LY2608204

GK

559, 81

Male Wistar rats at a weight of 225-250 g

LY2608204 is suspended in a 1:1 mixture of solutol/ethanol in a bath sonicator (10% of total volume). The obtained suspension is then diluted with 9 volumes of 10% aqueous solutol solution.

LY2608204 activates glucokinase (GK) with EC50 of 42 nM at 10 mM glucose with a concentration dependent manner at lower glucose concentrations. LY2608204 also stimulates glucose metabolism in rat insulinoma INS1-E cells with EC50 of 579 nM. [1]

Glucokinase assay, The human islet GK isoform is expressed in E. coli as (His)6-tagged fusion protein and purified with metal chelate affinity chromatography. After purification the enzyme is stored in aliquots at concentration 0.8 mg/mL in 25 mM sodium phosphate, 150 mM sodium chloride, 100 mM imidazole, 1 mM dithiothreitol, 50% glycerol at 80 C. The assay is performed in flat bottom 96-well plates in a final incubation volume of 100 uL. The incubation mixture consists of 25 mM HEPES (pH7.4), 50 mM potassiumchloride, 2.5 mM magnesiumchloride, 2 mM dithiothreitol, 4 U/mL glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, 5 mM ATP, 1 mM NAD and a set concentration of glucose. LY2608204 is dissolved in DMSO and then added to the reaction mixture giving the final DMSO concentration of 10%. The reaction is initiated by addition of 20 uL GK and runs for 20 min at 37 C. The amount of formed NADH is measured as an increase in absorbance at 340 nm using a microplate reader. Absorbance values are used for EC50 calculations.

LY2608204 activates glucokinase (GK) with EC50 of 42 nM. Phase 2.