VU 0365114 is a highly selective positive allosteric modulator (PAM) for the muscarinic acetylcholine receptor subtype M5 (M5 mAChR). (M5 EC50 = 2.7 µM, >30 µM vs. M1-M4). [1]
Muscarinic acetylcholine receptor M5 (M5 receptor) positive allosteric modulator (PAM). (EC50 = 2.7 µM, % ACh Max = 85%). [3]
The compound is selective for M5 over M1, M3, M4 receptors (M1 EC50 > 30 µM). [3]

Human beta cells activated with ACh secrete more insulin when exposed to 10 μM of VU 0365114 [2]. Human beta cells' ACh-stimulated somatostatin production is unaffected by VU 0365114 (10 μM) [2].

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VU 0365114 (also referred to as compound 4) is a potent and selective M5 PAM. In M5-CHO cells, it potentiated acetylcholine (ACh)-induced intracellular calcium mobilization with an EC50 of 2.7 µM. It exhibited >30 µM activity at M1, M2, M3, and M4 mAChR subtypes, indicating high selectivity. In an acetylcholine fold-shift assay at a standard 30 µM concentration, VU 0365114 elicited a 10-fold leftward shift of the ACh concentration-response curve. [1]
In human pancreatic islets, VU 0365114 (10 μmol/L) amplified the insulin secretion stimulated by acetylcholine (10 μmol/L). [2]
VU 0365114 (10 μmol/L) further increased insulin secretion at a basal glucose concentration (3 mmol/L) in the absence of exogenous acetylcholine, indicating potentiation of endogenous cholinergic signaling. This insulin response to VU 0365114 was blocked by atropine. [2]
VU 0365114 (10 μmol/L) did not alter somatostatin secretion in response to acetylcholine or at basal glucose concentration. [2]
VU0365114 was identified as a highly selective M5 positive allosteric modulator (PAM). In concentration-response curves (CRCs) using M5-CHO cells, it exhibited an M5 EC50 of 2.7 µM and potentiated the acetylcholine (ACh) response to 85% of the maximum ACh response. [3]
VU0365114 showed significant selectivity versus M1 and M3 receptors. At 30 µM, it only modestly activated M3 and was inactive at M1 (EC50 > 30 µM). [3]
In M5 fold-shift experiments, VU0365114 at 30 µM elicited a greater than 50-fold leftward shift of the ACh concentration-response curve, indicating strong positive allosteric modulation. [3]
The compound also displayed moderate intrinsic allosteric agonism at 30 µM in the absence of ACh. [3]

The functional activity of VU 0365114 as an M5 PAM was determined using an intracellular calcium mobilization assay in Chinese Hamster Ovary (CHO) cells expressing the human M5 muscarinic receptor. Cells were loaded with a calcium-sensitive fluorescent dye. Increases in intracellular calcium in response to acetylcholine (ACh) were measured fluorometrically in the presence or absence of the test compound. The assay was used to determine the compound's ability to potentiate the response to a submaximal concentration (EC20) of ACh (for initial screening) and to generate full concentration-response curves for calculating EC50 values and maximal potentiation (ACh Max %). The fold-shift of the ACh concentration-response curve was also assessed at a fixed compound concentration. [1]
Hormone secretion from human pancreatic islets was dynamically measured using a high-capacity, automated perfusion system. A peristaltic pump pushed HEPES-buffered solution (containing 125 mmol/L NaCl, 5.9 mmol/L KCl, 2.56 mmol/L CaCl2, 1 mmol/L MgCl2, 25 mmol/L HEPES, and 0.1% BSA, pH 7.4) at a rate of 100 μL/min through a column containing 100 human islets immobilized in gel. The column and solutions were maintained at 37°C. The perfusate was collected every minute into a cooled fraction collector. [2]
For experiments, the glucose concentration was adjusted to 3 mmol/L. Drugs, including acetylcholine and VU 0365114, were applied via the perfusion buffer. Insulin in the collected fractions was quantified using a multiplex immunoassay kit. Somatostatin secretion was determined using a fluorescent enzyme-linked immunoassay kit. [2]
Functional activity (potentiation of an EC20 concentration of acetylcholine) was assessed in Chinese Hamster Ovary (CHO) cells stably expressing human muscarinic acetylcholine receptor subtypes (M1, M3, M5). For M2 and M4 receptor assays, which couple to Gαi/o proteins, the cells were co-transfected with a chimeric Gαq/i5 protein to redirect signaling to the phospholipase C pathway, allowing detection via intracellular calcium mobilization. [3]
Intracellular calcium mobilization was used as the functional readout for receptor activation. Compound activity was initially triaged in a single-point screen at 10 µM for its ability to potentiate an EC20 of ACh in M5-CHO cells. [3]
Selected compounds, including VU0365114, were further characterized in full 8-point concentration-response curves to determine EC50 values and the percentage of maximal ACh response (% ACh Max). [3]
Fold-shift experiments were performed by evaluating the acetylcholine concentration-response curve in the presence of a fixed concentration (30 µM) of the test compound (VU0365114) to determine the magnitude of leftward shift. [3]

The compounds in this series (including VU0365114) exhibited moderate to poor pharmacokinetic (PK) characteristics in rats. [3] Limited brain exposure was observed, with a brain/plasma AUC ratio (AUCBrain/AUCPlasma) of approximately 0.25. This was attributed to the presence of a dicarbonyl group in the indigo moiety of the compounds. [3] Notably, brain exposure was not detected when using solvents containing DMSO, which may contribute to increased brain concentrations. [3]

[1]. Heterobiaryl and heterobiaryl ether derived M5 positive allosteric modulators. Bioorg Med Chem Lett. 2010 Oct 1;20(19):5617-22.

[2]. Control of insulin secretion by cholinergic signaling in the human pancreatic islet. Diabetes. 2014 Aug;63(8):2714-26.

[3]. Chemical lead optimization of a pan G(q) mAChR M(1), M(3), M(5) positive allosteric modulator (PAM) lead. Part I: Development of the first highly selective M(5) PAM. Bioorg Med Chem Lett. 2010 Jan 15;20(2):558-62.

1-[(4-phenylphenyl)methyl]-5-(trifluoromethoxy)indole-2,3-dione belongs to the biphenyl class of compounds.

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VU 0365114 is a lipophilic compound with logP > 4.5, which results in limited solubility and poor overall physicochemical properties. It was developed by optimizing the earlier M5 preferential coordination PAM compound VU0238429. Structure-activity relationship (SAR) studies showed that the 5-trifluoromethoxy (5-OCF3) group on the indigo core is crucial for the activity and selectivity of M5 PAM. This compound was used as an important chemical tool and benchmark for further optimization, aiming to improve its physicochemical properties by introducing a basic heterocycle, thereby promoting salt formation and reducing lipophilicity. [1] VU 0365114 was identified as an allosteric modulator of the M5 muscarinic acetylcholine receptor. [2] This study used VU 0365114 as a pharmacological tool to reveal the existence of a functional M5 receptor on human pancreatic β cells, indicating that human β cells express M5 receptors in addition to M3 receptors. [2] The results showed that endogenous acetylcholine in human pancreas acts on β cell M5 receptors, and regulation of this receptor can affect insulin secretion. [2]
VU0365114 (compound 6a in this study) was developed through chemical lead compound optimization, with the starting compounds being pan-Gq mAChR PAM (VU0119498) and M5-preferred PAM (VU0238429). The optimization strategy involved the introduction of a biaryl structure. [3]
It is one of the first highly selective M5 PAM tool compounds, making it possible to study the role of M5 receptors in the central nervous system (CNS), which had been hampered by the lack of selective pharmacological reagents. [3]
This compound is considered an important tool for studying M5 function and can be used in cell experiments, electrophysiological experiments, and intravenous ventricle (ICV) injection. [3]

C22H14NO3F3

397.34666

397.093

1208222-39-2

45281794

Light yellow to orange solid powder

5.046

0

6

4

29

609

0

SPBGRXOPAXZSER-UHFFFAOYSA-N

InChI=1S/C22H14F3NO3/c23-22(24,25)29-17-10-11-19-18(12-17)20(27)21(28)26(19)13-14-6-8-16(9-7-14)15-4-2-1-3-5-15/h1-12H,13H2

1-[(4-phenylphenyl)methyl]-5-(trifluoromethoxy)indole-2,3-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 77.5 mg/mL (~195.04 mM)

Solubility in Formulation 1: ≥ 2.58 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 25.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.58 mg/mL (6.49 mM) 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 25.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)