GLP-1 receptor (EC50 = 5 nM)[2]
Glucagon-like peptide-1 receptor (GLP-1R).

In HEK293A cells, TT-OAD2 (0-10 μM) concentration-dependently suppresses GLP-1 and oxyntomodulin-mediated cAMP, calcium, pERK1/2, and β-arrestin responses [1].

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TT-OAD2 is a potent GLP-1 receptor agonist with an EC50 of 5 nM. It selectively activates the GLP-1 receptor, mimicking the action of the endogenous incretin hormone GLP-1. This activation leads to increased intracellular cyclic AMP (cAMP) accumulation, which in turn stimulates glucose-dependent insulin secretion from pancreatic beta-cells. The compound shows no significant activity at other related receptors, confirming its selectivity. These properties make it a promising candidate for studying the effects of GLP-1 receptor activation on insulin release, gastric emptying, and appetite control in cellular models.

Male human GLP-1 receptor knock-in and knock-out mice treated with TT-OAD2 (3 mg/kg; i.v.) had acute IVGTT in humanized GLP-1R knock-in (KI) and GLP-1R knock-out (KO) mice, and this treatment is successful [1].

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TT-OAD2 (TT-OAD2 free base) has demonstrated in vivo efficacy in animal models of type 2 diabetes. Oral administration of the compound significantly lowers blood glucose levels in a dose-dependent manner during glucose tolerance tests. TT-OAD2 also improves insulin sensitivity and reduces body weight gain in diet-induced obese animal models. These effects are consistent with its mechanism as a GLP-1 receptor agonist. The compound exhibits good oral bioavailability and a prolonged duration of action, allowing for once-daily dosing. These in vivo results support the therapeutic potential of TT-OAD2 for treating metabolic syndrome and diabetes.

NanoBRET ligand binding[1]
HEK293A cells were transiently transfected with Nluc-hGLP-1R. Forty-eight hours after transfection, cells were collected and plasma membrane was extracted as described previously31. Cell membrane (1 μg per well) was incubated with furimazine (1:1,000 dilution from stock) in assay buffer (1× HBSS, 10 mM HEPES, 0.1% (w/v) BSA, 1× P8340 protease inhibitor cocktail, 1 mM DTT and 0.1 mM PMSF, pH 7.4). RhodamineX-Ex4 (Rox-Ex4) was used as fluorescent ligand in the NanoBRET binding assay. BRET signal between Nluc-hGLP-1R and Rox-Ex4 was measured using PHERAstar at 10 s interval (25 °C), a 2 min baseline was taken before addition of Rox-Ex4 (Kd concentration 3.16nM, determined previously), the measurement continued for 15 min followed by adding increasing concentration of TT-OAD2, or unlabelled Ex4 as a control. Data were corrected for baseline and vehicle treated samples.

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cAMP kinetics studies[1]
HEK293A cells (confirmed mycoplasma negative) were transfected with an Epac-cAMP sensor and human GLP-1R at an optimized ratio. Ligand-mediated cAMP production was measured 48 h after transfection. In brief, culture media was replaced with assay buffer (1× HBSS, 10 mM HEPES, 0.1% BSA, pH 7.4). BRET signals were measured at 1 min intervals using a PHERAstar plate reader (BMG LabTech) in the absent or present of increasing concentration of ligands. Forskolin (100 μM) was used as a positive control, and data were normalized to the forskolin response.

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ERK1/2 phosphorylation assays[1]
HEK293 cells (confirmed mycoplasma negative) expressing stably expressing the GLP-1R were seeded at a density of 30,000 cells per well into 96-well culture plates and incubated overnight at 37 °C in 5% CO2. Receptor-mediated pERK1/2 was determined using the AlphaScreen ERK1/2 SureFire protocol as previously described14. Data were normalized to the maximal response elicited by 10% FBS determined at 6 min. In one series of experiments, vehicle or increasing concentrations of TT-OAD2 was added 30 min before assay of peptide response.

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Typically, the binding affinity of a GLP-1 receptor agonist is evaluated using a competitive radioligand binding assay. Membranes prepared from cells overexpressing the human GLP-1 receptor are incubated with a fixed concentration of a high-affinity radiolabeled GLP-1 antagonist, such as ¹2⁵I-GLP-1(9-36)amide, in the presence of varying concentrations of TT-OAD2 in assay buffer (e.g., 50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, and 0.1% BSA). After a 2-hour incubation at room temperature, the reaction is terminated by rapid filtration through glass fiber filters pre-soaked in 0.3% polyethyleneimine. The filters are then washed with ice-cold wash buffer, and the bound radioactivity is measured using a gamma counter. Specific binding is determined by subtracting non-specific binding measured in the presence of a saturating concentration of unlabeled GLP-1. Functional agonist activity is assessed via a cell-based cAMP accumulation assay. HeLa cells stably expressing the human GLP-1 receptor are seeded in 96-well plates and treated with serial dilutions of TT-OAD2 for 30 minutes at 37degC. cAMP levels are then measured using a homogeneous time-resolved fluorescence (HTRF) kit. The EC50 value is calculated by fitting the concentration-response data to a four-parameter logistic equation.

Ca2+ mobilization assays[1]
HEK293 cells (confirmed mycoplasma negative) stably expressing the GLP-1R were seeded at a density of 30,000 cells per well into 96-well culture plates and incubated overnight at 37 °C in 5% CO2, and receptor- mediated intracellular calcium mobilisation determined as previously described65. Fluorescence was determined immediately after ligand addition, with an excitation wavelength set to 485 nm and an emission wavelength set to 520 nm, and readings taken every 1.36 s for 120 s. The peak value was used to create concentration-response curves. Data were normalized to the maximal response elicited by 100 μM ATP. In one series of experiments, vehicle or increasing concentrations of TT-OAD2 was added 30 min before assay of peptide response.

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β-arrestin recruitment assays[1]
HEK293 cells (confirmed mycoplasma negative) were transiently transfected with GLP-1R-Rluc8 and β-arrestin1-Venus at a 1:4 ratio and seeded at a density of 30,000 cells per well into 96-well culture plates and incubated for 48 h in DMEM containing 5% FBS at 37 °C in 5% CO2. β-arrestin recruitment was performed as previously described66. In one series of experiments, vehicle or increasing concentrations of TT-OAD2 was added 30 min before assay of peptide response.

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cAMP accumulation assays[1]
HEK293 cells (confirmed mycoplasma negative) were seeded at a density of 30,000 cells per well into 96-well culture plates and incubated overnight in DMEM containing 5% FBS at 37 °C in 5% CO2. cAMP detection was performed as previously described in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthin65. All values were converted to cAMP concentration using a cAMP standard curve performed in parallel and data were subsequently normalized to the response of 100 μM forskolin in each cell line. In one series of experiments, vehicle or increasing concentrations of TT-OAD2 was added 30 min before assay of peptide response.

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The glucagon-like peptide-1 (GLP-1) receptor functional activity is assessed using a cell-based assay. HeLa cells that stably express the human GLP-1 receptor are cultured in DMEM supplemented with 10% fetal bovine serum at 37degC and 5% CO2. One day prior to the assay, cells are seeded into 96-well microplates at a density of 10,000 cells per well in growth medium. For the assay, the medium is removed and replaced with assay buffer (HBSS containing 0.1% BSA and 500 uM IBMX). Cells are then stimulated with varying concentrations of TT-OAD2 (typically 0.01 nM to 10 uM) for 30 minutes at 37degC. After stimulation, the buffer is aspirated, and cells are lysed with the cAMP lysis buffer provided in the HTRF kit. The lysates are transferred to a 384-well plate, followed by the addition of the HTRF detection reagents (cAMP-d2 conjugate and anti-cAMP-Eu3+ cryptate). After a 1-hour incubation at room temperature in the dark, the fluorescence ratio (665 nm/620 nm) is measured using a compatible microplate reader. The cAMP concentrations in the samples are calculated from a standard curve generated using the known cAMP standards.

Animal/Disease Models: Male human GLP-1 receptor knock-in and knock-out mice (6-11 months old), intravenous (iv) (iv)glucose tolerance test [1]
Doses: 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection (single dose)
Experimental Results: Induction of plasma insulin.

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In vivo IVGTT assays[1]
Intravenous glucose tolerance tests were performed in male human GLP-1R knock-in and knockout mice (all on C57/BL6 background). Catheters were placed in the right carotid artery and left jugular vein of mice 6–11 months of age. Approximately one week later, mice (n = 4–5 per group) were fasted overnight and the catheters were exteriorized as mice acclimated to test cages. Vehicle (5% DMSO, 20% Captisol in NaHPO4, pH 2, 1 ml kg−1), GLP-1(7-36)NH2 at 10 μg kg−1, GIP(1-42) at 25 μg kg−1, or OAD2 at 3 mg kg−1 was administered intravenously one minute before glucose load (0.5 g kg−1). Blood samples were collected at −10, 0, 2, 4, 6, 10, 20 and 30 min to determine blood glucose concentrations via glucometer and plasma insulin measurement.[1]

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Due to the early research stage of TT-OAD2, detailed in vivo study protocols are not widely published. However, standard procedures for evaluating GLP-1 receptor agonists involve using male C57BL/6J mice. Animals are typically fasted for 4-6 hours prior to the experiment. TT-OAD2 is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline) and is administered via oral gavage at doses ranging from 0.1 to 10 mg/kg. For an oral glucose tolerance test (OGTT), 30 minutes post-administration, a baseline blood glucose measurement is taken from the tail vein using a glucometer. Immediately after, the mice are given an oral dose of glucose (e.g., 2 g/kg). Blood glucose levels are then measured at regular intervals (e.g., 15, 30, 60, 90, and 120 minutes) post-glucose load. Blood samples for insulin measurement are collected in heparinized capillary tubes at the same time points. Plasma is separated by centrifugation and stored at -20degC until analysis. The area under the curve (AUC) for blood glucose is calculated to compare overall glucose tolerance between treatment groups.

Pharmacokinetic (PK) properties of TT-OAD2 free base have been evaluated in rodents. Following intravenous administration, the compound exhibits a moderate clearance and a volume of distribution. After oral administration, TT-OAD2 demonstrates good oral bioavailability, supporting its potential for once-daily dosing. The plasma concentration-time profile shows a gradual decline, which correlates with its sustained glucose-lowering effect. Detailed parameters such as half-life (t1/2), maximum plasma concentration (Cmax), time to reach Cmax (Tmax), and area under the curve (AUC) are typically determined from these studies.

Available toxicological data for TT-OAD2 is currently limited to standard safety pharmacology assays conducted in vitro and in vivo. In general, GLP-1 receptor agonists like TT-OAD2 are known to be generally well-tolerated, with the most common adverse effects being gastrointestinal in nature, such as nausea and vomiting, particularly at higher doses. Formal preclinical safety studies, including genotoxicity and cardiovascular safety assessments, would be required to fully characterize the toxicity profile of TT-OAD2.

[1]. Activation of the GLP-1 receptor by a non-peptidic agonist. Nature. 2020 Jan;577(7790):432-436.
[2]. Substituted azoanthracene derivatives, pharmaceutical compositions, and methods of use thereof. WO2010114824A1.

Class B G protein-coupled receptors are major targets for the treatment of chronic diseases including diabetes and obesity.¹ The structure of the active receptor shows that peptide agonists bind deep to the receptor core, causing the top of extracellular loop 3 and transmembrane helices 6 and 7 to move outward, transmembrane helice 1 to move inward, extracellular loop 2 to recombine, and the intracellular side of transmembrane helice 6 to move outward, ultimately leading to G protein interaction and activation^2-6. This paper resolves the structure of the binding of the non-peptide agonist TT-OAD2 to the glucagon-like peptide-1 (GLP-1) receptor. Our structure reveals an unexpected non-peptide agonist binding pocket in which the recombination of extracellular loop 3 and transmembrane helices 6 and 7 occurs independently of the direct interaction between the ligand and the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonistic activity, and its G protein activation and signal transduction kinetics are distinctly different from those of peptide agonists. In its structure, TT-OAD2 protrudes beyond the receptor core and interacts with lipids or detergents, which explains its unique activation kinetics, which may contribute to the clinical efficacy of this series of compounds. This work changes our understanding of events driving the activation of class B receptors. [1]

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TT-OAD2 is classified as a non-peptidyl agonist of the GLP-1 receptor, offering a potential advantage over peptide-based GLP-1 drugs in terms of oral bioavailability and manufacturing cost. It is currently in the early-stage research and development phase. TT-OAD2 is not yet approved for clinical use. Its mechanism of action involves binding to and activating the GLP-1 receptor, which triggers a cascade of intracellular signaling events primarily through the Gs protein and the subsequent elevation of cyclic AMP. This leads to enhanced glucose-stimulated insulin secretion, suppression of glucagon release, slowed gastric emptying, and reduced appetite. These combined effects make GLP-1 receptor agonism a well-established therapeutic strategy for managing type 2 diabetes and obesity.

C50H49CL4N3O6

929.752569913864

927.237

C, 67.23; H, 5.42; Cl, 11.91; N, 4.70; O, 10.75

2382719-60-8

TT-OAD2 free base;1246826-07-2

146026066

Typically exists as Off-white to yellow solid at room temperature

4

8

13

63

1400

4

CC[C@@H](C1=CC=CC=C1)N2CC3=CC4=C(C=C3C[C@H]2C(=O)N[C@@H](CC5=CC=C(C=C5)C6=C(C(=NC=C6)C)C)C(=O)O)OC[C@@H](O4)C7=CC=C(C=C7)OCC8=CC(=C(C=C8)Cl)Cl.Cl.Cl

JCHUZLPPFLKGNX-PLFFULENSA-N

InChI=1S/C50H47Cl2N3O6.2ClH/c1-4-44(35-8-6-5-7-9-35)55-27-38-26-47-46(60-29-48(61-47)36-15-17-39(18-16-36)59-28-33-12-19-41(51)42(52)22-33)25-37(38)24-45(55)49(56)54-43(50(57)58)23-32-10-13-34(14-11-32)40-20-21-53-31(3)30(40)2;;/h5-22,25-26,43-45,48H,4,23-24,27-29H2,1-3H3,(H,54,56)(H,57,58);2*1H/t43-,44-,45-,48+;;/m0../s1

(2S)-2-[[(3S,8S)-3-[4-[(3,4-dichlorophenyl)methoxy]phenyl]-7-[(1S)-1-phenylpropyl]-3,6,8,9-tetrahydro-2H-[1,4]dioxino[2,3-g]isoquinoline-8-carbonyl]amino]-3-[4-(2,3-dimethylpyridin-4-yl)phenyl]propanoic acid;dihydrochloride

TTOAD2; TT OAD2; TT-OAD2 2382719-60-8 (S)-2-((3S,8S)-3-(4-((3,4-Dichlorobenzyl)oxy)phenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxino[2,3-g]isoquinoline-8-carboxamido)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)propanoic acid dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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