Previous studies have shown that activation of mouse MrgprC11, a G-protein coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large scale screen has been carried out to isolate small molecule allosteric agonists of MrgprX1, the human homologue of MrgprC11. The goal of this study is to improve the efficacy and potency of the allosteric agonists with therapeutic implications of anti-chronic pain. From compounds identified through the high-throughput screening effort, a structure-activity relationship of a series of arylsulfonamides led to the discovery of the first allosteric agonist of MrgprX1, ML382.

Probe Structure & Characteristics

ML382

Recommendations for scientific use of the probe

ML382 (CID 71598556) is a potent allosteric agonist of MrgprX1 when tested in cell based Ca2+ imaging assay (EC₅₀ = 190 nM; using 10 nM BAM8-22 as an agonist of MrgprX1). The action of ML382 on enhancing MrgprX1 activation by BAM8-22 is highly selective since it did not have any effect on MrgprX2, a closely related gene to MrgprX1. Since this is the first allosteric agonist of MrgprX1, ML382 is an invaluable research tool to study the role of MrgprX1 in chronic pain. The expected effect of this compound is to inhibit chronic pain.

2. Materials and Methods

Cell based Ca2+ imaging assay to detect MrgX1 activation. The purpose of this assay is to identify test compounds that act as an allosteric agonist for MrgX1. This assay employs a HEK293 cell line that stably expresses MrgX1 protein. The cells, which loaded with fluorescent dye-Flou4, are treated with test compounds, followed by measurement of calcium flux. Those HEK293 cells stably expressing MrgX1 were plated into 96-well plates. On the following day, cells were incubated with Fluo4 solution at 37 °C for 30min and at RT for 30 min after removing media. 10 μM compounds were added to the assay buffer with dye for 80 sec followed by adding 10 nM BAM8-22 for 75 sec and recorded the change of fluorescence by Flexstation3 imaging plate reader. Compound effect was evaluated by the calculated fluorescence ratio. If the compound causes more than 3 times the standard deviation of the B-scores of the library compounds, the compound is then considered to be active as an agonist of the MrgX1 protein.

DMPK Methods. In vitro: The metabolism of ML382 was investigated in human, rat and mouse hepatic microsomes (BD Biosciences, Billerica, MA) using substrate depletion methodology (% test article remaining). A potassium phosphate-buffered reaction mixture (0.1 M, pH 7.4) of test article (1 μM) and microsomes (0.5 mg/mL) was pre-incubated (5 min) at 37°C prior to the addition of NADPH (1 mM). The incubations, performed in 96-well plates, were continued at 37 °C under ambient oxygenation and aliquots (80 μL) were removed at selected time intervals (0, 3, 7, 15, 25 and 45 min). Protein was precipitated by the addition of chilled acetonitrile (160 μL), containing glyburide as an internal standard (50 ng/mL), and centrifuged at 3000 rpm (4°C) for 10 min. Resulting supernatants were transferred to new 96-well plates in preparation for LC/MS/MS analysis. The in vitro half-life (t_1/2, min, Eq. 1), intrinsic clearance (CL_int, mL/min/kg, Eq. 2) and subsequent predicted hepatic clearance (CL_hep, mL/min/kg, Eq. 3) were determined employing the following equations:

Plasma Protein Binding. Protein binding of ML382 was determined in human, rat and mouse plasma via equilibrium dialysis employing Single-Use RED Plates with inserts (ThermoFisher Scientific, Rochester, NY). Briefly plasma (220 μL) was added to the 96 well plate containing test article (5 μL) and mixed thoroughly. Subsequently, 200 μL of the plasma-test article mixture was transferred to the cis chamber (red) of the RED plate, with an accompanying 350 μL of phosphate buffer (25 mM, pH 7.4) in the trans chamber. The RED plate was sealed and incubated 4 h at 37 °C with shaking. At completion, 50 μL aliquots from each chamber were diluted 1:1 (50 μL) with either plasma (cis) or buffer (trans) and transferred to a new 96 well plate, at which time ice-cold acetonitrile (2 volumes) was added to extract the matrices. The plate was centrifuged (3000 rpm, 10 min) and supernatants transferred to a new 96 well plate. The sealed plate was stored at -20 °C until LC/MS/MS analysis.

Liquid Chromatography/Mass Spectrometry Analysis. In vitro experiments. ML382 was analyzed via electrospray ionization (ESI) on an AB Sciex API-4000 (Foster City, CA) triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 2.1 × 50 mm, 3.5 μm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40 °C. HPLC mobile phase A was 0.1% NH₄OH (pH unadjusted), mobile phase B was acetonitrile. The gradient started at 30% B after a 0.2 min hold and was linearly increased to 90% B over 0.8 min; held at 90% B for 0.5 min and returned to 30% B in 0.1 min followed by a re-equilibration (0.9 min). The total run time was 2.5 min and the HPLC flow rate was 0.5 mL/min. The source temperature was set at 500°C and mass spectral analyses were performed using multiple reaction monitoring (MRM) utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage). LC/MS/MS analysis was performed employing a TSQ Quantum^ULTRA that was coupled to a ThermoSurveyor LC system (Thermoelectron Corp., San Jose, CA) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Chromatographic separation of analytes was achieved with an Acquity BEH C18 2.1 × 50 mm, 1.7 μm column (Waters, Taunton, MA).

2.2. Probe Chemical Characterization

Probe compound ML382 (CID: 71598556, SID: 164175119) was prepared according to scheme 1 and had the following characterization. 2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382. LCMS: R_T = 1.13 min, >98% @ 215 and 254 nm, m/z = 360.9 [M]⁺. ¹H NMR (400.1 MHz, CDCl₃): δ 10.45 (s, 1H), 8.67 (s, 1H), 8.45-8.43 (m, 1H), 7.85 (d, J = 8.38 Hz, 1H), 7.64 (d, J = 8.02 Hz, 1H), 7.56-7.52 (m, 1H), 7.28-7.22 (m, 1H), 7.15-7.11 (m, 1H), 7.06-7.02 (m, 1H), 6.96-6.94 (m, 1H), 4.22-4.16 (m, 2H), 2.56-2.50 (m, 1H), 1.51 (t, J = 6.97 Hz, 3H), 1.27-1.25 (m, 2H), 0.95-0.92 (m, 2H).

Scheme 1

Chemical characterization of Probe ML382.

Solubility. Solubility for ML382 in PBS was determined to be 73.0 ± 3.5 μM, which is ∼380-fold higher than the cellular EC₅₀ for MrgX1 activation.

Stability. Stability was determined for ML382 at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%). After 48 hours, ∼91% of the initial concentration of ML382 remained in solution, signifying that ML382 is a stable compound under these conditions.

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	Percent Remaining (%)
Compound	1 min	18 min	34 min	90 min	24 Hour	48 Hour
ML347, CID 44577753	100 ± 0.0	98.2 ± 0.2	97.8 ± 0.3	97.1 ± 1.0	93.9 ± 3.9	91.2 ± 4.6

Compounds added to the SMR collection (MLS#s): MLS005527261 (ML382, CID 71598556, 23.5 mg); MLS005527262 (CID 2712184, 7.5 mg); MLS005527263 (CID 951628, 7.7 mg); MLS005527264 (CID 71598554, 7.6 mg); MLS005527265 (CID 838665, 7.0 mg); MLS005527266 (CID 1260775, 6.1 mg).

2.3. Probe Preparation

Probe compound ML382 (CID 71598556, SID 164175119) was prepared according to scheme 2 and had the following characterization. 2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382.

Scheme 2

Preparation of Probe ML382.

2-amino-N-(2-ethoxyphenyl)benzamide (3). To a dry argon-filled flask was added anthrinilic acid, 1, (1 eq), and 3 drops of dimethylformamide (DMF) and ether as solvent. To the solution, thionyl chloride (2 eq) was added dropwise at 0 °C. The mixture was refluxed for 2 hours. On completion, ether and remaining thionyl chloride were removed under reduced pressure. The mixture was cooled to 0 °C, followed by addition of 0.5 ml pyridine, 2,4-dimethoxyaniline, 2, (1.2 eq) and ether as solvent. The solution was allowed to warm up to room temperature and stir for 2 h. After removing solvents under reduced pressure, the crude product was purified via reverse phase preparative chromatography (Gilson, Acetonitrile-0.5% NH₄OH in water), which yielded 2-amino-N-(2-ethoxyphenyl)benzamide, 3, as yellow solid, 77% yield. 1H NMR (400.1 MHz, CDCl₃): δ 8.48-8.46 (m, 1H), 7.52-7.50 (m, 1H), 7.30-7.26 (m, 1H), 7.10-7.00 (m, 2H), 6.94-6.92 (m, 1H), 6.75 (t, 2H, J = 6.87 Hz), 5.62 (s, 2H), 4.18-4.13 (m, 2H), 1.50 (t, 3H, J = 6.94 Hz) ¹³C NMR (100.6 MHz, CDCl₃): δ 167.3, 149.3, 147.7, 132.6, 128.0, 127.2, 123.7, 121.0, 119.9, 117.6, 116.9, 116.7, 111.0, 64.3, 15.0. LCMS: R_T = 1.01 min, >98% @ 215 and 254 nm, m/z = 257.0 [M + H]⁺.

2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382. To dry vial was added 2-amino-N-(2-ethoxyphenyl)benzamide, 3, (1 eq), cyclopropanesulfonyl chloride (2 eq), pyridine (4 eq) and DCM as solvent. The mixture was stirred for 16 h at room temperature. On completion, the crude product was purified by reverse phase preparative chromatography (Gilson, Acetonitrile-0.5% NH₄OH in water). LCMS: R_T = 1.13 min, >95% @ 215 and 254 nm, m/z = 360.9 [M]⁺. ¹H NMR (400.1 MHz, CDCl₃): δ 10.45 (s, 1H), 8.67 (s, 1H), 8.45-8.43 (m, 1H), 7.85 (d, J = 8.38 Hz, 1H), 7.64 (d, J = 8.02 Hz, 1H), 7.56-7.52 (m, 1H), 7.28-7.22 (m, 1H), 7.15-7.11 (m, 1H), 7.06-7.02 (m, 1H), 6.96-6.94 (m, 1H), 4.22-4.16 (m, 2H), 2.56-2.50 (m, 1H), 1.51 (t, J = 6.97 Hz, 3H), 1.27-1.25 (m, 2H), 0.95-0.92 (m, 2H).

3. Results

3.1. Dose Response Curves for Probe

Figure 1In vitro molecular pharmacology characterization of ML382

Concentration-response curves of ML382 in Ca2+ imaging assay in HEK293 cells expressing either MrgX1 (black squares) or MrgX2 (open cicle). EC50 of ML382 enhancing MrgX1 activation by BAM8-22 is 190 nM. 5μM ML382 did not produce a significant increase in activation of MrgX2 by its specific agonist peptide PAMP (5μM).

4. Discussion

5. References

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