Characterization of an agonist probe for opioid receptor mu 1 (OPRM1)-opioid receptor delta 1 (OPRD1) heterodimerization

Pinello C, Guerrero M, Eberhart C, et al.

Opiates such as morphine are the choice analgesic in the treatment of chronic pain due to their potent and rapid action. Opioid receptors belong to the family of G protein-coupled receptors (GPCRs), one of the largest gene families in the mammalian genome. The OPRM1 gene encodes the mu opioid receptor, which is the primary site of action for morphine and other commonly used opioids such as heroin, fentanyl, and methadone. The long-term use of opiates is limited because of the development of tolerance and dependence, as well as respiratory suppression and constipation. Due to their clinical importance, various strategies have been considered for making opiates more effective while curbing liabilities such as addiction. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, delta opioid receptor (OPRD1) ligands have been useful in enhancing morphine’s potency, but the underlying molecular basis is not understood. It has been shown that modulation of receptor function by physical association between OPRM1 and OPRD1 is a potential mechanism; heteromerization of OPRM1 with OPRD1 leads to the modulation of receptor binding and signaling properties. It has further been shown that the selective activation of the OPRM1-OPRD1 heteromer by a combination of OPRM1 agonist with OPRD1 antagonist can be blocked by antibodies that selectively recognize the heteromer. Therefore, the identification of compounds that selectively activate OPRM1-OPRD1 heterodimerization may have potential in the treatment of pain and alleviate unwanted effects associated with opiate use. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports here an agonist for OPRM1-OPRD1 heterdimerization, ML335, with an EC50 of 403 nM, and selectivities vs. OPRM1, OPRD1, and HTR5A of 37, 2.7, and >99, respectively.

Assigned Assay Grant #: R03NS053751

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

Chemistry Center Name & PI: SRIMSC, H Rosen

Assay Submitter & Institution: Lakshmi Devi, Mount Sinai School of Medicine

PubChem Summary Bioassay Identifier (AID): 504355

Probe Structure and Characteristics

ML335.

ML335

1. Recommendations for Scientific Use of the Probe

Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, OPRD1 opioid receptor ligands have been useful in enhancing morphine’s potency. The underlying molecular basis for these observations is not understood. The modulation of receptor function by physical association between OPRM1 and OPRD1 opioid receptors has been postulated as a potential mechanism. In support of this hypothesis, OPRM1-OPRD1 interacting complexes have been shown to exist in live cells and native membranes and the occupancy of OPRD1 receptors (by antagonists) is sufficient to enhance OPRM1 opioid receptor binding and signaling activity. Furthermore, OPRD1 receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus heterodimeric associations between OPRM1-OPRD1 opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies.

Accumulating evidence indicates that the majority of GPCRs form constitutive dimers in the endoplasmic reticulum and that dimerization is necessary for GPCR trafficking to the cell surface. Association between receptors from two distinct GPCR subfamilies has also been shown to lead to changes in receptor pharmacology. A growing number of studies point towards a role for GPCR heterodimerization in modulating receptor pharmacology and suggest that heterodimers could represent a functional unit, distinct from their cognate protomers. In essence, the association of two receptor molecules would allow the allosteric modulation of one monomeric unit by the other. The resulting cooperativity could be negative, neutral or positive, often resulting in changes in observed receptor pharmacology. This would be an extension of the concept of allosteric modulation of GPCRs by small molecules. This probe will be useful for investigations into the biology of OPRM1-OPRD1 heterodimers.

2. Materials and Methods

Chemistry

Solvents for extraction were ACS grade. Solvents for reaction were reagent grade. Unless otherwise noted, chemistry reagents were from Alfa Aesar, Fisher and Aldrich; highest quality available. TLC: silica gel 60 F254 aluminum plates, (Whatman, type Al Sil G/UV, 250 μm layer); visualization by UV absorption. Flash column chromatography (CC) was performed on silica gel 60 (0.40–0.63 mm, 230–440 mesh, EM Science). Biotage Flash+ systems were used for medium-pressure column chromatography. NMR: 1H and 13C spectra were obtained on a Bruker DRX-600 MHz spectrometer. Chemical shifts are given as δ values (ppm) using trimethylsilane as the internal standard. Reactions were monitored by LC/MS Shimadzu 210A.

2.1. Assays

Probe Characterization Assays

Solubility in PBS: The solubility of compounds are tested in triplicate in phosphate buffered saline (PBS), pH 7.4. per well, 198 μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well filter plate: pore size: 0.45um (MSRLN0450). Test compounds are introduced from 10 mM DMSO stock solutions (2 μl). The final concentration of DMSO was 1 percent. Samples are allowed to incubate at 22ºC for 18 hours. In the morning the plate is centrifuged where the soluble portion passes through the filter and is collected in a capture plate. Clotrimazole is included as a control to assure the assay is working properly. The samples are analyzed by HPLC. Peak area is compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS-MS analysis is used.

Solubility in Media: The solubility of compounds are tested in triplicate in complete media (Dulbecco’s Modified Eagle Media [DMEM] plus 10% FBS). Per well, 198 μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well filter plate: pore size: 0.45um (MSRLN0450). Test compounds are introduced from 10 mM DMSO stock solutions (2 μl). The final concentration of DMSO was 1 percent. Samples are allowed to incubate at 22ºC for 18 hours. In the morning the plate is centrifuged where the soluble portion passes through the filter and is collected in a capture plate. The samples are analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area is compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS-MS analysis is used.

Stability in PBS: Demonstration of stability in PBS was conducted by addition of 10 μM compound from a DMSO stock to PBS in HPLC autosampler vials. Samples are held in the HPLC autosampler at ambient temperature. At approximately 0, 1, 2, 4, 8, 24, and 48 hours the samples are injected on the HPLC. Peak area and retention time are compared between injections. Data are log transformed and represented as half-life. DMSO is added as a co-solvent as needed for solubility.

Determination of Glutathione reactivity: One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 50 μM reduced glutathione (GSH). Final compound concentration is 10 μM unless limited by solubility. The solution was allowed to incubate at 37°C for 6 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 (4243).

Primary Ultrahigh-Throughput Screening (uHTS) Assays

Primary uHTS assay to identify agonists of OPRD1-OPRM1 heterodimerization (AID 504326, AID 504904, and AID 588407)

Assay Overview: The purpose of this assay is to identify compounds that activate heterodimer formation between the OPRM1 and OPRD1 opioid receptors, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express OPRD1, OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers or OPRM1-OPRD1 heterodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B is used as the high control for agonists, and wells containing cells treated with DMSO are used as the low control. Compounds were tested in singlicate (AID 504326) or triplicate (AID 504904) at a final nominal concentration of 9.3 μM, or in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 93 μM (AID 588407).

Protocol Summary:The U2OS-OPRM1-OPRD1 cell line was routinely cultured in T-175 sq cm flasks at 37°C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham’s F-12 Nutrient Media (F-12) and Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 μg/mL geneticin, 250 μg/mL hygromycin B, 0.25 μg/mL puromycin and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay, 1000 cells in 3 μL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 28 nL of test compound in DMSO, Deltorphin B (0.9 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 3 hours at 37°C, 5% CO2, and 95% RH. The assay was started by adding 2 μL of PathHunter™ reagent (prepared according to the manufacturer’s protocol); followed by 1 hour incubation at room temperature. Well luminescence was then read on the ViewLux plate reader. Assay Cutoff: Compounds that exhibited ≥18.04% activation (AIDs 504326 and 504904) or an EC50 of <10 μM (AID 588407) were considered active.

Counterscreen uHTS assay to identify agonists of HTR5A (AID 504692, AID 504905 and AID 588408)

Assay Overview: The purpose of this assay is to identify compounds that activate the human serotonin receptor HTR5A, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-gal. This assay employs U2OS cells which express HTR5A fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that activate HTR5A will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Serotonin (5HT) is used as the high control for agonists, and wells containing cells treated with DMSO are used as the low control. Compounds were tested in singlicate (AID 504692) or triplicate (AID 504905) at a final nominal concentration of 9.3 μM, or in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 93 μM (AID 588408).

Protocol Summary: The U2OS-HTR5A cell line was routinely cultured in T-175 sq cm flasks at 37°C and 95% RH. The growth media consisted of a 1:1 mixture of Ham’s F-12 Nutrient Media (F-12) and DMEM supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL geneticin, 250 ug/mL hygromycin B, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay, 1000 cells in 3 μL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 28 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AID 504326, AID 504904, and AID 588407). Assay Cutoff: Compounds that exhibited ≥18.04% activation (AID 504692 and AID 504905) or an EC50 of <10 μM (AID 588408) were considered active.

Counterscreen uHTS assay to identify agonists of OPRD1 (AID 588411)

Assay Overview: The purpose of this counterscreen assay is to identify compounds that are agonists of OPRD1 homodimerization, resulting in membrane recruitment of beta-arrestin. This assay employs U2OS cells which express OPRD1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 93 μM. Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Protocol Summary: The U2OS-OPRD1 cell line was routinely cultured as described above (AID 504692, AID 504905 and AID 588408). The day before the assay, 1000 cells in 3 μL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 28 nL of test compound in DMSO, Deltorphin B (0.9 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AID 504326, AID 504904, and AID 588407). Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Counterscreen uHTS assay to identify agonists of OPRM1 (AID 588435)

Assay Overview: The purpose of this counterscreen assay is to identify compounds that are agonists of OPRM1 homodimerization, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-gal. This assay employs U2OS cells which express OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRM1 homodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 93 μM.

Protocol Summary: The U2OS-OPRM1 cell line was routinely cultured as described above (AIDs 504692, 504905 and 588408). The day before the assay, 1000 cells in 3 μL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 28 nL of test compound in DMSO, DAMGO (9.2 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AID 504326, AID 504904, and AID 588407). Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Secondary Assays

Assay to identify activators of OPRM1 and OPRD1 heterodimer formation (AID 651685, AID 651762)

Assay Overview: The purpose of this assay is to test powder samples of compounds that activate heterodimer formation between the OPRM1 and OPRD1 opioid receptors, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-gal. This assay employs U2OS cells which express OPRD1, OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers or OPRM1-OPRD1 heterodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B is used as the high control for agonists, and wells containing cells treated with DMSO are used as the low control. Compounds are tested in triplicate in a 10-point dilution series starting at a nominal concentration of 40 μM.

Protocol Summary: The U2OS-OPRM1-OPRD1 cell line was routinely cultured as described above (AID 504326, AID 504904, and AID 588407). The day before the assay 1000 cells in 20 μL of cell plating media were seeded into each well of 384 well plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 100 nL of test compound in DMSO, Deltorphin B (1 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AID 504326, AID 504904, and AID 588407). Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Counterscreen assay to identify activators of OPRM1 homodimer formation (AID 651689, AID 651769)

Assay Overview: The purpose of this counterscreen assay is to test powder samples of compounds that are agonists of OPRM1-OPRD1 heterodimerization for agonist activity of OPRM1 homodimerization, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-gal. This assay employs U2OS cells which express OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRM1 homodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. DAMGO is used as the high control for agonists, and wells containing cells treated with DMSO is used as the low control. Compounds are tested in triplicate in a 10-point dilution series starting at a nominal concentration of 40 μM.

Protocol Summary: The U2OS-OPRM1 cell line was routinely cultured as described above (AID 504326, AID 504904, and AID 588407). The day before the assay, 5000 cells in 20 μL of cell plating media were seeded into each well of 384 well plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 100 nL of test compound in DMSO, DAMGO (10 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AID 504326, AID 504904, and AID 588407). Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Counterscreen assay to identify activators of OPRD1 homodimer formation (AID 651686, AID 651764)

Assay Overview: The purpose of this counterscreen assay is to test powder samples of compounds that are agonists of OPRM1-OPRD1 heterodimerization for agonist activity of OPRD1 homodimerization, resulting in membrane recruitment of beta-arrestin. This assay employs U2OS cells which express OPRD1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B is used as the high control for agonists, and wells containing cells treated with DMSO are used as the low control. Compounds are tested in triplicate in a 10-point dilution series starting at a nominal concentration of 40 μM.

Protocol Summary: The U2OS-OPRD1 (Delta) cell line was routinely cultured as described above (AID 504326, AID 504904, and AID 588407). The day before the assay, 5000 cells in 20 μL of cell plating media were seeded into each well of 384 well plates and allowed to incubate at 37°C, 5% CO2, and 95 % RH for 23 hours. Next, 100 nL of test compound in DMSO, Deltorphin B (1 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AIDs 504326, 504904, and 588407). Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Counterscreen assay to identify activators of HTR5A (AID 651688, AID 651765)

Assay Overview: The purpose of this counterscreen assay is to test powder samples of compounds that are agonists of OPRM1-OPRD1 heterodimerization for activation of the human serotonin receptor HTR5A, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-gal. This assay employs U2OS cells which express HTR5A fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that activate HTR5A will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Serotonin is used as the high control for agonists, and wells containing cells treated with DMSO is used as the low control. Compounds were tested in singlicate at a final nominal concentration of 9.3 μM. Compounds were tested in triplicate in a 10-point dilution series starting at a nominal concentration of 40 μM.

Protocol Summary: The U2OS-HTR5A cell line was routinely cultured as described above (AID 504326, AID 504904, and AID 588407). The day before the assay, 5000 cells in 20 μL of cell plating media were seeded into each well of 384 well plates and allowed to incubate at 37°C, 5% CO2, and 95% RH for 23 hours. Next, 100 nL of test compound in DMSO, serotonin (5-CT) (3 μM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was performed as described above (AID 504326, AID 504904, and AID 588407). Assay Cutoff: Compounds that exhibited an EC50 of <10 μM were considered active.

Cytotoxicity assay to identify cytotoxic compounds (AID 651904)

Assay Overview: The purpose of this assay is to determine cytotoxicity of powder compounds that activate heterodimer formation between the OPRM1 and OPRD1 opioid receptors. In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 13-point 1:3 dilution series starting at a nominal test concentration of 10 uM.

Protocol Summary: This assay was started by dispensing PathHunter™ OPRM1/OPRD1-β-Arrestin U2OS cells in McCoy’s 5A medium plus 10% FBS, penicillin 100 U/mL and streptomycin 100 μg/mL (20 μL, 4 × 1000 cells/well) into the wells of a 384-well plate. Thirteen 1:3 serial dilutions of compound (100 μM in growth media) were made. 5 μL of diluted compound or media were added to wells, giving final compound concentrations of 0–10 μM. The plate was incubated at 37°C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 μL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader. Assay Cutoff: Compounds that exhibited a CC50 (defined as the concentration of compound that induces 50% cell death) of >10 μM were considered non-cytotoxic.

2.2. Probe Chemical Characterization

CID 23723457SID 134220670ML335.

CID 23723457
SID 134220670
ML335

The probe structure was verified by 1H and 13C NMR (see Section 2.3) and high resolution LC/MS (Figure 1). Purity was assessed to be greater than 95% by LC-MS and NMR.

Figure 1. LC/MS analysis for ML335.

Figure 1

LC/MS analysis for ML335.

Solubility (at room temperature) for ML335 in PBS and in DMEM plus 10% FBS media was determined to be 58.6 μM and 66.1 μM, respectively. ML335 has a half-life of >48 hours in PBS at room temperature (94.6% compound remaining at 48 hours) (Figure 2).

Figure 2. Stability of ML335 (CYM51010) in PBS.

Figure 2

Stability of ML335 (CYM51010) in PBS.

No Michael acceptor adducts were observed when a sample of the probe was incubated with 50 μM glutathione and analyzed by LC-MS.

The following compounds have been submitted to the SMR collection (Table 1).

Table 1. Compounds submitted to the SMR collection (12-17-2012).

Table 1

Compounds submitted to the SMR collection (12-17-2012).

2.3. Probe Preparation

Figure 3. Synthetic scheme for ML335.

Figure 3Synthetic scheme for ML335

To a stirred solution of LDA (2M) (4.22 mmol, 1.09 equiv.) in THF at −78°C was added dropwise a solution of 1 (3.88 mmol, 1 equiv.) in THF (8 mL) and the reaction was stirred for 1 hour at −78°C. A solution of 2 (4.03 mmol, 1.04 equiv.) in anhydrous THF (8 mL) was added over a period of 30 minutes. The reaction mixture was slowly warmed to room temperature and the reaction was stirred for an additional 8 hours. The mixture was poured onto ice-water and the product extracted with ethyl acetate (3X). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The product was purified by flash column chromatography using hexanes/ethyl acetate. Product 3 was obtained as an oil in 35% yield (1.36 mmol, 490 mg). MS (EI) m/z: 362 (M+).

To a stirred solution of 3 (1.1 mmol, 1 equiv.) in CH2Cl2, TFA (22.1 mmol, 20 equiv.) was added at room temperature and the reaction mixture was stirred for 30 minutes. The mixture was concentrated under reduced pressure. The crude was diluted in water and slowly basified with NaOH (1M) (~pH 12). The product was extracted using ethyl acetate (5X) and the organic phase dried over anhydrous sodium sulfate and concentrated under reduced pressure. The product was used without further purification.

A mixture of the crude (0.057 mmol, 1 equiv.) chloride 4 (0.063 mmol, 1.1 equiv.) and sodium carbonate (0.063 mmol, 1.1 equiv.) in acetonitrile was stirred at room temperature overnight. The mixture was quenched with silica gel and the product purified by flash column chromatography using CH2Cl2/MeOH. The product was obtained as yellow oil in 60% yield (13.7 mg, 0.034mmol) and judged to be >95% pure by LC-MS (Figure 1).

1H NMR (600 MHz, CD3OD): δ 7.51 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 7.5 Hz, 2H), 7.15-7.10 (m, 3H), 4.15 (q, J = 7.12 Hz, 2H), 3.47 (s, 2H), 2.77-2.44 (m, 2H), 2.50-2.46 (m, 2H), 2.21-2.11 (m, 4H), 2.11 (s, 3H), 1.81-1.76 (m, 2H), 1.59-1.52 (m, 2H), 1.26 (t, J = 7.12 Hz, 3H); 13C NMR (150 MHz, CDCl3): δ 174.12, 169.68, 140.58 (2C), 140.16, 131.64, 128.52, 128.21, 126.24, 120.27, 61.47, 60.53, 50.04, 44.19, 41.91, 30.54, 30.31, 24.26, 14.27 MS (EI) m/z: 409 (M+H). HRMS (EI) for C25H33N2O3 (M+H): calcd 409.2486, found 409.2481.

3. Results

3.1. Dose Response Curves for Probe

Figure 4 shows the dose response curve for binding of ML335 to U2OS cells which express OPRD1 and OPRM1.

Figure 4. IC50 curve for probe ML335 determined in assay AID 651685.

Figure 4

IC50 curve for probe ML335 determined in assay AID 651685.

3.2. Cellular Activity

ML335 has been evaluated in a series of cell-based assays (the OPRM1-OPRD1 screen, the OPRM1, OPRD1, 5HTRA counterscreens, and the cytotoxicity assay) and has been shown to have activity in a cell-based system.

3.3. Profiling Assays

To date, ML335 has been tested in 438 bioassays deposited in PubChem, and has shown activity in 10 of those assays, four of which are for this project. The other six assays give a hit rate of 1.4%, indicating that this molecule is generally inactive across a broad range of cell-based and non–cell-based assays. Five of the six cross-reactive assays are for transient receptor potential cation channel (TRPC) 4 and C6. These assays are performed with HEK cells expressing both mu opioid and TRPC. It is likely the cross-reactivity is due to activity on the mu receptor. The remaining assay (AID 504834) is “Primary qHTS for delayed death inhibitors of the malarial parasite plastid, 96 hour incubation”. It is likely the probe is active (4.1 μM) in this assay, but the species and dosing are not relevant to the current project.

4. Discussion

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

There are currently no known potent and selective small molecule agonists of OPRM1-OPRD1 heterodimer formation.

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