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note
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33P-MeSADP, and serial dilutions of test compounds, vehicle or 300 nM 2-MeSADP for the definition of non-specific binding. Dry compounds were prepared as 10 mM DMSO stocks and were diluted in seven-point, threefold dilution series in assay buffer with 0.02% BSA. Each concentration was run in triplicate beginning at 10 mM, final concentration in the assay. Binding reactions were incubated at room temperature for 1 h and stopped by dilution and transfer/aspiration of the mixture onto GF/B UniFilter 96 Well Plates (Perkin-Elmer), and washed 3× with ice-cold 50 mM Tris, pH 7.4. The filter plates were counted on a Top Count (Perkin-Elmer) and data were analyzed using GraphPad Prism using a single site binding equation.
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30
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71749102677
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note
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50 data was normalized against these two standards to minimize donor-to-donor variability.
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31
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71749099211
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32
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71749106754
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note
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50 value of the functional PRP assay was attributed to protein binding. These compounds showed significantly reduced binding affinity when 0.4% human serum albumin was added to the binding assay.
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33
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71749085692
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note
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Compounds were incubated in human liver and rat liver microsomes for 30 min and the percent remaining was measured. These compounds can be categorized as having good metabolic stability with percent remaining values of >80%.
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34
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71749094978
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note
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Compounds were examined at a single concentration (3 μM) and conducted in duplicate using a minimum of one positive control inhibitor per CYP-specific assay which included CYP1A2, CYP2C9, CYP2D6, and CYP3A4. Incubation time for the assays were 30 min at 37 °C and end-point measurements were used to assess the degree of inhibition. These compounds can be categorized as weak inhibitors with percent inhibition values of <25%.
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35
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71749103483
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note
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Compounds were examined at a single concentration (10 μM) in a 384 well fluorescence polarization assay using Cy3B tagged N-desmethyl dofetilide to competitively bind to HEK-hERG membrane homogenates. Incubation time for the assay was 120 min and end-point measurements were used to assess the degree of inhibition. These compounds can be categorized as weak inhibitors with percent inhibition values of <25%.
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