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Inhibition of mTOR kinase was assayed using full-length mTOR (extracted from HeLa cytoplasmic extract by immunoprecipitation) with a recombinant 4E-BP1 protein substrate and an ATP concentration of 50 μM. Detection of phosphorylated product through an ELISA format was essentially as described in Ref. 8 Figures quoted are the mean of at least 2 determinations and variability around the mean was <50%.
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mTOR Kinase inhibition experiments were conducted at increasing concentrations of inhibitor (1) (50, 100, 200, 350, 500 nM) in varying concentrations of ATP (10-100 μM). The data was plotted using a Lineweaver-Burke double reciprocal plot and revealed characteristic behaviour of competitive inhibition.
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mTOR Kinase inhibition experiments were conducted at increasing concentrations of inhibitor (1) (50, 100, 200, 350, 500 nM) in varying concentrations of ATP (10-100 μM). The data was plotted using a Lineweaver-Burke double reciprocal plot and revealed characteristic behaviour of competitive inhibition.
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Monolayers of MDCKII (Madin Darby Canine Kidney Cells) cells transfected with the human MDR1 (Multi-Drug Resistance 1) transporter protein were cultured in transwells and used to study the permeability and efflux potential of compounds (17) and (19) at a concentration of 10 μM. Since neither compound showed significant levels of efflux in this assay, the Papp in the A to B direction is considered to provide a measure of the passive permeability of the compounds in these cells.
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