Highly Selective Protein Tyrosine Phosphatase Inhibitor, 2,2â²,3,3

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Highly selective protein tyrosine phosphatase inhibitor, 2, 2’, 3, 3’-tetrabromo-4, 4’, 5, 5’-tetrahydroxydiphenylmethane, ameliorates type 2 diabetes mellitus in BKS db mice Chao Li, Jiao Luo, Shuju Guo, Xiaoling Jia, Chuanlong Guo, Xiangqian Li, Qi Xu, and Dayong Shi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01106 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 14, 2019

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Molecular Pharmaceutics

Highly selective protein tyrosine phosphatase inhibitor, 2, 2’, 3, 3’-tetrabromo-4, 4’, 5, 5’-tetrahydroxydiphenylmethane, ameliorates type 2 diabetes mellitus in BKS db mice Chao Li, †,‡,§,

∥

Jiao Luo, †,‡,§,

∥

Shuju Guo, †,‡,§ Xiaoling Jia, †,‡,§ Chuanlong Guo, †,‡,§,

Xiangqian Li, †,‡,§ Qi Xu, †,‡,§, †CAS

∥

and Dayong Shi *,†,‡,§,

∥

∥, #

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese

Academy of Sciences, Qingdao 266071, China; ‡Laboratory

for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine

Science and Technology, Qingdao 266237, China; §Center

for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao,

266071, P. R. China; ∥University #State

of Chinese Academy of Sciences, Beijing, China

Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

ABSTRACT: Protein tyrosine phosphatase 1B (PTP1B) is a widely confirmed target of the Type 2 Diabetes mellitus (T2DM) treatment. Herein, we reported a highly specific PTP1B inhibitor 2, 2’, 3, 3’-tetrabromo-4, 4’, 5, 5’-tetrahydroxydiphenylmethane (compound 1) which showed promising hypoglycemic activity in diabetic BKS db mice. With the IC50 value of 2.4 μM, compound 1 could directly bind to the catalytic pocket of PTP1B through a series of hydrogen bonds. SPR analysis revealed that the target affinity [KD (equilibrium dissociation constant) value] of compound 1 binding to PTP1B was 2.90 μM. Moreover, compound 1 could activate the insulin signaling pathway in C2C12 skeletal muscle cells. We further evaluated the long-term effects of compound 1 in diabetic BKS db mice. Notably, oral administration of compound 1 significantly reduced the blood glucose levels of diabetic mice with increasing insulin sensitivity. In addition, the dyslipidemia of diabetic mice was also significantly improved by compound 1 gavage. The histological experiments showed that compound 1 treatment significantly ameliorated the

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disordered hepatic and pancreatic architecture and increased the glycogen content in the liver tissues as well as improved the insulin secretion function of pancreas. Taken together, our results manifested that the natural product compound 1 was a highly specific PTP1B inhibitor which could activate insulin signaling pathway and ameliorate hyperglycemia and dyslipidemia in diabetic BKS db mice. KEYWORDS: Type 2 Diabetes mellitus, Protein tyrosine phosphatase 1B, Insulin resistance, Hypoglycemic activity, Dyslipidaemia

INTRODUCTION Type 2 diabetes mellitus, a severe epidemic which primarily caused by inadequate insulin secretion and insulin resistance, is featured by hyperglycemia and hyperinsulinemia.1 It is estimated that about 425 million people with diabetes worldwide in 2017, with T2DM making up approximate 90% of the cases. And 12% of global health expenditure, 727 billion dollars, is spent on diabetes. Notably, it is predicted that the number of people suffering from diabetes would rise to 629 million by 2045.2 Confronted with the alarming worldwide growth in the incidence of type 2 diabetes mellitus, developing effective treatment and precaution measures is increasingly urgent.3 At present, several antidiabetic drugs such as metformin (Met), a classic drug that promote insulin secretion, coupled with sulfonylurea and non-sulfonylurea drugs have been used to prevent type 2 diabetes. In addition, other compounds are also available in T2DM treatment including peroxisome proliferator activated receptor-γ (PPARγ) activators, dipeptidyl peptidase-4 (DPP-4) inhibitors and glucogan-like peptide-1 (GLP-1) analogs. Although the mentioned drugs above are all significant for type 2 diabetes therapy, certain side-effects associated with the use of them inevitably exist such as hypoglycemia, weight gain, flatulence, congestive heart failure, abdominal discomfort and acute pancreatitis.4-7 Therefore, there has been an increasingly growing interest in exploring novel antidiabetic drugs with content efficacy and slight even no side-effect for type 2 diabetes treatment and precaution. Protein tyrosine phosphatase 1B (PTP1B), a ubiquitously expressed protein, has been reported as a potential therapeutic target for T2DM as it negatively regulates insulin signal transduction by


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dephosphorylating insulin receptor-β (IRβ) and insulin receptor substrate (IRS) on tyrosine residues.8-10 In the insulin signaling pathway, IRS as a member of the insulin receptor substrate family, it plays a significant role in insulin signal transduction. Without the dephosphorylation of PTP1B, the phosphorylated IRS would serve as an adaptor protein which recruits phosphatidylinositol 3-kinase (PI3K) by the regulatory subunit. Then PI3K would stimulate the activity of protein kinase B (Akt or PKB) via catalyzing the phosphorylation of a critical serine and threonine residue of it. Subsequently, the phosphorylated Akt promotes the translocation of intracellular glucose transporter 4 (Glut-4), leading to increased glucose intake of the skeletal muscle cells, which plays a vital role in glucose metabolism homeostasis. Furthermore, early cell culturing studies reported that overexpression of PTP1B inhibited phosphorylation of IR and IRS leading to insulin resistance.11, 12 And the T2DM animal models treated with a PTP1B-specific antisense oligonucleotide possessed improved hyperglycemia and insulin sensitivity. In addition, increased tyrosine phosphorylation of the IR in the muscle and liver were observed in PTP1B knockout mice, and PTP1B-deficient mice showed increase insulin sensitivity as well as altered glucose homeostasis.13-15 On the basis of these findings, the inhibition of PTP1B has emerged as a potential therapeutic strategy to treat T2DM, and endeavors on developing effective PTP1B inhibitors are underway. However, it is intractable to discover clinically available PTP1B inhibitors due to their low cell permeability, limited inhibitor selectivity and poor bioavailability.16, 17

Compound 1: 2’, 3, 3’-tetrabromo-4, 4’, 5, 5’-tetrahydroxydiphenylmethane, a natural PTP1B inhibitor which previously isolated from the red alga Rhodomela confervoides in our lab, exhibited potent PTP1B inhibitory activity with the IC50 value of 2.4 μM.18, 19 In order to further investigate the antidiabetic effect of compound 1 and the underlying mechanism, the hypoglycemic profiles with related characters of it were evaluated both in vitro and in vivo. EXPERIMENTAL SECTION Materials. Compound 1 (molecular formula C13H8Br4O4, molecular weight 547.8190) was isolated and identified in our lab with the purity of 95 %. The chemical structure of compound 1 is shown in Figure 1A. Human PTP1B1-321 (hPTP1B1-321) was expressed and purified in our lab.20 Biacore T200, Sensor Chips Series S CM5, and relevant regents EDC, NHS and


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ethanolamine HCl (pH 8.5) were obtained from GE Healthcare Life Sciences. Recombinant human leucocyte common antigen-related (LAR) PTP, T-cell protein tyrosine phosphatase (TCPTP), tyrosine-protein phosphatase non-receptor type 11 (SHP2), insulin and Met were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from PAN (Adenbach, Bavaria, Germany). DMEM, penicillin-streptomycin and horse serum (HS) were bought from Hyclone (Logan, Utah, USA). 4-nitrophenyl phosphate disodium salt (pNPP) was purchased from Solarbio (Beijing, China). The BCA Protein Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China). IRS1 antibody, IRβ (L55B10) mouse mAb,， anti-Akt rabbit mAb and phosphor-Akt (Ser473) (D9E) XP rabbit mAb were bought from Cell Signaling Technology (Danvers, MA, USA), while the antibody of p-IRβ (Tyr1185) were purchased from Abcam (Cambridge, UK). The antibody directed against p-IRS1 (Tyr608) and PVDF membrane were bought from Milipore (Bedford, MA, USA). The PTP1B antibody, all secondary antibodies and anti-β-actin mouse monoclonal IgG were purchased from Proteintech Group (Wuhan, Hubei, China). PierceTM ECL western Blotting Substrate was obtained from Thermo Scientific (Waltham, MA, USA). The kits for the measurement of glycated serum protein (GSP), triglyceride (TG), total cholesterol (TC), non-esterified fatty acid (NEFA), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were purchased from Nanjing Jianchen Bioengineering Institute (Nanjing, Jiangsu, China). The protease inhibitor cocktail as well as fast blood glucose meter and blood glucose test strips were bought from Roche (Basel, Switzerland). Determination in PTP1B inhibition mode of compound 1. The substrate used to measure the activity of PTP1B was 4-nitrophenyl phosphate disodium salt (p-NPP).21 After compound 1 pre-incubated with recombinant hPTP1B1-321 at room temperature for 5 min, the experiment was performed in a final volume of 100 μL reaction system containing 10 mM Tris-Hcl (pH 7.5), 25mM NaCl and 1 mM EDTA in a 96 well plate at 37 °C for 10 min. Then the amount of pnitrophenol generated was measured by reading the absorbance value at 405 nm in a microplate reader (Tecan, Mannedorf, Switzerland). On the basis of the intersection characteristics of approximate straight lines obtained, the inhibitory mode of compound 1 was determined by Lineweaver-Burk analysis. In order to evaluate the specificity of compound 1 to PTP1B, enzyme assay was conducted with p-NPP as a substrate to determine the IC50 values of the compound


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against other typical protein tyrosine phosphatases (PTPs) which includes SHP2, LAR and TCPTP due to their high degree of sequential similarity with PTP1B.22 SPR assay. The binding kinetics research were developed by surface plasmon resonance (SPR) experiments. Human PTP1B1-321 at final concentration of 50 μg/mL was dissolved in 10 mM sodium buffer (pH 5.0), followed by immobilizing the protein to CM5 chips employing amine coupling procedure.23 The running buffer was Phosphate-buffered saline (PBS) adding 0.05 % P20. Momently, adopting a 10 μL/min flow rate, the surface of the flow cell was activated employing an equal volume mixture of 200 mM N-ethyl-N’-(dimethylaminopropyl)-carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). 1 M ethanolamine (pH 8.5) were used to block excess reactive esters on the sensor chip surface. The final immobilization level was about 13000 response units (RU). Flow cell used for reference was activated and blocked as described above but reminded uncoupled. Binding was expressed as relative RU defined as the response gained from the flow cell which contained the immobilized receptor minus the response obtained from the reference flow cell. Simulation of molecular docking. Based on the previously published method,24 the molecular docking simulation studies were performed by means of the SYBYL-X 2.0 software and the crystal structure of PTP1B (PDB code: 2HNP), with the molecular structure of compound 1 drown by using the standard parameters of SYBYL-X. Applying the Tripos force field for 1000 steps, their geometric conformations were energy minimized. In addition, Gasteriger-Huckel charges were calculated. Then the protein receptor was prepared by clearing the ligand and all the water molecules. The radius of active site was set equal to 5 Å. With the standard default settings, compound 1 was docked into the PTP1B protein model employing SYBYL-X 2.0. Cell culture. The mouse myoblast cell (C2C12) lines in our researches were purchased from COBIOER BIOSCIENCES (Nanjing, Jiangsu, China). Cells were cultured in DMEM (4500 mg/L glucose) supplemented with 10 % fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 units/mL streptomycin at 37 °C incubator with 5 % CO2.12 For C2C12 cells differentiation, the composition of 10 % FBS was replaced by 10 % horse serum after the cells reaching confluency.25 The differentiation medium was replaced every 48 h, and the myotubes were prepared for the subsequent experiments 4 days after cell differentiation.


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Cytotoxicity test of compound 1. C2C12 cells were seeded in 96 well plates with the cell density of 5×103 per well. Cells were starved in serum-free DMEM medium for 24 h when they reached confluence and were completely differentiated to myotubes. The myotubes subsequently grew in serum-free DMEM culture medium in the presence of compound 1 at a series of concentrations. After starvation, the culture medium was removed from the plates and myotubes were incubated with serum-free DMEM medium containing MTT at final concentration of 0.5 mg/mL for 4 h. The medium was discarded and the produced formazan crystals were dissolved by adding to each well 150 μL DMSO. The cell viability was determined on the basis of the absorbance at 570 nm detected from the microplate reader (Tecan, Mannedorf, Switzerland). Target validation of compound 1. Drug affinity responsive target stability (DARTS) experiment was performed and it is a relatively straightforward approach to identify potential targets for small molecules.26 The differentiated C2C12 myotubes were lysed with an appropriate volume of M-PER mammalian protein extraction lysis buffer containing protease inhibitor cocktail. The total protein was extracted and the concentration was determined using BCA kits, followed by incubation with compound 1 of different concentration for 30 min in room temperature. Then appropriate volume of pronase was added to digest the protein for 20 min by the end of mixing with appropriate amount of protease inhibitor cocktail to stop each digestion reaction. The samples were stored at -20 °C for western blot test.27 Effects of compound 1 on C2C12 myotubes. Compound 1 were dissolved in DMSO at the concentration of 10 mM for use. Differentiated C2C12 myotubes grew in a 6 well plate were starved in serum-free DMEM medium for 24 h before compound treatment. After replacing with fresh serum-free medium, the myotubes were exposed to varying concentrations of compound 1. After 8 h of treatment, the insulin with the final concentration of 100 nM was added to treat the myotubes for 5 min. Then the cells lysates were collected and the proteins were extracted. Animal experiments. All studies in mice were approved by IOCAS (Institute of Oceanology, Chinese Academy of Sciences) Laboratory Animal Care and Ethics Committee in accordance with the animal care and use guidelines. Male BKS.Cg-Dock7m+/+Leprdb/J mice of and the lean C57BLKS/J (BKS, the Jackson Laboratory stock number 000662) wild type controls (6-7 weeks of age) were purchased from the Model Animal Research Center of Nanjing University (MARC). All mice were fed a normal rodent chow diet and housed in isolated ventilated cages which were


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in a standard pathogen-free room with the control temperature of 24 ± 2 °C and humidity of 50 %, a condition of 12-h light/12-h dark cycle as well as ad libitum access to diet and water. One week of environmental acclimation was given to all mice before the experiments. After that, the blood glucose levels of all the mice were measured. Then, the diabetic mice were randomly divided into four groups (n=8): model control group (BKS db), Met-treated group (Met), high-dose compound 1-treated group (compound 1 (H)) and low-dose compound 1-treated group (compound 1 (L)). The male BKS mice of the same age (BKS) were fed as the normal control. The groups of BKS db and BKS were given 0.5 % CMC-Na (vehicle), while the Met-treated, high-dose compound 1-treated and low-dose compound 1-treated mice were respectively given Met-0.5 % CMC-Na (100 mg/kg/day Met), compound 1-0.5 % CMC-Na (100 mg/kg/day compound 1) and compound 1-0.5 % CMC-Na (50 mg/kg/day compound 1) by gavage administration (10 mL/kg body weight). Fasting plasma glucose levels were evaluated from tail blood by using fast blood glucose meters once a week. And the weight, food consumption and water intake of all the mice were also assessed every week. Three-week of gavage administration later, an insulin tolerance test (ITT) was carried out. After 6 weeks of feeding, the mice were executed by decapitation after 15 min of the insulin injection and the blood samples were drawn from orbits. The animal tissues were dissected and quickly frozen in liquid N2 for subsequent experiments. And the abdominal fat of all groups of mice was weighed. The indicators related to blood glucose and lipid such as glycated serum protein (GSP), triglyceride (TG), total cholesterol (TC), non-esterified fatty acid (NEFA), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) of the serum separated from the blood samples were determined using corresponding kits. Histological and immunohistochemical analysis. Pieces of liver, pancreas, heart and kidney of the mice were fixed in formalin fixation fluid and embedded in paraffin. Paraffin sections of the tissues with the thickness of 5 μm were prepared for subsequent analysis. Morphological changes were observed by staining with haematoxylin and eosin (H&E). Immunohistochemical studies were performed to identify the changes of glycogen content and insulin secretion function of different mice. Periodic acid-Schiff (PAS) stain was used to identify the changes of glycogen content in liver tissues in different mice. For insulin immunochemical analysis of the pancreas, the sections were incubated overnight at 4 °C with insulin Antibody #4590 (1:100, Cell Signaling Technology, Beverly, MA, USA).


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Western blot analysis. For cells protein western blot, the myotubes were rinsed twice with precooled PBS after compounds treatments. Ice-cold RIPA cell lysis buffer containing refresh phenylmethanesulfonyl fluoride (PMSF) were used to lyse the cell. The tissue protein of mice was extracted using the Tissue or Cell Total Protein Extraction Kit from Sangon Biotech (Shanghai, China). Using the BCA Protein Assay Kit, the concentration of the total protein was measured. The protein obtained was separated on SDS polyacrylamide gels, and then transferred onto PVDF membranes. After transfer, the membranes obtained were blocked in 5% defatted milk which was dissolved in TBST for 1 h at room temperature, followed by incubation with the primary antibodies at 4 °C overnight. The membranes were rinsing with TBST for three times and incubated with suitable secondary antibodies for 1 h at room temperature the following day. After washing the membranes with TBST for three times again, the bands were detected by employing the chemiluminescence method and using PierceTM ECL Western Blotting Substrate. Statistical analysis. The results are expressed as the mean ± SEM. Statistical differences between the model and drug-treated groups were determined using the unpaired two-tailed Student’s t test by comparing with the control group and P ＜ 0.05 was considered statistically significant for all the analyses. The software GraphPad Prism 7.0 was used for all statistical evaluations. RESULTS Compound 1 is a competitive and highly specific PTP1B inhibitor. First, the inhibitory effect of compound 1 on the phosphatase activity of PTP1B was determined. The result showed that the lines intersected at the y-axis and the Km values increased in a dose-dependent manner with constant Vmax value, the Lineweaver-Burk plot in the Figure 1B demonstrated that compound 1 was a competitive inhibitor of PTP1B. In addition, the IC50 values of compound 1 on PTPs including TCPTP, LAR and SHP2 were determined and the data was displayed in Table 1. The IC50 value of compound 1 against PTP1B was 2.4 μM consulted our previous results, while the values to TCPTP, SHP2 and LAR were respectively 43.25, 167.88 and more than 350 μM. To explore whether compound 1 could directly interact with PTP1B, SPR biosensor-based technology was applied to evaluate the binding kinetics. As shown in Figure 1C, compound 1 was


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showed significant binding ability to PTP1B with the KD value of 2.90 μM, while a blank injection of buffer did not show this kind of binding. In addition, the precise predictions of the protein-small molecule interaction geometries of PTP1B and compound 1 were obtained by the molecular docking studies. As shown in Figure 1D, compound 1 could embed into the catalytic pocket of PTP1B. Furthermore, the compound was able to form five hydrogen bonds with the four amino acid residues, including Asp181, Lys116, Ala217 and Ser218, of PTP1B (Figure 1E), which play an important role in consolidating the ligandreceptor protein interaction between compound 1 and PTP1B. DARTS detection by western blotting showed that the protein exhibited more strong ability to resist the lysis effect of pronase after incubating with compound 1 (Figure 1F, G), which indirectly confirmed that compound 1 could bind to PTP1B and induce increased resistance to pronase. Table 1. The IC50 values of compound 1 against PTPs

a

PTPs

IC50 values (μM)

PTP1B

2.4

TCPTP

43.25±4.02

SHP2

167.88±36.65

LAR

＞350

IC50 values, half maximal inhibitory concentration of compound 1 against the PTPs.


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Figure 1. Enzymatic effects and interaction of compound 1 on PTP1B. (A) The chemical structure of compound 1. (B) Lineweaver-Burk analysis of the inhibitory model of compound 1 on PTP1B. The reciprocal of initial reaction rate (1/[V]) was plotted against the reciprocal of p-NPP concentrations (1/[p-NPP]) in the presence of a series of concentrations of compound 1. (C) The interaction between compound 1 and PTP1B. Compound 1 concentrations of 12.50, 6.25, 3.13 and 1.56 μM in PBSP containing 5 % DMSO were injected over a CM5 sensor chip surface which was immobilized with PTP1B. Binding was measured by a flow rate of 30 μL·min-1 for 60 s and the response units (RU) were corrected to a reference flow cell. (D) Surface representation and (E)


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stick representation of predicted binding models of compound 1 with PTP1B (PDB code: 2HNP). Hydrogen bonds are depicted as green dot lines. (F) Western blotting of PTP1B after digested by pronase. The protein samples were extracted from the lysis of C2C12 myotubes. (G) Quantification of PTP1B in DARTS western blotting. Band density was quantified and then normalized to the control (pronase digest without incubating with compound 1) signal. Data are expressed as mean ± SEM (n = 3). ** p < 0.01 and *** p < 0.001 vs. non-insulin-treated groups. Cellular effects of compound 1. MTT assay was performed to determine the cell toxicity of compound 1 to C2C12 myotubes. After C2C12 myotubes treated with a series of concentration of compound 1 (range from 1.2 to 40 μM), the cell viability was measured using MTT assay. Varying concentration of compound 1 had no significant effect on the viability of the C2C12 cells, indicating that compound 1 was almost non-toxic to C2C12 cells (Figure 2A). Coordinated tyrosine phosphorylation is essential in insulin signaling pathway.28 To determine whether compound 1 could improve insulin signaling in C2C12 myotubes, the phosphorylation levels of IRβ, IRS1 and Akt were examined via western blotting. As shown in Figure 2B-E, activation of insulin signaling was observed in the cells treated with compound 1 at the final concentration of 1 and 5 μM for the significant increase of the phosphorylation levels of IRβ, IRS1 and Akt.


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Figure 2. Cellular effects of compound 1 in C2C12 myotubes. (A) Relative cell viability of C2C12 myotubes. Cells were incubated with compound 1 for 24 h, and then the cell viability was measured by MTT method. The viability of vehicle treated cells was normalized as 100 %. (B) compound 1 activated insulin signaling in C2C12 myotubes during exposure of C2C12 to compound 1. pAkt (Ser473), pIRβ (Tyr1185) and pIRS1(Tyr608) as well as the corresponding total protein were detected by western blotting. (C) Quantification of Akt phosphorylation. Band density was quantified and then normalized to a total Akt signal. (D) Quantification of IRβ phosphorylation. Band density was quantified and then normalized to a total IRβ signal. (E) Quantification of IRS1 phosphorylation. Band density was quantified and then normalized to a total IRS1 signal. Data are expressed as mean ± SEM (n = 3). * p

Highly Selective Protein Tyrosine Phosphatase Inhibitor, 2,2â²,3,3

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