Letters

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Abbreviations !

Ki: pAkt: p-NPP: PTP1B: SHP-1:

dissociation constant phosphorylated Akt para-nitrophenylphosphate protein tyrosine phosphatase 1B Src homology domain 2-containing protein tyrosine phosphatase 1 Src homology domain 2-containing protein tyrosine phosphatase 2 selective index T cell protein tyrosine phosphatase vaccinia H1-related phosphatase

Tatsunori Sasaki 1, Wei Li 1, Koji Higai 1, Tran Hong Quang 2, Young Ho Kim 2, Kazuo Koike 1 1 Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Japan 2 College of Pharmacy, Chungnam National University, Daejeon, Korea

SHP-2:

Abstract

PTP1B is a non-transmembrane protein tyrosine phosphatase that is expressed ubiquitously in the classical insulin-targeted tissues and plays critical roles in negatively regulating both insulin and leptin signaling cascades [1, 2]. Pharmacological studies have shown that PTP1B knockout mice exhibited increased insulin sensitivity and obesity resistance [3]. These findings have therefore led to an intense interest in developing PTP1B inhibitors as potential therapies for diabetes and obesity [4, 5]. The use of natural products has received much attention as an approach to the discovery of novel PTP1B inhibitors because of their advantage in large structure diversity [6]. As part of our ongoing studies on the discovery of novel PTP1B inhibitors in natural products [7, 8], herein we report the discovery of five lavandulyl flavonoids from the roots of Sophora flavescens Ait. (Leguminosae) as new PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2′" Fig. 1). The inhibimethoxykurarinone (4), and kushenol T (5) (l tion mechanism and selectivity as well as the cellular activities of these compounds in the insulin signaling pathway are also reported. A small compound library consisting of 82 natural compounds was evaluated for their inhibitory activities against PTP1B at a final concentration of 100 µM. A known PTP1B inhibitor, RK-682, was used as the positive control (IC50 4.49 µM) in this bioassay. As a result, five lavandulyl flavonoids, kuraridin (1), norkurarinone (2), kurarinone (3), 2′-methoxykurarinone (4), and kushenol T (5), from the roots of S. flavescens [9] produced more than 80 % inhibition. These compounds inhibited PTP1B in a concen-

!

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and much attention has been paid to protein tyrosine phosphatase 1B inhibitors as potential therapies for diabetes. The screening of a natural compound library led to the discovery of five lavandulyl flavonoids, which were isolated from the roots of Sophora flavescens, as novel PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2′-methoxykurarinone (4), and kushenol T (5). The three most potent compounds, 1, 2, and 4 (IC50 < 30 µM), were demonstrated to be noncompetitive inhibitors of protein tyrosine phosphatase 1B based on a kinetic analysis, and they exhibited different inhibitory selectivities against four homologous protein tyrosine phosphatases (T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2). Compounds 1, 2, and 4 also exhibited cellular activity in the insulin signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, suggesting their potential for new anti-insulin-resistant drug developments.

Key words Sophora flavescens · Leguminosae · lavandulyl flavonoids · protein tyrosine phosphatase 1B · insulin signaling pathway

SI: TCPTP: VHR:

Fig. 1 Five protein tyrosine phosphatase 1B inhibitory flavonoids.

Sasaki T et al. Protein Tyrosine Phosphatase …

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Protein Tyrosine Phosphatase 1B Inhibitory Activity of Lavandulyl Flavonoids from Roots of Sophora flavescens

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Table 1 Inhibition effects of compounds 1–5 and RK-682 against protein tyrosine phosphatase 1B.

a

Sample

IC50 (µM)a

Inhibition mode

Ki (µM)

1 2 3 4 5 RK-682b

16.86 ± 0.29 28.94 ± 0.23 41.68 ± 0.11 5.26 ± 0.24 49.59 ± 0.16 4.49 ± 0.15

Non-competitive Non-competitive

6.53 14.85

Non-competitive

3.15

Values are mean ± SD from three separate experiments. Nine concentration points

were set for establishment of the inhibition curve of PTP1B to calculate the IC50 value. b

Positive control

tration-dependent manner, and their IC50 values were obtained " Table 1). Among the five, flavanone 4 by regression analyses (l was the most potent. The C-2′ methoxy moiety in compound 4 seems to play an important role in the activity, which is evidenced by the greatly decreased activity produced by demethylated compound 3. Kuraridin (1), norkurarinone (2), and 2′-methoxykurarinone (4), which showed more potent inhibitory activity (IC50 < 30 µM), were chosen for further evaluation. Their inhibition mechanisms

were elucidated by kinetics analysis with various concentrations of compounds and the substrate p-NPP. Lineweaver-Burk plots suggested that these compounds inhibited PTP1B activity by a " Fig. 2). The secondary plots were noncompetitive mechanism (l created by the slopes from the Lineweaver-Burk plots on the yaxis against the concentration of the compounds on the x-axis, showing a good linear relationship, and the Ki values of compounds 1, 2, and 4 were calculated as 6.53, 14.85, and 3.15 µM, respectively. Because of the high structural similarity of the catalytic center among the family of protein tyrosine phosphatases [10], the inhibitory selectivity of kuraridin (1), norkurarinone (2), and 2′methoxykurarinone (4) were evaluated by comparison of their inhibitory activity against PTP1B and four homologous protein tyrosine phosphatases, TCPTP, VHR, SHP-1, and SHP-2. These " Table 2). Among compounds exhibited SI values of 2.2–7.5 (l them, kuraridin (1) possessed better inhibitory selectivity in tested compounds because its SI values between PTP1B and all four PTPs of compound 1 were more than 3.2. Norkurarinone (2) obtained a higher selective index value between PTP1B and TCPTP (SI = 4.9), and PTP1B and SHP-2 (SI = 7.5). Based on the enzymatic inhibition results, kuraridin (1), norkurarinone (2), and 2′-methoxykurarinone (4) were evaluated for

Fig. 2 Inhibition of protein tyrosine phosphatase 1B-catalyzed hydrolysis of para-nitrophenylphosphate by compounds 1, 2, and 4. For each compound, A represents the Lineweaver-Burk plots, and B is the secondary plots from A.

Sasaki T et al. Protein Tyrosine Phosphatase …

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Letters

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559

Table 2 Inhibition rate (%) and selective index of compounds 1–5 and RK-682 against protein tyrosine phosphatase 1B, T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2. PTP

Compound 1 Inhibition rate (%)

PTP1B TCPTP VHR SHP-1 SHP-2 a

91.8 ± 1.2 24.9 ± 1.4 29.0 ± 2.7 22.8 ± 0.3 18.9 ± 0.7

Compound 2 a

b

S. I.

Inhibition rate (%)

3.7 3.2 4.0 4.9

92.2 ± 1.1 18.7 ± 1.4 27.1 ± 1.9 40.3 ± 1.1 12.3 ± 0.5

RK-682c

Compound 4 a

S. I.

b

Inhibition rate (%)

a

90.6 ± 1.3 26.1 ± 2.4 24.4 ± 2.5 41.5 ± 0.7 14.6 ± 0.7

4.9 3.4 2.3 7.5

S. I. 3.5 3.7 2.2 6.2

b

Inhibition rate (%)a

S. I.b

89.3 ± 1.9 9.8 ± 0.7 93.1 ± 2.7 15.5 ± 1.1 10.9 ± 1.1

9.1 1,0 5.8 8.2

Inhibition rate (%) are mean ± SD from three separate experiments; b S. I.: selective index value (Inhibition rate (%) against PTP1B/Inhibition rate (%) against other PTPs); c Positive

Fig. 3 Cellular activity of compounds 1, 2, and 4 (at 10 µM), sodium orthovanadate (positive control, at 200 µM), and DMSO (negative control) on Akt phosphorylation in HepG2 cells.

their cellular activity in the insulin signal transduction pathway in the human hepatocellular carcinoma cell line HepG2 by measuring the phosphorylation level of Akt, a key downstream effector of the insulin signaling cascade. With a sample final concentration of 10 µM, none of the compounds showed cytotoxicity. In the Akt phosphorylation assay, after the cells were stimulated with insulin, the pAkt levels were analyzed by Western blotting at different times (0, 5, and 60 min); dimethyl sulfoxide was used as a negative control and sodium orthovanadate was used as a " Fig. 3, the rapid increase in pAkt positive control. As shown in l levels after insulin stimulation was promoted by the administration of compounds 1, 2, and 4. 2′-Methoxykurarinone (4) exhibiting the most effective cellular activity is in good accordance with " Fig. 3). In addition to PTP1B, its potency in the enzymatic assay (l TCPTP and SHP-1 were also reported as negative regulators of the insulin signal transduction pathway [11, 12]. Taking into account the inhibitory selectivity of these compounds against various phosphatases, the increased insulin signal may be caused not only by PTP1B inhibition but by the sum of inhibitory action on various phosphatases. In conclusion, in a screening program of natural occurring PTP1B inhibitors, five lavandulyl flavonoids from the roots of S. flavescens were discovered as new PTP1B inhibitors, kuraridin (1), norkurarinone (2), kurarinone (3), 2′-methoxykurarinone (4), and kushenol T (5). Kuraridin (1), norkurarinone (2), kurarinone (3), and 2′-methoxykurarinone (4) were clarified to be noncompetitive PTP1B inhibitors with inhibitory selectivity between PTP1B and other PTPs. Compounds 1, 2, and 4 also showed promoting activity on the insulin signaling pathway in cellular-based assays. Numerous prenylated flavanoids have been reported as natural PTP1B inhibitors [6]. Compared with previous reported compounds, the structure of 2′-methoxykurarinone (4) is characterized by the presence of an 8-lavandulyl moiety and 5,2′-dime-

thoxy moieties in its flavanone skeleton. To our knowledge, the PTP1B inhibitory activity of lavandulyl flavonoids is reported for the first time. Lavandulyl flavonoids are characteristic bioactive constituents of the roots of S. flavescens, and have been reported to have a wide range of bioactivities such as antioxidant, anti-inflammatory, antidiabetic complications, and anti-Alzheimer activities [13, 14]. Lavandulyl flavonoids from the roots of S. flavescens revealing PTP1B inhibitory activity expended our understanding on their multifunctional properties and suggested their potential for further development for the treatment of lifestylerelated diseases.

Materials and Methods !

Chemicals and reagents: TCPTP and VHR were purchased from Enzo Life Sciences. SHP-1, SHP-2, p-NPP, and sodium orthovanadate (> 90 %) were purchased from Sigma-Aldrich. RK-682 (> 95 %) was purchased from Wako. p-Akt1/2/3 (Ser 473) and Akt1/2/3 (H-136) antibodies were purchased from Santa Cruz Biotechnology. The goat anti-rabbit IgG heavy and light chain antibody was purchased from Bethyl Laboratories. Kuraridin (1), norkurarinone (2), kurarinone (3), 2′-methoxykurarinone (4), and kushenol T (5) were isolated from the roots of S. flavescens in a previous study [9], and their purity was confirmed to be > 98 % by 1H‑NMR and HPLC analyses. All of the other solvents and reagents were purchased commercially. PTP1B and other PTP inhibitory activity assays: Protein tyrosine phosphatase activity was measured using p-NPP as the substrate. A mixture consisting of p-NPP and PTP1B or VHR in a buffer containing 0.06 M citrate (pH 6.0), 0.1 M NaCl, 1 mM ethylenediaminetetraacetic acid, and 1 mM dithiothreitol with or without a tested compound solution (prepared in the above buffer solution containing 3% dimethyl sulfoxide) was incubated at 37 °C for

Sasaki T et al. Protein Tyrosine Phosphatase …

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Letters

30 min. For TCPTP, SHP-1, and SHP-2, an assay buffer (pH 7.0) was prepared using 25 mM Tris/HCl, 50 mM NaCl, 2 mM ethylenediaminetetraacetic acid (Dojindo), 5 mM dithiothreitol, 0.01 % Brij35 (Wako), and 1 mg/mL bovine serum albumin (Sigma Aldrich). The substrate (p-NPP) was used at a concentration of 2 mM for PTP1B, TCPTP, VHR, and SHP-2 and 16 mM for SHP-1. The reaction was terminated by adding 20 µL of 10 M NaOH. The reaction mixture was blended by a microplate mixer for 5 min and the amount of produced p-nitrophenol was tested by measuring the absorbance at 405 nm (Shimadzu Biospec-mini). The blank was measured in the same way except buffer solution was added instead of the enzyme. The inhibitory activities were further measured at three different concentrations to obtain the IC50 values by regression analyses. The IC50 values were obtained by regression analyses with measuring the inhibitory activity at three different concentrations. RK-682 was used as the positive control. Cytotoxicity assay: For the human hepatocellular carcinoma cell line, HepG2 cells were purchased from the Japanese Cancer Research Resources Bank/Health Science Research Resources Bank. The cells were cultured in Dulbeccoʼs modified Eagleʼs medium (Nissui Pharmaceutical), supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences), at 37 °C in a humidified atmosphere containing 5 % CO2. After the HepG2 cells were seeded (100 µL, 15 × 104 cell/mL) into a 96-well plate, the cells were incubated for 4 h at 37 °C. HepG2 cells were exposed to each concentration of the compounds (0–10 µmol/L) for 48 h. The number of living cells was determined with the Premix WST-1 cell proliferation assay system (TaKaRa), according to the manufacturerʼs instructions. Insulin-stimulated Akt phosphorylation assay: HepG2 cells were cultured in Dulbeccoʼs modified Eagleʼs medium (Nissui Pharmaceutical), supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences), at 37 °C in a humidified atmosphere containing 5% CO2. After pretreatment with each compound at 37 °C for 60 min, HepG2 cells (500 µL, 5 × 105 cells/well) in a 48well plate were stimulated with 50 nmol/L insulin (Wako) for 5 min at 37 °C. After washing with cold phosphate buffered saline, the cells were lysed by sonication. The lysate was centrifuged at 15 000 rpm for 15 min, and the supernatant was separated on 12 % SDS-PAGE (Wako). After transfer to a Hybond-P PVDF membrane (GE Healthcare Bio-Science), the membrane was blocked with PVDF blocking reagent (Toyobo) at 4 °C overnight and was probed with 0.4 µg/mL anti-AKT1/2/3 rabbit polyclonal antibody (sc-8312, Santa Cruz Biotechnology) or antiphosphorylated AKT1/2/3 rabbit polyclonal antibody (Ser-473, sc-101629, Santa Cruz Biotechnology) in Can Get Signal Solution-I (Toyobo) for 1 h at room temperature, followed by further incubation with 0.025 µg/mL HRP-labeled anti-rabbit IgG‑h+l goat antibody (Bethyl Laboratories) in Can Get Signal Solution-II (Toyobo) for 1 h at room temperature. The membrane was then washed 3 times with phosphate buffered saline-T and then incubated with ECL-plus (GE Healthcare Bio-Science) for 5 min at room temperature and analyzed with a Typhoon 9410 (GE Healthcare Bio-Science). Sodium orthovanadate was used as the positive control.

Acknowledgements The research was partially supported by a Grant-in-Aid for Young Scientists (B) (No. 23790025) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Sasakawa Scientific Research Grant (No. 25–316) from The Japan Science Society.

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Conflict of Interest !

The authors declare no conflict of interest.

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Bibliography DOI http://dx.doi.org/10.1055/s-0034-1368400 Published online April 29, 2014 Planta Med 2014; 80: 557–560 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Wei Li Faculty of Pharmaceutical Sciences Toho University Chiba 274–8510 Japan Phone: + 81 4 74 72 11 61 Fax: + 81 4 74 72 14 04 [email protected]

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Protein tyrosine phosphatase 1B inhibitory activity of lavandulyl flavonoids from roots of Sophora flavescens.

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and mu...
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