In Silico Structure-Based Design of a Potent and Selective Small Peptide Inhibitor of Protein Tyrosine Phosphatase 1B,A Novel Therapeutic Target for Obesity and Type 2 Diabetes Mellitus: A Computer Modeling Approach

Abstract

Protein Tyrosine Phosphatase 1B (PTP1B) has been shown to be a negative regulator of insulin signaling by dephosphorylating key tyrosine residues within the regulatory domain of the β-subunit of the insulin receptor. Recent gene knockout studies in mice have shown the mice to have increased insulin sensitivity and improved glucose tolerance. Furthermore, these mice also exhibited a resistance to diet induced obesity. Inhibitors of PTP1B would have the potential of enhancing insulin action by prolonging the phosphorylated state of the insulin receptor. In addition, recent clinical studies have shown that the haplotype ACTTCAG0 of the PTPN1 gene, which encodes PTP1B, is a major risk contributor to type 2 diabetes mellitus (T2DM). Thus, there is compelling evidence that small molecule inhibitors of PTP1B may be effective in treating insulin resistance at an early stage, thereby leading to a prevention strategy for T2DM and obesity.

Based on the crystal structure of the complex of PTP1B with a known inhibitor, we have identified a tetrapeptide inhibitor with the sequence WKPD. Docking calculations indicate that this peptide is as potent as the existing inhibitors. Moreover, the peptide is also found to be selective for PTP1B with a greatly reduced potency against other biologically important protein tyrosine phosphatases such as PTP-LAR, Calcineurin, and the highly homologous T-Cell Protein Tyrosine Phosphatase (TCPTP). Thus the designed tetrapeptide is a suitable lead compound for the development of new drugs against type 2 diabetes and obesity.     

In silico structure-based design of a potent and selective small peptide inhibitor of protein tyrosine phosphatase 1B, a novel therapeutic target for obesity and type 2 diabetes mellitus: a computer modeling approach

Protein Tyrosine Phosphatase 1B (PTP1B) has been shown to be a negative regulator of insulin signaling by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor. Recent gene knockout studies in mice have shown the mice to have increased insulin sensitivity and improved glucose tolerance. Furthermore, these mice also exhibited a resistance to diet induced obesity. Inhibitors of PTP1B would have the potential of enhancing insulin action by prolonging the phosphorylated state of the insulin receptor. In addition, recent clinical studies have shown that the haplotype ACTTCAG0 of the PTPN1 gene, which encodes PTP1B, is a major risk contributor to type 2 diabetes mellitus (T2DM). Thus, there is compelling evidence that small molecule inhibitors of PTP1B may be effective in treating insulin resistance at an early stage, thereby leading to a prevention strategy for T2DM and obesity. Based on the crystal structure of the complex of PTP1B with a known inhibitor, we have identified a tetrapeptide inhibitor with the sequence WKPD. Docking calculations indicate that this peptide is as potent as the existing inhibitors. Moreover, the peptide is also found to be selective for PTP1B with a greatly reduced potency against other biologically important protein tyrosine phosphatases such as PTP-LAR, Calcineurin, and the highly homologous T-Cell Protein Tyrosine Phosphatase (TCPTP). Thus the designed tetrapeptide is a suitable lead compound for the development of new drugs against type 2 diabetes and obesity.     

Involvement of the small protein tyrosine phosphatases TC-PTP and PTP1B in signal transduction and diseases: from diabetes, obesity to cell cycle, and cancer

As in other fields of biomedical research, the use of gene-targeted mice by homologous recombination in embryonic stem cells has provided important findings on the function of several members of the protein tyrosine phosphatase (PTP) family. For instance, the phenotypic characterization of knockout mice has been critical in understanding the sites of action of the related PTPs protein tyrosine phosphatase 1B (PTP1B) and T-cell-PTP (TC-PTP). By their increased insulin sensitivity and insulin receptor hyperphosphorylation, PTP1B null mice demonstrated a clear function for this enzyme as a negative regulator of insulin signaling. As well, TC-PTP has also been recently involved in insulin signaling in vitro. Importantly, the high identity in their amino acid sequences suggests that they must be examined simultaneously as targets of drug development. Indeed, they possess different as well as overlapping substrates, which suggest complementary and overlapping roles of both TC-PTP and PTP1B. Here, we review the function of PTP1B and TC-PTP in diabetes, obesity, and processes related to cancer.     

蛋白质酪氨酸磷酸酶1B（PTP1B）与2型糖尿病及肥胖的关系

蛋白质酪氨酸磷酸酶1B（PTP1B）是一种在体内广泛表达的胞内蛋白质酪氨酸磷酸酶,在调节胰岛素敏感性和能量代谢的过程中起着重要作用。通过抑制PTP1B可增加胰岛素和瘦蛋白（leptin）的活性, 为寻找2型糖尿病、肥胖的治疗提供了光明前景。     

Effects of small interference RNA against PTP1B and TCPTP on insulin signaling pathway in mouse liver: evidence for non-synergetic cooperation

Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Two closely-related protein tyrosine phosphatases, PTP1B and TCPTP both showed abilities to negatively regulate insulin receptor signaling. In order to test whether these two phosphatases can act synergistically, hydrodynamic injection was applied to deliver small interfering RNA (siRNA) of PTP1B and/or TCPTP to mouse liver. By measuring insulin-sensitive reporter gene expression and plasma glucose of diabetic mice, we found siRNA of PTP1B or TCPTP alone can sensitize insulin signal transduction, but combined treatment of both siRNAs had no better effects than siRNA of PTP1B. These results suggested siRNA of PTP1B and TCPTP can strengthen insulin signaling, but their effects do not appear to be synergistic in mouse liver.     

Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/-)) were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/-) mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD)-fed adip-crePTP1B(-/-) mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR) and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α) expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.     

Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake

Protein tyrosine phosphatase 1B (PTP1B) is a key element in the negative regulation of the insulin signaling pathway and may play an important role in diabetes and obesity. We identified ursolic acid, a natural pentacyclic triterpenoid that occurs widely in traditional Chinese medicinal herbs, as an inhibitor of PTP1B by screening an extract library of the traditional Chinese medicinal herbs used a diabetes clinic. By modifying urosolic acid, we designed and synthesized a derivative with a K(i) of 283 nM. As competitive inhibitors of PTP1B, ursolic acid and its derivative also inhibit T-cell protein tyrosine phosphatase and src homology phosphatase-2 but not leucocyte antigen-related phosphatase or protein tyrosine phosphatase alpha and epsilon, which are all possibly involved in the insulin pathway. The ursolic acid derivative enhanced insulin receptor phosphorylation in CHO/hIR cells and stimulate glucose uptake in L6 myotubes.     

Protein-tyrosine phosphatase 1B deficiency reduces insulin resistance and the diabetic phenotype in mice with polygenic insulin resistance

Mice heterozygous for insulin receptor (IR) and IR substrate (IRS)-1 deficiency provide a model of polygenic type 2 diabetes in which early-onset, genetically programmed insulin resistance leads to diabetes. Protein-tyrosine phosphatase 1B (PTP1B) dephosphorylates tyrosine residues in IR and possibly IRS proteins, thereby inhibiting insulin signaling. Mice lacking PTP1B are lean and have increased insulin sensitivity. To determine whether PTP1B can modify polygenic insulin resistance, we crossed PTP1B-/- mice with mice with a double heterozygous deficiency of IR and IRS-1 alleles (DHet). DHet mice weighed slightly less than wild-type mice and exhibited severe insulin resistance and hyperglycemia, with approximately 35% of DHet males developing diabetes by 9-10 weeks of age. Body weight in DHet mice with PTP1B deficiency was similar to that in DHet mice. However, absence of PTP1B in DHet mice markedly improved glucose tolerance and insulin sensitivity at 10-11 weeks of age and reduced the incidence of diabetes and hyperplastic pancreatic islets at 6 months of age. Insulin-stimulated phosphorylation of IR, IRS proteins, Akt/protein kinase B, glycogen synthase kinase 3beta, and p70(S6K) was impaired in DHet mouse muscle and liver and was differentially improved by PTP1B deficiency. In addition, increased phosphoenolpyruvate carboxykinase expression in DHet mouse liver was reversed by PTP1B deficiency. In summary, PTP1B deficiency reduces insulin resistance and hyperglycemia without altering body weight in a model of polygenic type 2 diabetes. Thus, even in the setting of high genetic risk for diabetes, reducing PTP1B is partially protective, further demonstrating its attractiveness as a target for prevention and treatment of type 2 diabetes.     

Berberine inhibits PTP1B activity and mimics insulin action

Type 2 diabetes patients show defects in insulin signal transduction that include lack of insulin receptor, decrease in insulin stimulated receptor tyrosine kinase activity and receptor-mediated phosphorylation of insulin receptor substrates (IRSs). A small molecule that could target insulin signaling would be of significant advantage in the treatment of diabetes. Berberine (BBR) has recently been shown to lower blood glucose levels and to improve insulin resistance in db/db mice partly through the activation of AMP-activated protein kinase (AMPK) signaling and induction of phosphorylation of insulin receptor (IR). However, the underlying mechanism remains largely unknown. Here we report that BBR mimics insulin action by increasing glucose uptake ability by 3T3-L1 adipocytes and L6 myocytes in an insulin-independent manner, inhibiting phosphatase activity of protein tyrosine phosphatase 1B (PTP1B), and increasing phosphorylation of IR, IRS1 and Akt in 3T3-L1 adipocytes. In diabetic mice, BBR lowers hyperglycemia and improves impaired glucose tolerance, but does not increase insulin release and synthesis. The results suggest that BBR represents a different class of anti-hyperglycemic agents.     

Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1B with cellular activities

Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were optimized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with a K(i) of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the derivatives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in L6 myotubes with or addition of without insulin.     

Protein Tyrosine Phosphatase 1B and Insulin Resistance: Role of Endoplasmic Reticulum Stress/Reactive Oxygen Species/Nuclear Factor Kappa B Axis

Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [³H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH₂-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.     

Leptin increases hepatic insulin sensitivity and protein tyrosine phosphatase 1B expression

Leptin has been shown to improve insulin sensitivity and glucose metabolism in obese diabetic ob/ob mice, yet the mechanisms remain poorly defined. We found that 2 d of leptin treatment improved fasting but not postprandial glucose homeostasis, suggesting enhanced hepatic insulin sensitivity. Consistent with this hypothesis, leptin improved in vivo insulin receptor (IR) activation in liver, but not in skeletal muscle or fat. To explore the cellular mechanism by which leptin up-regulates hepatic IR activation, we examined the expression of the protein tyrosine phosphatase PTP1B, recently implicated as an important negative regulator of insulin signaling. Unexpectedly, liver PTP1B protein abundance was increased by leptin to levels similar to lean controls, whereas levels in muscle and fat remained unchanged. The ability of leptin to augment liver IR activation and PTP1B expression was also observed in vitro in human hepatoma cells (HepG2). However, overexpression of PTP1B in HepG2 cells led to diminished insulin-induced IR phosphorylation, supporting the role of PTP1B as a negative regulator of IR activation in hepatocytes. Collectively, our results suggest that leptin acutely improves hepatic insulin sensitivity in vivo with concomitant increases in PTP1B expression possibly serving to counterregulate insulin action and to maintain insulin signaling in proper balance.     

Liver-specific protein-tyrosine phosphatase 1B (PTP1B) re-expression alters glucose homeostasis of PTP1B-/-mice

Protein-tyrosine phosphatase 1B (PTP1B) is an important negative regulator of insulin and leptin signaling in vivo. Mice lacking PTP1B (PTP1B-/- mice) are hyper-responsive to insulin and leptin and resistant to diet-induced obesity. The tissue(s) that mediate these effects of global PTP1B deficiency remain controversial. We exploited the high degree of hepatotropism of adenoviruses to assess the role of PTP1B in the liver. Liver-specific re-expression of PTP1B in PTP1B-/- mice led to marked attenuation of their enhanced insulin sensitivity. This correlated with, and was probably caused by, decreased insulin-stimulated tyrosyl phosphorylation of the insulin receptor (IR) and IR substrate 2-associated phosphatidylinositide 3-kinase activity. Analysis using phospho-specific antibodies for the IR revealed preferential dephosphorylation of Tyr-1162/1163 compared with Tyr-972 by PTP1B in vivo. Our findings show that the liver is a major site of the peripheral action of PTP1B in regulating glucose homeostasis.     

Allosteric inhibition of protein tyrosine phosphatase 1B

Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.     

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.     

Reduction of PTP1B by RNAi upregulates the activity of insulin controlled fatty acid synthase promoter

Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of IR autophosphorylation and thus has been considered as a major therapeutic target for the treatment of type II diabetes. We use RNA interference technique to downregulate PTP1B expression in hepatoma cell line. A secretory HBV s-antigen was introduced as reporter and driven by mouse fatty acid synthase promoter, which is positively controlled by insulin signaling. Liver-targeted hydrodynamic injection in tail vein was introduced to transfer siRNA (or siRNA expression vector) and reporter plasmid into mouse liver. On fasted/refed and glucose stimulation condition, the HBV s-antigen in sera in RNAi group was higher than that in the negative group. Our results provided evidence that upregulation of insulin signaling by reducing PTP1B liver with RNAi can be a potent diabetes treatment method.     

Coordinated regulation of insulin signaling by the protein tyrosine phosphatases PTP1B and TCPTP

The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP-/- and PTP1B-/- immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR beta-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B-/- MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP-/- MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B-/- MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell.     

Essential role of protein tyrosine phosphatase 1B in obesity-induced inflammation and peripheral insulin resistance during aging

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes (T2DM). In this study, we have evaluated the role of PTP1B in the development of aging-associated obesity, inflammation, and peripheral insulin resistance by assessing metabolic parameters at 3 and 16 months in PTP1B(-/-) mice maintained on mixed genetic background (C57Bl/6J × 129Sv/J). Whereas fat mass and adipocyte size were increased in wild-type control mice at 16 months, these parameters did not change with aging in PTP1B(-/-) mice. Increased levels of pro-inflammatory cytokines, crown-like structures, and hypoxia-inducible factor (HIF)-1α were observed only in adipose tissue from 16-month-old wild-type mice. Similarly, islet hyperplasia and hyperinsulinemia were observed in wild-type mice with aging-associated obesity, but not in PTP1B(-/-) animals. Leanness in 16-month-old PTP1B(-/-) mice was associated with increased energy expenditure. Whole-body insulin sensitivity decreased in 16-month-old control mice; however, studies with the hyperinsulinemic-euglycemic clamp revealed that PTP1B deficiency prevented this obesity-related decreased peripheral insulin sensitivity. At a molecular level, PTP1B expression and enzymatic activity were up-regulated in liver and muscle of 16-month-old wild-type mice as were the activation of stress kinases and the expression of p53. Conversely, insulin receptor-mediated Akt/Foxo1 signaling was attenuated in these aged control mice. Collectively, these data implicate PTP1B in the development of inflammation and insulin resistance associated with obesity during aging and suggest that inhibition of this phosphatase by therapeutic strategies might protect against age-dependent T2DM.     

Inhibition of PTP1B by Trodusquemine (MSI‐1436) Causes Fat‐specific Weight Loss in Diet‐induced Obese Mice

Trodusquemine (MSI‐1436) causes rapid and reversible weight loss in genetic models of obesity. To better predict the potential effects of trodusquemine in the clinic, we investigated the effects of trodusquemine treatment in a murine model of diet‐induced obesity (DIO). Trodusquemine suppressed appetite, reduced body weight (BW) in a fat‐specific manner, and improved plasma insulin and leptin levels in mice. Screening assays revealed that trodusquemine selectively inhibited protein‐tyrosine phosphatase 1B (PTP1B), a key enzyme regulating insulin and leptin signaling. Trodusquemine significantly enhanced insulin‐stimulated tyrosine phosphorylation of insulin receptor (IR) β and STAT3, direct targets of PTP1B, in HepG2 cells in vitro and/or hypothalamic tissue in vivo. These data establish trodusquemine as an effective central and peripheral PTP1B inhibitor with the potential to elicit noncachectic fat‐specific weight loss and improve insulin and leptin levels.