Indeed, even the endogenous substrate of PTPMT1 in the ��-cell is still being investigated because, in spite of the homology of its catalytic motif to that of PTEN and its ability to selleck Tubacin use phospholipid substrates in vitro (Pagliarini et al., 2004), such activity has not yet been shown in cells (Pagliarini et al., 2005). Thus, to facilitate further study of PTPMT1 and its role in ��-cell metabolism in particular, we undertook a search for inhibitors of the enzyme. There is good precedence for the use of small-molecule inhibitors of phosphatases in the interrogation of the biology of these enzymes, and selective inhibitors of phosphatases may well prove valuable in the treatment of diseases affected by their dysregulation (Lai et al., 2009).

Because the absence of a crystal structure for PTPMT1 limited the applicability of rational drug design, we adopted an unbiased screen of diverse chemical structures as the best approach toward identifying an inhibitor of the enzyme. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective inhibitor of PTPMT1. Kinetic studies suggested that alexidine dihydrochloride bound cooperatively and inhibited PTPMT1 in a predominantly uncompetitive manner. In isolated rat pancreatic islets, alexidine dihydrochloride induced insulin secretion in a dose-dependent manner, whereas in a pancreatic ��-cell line it affected the mitochondrial phosphoprotein profile, thus phenocopying the effect of knockdown of cellular expression of PTPMT1.

Taken together, these studies not only demonstrate the ability of alexidine dihydrochloride to inhibit PTPMT1 and induce increased insulin secretion, thus supporting the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes, but they also support the use of alexidine dihydrochloride as a tool to facilitate further study of PTPMT1. Materials and Methods Materials. Recombinant VHR (Vaccinia virus VH1-related phosphatase), PTEN, and PTPMT1 were prepared as described previously (Denu et al., 1995; Maehama and Dixon, 1998; Pagliarini et al., 2004). T-cell PTP and �� protein phosphatase and accompanying buffers were purchased from New England Biolabs (Ipswich, MA). Alexidine dihydrochloride was purchased from Toronto Research Chemicals Inc.

(North York, ON, Canada), and chlorhexidine dihydrochloride, phenformin, metformin, and 3-O-methylfluorescein phosphate cyclohexyl ammonium salt were purchased from Sigma-Aldrich Entinostat (St. Louis, MO). N-(2-ethylhexyl)-N��-proplyimidodicarbonimidic diamide (half alexidine) was synthesized by the Duke Center for Chemical Biology (Durham, NC) with purity of the final product (approximately 95%) being determined by mass spectrometry and NMR elemental analysis. PTPMT1-targeted shRNA adenovirus was prepared as described previously (Pagliarini et al., 2005).