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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Optimization and characterization of a triazole urea dual inhibitor for lysophospholipase 1 (LYPLA1) and lysophospholipase 2 (LYPLA2)

, , , , , , , , , and .

Author Information and Affiliations

Received: ; Last Update: March 7, 2013.

Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. We [1] and others [2] have previously identified lysophospholipase 1 (LYPLA1) as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis, we conducted a fluorescence polarization-based competitive activity-based protein profiling (FluoPol ABPP) [3] high throughput screening (HTS) campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a micromolar triazole urea inhibitor, which we successfully optimized via several rounds of structure activity relationship (SAR)-by-synthesis to produce ML211 (SID 99445338), a low nanomolar dual inhibitor of LYPLA1 and LYPLA2. The reported probe operates by a covalent mechanism of action and is active both in vitro and in situ. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML211 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11). However, during our SAR campaign, we fortuitously discovered a selective ABHD11 inhibitor from among the synthetic triazole urea library compounds. This compound, ML226, is presented as an anti-probe for control studies.

Assigned Assay Grant #: 1 R01 CA132630

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): 2202 (LYPLA1), 2203 (LYPLA2)

Probe Structure & Characteristics

Image ml211fu1
CID/ML#Target NameIC50 (nM) [SID, AID]Anti-target Name(s)IC50 (nM) [SID, AID]Fold Selective†Secondary Assay(s) Name: IC50 (nM) [SID, AID]
CID 56593118/ML211LYPLA1 (a.k.a. APT1)17 [SID 99445338, AID 493110]ABHD11 and > 20 SHs*10 vs. ABHD11 [SID 99445338, AID 493154]

> 1500 vs. all other SHs [SID 99445338, AID 493111]**		0 vs. ABHD11

≥ 50 vs. all other SHs		Inhibition Assay: [SID 99445338, AID 493105]
Selectivity Assay: [SID 99445338, AID 493111]
In Situ Assay: [SID 99445338, AID 493108]
IC50 Assay: 17 nM LYPLA1, 30 nM LYPLA2 [SID 99445338, AID 493110]
IC50 anti-target Assay: 10 nM ABHD11 [SID 99445338, AID 493154]
Cytox assay: [SID 99445338, AID 493161]
LC-MS/MS assay: [SID 99445338, AID 493109]
LYPLA2 (a.k.a. APT2)30 [SID 99445338, AID 493110]
*

As assessed by gel-based competitive ABPP in a soluble proteome derived from murine T cells with the serine hydrolase probe FP-Rhodamine.

**

IC50 of the anti-target is defined as greater than the test compound concentration at which less than or equal to 50% inhibition of the anti-target is observed, which is reported in AID 493111. For SID 99445338, no anti-targets were observed for all other serine hydrolases (SHs) besides ABHD11 at 1.5 μM, so the IC50 is reported as > 1.5 μM.

†

Fold-selectivity was calculated as: > IC50 for anti-target/IC50 for target.

Recommendations for Scientific Use of the Probe

Protein palmitoylation is an essential post-translational modification (PTM), and identification of enzymes responsible for the dynamic modulation of palmitoylation is paramount to understanding its patho/physiological roles. We [1] and others [2] have previously identified LYPLA1 as a putative protein palmitoyl thioesterase capable of regulating HRAS palmitoylation in mammalian cells. LYPLA2 is 65% identical to LYPLA1, but its potential role as a thioesterase is unknown. The probe described in this report can inhibit LYPLA1 and LYPLA2 both in vitro (complex proteome lysates) and in situ (cells in culture) at low nanomolar concentration. The probe is observed to have one anti-target, the serine hydrolase ABHD11; however, a specific inhibitor of ABHD11 is also described herein (ML226), and is to be used as a control for identification of ABHD11-selective phenotypes. These inhibitors are intended for use in primary research studies aimed at elucidating the patho/physiological roles of LYPLA1 and LYPLA2.

1. Introduction

Protein palmitoylation is an essential PTM necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Numerous proteins have been identified as targets of palmitoylation, including cytoskeletal proteins, kinases, receptors, and other proteins involved in various aspects of cellular signaling and homeostasis [4]. Using a global chemo-proteomic method for the metabolic incorporation and identification of palmitoylated proteins, we were able to identify hundreds of palmitoylated proteins, revealing palmitoylation as a widespread PTM [1]. Palmitoylation involves an acyl-thioester linkage to specific cysteines [5–6]. Given the labile properties of thioesters, palmitoylation is potentially reversible and may be regulated in a manner analogous to other PTMs (e.g., phosphorylation). As such, identification of proteins responsible for the dynamic modulation of palmitoylation is paramount to understanding its patho/physiological roles. For example, multiple oncogenes, including HRAS and SRC, require palmitoylation-mediated localization for signaling and subsequent malignant transformation [7]. It has been suggested by Waldmann et al. that preventing depalmitoylation of RAS by inhibiting thioesterase activity may disrupt targeted distribution (e.g., to Golgi), thereby downregulating oncogenic signaling [2]. This suggests that inhibitors of protein palmitoyl thioesterases may act as tumor suppressors by preventing aberrant growth signaling. More than a decade ago, the cytosolic serine hydrolase (SH) acyl-protein thioesterase 1 (APT1) was identified as an in vitro HRAS palmitoyl thioesterase [8]. Initially classified as lysophospholipase 1 (LYPLA1) [9], the enzyme has since been demonstrated to have several hundred-fold higher activity as a protein thioesterase. While the in vitro data [8, 10] provided an intriguing clue to its possible role in vivo, much less is known about the in vivo thioesterase activity of LYPLA1. Upon retroviral shRNA knockdown of LYPLA1, we found that over-expressed HRAS was hyper-palmitoylated, providing evidence that the endogenous enzyme is a functional protein palmitoyl thioesterase capable of regulating over-expressed HRAS palmitoylation in mammalian cells. However, shRNA resulted in only an 80% reduction in LYPLA1 expression (unpublished). LYPLA2 (a.k.a. APT2) is 65% identical to LYPLA1, and also exhibits lysophospholipase activity in vitro, but its potential role as a thioesterase is unknown [11]. shRNA knockdown studies of LYPLA2 revealed only partial knockdown of the enzyme, making substrate identification inconclusive (unpublished). A principle goal of post-genomic research is the determination of the molecular and cellular role of uncharacterized enzymes like LYPLA1 and LYPLA2. As such, a dual inhibitor selective for both LYPLA1 and LYPLA2 would greatly aid investigations into the biological function of these related enzymes.

Several inhibitors of LYPLA1 have been described [2, 12–13] (see section 4.1 for further discussion), but none of these agents have proven capable of selectively inhibiting LYPLA1 activity in cells, and no selective inhibitors of LYPLA2 have been reported to date. To comprehensively identify LYPLA1 and LYPLA2 substrates and functionally test the role of these enzymes in dynamic de-palmitoylation and tumorigenesis, development of a high affinity and selective inhibitor capable of achieving temporal and more complete control over activity is critical.

As SHs, catalytically active LYPLA1 and LYPLA2 are readily labeled by fluorescent activity-based protein profiling (ABPP) probes bearing a fluorophosphonate (FP) reactive group [14]. 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 [15]. Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP [3]. In conjunction with the SRIMSC, we applied FluoPol-ABPP to LYPLA1 and LYPLA2 inhibitor discovery. Initial efforts focused on deriving a dual LYPLA1/2 inhibitor based on a triazole urea scaffold, which has also been developed into inhibitors of other SHs (e.g, see Probe Reports for ML225, ML294, and ML295 and ref. [16]. The reported compound (ML211) inhibits both target enzymes with high potency (IC50 17 nM vs. LYPLA1, 30 nM vs. LYPLA2), and is active in situ, inhibiting LYPLA1 and LYPLA2 completely at 30nM concentration in serum-containing media. ML211 exhibits good selectivity (50-fold) among SHs, with the exception of the SH ABHD11 (IC50 10 nM), which was a common anti-target for all triazole ureas tested; however, we were able to derive a highly potent ABHD11-selective anti-probe, ML226 (IC50 15 nM against ABHD11, IC50 >10,000 nM against LYPLA1 and LYPLA2), that is designed to be used in parallel with the dual LYPLA1/2 inhibitor for identification of ABHD11-inhibition specific effects. Selective inhibitors for LYPLA1 and/or LYPLA2 with nanomolar potency and demonstrated in situ activity have not been described to date.

2. Materials and Methods

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

2.1. 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 were in MRM mode where the parent ion was selected in Q1 of the mass spectrometer. The parent ion was fragmented and a characteristic fragment ion was 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 were rapidly set up using Automaton® (Applied Biosystems), where the compounds were listed with their name and mass in an Excel datasheet. Compounds were submitted in a 96-well plate to the HPLC autosampler and slowly injected without a column present. A narrow range centered on the indicated mass was scanned to detect the parent ion. The software then evaluated a few pre-selected parameters to determine conditions that maximized the signal for the parent ion. The molecule was then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass were determined. Three separate collision energies were evaluated to fragment the parent ion and the largest three ions were selected. Each of these three fragment ions was further optimized and the best fragment was chosen. The software then inserted the optimized masses and parameters into a template method and saved it with a unique name that indicated the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern were saved and could 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 was 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 [17–18].

Primary Assays

Primary uHTS assay to identify LYPLA1 inhibitors (AID 2174)

Assay Overview: The purpose of this assay was to identify compounds that act as LYPLA1 inhibitors. This assay also served as a counterscreen for a set of previous experiments entitled, “Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1)” (AID 2130). This competitive activity-based protein profiling (ABPP) assay uses fluorescence polarization to investigate enzyme-substrate functional interactions based on active site-directed molecular probes [3, 15]. A fluorophosphonate-rhodamine (FP-Rh) probe, which broadly targets enzymes from the serine hydrolase family [14] was used to label LYPLA1 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 LYPLA1 inhibitors prevent LYPLA1-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) served as a positive control. Compounds were tested at a nominal concentration of 5.9 μM.

Protocol Summary: Prior to the start of the assay, Assay Buffer (4.0 μL; 0.01% Pluronic acid, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1mM DTT) containing LYPLA1 protein (6.25 nM) was dispensed into 1536-well microtiter plates. Next, test compound (30 nL in DMSO) or DMSO alone (0.59% final concentration) was added to the appropriate wells and incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing FP-Rh probe (1.0 μL of 375 nM in Assay Buffer) to all wells. Plates were centrifuged and, after 10 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software. Assay Cutoff: Compounds that inhibited LYPLA1 greater than 14.15% were considered active.

Confirmation uHTS assay to identify LYPLA1 inhibitors (AID 2233)

Assay Overview: The purpose of this assay was to confirm activity of compounds identified as active in the primary uHTS screen (AID 2174). In this assay, the FP-Rh probe was used to label LYPLA1 in the presence of test compounds and analyzed as described above (AID 2174). Compounds were tested in triplicate at a nominal concentration of 5.9 μM.

Protocol Summary: The assay was performed as described above (AID 2174), except that compounds were tested in triplicate. Assay Cutoff: Compounds that inhibited LYPLA1 greater than 14.15% were considered active.

Primary uHTS assay to identify LYPLA2 inhibitors (AID 2177)

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

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

Confirmation uHTS assay to identify LYPLA2 inhibitors (AID 2232)

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

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

Secondary Assays

Inhibition of LYPLA1 and LYPLA2 by top HTS hits (AID 493105)

Assay Overview: The purpose of this assay was to determine whether test compounds can inhibit LYPLA1 and LYPLA2 in a complex proteomic lysate using an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome, containing endogenous and spiked-in recombinant forms of LYPLA1 or LYPLA2, was incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining was determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as LYPLA1 and/or LYPLA2 inhibitors prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition was calculated relative to a DMSO (no compound) control.

Protocol Summary: To soluble (Assay 1) or membrane (Assays 2–4) proteome prepared from mouse brain (1 mg/ml in DPBS) was added either 20 nM purified recombinant human (rh) LYPLA1 (Assays 1 and 3) or 20 nM purified rhLYPLA2 (Assays 2 and 4). For each Assay, proteome was treated with 20 μM test compound (1 μL of a 50× stock in DMSO) 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 bands: rhLYPLA1 (Assay 1), rhLYPLA2 (Assay 2), endogenous mouse (em) LYPLA1 (Assay 3) and emLYPLA2 (Assay 4) relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥30% inhibition of target band in each assay were considered active.

Triazole urea library potency and selectivity analysis (AID 493111)

Assay Overview: The purpose of this assay was to determine whether powder samples of test compounds could inhibit LYPLA1 and LYPLA2 in a complex proteomic lysate and to estimate compound selectivity in an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome was incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining was determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as LYPLA1 and/or LYPLA2 inhibitors prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition were 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 30 nM, 200 nM, or 1.5 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 (LYPLA1 and LYPLA2) and anti-target (alpha/beta hydrolase domain-containing protein 11 [ABHD11], esterase D/formylglutathione hydrolase [ESD], and N-acylaminoacyl-peptide hydrolase [APEH]) bands relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥50% inhibition at 200 nM were considered active. Compounds that were active for inhibition of LYPLA1 and LYPLA2 and inactive for inhibition of anti-targets ESD and APEH were considered active regardless of the ADHD11 inhibition in the overall outcome.

Inhibition of LYPLA1 in situ (AID 493108)

Assay Overview: The purpose of this assay was to determine whether or not powder samples of test compounds could inhibit LYPLA1, LYPLA2 and anti-target ABHD11 activity in situ. In this assay, cultured BW5147-derived murine T-cells were incubated with test compound. Cells were harvested and the soluble fraction was isolated and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining was determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as LYPLA1, LYPLA2 and/or ABHD11 inhibitors 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 30 nM test compound (5 μL of a 1000× stock in DMSO) for 2 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 LYPLA1 band, the LYPLA2 band, and the ABHD11 band relative to a DMSO-only (no compound) control. Assay Cutoff: Compounds with ≥90% inhibition were considered active. Compounds active for both LYPLA1 and LYPLA2 were considered active in the overall outcome.

Determination of IC50 values against LYPLA1 and LYPLA2 (AID 493110)

Assay Overview: The purpose of this assay was to determine the IC50 values of powder samples of test compounds for LYPLA1 and LYPLA2 inhibition in a complex proteome. In this assay, a fluorophosphonate-conjugated rhodamine (FP-Rh) activity-based probe was used to label LYPLA1 and LYPLA2 in the presence of test compounds. The reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining was determined by measuring the integrated optical density of the bands. As designed, test compounds that act as LYPLA1 and LYPLA2 inhibitors 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 LYPLA1 and LYLA2 were determined from dose-response curves from three replicates at each inhibitor concentration (7-point 1:3 dilution series from 10 μM to 10 nM). Assay Cutoff: Compounds with an IC50 <1 μM were considered active. Compounds active against both LYPLA1 and LYPLA2 were considered active in the overall outcome.

Determination of IC50 values against anti-target ABHD11 (AID 493154)

Assay Overview: The purpose of this assay was to determine the IC50 values of powder samples of test compounds for anti-target ABHD11 (alpha/beta hydrolase domain-containing protein 11) inhibition in a complex proteome. In this assay, a fluorophosphonate-conjugated rhodamine (FP-Rh) activity-based probe was used to label ABHD11 in the presence of test compounds. The reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining was determined by measuring the integrated optical density of the bands. As designed, test compounds that act as ABHD11 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. An IC50 value for inhibition of ABHD11 was determined from a dose-response curve from three replicates at each inhibitor concentration (7-point 1:3 dilution series from 10 μM to 10 nM). Assay Cutoff: Compounds with an IC50 <1 μM were considered active.

Analysis of Cytotoxicity (AID 493161)

Assay Overview: The purpose of this assay was to determine cytotoxicity of inhibitor compounds belonging to the triazole urea scaffold. In this assay, BW5147-derived murine T-cells in either serum-free media (Assay 1) or media containing FCS (Assay 2) were 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 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 50μM.

Protocol Summary: This assay was started by dispensing BW5147-derived murine T-cells in RPMI media (100μL, 10×104 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 0–200 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). Compounds with a CC50 value ≥ 5 μM were considered inactive (non-cytotoxic). Compounds that were inactive in both assays were considered inactive in the overall outcome.

LC-MS/MS Analysis of Inhibitor Binding Mode (AID 493109)

Assay Overview: The purpose of this assay was to assess the covalent nature of an inhibitor compound belonging to the triazole urea scaffold and determine whether or not it labels the active site serine of LYPLA1. In this assay, purified enzyme was reacted with inhibitor compound, digested with trypsin, and the resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting data were analyzed to identify sites of covalent labeling.

Protocol Summary: Two aliquots (25 μL) of 50 μM LYPLA1 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 Celsius 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 Celsius 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 +167.131 was specified for serine to account for possible probe labeling by AA64-2. 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 LYPLA1 were considered active.

2.2. Probe Chemical Characterization

CID 56593118, SID 99445338, ML211.

CID 56593118, SID 99445338, ML211

The probe structure was verified by 1H NMR (see section 2.3) and high resolution MS (m/z calculated for C25H30N4O2 [M+H]+: 418.2369; found: 418.2382). 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 [16]. Solubility in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4) at room temperature was determined by UV trace to be 6.6 μM. Stability could not be assessed using the standard LC-MS assay, as the probe was found to be unstable under ESI conditions.

Table 1Compounds submitted to the SMR collection

DesignationCompound Lab NameCIDSIDSRIDMLS
ProbeAA64-2CID 56593118SID 99445338SR-02000000958-1MLS003336859
Analog 1AA69-2CID 46937237SID 99445340SR-02000000960-1MLS003336860
Analog 2AA69-1CID 46937229SID 99445339SR-02000000959-1MLS003336861
Analog 3AA44-1CID 46937236SID 99445332SR-02000000952-1MLS003336862
Analog 4AA62-2CID 46937243SID 99445337SR-02000000957-1MLS003336863
Analog 5AA50-1CID 46937231SID 99445336SR-02000000956-1MLS003336864

2.3. Probe Preparation

Figure 1. Synthesis of ML 211.

Figure 1Synthesis of ML 211

Synthesis of 4-(tert-butyl)piperidine-1-carbonyl chloride (B): 4-(tert-butyl)piperidine A (1 equiv) was dissolved in dry CH2Cl2 (10 mL/mmol) and cooled to 0 ºC. Triphosgene (0.6 equiv) was added and the reaction was stirred for 10 min at 0 ºC and for further 15 min at rt. The reaction was carefully quenched by dropwise addition of sat. aq. 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 ºC). 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 diphenyl(1H-1,2,3-triazol-4-yl)methanol (D): NH-1,2,3-triazole D was prepared following a slightly modified procedure of Fokin et al. [19]. Mixture of 37% HCHO (10 equiv), glacial AcOH (1.5 equiv), and THF (1 mL/mmol C) was stirred for 15 min. Sodium azide was added (1.5 equiv), followed by 1,1-diphenyl-2-propyn-1-ol C (562 mg, 2.7 mmol, 1 equiv). The mixture was stirred for 10 min and sodium ascorbate (0.2 equiv) was added, followed by CuSO4 solution (200 mg/mL H2O; 5 mol %). The reaction was stirred for 24 h at 60 ºC. The solvents were removed and the residue was re-dissolved in 3:1 MeOH/2N NaOH (1 mL/mmol C). After stirring for 24 h at rt, the solvents were azeotropically removed and the residue was purified by silica gel chromatography (15:85:1 MeOH/CH2Cl2/NEt3) to yield diphenyl(1H-1,2,3-triazol-4-yl)methanol D (495 mg, 2.0 mmol, 73%). 1H-NMR (400 MHz, d6-DMSO): δ = 7.82 (s, 1H), 7.54-7.27 (m, 10H). HRMS: m/z calculated for C15H14N3O [M+H]+: 252.1131; found: 252.1135. 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-(tert-butyl)piperidin-1-yl)(4-(hydroxydiphenylmethyl)-2H-1,2,3-triazol-2-yl)methanone (ML211): Mixture of carbonyl chloride B (183 mg, 0.9 mmol, 1 equiv), NH-1,2,3-triazole D (271 mg, 1.08 mmol, 1.2 equiv), and 4-DMAP (cat) in 5:1 THF/NEt3 (2 mL/mmol B) was stirred for 10 h at 60 ºC. The solvents were removed to yield the desired triazole urea AA64-2 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 [16]. The N2-carbamoyl triazole was isolated by silica gel chromatography (3:1 hexanes/ethyl acetate -> ethyl acetate) to afford ML211 (193 mg, 0.46 mmol, 51%). 1H-NMR (400 MHz, CDCl3): δ = 7.58 (s, 1H), 7.38-7.23 (m, 10H), 3.71-3.38 (m, 4H), 1.29-1.12 (m, 5H), 0.88 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), purity >95%. HRMS: m/z calculated for C25H30N4O2 [M+H]+: 418.2369; found: 418.2382.

3. Results

IC50 values of 17 nM vs. LYPLA1 and 30 nM vs. LYPLA2 were derived from gel-based competitive ABPP data (see Section 3.2) for probe ML211 (compound 1.18, SAR Table 3). A search of more than 20 SHs yielded data for target/anti-target selectivity, revealing one anti-target: ABHD11 (IC50 of 10 nM based on gel-based competitive ABPP data). ML211 was 50-fold or more selective for all other SHs visualized by gel-based ABPP (IC50 ≥ 1.5 μM). A potent and selective inhibitor of ABHD11 (IC50 15 nM for ABHD11, IC50 > 10,000 nM for LYPLA1 and LYPLA2), compound 1.12 (SID 99445332), (ML226) is provided as a control anti-probe. Both ML211 and ML226 were shown to be highly active in situ against their targets (see Section 3.5).

Table 3. Target SAR Analysis.

Table 3

Target SAR Analysis.

3.1. Summary of Screening Results

In the primary FluoPol HTS assay for LYPLA1 (AID 2174), ~315K compounds were screened with the SH-specific FP-Rh probe [14]. A total of 499 compounds (0.16%) were active, passing the set threshold of 14.15% LYPLA1 inhibition. For the confirmation HTS screen (AID 2233), 478 active compounds were retested in triplicate, and 331 compounds (69%) were confirmed as active (Figure 2).

Figure 2. Flow chart describing HTS results.

Figure 2

Flow chart describing HTS results.

Prior to secondary screening, the active compounds were filtered to remove compounds with at least 30% activity against previous FluoPol ABPP enzyme inhibitor screens: GSTO1 (AID 1974; 48 compounds), FAM108b (AID 1947, 14 compounds), and PME-1 (AID 2130, 16 compounds). Additionally, 23 compounds with greater than 5% hit rate in all bioassays tested were removed, as well as the majority of compounds (30) that contained esters. Of the remaining 200 compounds, all hits with at least 50% inhibition against LYPLA1 (37 compounds) were chosen for secondary screening along with 54 additional compounds cherry-picked from the remaining hits (total compounds for secondary screening = 91). Of these 91 hits, the majority (62) also showed activity against LYPLA2 in the primary (AID 2177) and/or confirmation (AID 2232) FluoPol ABPP HTS assay for LYPLA2 inhibitors (activity cutoff: 25.78% inhibition).

The 91 compounds selected for secondary screening were assayed for their ability to inhibit both the recombinant human (rh) and endogenous mouse (em) isoforms of LYPLA1 and LYPLA2 by gel-based competitive ABPP in the context of a complex proteome (AID 493105; See Supplemental Figures S1, S2, S3 and Table S1 at the end of this report). Compounds with at least 30% inhibition were scored as active. In this assay, a complex proteome, containing endogenous and spiked-in recombinant forms of LYPLA1 or LYPLA2, was incubated with test compound (20 μM) followed by reaction with the FP-Rh activity-based probe. The reaction products were separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. Test compounds that act as LYPLA1/LYPLA2 inhibitors prevent enzyme-probe interactions, thereby decreasing the fluorescence intensity of the protein bands. Nineteen compounds were active against rhLYPLA1, 10 compounds were active against rhLYPLA2, and 5 compounds were active against both human enzymes. Of the five compounds active against both rhLYPLA1 and rhLYPLA2, three were also active against emLYPLA1 and emLYPLA2.

The top lead (Compound 55, SID 7974398; see Table 2) has a triazole urea scaffold that appeared readily amenable to medchem optimization. Interestingly, the second best compound (Compound 19, SID 57261525; see Table 2) is an amine that bears a strikingly similar structure.

Table 2. Top Inhibitors from 2ary Screen.

Table 2

Top Inhibitors from 2ary Screen. (See supplemental information for complete results.)

Additionally, as discussed in ref. [16], while previously characterizing agents that perturb endocannabinoid uptake and metabolism, we discovered that the tetrazole urea LY2183240 (92, Figure 3) [20] was a potent inhibitor of numerous SHs [21], including the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL or MGLL), and α/β-hydrolase 6 (ABHD6), and have confirmed that 92 inhibits FAAH by covalent, carbamoylation of the enzyme’s serine nucleophile [21]. There are a handful of other reports of N-heterocyclic ureas as SH inhibitors, including the isoxazolonyl urea 93 [22], and 1,2,4-triazole urea 94 [23], 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 3. N-Heterocyclic Urea SH Inhibitors.

Figure 3

N-Heterocyclic Urea SH Inhibitors.

3.2. Dose Response Curves for Probe

IC50 values were obtained from gel-based competitive-ABPP data (Figure 4) as described in AID 493110.

Figure 4. IC50s Curves for Probe ML211 (Compound 1.18, SID 99445338) as determined by gel-based competitive-ABPP with FP-Rh (AID 493110) against targets LYPLA1 and LYPLA2.

Figure 4

IC50s Curves for Probe ML211 (Compound 1.18, SID 99445338) as determined by gel-based competitive-ABPP with FP-Rh (AID 493110) against targets LYPLA1 and LYPLA2.

3.3. Scaffold/Moiety Chemical Liabilities

The probe compound was determined to covalently modify the catalytic serine (Ser114) of LYPLA1 (AID 493109). The observed mass shift of the active site peptide corresponds to the adduct depicted in Figure 5, formed by serine nucleophilic attack at the carbonyl followed by loss of the triazole moiety. Given the high structural homology between LYPLA1 and LYPLA2 (65% overall sequence homology; both exhibit the SH Ser-His-Asp catalytic triad signature), it is anticipated that ML211 modifies LYPLA2 in an analogous manner.

Figure 5. Covalent modification of LYPLA1 by Probe ML211 (Compound 1.18, SID 99445338).

Figure 5

Covalent modification of LYPLA1 by Probe ML211 (Compound 1.18, SID 99445338). Active Site Peptide: R.IILGGFS114QGGALSLYTALTTQQK.L

The probe compound showed some reactivity with glutathione (100 μM); however, its general proteome reactivity profile (see Figure 6, last lane panels A-C) suggests that it is not a broadly reactive compound.

Figure 6. Potency and Selectivity of 20 Triazole Urea Synthetic Library Compounds at three compound concentrations: 1.5 μM (A), 200 nM (B), and 30 nM (C).

Figure 6

Potency and Selectivity of 20 Triazole Urea Synthetic Library Compounds at three compound concentrations: 1.5 μM (A), 200 nM (B), and 30 nM (C). Gel-based competitive ABPP assay conducted as described in AID 493111. Control = DMSO only (no compound). (more...)

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 [24]. 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 [25].

Indeed, over a third of enzymatic drug targets are irreversibly inhibited by currently marketed drugs [26]. 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 [26].

3.4. SAR Tables

We re-synthesized the initial HTS hit compound 55 (Table 2, SID 7974398) as compound 1.1 (Table 3; SID 92709166) and synthesized an additional 19 analogs with variable structures at three positions: the substituent at 4-position of the triazole (R1), the substituent of the piperidine ring (R2), and the identity of the atom at the 4-position of the piperidine ring (X) (See Table 3). Note: compounds are given a prefix of “1” to distinguish them from the numbered compound in Supplemental Figures S1–3 and Table 2. The synthetic compounds were subject to analysis as outlined in AID 493111 to determine potency and anti-target selectivity (see Figure 6). As demonstrated for other triazole urea inhibitors [16], this type of gel-based competitive ABPP profiling allows for simultaneous assessment of both potency and selectivity of several dozen FP-sensitive SHs. The only observed anti-targets (indicated by disappearance of band in compound treated lane relative to the DMSO control) were ABHD11, esterase D/formylglutathione hydrolase (ESD), and N-acylaminoacyl-peptide hydrolase (APEH). ABHD11 was a common anti-target for all library compounds, with inhibition of ESD and APEH more easily tailored by structural modification (see discussion below).

First Round SAR (variation of R1): The initial round of SAR preserved the triazole urea core and the morpholine ring of the HTS hit structure 1.1 (SID 92709166), varying only the substituent at R1. Of the five new compounds synthesized (1.2 – 1.6 in SAR Table 2), only compound 1.6 demonstrated improved potency against the dual targets as judged by percent inhibition against LYPLA1 and LYPLA2. Of this class, compound 1.6 had the largest substituent at R1, a bulky di-phenyl methanol.

Second Round SAR (variation of X): The second round of SAR involved conversion of the morpholine to a piperdine, with the synthesis of compounds 1.7 (analog of 1.2) and 1.8 (analog of 1.6). Both piperidine analogs showed a marked improvement in potency as compared to their morpholine counterparts, with greater inhibition of LYPLA1 for 1.7 and LYPLA1/LYPLA2 for 1.8.

Third Round of SAR (variation of R1 – second iteration): Given the successful potency improvement with of compounds 1.7 and 1.8, the third round of SAR preserved the piperidine ring and focused on a second iteration of R1 variation, with groups similar to the di-phenyl methanol of compound 1.8 synthesized: compounds 1.9 (4-phenyoxyphenyl), 1.10 (7-methoxy naphthal), and 1.11 (phenyl methanol). However, none of these compounds were as potent as 1.8 for LYPLA1 and LYPLA2, as judged by percent inhibition, although it is worth noting that the single phenyl analog, compound 1.11, came closest to matching the potency of 1.8.

Fourth Round of SAR (variation of R2): With the di-phenyl methanol substituent as the most potent R1 derivative (compound 1.8), attention then turned to substitution of the piperidine ring (R2). Seven di-phenyl methanol analogs (compounds 1.12 to 1.18) with various aliphatic or aromatic substitutions at positions 2, 3, and 4 were synthesized. The ethyl substituent at position 2 in compound 1.12 completely abolished inhibition of LYPLA1 and LYPLA2, but, interestingly, gave rise to a selective inhibitor of the anti-target ABHD11. As such, compound 1.12 was designated as an anti-probe (ML226). A benzyl at the 3 position (compound 1.13) was tolerated with only a modest loss of potency. As compared to compound 1.8, potency was improved slightly with a methyl at the 4 position (compound 1.14) but diminished when the substituent was a methoxy (compound 1.15). This effect was likely due to electronic, rather than steric effects, as larger benzyl (compound 1.16) and di-phenyl methanol (compound 1.17) moieties was tolerated, and a tert-butyl group (compound 1.18, ML211) significantly improved potency for LYPLA1 and LYPLA2 when introduced to position 4.

Fifth Round of SAR (variation of R1 – third iteration): Starting with the most potent analog thus far, compound 1.18, variation at R1 was revisited holding the tert-butyl group at R2 constant. Two additional analogs were synthesized, compound 1.19, with a cyclohexan(1)ol at R1, and compound 1.20, with a 2-propanol at R1. However, both compounds showed reduced potency as compared to compound 1.18.

Selectivity Analysis: Selectivity for LYPLA1 and LYPLA2 seemed to track largely with one another, with most compounds exhibiting slightly increased potency for LYPLA1 over LYPLA2. In terms of anti-targets, with the exception of compound 1.20 (which was two to three fold less potent than compound 1.18), all compounds inhibited the anti-target ABHD11 with equal or greater potency than LYPLA1 and LYPLA2, suggesting that elimination of ABHD11 as an anti-target would involve synthesis of a significantly larger library (future directions) or investigation of alternative scaffolds. In general, the larger groups at position R2 tended to increase selectivity for LYPLA1 and LYPLA2 relative to the anti-targets APEH and ESD.

Summary: Both the acylating group and leaving group of urea triazole inhibitors appear to play a key role in target selectivity. The electrophile has sufficiently tempered reactivity that carbamoylation is dependent upon the initial binding interaction, which can be strongly influenced by both halves of the molecule.

As compared to the initial HTS hit with an IC50 of 795 nM for LYPLA1 and 5.2 μM for LYPLA2 (compound 1.1, SID 92709166), after five rounds of SAR, compound 1.18 represents an almost 50-fold improvement for LYPLA1 inhibition (IC50 17 nM) and greater than a 150-fold improvement for LYPLA2 inhibition (IC50 30 nM). Given the ubiquitous presence of the anti-target ABHD11 and the fortuitous discovery of the ABHD11-selective inhibitor 1.12, we declared compound 1.18 as probe ML211 and compound 1.12 as the anti-probe ML226 for use in parallel experiments as a control for ABHD11-selective phenotypes.

3.5. Cellular Activity

Probe compound 1.18 (ML211) and analog 1.20 are highly active against both LYPLA1 and LYPLA2 in situ (AID 493108), completely inhibiting enzymatic activity at 30 nM compound concentration after 2 hours (media + 10% serum) as assayed by gel-based competitive ABPP (Figure 7A). In this context, both compounds also completely inhibited anti-target ABHD11. However, anti-probe ML226 is also active in situ against ABHD11 under the same conditions (Figure 7B), and thus can serve as an effective control for live-cell experiments. This result indicates that compound 1.18, anti-probe ML226, and analog 1.20 are free to cross cell membranes and inhibit their targets in the cytoplasm of cells.

Figure 7. In Situ inhibition of target enzymes.

Figure 7

In Situ inhibition of target enzymes. A. Selective Inhibition of LYPLA1, LYPLA2, and ABHD11 activity by Probe ML211 (compound 1.18, AA64-2, SID 99445338) and Analog 1.20 (AA69-2, SID 99445340). B. Selective inhibition of ABHD11 by Anti-Probe ML226 (compound (more...)

The probe ML211 (compound 1.18), analog 1.20, and anti-probe ML226 were evaluated for cell toxicity (AID 493161) using both serum-free and serum-supplemented media. As shown in Figure 8, all three compounds have a CC50 greater than 6 μM, which is 200-fold greater than the concentration (30 nM) necessary for complete inhibition of their respective target enzyme(s) in situ.

Figure 8. Cytotoxicity of probe and probe analogs in serum-free (A) and serum-supplemented (B) media.

Figure 8

Cytotoxicity of probe and probe analogs in serum-free (A) and serum-supplemented (B) media. See AID 493161 for details.

3.6. Profiling Assays

To date, the HTS hit 55 (a.k.a. compound 1.1; 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 ML211 (compound 1.18) or any of the synthetic analogs.

4. Discussion

Probe ML211 (Compound 1.18) was identified as a highly potent and selective covalent dual inhibitor of the target enzymes LYPLA1 (IC50 17 nM, AID 493110) and LYPLA2 (IC50 30 nM, AID 493110) that carbamoylates the catalytic serine (section 3.3). The probe was observed to have one anti-target, the SH ABHD11 (IC50 10 nM, AID 493154) and at least 50-fold selectivity against all other anti-targets surveyed. The anti-probe ML226 has an IC50 of 15 nM for ABHD11 (AID 493154), and greater than 100-fold selectivity against potential SH anti-targets as assessed by competitive ABPP profiling (AID 493111). Due to the presence of the ABHD11 anti-target, the anti-probe ML226 should be used in parallel experiments as a control for ABHD11-specific phenotypes. Both probe ML211 and anti-probe ML226 are active in situ (section 3.5), completely inhibiting their target enzymes in serum-containing media after two hours at 30 nM concentration. ML211 is soluble in PBS at up to 6.6 μM, 200 times its active in situ concentration. Though the probe does show some reactivity with glutathione, the complex proteome profiles (Figure 6) do not indicate general reactivity, and HTS analog 55 (a.k.a 1.1; CID 735660) has low (1.3%) activity in other PubChem bioassays. Taken together, these findings suggest that it is very possible to develop potent and selective probes based on tempered electrophilic scaffolds, and that ML211 will be a highly successful probe for biochemical investigation of LYPLA1 and LYPLA2, provided the anti-probe ML226 is used as an appropriate control.

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

Several substrate-mimetic inhibitors of LYPLA1 have been described by Waldmann et al. [12–13], but none of these agents have proven capable of inhibiting LYPLA1 activity in cells, and selectivity has not been reported. The most potent of this class of LYPLA1 inhibitors (IC50 27 nM) is shown in Figure 9A; the compound has a MW of 928 and a synthetic route of more than a dozen steps. By comparison, ML211 has been demonstrated to inhibit LYPLA1 in live cells (section 3.5), can be synthesized in three steps (section 2.3), and has a MW below 500.

Figure 9. Structures of existing LYPLA1-inhibitors.

Figure 9

Structures of existing LYPLA1-inhibitors.

More recently, a small library of beta-lactone compounds, termed palmostatins, have also been reported as LYPLA1 inhibitors [2]. They have a scaffold similar to the marketed drug tetrahydrolipstatin (THL a.k.a. Orlistat®, Roche). The most potent of this class is Palmostatin B, depicted in Figure 9B. In contrast to the previous class of substrate-mimetic LYPLA1 inhibitors, the palmostatins are active against LYPLA1 in situ and synthesis is achieved following a more tractable route. Palmostatin B has a reported IC50 of 300 nM and is active in situ at 1 μM concentration. By comparison, ML211 is 17-fold more potent in vitro (IC50 17 nM) and active at >30-fold lower concentration (30 nM) in situ. Additionally, we have previously profiled THL by gel-based ABPP and found that it inhibits numerous serine hydrolases [27]. We therefore expect that palmostatins will similarly suffer from a wide range of anti-targets. Thus, ML211 represents a significant improvement with regard to potency, and potentially selectivity as well.

No selective LYPLA2 or dual LYPLA1/2 inhibitors have been reported to date. As such, ML211 represents the first LYPLA1 and/or LYPLA2 inhibitor that has demonstrated activity in living cells at sub-micromolar concentration and the first dual LYPLA1/LYPLA2 inhibitor.

4.2. Mechanism of Action Studies

As determined from LC-MS/MS analysis (AID 493109), the probe is an activity-based inhibitor that covalently labels the active site serine nucleophile, Ser114, of LYPLA1 (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 5). Given the high structural homology between LYPLA1 and LYPLA2 (65% overall sequence homology; both exhibit the SH catalytic triad signature), it is anticipated that ML211 modifies LYPLA2 in an analogous manner.

4.3. Planned Future Studies

We plan to continue expanding the triazole urea library for identification of additional LYPLA1, LYPLA2, and dual LYPLA1/LYPLA2 probes with optimized selectivity. Additionally, we plan to more comprehensively establish parameters for in situ and in vivo use and explore the target specificity of ML211 via application of alkyne analogs. For biological application, we plan to use ML211 to investigate the link between LYPLA1, LYPLA2, palmitoylation, and cancer progression by 1) global proteomic and metabolomic profiling of cultured cancer cells treated with and without probe/anti-probe to identify potential metabolic pathway involvement, 2) global profiling of palmitoylation events differentially regulated in cancer cells treated with and without probe/anti-probe, and 3) evaluating the role of LYPLA1 and LYPLA2 in cancer pathogenesis using assays that measure proliferation, migration, and invasion in situ. Future studies may also extend to the nervous system, where LYPLA1 has been implicated in regulating dendritic spine morphogenesis, presumably through regulating dynamic palmitoylation of key dendritic signaling and scaffolding proteins [28].

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