MBX-2982

Kinase experiment:

HEK-GPR119 cells are transfected with GloSensor 22F plasmid and used for dynamic cAMP measurements 24-30 h later. Cell suspensions are made by dislodging the cells using PBS wash and Accutase treatment followed by resuspension in culture media. Cells are then washed twice by pelleting through centrifugation (300g, 5 min) and resuspension in assay buffer (Hank's Balanced Salt Solution supplemented with 20 mM HEPES and 0.01% fatty acid free BSA, pH 7.4). Cells are then counted and diluted to 600,000 cells/mL in buffer, before GloSensor cAMP reagent is added (2% v/v) and equilibrated with the cells for 2 h at 20°C with periodic mixing. 50 µl/well of cells are added to white-bottomed 384 well plates (30,000 cells/well) in triplicate and baseline luminescence is measuring using an Envision plate-reader. 5 μL of MBX-2982 (serially diluted in DMSO and then diluted 1:100 in assay buffer to obtain ×10 concentrated solution) is manually added to the assay wells to achieve the stated final concentration. Plates are incubated at 20°C with luminescence read at regular intervals to detect dynamic cAMP changes over time within the same wells. cAMP responses at each time-point are expressed as fold over control (vehicle-treated cells)[1].

Cell experiment:

HEK-GPR119 cells are grown to confluency in flasks, and cell suspensions are made by dislodging cells using PBS wash and accutase treatment followed by resuspension in culture media. Cells are then washed twice by pelleting through centrifugation (227g, 7 min, 20°C) and resuspension in warm assay buffer (Hank's Balanced Salt Solution supplemented with 20 mM HEPES and 0.01% fatty acid free BSA, pH 7.4), with a 5 min incubation at 37°C after the second wash. Cells are then counted and diluted to 200,000 cells/mL in warm assay buffer[1].

Animal experiment:

Mice[2] C57BL/6 male mice are used. Overnight fasted, 10 week-old male mice (n=20 per group) are given either vehicle (15% polyethylene glycol 400+85% of 23.5% hydroxypropyl-β-cyclodextrin) or MBX-2982 at 10 mg/kg via oral gavage. Half of the animals (n=10 per group) are killed by CO2 asphyxiation 30 min after compound dosing, and blood is collected by cardiac puncture. To preserve active GLP-1, a DPP-IV inhibitor (10 µL per 1 mL of blood) is pre-added to the blood collection tubes and, before the cardiac puncture, the walls of the syringes are rinsed with the DPP-IV inhibitor. The other half of the animals (n=10 per group) received a bolus of oral glucose (3 g/kg) 30 min after compound dosing, and are killed for blood collection 10 min after the glucose load. GLP-1 levels in the plasma samples are measured using the active GLP-1 (ver 2) kit.

References:

[1]. Hothersall JD, et al. Sustained wash-resistant receptor activation responses of GPR119 agonists. Eur J Pharmacol. 2015 Sep 5;762:430-42.
[2]. Lan H, et al. Agonists at GPR119 mediate secretion of GLP-1 from mouse enteroendocrine cells through glucose-independent pathways. Br J Pharmacol. 2012 Apr;165(8):2799-807.
[3]. Yang JW, et al. GPR119: a promising target for nonalcoholic fatty liver disease. FASEB J. 2016 Jan;30(1):324-35.