Pharmacological targeting of the mitochondrial phosphatase PTPMT1

Abstract

The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 microM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic beta-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes.

Fig. 1.

Alexidine dihydrochloride is a selective inhibitor of PTPMT1. A, structure of alexidine dihydrochloride. B, inhibition of selected phosphatases by alexidine dihydrochloride. PTPMT1, VHR phosphatase, λ-Ppase, and T-cell PTPase assays were carried out with O-MFP as the substrate (n = 3), and PTEN assays were carried out with lipid substrate (n = 2). All assays were carried out by using optimum buffer and pH for each enzyme. The IC₅₀ with PTPMT1 was 1.08 μM ± 0.08 with a Hill coefficient of 2.16 ± 0.31. Data are presented as the mean ± S.E.M. of independent experiments. C, comparison of the sequences of the catalytic motif of the protein tyrosine phosphatases assayed. Boxed residues are those conserved within the catalytic motif.

Fig. 2.

The dibiguanide structure of alexidine dihydrochloride is important for its inhibition of PTPMT1. A, inhibition of PTPMT1 by various biguanide and dibiguanide compounds using O-MFP as the substrate. Inhibition by alexidine dihydrochloride yielded IC₅₀ of 1.08 ± 0.08 with Hill coefficient of 2.16 ± 0.31, whereas inhibition by chlorhexidine dihydrochloride yielded IC₅₀ of 19.7 ± 3.3 with Hill coefficient of 1.3 ± 0.3, and inhibition by the half alexidine molecule yielded IC₅₀ of 207.1 ± 43.1 with Hill coefficient of 0.7 ± 0.1. Data represent the mean ± S.E.M. of three independent experiments. B, structures of dibiguanide and biguanide compounds tested for inhibition of PTPMT1.

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 substrate. Alexidine dihydrochloride reduced both V_max and K_m. Data represent mean ± S.E.M. of three independent experiments. B, Lineweaver–Burk plot of the data presented in A.

Fig. 4.

Alexidine dihydrochloride induces a dose-dependent increase in insulin secretion from rat islets. Rat islets were treated with the indicated concentrations of alexidine dihydrochloride, first in the presence of buffer containing basal (2.8 mM) glucose concentration and subsequently in the presence of buffer containing stimulatory (16.7 mM) glucose concentration. The alexidine dihydrochloride-induced dose-dependent increase in insulin secretion (bar graph plotted on the left y-axis), was significant at both basal and stimulatory glucose concentrations. At both basal and stimulatory glucose concentrations the effect of 4 μM alexidine dihydrochloride was significant compared with untreated controls. Data represent the mean ± S.E.M. of six independent experiments. Data were analyzed by ANOVA with post hoc Dunnett test for significance. A cell membrane integrity-based cytotoxicity assay, using adenylate kinase release from cells as a measure of increased cytotoxicity, was used alongside the insulin secretion assay. The alexidine dihydrochloride-induced cytotoxicity (line graph plotted on the right y-axis) did not exceed 4% and was not significant. Data represent the mean ± S.E.M. of four independent experiments.

Fig. 5.

Alexidine dihydrochloride treatment phenocopies the effect of knockdown of PTPMT1 on threonine phosphorylation of mitochondrial proteins. A, representative immunoblot showing the impact of alexidine dihydrochloride on the threonine phosphorylation of mitochondrial proteins. INS-1 cells were treated with PTPMT1-targeted shRNA (48 h) or alexidine dihydrochloride (2 h). Threonine phosphorylation of proteins in the mitochondria-enriched fraction of INS-1 cell lysate was then assessed by immunoblot using a phosphothreonine antibody. Both alexidine dihydrochloride and knockdown of PTPMT1 increased threonine phosphorylation of an 80-kDa protein and decreased threonine phosphorylation of a 90-, 55-, and 45-kDa protein (indicated by arrows). B, quantification of relative amounts of PTPMT1 and threonine-phosphorylated 90-, 80-, 55-, and 45-kDa proteins in the mitochondrial enriched fraction of INS-1 cell lysate after shRNA-mediated knockdown of PTPMT1 protein levels or treatment of cells with alexidine dihydrochloride. Protein was quantified from immunoblots like the representative one shown in A. The amount of each protein of interest was quantified relative to the amount of VDAC protein in the mitochondria-enriched cell lysate and normalized to the amount of that particular protein in the lysate from cells treated with the control shRNA. Data represent mean values ± S.E.M. from four independent experiments. Data were analyzed by ANOVA with post hoc Dunnett test for significance.

Fig. 6.

Stimulation of insulin secretion by alexidine dihydrochloride depends on the presence of PTPMT1. A, representative immunoblot analysis of the level of knockdown of PTPMT1 achieved with PTPMT1-targeted shRNA in islets after 72 h. Typical knockdown of PTPMT1 was 45 to 60% of the endogenous protein level. B, impact of alexidine dihydrochloride on insulin secretion from islets in which PTPMT1 expression is reduced. Rat islets were treated with PTPMT1-targeted shRNA or a control shRNA (72 h). Islets were then treated with 4 μM alexidine dihydrochloride for 1 h, first in the presence of basal (2.8 mM) glucose concentration, then in the presence of stimulatory (16.7 mM) glucose concentration. In rat islets in which the level of PTPMT1 expression was unperturbed, the effect of alexidine dihydrochloride on insulin secretion was significant at both basal and stimulatory glucose concentrations; however, when the level of PTPMT1 expression was reduced, the effect of alexidine dihydrochloride was no longer significant. Data represent the mean ± S.E.M. of six independent experiments and were analyzed using ANOVA with the Bonferroni test applied post hoc.