Phosphatase catalytic activity was measured by monitoring dephosphorylation of p-Nitrophenyl phosphate (pNPP). All assays were performed in Tris buffer containing 50 mM Tris adjusted to an ionic strength of 150 mM with NaCl at pH 7.5. All measurements were taken at 30 ºC. Each enzyme was diluted to 250 nM in Tris buffer. Enzymes were pretreated with compound: for each enzyme, an aliquot (2.2 µL) of each compound in DMSO stock was dispensed into a well of a 96-well plate. A DMSO group was included to serve as an uninhibited control. Subsequently, enzyme solution (217.8 µL, final DMSO concentration: 1% v/v) was dispensed and mixed in each well. Similarly, a well was prepared with DMSO and Tris buffer without enzyme to serve as a substrate-only control. At 1, 2, and 4 hour time points, the pretreated enzyme solutions were aliquot (20 µL) in triplicate to a 96-well plate, and pNPP solution (180 µL) was added to each well. pNPP solution was prepared fresh with 100 mM DTT, and 0.1% (w/v) BSA in Tris buffer for a final pNPP concentration of 20mM during measurement. For CD148 only, this solution was diluted 10x for a final pNPP concentration of 2 mM. Absorbance measurements at 405nm were acquired every 30 seconds for 10 min in a microplate reader. The data were fit to a linear regression to obtain an initial rate from the slope, and the slope of the substrate-only control was subtracted from each group. The residual activity of each enzyme after incubation with compound was calculated from the slope of the compound-treated group as a percentage of the slope of the uninhibited group. pNPP dephosphorylation assays were conducted against each enzyme for each compound across 6 concentrations (3.3 µM, 10 µM, 33 µM, 100 µM, 330 µM, 1 mM). The IC50 value for the inhibition of phosphatase activity was determined by fitting the plot of residual enzyme activity against the inhibitor concentrations using a four-parameter dose-response nonlinear regression in GraphPad Prism. Analysis of KI and kinact for phosphatase inhibition using a colorimetric assay Phosphatase catalytic activity was measured using p-nitrophenyl phosphate (pNPP) as described above. Enzymes were treated with compound in DMSO stock (2.4µL stock in 237.6µL enzyme, final DMSO concentration: 1% v/v). Concentration ranges were selected per compound to cover the range of measurable inhibition in a 1 hour period. The percent inhibition was determined as described above at each time point and the inhibition over time was fit to a first-order rate equation at each inhibitor concentration to obtain an observed first-order rate constant, kobs. Six time points were collected over the course of 1 hour. The double reciprocal plot of kobs versus inhibitor concentration was then fit to the following equation to obtain KI and kinact: