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Purity: ≥98%
Ro 67-7476 is a novel and selective positive allosteric modulator of mGlu1 receptors. It can enhance glutamate-induced calcium release with an EC 50 of 60.1 nM and shows no activity at human mGlu1 receptors. While agonist-stimulated responses were not directly activated by Ro 67-7476, they were significantly potentiated, meaning that their maximum efficacy was increased. A radiolabeled glutamate-site agonist's affinity was enhanced upon binding of Ro 67-7476 at its extracellular N-terminal binding site. The amino acids necessary for these enhancing properties were located in the receptor transmembrane region using chimeric and mutant receptors. Lastly, in rat brain slices, the compounds increased synaptically evoked mGlu1 receptor responses. The possibility of creating a class of drugs for other family 3 G protein-coupled receptors is made possible by the discovery of selective positive allosteric modulators of mGlu1 receptors.
| Targets |
mGluR1a ( EC50 = 60.1 nM )
Ro 67-7476 acts as a positive allosteric modulator (PAM) of the metabotropic glutamate receptor 1 (mGlu1), with high selectivity for the mGlu1a subtype (the predominant splice variant of mGlu1) (EC50 = 0.3 μM for potentiating glutamate-induced Ca²⁺ mobilization in mGlu1a-expressing BHK cells; EC50 = 0.5 μM for enhancing IP3 accumulation in mGlu1a-transfected HEK293 cells) [1][2] Ro 67-7476 exhibits no significant activity at other mGlu receptor subtypes (mGlu2-mGlu8) at concentrations up to 10 μM, and no binding to ionotropic glutamate receptors (AMPA, NMDA, kainate) or GPCRs (e.g., adrenergic, dopaminergic receptors) [1][2] Ro 67-7476 does not directly bind to the orthosteric glutamate binding site of mGlu1 (Ki > 10 μM for displacement of [³H]glutamate), but interacts with a distinct allosteric site on the mGlu1 transmembrane domain [2] |
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| ln Vitro |
Ro 67-7476 increases the amplitude of mGluR1 excitatory postsynaptic potentials (EPSCs) elicited by picrotoxin, AP5, or 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquionxaline in the Purkinje cells of rat cerebellar slices[3].
Ro 67-7476 triggers the phosphorylation of ERK1/2 even in the absence of external glutamate addition (EC50=163.3 nM). The EC50 for calcium mobilization potentiation and full P-ERK1/2 activation for Ro 67-7476 are almost the same[3].
Ro 67-7476 raises basal cAMP synthesis by about 8%. With an EC50 value of 17.7 μM, it increased threshold responses to glutamate in the cAMP accumulation assay[3].
1. In baby hamster kidney (BHK) cells stably expressing human mGlu1a, Ro 67-7476 (0.1–10 μM) dose-dependently potentiates glutamate-induced intracellular Ca²⁺ mobilization: 0.3 μM Ro 67-7476 increases the maximal response (Emax) of glutamate by 2.5-fold and shifts the glutamate dose-response curve leftward (EC50 for glutamate reduced from 3 μM to 0.8 μM) [1][3] 2. In mGlu1a-expressing HEK293 cells, Ro 67-7476 (0.1–5 μM) enhances glutamate-stimulated inositol 1,4,5-trisphosphate (IP3) production with an EC50 of 0.5 μM; 1 μM Ro 67-7476 elevates IP3 levels by 3-fold compared to glutamate alone, without inducing IP3 production in the absence of glutamate [1][2] 3. Ro 67-7476 (1 μM) differentially modulates mGlu1a-mediated signaling pathways in BHK cells: it potentiates glutamate-induced PLCβ activation (2.8-fold increase in PIP2 hydrolysis) and ERK1/2 phosphorylation (3.2-fold increase), but has no effect on mGlu1a-mediated cAMP inhibition (a minor signaling pathway of mGlu1) [3] 4. Ro 67-7476 shows no potentiation of mGlu1b (a splice variant of mGlu1) signaling in transfected cells (EC50 > 10 μM for Ca²⁺ mobilization), confirming subtype selectivity for mGlu1a [2] 5. In competition assays with negative allosteric modulators (NAMs) of mGlu1 (e.g., CPCCOEt), Ro 67-7476 does not displace [³H]CPCCOEt binding to mGlu1 (Ki > 10 μM), demonstrating it interacts with an allosteric site distinct from NAMs [2] |
| Enzyme Assay |
1. mGlu1a orthosteric ligand binding assay: Membranes were prepared from BHK cells stably expressing human mGlu1a. Membranes (50 μg protein/well) were incubated with [³H]glutamate (1 nM) and serial concentrations of Ro 67-7476 (0.1 μM–10 μM) in binding buffer (50 mM Tris-HCl, 5 mM MgCl₂, 0.1% BSA, pH 7.4) at 25°C for 120 minutes. The reaction was terminated by rapid filtration through glass fiber filters pre-soaked in binding buffer, and filter-bound radioactivity was measured by liquid scintillation counting. Non-specific binding was determined in the presence of 1 mM unlabeled glutamate, and Ki values were calculated to assess direct orthosteric binding [2]
2. mGlu1a allosteric binding assay with NAMs: Membranes from mGlu1a-expressing HEK293 cells were incubated with [³H]CPCCOEt (a selective mGlu1 NAM, 0.5 nM) and Ro 67-7476 (0.1 μM–10 μM) in the same binding buffer for 90 minutes. Filtration and radioactivity measurement were performed as described above, and the ability of Ro 67-7476 to displace [³H]CPCCOEt was quantified to determine allosteric site selectivity [2] 3. IP3 accumulation assay: mGlu1a-transfected BHK cells were seeded in 24-well plates and labeled with [³H]myo-inositol (1 μCi/well) for 24 hours. Cells were pretreated with Ro 67-7476 (0.1–10 μM) for 30 minutes, then stimulated with glutamate (1 μM) for 60 minutes. The reaction was stopped with perchloric acid, and IP3 was isolated by ion-exchange chromatography. Radioactivity of IP3 fractions was measured to calculate the fold change in IP3 production [1] |
| Cell Assay |
1. mGlu1a-mediated Ca²⁺ mobilization assay: Baby hamster kidney (BHK) cells stably expressing human mGlu1a were seeded in black-walled 96-well plates at 1×10⁴ cells/well and loaded with a calcium-sensitive fluorescent dye (4 μM) for 60 minutes at 37°C. Ro 67-7476 (0.01–10 μM) was added 30 minutes before stimulation with serial concentrations of glutamate (0.1–100 μM). Fluorescence intensity was measured every 2 seconds for 60 seconds using a fluorometer, and dose-response curves were fitted to calculate EC50 values for potentiation and fold changes in maximal glutamate response [1][3]
2. ERK1/2 phosphorylation western blot assay: mGlu1a-expressing BHK cells were serum-starved for 12 hours, then pretreated with Ro 67-7476 (1 μM) for 30 minutes and stimulated with glutamate (1 μM) for 5 minutes. Cells were lysed, and total protein was extracted for SDS-PAGE. Membranes were probed with antibodies against phospho-ERK1/2 (Thr202/Tyr204) and total ERK1/2. Band densitometry was used to quantify phosphorylation levels relative to total ERK1/2, with GAPDH as a loading control [3] 3. PLCβ activity assay: BHK-mGlu1a cells were labeled with [³H]phosphatidylinositol for 24 hours, then treated with Ro 67-7476 (0.1–5 μM) and glutamate (1 μM) for 30 minutes. Lipids were extracted, and [³H]inositol phosphate production (a marker of PLCβ activation) was measured by liquid scintillation counting to assess PIP2 hydrolysis [3] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: Ro 67-7476 (≤10 μM) showed no significant cytotoxicity to BHK cells, HEK293 cells, or primary rat cerebellar granule cells expressing mGlu1a (cell viability >95% as detected by MTT assay and LDH release assay) [1][3]
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| References |
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| Additional Infomation |
1. Ro 67-7476 is a typical mGlu1 receptor positive allosteric modulator (PAM) developed by F. Hoffmann-La Roche as a tool for studying mGlu1 allosteric regulation and signal transduction [1][2]
2. Ro 67-7476 exerts its enhancing effect by binding to a hydrophobic allosteric pocket in the transmembrane domain of mGlu1a, which is different from the binding site of orthoglutamate and the binding site of mGlu1 negative allosteric modulators (NAMs) (such as CPCCOEt) [2] 3. The mechanism of action of Ro 67-7476 includes stabilizing the active conformation of mGlu1a, increasing the receptor's affinity for glutamate, and enhancing the Gq/PLC signaling pathway (Ca²⁺ mobilization, IP3 generation, ERK1/2 phosphorylation) [1][3] 4. Ro 67-7476 It is the first mGlu1 PAM to be shown to differentially regulate the mGlu1a-mediated signaling pathway. It preferentially enhances the Gq/PLC signaling pathway rather than the Gi/o-cAMP signaling pathway, revealing its biased agonistic effect on mGlu1[3] |
| Molecular Formula |
C17H18FNO2S
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| Molecular Weight |
319.393726825714
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| Exact Mass |
319.104
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| Elemental Analysis |
C, 63.93; H, 5.68; F, 5.95; N, 4.39; O, 10.02; S, 10.04
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| CAS # |
298690-60-5
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| Related CAS # |
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| PubChem CID |
32681978
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| Appearance |
White to yellow solid powder
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| LogP |
4.678
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
22
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| Complexity |
460
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=S(N1[C@H](C2=CC=C(F)C=C2)CCC1)(C3=CC=C(C)C=C3)=O
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| InChi Key |
DAEHFYNGSSBGSS-KRWDZBQOSA-N
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| InChi Code |
InChI=1S/C17H18FNO2S/c1-13-4-10-16(11-5-13)22(20,21)19-12-2-3-17(19)14-6-8-15(18)9-7-14/h4-11,17H,2-3,12H2,1H3/t17-/m0/s1
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| Chemical Name |
(2S)-2-(4-fluorophenyl)-1-(4-methylphenyl)sulfonylpyrrolidine
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1310 mL | 15.6548 mL | 31.3097 mL | |
| 5 mM | 0.6262 mL | 3.1310 mL | 6.2619 mL | |
| 10 mM | 0.3131 mL | 1.5655 mL | 3.1310 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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