MK-3328

Kinase experiment:

[3H]-DMAB is synthesized at a specific activity of ~80 Ci/mmol. The final concentration of radioligand for tissue homogenate binding assay is 1.5nM. Brain homogenates are diluted with PBS to 0.4 mg/mL from original 10 mg/mL volume and 200 μL is used in assay for a final concentration of 50 μg/assay tube. Unlabeled test compounds are dissolved in DMSO at 1 mM. Dilution of test compound (e.g., MK-3328) to various concentrations is made with PBS containing 2% DMSO. Total binding is defined in the absence of competing compound, and non-displaceable binding is determined in the presence of 1 μM unlabeled self block. Compound dilutions (10×) are added into the assay tube (25 μL each/per tube, separately) containing 200 μL brain homogenate dilution, and the tubes are pre-incubated at room temperature for 10 minutes. Then radioligand dilutions (10×) are added into the assay tube (25 μL each/per tube, separately) to a final volume of 250 μL per tube. Incubation is carried out at room temperature (25°C) for 90 minutes, and then the assay samples are filtered onto GF/C filters using Skatron 12 well harvester, washing on setting 5-5-5 (~ 3×2 mL) ice cold buffer (PBS, pH 7.4). GF/C filter papers for the Skatron harvester are pre-soaked in 0.1% BSA for 1 hour at room temperature before use. Filters are punched into scintillation vials and counted in 2 mL Ultima Gold on Perkin Elmer Tri-Carb 2900TR for 1 minute. The data analysis is done with Prism software. All assays are done in triplicate, and in the laboratory designated for studies using human tissues[1].

References:

[1]. Harrison ST, et al. Synthesis and Evaluation of 5-Fluoro-2-aryloxazolo[5,4-b]pyridines as β-Amyloid PET Ligands and Identification of MK-3328. ACS Med Chem Lett. 2011 Apr 18;2(7):498-502.