Christoph Grundner (University of Washington) for generously providing em m /em PTPA protein and plasmid, Dr

Christoph Grundner (University of Washington) for generously providing em m /em PTPA protein and plasmid, Dr. resulted in increased phagosomeClysosome fusion and activity. Notably, because cell wall and drug efflux mechanisms. Our lab previously identified active site directed reversible inhibitor 1 with single-digit micromolar inhibition of 0.05; ** 0.01 one-way ANOVA, Dunnetts = 3C5 in each group). Following evaluation of GSH-exchange and inhibitor potency against purified enzyme, prodrug inhibitors 9 and 10 were evaluated for their ability to inhibit STEP in rat cortical neurons. Inhibition was evaluated by monitoring phosphorylation levels of known STEP substrates pNR2B, pPyk2, and pERK.50?54 Statistically significant increases in the phosphorylation levels of all three STEP substrates were observed upon treatment with 9 (Figure ?Figure44c), while inhibitor 10 showed more modest increases in phosphorylation levels (data not shown). Finally, in preliminary studies for future in vivo evaluation, 9 was determined to have a reasonable 73% stability to rat plasma over 1 h as well as good stability to rat liver microsomes (77% and 82% remaining after 1 h with and without NADPH).55 Inhibitor Synthesis As a general approach toward the preparation of the selenosulfide PTP inhibitors described in this article, appropriately functionalized alkyl halide precursors 12 can be transformed EC1167 to the corresponding selenate esters 13 (Scheme 1). Using a procedure developed by Zhang, Knapp, and co-workers, oxidation of the selenate esters 13 with dimethyl dioxirane (DMDO) results in the seleninic acids 14.21 Treatment of seleninic acids 14 with an excess of the desired thiol then provides the selenosulfide prodrug inhibitors 15. An advantageous aspect of this approach is that the physicochemical-modulating thiol component of an inhibitor is installed in the final step of the sequence. This enables rapid preparation of inhibitors with a range of thiol physicochemical modulators appended to a given inhibitor core. Open in a separate window Scheme 1 General Approach Towards the Preparation of Selenosulfide Inhibitors Conclusions A GSH-responsive prodrug strategy has been developed to facilitate the efficient intracellular delivery of a novel class of selenosulfide PTP inhibitors. As an initial exploration into this prodrug strategy, we EC1167 have developed inhibitors for two PTPs: the virulence factor em m /em PTPA and the brain-specific tyrosine phosphatase STEP. The lead molecules described in this work enable potent and selective inhibition of em m /em PTPA and STEP, respectively. We have further characterized the expected covalently modification resulting from selenosulfide inhibitor treatment by mass spectrometry and have demonstrated cellular activity. These studies demonstrate the feasibility of the selenosulfide prodrug approach, EC1167 which potentially could be applied to many other PTPs. Acknowledgments We thank Dr. Christoph Grundner (University of Washington) for generously providing em m /em PTPA protein and plasmid, Dr. Haya Jamali CDK4 (Yale University) for assistance with the expression and purification of STEP, and Dr. Rebecca Wissner (Yale University) for assistance with protein mass spectrometry experiments. The authors gratefully acknowledge the support of the NIH (R35GM122473). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.7b00486. All synthetic procedures, characterization, analytical data, reversibility of inhibition by dialysis and thiols, details on generation of mutant em m /em PTPA, mass spectrometry experiments, rate of GSH-exchange of selenosulfide 11, cell culture and Western blotting. (PDF) Author Contributions C.C.T. and K.D.O. contributed equally to this work. Notes The authors declare no competing financial interest. Supplementary Material oc7b00486_si_001.pdf(3.0M, pdf).