GLP-1 receptor
Danuglipron (PF-06882961) targets the human glucagon‑like peptide‑1 receptor (GLP‑1R).
Binding affinity (Ki): 80 nM using [³H]‑PF‑06883365 radioligand; 360 nM using [¹²⁵I]‑GLP‑1 radioligand.
cAMP potency (EC50): 13 nM in candidate selection (CS) cell line; 0.38 nM in screening assay (SA) with BETP; 4.6 nM in HEK293 cells expressing FAP‑GLP‑1R.
β‑arrestin recruitment (EC50): 490 nM (β‑arrestin2), 500 nM (β‑arrestin1) with Emax 36% and 32%, respectively.
Receptor internalization (EC50): 230 nM (Emax 83%). [2]

PF-06882961 promotes the accumulation of cAMP in CHO cells expressing the GLP-1Rs from both humans and monkeys with comparable EC50 values. On the other hand, PF-06882961 does not raise cAMP levels in GLP-1R-expressing mouse, rat, or rabbit cells.[2]

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Danuglipron (PF-06882961) acts as a full agonist of the cAMP signaling pathway and a partial agonist for β‑arrestin recruitment and receptor internalization. [2]
In CHO cells stably expressing human GLP‑1R, the compound increased cAMP production with an EC50 of 13 nM in the CS cell line (lower receptor density, ~500 fmol/mg) and 0.38 nM in the SA cell line (higher density, ~2200 fmol/mg) in the presence of BETP. [2]
In a β‑arrestin recruitment assay (PathHunter), Danuglipron (PF-06882961) gave EC50 values of 490 nM (β‑arr2) and 500 nM (β‑arr1), with Emax of 36% and 32%, respectively, compared to liraglutide (Emax 99% for β‑arr2). [2]
Using HEK293 cells stably expressing fluorogen‑activated protein (FAP)‑tagged GLP‑1R, the compound induced receptor internalization with an EC50 of 230 nM and Emax of 83%; exenatide and liraglutide produced greater internalization (Emax 125% and 117%). [2]
Confocal microscopy of HEK293 cells expressing GFP‑tagged GLP‑1R showed that 30‑minute stimulation with Danuglipron (PF-06882961) triggered marked intracellular accumulation of the receptor, which was reversible after a 2‑hour washout. [2]
The compound did not increase cAMP in cells expressing mouse, rat, or rabbit GLP‑1R, but activity was restored in mouse GLP‑1R S33W mutant, confirming species selectivity dependent on tryptophan 33. [2]

PF-06882961 decreases food intake and increases insulin release in response to glucose stimulation in monkeys.[2]
Danuglipron Is Orally Bioavailable and Efficacious in Decreasing the Level of Glucose and Food Intake in Monkeys. Following iv administration, Danuglipron demonstrated moderate to high plasma clearance (CLp) values in rats (CLp = 57.3 mL/min/kg) and monkeys [CLp = 13.8 mL/min/kg] with relatively short elimination half-lives of 1.1 and 1.9 h in rats and monkeys, respectively. The oral bioavailability of a Tris salt form of danuglipron in animals was low to moderate and increased in a dose-dependent manner, which was adequate for studying the preclinical in vivo efficacy and safety of Danuglipron delivered via oral gavage in a standard 0.5% methyl cellulose formulation containing 2% Tween 80.[3]
Because danuglipron does not activate the rodent GLP-1R, the therapeutic effects of danuglipron on insulin and glucose were examined in an intravenous glucose tolerance test (IVGTT) in cynomolgus monkeys following iv infusion and po administration. Infusion rates and po doses were projected using systemic concentrations from monkey pharmacokinetic (PK) studies that were required to achieve receptor occupancies (RO) bracketing the RO estimated from the reported human plasma exposures for liraglutide at its clinically efficacious dose (1.8 mg once daily). The iv infusion of danuglipron during the IVGTT led to an increase in the rate of insulin secretion and the rate of glucose disappearance (K value) (Figure ​Figure88B–D). Enhancement of glucose-stimulated insulin secretion by danuglipron was concentration-dependent and comparable following the po dosing and iv infusion routes (Figure ​Figure88E). Once-daily subcutaneous administration of danuglipron for 2 days also inhibited food intake compared with that of vehicle-treated monkeys (Figure ​Figure88F). The subcutaneous route of administration was chosen to reduce variability in systemic exposure noted upon po administration and was more convenient than iv administration.[3]

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Danuglipron (PF-06882961) administered intravenously (IV) during an IV glucose tolerance test (IVGTT) in cynomolgus monkeys increased glucose‑stimulated insulin secretion and the rate of glucose disappearance (K‑value) in a concentration‑dependent manner. [2]
Oral administration of Danuglipron (PF-06882961) in monkeys also potentiated glucose‑stimulated insulin release (AUC0‑30 min) with similar efficacy to IV infusion. [2]
Once‑daily oral dosing for 2 days significantly reduced food intake compared to vehicle‑treated monkeys. [2]
In C57BL/6 mice, a subcutaneous dose of 10 mg/kg Danuglipron (PF-06882961) did not improve glucose tolerance during an IPGTT, consistent with its lack of activity at rodent GLP‑1R. [2]
In a phase 1 human study, single oral doses (3‑300 mg) of Danuglipron (PF-06882961) showed a trend for dose‑dependent lowering of fasting serum glucose at 24 hours post‑dose; the 300 mg dose produced a statistically significant reduction versus placebo. [2]

Biological Assays. Min6 Ca2+ Mobilization Assay[3]
\nMIN6-c4 cells were plated at a density of 5 × 104 cells per well into black 96-well plates and cultured for 20–24 h at 37 °C and 5% CO2. For cell loading, culture supernatants were aspirated and 100 μL of assay buffer (Krebs–Ringer buffer, 10 mM HEPES, 0.1% BSA, 2.5 mM glucose) and an equal volume of calcium 6 dye (FLIPR calcium 6 assay kit, Molecular Devices, R8191) dissolved in the same buffer according to instructions of the manufacturer were added to each well. Cells were incubated for 70 min at 37 °C/5% CO2 and equilibrated for an additional 10 min at room temperature in the dark. To assess the effect of test compounds on glucose-mediated increase in intracellular Ca2+, a volume of 50 μL assay buffer containing 75 mM glucose (resulting in a final concentration of 15 mM glucose) and test compounds or DMSO was added per well during detection on a FLIPR Tetra instrument (molecular devices). For low glucose controls, 50 μL of starvation buffer without additional glucose was added to keep the final glucose concentration at 2.5 mM. Calcium flux was quantified by calculating the area under the curve of fluorescence readings from 3 s to 372 s.

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\ncAMP Stimulation Assay in PSC-HEK293 Cell Line Stably Expressing Human GLP-1R[3]
\nIn vitro cellular assays for GLP-1R agonists and positive allosteric modulators were conducted in 1536-well plates using thaw-and-use frozen cells. Prior to use, frozen cells were thawed quickly at 37 °C and washed (5 min at 900 rpm) with 20 mL of cell buffer (1× HBSS; 20 mM HEPES, 0.1% BSA). Cells were resuspended in assay buffer (cell buffer plus 2 mM IBMX) and adjusted to a cell density of 1 million cells/mL. To a 1536-well microtiter plate, an amount of 2 μL of cells is added (final 2000 cells/well) and 2 μL compound for an agonist assay. For a PAM assay, two assay formats were applied, namely, (a) an enhancer assay with 1 μL of different doses of the compound and 1 μL of a fixed concentration (EC20) of GLP1(9–36)NH2 and (b) a shift assay with 1 μL of different doses of GLP1(9–36)NH2 and 1 μL of 10 μM and 3 μM compound. The mixtures containing 2 μL of each cells and compounds were incubated for 30 min at room temperature.
\nThe cAMP content of cells was determined using a kit from Cisbio Corp. (catalog no. 62AM4PEC) based on HTRF (homogeneous time resolved fluorescence). After addition of HTRF reagents diluted in lysis buffer (kit components), plates were incubated for 1 h, followed by measurement of the fluorescence ratio at 665/620 nm. Dose–response results were calculated with the internal software Biost@t-Speed version 2.0 HTS using a four-parameter logistic model.

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\ncAMP Stimulation Assay in the Human Pancreatic β-Cell Line 1.1B4[3]
\nIn vitro cellular assays of GLP-1(7–36)NH2, GLP1(9–36)NH2, and test compounds were conducted using the human pancreatic β-cell line 1.1B4. Upon GLP-1R activation, the 1.1B4 cells accumulate intracellular cyclic adenosine monophosphate (cAMP). Cyclic AMP formation was measured using a commercial immunoassay technology with HTRF readout. In these experiments, all reagents necessary for quantification of cAMP were supplied in a kit (catalog no. 62AM4PEC from Cisbio Corp., France) and applied according to protocols supplied by the vendor. Two assay formats were applied, namely, (a) an enhancer assay with a compound concentration–response curve and a fixed concentration of 10 nM GLP1(9–36)NH2 and (b) a shift assay with a GLP1(9–36)NH2 concentration–response curve and a fixed concentration of 1 μM compound. 20 000 cells were seeded into a 96-well microtiter plate. Following overnight culturing, cells were washed twice, and serial dilutions of GLP-1R ligand or test compound with or without the respective fixed concentration of either test compound or GLP1(9–36)NH2 were transferred to the cells. After incubation for 30 min with the test agents, the cells were lysed and prepared for cAMP determination according to the manufacturer’s description. Data points were obtained by fluorescence measurement at 665 and 620 nm, calculation of 665/620 nm ratio, and expression in percentage of effect relative to negative (0%) and positive (100%) controls. The negative control was assay buffer (1× HBSS, 0.1% BSA, 1 mM IBMX), and the positive control was GLP-1(7–36)NH2. Concentration–response results were calculated with internal software Biost@t-Speed version 2.0 LTS using a four-parameter logistic model. The adjustment was obtained by nonlinear regression using the Marquardt algorithm in SAS version 9.1.3.

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To assess binding affinity, competition binding experiments were performed using either [¹²⁵I]‑GLP‑1 or a novel radiolabeled small‑molecule probe [³H]‑PF‑06883365 (expected to bind in the same pocket as Danuglipron (PF-06882961)). Membranes from CHO cells expressing human GLP‑1R were incubated with increasing concentrations of unlabeled Danuglipron (PF-06882961) and a fixed concentration of radioligand. The inhibition constant (Ki) was calculated using the Cheng‑Prusoff equation. The Ki of Danuglipron (PF-06882961) was 360 nM against [¹²⁵I]‑GLP‑1 and 80 nM against [³H]‑PF‑06883365. [2]
A cryogenic electron microscopy (cryo‑EM) structure was determined for a close analog (PF‑06883365) bound to human GLP‑1R. The complex was prepared using a stabilized GLP‑1R construct (StaR® technology). The structure revealed that tryptophan 33 (W33) on the extracellular domain moves ~14 Å to close the top of the small‑molecule binding pocket, and arginine 380 (R380) interacts with the carboxylic acid substituent of the agonist. This structural information explained the species selectivity and binding mode. [2]

HEK293 cells that express hGLP-1R fused to green fluorescent protein (GFP) steadily (400,000 cells/well) are grown on 6-well plates for a full day and then stimulated for half a minute with PF-06882961. For these investigations, an agonist concentration of 1 μM that has been shown to cause maximal internalization is used. To test the endocytosis process' reversibility, cells are placed in specific wells, rinsed three times with PBS containing 0.1% BSA, and then incubated for a further two hours at 37 °C. After fixing the cells for 15 minutes at room temperature with 4% paraformaldehyde, the cells are cleaned three times using PBS containing 0.1% BSA.

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For cAMP functional assays, Chinese hamster ovary (CHO) cells stably expressing human GLP‑1R (either high‑density SA line or low‑density CS line) were seeded into 384‑well plates. Cells were incubated with test compound in the presence or absence of 10 μM BETP (positive allosteric modulator) for 30 minutes. cAMP levels were measured using a homogeneous time‑resolved fluorescence (HTRF) kit. Danuglipron (PF-06882961) was tested at various concentrations to generate dose‑response curves and EC50 values. [2]
For β‑arrestin recruitment assays, CHO cells expressing human GLP‑1R and β‑arrestin2‑ or β‑arrestin1‑enzyme acceptor fragment were used (PathHunter technology). Cells were incubated with compound for 90 minutes, and recruitment was detected by chemiluminescence after addition of substrate. EC50 and Emax were calculated. [2]
For receptor internalization studies, HEK293 cells stably expressing a fluorogen‑activated protein (FAP)‑tagged human GLP‑1R were used. Cells were treated with compound for 30 minutes, then internalization was quantified using a FAP‑binding dye and a plate reader. Confocal microscopy was performed on HEK293 cells expressing GFP‑tagged human GLP‑1R; cells were treated with Danuglipron (PF-06882961) for 30 minutes, washed, and imaged after 2 hours of recovery. [2]
To test species selectivity, CHO cells expressing human, monkey, mouse, rat, rabbit GLP‑1R or mutant receptors (mouse S33W, human W33S) were exposed to Danuglipron (PF-06882961) or GLP‑1, and cAMP accumulation was measured. [2]

male cynomolgus monkeys
1 mg/kg, 5 mg/kg, 100 mg/kg
IV, Oral gavage

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Animal Pharmacokinetic Studies[3]
Jugular vein/carotid artery doubly cannulated male Wistar-Han rats (∼250 g), and male cynomolgus monkeys (∼7 kg) were used for these studies. Animals were fasted overnight and through the duration of the study (1.0 or 2.0 h), whereas access to water was provided ad libitum. Danuglipron was administered by slow iv bolus as a solution (1 mg/mL) in a 5:95 (v/v) polyethylene glycol 400/12% (w/v) sulfobutyl-β-cyclodextrin in water mixture or a 10:50:40 (v/v/v) DMSO/polyethylene glycol 400/water mixture via the tail vein in rats (n = 4) or the femoral vein in monkeys (n = 2) at a dose of 1.0 mg/kg in a dosing volume of 1 mL/kg. Serial blood samples were collected before dosing and 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 7.0, and 24 h after dose administration. The crystalline 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) salt form of Danuglipron was also administered by oral gavage to rats (5 and 100 mg/kg at 10 mL/kg) and monkeys [5 mg/kg (5.0 mL/kg) and 100 mg/kg (10 mL/kg)] as a homogeneous suspension in a 2:98 (v/v) Tween 80/0.5% (w/v) methylcellulose A4M in distilled water mixutre. Blood samples were taken prior to po administration, and then serial samples were collected 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h after dosing. Blood samples from the pharmacokinetic studies were centrifuged to generate plasma. All plasma samples were kept frozen until analysis. For rat and monkey samples, aliquots of plasma (20–50 μL) were transferred to 96-well blocks, and acetonitrile (150–200 μL) containing verapamil (monkeys) or terfenadine (rat) as internal standard was added to each well. The supernatant was dried under nitrogen and reconstituted with 100 μL of water without evaporation. Following extraction, the samples were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and concentrations of Danuglipron in plasma were determined by interpolation from a standard curve as described in the Supporting Information.

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Determination of Preclinical Pharmacokinetic Parameters[3]
Pharmacokinetic parameters in animals were determined using noncompartmental analysis. Maximum plasma concentrations (Cmax) of Danuglipron in plasma after po dosing in rats and monkeys were determined directly from the experimental data, with Tmax defined as the time of first occurrence of Cmax. The area under the plasma concentration–time curve from time zero to infinity (AUC0–∞) was estimated using the linear trapezoidal rule. Systemic plasma clearance (CLp) was calculated as the intravenous dose divided by AUC0–∞iv. The terminal rate constant (kel) was calculated by a linear regression of the log-linear concentration–time curve, and the terminal elimination t1/2 was calculated as 0.693/kel. The apparent steady-state distribution volume (Vdss) in animals was determined as the iv or po dose divided by the product of AUC0–∞ and kel. The absolute bioavailability (F) of the po doses in animals was calculated using the equation F = AUC0–∞po/AUC0–∞iv × doseiv/dosepo.Detailed protocols used in animal pharmacology studies are provided in the Supplementary Methods.

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For the mouse intraperitoneal glucose tolerance test (IPGTT), C57BL/6 mice were fasted overnight and then given a subcutaneous injection of Danuglipron (PF-06882961) (10 mg/kg) or liraglutide (0.2 mg/kg) 60 minutes before an intraperitoneal glucose load (2 g/kg). Blood glucose was measured at various time points, and area under the curve (AUC) was calculated. [2]
In cynomolgus monkey studies, animals were fasted overnight. For IV glucose tolerance tests (IVGTT), Danuglipron (PF-06882961) was infused intravenously to achieve target serum concentrations (e.g., 3.0 μM total, 55 nM unbound) or administered orally (doses not specified). Dextrose (250 mg/kg as 50% solution) was injected intravenously, and blood samples were collected for glucose and insulin measurements. The glucose disappearance rate (K‑value) was calculated. For comparison, liraglutide was given subcutaneously. [2]
For food intake studies, monkeys were treated once daily with oral Danuglipron (PF-06882961) or vehicle for 2 days, and food consumption was measured. [2]
For pharmacokinetic studies, Danuglipron (PF-06882961) was administered intravenously (dose not specified) and orally to rats and monkeys (n=2 each) to determine bioavailability. [2]
In the human phase 1 study (NCT03309241), healthy adult participants received single oral doses of Danuglipron (PF-06882961) (3‑300 mg) or placebo in tablet form under fasted conditions (except 100 mg also under fed conditions). Blood samples were collected for pharmacokinetic and glucose analysis. [2]

In rats and monkeys, the oral bioavailability of Danuglipron (PF-06882961) was low to moderate and increased in a dose‑dependent manner. [2]
In healthy human volunteers, after single oral doses of 3 to 300 mg under fasted conditions, plasma exposure (AUCinf and Cmax) increased in an approximately dose‑proportional manner. Mean terminal half‑life (t½) ranged from 4.3 to 6.1 hours. Median time to maximum concentration (Tmax) ranged from 2.0 to 6.0 hours post‑dose. [2]
When administered with food (100 mg dose), exposure (AUCinf) and half‑life were similar to fasted conditions, indicating that food does not significantly affect pharmacokinetics. [2]
Metabolic stability: Danuglipron (PF-06882961) showed low intrinsic clearance (CLint) in human liver microsomes and human hepatocytes (exact values not provided). [2]
Lipophilicity: logD7.4 was not explicitly given for PF‑06882961, but its acid‑containing analogs had logD7.4 as low as 2.3, indicating improved polarity. [2]

In vitro hERG (human ether‑à‑go‑go‑related gene) channel inhibition: IC50 = 4.3 μM for Danuglipron (PF-06882961). [2]
In the phase 1 human study, single doses of 3‑300 mg were generally well‑tolerated. No serious or severe adverse events were reported, and no discontinuations due to adverse events. The most common treatment‑related adverse events were nausea, vomiting, and decreased appetite, consistent with GLP‑1R agonist mechanism. A higher proportion of participants reported adverse events at the 300 mg dose compared to lower doses or placebo. No hypoglycemia events were reported, and all post‑dose glucose levels remained within normal range. [2]
Selectivity: Danuglipron (PF-06882961) showed good selectivity against related class B GPCRs (Table S9, exact values not provided). [2]

[1]. Endocrinol Metab (Seoul). 2021 Feb;36(1):22-29

[2]. bioRxiv. 2020 Sep 30.

[3]. J Med Chem . 2022 Jun 23;65(12):8208-8226.

[4]. J Med Chem. 2020 Mar 12;63(5):2292-2307.

PF-06882961 is being studied in the clinical trial NCT03985293 (a 16-week study designed to evaluate the efficacy and safety of PF-06882961 in adult patients with type 2 diabetes).

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Danuglipron (PF-06882961) is a non‑peptidic, small‑molecule GLP‑1R agonist with a molecular weight of 555.6 g/mol. [1]
It exhibits biased agonism: full agonist for the cAMP/protein kinase A (PKA) pathway and partial agonist for the β‑arrestin pathway (partial activity at recruiting β‑arrestin). [1][2]
The compound requires tryptophan 33 (W33) on the extracellular domain of GLP‑1R for activity, a primate‑specific residue. This explains its lack of activity in rodents and enables species selectivity. [2]
Clinical development: Danuglipron (PF-06882961) has completed a phase 1 first‑in‑human study (NCT03309241) showing safety, tolerability, and glucose‑lowering effects. Further studies are warranted to assess long‑term efficacy on body weight and cardiovascular outcomes. [2]
The cryo‑EM structure of an analog (PF‑06883365) bound to GLP‑1R revealed that the carboxylic acid substituent interacts with arginine 380, and W33 closes the top of the binding pocket, stabilizing the agonist conformation. [2]
Comparison with oral semaglutide: Danuglipron (PF-06882961) does not require a permeation enhancer (SNAC) and can be dosed without regard to food, potentially offering a more convenient oral regimen. [1][2]

C31H30FN5O4

555.599410533905

555.23

C, 67.01; H, 5.44; F, 3.42; N, 12.61; O, 11.52

2230198-02-2

2230198-02-2 (free acid); 2230198-03-3 (tris)

134611040

Off-white to light yellow solid powder

1.4

1

9

9

41

941

1

C1CO[C@@H]1CN2C3=C(C=CC(=C3)C(=O)O)N=C2CN4CCC(CC4)C5=NC(=CC=C5)OCC6=C(C=C(C=C6)C#N)F

HYBAKUMPISVZQP-DEOSSOPVSA-N

InChI=1S/C31H30FN5O4/c32-25-14-20(16-33)4-5-23(25)19-41-30-3-1-2-26(35-30)21-8-11-36(12-9-21)18-29-34-27-7-6-22(31(38)39)15-28(27)37(29)17-24-10-13-40-24/h1-7,14-15,21,24H,8-13,17-19H2,(H,38,39)/t24-/m0/s1

2-[[4-[6-[(4-cyano-2-fluorophenyl)methoxy]pyridin-2-yl]piperidin-1-yl]methyl]-3-[[(2S)-oxetan-2-yl]methyl]benzimidazole-5-carboxylic acid

PF06882961; Danuglipron; PF 06882961; PF-06882961

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)

Ethanol: ~100 mg/mL
DMSO: ~10 mg/mL (~18 mM)

5%DMSO + 40%PEG300 + 5%Tween 80: 5.0mg/ml (9.00mM) (Please use freshly prepared in vivo formulations for optimal results.)