BMS-819881

Kinase experiment:

Membranes from stably transfected HEK-293 cells expressing a mutated (E4Q, A5T) hMCHR1 receptor are prepared and differential centrifugation. Binding experiments are carried out with 0.5-1.0 μg of membrane protein incubated in a total of 0.2 mL in 25 mM HEPES (pH 7.4) with 10 mM MgCl2, 2 mM EGTA, and 0.1% BSA (binding buffer) for 90 min. For competition binding assays, reactions are carried out in the presence of 0.06–0.1 nM [Phe13, [125I]Tyr19]MCH and increasing concentrations of unlabeled test molecules. Reactions are terminated by rapid vacuum filtration over 96-well GFC Unifilter plates precoated with 0.075 mL of binding buffer containing 1% BSA and washed 3 times with 0.4 mL of PBS (pH 7.4) containing 0.01% TX-100. Filters are dried, 0.05 mL of MicroScint 20 is added to each well, and radioactivity is subsequently quantified by scintillation counting on a TopCount microplate scintillation counter. Inhibitory constants are determined by nonlinear least-squares analysis using a four-parameter logistic equation[1].

Cell experiment:

Stable HEK-293 cells expressing human MCHR1 or MCHR2 receptor are plated at a density of 50 000 cells/well in 96-well polylysine coated plates and cultured overnight in DMEM (high glucose (4.5 g/mL), 25 mM HEPES, pH 7.4, 10% fetal bovine serum, 1 mM NaCl) at 37°C, 5% CO2 conditions. For assay, the medium is replaced with 90 mL per well dye solution consisting of 3.8 mM Fluo4 AM, 0.04% Pluronic F-127, and 2.5 mM Probencid in base buffer (Hank’s balanced salt solution, 25 mM HEPES, 0.1% BSA). Dye solution is allowed to “load” for 1 h at room temperature in subdued light. Dye is subsequently removed and replaced with 75 mL of base buffer and 75 mL of diluted test compound (e.g., BMS-819881; 10 μM) and incubated for an additional 15 min. Test compound dilution plates are prepared by serial diluting test and reference compounds from 100% DMSO stocks first 1:50 in base buffer and then serially (1:3.26) in base buffer containing 2% DMSO to generate 12 half log test concentrations[1].

Animal experiment:

Rats, Dogs, and Cynomologous monkeys [1]PK studies using three species (rat, dog, and cynomologous monkey) are conducted with BMS-819881 administered iv at 1 mg/kg[1].

References:

[1]. Washburn WN, et al. Identification of a nonbasic melanin hormone receptor 1 antagonist as an antiobesity clinical candidate. J Med Chem. 2014 Sep 25;57(18):7509-22.