Optimization and characterization of a triazole urea inhibitor for platelet-activating factor acetylhydrolase type 2 (PAFAH2)

Adibekian A, Hsu KL, Speers AE, et al.

Publication Details

Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. In support of this theory, over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death [1]. However, no selective inhibitors of PAFAH2 are known for investigation of PAFAH2 biology. We initiated a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) HTS campaign to identify potential inhibitors of PAFAH2 (AIDs 492956 and 493030). The assay also served as a counterscreen for inhibitor discovery for the related enzyme, plasma PAFAH (pPAFAH; AIDs 463082, 463230).

Interestingly, the triazole urea SID 7974398—a top lead in the lysophospholipase (LYPLA1) inhibitor screen from which we derived a dual inhibitor of LYPLA1/LYPLA2 (ML211) and inhibitor of ABHD11 (ML226)—was also a top hit in the PAFAH2 HTS assay. Given that triazole ureas were previously found to have tunable potency and selectivity, low cytotoxicity, and good activity in situ, we endeavored to derive a PAFAH2-selective probe from the triazole urea scaffold. The medchem optimized probe (ML225, SID 103913572) is highly potent against its target enzyme (IC50 = 3 nM), and is active in situ at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling, and is selective for other PAFAH enzymes. ML225 inhibits PAFAH2 by carbamoylating the active site serine. The complete properties, characterization, and synthesis of ML225 are detailed in this Probe Report.

Assigned Assay Grant #: 1 R01 HL084366

Screening Center Name & PI: The Scripps Research Institute Molecular Screening Center (SRIMSC), H Rosen

Chemistry Center Name & PI: SRIMSC, H Rosen

Assay Submitter & Institution: BJ Bahnson, Univ. of DE; BF Cravatt, TSRI, La Jolla

PubChem Summary Bioassay Identifier (AID): 492969

Probe Structure & Characteristics

Image ml225fu1

Recommendations for Scientific Use of the Probe

Oxidative stress has been implicated as an underlying inflammatory factor in numerous disease pathologies, including cancer, atherosclerosis, aging, and neurodegenerative disorders [15]. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains [1, 6]. ML225, described herein, is a potent and specific inhibitor of PAFAH2 both in vitro and in situ. As such, it is recommended for use in primary research studies aimed at elucidating the patho/physiological roles of PAFAH2 and its contribution to inflammatory disease processes.

1. Introduction

Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders [15]. Phospholipids in particular are susceptible to oxidative damage, and (per)oxidized phospholipids can have deleterious effects, including disruption of membrane bilayers and production of toxic byproducts [1, 79]. One hypothesized pathway for removal of oxidatively damaged lipids involves hydrolysis by phospholipase A2-type enzymes. Candidate hydrolytic enzymes include the platelet-activating factor acetylhydrolases (PAFAHs) [1, 6]. The initial role assigned to the PAFAHs was the hydrolysis of platelet activating factor (PAF) [1011], a potent pro-inflammatory phospholipid signaling molecule [12], which plays a role in myriad physiological processes including inflammation, anaphylaxis, fetal development, and reproduction [1, 13]. The PAFAHs are subdivided into three classes: plasma (p)PAFAH, and intracellular types 1 and 2. In terms of sequence homology, pPAFAH and PAFAH2 are close homologs and show little similarity to type 1 enzymes.

PAFAH2 has been shown to play a role in inflammatory processes via hydrolysis of oxidized phospholipids. Over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death [1415] and to mediate repair of oxidative-stress induced tissue injury [1]. The enzyme also undergoes N-terminal myristoylation and subsequent translocation to the membrane under conditions of oxidative stress [1415]. This evidence suggests that PAFAH2 functions as an important, and perhaps primary, antioxidant enzyme in certain tissues [1]; however, its substrate specificity and pathway involvement are far from being fully elucidated. Currently no suitable inhibitors exist for co-crystallization or biochemical studies of PAFAH2.

Chemical tools capable of interrogating enzyme architecture and providing precise temporal control over PAFAH activity are necessary for complete characterization of patho/physiological roles of the PAFAHs in phospholipid metabolism and inflammatory disease processes. Towards that goal, we developed a HTS assay for inhibitor discovery for four PAFAH enzymes: pPAFAH, PAFAH2, PAFAH1b2, and PAFAH1b3, based on their reactivity with the serine-hydrolase-specific fluorophosphonate (FP) [16] activity-based protein profiling (ABPP) probe. 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 [17]. Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP [18]. Following the FluoPol-ABPP HTS campaign for PAFAH2, we identified a triazole urea hit compound with an IC50 of 1.4 μM. After several rounds of medchem optimization the derived probe ML225 (SID 103913572) is highly potent against its target enzyme (IC50 3 nM), and is active in situ at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (SHs) (~20) assessed by gel-based competitive ABPP, and is selective for the counterscreening enzymes pPAFAH, PAFAH1b2, and PAFAH1b3. As with the other triazole urea probes (ML211 and ML226; see also [19]), ML225 inhibits its target by carbamoylating the active site serine. This is the first reported selective inhibitor of PAFAH2.

2. Materials and Methods

All reagents for chemical synthesis were obtained from ThermoFisher or SigmaAldrich. All other protocols are summarized below.

2.1. Assays

Probe Characterization Assays

Solubility

The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration.

Stability

Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80°C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed in a room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.

LC-MS/MS for stability assay

All analytical methods are in MRM mode where the parent ion is selected in Q1 of the mass spectrometer. The parent ion is fragmented and a characteristic fragment ion is monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods will be rapidly set up using Automaton® (Applied Biosystems), where the compounds are listed with their name and mass in an Excel datasheet. Compounds are submitted in a 96-well plate to the HPLC autosampler and are slowly injected without a column present. A narrow range centered on the indicated mass is scanned to detect the parent ion. The software then evaluates a few pre-selected parameters to determine conditions that maximize the signal for the parent ion. The molecule is then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass are determined. Three separate collision energies are evaluated to fragment the parent ion and the largest three ions are selected. Each of these three fragment ions is further optimized and the best fragment is chosen. The software then inserts the optimized masses and parameters into a template method and saves it with a unique name that indicates the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern are saved and can be reviewed later.

Determination of glutathione reactivity

One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 100 μM reduced glutathione. Final compound concentration is 10 μM unless solubility limited. The solution was allowed to incubate at 37°C for two hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized: a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of −129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [2021].

Primary Assays

Primary uHTS assay to identify PAFAH2 inhibitors (AID 492956)

Assay Overview: The purpose of this assay is to identify compounds that act as inhibitors of the platelet activating factor acetylhydrolase 2 (PAFAH2). In this assay, recombinant PAFAH2 protein is incubated with test compounds and a Rh-conjugated activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. The assay is performed by incubating test compounds with PAFAH2 for a defined period, followed by addition of the FP-rhodamine probe and measurement of fluorescence polarization at a specific time point. As designed, test compounds that act as PAFAH2 inhibitors will prevent PAFAH2-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 3.39 μM

Protocol Summary: Prior to the start of the assay, 4.0 μL of Assay Buffer (0.01% pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT) containing 25 nM of PAFAH2 protein were dispensed into 1536 microtiter plates. Next, 17 nL of test compound in DMSO or DMSO alone (0.34% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 C. The assay was started by dispensing 1.0 μL of 250 nM FP-Rh probe in Assay Buffer to all wells. Plates were incubated for 35 minutes 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) for 15 seconds for each polarization plane (parallel and perpendicular). Assay Cutoff: Compounds that inhibited PAFAH2 greater than 29.65% were considered active.

Confirmation uHTS assay to identify PAFAH2 inhibitors (AID 493030)

Assay Overview: The purpose of this assay was to confirm activity of compounds identified as active in the primary uHTS screen (AID 492956). In this assay, the FP-Rh probe was used to label PAFAH2 in the presence of test compounds and analyzed as described above (AID 492956). Compounds were tested in triplicate at a nominal concentration of 3.39 μM.

Protocol Summary: The assay was performed as described above (AID 492956), except that compounds were tested in triplicate. Assay Cutoff: Compounds that inhibited PAFAH2 greater than 29.65% were considered active.

Secondary Assays

Inhibition and selectivity of triazole urea library members (AID 504513)

Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds can inhibit PAFAH2 in a complex proteomic lysate and to estimate compound selectivity in 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 fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as PAFAH2 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.

Protocol Summary: Soluble proteome (1 mg/mL in DPBS) of BW5147-derived murine T cells was treated with 20 nM, 200 nM, or 1 μM test compound (1 μL of a 50× stock in DMSO). Test compounds were incubated for 30 minutes at 25 degrees Celsius (50 μL reaction volume). FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 2 μM. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the target (PAFAH2) and anti-target (N-acylaminoacyl-peptide hydrolase [APEH], alpha/beta hydrolase domain-containing protein 11 [ABHD11], esterase D/formylglutathione hydrolase [ESD], and lysophospholipase 1 [LYPLA1]) bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition of PAFAH2 at 200 nM were considered active.

Inhibition of PAFAH2 in situ (AID 504495)

Assay Overview: The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit PAFAH2 activity in situ. In this assay, cultured BW5147-derived murine T-cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as PAFAH2 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: BW5147-derived murine T cells (5 mL total volume; supplemented with FCS) were treated with 30 nM test compound (5 μL of a 1000× stock in DMSO) for 4 hours at 37 degrees Celsius. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K × g, 45 minutes) and 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 2 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× 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 PAFAH2 band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥90% inhibition of PAFAH2 were considered active.

Determination of IC50 values against PAFAH2 in vitro (AID 504494)

Assay Overview: The purpose of this assay is to determine the IC50 values of powder samples of test compounds for PAFAH2 inhibition in a complex proteome. In this assay, a fluorophosphonate-conjugated rhodamine (FP-Rh) activity-based probe is used to label PAFAH2 in the presence of test compounds. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as PAFAH2 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: Soluble proteome (1 mg/mL in DPBS) of BW5147-derived murine T cells was incubated with DMSO or compound for 30 minutes at 37 degrees Celsius before the addition of FP-Rh at a final concentration of 2 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the bands. IC50 values for inhibition of PAFAH2 were determined from dose-response curves from three replicates at each inhibitor concentration (6-point series from 5000 nM to 1 nM). Assay Cutoff: Compounds with an IC50 <100 nM were considered active.

Determination of IC50 values against PAFAH2 in situ (AID 504496)

Assay Overview: The purpose of this assay is to determine the IC50 values of powder samples of test compounds for PAFAH2 inhibition in situ. In this assay, cultured cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as PAFAH2 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: BW5147-derived murine T-cells (5 mL total volume; supplemented with 10% FCS) were treated with DMSO or test compound (5 μL of a 1000× stock in DMSO) for 4 hours at 37 degrees Celsius. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K × g, 45 minutes) and 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 2 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× 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 PAFAH2 band relative to a DMSO-only (no compound) control. IC50 values for inhibition of PAFAH2 were determined from dose-response curves from three replicates at each inhibitor concentration (4-point 1:3 dilution series from 2.5 nM to 75 pM). Assay Cutoff: Compounds with an IC50 <100 nM were considered active

Analysis of Cytotoxicity (AID 504511)

Assay Overview: The purpose of this assay is to determine cytotoxicity of inhibitor compounds belonging to the urea triazole scaffold. In this assay, BW5147-derived murine T-cells in either serum-free media (Assay 1) or media containing FCS (Assay 2) are incubated with test compounds, followed by determination of cell viability. The assay utilizes the WST-1 substrate that 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. Compounds were tested in quadruplicate in a 7-point 1:5 dilution series starting at a nominal test concentration of 50 μM.

Protocol Summary: This assay was started by dispensing BW5147-derived murine T cells in RPMI media (100 μL, 10E4 cells/well) into a 96-well plate. Both serum-free media (Assay 1) and media supplemented with fetal calf serum (FCS) (Assay 2) were tested. Compound (5 μL of 0–1000 μM in media containing 5% DMSO) was added to each well, giving final compound concentrations of 0–50 μM. Cells were incubated for 48 hours at 37 degrees Celsius in a humidified incubator and cell viability was determined by the WST-1 assay (Roche) according to manufacturer instructions. Assay Cutoff: Compounds with a CC50 value of <5 μM were considered active (cytotoxic).

LC-MS/MS Analysis of Inhibitor Binding Mode (AID 504486)

Assay Overview: The purpose of this assay is to assess the covalent nature of an inhibitor compound belonging to the urea triazole scaffold and determine whether or not it labels the active site serine of PAFAH2. In this assay, purified enzyme is reacted with inhibitor compound, digested with trypsin, and the resulting peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting data are analyzed to identify sites of covalent labeling.

Protocol Summary: Two aliquots (25 μL) of 50 μM PAFAH2 were prepared. To one aliquot was added inhibitor (0.5 μL of 10 mM in DMSO), giving a final concentration of 200 μM. To the second (control) aliquot was added DMSO (0.5 μL). Reactions were gently vortexed and incubated at room temperature for 30 minutes. To each reaction was added solid urea (50 mg), followed by freshly prepared aqueous ammonium bicarbonate (75 μL of 25 mM). The reactions were vortexed until the urea was dissolved. Final urea concentration was approximately 8 M. To each reaction was added freshly prepared TCEP (5 μL of 100 mM in water), and the reactions were incubated at 30 degrees C for 30 minutes. To each reaction was then added freshly prepared IAA (10 μL of 100 mM in water), and the reactions were incubated for 30 min at room temperature in the dark. Aqueous ammonium bicarbonate (375 μL of 25 mM) was added to reduce the urea concentration to 2 M. To each reaction was added sequencing grade modified trypsin (1 μg), and reactions were incubated at 37 degrees C for 12 hours. Formic acid was added to 5% (v/v) final.

An Agilent 1200 series quaternary HPLC pump and Thermo Scientific LTQ-Orbitrap mass spectrometer were used for sample analysis. A fraction (10 μL) of the protein digest for each sample was pressure-loaded onto a 100 micron fused-silica column (with a 5 micron in-house pulled tip) packed with 10 cm of Aqua C18 reversed-phase packing material. Chromatography was carried out using an increasing gradient of aqueous acetonitrile containing 0.1% formic acid over 125 minutes. Mass spectra were acquired in a data-dependent mode with dynamic exclusion enabled.

The MS/MS spectra generated for each run were searched against a human protein database concatenated to a reversed decoy database using Sequest. A static modification of +57.021 was specified cysteine, and a variable modification of +97.053 was specified for serine to account for possible probe labeling by AA39-2. The resulting peptide identifications were assembled into protein identifications using DTASelect, and filters were adjusted to maintain a false discovery rate (as determined by number of hits against the reversed database) of <1%. Any modified peptides identified in the DMSO-treated sample were discarded as spurious hits. Assay Cutoff: Compounds observed to covalently modify the active site serine of PAFAH2 were considered active.

SILAC-ABPP Analysis of Inhibition and Selectivity (AID 504519)

Assay Overview: The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using stable isotope labeling with amino acids in cell culture (SILAC) ABPP. In this assay, cultured BW5147-derived murine T-cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with inhibitor and DMSO, respectively, in situ. Cells are lysed, proteomes are treated with FP-biotin, and combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block FP-biotin labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, giving a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio of ~1.

Protocol Summary

Stable isotope labeling with amino acids in cell culture (SILAC). BW5147-derived murine T-cell hybridoma cells were initially grown for 6 passages in either light or heavy SILAC RPMI 1640 media supplemented with 10% dialyzed FCS and 1× PenStrep Glutamine. Light media was supplemented with 100 μg/mL L-arginine (Sigma) and 100 μg/mL L-lysine (Sigma). Heavy media was supplemented with 100 μg/mL [13C615N4]-L-Arginine (Isotek) and 100 μg/mL [13C615N2]-L-Lysine (Isotek). Cells were treated with 3 nM test compound (5 μL of a 1000× stock in DMSO) for 4 hours at 37 degrees Celsius. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble and membrane fractions were isolated by centrifugation (100K × g, 45 minutes) and the protein concentration was adjusted to 1 mg/mL with DPBS.

Sample preparation for ABPP-SILAC. The light and heavy proteomes were labeled with 7 μM of FP-biotin (500 μL total reaction volume) for 1.5 hours at 25 degrees Celsius. 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 FP-labeled proteins were enriched with streptavidin beads. The beads were washed with 1% SDS in PBS (1x), PBS (3x), and H2O (3x), then resuspended in 6 M urea, reduced with DTT for 15 minutes at 60 degrees Celsius, and alkylated with iodoacetamide for 30 minutes at 25 degrees Celsius in the dark. On-bead digestions were performed for 12 hours at 37 degrees Celsius with trypsin (Promega; 4 μL of 0.5 μg/μL) in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% formic acid, pressure-loaded on to a biphasic (strong cation exchange/reverse 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 1100-series quaternary pump and Thermo Scientific LTQ Orbitrap ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (60 s, repeat of 1). Specifically, one full MS (MS1) scan (400–1800 m/z) was followed by 7 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.1; publicly available at http://fields.scripps.edu/?q=content/download). MS2 spectra data were searched using the SEQUEST algorithm (Version 3.0) against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. SEQUEST searches allowed for variable oxidation of methionine (+16), static modification of cysteine residues (+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 option, which applies different statistical models for the analysis of tryptic, half-tryptic, non-tryptic peptides. DTASelect 2.0 uses a quadratic discriminant analysis to achieve a user-defined maximum peptide false positive rate; the default parameters (maximum false positive rate of 2%) was used for the search; however, the actual false positive rate was much lower (1%). Ratios of Light/Heavy peaks were calculated using in-house software; reported ratios represent the mean of all unique, quantified peptides per protein. Assay Cutoff: A compound was considered active for a particular target/anti-target with a light/heavy ratio of ≤0.5

Analysis of General Serine Hydrolase Inhibition and Selectivity (AID 504527)

Assay Overview: The purpose of this assay is to assess the general inhibition profiles of powder samples of test compounds in a complex proteome by competitive activity-based proteomic profiling (ABPP). In Assay 1, a complex proteome is incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control. In Assay 2, the role of competitor and probe is reversed. A complex proteome is incubated with FP-biotin followed by reaction with test compound. Click chemistry is then used to append a fluorophore-azide to the alkyne-functionalized test compounds. As designed, labeling that is successfully competed by the FP-biotin probe is indicative of serine hydrolase targets. Bands that are not competed represent non-serine hydrolase targets of test compounds.

Protocol Summary

Assay 1: Soluble proteome (1 mg/mL in DPBS) of mouse brain soluble proteome was treated with 20 μM of test compound (1 μL of a 50× stock in DMSO). Test compounds were incubated for 30 minutes at 25 degrees Celsius (50 μL reaction volume). FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 2 μM. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition of two or more bands were considered “active”.

Assay 2: Mouse brain soluble proteome (1 mg/mL in DPBS) was treated with 20 μM of FP-biotin (1 μL of a 50× stock in DMSO) for 30 minutes at 25 degrees Celsius (50 μL reaction volume). Test compound (1 μL of 50× stock in DMSO) was added to a final concentration of 20 μM for 30 minutes at 25 degrees Celsius. Click chemistry with a rhodamine-azide tag (Rh-N3; 50μM) was carried out under standard conditions (1mM TCEP, 100μM TBTA ligand, 1mM Cu(II)sulfate) to append the rhodamine fluorophore to the alkyne-functionalized test compounds for visualization. Reactions were quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition by FP-biotin of all target bands were considered “active” (showing only reactivity within the serine hydrolase enzyme class).

Analysis of pPAFAH Selectivity In vitro (AID 504483)

Assay Overview: The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit the anti-target pPAFAH, a serine hydrolase with high sequence homology to the target enzyme PAFAH2, in vitro. In this assay, soluble proteome of COS-7 cells over-expressing recombinant murine pPAFAH are incubated with test compound and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as pPAFAH 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: COS-7 soluble proteome (1 mg/mL in DPBS, containing recombinantly expressed murine pPAFAH) was treated with 3, 10, 30, 100, or 1000 nM test compound (1 μL of a 50× stock in DMSO) for 30 minutes at 25 degrees Celsius. FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 2 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× 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 pPAFAH band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition of pPAFAH at 100 nM were considered active.

Analysis of pPAFAH Selectivity In situ (AID 504531)

Assay Overview: The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit the anti-target pPAFAH, a serine hydrolase with high sequence homology to the target enzyme PAFAH2, in situ. In this assay, cultured murine mast cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as PAFAH2 or pPAFAH 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: Murine mast cells (5 mL total volume; supplemented with FCS) were treated with 10 nM test compound (5 μL of a 1000× stock in DMSO) for 4 hours at 37 degrees Celsius. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K × g, 45 minutes) and 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 2 μM in 50 μL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× 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 PAFAH2 and pPAFAH bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition were considered active.

Analysis of Carbamate vs. Triazole Urea Compound Activity (AID 504491)

Assay Overview: The purpose of this assay is to determine whether powder samples of test compounds of different chemotypes—carbamate and triazole urea—can inhibit PAFAH2 in a complex proteomic lysate and to estimate compound selectivity in 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 fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as PAFAH2 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: Soluble proteome (1 mg/ml in DPBS) of BW5147-derived murine T cells was treated with 20 μM test compound (1 μL of a 50× stock in DMSO). Test compounds were incubated for 30 minutes at 25 degrees Celsius (50 μL reaction volume). FP-Rh (1 μL of 50× stock in DMSO) was added to a final concentration of 2 μM. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the target (PAFAH2) and anti-target (N-acylaminoacyl-peptide hydrolase [APEH], alpha/beta hydrolase domain-containing protein 11 [ABHD11], esterase D/formylglutathione hydrolase [ESD], lysophospholipase 1 [LYPLA1], and lysophospholipase 2 [LYPLA2]) bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition were considered active.

2.2. Probe Chemical Characterization

CID 56593029, SID 103913572, ML225.

CID 56593029, SID 103913572, ML225

The probe structure was verified by 1H NMR (see section 2.3) and high resolution MS (m/z calculated for C19H19N4O [M+H]+: 319.1553; found: 319.1556). Purity was assessed to be greater than 95% by NMR. The 2,4-regiostereochemistry was assigned by NMR by comparison with NMR shifts of triazole urea compounds of known 1,4 and 2,4 triazole substitution based on crystal structure data [19]. Solubility in PBS at room temperature was determined by UV trace to be 1.7 μM and stability in PBS was determined by LC-MS to be >48 hours.

Table 1 lists the compounds submitted to the SMR collection.

Table 1. Compounds submitted to the SMR collection.

Table 1

Compounds submitted to the SMR collection.

2.3. Probe Preparation

Image ml225fu3

Synthesis of pyrrolidine-1-carbonyl chloride (B): pyrrolidine A (1 equiv) was dissolved in dry CH2Cl2 (10 mL/mmol) and cooled to 0 degrees Celsius. Triphosgene (0.6 equiv) was added and the reaction was stirred for 10 minutes at 0 degrees Celsius and for a further 15 minutes at room temperature. The reaction was carefully quenched by dropwise addition of saturated aqueous NaHCO3, diluted with CH2Cl2, and washed with brine. The organic phase was dried over Na2SO4 and the solvent was removed under reduced pressure (water bath temperature <30 degrees Celsius). The crude carbamoyl chloride B was used for the next step without further purification. CAUTION: Triphosgene is very toxic. This reaction should be performed in a well-ventilated fume hood. Any object that comes into contact with triphosgene should be rinsed with 10% NaOH solution.

Synthesis of 4-([1,1′-biphenyl]-4-yl)-1H-1,2,3-triazole (D): NH-1,2,3-triazole D was prepared following a slightly modified procedure of Fokin et al. [22]. A mixture of 37% HCHO (10 equiv), glacial AcOH (1.5 equiv), and THF (1 mL/mmol C) was stirred for 15 minutes. Sodium azide was added (1.5 equiv), followed by (4-phenyl)phenyl acetylene C (481 mg, 2.7 mmol, 1 equiv). The mixture was stirred for 10 minutes and sodium ascorbate (0.2 equiv) was added, followed by CuSO4 solution (200 mg/mL H2O; 5 mol %). The reaction was stirred for 24 hours at 25 degrees Celsius. The solvents were removed and the residue was re-dissolved in 3:1 MeOH/2N NaOH (1 mL/mmol C). After stirring for 24 hours at room temperature, the solvents were azeotropically removed and the residue was purified by silica gel chromatography (15:85:1 MeOH/CH2Cl2/NEt3) to yield NH-1,2,3-triazole D (525 mg, 2.4 mmol, 88%). 1H-NMR (400 MHz, d6-DMSO): δ = 8.26 (s, 1H), 7.88-7.27 (m, 9H). HRMS: m/z calculated for C14H12N3 [M+H]+: 222.1026; found: 222.1028. CAUTION:This reaction may result in the formation of hydrazoic acid and should be performed in a well-ventilated fume hood and behind a blast shield. Sodium azide should not be mixed with strong acids.

Synthesis of (4-([1,1′-biphenyl]-4-yl)-2H-1,2,3-triazol-2-yl)(pyrrolidin-1-yl)methanone (ML225): A mixture of carbonyl chloride B (40 mg, 0.3 mmol, 1 equiv), NH-1,2,3-triazole D (80 mg, 0.36 mmol, 1.2 equiv), and 4-DMAP (cat) in 5:1 THF/NEt3 (2 mL/mmol B) was stirred for 10 hours at 60 degrees Celsius. The solvents were removed to yield the crude triazole urea ML225 as a 3:1 mixture of N2- and N1-carbamoylated regioisomers. Regioisomers were easily distinguishable by 1H-NMR shift of the triazole ring proton by comparison to NMRs for triazole ureas of known regiochemistry based on solved crystal structures [19]. The N2-carbamoyl triazole was isolated by silica gel chromatography (3:1 hexanes/ethyl acetate → ethyl acetate) to afford pure ML225 (83 mg, 0.26 mmol, 87%). 1H-NMR (400 MHz, CDCl3): δ = 8.10 (s, 1H), 7.90-7.29 (m, 9H), 4.07 (m, 1H), 3.94 (m, 1H), 3.73 (m, 2H), 2.01 (m, 4H), purity > 95%. HRMS: m/z calculated for C19H19N4O [M+H]+: 319.1553; found: 319.1556.

3. Results

Probe ML225 (Compound 29, SAR Table 5) has an IC50 value of 3 nM (section 3.2) vs. PAFAH2 and selectivity of >333 fold vs. more than 20 SHs and selectivity of >33-fold vs. pPAFAH (see section 3.6). Selectivity against pPAFAH was confirmed by gel-based ABPP both in vitro and in situ, and selectivity was confirmed for PAFAH1b2 and PAFAH1b3 by SILAC-ABPP (see section 3.6). ML225 was also shown to be highly active in situ against PAFAH2, with an IC50 of 170 pM (see section 3.5 and AID 504496).

3.1. Summary of Screening Results

In the Primary FluoPol HTS Assay for PAFAH2 (AID 492956), ~337K compounds were tested for inhibition of PAFAH2 labeling by the SH-specific activity-based probe FP-Rh [16]. The assay was conducted in singlicate at 3.39 μM compound concentration using purified, recombinant enzyme. A total of 3,330 compounds (0.99%) were active, passing the set threshold of 29.65% inhibition. The HTS Primary Assay for pPAFAH served as a counterscreen for PAFAH2. In silico comparison yielded 2,412 selective compounds for PAFAH2 and 915 overlapping hits with the other PAFAH assays. Of the 2,412 compounds, 2,341 were available to be tested in the Confirmation Assay. In the Confirmation Assay (AID 493030), compounds were tested at a single concentration (3.39 μM) in triplicate and 1,510 compounds (65%) were confirmed as active (Figure 1).

Figure 1. Flow chart describing HTS results.

Figure 1

Flow chart describing HTS results.

Of the 3,330 compounds that were considered active in the Primary Assay, 327 inhibited the target enzyme by at least 80.0%. Of those 327 compounds, 149 exhibited at least 2.5% activity in all bioassays tested and were removed. A further 13 compounds were removed because they registered as inactive in the Confirmation Assay (AID 493030). Of the remaining 165 compounds, 150 were discarded as having unwanted functional groups (esters, oximes, Michael acceptors, hydrazines, hydroxylamines, etc.), the vast majority (129) being esters. Interestingly, the trizole urea (SID 7974398, CID 735660; Compound 1 in Table 2) was a top lead in a previous screen for LYPLA1 inhibitors (AID 2174), and was used as a starting point to derive two successful probe compounds (ML211 and ML226). As a class, the triazole ureas were found to have tunable potency and selectivity, low cytotoxicity, and good activity in situ. Two other urea compounds (SID 26729517 and SID 24832496) and a structurally related bis-aryl compound (SID 49732109) (see Table 2) were also potent PAFAH2 inhibitors, suggesting that the urea triazole scaffold could likely be a promising starting point for inhibitor optimization for this enzyme as well. It is worth noting that SID 7974398 also inhibits anti-target pPAFAH; however, given past medchem success with the triazole urea scaffold, we thought it should be possible to improve selectivity via structural modification.

Table 2. Most promising leads from HTS Campaign for PAFAH2 inhibitor discovery.

Table 2

Most promising leads from HTS Campaign for PAFAH2 inhibitor discovery.

Additionally, as discussed in ref. [19], while previously characterizing agents that perturb endocannabinoid uptake and metabolism, we discovered that the tetrazole urea LY2183240 (92, Figure 2) [23] was a potent inhibitor of numerous SHs [24], including the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL or MGLL), and alpha/beta-hydrolase 6 (ABHD6), and have confirmed that 92 inhibits FAAH by covalent, carbamoylation of the enzyme’s serine nucleophile [24]. There are a handful of other reports of N-heterocyclic ureas as SH inhibitors, including the isoxazolonyl urea 93 [25], and 1,2,4-triazole urea 94 [26], which are potent inhibitors of hormone-sensitive lipase (LIPE); however, selectivity profiles have not been reported. Taken together, these data indicate that the triazole urea scaffold might be tolerant to modification without complete loss of binding efficiency, and would yield a probe with good in vitro and in situ properties.

Figure 2. N-Heterocyclic urea SH inhibitors.

Figure 2

N-Heterocyclic urea SH inhibitors.

Because the FluoPol-ABPP assay was conducted with purified, recombinant enzyme, we first confirmed that the triazole urea 1 (CID 735660 resynthesized as SID 103913565, Table 4) could inhibit endogenous PAFAH2 in a complex proteome by gel-based competitive ABPP. In this assay, a complex proteome containing endogenous PAFAH2 is incubated with test compound followed by reaction with the FP-Rh activity-based probe [16]. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. Test compounds that act as PAFAH2 inhibitors will prevent enzyme-probe interactions, thereby decreasing the fluorescence intensity of the protein bands. As reported in AID 504494, we determined that the triazole urea 1 (CID 735660, SID 103913565) had an IC50 of 1.4 μM for PAFAH2 using this method. Following confirmation, we embarked on a medchem campaign to improve the potency of compound 1, as detailed in section 3.4.

3.2. Dose Response Curves for Probe

An IC50 value was obtained from gel-based competitive-ABPP data (Figure 3) as described in AID 504494.

Figure 3. IC50 curve for probe ML225 (compound 29, SID 103913572) as determined by gel-based competitive-ABPP with FP-Rh (AID 504494) against target PAFAH2 in a complex proteome lysate.

Figure 3

IC50 curve for probe ML225 (compound 29, SID 103913572) as determined by gel-based competitive-ABPP with FP-Rh (AID 504494) against target PAFAH2 in a complex proteome lysate. IC50 = 3 nM

3.3. Scaffold/Moiety Chemical Liabilities

The probe compound was determined to covalently modify the catalytic serine (Ser236) of PAFAH2 by LC-MS/MS analysis (AID 504486). 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 followed by loss of the triazole moiety to carbamoylate the enzyme.

Figure 4. Covalent modification of PAFAH2 by probe ML225 (Compound 29, SID 103913572).

Figure 4

Covalent modification of PAFAH2 by probe ML225 (Compound 29, SID 103913572). Active Site Peptide: R.VAVMGHS236FGGATAILALAK.E

The probe compound showed no reactivity with glutathione (100 μM), indicating that it is not generally cysteine reactive, but rather has a tempered electrophilicity and specific structural elements that direct reactivity towards PAFAH2.

An irreversible probe has some distinct advantages over reversible analogs. Targets can be readily characterized by methods such as mass spectrometry and click chemistry-ABPP, required dosing is often lower, irreversible compounds are not as sensitive to pharmacokinetic parameters, and administration can induce long-lasting inhibition [27]. 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 [28].

Indeed, over a third of enzymatic drug targets are irreversibly inhibited by currently marketed drugs [29]. 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, esmoeprazole, and lansoprazole, and triacylglycerol lipase, whose serine nucleophile is targeted by orlistat [29].

3.4. SAR Tables

We first wanted to compare different N-heterocyclic ureas for their potential to act as SH inhibitors, as the electrophilicity of different compound would likely affect both reactivity with members of the SH superfamily as well as potential cross-reactivity with other classes of proteins. We synthesized alkyne-modified agents 610 (AA6-10, Table 3), which differ in electrophilicity due to variations in the leaving group. We then performed a competitive ABPP experiment by treating a mouse brain membrane proteome with 610 (20 μM, 30 min), followed by the SH-directed activity-based probe FP-Rh (2 μM, 30 min) [16], separation by SDS-PAGE, and detection of FP-Rh-labeled proteins by in-gel fluorescence scanning (Figure 3A, see also AID 504527 and ref. [19]). The carbamate 6 (AA6) and imidazole 7 (AA7) showed little to no detectable inhibition of SHs, whereas 1,2,3-triazoles 810 (AA8-AA10) blocked the FP-Rh-labeling of several proteins. As expected, the reactivity of these compounds followed the trend of electrophilicity imparted by their leaving groups, with the pyridyl triazole 10 (AA10) being the most acidic and reactive N-heterocyclic urea.

Table 3. N-heterocyclic urea compounds tested for general SH inhibition.

Table 3

N-heterocyclic urea compounds tested for general SH inhibition.

To assess the cross-reactivity of 1,2,3-triazole ureas 810 (AA8–AA10) with other protein classes, we performed a second, complementary competitive ABPP experiment (AID 504527 and ref. [19]). Here, we used the N-heterocyclic ureas themselves as probes and asked whether their proteome reactivity profiles could be blocked by pre-incubation with the non-fluorescent SH-directed activity based probe FP-biotin [30] (20 μM). Visualization of labeled proteins was achieved by click chemistry conjugation of the alkyne-functionalized triazole urea probes to an azide-Rh reporter tag [31]. FP-biotin competed the labeling of all proteins modified by triazoles 8 (AA8) and 9 (AA9) (Figure 5B), whereas several of the protein targets of the most electrophilic compound 10 (AA10) were not sensitive to FP-biotin competition (Figure 5B, red boxes), suggesting that they were not SH proteins. These data designated the monocyclic triazole ureas 8 (AA8) and 9 (AA9) as possessing the desired degree of electrophilicity to inhibit a number of SHs in proteomes, but, at the same time, avoid modification of proteins outside of the SH class.

Figure 5. Competitive ABPP with clickable N-heterocyclic urea activity-based probes 6–10 (AA6–AA10) to assess general SH reactivity.

Figure 5

Competitive ABPP with clickable N-heterocyclic urea activity-based probes 610 (AA6–AA10) to assess general SH reactivity. A. Competitive ABPP of compounds 610 (AA6–AA10) in the mouse brain membrane proteome with FP-Rh. (more...)

The SAR Tables 4 and 5 include 25 triazole urea analogs, including the resynthesized top triazole hit compound 1 (CID 735660, Table 2) as SID 103913565. The analogs are variable at three positions: the substituents of the urea nitrogen (R1 and R2) and the substituent at 4-position of the triazole (R3). (Note: when the substituent of the urea nitrogen is a ring system, the group is denoted R1/R2 in the SAR Tables 4 and 5, and the nomenclature includes the urea nitrogen). The synthetic compounds were subject to gel-based competitive ABPP profiling to assess both potency and selectivity against several dozen FP-sensitive SHs. Compounds 1 and 1130 were tested at 20, 200, and 1000 nM concentration as outlined in AID 404513 (see Tables 4 and 5 and Figure 8) and compounds 3134 were tested at 30, 150, 750 nM concentration as outlined in AID 504520 (see Table 6 in this report and Figure 9 of the ML226 probe report). The only observed anti-targets up to 1000 nM (indicated by disappearance of band in compound treated lane relative to the DMSO control) were N-acylaminoacyl-peptide hydrolase (APEH), alpha/beta hydrolase domain containing protein 11 (ABHD11), esterase D/formylglutathione hydrolase (ESD), and lysophospholipase 1 (LYPLA1). Compounds 1114, 16, 17, 20 are inactive (0% inhibition) at all concentrations tested.

Table 4. Target SAR Analysis.

Table 4

Target SAR Analysis.

Table 5. Target SAR Analysis.

Table 5

Target SAR Analysis.

Figure 8. Potency and selectivity of triazole urea synthetic library compounds at three compound concentrations.

Figure 8

Potency and selectivity of triazole urea synthetic library compounds at three compound concentrations. 1 μM (A), 200 nM (B), and 20 nM (C). Gel-based competitive ABPP assay conducted as described in AID 504513. Control = DMSO only (no compound). (more...)

Table 6. Target SAR Analysis.

Table 6

Target SAR Analysis.

First Round SAR (variation of R1/R2): The initial round of SAR preserved the triazole urea core of the HTS hit structure 1 (SID 103913565) and maintained R3 as a hydrogen, varying only the substituents at R1 and R2. Of the 10 new compounds synthesized (1120 in SAR Table 4), only compounds with small ring systems at R1/R2 – morpholine (compound 15), pyrrolidine (compound 18), and, to a lesser extent, piperidine (compound 19) showed any potency for PAFAH2, as judged by percent inhibition.

Second Round SAR (variation of R3): Based on results of the first round of SAR, for second generation compounds, we synthesized analogs bearing either morpholine (compounds 2124) or pyrrolidine (compounds 2530) at R1/R2 and introduced a series of (substituted) phenyl and diphenyl moieties at R3. As a class, the pyrrolidine compounds were more potent than their morpholine analogs (e.g., compare inhibition of 27 vs. 24; 26 vs. 23), and included two ultrapotent derivatives, compounds 28 and 29, with 4-phenoxyphenyl and 4-biphenyl substituents, respectively, at R3. While compound 28 showed evidence of anti-target reactivity with APEH, compound 29 (ML225) showed no evidence of anti-target activity at up to 1000 nM, giving >333-fold selectivity vs. more than 20 SH targets (IC50 vs. PAFAH2 = 3 nM) as analyzed by gel-based ABPP (see section 3.6).

Third Round of SAR (second iteration variation of R1/R2): To test the inter-dependence of the R1/R2 and R3 functional groups, we evaluated analogs bearing four of the most potent R3 groups identified in the second round of SAR and phenyl substituents at R1 and R2. As expected from the results with compound 13 (round 1), these derivatives (compounds 3134) showed significantly reduced potency as compared to the pyrrolidine and morpholine series (e.g., compare inhibition of 31 vs. 22; 32 vs. 28; 33 vs. 29; 34 vs. 30).

Summary: The SAR around PAFAH2 inhibition is observed to be rather steep, with several of the library members (i.e., 1114, 16, 17, 20) showing no inhibition of the target enzyme up to 1 μM compound concentration. This is likely a property unique to the target, rather than the inhibitor class, as triazole urea inhibitors of similar structure developed for LYPLA1/2 (ML211) and ABHD11 (ML226) do not evince this steep SAR profile. As compared to the initial HTS hit (compound 1) with an IC50 of 1400 nM for PAFAH2, probe ML225 (IC50 3 nM) represents more than a 450-fold improvement in potency. As assessed by gel-based ABPP (see section 3.6), ML225 shows no evidence of anti-target reactivity up to 1 μM, giving a fold-selectivity vs. FP-sensitive SHs of more than 333-fold.

3.5. Cellular Activity

In Situ Inhibition: Probe ML225 (compound 29, AA39-2) is highly active against PAFAH2 in situ (AID 504495), completely inhibiting enzymatic activity at 30 nM compound concentration after 4 hours (media + 10% fetal calf serum) as assayed by gel-based competitive ABPP (Figure 6). Further analysis revealed complete inhibition at 10 nM (Figure 9B) and an IC50 of 170 pM for in situ inhibition of PAFAH2 (AID 504496). These results indicate that ML225 is free to cross cell membranes and inhibit its target in the cytoplasm of cells, even in the presence of serum-containing medium.

Figure 6. Selective inhibition of PAFAH2 activity in situ by probe ML225 (compound 29, band.).

Figure 6

Selective inhibition of PAFAH2 activity in situ by probe ML225 (compound 29, band.).

Figure 9. Potency and selectivity of ML225 against anti-target pPAFAH.

Figure 9

Potency and selectivity of ML225 against anti-target pPAFAH. A.In Vitro: recombinant pPAFAH expressed in COS-7 cells; ML225 has an IC50 ≥100 nM for pPAFAH, corresponding to >33-fold selectivity. B.In Situ: endogenous pPAFAH in compound-treated (more...)

Cytotoxicity: The probe ML225 (compound 29, AA39-2) and analog 28 (AA39-1) were evaluated for cytotoxicity (AID 504511) using both serum-free and serum-supplemented media. As shown in Figure 7, both compounds have a CC50 of at least 35 μM, which is five orders of magnitude greater than the in situ IC50 value of 170 pM (AID 504496). It should be noted, however, that based on the solubility calculation of 1.7 μM for ML225 (section 2.2), the CC50 number may not be accurate. However, even if a strict lower limit of 1.7 μM is imposed on the CC50 value, there is still a >10,000-fold difference between the active and cytotoxic in situ concentrations.

Figure 7. Cytotoxicity of probe ML225 and analog 28.

Figure 7

Cytotoxicity of probe ML225 and analog 28. In serum-free (A) and serum-supplemented (B) media. See AID 504511 for details.

3.6. Profiling Assays

HTS Analysis: To date, the HTS hit (Compound 1, CID 735660) has been tested in 504 other cell-based and non-cell based bioassays deposited in PubChem, and has shown activity in only 11 of those assays, giving a hit rate of 2.0%. This low hit rate indicates that this compound class may not be generally active. No HTS activity data is yet available for probe ML225 or any of the synthetic analogs, nor has compound ML225 been submitted for commercial or non-commercial broad panel screening.

Gel-based Competitive ABPP: Probe ML225 and analogs listed in Section 3.4 have been subject to gel-based competitive ABPP screening to assess SH reactivity (see Section 3.4 for summary of data below). Competitive ABPP (Figure 8) was utilized for medchem optimization, allowing rapid assessment of anti-target inhibition (as visualized by disappearance of bands in compound-treated lanes) of analogs against >20 distinct SHs. The optimized probe ML225 shows >333-fold selectivity against all other FP-sensitive SHs (Figure 8A, compound AA39-2, see also AID 504513).

Selectivity vs. pPAFAH: ML225 was shown to selectively inhibit PAFAH2 over its closest sequence homologue pPAFAH, showing >33-fold selectivity in vitro (Figure 9A, IC50 3 nM for PAFAH2, ~100 nM for pPAFAH) and in situ, where 10 nM compound shows complete inhibition of PAFAH2 and no inhibition of pPAFAH (Figure 9B).

SILAC-ABPP: 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 is a merger of ABPP-MudPIT method [32] with the stable isotope labeling of amino acids in culture (SILAC) technique [33]. Competitive ABPP-SILAC allows for precise quantitation of inhibited enzymes by calculating the isotopic ratios of peptides from control- and inhibitor-treated cells. As described in AID 504519 and ref. [19], mouse T cells were cultured in ‘light’ medium (with 12C614N2-lysine and 12C614N4-arginine) or ‘heavy’ medium (with 13C615N2-lysine and 13C615N4-arginine). After 6 passages, near-complete (>97%) enrichment was achieved. Light and heavy cells were treated with 3 nM of ML225 and DMSO, respectively, for four hours and then harvested, lysed, separated into soluble and membrane fractions, and treated with the affinity-tagged FP-biotin activity-based probe (5 μM, 90 min) [31]. Light and heavy fractions were then mixed (1:1 w/w), enriched with avidin, digested on-bead with trypsin, and analyzed by MudPIT [3435] LC/LC-MS/MS using an LTQ-Orbitrap instrument. Light and heavy signals were quantified from parent ion peaks (MS1) and the corresponding proteins identified from product ion profiles (MS2) using the SEQUEST [36] search algorithm and filtered using DTASelect [37]. The depicted bar graphs represent the average ratios of light/heavy tryptic peptides for each of the SHs identified in mouse T cells. Enzymes susceptible to inhibition upon compound treatment would be expected to have light/heavy ratios significantly less than one, while unaffected enzymes would have a ratio close to one. The results (Figure 10B) demonstrate that ML225 exhibits significant selectivity for PAFAH2, blocking >95% of activity while not affecting any of the other 40 SHs detected in T cells, with the exception of 40% inhibition of ABHD6. The PAFAH enzymes PAFAH1b2 and PAFAH1b3 also showed no evidence of ML225 inhibition.

Figure 10. Potency and selectivity of ML225 assessed by SILAC-ABPP.

Figure 10

Potency and selectivity of ML225 assessed by SILAC-ABPP. A) Overview of the SILAC-ABPP method. B) Inhibition profile of ML225 shows significant inhibition of only PAFAH2 and 40% inhibition of ABHD6. See AID 504519 and ref. [19] for more details.

4. Discussion

Probe ML225 (Compound 29) was identified as a highly potent and selective covalent dual inhibitor of the target enzyme PAFAH2. The reported probe has an IC50 of 3 nM, >33-fold selectivity against the close homologue pPAFAH, and >333-fold selectivity against all other SH anti-targets surveyed by gel-based competitive ABPP (section 3.6). A more in-depth SILAC-ABPP analysis revealed no anti-target reactivity for more than 40 SHs, with the exception of ABHD6, for which ML225 showed some modest cross-reactivity (section 3.6). In the event that this property proves problematic, our previously reported ABHD6-selective inhibitor, WWL70 [38] (IC50 70 nM, selective for ABHD6 over other SHs by SILAC ABPP) could be used as a control. ML225 is active in situ (sections 3.5 and 3.6) with an IC50 of 170 pM, shows no evidence of cytotoxicity up to 35 μM, and is stable in PBS for more than 48 hours. Taken together, these findings suggest that it is very possible to develop potent and selective probes based on tempered electrophilic scaffolds, and that ML225 will be a highly successful probe for investigation of PAFAH2 biology.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

No selective PAFAH2 inhibitors have been reported.

4.2. Mechanism of Action Studies

As determined from LC-MS/MS analysis (AID 504486), the probe is an activity-based inhibitor that covalently labels the active site serine nucleophile, Ser236, of PAFAH2 (section 3.3). The observed mass shift of the active site peptide suggests that reaction occurs via serine nucleophilic attack on the carbonyl followed by loss of the triazole to carbamoylate the enzyme (Figure 1).

4.3. Planned Future Studies

Given the success of the triazole urea library, we plan to expand the library for identification of additional PAFAH2 and other SH probes. Additionally, we plan to more comprehensively establish parameters for in situ and in vivo use and explore the target specificity of ML225 via application of more homologous alkyne analogs. For biological application, we plan to use ML225 as a probe for investigation of the potential role of PAFAH2 in inflammatory processes by global proteomic and metabolomic profiling of cells cultured under conditions of oxidative stress to identify potential metabolic pathway involvement. We will also use ML225 as a tool for structural studies—to date, a three-dimensional structure of PAFAH2 has not been solved.

5. References

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