Human GLP-1 receptor ( EC50 = 0.66 μM ); Rat GLP-1 receptor ( EC50 = 0.755 μM )
Glucagon-like peptide-1 receptor (GLP-1R) allosteric modulator/partial agonist (EC50 = 740 nM for intrinsic partial agonism in a cAMP accumulation assay using HEK293 cells expressing the human GLP-1 receptor; efficacy was 7% relative to maximal GLP-1(7–36)-NH2 effect).[2]

BETP is an agonist of the GLP-1 receptor; its EC50s for the rat and human GLP-1 receptors are 0.755 and 0.66 μM, respectively. In cells expressing GLP-2, GIP, PTH, or glucagon receptors, BETP (Compound B) is inactive. In both normal and diabetic human islets, BETP (1-10 μM) increases insulin secretion. Furthermore, there are additive effects of BETP and GLP-1 on enhancing GLP-1 receptor signaling[1]. Oxyntomodulin's potency is ten times higher with BETP (EC50 of 80 pM). At the glucagon receptor, GLP-1 has no effect on the potencies and efficacies of either oxyntomodulin or glucagon. BETP (0-30 μM) raises oxyntomodulin's affinity for binding to the GLP-1 receptor[2].

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BETP potentiated oxyntomodulin-induced cAMP accumulation in HEK293 cells expressing the human GLP-1 receptor. In the presence of 4 µM BETP, the potency (EC50) of oxyntomodulin increased 10-fold (from 770 pM to 76 pM) while retaining full agonist efficacy.[2]
BETP showed no effect on oxyntomodulin or glucagon potency or efficacy at the closely related glucagon receptor in HEK293 cells, demonstrating its selectivity for the GLP-1 receptor.[2]
Radioligand binding studies using GLP-1 receptor-expressing membranes showed that BETP dose-dependently increased the ability of oxyntomodulin to compete with 125I-exendin-4(9-39). An operational model quantified a binding cooperativity factor (α) of 15, indicating BETP increases oxyntomodulin's affinity for the GLP-1 receptor by 15-fold.[2]
In a functional Gαs-specific GTPγS binding assay using GLP-1 receptor-expressing membranes, BETP dose-dependently increased the potency of oxyntomodulin-stimulated G protein activation. At saturation, the potentiated oxyntomodulin potency was within 2-fold of the observed potency of GLP-1(7-36)-NH2.[2]
BETP alone acted as a low-potency partial agonist in the GTPγS binding assay.[2]
BETP did not enhance the potency or efficacy of the full agonist GLP-1(7-36)-NH2 for GLP-1 receptor activation.[2]
In CHO cells expressing the human GLP-1 receptor, BETP engendered biased signaling when co-applied with oxyntomodulin. It showed strong positive cooperativity (αβ = 12.6) for cAMP accumulation, neutral cooperativity for intracellular Ca2+ mobilization (αβ = 1.70) and β-arrestin1/2 recruitment, and efficacy-driven negative cooperativity (β = 0.03) for ERK1/2 phosphorylation.[2]
BETP showed intrinsic partial agonist activity for β-arrestin1 recruitment (Emax = 45% of GLP-1(7–36)-NH2).[2]

BETP BETP has an insulinotropic effect in SD rats. In the intravenous glucose tolerance test (IVGTT) model, BETP (10 mg/kg, jugular vein cannula) demonstrates insulin secretagogue activity. Rats treated with BETP (10 mg/kg, i.v.) require 20% higher glucose infusion rates and exhibit higher plasma insulin levels in the hyperglycemic clamp model of SD rats[1].
BETP (5 mg/kg) increases insulin release that is triggered by oxyntomodulin[2].

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In an intravenous glucose tolerance test (IVGTT) in fasted, anesthetized Wistar rats, a single intravenous dose of BETP (5 mg/kg) alone caused only a minimal degree of insulin secretion above vehicle.[2]
Coadministration of BETP (5 mg/kg) with a subsaturating dose of oxyntomodulin (30 nmol/kg) markedly and synergistically enhanced the insulinotropic effect of oxyntomodulin compared to oxyntomodulin alone. The increase in insulin AUC was non-additive and greater than the sum of individual effects.[2]

For the Gαs-specific GTPγS binding assay, membranes were prepared from HEK293 cells stably expressing the human GLP-1 receptor. Reactions contained membrane protein in a buffer with HEPES, NaCl, MgCl2, saponin, BSA, and [35S]GTPγS. Peptide and BETP were diluted and co-treated. Binding was induced for 30 min at ambient temperature before solubilization with detergent, followed by addition of a rabbit anti-Gαs polyclonal antibody and anti-rabbit polyvinyltoluene beads. The detection mixtures were developed, centrifuged, and counted using a scintillation counter.[2]
For radioligand binding, a competition binding assay was performed using 125I-exendin-4(9-39) as the radioligand and GLP-1 receptor-expressing membranes or whole cells. The ability of oxyntomodulin to displace radioligand binding was measured in the presence of fixed concentrations of BETP. Data were fit to an operational model of allosteric agonism to determine cooperativity factors.[2]

For cAMP accumulation in HEK293 cells, cells transiently expressing the human GLP-1 receptor or glucagon receptor were used. Cells were transfected, then seeded into 96-well plates. Prior to assay, cells were lifted and resuspended in low-serum medium. Test compounds, intermediately diluted in assay medium containing BSA and a phosphodiesterase inhibitor (IBMX), were added to the cells. After a 20-min incubation, intracellular cAMP levels were assayed using a homogeneous time-resolved fluorescence technology kit. Fluorescence was measured using a plate reader, and data were expressed as a percentage of the response induced by reference peptide agonists.[2]
For β-arrestin recruitment assays, Flp-In CHO cell lines stably expressing a GLP-1 receptor-Renilla luciferase 8 (Rluc8) fusion protein and either β-arrestin1- or β-arrestin2-Venus fusion protein were generated using BRET (Bioluminescence Resonance Energy Transfer). Cells were seeded in 96-well plates and cultured. Before assay, cells were rinsed and incubated in buffer. The Rluc substrate coelenterazine-h was added. After incubation, agonist was added, and BRET readings were collected using a luminometer that sequentially integrates signals at two emission windows. The BRET signal was calculated by subtracting the ratio for a vehicle-treated sample from the ratio for the ligand-treated sample, yielding ligand-induced BRET.[2]
Measurement of intracellular Ca2+ mobilization and ERK1/2 phosphorylation in CHO cells expressing the human GLP-1 receptor was performed as described previously in the literature.[2]

Rats: The IVGTT research is carried out. Three male SD rats per cage are kept in groups in polycarbonate cages with filter tops. Rats are kept at 21°C on a 12:12 h light-dark cycle (lights come on at 6:00 a.m.), where they are given an unlimited supply of food and deionized water. Rats are fasted for the entire experiment and given 60 mg/kg of pentobarbital to put them to sleep. The jugular vein is punctured with a 0.84 mm catheter to administer glucose and compounds (BETP, etc.). A larger, 1.02 mm-diameter catheter is placed into the carotid artery to collect blood quickly. At times 0, 2, 4, 6, 10, and 20 minutes following intravenous administration of the BETP, blood is drawn for the measurement of glucose and insulin levels. An intravenous glucose bolus of 0.5 g/kg is then administered right away. Insulin and glucose plasma levels are measured[1].

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For the IVGTT, male Wistar rats were fasted overnight and anesthetized. Catheters were inserted into the jugular vein for compound/glucose administration and into the carotid artery for blood sampling. BETP was solubilized in a dosing solution containing ethanol-Solutol, polyethylene glycol-400, and phosphate-buffered saline (pH 7.4). A single intravenous dose of BETP (5 mg/kg), oxyntomodulin, GLP-1(7–36)-NH2, or their combinations was administered via the jugular vein catheter, immediately followed by an intravenous glucose bolus (0.5 g/kg). Blood samples were collected from the arterial catheter at specified time points (0, 2, 4, 6, 10, 20 min) for insulin measurement.[2]
ADME/Pharmacokinetics: The physicochemical liabilities of BETP (and a related quinoxaline compound) limit longer-term studies; both are reported to be unstable in the presence of nucleophiles.[2]

[1]. Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets. Diabetes. 2010 Dec;59(12):3099-107.

[2]. Small molecule allosteric modulation of the glucagon-like Peptide-1 receptor enhances the insulinotropic effect of oxyntomodulin. Mol Pharmacol. 2012 Dec;82(6):1066-73.

BETP is a low molecular weight pyrimidine positive allosteric modulator (PAM) and a partial agonist of the GLP-1 receptor. [2] It enhances the binding affinity of endogenous peptide hormone regulators to the GLP-1 receptor, which is an affinity-driven mechanism of action. [2] It is a highly selective allosteric modulator whose action depends on the GLP-1 receptor without enhancing the effect of hormone regulators on the glucagon receptor. [2] BETP generates biased signaling on the GLP-1 receptor bound to hormone regulators, selectively enhancing the cAMP pathway rather than Ca2+ mobilization, ERK1/2 phosphorylation, or β-arrestin recruitment pathway. [2] It demonstrates that small molecule allosteric modulators can enhance the activity of endogenous hormones. (Oxyregulators) act on the GLP-1 receptor to enhance insulin secretion, suggesting a new approach to treating type 2 diabetes. [2]

C20H17N2O2F3S

406.42138

406.096

1371569-69-5

49868481

Light yellow to yellow solid powder

5.734

0

8

6

28

514

0

FC(C1=CC(C2=CC=CC(OCC3=CC=CC=C3)=C2)=NC(S(CC)=O)=N1)(F)F

NTDFYGSSDDMNHI-UHFFFAOYSA-N

InChI=1S/C20H17F3N2O2S/c1-2-28(26)19-24-17(12-18(25-19)20(21,22)23)15-9-6-10-16(11-15)27-13-14-7-4-3-5-8-14/h3-12H,2,13H2,1H3

2-ethylsulfinyl-4-(3-phenylmethoxyphenyl)-6-(trifluoromethyl)pyrimidine

BETP

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: ~25 mg/mL (~61.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 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 25.0 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: ≥ 2.5 mg/mL (6.15 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.

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