Discovery of Orally Bioavailable Purine-Based Inhibitors of the Low-Molecular-Weight Protein Tyrosine Phosphatase

Abstract

Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.

Figure 1.

Chemical structure of previously reported LMPTP inhibitor compd 23 (1).

Figure 2.

Chemical structures of purine-based LMPTP inhibitors

Figure 3.

Analog 5d inhibits LMPTP with an uncompetitive MOA. (a–b) Activity of 20 nM human LMPTP-A on increasing concentrations of OMFP in the presence of increasing concentrations of 5d. (a) Mean±SEM reaction rate vs. OMFP concentration is shown. Lines show fitting to the Michaelis-Menten equation with 95% confidence interval. Mean±SEM K_i’ and α are shown. (b) Lineweaver-Burk plot of data from (a). Lines show fitting to a linear regression, (c-d) Mean±SEM K_m (c) and V_max (d) values for each concentration of inhibitor from the Michaelis-Menten curves in (a) are shown, (e) IC₅₀ values were calculated for 5d on 20 nM human LMPTP-A-catalyzed hydrolysis of increasing concentrations of OMFP. Mean±SEM IC₅₀ from 3 independent experiments performed in triplicate is shown. Lines show fitting to the one-phase decay equation with 95% confidence interval shown, (a-e) Data from 3 independent experiments performed in triplicate is shown.

Figure 4.

Crystal structure of 5d with human LMPTP. Model of 5d in the substrate binding site of LMPTP. The ligand is shown in light blue. The protein is shown as solvent accessible surface and stick representation (gray). Only one conformation of the o-tolyl group is shown for clarity. The figure was generated using Chimera. PDB: 7KH8.

Figure 5.

Selectivity of analog 6g. (a-b) PTPs were incubated with (a) 0.4 mM 3-O-methylfluorescein phosphate (OMFP) or (b) 5 mM para-nitro-phenylphosphate (pNPP) in the presence of dimethyl sulfoxide (DMSO) or 40 μM 6g. Mean±SEM % activity of PTPs incubated with inhibitors compared to DMSO is shown. Dotted line indicates 50% activity. Data from 2 independent experiments perfonned in triplicate is shown.

Figure 6.

Compd 6g augments insulin-stimulated AKT phosphorylation in hepatocytes. HepG2 hepatocytes were incubated overnight with dimethyl sulfoxide (DMSO) or 500 nM 6g solubilized in DMSO and stimulated with 10 nM insulin for 5 min or left unstimulated (Unstim). Left, representative Western blots (cropped) of phospho-AKT (pAKT) Thr308 from HepG2 cell homogenates. Right, quantification of pAKT Thr308/AKT and pAKT Thr308/GAPDH from 8 independent experiments. Mean±SEM is shown. *, p<0.05. two-tailed unpaired t-test with Welch’s correction.

Figure 7.

Oral administration of 6g reverses diabetes and enhances liver insulin signaling in obese mice. Diet-induced obese (DIO) male B6 litter-mate mice were treated with 0.03% w/w 6g in high-fat diet or high-fat diet alone for 2 weeks, (a) Intraperitoneal glucose tolerance test (IPGTT). (b) Fasting plasma insulin levels of mice as assessed by ELISA, (c-d) Mice were injected intraperitoneally with insulin and livers harvested after 10 min. (c) IR tyrosine phosphorylation in liver homogenates was assessed by phospho-IR (pIR) ELISA, (d) pAKT Thr308 was assessed by Western blotting of liver homogenates. Left, representative Western blots (cropped). Right, quantification of pAKT Thr308/AKT. (a-d) HFD. n=8; HFD + 6g, n=9. Mean±SEM is shown. *, p<0.05: (a) two-way ANOVA, (b) Mann-Whitney test, (c-d) two-tailed unpaired t-test with Welch’s correction.

Scheme 1.

Representative synthesis of purine compounds ^aReagents and conditions: (a) Br₂. H₂O, rt, 60%; (b) K₂CO₃, DMF, rt, 36%; (c) Pd(dppf)Cl₂, dioxane/H₂O, 90 °C, 22-55%