Incubation of PTPMT1 selleck kinase inhibitor with alexidine dihydrochloride resulted in reductions in both Vmax and Km with increasing inhibitor concentration (Fig. 3), suggesting that alexidine did not inhibit PTPMT1 in purely competitive or noncompetitive manners. In addition, fitting of the enzyme activity in the presence of varying concentrations of the inhibitor to models for competitive, noncompetitive, uncompetitive, and mixed inhibition revealed that the data fit the uncompetitive and mixed inhibition models best (Fig. 3; Table 1). Using GraphPad Prism 5.0 to fit the data to a model for mixed inhibition, which is a general one incorporating competitive, noncompetitive and uncompetitive inhibition, gave a value of �� = 0.097 �� 0.071, suggesting significant uncompetitive character. Fig. 3.

Alexidine dihydrochloride is a predominantly uncompetitive inhibitor of PTPMT1. A, PTPMT1 phosphatase activity in the presence of varying concentrations of alexidine dihydrochloride using O-MFP as … TABLE 1 Comparison of the fit of the data for inhibition of PTPMT1 by alexidine dihydrochloride to various kinetic models Alexidine Dihydrochloride Stimulates Insulin Secretion from Isolated Rat Pancreatic Islets and Affects Protein Phosphorylation in a Pancreatic ��-Cell Line in a Fashion Similar to Genetic Inhibition of PTPMT1. Having determined that alexidine dihydrochloride was an effective chemical inhibitor of PTPMT1 in vitro, we were keen to explore whether the compound would inhibit PTPMT1 in cells.

We were hopeful of this in part due to PTPMT1 being resident on the matrix-facing side of the inner mitochondrial membrane, and lipophilic dications (a structural class of which alexidine dihydrochloride is a member) have previously been shown to target the mitochondria, their hydrophobicity allowing them to cross the membranes while their positive charge encourages accumulation in the matrix (Ross et al., 2006; Murphy and Smith, 2007). As the role of PTPMT1 had previously been studied in the pancreatic ��-cell, and genetic perturbation had been shown to affect insulin secretion, we used this system as our model. Because lipophilic dications have also been reported to show cellular toxicity at high concentrations (Severina et al., 2007) and most previous studies of alexidine dihydrochloride have focused on its antimicrobial and cytotoxic effects (Gilbert and Moore, 2005; Yip et al.

, 2006), we first ensured that the assays were performed at concentrations that did not affect cell viability. Through use of a cell membrane integrity-based cytotoxicity assay, we found that alexidine dihydrochloride did not significantly affect membrane permeability and viability of the cells of pancreatic islets at concentrations up to 4 ��M, hence this was the maximum concentration Cilengitide used in the assays.