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| | In Vitro Biochemical Evaluation |
| Description: | The in vitro biochemical evaluation of all compounds was carried out using recombinant human PTP4A3, overexpressed as a His6-tag fusion protein in E. coli and purified on a metal affinity column. Assays were performed using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as an artificial substrate at 25° C. for 30 min in 40 mM Tris-HCl (pH 7.0), 75 mM NaCl, 2 mM EDTA, and 4 mM DTT buffer. The reaction was carried out in 45 μL total volume per well of a black 384-well plate and initiated upon addition of DiFMUP at a final concentration of 12 μM (3× the Km of PTP4A3 for DiFMUP, to ensure that the reaction velocity remained constant throughout the assay) to each well containing 1 μg of full-length protein. The fluorescence was measured using a SpectraMax M5 plate reader at 358 nm excitation and 455 nm emission. Fluorescence values were used to calculate the percent inhibition of enzyme activity relative to maximal activity, PTP4A3 in the absence of inhibitor, and maximal inhibition, PTP4A3 in the presence of 2 mM Na3VO4. |
| Affinity data for this assay | |
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