4 Hz, 1H), 2.03 (s, 3H), 1.89 (ddq, J = 14.7, 7.4, 7.4 Hz, 1H), 1.55 (ddq, J = 14.9, 7.5, 7.5 Hz, 1H), 0.60 (app. t, J = 7.4 Hz, 3H)
To a 0°C solution of the distilled acid chloride (10.42 g, 53.00 mmol, 1.0 equiv) in THF (66 mL) was added N,N-dimethylethylamine (28.7 mL, 265.01 mmol, 5.0 equiv) over 10 min. A white precipitate formed immediately and the liquid phase became bright yellow. The mixture was stirred at 0°C for 16 hours, and then warmed to ambient temperature. The solids were removed by filtration under an atmosphere of dry N2 using a flip-frit apparatus with a medium porosity sintered glass frit. The solids were washed with a small amount of dry Et2O. The yellow-orange filtrate solution was concentrated by rotary evaporation at ambient temperature. The residue was immediately distilled through a short path distillation head to yield ethyl p-tolyl ketene as an orange liquid (bp 44 °C [235 mTorr], 75°C oil bath temp). The distillate was immediately transferred to a nitrogen-atmosphere glovebox where the mass was measured (4.6313 g, 54.5% yield). The ketene was divided into small vials that were sealed with Teflon-lined caps and tape to exclude air and moisture. The neat ketene was stored outside the glovebox in a −20°C freezer and handled exclusively in the glovebox.
1H NMR (500 MHz, CDCl3) δ 7.13 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 2.42 (q, J = 7.4 Hz, 2H), 2.31 (s, 3H), 1.21 (t, J = 7.4 Hz, 3H)
(±)-Diisopropyl 3-ethyl-4-oxo-3-(p-tolyl)-1,2-diazetidine-1,2-dicarboxylate, (±)-ABL303 (ML257 and 303Bb): In a nitrogen-atmosphere glovebox, a solution of ethyl p-tolyl ketene (160.2 mg, 1.00 mmol, 1.0 equiv) in CH2Cl2 (59 mL) was prepared in a 200 mL round bottom flask. A solution of diisopropylazodicarboxylate (202.2 mg, 1.00 mmol, 1.0 equiv) in CH2Cl2 (4 mL) was added and the vial containing the azo-compound was rinsed with additional CH2Cl2 (3 × 3 mL). The flask was sealed with a rubber septum and taped. In a separate vial, a solution of (±)-PPY* (18.8 mg, 0.05 mmol, 0.05 equiv) in CH2Cl2 (2 mL) was prepared and the vial was closed with a septum cap. The flask and vial were removed from the glovebox and the flask containing the yellow-orange ketene/azodicarboxylate solution was cooled to −30 °C in a dry ice/CHCl3 bath. The dark purple catalyst solution was added via syringe in one portion leading to an immediate color change to green. The mixture was stirred overnight, warming gradually to ambient temperature. After 14 hours, the mixture was concentrated under vacuum to an oil. The residue was purified by automated silica gel chromatography using a Biotage Isolera Four (25 g SNAP SiO2 cartridge, linear gradient from 10–100% Et2O in hexanes) to provide ABL303 as a colorless oil (241.2 mg, 67% yield).
Separation of enantiomers was achieved by semi-preparative HPLC using a Gilson PLC 2020 and a Chiralpak IB column (20 × 250 mm) using 2% i-PrOH in hexanes as eluent (isocratic 17.0 mL/min flow rate, retention times: 6.6 min, 8.4 min). Yield of fast-eluting enantiomer: 84.9 mg; yield of slow-eluting enantiomer: 97.3 mg. Both isolates from the chromatographic resolution were >99% ee by analytical HPLC (4.6 × 250 mm Chiralpak IB-3 column, 2% i-PrOH in hexanes eluent, isocratic 1 mL/min flow rate, retention times: 8.834 min, 11.724 min or 4.6 × 250 mm Chiralpak OD-H column, 2% i-PrOH in hexanes eluent, isocratic 1 mL/min flow rate, retention times: 9.295 min, 14.389 min).
Gas chromatographic analysis (J & W Scientific HP-5 column, 100–310 °C ramp) of the resolved enantiomers found a single nonsolvent component (>99% purity).
1H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.9 Hz, 2H), 5.09 (septet, J = 6.2 Hz, 1H), 4.96 (septet, J = 5.9 Hz, 1H), 2.40 (dq, J = 15.1, 7.7 Hz, 1H), 2.35 (s, 3H), 2.25 (dq, J = 14.6, 7.2 Hz, 1H), 1.36 (d, J = 6.2 Hz, 3H), 1.35 (d, J = 6.2 Hz, 3H), 1.27 (d, J = 6.3 Hz, 3H), 1.16–1.07 (br s, 3H), 1.08 (app t, J = 7.3 Hz, 3H)
13C NMR (125 MHz, CDCl3) δ 165.2, 157.3, 147.8, 139.0, 132.2, 129.5, 126.4, 90.3, 72.8, 71.6, 28.4, 22.0, 21.9, 21.3, 8.7
IR (neat film, NaCl) 2983, 2940, 2884, 1835, 1767, 1739, 1467, 1376, 1358, 1301, 1267, 1233, 1180, 1146, 1102, 1051, 920 cm−1
LCMS (ES+) m/z: cacld for C19H27N2O5 [M + H]+: 363.2, found: 363.1
HRMS (ES+) m/z: cacld for C19H27N2O5 [M + H]+: 363.1914, found: 363.1916
Fast-eluting enantiomer (ML257): [α]23.9D −7.2° (c 1.27, CH2Cl2, >99% ee)
Slow-eluting enantiomer (303Bb): [α]23.8D +7.0° (c 1.17, CH2Cl2, >99% ee)
The ABHD10 probe ML257 was derived from lead inhibitors identified during our medchem campaign to optimize a probe for the methyltransferase PME-1. The following results section reveal how our initial PME-1 inhibitor scaffold was readily adapted for selective inhibition of an orthogonal target enzyme with only modest structural modifications, highlighting the versatile nature of the ABL scaffold for SH inhibition. ML257 has IC50 values of 17 nM and 28 nM for inhibition of ABHD10 in vitro and in situ, respectively (Section 3.2), is not overtly cytotoxic (Section 3.5), and exhibits high selectivity against all other SHs (~20) tested by gel-based competitive ABPP in vitro (>59-fold; Sections 3.4 and 3.6), and in situ (≥19-fold; Section 3.5), and selectivity vs. all other SHs (~40) assayed by in-depth proteome profiling by ABPP-SILAC (Section 3.6). We have also verified that ML257 is active in vivo, completely inhibiting ABHD10 in mice administered 25 mg/kg compound (Section 3.5).
3.1. Summary of Screening Results
In the primary uHTS assay for PME-1 inhibitors (AID 2130), ~302K compounds were screened by fluopol-ABPP with a SH-specific FP-Rh probe. A total of 1683 compounds (0.6%) were active, passing the set threshold (mean + 3x standard deviation) of 26.13% PME-1 inhibition. For the confirmation uHTS screen (AID 2171), 1514 active compounds were retested in triplicate (same inhibitor concentration), and 1068 compounds (70.5%) confirmed as active (Figure 1).
One of the most interesting chemotypes to emerge from the fluopol-ABPP uHTS campaign was the aza-beta-lactam (ABL). Out of the 26 ABLs in the screening library, only 2 compounds, 127A (SID 92709579) and 103A (SID 92709580), inhibited more than 50% of PME-1 activity (see Table 3 for structures). Because the uHTS assay was conducted with purified enzyme, we first tested whether these hits were also active against PME-1 in a complex proteome. Both confirmed as active, completely inhibiting activity at 100 nM concentration in soluble proteome preparations of two different human cancer cell lines (see AID 463146 and Probe Report for ML174). Through a collaboration with Dr. Greg Fu (Massachusetts Institute of Technology; original depositor of ABLs to MLSMR), we obtained powder samples of 24 ABLs in the MLSMR collection for gel-based competitive ABPP analysis (AID 463149). Reflective of the uHTS results, 127A (SID 99206500) was the most potent PME-1 inhibitor, with an IC50 of 10 nM (AID 463124) and high selectivity (>100-fold) vs. all other SHs assayed by gel-based competitive ABPP (AID 463149). As such, 127A was declared probe ML174 (SID 99206500) for PME-1.
Among the initial 24 ABLs screened by gel-based competitive ABPP, four compounds—117A (SID 99206498), 091C (SID 99206515), 143A (SID 99206509), and 115A (SID 99206518)—showed at least 50% inhibition of the uncharacterized SH anti-target ABHD10 when tested at 20 μM compound concentration (Table 3 and Figure 2; see also ML174 Probe Report). All compounds had “R” stereochemistry, showing the same potency profile of R >> S as the PME-1 inhibitors. In contrast to the PME-1-active compounds, which both had cyclo group substitutions at the “up” position of the chiral center (* in Table 4), the majority (3 of 4) ABHD10 leads had ethyl groups at that position. Methyl and ethyl carbamate substituents seemed equally potent, as 091C and 143A show similar inhibition profiles in Figure 2. However, a change from phenyl to m-tolyl at the “down” position of the chiral center (* in Table 4) seemed to significantly improve potency, as evinced by greater inhibition of ABHD10 for 117A vs. 091C in Figure 2. To improve potency and selectivity of these compounds towards ABHD10 we, in collaboration with Dr. Fu, initiated a medchem campaign based around these lead hit leads (Section 3.4), ultimately leading us to the optimized ABHD10 probe ML257 (SID 125311344).
3.2. Dose Response Curves for Probe
IC50 values for ML257 (SID 125311344) were obtained from gel-based competitive-ABPP data with endogenous ABHD10 in native mouse proteomes both in vitro (17 nM; AID 588802) and in situ (28 nM; AID 588801) (Figure 3).
3.3. Scaffold/Moiety Chemical Liabilities
PME-1 probe ML174 was previously determined to covalently modify the enzyme’s catalytic serine (Ser156; AID 463090). The observed mass shift of the active site peptide corresponds to the adduct depicted in Figure 4, formed by serine nucleophilic attack at the carbonyl to open the lactam ring. Given the similar structures of ML174 and ML257, and the fact that PME-1 and ABHD10 both share the Ser-His-Asp catalytic triad characteristic of SHs, it is likely that ML257 operates by a similar mechanism to irreversibly inactivate ABHD10.
Glutathione (GSH) reactivity studies with ML257 (SID 125311344) following the revised MLPCN-protocol (monitoring disappearance of parent compound) suggest that the probe may be reactive with GSH. However, in the absence of data confirming adduct formation, reactivity is purely speculative (e.g., retesting of ML174 (SID 99206500) using the revised protocol indicates reactivity, whereas, in previous LC-MS/MS studies, no adduct formation was observed, indicating a lack of reactivity). Regardless, both ML257 and ML174) show clean selectivity profiles both in vitro (Sections 3.4 and ML174 Probe Report, respectively) and in situ (Section 3.6), 2) are active in cells at very low concentrations (100 nM or less, Section 3.5 and ML174 Probe Report, respectively), and 3) are also active in vivo (Section 3.5 and ref , respectively). As such, GSH reactivity, if accurate, does not appear to be a good indicator of general reactivity/instability in this case. More generally, it should be noted that the dual LYPLA1/2 probe ML211 (a triazole urea covalent inhibitor) also showed evidence of GSH reactivity, however, its clean proteome reactivity profiles and in situ activity at low concentration (30 nM) suggest that it, likewise, is not a broadly reactive compound. Taken together, this evidence suggests that, in the presence of more relevant selectivity and in situ and/or in vivo activity data, GSH reactivity may be a poor indicator of general probe instability. Rather, covalent inhibitors featuring “privileged” core scaffolds like the ABL of ML257 and ML174 or the triazole urea of ML211 appear have a tempered electrophilicity and specific structural elements that direct reactivity towards their intended target enzyme.
We , and others [29–31], have found that irreversible inhibitors such as ML174 and, by analogy, ML257, offer many advantages over reversible inhibitors as pharmacologic probes for enzyme characterization in biological systems. Perhaps most importantly, it is technically straightforward to confirm the irreversible inhibition of enzymes in living systems (including mice)  using competitive ABPP and click chemistry ABPP; as such, we can define the precise quantity of inhibitor and treatment time required to selectively inactivate an enzyme of interest for biological studies. This information is extremely valuable for guiding the functional characterization of SHs. Additionally, required dosing is often lower, irreversible compounds are not as sensitive to pharmacokinetic parameters, and administration can induce long-lasting inhibition . In the case of the EGFR inhibitor PD 0169414, its irreversibility and high selectivity were credited with producing prolonged inhibition of the target, alleviating concerns over short plasma half-lives and reducing the need for high peak plasma levels, thus minimizing potential nonspecific toxic effects .
Indeed, over a third of enzymatic drug targets are irreversibly inhibited by currently marketed drugs . Examples of covalent enzyme-inhibitor pairs include serine type D-Ala-D-Ala carboxypeptidase, which is covalently modified by all B-lactam antibiotics, acetylcholinesterase, whose active site serine undergoes covalent modification by pyridostigmine, prostaglandin-endoperoxide synthase, which is the target of the ubiquitously prescribed aspirin, aromatase, which is irreversibly modified by exemestane, monoamine oxidase, which is covalently modified by L-deprenyl, thymidylate synthase, which is covalently modified by floxuridine, H+/K+ ATPase, which undergoes covalent modification by omeprazole, esomeprazole, and lansoprazole, and triacylglycerol lipase, whose serine nucleophile is targeted by orlistat .
3.4. SAR Tables
The SAR Table 4 includes 43 ABLs: the PME-1 probe ML174 (entry 1), five ABLs from the original PME-1 library (entries 2–6), and 37 new synthetic compounds (entries 7–43). The relative potency and selectivity of the ABL library members was assessed by gel-based competitive ABPP with FP-Rh (see Figure 8 for gel images and AID 588807 for protocol details). This assay was conducted using mouse brain membrane proteome owing to its rich diversity of potential anti-target SHs, and compounds were tested at three concentrations (10 μM, 1 μM, and 0.1 μM). SHs are listed as anti-targets if at least 50% inhibition is observed (relative to DMSO-only control) for a given compound concentration. The vast majority of anti-targets were only observed at the highest (10 μM) concentration tested; no anti-targets were observed at 0.1 μM, and only two anti-targets (APEH for entry 28 and PREP for entry 38) were observed at 1 μM concentration. See Table S1 for list of anti-target names and abbreviations.
First Round SAR
Variation of the carbamate substituents (R3 and R4): Based on initial SAR analysis of ABHD10 lead hits (Section 3.1), we first tested a series of m-tolyl analogs (entries 7–18) of 117A (entry 4). We fixed R2 as ethyl since that group was prevalent in the initial hits (Table 3), and varied carbamate substituents R3 and R4. Nearly all compounds tested were less potent than 117A, with the exception of the 242C (entry 7), with matched ethyl carbamates, which showed a marked improvement in potency (compare percent inhibition ABHD10 at 0.1 μM in Table 4). The next most potent analog (025C, entry 18) featured allyl carbamate groups, and was ~equally potent as compared to 117A, and a compound with match isopropyl groups (091Ca, entry 10) was a close contender as well. While some analogs in this series had fewer anti-targets, these also tended to be the least potent analogs (i.e., entries 9, 11, 12, and 14, Table 4). The same general trend of ethyl exhibiting superior potency over other, larger substituents like benzyl (250Ea, entry 21) is also observed when R2 is fixed as methyl. The isopropyl carbamate compound (248BbD, entry 20) exhibited decent potency as well (compare percent inhibition at 0.1 μM, Table 4). Thus ethyl, allyl, and isopropyl carbamate substituents seemed to impart the best potency for ABHD10 inhibition.
Variation of the alkyl substituent (R2): A switch from ethyl (242C, entry 7) to methyl (247D, entry 19) alkyl substituents does not have a dramatic effect on potency, with the latter being slightly less potent. Exchanging ethyl for n-propyl (222C, entry 22), however, almost completely abolishes activity (compare percent inhibition at 0.1 μM, Table 4). As such, we left the alkyl substituent as ethyl in subsequent rounds of SAR exploration.
Variation of of the aryl substituent (R1): While we knew from the initial ABL library that an ortho-tolyl group (123A, entry 6) had significantly reduced potency as compared to the meta-tolyl of 117A (entry 4, compare inhibition of ABHD10 at 1 μM in Table 4), para-tolyl functionality was not explored in the first generation ABL collection. However, with the current library, a comparison of percent inhibition at 0.1 μM (Table 4) of meta-tolyl (025C, entry 18), to para-tolyl (014Ba, entry 23), and phenyl (021C, entry 24) reveals that para-tolyl (014Ba) afforded significantly greater potency for ABHD10.
Second Round SAR
Variation of the carbamate substituents (R3 and R4): Based on the first round of SAR analysis, we fixed the aromatic position as para-tolyl (R1 = p-Me) and R2 as ethyl and again investigated substitution of the carbamate (entries 27–31). The best analogs—and most potent compounds tested thus far—featured n-propyl (015Ba, entry 29) and isopropyl (303Ba, entry 30) carbamates. In vitro IC50 determination for these compounds confirmed high potency (IC50s of 12 nM and 17 nM, respectively).
Variation of of the aryl substituent (R1): One final investigation of the aryl position (R1) was conducted with the R2 alkyl group fixed as ethyl and the carbamate substituent fixed as either isopropyl (entries 32–37) or methyl (entries 38–43). However, neither methoxy (entries 33, 34, 38, 39) nor halogen (entries 35–37 and 40–43) groups at the meta and/or para positions of the phenyl ring showed even comparable potency to the para-tolyl compounds 015Ba (entry 29) and 303Ba (entry 30) (compare percent inhibition at 0.1 μM, Table 4). IC50 values for two of the most potent compounds from this series, 092D (entry 32) and 220Ea (entry 41), were determined for confirmation, and both were at least 6-fold less potent than the top compounds 015Ba and 303Ba.
Of the 40+ ABLs in the ABHD10-directed library, the most potent compounds were 015Ba and 303Ba (entries 29 and 30, ≥90% inhibition at 0.1 μM and IC50s <20 nM, Table 4). Not surprisingly, given the R >> S trend observed with initial ABHD10 hits in the 24-member PME-1-directed library (see Probe Report for ML174), the S enantiomer of 303Ba (303Bb, entry 31) is at least 2 orders of magnitude less potent than the R counterpart. Other top ABHD10 inhibitors were the meta-tolyl compound 242C (entry 7), with an IC50 of 60 nM (~4-fold less potent), and the para-tolyl compound featuring allyl carbamates, 014Ba (entry 23), which showed good inhibition (80%) at 0.1 μM compound concentration (Table 4); however, as compared to 015Ba and 303Ba, it was shown to be less active in situ (Figure S2; AID 588806). All four top hits showed some evidence of anti-target activity at 10 μM compound concentration, with PREP and ABHD6 being the most ubiquitous anti-targets. However, no anti-targets were observed at 1 μM compound concentration, leaving a large selectivity window (>50) for both 015Ba and 303Ba. The latter compound, 303Ba, was designated as ABHD10 Probe ML257 (SID 125311344) for further investigation (Sections 3.5 and 3.6).
It is promising that, with only a modest-sized ABL library, we were able to identify lead inhibitors and then rapidly derive a potent and highly selective probe for an orthogonal “anti-target” enzyme. Given this target flexibility, the ABL class can rightly be considered a “privileged” scaffold for SH inhibitor development along with carbamates [ML256 Probe Report, submitted, and ref. ) and triazole ureas (ML211, ML225, and ML226 Probe Reports, and ref. ).
3.5. Cellular Activity
In Situ Inhibition: Probe ML257 (SID 125311344) is highly active against ABHD10 in situ, with an IC50 of 28 nM (Figure 5). For this experiment, cultured Neuro-2A murine neuroblastoma cells were treated with ML257 for 2 hours, washed, harvested and homogenized for gel-based competitive ABPP with FP-Rh as described (AID 588801 and ref. ). This result indicates that, like ML174 (see ML174 Probe Report), ML257 is able to cross cell membranes and inhibit its target in living cells. Consistent with in vitro analysis (see Table 5 and Section 3.6), there is evidence of anti-target inhibition of PREP (IC50 = 2.3 μM, AID 588835) and ABHD6 (IC50 = 532 nM, AID 588835) at higher compound concentrations. However, IC50 differences still provide a substantial selectivity window (≥19-fold), and we were able to establish an ML257 concentration (100 nM) where we could achieve selective inhibition of ABHD10 in Neuro-2A cells without significant inhibition of either PREP, ABHD6, or ~40 other SHs (see ABPP-SILAC profiling results in Figure 10B). It should be noted that, should inhibition of either of these anti-targets become problematic, there are existing in situ and/or in vivo active ABHD6 (e.g., WWL70 [34, 36]) and PREP (e.g., JTP-4819 , KYP-2047 ) inhibitors that could be used as controls for anti-target inhibition effects in biological experiments. No inhibition of any other ~20 SHs profiled in this experiment is apparent even at the higher (1 and 10 μM) concentrations tested.
In Vivo Inhibition: Probe ML257 (SID 125311344) is also active against ABHD10 in vivo (Figure 6. and AID 602485). For gel-based in vivo analysis (e.g., see ref ), mice were administered ML257 (6, 13, or 25 mg/kg, i.p.). After six hours, mice were sacrificed and heart tissue removed. The soluble proteome was isolated and analyzed by gel-based ABPP with FP-Rh, with near-complete inhibition of ABHD10 observed at 25 mg/kg. For comparison, in vitro treatment with ML257 (Figure 6, first lane) is shown as a control. No inhibition of any other ~20 SHs profiled in this experiment is apparent at any concentration tested, indicating a clean selectivity profile in vivo. These results suggest that, despite concerns over potential reactivity and chemical instability, ML257 is a highly useful tool for inhibition of ABHD10 in living animals.
Cytotoxicity: The probe ML257 and two representative analogs, 015Ba (entry 29) and 220Ea (entry 41) were evaluated for toxicity (AID 588804) to Neuro-2A murine neuroblastoma cells cultured in both serum-free and serum-supplemented medium (Figure 7). All three compounds were calculated to have CC50s >10 μM, affording a large dosing window for ML257 (>350-fold) over the in situ IC50 (28 nM, Figure 5). Even if solubility (3.3 μM) is considered the lower limit for cytotoxicity, that value would still leave a more than adequate dosing window (118-fold) over the in situ IC50 of ML257.
3.6. Profiling Assays
HTS ABLs: To date, there are 26 ABLs in the MLSMR library that have been tested in several hundred bioassays (Table 5). On average, these compounds have a hit rate of 2.4%, indicating that the ABL compound class is not generally active across a broad range of cell-based and non-cell based assays. The most bioactive compounds (CIDs 24856323 and 24856233) both have para-Cl substitution (R1) on the phenyl ring, suggesting that this particular chemotype has a somewhat heightened reactivity. No HTS activity data is yet available for probe ML257 or any of the synthetic analogs, nor has ML257 been submitted for commercial or non-commercial broad panel screening.
Gel-based Competitive ABPP: As summarized in Table 4, probe ML257 (SID 125311344) and ABL analogs have been subject to gel-based competitive ABPP  to assess ABHD10-directed potency and selectivity against >20 FP-sensitive SHs (including lipases, esterases, proteases, and uncharacterized hydrolases) visible by 1D SDS-PAGE separation and fluorescent detection.
Gel-based competitive ABPP is a medium-throughput proteome-wide screening technique that was instrumental in our medchem optimization of the probe compound (Section 3.4), allowing rapid assessment of potency and selectivity, as visualized by disappearance of target and anti-target bands in compound-treated lanes relative to the DMSO-only control. For simultaneous assessment of compound potency for ABHD10 and selectivity among the SH superfamily (AIDs 588807), Figure 8 shows gel-based competitive ABPP in the mouse brain membrane proteome at three compound concentrations (10 μM, 1 μM, and 0.1 μM). Based on this analysis, we were able to rapidly focus on two compounds, 015Ba (entry 29, Table 4) and 303Ba (entry 30, Table 4) as inhibitors with excellent potency and selectivity (>50-fold) profiles, one of which, 303Ba, was subsequently designated as probe ML257 for ABHD10.
To assess selectivity outside of the SH class, we also conducted both in vitro and in situ profiling with a cysteine-reactive chloroacetamide-rhodamine (CA-Rh)  activity based probe (Figure 9, AID 602468). As depicted in Figure 9A, even at 10 μM concentration of ML257 (SID 125311344), there is no evidence of anti-target effects in vitro in the mouse brain proteome. It should be noted that 10 μM is above the determined solubility limit of ML257 (3.3 μM). However, given that no anti-targets are observed at 1 μM, there still exists a wide (>50-fold) selectivity window vs. the in vitro IC50 value of 17 nM (Figure 3). Likewise, in Figure 9B, there are no significant thiol-based anti-targets in situ at either concentration tested (100 nM and 250 nM, cultured Neuro-2A murine neuroblastoma cells, 2 hr incubation). These data suggest that ML257 is not broadly reactive even outside the SH class.
ABPP-SILAC: To more comprehensively identify potential anti-targets, we utilized an advanced quantitative mass spectrometry (MS)-based platform termed competitive ABPP-SILAC (Figure 10A). Competitive ABPP-SILAC  combines competitive ABPP  with stable isotope labeling of cells (SILAC) , and allows for precise quantitation of enzyme inhibition by calculating the isotopic ratios of peptides from inhibitor-treated and control cells. As described in AIDs 588803 and 588805, Neuro-2A murine neuroblastoma cells were cultured in ‘light’ medium (with 12C614N2-lysine and 12C614N4-arginine) or ‘heavy’ medium (with 13C615N2-lysine [+8] and 13C615N4-arginine [+10]). After 10 passages, near-complete (>97%) enrichment was achieved. Heavy cells were treated with ML257 (SID 125311344, AID 588805) or ML174 (SID 125311304, AID 588803) and light cells were treated with DMSO in situ. After two hours, cells were harvested, lysed, separated into soluble and membrane fractions, and treated with the affinity-tagged SH-specific probe FP-biotin . Heavy (probe-labeled) and light (DMSO-treated) fractions were then mixed (1:1 w/w), enriched with avidin, digested on-bead with trypsin, and analyzed by MudPIT LC-MS/MS [20, 21] using an LTQ-Orbitrap Velos instrument. Light and heavy signals were quantified from parent ion peaks (MS1) and the corresponding proteins identified from product ion profiles (MS2) using the ProLucid search algorithm. The depicted bar graphs represent the average ratios of heavy/light tryptic peptides for each of the SHs identified. Enzymes susceptible to inhibition upon compound treatment would be expected to have heavy/light ratios significantly less than one, while uninhibited enzymes would be expected to have a ratio close to one. The results (Figure 10B and 10C) demonstrate that both ML257 and ML174 display remarkable selectivity for their intended targets in situ, blocking ~100% of activity at 100 nM concentration while not affecting activity of 40+ other SHs. Importantly for ML257, this ABPP-SILAC analysis reveals no inhibition of either ABHD6 or PREP, potential anti-targets identified by gel-based competitive ABPP profiling in vitro (Table 4 and Figure 8) and in situ (Figure 5). We have previously determined that we could detect the SH fatty acid amide hydrolase (FAAH) at concentrations as low as 0.0005% of the total proteome (~200 copies per cell) by gel-based ABPP . By comparison, we estimate that the sensitivity of a standard ABPP-MudPIT assay is at least 10-fold higher (0.00005% of the total cell proteome, or 20 copies per cell). As such, gel-based and LC-MS/MS-based methods offer detection of low abundance SHs. These clean selectivity profiles highlight the value of specific chemotypes, like the ABLs, from which it is possible to derive potent and selective chemical probes for multiple enzyme targets.
Probe ML257 (SID 125311344) was identified as a highly potent and selective inhibitor of the target enzyme ABHD10. The probe has an IC50 of 17 nM in vitro and 28 nM in situ against endogenous ABHD10 (Section 3.2), and shows no evidence of cytotoxicity up to 10 μM (Section 3.5). The probe is also active in vivo, inhibiting murine ABHD10 activity upon administration of 25 mg/kg compound (Section 3.5). The compound exhibits high selectivity (>59-fold in vitro, ≥19-fold in situ) against all other (~20) SH anti-targets surveyed by gel-based competitive ABPP (Sections 3.4, 3.5 and 3.6), and in-depth in situ ABPP-SILAC analysis revealed near-complete inhibition of ABHD10 with no anti-target reactivity for more than 40 SHs at 100 nM compound concentration (Section 3.6). ML257 does have rather low (< 3.3 μM) solubility (Section 2.2). However, experimentally, it is sufficiently soluble, stable, and tempered in reactivity for in vitro, in situ, and in vivo inhibition of ABHD10. As such, ML257 should be invaluable for investigation of the role of ABHD10 in lipid metabolism in native cellular systems.
Development of a second SH inhibitor based on the ABL scaffold is yet another example of the wealth of knowledge that can be derived from HTS inhibitor discovery assays, and emphasizes the importance of 1) populating screening libraries with compounds from academic chemistry laboratories, such as that of our co-author, Dr. Gregory Fu, who synthesized the ABLs while investigating synthetic applications of 4-pyrrolidinopyridine chiral catalysts , and 2) providing an avenue, like the MLPCN, for HTS inhibitor discovery for academic biology laboratories, whose enzymatic targets of interest may lie beyond the scope/interest of the pharmaceutical industry. By following up on a fortuitous lead inhibitor for an “anti-target” of our original target enzyme, PME-1, we have significantly increased the output of useful chemical tools derived from the initial HTS campaign, and established the ABL as a key scaffold for SH inhibitor development, which we hope to expand to other targets in the future.
4.1. Comparison to Existing Art and How the New Probe is an Improvement
ML257 is the first selective ABHD10 inhibitor reported in the literature.
4.2. Mechanism of Action Studies
As determined from LC-MS/MS analysis (AID 463090), the PME-1 probe ML174 is an activity-based inhibitor that covalently labels the active site serine nucleophile, Ser156, of PME-1. The observed mass shift of the active site peptide suggests that reaction occurs via serine nucleophilic attack on the carbonyl to open the lactam ring (Figure 4). It is hypothesized that this reactivity is a general mechanism for ABL inactivation of SH enzymes. As such, ML257 is expected to inactivate ABHD10 in an analogous fashion.
4.3. Planned Future Studies
We plan to use probe ML257 to investigate the metabolic role of ABHD10 in dynamic regulation of palmitoylation and other lipid signaling networks by 1) generating a metabolic fingerprint of ABHD10 small molecule substrates through the non-directed discovery metabolite profiling (DMP) platform  and 2) profile protein targets of ABHD10 using advanced competitive ABPP-SILAC methods to globally monitor dynamic protein palmitoylation . We will also investigate the potential for in vivo inhibition of ABHD10 by ML257.
As we to expand the ABL library, we will continue to seek improved inhibitors for existing targets and generate novel inhibitors for additional SH targets. With this approach, we hope to turn the outcome of an isolated HTS assay into a bounty of highly potent and specific ABL SH inhibitors for employ by the biological community for patho/physiological investigation of members of this important enzyme class.
- Oliver CJ, Shenolikar S. Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 1998;3:D961–72. [PubMed: 9727084]
- Janssens V, Goris J, Van Hoof C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005;15(1):34–41. [PubMed: 15661531]
- Chen J, Martin BL, Brautigan DL. Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science. 1992;257(5074):1261–4. [PubMed: 1325671]
- Favre B, et al. The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo. J. Biol. Chem. 1994;269(23):16311–7. [PubMed: 8206937]
- Leung D, et al. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 2003;21(6):687–91. [PubMed: 12740587]
- Schlesinger MJ, Magee AI, Schmidt MF. Fatty acid acylation of proteins in cultured cells. J. Biol. Chem. 1980;255(21):10021–4. [PubMed: 7430112]
- Smotrys JE, Linder ME. Palmitoylation of intracellular signaling proteins: regulation and function. Annu. Rev. Biochem. 2004;73:559–87. [PubMed: 15189153]
- Duncan JA, Gilman AG. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS) J. Biol. Chem. 1998;273(25):15830–7. [PubMed: 9624183]
- Camp LA, Hofmann SL. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J. Biol. Chem. 1993;268(30):22566–74. [PubMed: 7901201]
- Ong SE, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics. 2002;1(5):376–86. [PubMed: 12118079]
- Zuhl AM, Mohr JT, Bachovchin DA, Niessen S, Hsu KL, Berlin JM, Dochnahl M, López-Alberca MP, Fu GC, Cravatt BF. Competitive Activity-Based Protein Profiling Identifies Aza-β-Lactams as a Versatile Chemotype for Serine Hydrolase Inhibition. J. Am. Chem. Soc. 2012;134(11):5068–71. [PMC free article: PMC3326416] [PubMed: 22400490]
- Washburn MP, Wolters D, Yates JR 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001;19(3):242–7. [PubMed: 11231557]
- Wolters DA, Washburn MP, Yates JR 3rd. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 2001;73(23):5683–90. [PubMed: 11774908]
- Wurz RP, et al. Synthesis and resolution of planar-chiral derivatives of 4-(dimethylamino)pyridine. Adv. Synth. Catal. 2007;349:2345–2352.
- Lv H, et al. Asymmetric dimerization of disubstituted ketenes catalyzed by N-heterocyclic carbenes. Adv. Synth. Catal. 2008;350:2715–2718.
- Kinbara K, Kobayashi Y, Saigo K. Systematic study of chiral discrimination upon crystallisation. Part 2.1 Chiral discrimination of 2-arylalkanoic acids by (1R,2S)-2-amino-1,2-diphenylethanol. J. Chem. Soc. Perkin Trans. 1998;2:1767–1775.
- Rupe H, Wiederkehr F. Die konstitution des curcumons aus dem curcumal-öl. Helv. Chim. Acta. 1924;7:654–669.
- Potashman MH, Duggan ME. Covalent modifiers: an orthogonal approach to drug design. J. Med. Chem. 2009;52(5):1231–46. [PubMed: 19203292]
- Singh J, et al. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 2011;10(4):307–17. [PubMed: 21455239]
- Kodadek T. Rethinking screening. Nat. Chem. Biol. 2010;6(3):162–165. [PubMed: 20154660]
- Vincent PW, et al. Anticancer efficacy of the irreversible EGFr tyrosine kinase inhibitor PD 0169414 against human tumor xenografts. Cancer Chemother. Pharmacol. 2000;45(3):231–8. [PubMed: 10663641]
- Robertson JG. Mechanistic basis of enzyme-targeted drugs. Biochemistry. 2005;44(15):5561–71. [PubMed: 15823014]
- Li W, Blankman JL, Cravatt BF. A functional proteomic strategy to discover inhibitors for uncharacterized hydrolases. J. Am. Chem. Soc. 2007;129(31):9594–5. [PubMed: 17629278]
- Toide K, et al. Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on prolyl endopeptidase activity and substance P- and arginine-vasopressin-like immunoreactivity in the brains of aged rats. J. Neurochem. 1995;65(1):234–40. [PubMed: 7540663]
- Venalainen JI, et al. Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors. Biochem. Pharmacol. 2006;71(5):683–92. [PubMed: 16405869]
- Saghatelian A, Cravatt BF. Discovery metabolite profiling--forging functional connections between the proteome and metabolome. Life Sci. 2005;77(14):1759–66. [PubMed: 15964030]
Andrea M Zuhl,* Justin T Mohr,† Anna E Speers,* Daniel A Bachovchin,* Jacob M Berlin,† Timothy Spicer,‡ Virneliz Fernandez-Vega,‡ Steven J Brown,* Jill Ferguson,* Greg C Fu,† Benjamin F Cravatt,* Peter Hodder,‡ and Hugh Rosen*,1.
Received: December 6, 2011; Last Update: March 7, 2013.
National Center for Biotechnology Information (US), Bethesda (MD)
Zuhl AM, Mohr JT, Speers AE, et al. Probe Development Efforts to Identify Novel Inhibitors of ABHD10. 2011 Dec 6 [Updated 2013 Mar 7]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.