| Assay Method Information | |
| | Phosphatase (PTPase) Assay |
| Description: | First, the GST fusion of the PTP domains of SHP2, SHP1, and PTP1B were produced and purified as reported by us previously.32 The reactions were performed in a buffer containing 10 mM Tris-HCl (pH 7.2), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.01% Tween-20. Briefly, CNBCA or CNBBA was added first in a serial dilution ranging from 100 μM to 97 nM followed by the enzymes (SHP2, SHP1, or PTP1B) to a final concentration of 1 nM. After 5 min of incubation at room temperature, the reactions were started by adding the artificial substrate DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) to a final concentration of 20 μM for SHP2, 35 μM for SHP1, and 10 μM for PTP1B in a reaction volume of 100 μL; variations in DiFMUP concentrations reflect the reported Km values for the respective PTPs.33 The complete mixture was incubated at 37° C. for 20 min, and fluorescence intensity was measured by the Synergy 4 plate reader at the excitation and emission wavelengths of 360 and 460 nm, respectively. The half inhibitory concentration (IC50) value of each compound was calculated and plotted using Graphpad Prism software. The results showed inhibition of the SHP2 enzyme activity by CNBCA with an IC50 of 0.87 μM and by CNBBA with an IC50 of 5.1 μM (Table 1). While CNBCA showed significant improvement in potency over that of the parent compound (0.87 μM versus 5.0 μM), CNBBA exhibited similar potency as that of the parent compound (5.1 μM versus 5.0 μM). Comparative fold difference calculations showed that CNBCA is better than the parent compound CNBDA and the other new compound (CNBBA) by 5.7 fold and 5.8 fold, respectively. |
| Affinity data for this assay | |
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