His structure from the protein data bank and verified its utility by molecularly docking a phosphotyrosine peptide into the catalytically active D1 domain. We hypothesized that the active site could be exploited in the MCB-613 development of competitive inhibitors targeted to PTPs. To this end, we used the ZINC database to virtually screen a library of compounds for their ability to dock into the D1 domain of PTPs. From the top scoring compounds which were most favorably bound by the active site, we identified three compounds which represented structurally distinct scaffolds and demonstrated an ability to inhibit PTPs activity in preliminary in vitro assays. To expand these into a set of compounds for biochemical investigation, we performed a substructure search and retrieved 74 additional molecules similar to these three scaffolds from the ChemBridge compound library. This entire collection of molecules, along with the established pan-PTP inhibitor sodium orthovanadate, were analyzed for their ability to inhibit PTPs phosphatase activity in vitro. To measure the catalytic activity of PTPs in vitro, we utilized the chromogenic phosphatase substrate, para-nitrophenyl phosphate. The dephosphorylated product para-nitrophenol, yields an intense yellow color under alkaline conditions measurable at 405 nm absorbance on a spectrophotometer. We generated recombinant PTPs and determined an amount that yielded linear pNP formation during the course of the phosphatase reaction while producing a maximal signal at least five-fold above background. We then used initial velocities measured across a series of pNPP substrate concentrations to calculate the Km of PTPs. The Km of PTPs was determined to be 250 mM. When analyzing competitive inhibition, the mode of Digitoxin inhibition predicted for molecules binding the D1 active site, it is critical to use a substrate concentration at or below the Km. Accordingly, we used a pNPP substrate concentration less than 250 mM for inhibitor studies. To profile the inhibition of PTPs conferred by compounds, we pre-incubated recombinant PTPs with each compound for 30 minutes, then initiated phosphatase reactions with the addition of pNPP for an additional 30 minutes. We identified 25 active compounds which inhibited PTPs activity by 90 or more, a potency s
