Diuron → Benzimidazole

Membranes from CHO cells expressing the MCH receptor were prepared by lysing cells with 5 mM HEPES for 15 min at 4C. Cell lysates were centrifuged (12.5000×g, 15 min) and the pellet was resuspended in 5 mM HEPES. For each 96-well plate (Microlite, Dynex Technologies), 1 mg of cell membranes were incubated with 10 mg of wheat germ agglutinin SPA beads (Amersham) for 5 min at 4 C in a volume of 10 ml of binding buffer (25 mM HEPES, 10 mM MGCl2, 10 mM NaCl, 5 mM MnCl2, 0.1% BSA). The membrane/bead mixture was centrifuged (1500×g, 3.5 min), the supernatant was aspirated, and the pellet was resuspended in 10 ml binding buffer. The centrifugation, aspiration and resuspension were then repeated. The membrane/bead mixture (100 μl) was then added to 96-well plates containing 50 μl of 500 pM [125I]-MCH (NEN) and 50 ml of the appropriate concentration of compound (4× the desired final concentration). Nonspecific binding was determined by including 1 μM MCH in the binding reaction. The binding reaction was incubated at room temperature for 2 h. Plates were then analyzed in a TOPCOUNT microplate scintillation counter (Packard). Data was analyzed and Ki values were determined using GraphPad Prism.

1. customwere prepared
2. customfor 15 min
3. custom(12.5000×g, 15 min)
4. customfor 5 min
5. custom(1500×g, 3.5 min)
6. additioncontaining
7. concentration50 μl of 500 pM [125I]-MCH (NEN) and 50 ml of the appropriate concentration of compound (4× the desired final
8. concentrationconcentration)
9. customin the binding reaction
10. customThe binding reaction

ORD record

ord-931b01742a32446eb694fb362358701e

Source dataset

uspto-grants-2009_03

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07511146B2