Protein tyrosine phosphatase 1B inhibitory activity of alkaloids from Rhizoma Coptidis and their molecular docking studies.

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Q1 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Protein tyrosine phosphatase 1B inhibitory activity of alkaloids from Rhizoma Coptidis and their molecular docking studies Jae Sue Choi a,n, Md. Yousof Ali a, Hyun Ah Jung b,nn, Sang Ho Oh c, Ran Joo Choi d, Eon Ji Kim a a

Department of Food and Life Science, Pukyong National University, Busan 608-737, Republic of Korea Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea c Korean BioInformation Center (KOBIC), Daejeon 305-806, Republic of Korea d Angiogenesis & Chinese Medicine Laboratory, Department of Pharmacology, University of Cambridge, Cambridge, UK b

art ic l e i nf o

Keywords: Rhizoma Coptidis Protein tyrosine phosphatase 1B Alkaloids Molecular docking Enzyme kinetics

a b s t r a c t Ethnopharmacologic relevance: Rhizoma Coptidis (the rhizome of Coptis chinensis Franch) has commonly been used for treatment of diabetes mellitus in traditional Chinese medicine due to its blood sugarlowering properties and therapeutic benefits which highly related to the alkaloids therein. However, a limited number of studies focused on the Coptis alkaloids other than berberine. Materials and methods: In the present study, we investigated the anti-diabetic potential of Coptis alkaloids, including berberine (1), epiberberine (2), magnoflorine (3), and coptisine (4), by evaluating the ability of these compounds to inhibit protein tyrosine phosphatase 1B (PTP1B), and ONOO -mediated protein tyrosine nitration. We scrutinized the potentials of Coptis alkaloids as PTP1B inhibitors via enzyme kinetics and molecular docking simulation. Results: The Coptis alkaloids 1–4 exhibited remarkable inhibitory activities against PTP1B with the IC50 values of 16.43, 24.19, 28.14, and 51.04 μM, respectively, when compared to the positive control ursolic acid. These alkaloids also suppressed ONOO -mediated tyrosine nitration effectively in a dose dependent manner. In addition, our kinetic study using the Lineweaver-Burk and Dixon plots revealed that 1 and 2 showed a mixed-type inhibition against PTP1B, while 3 and 4 noncompetitively inhibited PTP1B. Moreover, molecular docking simulation of these compounds demonstrated negative binding energies (Autodock 4.0¼ 6.7 to 7.8 kcal/mol; Fred 2.0 ¼ 59.4 to 68.2 kcal/mol) and a high proximity to PTP1B residues, including Phe182 and Asp181 in the WPD loop, Cys215 in the active sites and Tyr46, Arg47, Asp48, Val49, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221 and Gln262 in the pocket site, indicating a higher affinity and tighter binding capacity of these alkaloids for the active site of the enzyme. Conclusion: Our results clearly indicate the promising anti-diabetic potential of Coptis alkaloids as inhibitors on PTP1B as well as suppressors of ONOO -mediated protein tyrosine nitration, and thus hold promise as therapeutic agents for the treatment of diabetes and related disease. & 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The incidence of type 2 diabetes mellitus (T2DM) has dramatically increased worldwide. T2DM is a chronic metabolic disease that is a serious global problem. In 2010, an estimated 285 million people had diabetes mellitus (DM), and within the next 20 years this value is expected to almost double (Nazaruk and BorzymKluczyk, 2014). A major subtype of T2DM is insulin-resistant T2DM (IR-T2DM), which mainly develops when insulin secretion

n

Corresponding author. Tel.: þ 82 51 629 5845; fax: þ82 51 629 5842. Corresponding author. Tel.: þ 82 63 270 4882; fax: þ82 63 270 3854. E-mail addresses: [email protected] (J.S. Choi), [email protected] (H.A. Jung).

nn

in peripheral tissues is unable to compensate for insulin resistance (Turkoski, 2006). IR-T2DM is of importance because it is associated with multiple complications such as cardiovascular anomalies (Rader, 2007). Although the first-line treatment for IR-T2DM usually includes a healthy diet and exercise, patients with DM, which cannot be controlled with healthy diet and exercise alone, are treated with drugs such as sulfonylureas, dipeptidyl peptidase (DPP)-4 inhibitors, biguanides and thiazolidine derivatives (Inzucchi, 2002; Duez et al., 2012). However, because of the adverse effects and the presence of non-responders, current treatments using these drugs have been limited. Protein tyrosine phosphatase 1B (PTP1B) is a major non-transmembrane protein tyrosine phosphatase and an important factor in non-insulindependent DM, namely T2DM. It is a negative regulator of the

http://dx.doi.org/10.1016/j.jep.2015.05.020 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Choi, J.S., et al., Protein tyrosine phosphatase 1B inhibitory activity of alkaloids from Rhizoma Coptidis and their molecular docking studies. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.020i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Q2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

J.S. Choi et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

insulin-signaling pathway and can hydrolyze the phosphotyrosines on the insulin receptor, which leads to the deactivation of the receptor. Overproduction of this enzyme has been implicated in the onset of T2DM (Elchebly et al., 1999; Kasibhatla et al., 2007). PTP1B inhibition has been shown in vivo to increase insulin and leptin activity and results in normalized blood glucose levels (Zinker et al., 2002). In addition, PTP1B inhibition has been studied from a broad perspective (González-Rodríguez et al., 2012). PTP1B inhibitors have gained considerable attention for their therapeutic value associated with their novel mode of action and are actively pursued in the development of new drugs. Although some PTP1B inhibitors are undergoing clinical trials, currently there are no PTP1B inhibitors available for clinical use. Hence, inhibition of PTP1B is an effective therapeutic approach to the treatment of T2DM, making PTP1B an attractive target for drug discovery (Koren and Fantus, 2007; Elchebly et al., 1999). Moreover, nitrotyrosine is a product of peroxynitrite (ONOO ) action, thus the production of ONOO can be indirectly inferred by the presence of nitrotyrosine residues (Ischiropoulos, 1998). Recently, much attention has been paid to the role of nitrotyrosine as a possible risk factor in DM, as increased levels of nitrotyrosine have been reported in the plasma of diabetic patients (Ceriello et al., 2001). So, inhibition of PTP1B and nitrotyrosine are attractive targets in the development of new treatments for DM and other related metabolic syndromes. Coptis chinensis Franch, of the Ranunculaceae family is commonly known as Huanglian in China, Ouren in Japan or Hwangryunhaedok-tang in Korea. C. chinensis has commonly been prescribed for treatment of various clinical effects, such as antidiabetic (Jung et al., 2008), anti-inflammatory (Schinella et al., 2002), anti-hypertensive (Ko et al., 2000), hypoglycemic and hypocholesterolemic (Yuan et al., 2006), anti-proliferative (Tse et al., 2006), antioxidant (Schinella et al., 2002; Yokozawa et al., 2005), anti-adipogenic (Choi et al., 2014), and anti-Alzheimer disease (Jung et al., 2009). In addition, Rhizoma Coptidis (the rhizome of C. chinensis) is one of 50 basic herbal medicinal materials used in traditional Chinese medicine (Li et al., 2009). Rhizoma Coptidis has proven to have various biological activities, such as suppression of fever and hyperhidrosis, detoxification, relaxation, and pyretic effects (Xu et al., 2009; Huang, 1999), and exhibits anti-microbial, anti-fungal, anti-viral (Huang, 1999) and anti-diabetic effects (Jung et al., 2008). Rhizoma Coptidis is known to harbor a diversity of alkaloids, including berberine, magnoflorine, epiberberine, and coptisine, which are considered to be its active constituents (Sun et al., 2006). In particular, berberine is the most predominant component and shows various pharmacological and biological effects, including anti-hypertensive (Ko et al., 2000), anti-diabetic, anti-adipogenic (Tang et al., 2006; Huang et al., 2006), anti-inflammatory (Kuo et al., 2004), hypolipidemic (Doggrell, 2005), antioxidant (Hsieh et al., 2007), hypoglycemic, and hypocholesterolemic (Yuan et al., 2006). Yokozawa et al. (2005) reported that coptisine, magnoflorine, and epiberberine might contribute to the protective effects of Rhizoma Coptidis on oxidative stress, including inhibition of cellular ONOO generation. Magnoflorine was also reported to exhibit significant antioxidant and antiradical capacities (Rackova et al., 2004; Hung et al., 2007). Despite the potential of Rhizoma Coptidis, there has been no detailed investigation on the possibility of developing antidiabetic drugs via enzyme kinetics and molecular docking evaluation. In this study, two kinetics methods, including LineweaverBurk and Dixon plots, have been used to determine the type of inhibition of test Coptis alkaloids 1–4. Since there is no detailed information on the mode of inhibition or on the molecular interactions of PTP1B-alkaloids 1–4, this study proposes an approach to develop alkaloids as a potent anti-diabetic drug

candidate by scrutinizing molecular docking predictions and enzyme kinetics.

2. Materials and methods 2.1. General experimental procedures The 1H and 13C NMR spectra were determined using a JEOL JNM ECP-400 spectrometer (Tokyo, Japan) at 400 MHz for 1H and 100 MHz for 13C. The 1H and 13C NMR chemical shifts were referenced to residual solvent peaks (δH 3.31 and δC 49.0 for CD3OD). The EI-MS spectrum was collected on a GC–MS QP-5050 A spectrometer (Shimadzu, Kyoto, Japan). The LR FAB-MS data were assessed using a JEOL JMS-HX110/110 A spectrometer (Tokyo, Japan). The IR spectrum (KBr) was recorded on a Shimadzu FT-IR spectrometer (Tokyo, Japan). Column chromatography was conducted using silica gel 60 (70–230 mesh, Merck, Darmstadt, Germany), RP-18 Lichroprep (40–63 mm, Merck, Germany), and Sephadex LH20 (20–100 mm, Sigma Co., St. Louis, MO, USA). Thin layer chromatography (TLC) was conducted on pre-coated Merck Kieselgel 60 F254 plates (20 20 cm2, 0.25 mm) and RP-18 F254 plates (5 10 cm2, Merck, Darmstadt, Germany), using 50% H2SO4 as a spray reagent. 2.2. Chemicals and reagents p-Nitrophenyl phosphate (pNPP) and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). PTP1B (human recombinant) was purchased from Biomols International LP (Plymouth Meeting, PA, USA), and dithiothreitol (DTT) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals and solvents were purchased from E. Merck, Fluka, and Sigma-Aldrich, unless otherwise stated. Peroxynitrate (ONOO ) was purchased from Cayman Chemicals Co. Anti-nitrotyrosine (clone 1A6, mouse-monoclonal primary antibody, IgG2b), horseradish peroxide-conjugated antirabbit, and anti-mouse antibodies were purchased from Millipore Co. (Billerica, MA, USA), as well as Polyvinylidene fluoride (PVDF) membrane (Immobilon-P). Supersignals West Pico Chemiluminescent Substrate was obtained from Pierce Biotechnology, Inc. (Rockford, IL, USA). 2.3. Plant materials The rhizome of C. chinensis was purchased from a local retailer and authenticated by Prof. J. H. Lee at Dongguk University, Gyeongbuk Province, Korea. A voucher specimen (No. 20060420) was deposited in the author's laboratory (Prof. J. S. Choi). 2.4. Extraction, fractionation, and isolation The powdered rhizome of C. chinensis (10 kg) was refluxed with methanol (MeOH) for 3 h (3 10 L). The total filtrate was then concentrated to dryness in vacuo at 40 1C, in order to render the MeOH extract (2.2 kg). This extract was suspended in distilled water (H2O) and then successively partitioned with methylene chloride (CH2Cl2) and n-butanol (BuOH), to yield CH2Cl2 (230 g) and n-BuOH (1.1 kg) fractions, respectively, as well as H2O residue (840 g). A portion of the n-BuOH fraction (316 g) was initially chromatographed over a Si gel column using a mixed solvent of EtOAc, MeOH, and H2O (EtOAc:MeOH:H2O 21:4:3-21:10:3EtOAc:MeOH: 2% HCl 21:10:3, gradient conditions) to yield 14 subfractions (BF01-BF18). BF03 (21.1 g) was recrystallized with MeOH, yielding (berberine, 1). BF12 (17.3 g) was chromatographed over an MCI gel column with aqueous MeOH (0%-100%), followed


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

by recrystallization with MeOH to obtain (coptisine, 4). A portion of BF13 (20 g) was chromatographed over an MCI gel column with aqueous MeOH (0%-100%), followed by recrystallization with MeOH to obtain (epiberberine, 2). (Magnoflorine, 3) was isolated from BF14 (13 g) by MCI gel column chromatography with aqueous MeOH (0%-100%). The chemical structures of isolated alkaloids (Fig. 1) were elucidated on the basis of spectroscopic evidence and by comparison with published data (Grycová et al., 2007; Lee and Kim, 1997). 2.5. Assay for PTP1B inhibition The PTP1B inhibitory activities of the alkaloids were evaluated using pNPP (Cui et al., 2006). To each well of a 96-well plate (final volume 100 mL), 40 mL of PTP1B enzyme [0.5 units diluted with PTP1B reaction buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM DTT] was added with or without a test sample (final concentration 50 mM, dissolved in 10% DMSO). The plate was pre-incubated at 37 1C for 10 min, and then 50 mL of 2 mM pNPP in PTP1B reaction buffer was added. After incubation at 37 1C for 20 min, the reaction was terminated by the addition of 10 M NaOH. The amount of p-nitrophenyl produced by enzymatic dephosphorylation of pNPP was estimated by measuring the absorbance at 405 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Non-enzymatic hydrolysis of 2 mM pNPP was corrected for by measuring the increase in absorbance at 405 nm in the absence of PTP1B enzyme. The percent inhibition (%) was obtained by the following equation: % inhibition ¼(Ac As)/Ac 100, where Ac is the absorbance of the control and As is the absorbance of the sample. Ursolic acid was used as a positive control. 2.6. Inhibition of ONOO -mediated protein tyrosine nitration ONOO -mediated protein tyrosine nitration was evaluated using the method of Aulak et al. (2001), with slight modifications. Various concentrations of alkaloids dissolved in 10% DMSO were added to 95 mL BSA (0.4 mg protein/mL) and mixed with 2.5 mL ONOO (200 μM). After incubation with shaking at 37 1C for 20 min, the sample was added to Bio-Rad gel buffer in a ratio of 1:1 and boiled for 5 min to denature the proteins. The total protein equivalent for the reactant was separated on 10% SDSpolyacrylamide minigel at 80 V for 30 min and 100 V for 1 h, and then transferred to a PVDF membrane at 80 V for 110 min using a wet transfer system (Bio-Rad, Hercules, CA, USA). The membrane was immediately placed in a blocking solution [5% non-fat dry 4

O O

5

2

H3CO

6

4a 13b

N+

H3CO

8

13a

1

8a

13

9

12a

10

12

O

OCH3 OCH3

O

Berberine (1) 3

H3CO HO

5

6b 11b 11a

HO

Epiberberine (2)

4 3a

1

+

N

N 6a

7a

O

+

O

+

N

7

O

11 10

H3CO

3

milk in TBS-Tween buffer (w/v), Bio-Rad TBS, and 0.1% Tween-20, pH 7.4] at room temperature for 1 h. The membrane was washed three times (10 min each wash) in TBS-Tween buffer and incubated with a monoclonal anti-nitrotyrosine antibody (diluted 1:2500 in TBS-Tween buffer with 5% non-fat dry milk) at 4 1C overnight. After three more washes in TBST buffer (10 min and 5 min), the membrane was incubated with horseradish peroxidase-conjugated sheep anti-mouse secondary antibody diluted 1:2000 in TBST buffer at room temperature for 1 h. After three washes in TBST buffer, antibody labeling was visualized using the Supersignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL, USA), according to the manufacturer's instructions, and the membrane was exposed to X-ray film (Kodak, Rochester, NY, USA). Pre-stained blue protein markers were used for molecular weight determination. 2.7. Kinetic parameters in PTP1B inhibition – Dixon and LineweaverBurk plots In order to determine the kinetic mechanism, two kinetic methods using Lineweaver-Burk and Dixon plots were complementarily used (Lineweaver and Burk, 1934; Cornish-Bowden, 1974; Dixon, 1953). Each enzymatic inhibition at various concentrations of four Coptis alkaloids was evaluated by monitoring the effects of different concentrations of the substrates in Dixon plots (single reciprocal plot). Dixon plots for PTP1B inhibition were obtained in the presence of different concentrations of p-NPP substrate: 0.5, 1.0, and 2.0 mM. The test concentrations of the alkaloids in the PTP1B kinetics analysis were as follows: 4.0, 20.0, and 100.0 mM for 1; 10.0, 50.0, and 100.0 mM for 2; 50.0, 100.0, and 150 mM for 3; 10.0, and 50.0 mM for 4. Using Lineweaver-Burk double reciprocal plots, the PTP1B inhibition mode was determined at various concentrations of p-NPP substrate (0.5, 1.0, and 2.0 mM) in the absence or presence of different test compound concentrations (4.0, 20.0, and 100.0 mM for 1; 10.0, 50.0, and 100.0 mM for 2; 50.0, 100.0, and 150 mM for 3; 10.0, and 50.0 mM for 4.). The enzymatic procedures consisted of the same, aforementioned PTP1B assay methods. The inhibition constants (Ki) were determined by interpretation of the Dixon plots. 2.8. Molecular docking simulation in PTP1B inhibition – Autodock 4.0 and Fred 2.0 In order to estimate the conformation of the protein–ligand complex and to increase accuracy, repeatability, and reliability of the docking results, two programs: Autodock 4.0 (AutoDock4 and AutoDockTools4) and Fred 2.0 (OpenEye Scientific Software, Santa Fe, NM, USA) were utilized. For docking studies, the crystal structures of the protein targets [protein data Bank (PDB ID: 1NNY for human PTP1B)] were allocated from the protein sequence alignment (sequence alignment tool: NCBI b12seq). The 3D structures of test alkaloids 1–4 were constructed and minimized using Chemsketch 3.5 and Omega 2.0 software (OpenEye Scientific Software, USA) for 2D and 3D conformations, respectively. The predicted protein–ligand complexes were optimized and ranked according to the empirical scoring function, ScreenScore, which estimates the binding free energy of the ligand receptor complex. The docking of the PTP1B-alkaloid molecules was successful, as indicated by statistically significant scores. 2.9. Statistics

8

O Magnoflorine (3)

Coptisine (4)

Fig. 1. Structures of compounds isolated from C. chinensis.

Statistical significance was analyzed by one-way ANOVA and Student's t-test (Systat Inc., Evanston, IL, USA) and considered significant at p o0.01. All results are presented as mean 7SEM.


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

3. Results 3.1. Enzyme kinetics in PTP1B inhibition As shown in Table 1, Coptis alkaloids 1–4 were potent PTP1B inhibitors with respective IC50 values of 16.43 70.93, 24.19 71.47, 28.14 71.79, and 51.04 72.49 μM when the ursolic acid positive control exhibited an IC50 value of 3.91 70.19 μM. As part of our continuous search for alkaloids derived from C. chinensis as potent PTP1B inhibitors, the type of inhibition and inhibition constants (Ki) of four active alkaloids 1–4 were investigated using Lineweaver-Burk and Dixon plots (Figs. 2 and 3). Each line of inhibitors intersected at the xy-side, indicating mixed-type inhibitors, while the lines penetrate the same point on the x-intercept, representing noncompetitive inhibitors in Lineweaver-Burk plots (Lineweaver and Burk, 1934). Therefore, 1 and 2 exhibited mixedtype inhibition against PTP1B, while 3 and 4 showed noncompetitive-type PTP1B inhibition (Fig. 2A–E). In addition, the Dixon plot is a common method for determining the type of enzyme inhibition and the dissociation or inhibition constant (Ki) for an enzyme–inhibitor complex, where the value of the x-axis implies Ki (Cornish-Bowden, 1974; Dixon, 1953). As shown in Fig. 3A–E, the Ki values of 1–4 were 18.51, 24.19, 28.14, and 51.04 μM, respectively. As the Ki value represents the concentration needed to form an enzyme–inhibitor complex, a lower Ki value may manifest more effective inhibitors against PTP1B in the development of preventive and therapeutic agents. 3.2. Inhibitory effect of the Coptis alkaloids on ONOO -mediated tyrosine nitration In order to determine the inhibitory effect of the Coptis alkaloids against ONOO -induced tyrosine nitration, Western blot analysis was performed using a 3-nitrotyrosine antibody, and results are presented in Fig. 4. As shown in Fig. 4A–C, pretreatment of the berberine, epiberberine and magnoflorine with different concentrations ranges of 12.5–100 μM resulted in dose-dependent inhibitory activities against ONOO -mediated tyrosine nitration. The coptisine showed a significant concentration-dependent inhibitory effect at even lower concentration ranges of 6.25–50 μM, and tyrosine nitration was barely detectable at the 50 μM concentration, as shown in Fig. 4D and E. 3.3. Molecular docking model of 1–4 in inhibition against PTP1B Based on molecular docking studies, Coptis alkaloids 1–4 were predicted using the Autodock and Fred program to evaluate the binding site-directed inhibition of PTP1B. As shown in Fig. 5A–D, PTP1B inhibitor complexes were formed with compounds 1–4 stably posed in the pocket of PTP1B Autodock 4.0 (red) and Fred 2.0 (blue). As summarized in Table 2, the binding sites of compounds 1–4 were predicted by Autodock 4.0 and Fred 2.0. Table 1 Protein tyrosine phosphatase 1B inhibitory activity of bioactive alkaloids. Test compounds

IC50 (mM)a

Ki value

Inhibition type

Berberine (1) Epiberberine (2) Magnoflroine (3) Coptisine (4) Ursolic acidb

16.43 7 0.93 24.197 1.47 28.147 1.79 51.047 2.49 3.917 0.19

18.51 25.11 38.74 64.74 6.43

Mixed Mixed Noncompetitive Noncompetitive Competitive

Values are expressed as mean7 S.E.M. of triplicates. a Test concentration of the samples were in the range of 4–150 mM, dissolved in 10% DMSO. b Positive control.

The Autodocking and Fred docking programs were used to dock the compounds into the binding sites of the crystallographic structures, with all residues defined as 5–6 Å from the inhibitor in the original complex. In addition to the active site residue, the docking analysis also showed that the respective docking energies of compounds 1–4 were –7.2, –6.9, –6.8 and –7.7 kcal/mol according to Autodock 4.0 and –67.3, –59.6, –60.1 and –64.3 kcal/mol according to Fred 2.0, when accounting for the lowest energy conformation of the most predicted complex. This result indicated that Coptis alkaloids 1–4 bound tightly at the active site of PTP1B.

4. Discussion Over the last two decades, tremendous amounts of effort have been devoted to developing active PTP1B inhibitors for the treatment of T2DM. The majority of known inhibitors possess tyrosine mimetic structures functionalized with negatively charged moieties. Although highly potent PTP1B inhibitors have been identified, most of the development of small molecule PTP1B inhibitors has emerged only recently as a rapidly growing area of investigation in natural product chemistry. Recently, there is an emerging trend to use potential plant constituents, especially alkaloids, to treat DM (Chen et al., 2014; Jung et al., 2008). C. chinensis, which is regarded as a very good source of various alkaloids and widely used in traditional Chinese medicine, has attracted considerable attention because of its multiple pharmacological effects, such as anti-diabetic, anti-inflammatory, antioxidant, hypoglycemic, hypocholesterolemic and anti-Alzheimer disease (Jung et al., 2008, 2009; Schinella et al., 2002; Yokozawa et al., 2005; Yuan et al., 2006). As part of our ongoing search to identify anti-diabetic agents present in natural sources, we explored the effects of alkaloids isolated from the C. chinensis. The four known alkaloids were identified as berberine (1), epiberberine (2), magnoflorine (3) and coptisine (4). Although previous reports have shown that berberine, epiberberine, magnoflorine and coptisine possess anti-diabetic activity through the inhibition of alpha glucosidase and aldose reductase (Zhou et al., 2014; Jung et al., 2008), there is no report of these four alkaloids on PTP1B inhibition. Berberine activates insulin signaling through the inhibition of PTP1B activity, decreases blood glucose, and enhances insulin sensitivity (Chen et al., 2010; Leng et al., 2004; Yin et al., 2004; Tang et al., 2006). Epiberberine and magnoflorine were previously shown to possess in vitro hypoglycemic effects (Jiang et al., 2011; Patel and Mishra, 2011). Magnoflorine, coptisine, and epiberberine were also reported to inhibit cellular ONOO generation (Rackova et al., 2004; Hung et al., 2007). However, the effects of these alkaloids in controlling DM have not yet been examined using the corresponding key enzyme of PTP1B. Until now, there has been no report on the mode of inhibition of PTP1B or the molecular interactions of corresponding enzymes of these four alkaloids. Therefore, the aim of this study was to identify an approach to develop potent anti-diabetic drugs using molecular docking predictions and enzyme kinetics of alkaloids. It has been hypothesized that a specific protein tyrosine phosphatase (PTP) is involved in the dephosphorylation and inactivation of the insulin receptor, which attenuates insulin signaling; disequilibrium between the insulin receptor and PTPs could be a contributing factor to the insulin resistance observed in T2DM (Tonks and Neel, 2001; Wu et al., 2003). Therefore, PTP1B inhibitors are potential therapeutic candidates to restore insulin sensitivity and treat T2DM. A series of PTP1B inhibitors containing highly charged, nonhydrolyzable phosphonate mimetic have been reported such as, difluoromethylene phosphonate, 2-carbomethoxybenzoic acid, and 2-oxalylaminobenzic acids (Ala et al., 2006; Johnson et al., 2002). Their development as drug candidates has been extremely difficult


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


1000

1200 800

1000 600

800

1/V

1/V 400

600

400 200

200 0

-1

0

1

2

3

-2

0

-1

1/[pNPP] (mM-1)

0

1

2

3

1/[pNPP] (mM-1)

1600 1400

1200

1200

1000

1000

800

800

1/V

1/V

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

5

600

400

400

200

200

-4

-2

0

600

0

2

4

1/[pNPP] (mM -1)

-2

-1

0

0

1

2

3

1/[pNPP] (mM-1)

Fig. 2. Lineweaver-Burk plot for PTP1B inhibition of Coptis alkaloids 1–4. PTP1B inhibition was analyzed in the presence of different concentration of samples as follows: 0 mM (△), 4 mM (▼), 20 mM (○) and100 mM (●) for Berberine (A); 0 mM (△), 10 mM (▼), 50 mM (○) and 100 mM (●) for epiberberine (B); 0 mM (△), 50 mM (▼), 100 mM (○) and 150 mM (●) for Magnoflorine (C); 0 mM (▼), 10 mM (○) and 50 mM (●) for coptisine (D).

due to poor cell membrane permeability, low oral bioavailability, and the difficulty in balancing the IR tyrosine kinase that is involved in downstream insulin signal phosphatases, such as PTP1B, that are required to shut down these signals. Therefore, small molecular and lipophilic PTP1B inhibitors with selectivity, bioavailability and acceptable pharmacokinetic properties have emerged as novel PTP1B drugs (Combs, 2010). Molecular docking has contributed important insights into drug discovery for many years. However, docking procedures aim to identify correct positions of ligands in the binding pocket of a protein and to predict the affinity between the ligand and the protein. In other words, docking describes a process by which two molecules fit together in three-dimensional space. Based on molecular docking studies, Coptis alkaloids 1–4 were predicted using the Autodock and Fred programs to evaluate the binding site-directed inhibition of PTP1B. Actually, the Autodock program uses a semi-empirical free energy force field to predict binding of protein–ligand complexes of a known structure and the binding energies for both the bound and unbound states (Morris et al., 2009). The other approach of the Fred software is to thoroughly dock the scores of all possible positions of each ligand in the binding site, exhaustively test all poses of the ligand within the defined binding site, and maintain the protein–ligand complex as

rigid during most of the docking process, leading to compensation for target flexibility (Bustanji et al., 2009). The main structural features of PTP1B have been well established and consist of 435 amino acid residues, including residues 30–278, which comprise the catalytic domain. The main structural properties of PTP1B are the catalytic loop, containing the catalytic residue Cys215, the secondary phosphate-binding loop (P-loop) mediated by residues His214–Arg221, and the WPD loop identified as residues Thr177–Pro185. The α3-helix is comprised of residues Glu186–Glu200, the S-loop is comprised of residues Ser201–Gly209, and the α6-helix is comprised of residues Ala264–Ile281 (Combs, 2010; Kamerlin et al., 2007). In particular, the WPD loop plays a much more important role in the specificity and affinity of the inhibitors. In the presence of the “open” confirmation of the WPD loop, the binding pocket of PTP1B is easily accessible to the substrate/inhibitor. After substrate binding, the WPD loop closes over the active site, forming a tight binding pocket for the substrate. The WPD loop closes onto the substrate and thereby positions the thiolate of Cys215 for nucleophilic attack of the phosphotyrosine. Asp181 acts as a general acid catalyst (Combs, 2010). Considering docking results of the PTP-inhibitor complex, Coptis alkaloids 1–4 were stably posed in similar pocket/catalytic domains of PTP1B residues, including Tyr46, Arg47, Asp48, Lys120, Asp181,


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


6

1200

-100

1000

1000

800

800

600

600

400

400

200

200

0

-50

1/V

1/V

1200

0

50

100

150

-100

0

-50

concentration concentration(µM) (µM)

0

50

100

150

concentration (µM)

1400 1600 1200

1400

1000

1200

800

1000

1/V

1/V

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

600

800 600

400

400

200

-100

-50

0

200

0

50

100

150

200

concentration (µM)

-100

-50

0

0

50

100

150

concentration (µM) concentration (µM)

Fig. 3. Dixon plots for PTP1B inhibition of Coptis alkaloids 1–4. Berberine (A), epiberberine (B), magnoflorine (C), and coptisine (D) were tested in the presence of different concentration of substrates (pNPP): 2.0 mM (●); 1.0 mM (○) and 0.5 mM (▼).

Fig. 4. The inhibitory effect of the Coptis alkaloids against ONOO -induced tyrosine nitration. Mixtures of samples and BSA were incubated at 25 1C for 10 min. Reactants were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Berberine (A), epiberberine (B), magnoflorine (C) and coptisine (D) were used at the indicated concentrations.

Phe182, Cys215, Ser216, Ala217, Ile219, Gly220, Arg221, and Gln262. As a result, Coptis alkaloids 1–4 showed negative binding energies and a high proximity to PTP1B residues, including Phe182 and Asp181 in the WPD loop, Cys215 in the active sites, and Tyr46, Arg47, Asp48, Val49, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221 and Gln262 in the pocket site. The binding energies of 1–4 were –7.2, –6.9, –6.8 and –7.7 kcal/mol, respectively, according to Autodock 4.0. These values were –67.3, –59.6, –60.1 and –64.3 kcal/mol, respectively, according to Fred 2.0, indicating high affinity to PTP1B residues. Alkaloids 1–4 interacted with Phe182 and Asp181 in the WPD loop and Cys215 in the active site, indicating that inhibitors 1–4 may reduce the mobility of the WPD loop toward a more rigid conformation, which inhibits WPD loop closure and prevents substrate binding (Popov, 2011). Previously, Bustanji et al. (2006) demonstrated the interaction between the positive charged nitrogen of berberine and the negative charged carboxylate moiety of Asp48 which also observed in our Coptis alkaloids 1–4. In the docking simulation, the electrostatic interaction of electron-rich aromatic ring of Coptis alkaloid 1–4 with the positive charged guanidine moiety in Arg24 was observed. In addition, the van der Waals' attraction between nonpolar dioxymethylene and bulky skeleton of Coptis alkaloids 1–4, and the phenyl moiety in Tyr46 was observed. The three key docking interactions might be crucial for reducing binding energy and tightening the complex of PTP1B ligand and compounds. Berberine, epiberberine and coptisine are quaternary protoberberine-type alkaloids, while magnoflorine is a quaternary aporphine-type alkaloid in Fig. 1. In the enzyme kinetic study,


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Q5 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

7

Fig. 5. Molecular docking models for PTP1B inhibition of Coptis alkaloids 1–4. Molecular docking models for PTP1B inhibition of berberine (5A), epiberberine (5B), magnoflorine (5C), and coptisine (5D). 3D docking molecule of the PTP1B and Coptis alkaloids: the yellow surface represent pocket site in the PTP1B enzyme and ligand interaction model of Coptis alkaloids: red and blue ligands were predicted using the Autodock 4.0 and Fred 2.0 programs, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 2 Binding sites of compounds 1–4 in PTP1B using two molecular docking programs. Test Binding sitesa compounds

1 2 3 4

a b

Docking scoreb (kcal/mol)

Autodock 4.0

Fred 2.0

Autodock 4.0

Fred 2.0

Tyr46, Arg47, Asp48, Lys120, Asp181, Phe182, Cys215, Ser216, Ala217, Ile219, Gly220, Arg221, Gln262 Tyr46, Arg47, Asp48, Val49, Lys120, Asp181, Phe182, Cys215, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221,Gln262 Tyr46, Asp48, Val49, Ser118, Lys120, Asp181, Phe182, Cys215, Ser216, Ala217, Gly218, Gly220, Arg221, Gln262 Tyr46, Arg47, Asp48, Val49, Asp181, Phe182, Cys215, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221, Gln262

Tyr46, Arg47, Asp48, Lys120, Asp181, Phe182, Cys215, Ser216, Ala217, Ile219, Gly220, Arg221, Gln262, Val49, Gly218 Tyr46, Arg47, Asp48,Val49, Lys120, Asp181, Phe182, Cys215, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221,Gln262 Tyr46, Asp48, Val49, Ser118, Lys120, Asp181, Phe182, Cys215, Ser216, Ala217, Gly218, Gly220, Arg221, Gln262 Tyr46, Asp48, Asp181, Phe182, Cys215, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221, Gln262

7.2

67.3

6.9

59.6

6.8

60.1

7.7

64.3

All amino residues located 5–6 Å from the original enzyme/inhibitor complex in the two programs. The empirical scoring function TMscore (shelba3.1), which estimates the binding free energy of the ligand receptor complex.

alkaloids 1 and 2 were mixed-type PTP1B inhibitors; alkaloids 3 and 4 were noncompetitive-type PTP1B inhibitors. In comparison to mode of inhibition of alkaloids 1, 2 and 4, alkaloid 1 has two functional groups, one is dioxymethylene group in the A ring and another dimethoxy group in the D ring, whereas alkaloid 2 contains same functional groups like as alkaloid 1 but in opposite rings, while alkaloid 4 contains two dioxymethylene functional groups in both A and D rings. So, the above structure relationship showed that substitutions greatly affected the inhibition mode, which is made clear by the fact that alkaloids 1 and 2 are mixed type PTP1B inhibitors but alkaloid 4 is noncompetitive PTP1B inhibitors. This fact was also supported by the binding energy. In comparison with the calculated binding energy of alkaloid 1 (Autodock 4.0 ¼–7.2 and Fred 2.0 ¼ 67.3 kcal/mol), alkaloid 2

(Autodock 4.0 ¼ 6.9 and Fred 2.0 ¼ 59.6 kcal/mol) and alkaloid 4 (Autodock 4.0 ¼ 7.7 and Fred 2.0 ¼ 64.3 kcal/mol), alkaloids containing dioxymethylene and dimethoxy groups substitutions greatly depressed the binding energy. Recently, Sasaki et al. (2015) and Jung et al. (2013) also reported that presence or lack of functional groups and its substitutions greatly affected the inhibition mode and binding energy, which is made clear by the fact that alkaloids 1 and 2 are similar type of inhibition but lack of functional groups greatly affected the inhibition mode and binding energy of alkaloid 4. Therefore, alkaloids 1 and 2 were stably posed in similar pocket/catalytic domains of PTP1B residues and alkaloid 4 showed different catalytic domains of PTP1B residues in Fig. 6. Considering both enzymatic kinetics and molecular docking results, alkaloids 3 and 4 bind to the free enzyme and inhibit the


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Fig. 6. Ligand interaction and comparative binding sites diagrams of the three alkaloids in the active site of PTP1B enzyme. Berberine (A), epiberberine (B) and coptisine (C).

formation of the enzyme/substrate complex, while 1 and 2 bind to both the allosteric sites of free enzyme and to the enzyme/ substrate complex. Wiesmann et al. (2004) also proposed a range of PTP1B inhibitor candidates that bind to a novel allosteric binding site 20 Å from the catalytic pocket. This allosteric site makes these inhibitors highly selective for PTP1B. The allosteric inhibitor may act by stabilizing the conformation that precludes the closure of the WPD loop (Kamerlin et al., 2007; Bharatham et al., 2008). Bialy and Waldmann (2005) reported that noncompetitive or allosteric inhibitors might oxidize the catalytic cysteine (Cys215). These findings led to the design of alkaloids as PTP1B inhibitors that interact with both catalytic and allosteric sites to achieve selectivity. Bustanji et al. (2006) previously reported that berberine exhibited competitive inhibition on human recombinant PTP1B. This inconsistency in the enzyme kinetic results may be due to difference of assay method such as substrate and buffer used.

5. Conclusion In conclusion alkaloids 1–4, isolates from Rhizoma Coptidis, displayed negative binding energies and a high proximity to PTP1B residues, including Phe182 and Asp181in the WPD loop, Cys215 in the active site, and Tyr46, Arg47, Asp48, Val49, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221 and Gln262 in the pocket site. Additionally, alkaloids 1 and 2 exhibited mixed-type inhibition against PTP1B, while 3 and 4 showed non-competitive-type PTP1B inhibition. In the present study, alkaloids 1–4 showed promising inhibitory potential against PTP1B and suppressed ONOO -mediated protein tyrosine nitration,

and thus hold promise as therapeutic agents for the treatment of DM and related disease.

Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A6A1028677).

References Ala, P.J., Gonneville, L., Hillman, M., Becker-Pasha, M., Yue, E.W., Douty, B., Wayland, B., Polam, P., Crawley, M.L., McLaughlin, E., Sparks, R.B., Glass, B., Takvorian, A., Combs, A.P., Burn, T.C., Hollis, G.F., Wynn, R., 2006. Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of proteintyrosine phosphatase 1B. J. Biol. Chem. 281, 38013–38021. Aulak, K.S., Miyagi, M., Yan, L., West, K.A., Massillon, D., Crabb, J.W., Stuehr, D.J., 2001. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc. Natl. Acad. Sci. USA 98, 12056–12061. Bharatham, K., Bharatham, N., Kwon, Y.J., Lee, K.W., 2008. Molecular dynamics simulation study of PTP1B with allosteric inhibitor and its application in receptor based pharmacophore modeling. J. Comput. Aided Mol. Des. 22, 925–933. Bialy, L., Waldmann, H., 2005. Inhibitors of protein tyrosine phosphatases: nextgeneration drugs? Angew. Chem. 44, 3814–3839. Bustanji, Y., Al-Masri, I.M., Qasem, A., Al-Bakri, A.G., Taha, M.O., 2009. In silico screening for non-nucleoside HIV-1 reverse transcriptase inhibitors using physicochemical filters and high-throughput docking followed by in vitro evaluation. Chem. Biol. Drug Des. 74, 258–265. Bustanji, Y., Taha, M.O., Yousef, A.M., Al-Bakri, A.G., 2006. Berberine potently inhibits protein tyrosine phosphatase 1B: investigation by docking simulation and experimental validation. J. Enzyme Inhib. Med. Chem. 21, 163–171.


67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ceriello, A., Mercuri, F., Quagliaro, L., Assaloni, R., Motz, E., Tonutti, L., Tabaga, C., 2001. Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia 44, 834–838. Chen, C., Zhang, Y., Huang, C., 2010. Berberine inhibits PTP1B activity and mimics insulin action. Biochem. Biophys. Res. Commun. 397, 543–547. Chen, Q.B., Xin, X.L., yang, Y., lee, S.S., Aisa, A.H., 2014. Highly conjugated norditerpenoid and pyrroloquinoline alkaloids with potent PTP1B inhibitory activity from Nigella glandulifera. J. Nat. Prod. 77, 807–812. Choi, J.S., Kim, J.H., Ali, M.Y., Byung, S.M., Kim, D.G., Jung, H.A., 2014. Coptis chinensis alkaloids exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBP-α and PPAR-γ. Fitoterapia 98, 199–208. Combs, A.P., 2010. Recent advances in the discovery of competitive protein tyrosine phosphatase 1B inhibitors for the treatment of diabetes, obesity, and cancer. J. Med. Chem. 53, 2333–2344. Cornish-Bowden, A., 1974. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137, 143–144. Cui, L., Na, M., Oh, H., Bae, E.Y., Jeong, D.G., Ryu, S.E., Kim, S., Kim, B.Y., Oh, W.K., Ahn, J.S., 2006. Protein tyrosine phosphatase 1B inhibitors from Morus root bark. Bioorg. Med. Chem. Lett. 16, 1426–1429. Dixon, M., 1953. The determination of enzyme inhibitor constants. Biochem. J. 55, 170–171. Doggrell, S.A., 2005. Berberine-a novel approach to cholesterol lowering. Expert Opin. Investig. Drugs 14, 683–685. Duez, H., Cariou, B., Staels, B., 2012. DPP-4 inhibitors in the treatment of type 2 diabetes. Biochem. Pharmacol. 83, 823–832. Elchebly, M., Payette, P., Michaliszyn, E., Cromlish, W., Collins, S., Loy, A.L., Normandin, D., Cheng, A., Himms-Hagen, J., Chan, C.C., Ramachandran, C., Gresser, M.J., Tremblay, M.L., Kennedy, B.P., 1999. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283, 1544–1548. González-Rodríguez, A., Más-Gutierrez, J.A., Mirasierra, M., Fernandez-Pérez, A., Lee, Y.J., Ko, H.J., Kim, J.K., Romanos, E., Carrascosa, J.M., Ros, M., Vallejo, M., Rondinone, C.M., Valverde, A.M., 2012. Essential role of protein tyrosine phosphatase 1B in obesity-induced inflammation and peripheral insulin resistance during aging. Aging Cell 11, 284–296. Grycová, L., Dostál, J., Marek, R., 2007. Quaternary protoberberine alkaloids. Phytochemistry 68, 150–175. Hsieh, Y.S., Kuo, W.H., Lin, T.W., Chang, H.R., Lin, T.H., Chen, P.N., Chu, S.C., 2007. Protective effects of berberine against low-density lipoprotein (LDL) oxidation and oxidized LDL-induced cytotoxicity on endothelial cells. J. Agric. Food Chem. 55, 10437–10445. Huang, C., Zhang, Y., Gong, Z., Sheng, X., Li, Z., Zhang, W., Qin, Y., 2006. Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARγ pathway. Biochem. Biophys. Res. Commun. 348, 571–578. Huang, K.C., 1999. The Pharmacology of Chinese Herbs. CRC Press Inc., Boca Raton: FL, pp. 381–382. Hung, T.M., Lee, J.P., Min, B.S., Choi, J.S., Na, M., Zhang, X., Ngoc, T.M., Lee, I., Bae, K., 2007. Magnoflorine from Coptidis Rhizoma protects high density lipoprotein during oxidant stress. Biol. Pharm. Bull. 30, 1157–1160. Inzucchi, S.E., 2002. Oral antihperglycemic therapy for type 2 diabetes. J. Am. Med. Assoc. 287, 360–372. Ischiropoulos, H., 1998. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch. Biochem. Biophys. 356, 1–11. Jiang, X.F., Wang, L.J., Li, X.G., Zhao, Z.Q., Zhu, J.Y., 2011. Isolation of jatrorrhizine and epiberberine in Coptis chinensis and their in vitro hypoglycemic effect. Guizhou Agric. Sci. 39, 44–46. Johnson, T.O., Ermolieff, J., Jirousek, M.R., 2002. Protein tyrosine phosphatase 1B inhibitors for diabetes. Nat. Rev. Drug Discov. 1, 696–709. Jung, H.A., Cho, Y.S., Oh, S.H., Lee, S., Min, B.S., Moon, K.H., Choi, J.S., 2013. Kinetics and molecular docking studies of pimarane-type diterpenes as protein tyrosine phosphatase (PTP1B) inhibitors from Aralia continentalis roots. Arch. Pharm. Res. 36, 360–372. Jung, H.A., Min, B.S., Yokazawa, T., Lee, J.H., Kim, Y.S., Choi, J.S., 2009. Anti-alzheimer and antioxidant activities of Coptidis Rhizoma alkaloids. Biol. Pharm. Bull. 32, 1433–1438. Jung, H.A., Yoon, N.Y., Bae, H.J., Min, B.S., Choi, J.S., 2008. Inhibitory activities of the alkaloids from Coptidis Rhizoma against aldose reductase. Arch. Pharm. Res. 31, 1405–1412. Kamerlin, S.C., Rucker, R., Boresch, S., 2007. A molecular dynamics study of WPDloop flexibility in PTP1B. Biochem. Biophys. Res. Commun. 356, 1011–1016. Kasibhatla, B., Wos, J., Peters, K.G., 2007. Targeting protein tyrosine phosphatase to enhance insulin action for the potential treatment of diabetes. Curr. Opin. Investig. Drugs 8, 805–813. Ko, W.H., Yao, X.Q., Lau, C.W., Law, W.I., Chen, Z.Y., Kwok, W., Ho, K., Huang, Y., 2000. Vasorelaxant and antiproliferative effects of berberine. Eur. J. Pharmacol. 399, 187–196.

9

61 Koren, S., Fantus, I.G., 2007. Inhibition of the protein tyrosine phosphatase PTP1 B: potential therapy for obesity, insulin resistance and type-2 diabetes mellitus. 62 Best Pract. Res. Clin. Endocrinol. Metab. 21, 621–640. 63 Kuo, C.L., Chi, C.W., Liu, T.Y., 2004. The anti-inflammatory potential of berberine 64 in vitro and in vivo. Cancer Lett. 203, 127–137. Lee, H.Y., Kim, C.W., 1997. Studies on the constituents of Berberis amurensis 65 Ruprecht. Korean J. Pharmacogn. 28, 257–263. 66 Leng, S.H., Lu, F.E., Xu, L.J., 2004. Therapeutic effects of berberine in impaired 67 glucose tolerance rats and its influence on insulin secretion. Acta. Pharmacol. Sin. 25, 496–502. 68 Li, C.Y., Tsai, S.I., Damu, A.G., Wu, T.S., 2009. A rapid and simple determination of 69 protoberberine alkaloids in Rhizoma Coptidis by 1H NMR and its application for 70 quality control of commercial prescriptions. J. Pharm. Biomed. Anal. 49, 1272–1276. 71 Lineweaver, H., Burk, D., 1934. The determination of enzyme dissociation constants. 72 J. Am. Chem. Soc. 56, 658–666. 73 Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., Olson, A.J., 2009. AutoDock4 and AutoDockTools4: automated docking with selective 74 receptor flexibility. J. Comput. Chem. 30, 2785–2791. 75 Nazaruk, J., Borzym-Kluczyk, M., 2014. The role of triterpenes in the management of Q3 76 diabetes mellitus and its complications. Phytochem. Rev. , http://dx.doi.org/ 77 10.1007/s11101-014-9369-x. Patel, M.B., Mishra, S., 2011. Hypoglycemic activity of alkaloidal fraction of 78 Tinospora cordifolia. Phytomedicine 18, 1045–1052. 79 Popov, D., 2011. Novel protein tyrosine phosphatase 1B inhibitors: interaction 80 requirements for improved intracellular efficacy in type 2 diabetes mellitus and obesity control. Biochem. Biophys. Res. Commun. 410, 377–381. 81 Rackova, L., Majekova, M., Kostalova, D., Stefek, M., 2004. Antiradical and antiox82 idant activities of alkaloids isolated from Mahonia aquifolium. Structural 83 aspects. Bioorg. Med. Chem. 12, 4709–4715. Rader, D.J., 2007. Effect of insulin resistance, dyslipidemia, and intra-abdominal 84 adiposity on the development of cardiovascular disease and diabetes mellitus. 85 Am. J. Med. 120, S12–S18. Sasaki, T., Li, W., Higai, K., Koike, K., 2015. Canthinone alkaloids are novel protein Q4 86 tyrosine phosphatase 1B inhibitors. Bioorg. Med. Chem. Lett.251979–1981 87 Schinella, G.R., Tournier, H.A., Prieto, J.M., Mordujovich de Buschiazzo, P., Ríos, J.L., 88 2002. Antioxidant activity of anti-inflammatory plant extracts. Life Sci. 70, 89 1023–1033. Sun, J., Ma, J.S., Jin, J., Wang, H.S., Wen, Q.H., Zhang, H.G., Zhou, Q.L., 2006. 90 Qualitative and quantitative determination of the main components of huan91 glianjiedu decoction by HPLC–UV/MS. Yao Xue Xue Bao 41, 380–384. 92 Tang, L.Q., Wei, W., Chen, L.M., Liu, S., 2006. Effects of berberine on diabetes 93 induced by alloxan and a high-fat/ high-cholesterol diet in rats. J. Ethnopharmacol. 108, 109–115. 94 Tonks, N.K., Neel, B.G., 2001. Combinatorial control of the specificity of protein 95 tyrosine phosphatases. Curr. Opin. Cell. Biol. 13, 182–195. 96 Tse, W.P., Che, C.T., Liu, K., Lin, Z.X., 2006. Evaluation of the anti-proliferative properties of selected psoriasis-treating Chinese medicines on cultured HaCaT 97 cells. J. Ethnopharmacol. 108, 133–141. 98 Turkoski, B.B., 2006. Diabetes and diabetes medications. Orthop. Nurs. 25, 227–231. 99 Wiesmann, C., Barr, K.J., Kung, J., Zhu, J., Erlanson, D.A., Shen, W., Fahr, B.J., Zhong, M., Taylor, L., Randall, M., McDowell, R.S., Hansen, S.K., 2004. Allosteric 100 inhibition of protein tyrosine phosphatase 1B. Nat. Struct. Mol. Biol. 11, 101 730–737. 102 Wu, X., Hardy, V.E., Joseph, J.I., Jabbour, S., Mahadev, K., Zhu, L., Goldstein, B.J., 2003. Protein-tyrosine phosphatase activity in human adipocytes is strongly corre103 lated with insulin-stimulated glucose uptake and is a target of insulin-induced 104 oxidative inhibition. Metabolism 52, 705–712. 105 Xu, H.-Y., Zhang, T.-J., Zhu, X.-Y., Li, Y.-B., 2009. Recent advance on chemical compositions and pharmacodynamic and pharmacokinetic studies of Rhizoma 106 Coptidis. Asian J. Pharmacodyn. Pharmacokinet. 9, 11–25. 107 Yin, J., Chen, M., Tang, J., 2004. Effects of berberine on glucose and lipid metabolism 108 in animal experiment. Chin. J. Diabetes 12, 215–218. Yokozawa, T., Satoh, A., Cho, E.J., Kashiwada, Y., Ikeshiro, Y., 2005. Protective role of 109 Coptidis Rhizoma alkaloids against peroxynitrite-induced damage to renal 110 tubular epithelial cells. J. Pharm. Pharmacol. 57, 367–374. 111 Yuan, L., Tu, D., Ye, X., Wu, J., 2006. Hypoglycemic and hypocholesterolemic effects 112 of Coptis chinensis Franch inflorescence. Plant Foods Hum. Nutr. 61, 139–144. Zhou, H., Jiang, T., Wang, Z., Ren, S., Zhao, X., Wu, W., Jiang, L., Liu, Z., Teng, L., 2014. 113 Screening for potential-α-glucosidase inhibitors in Coptis chinensis Franch 114 extract using ultrafiltration LC–ESI–MSn. Pak. J. Pharm. Sci. 27, 2007–2012. 115 Zinker, B.A., Rondinone, C.M., Trevillyan, J.M., Gum, R.J., Clampit, J.E., Waring, J.F., Xie, N., Wilcox, D., Jacobson, P., Frost, L., Kroeger, P.E., Reilly, R.M., Koterski, S., 116 Opgenorth, T.J., Ulrich, R.G., Crosby, S., Butler, M., Murray, S.F., McKay, R.A., 117 Bhanot, S., Monia, B.P., Jirousek, M.R., 2002. PTP1B antisense oligonucleotide 118 lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice. Proc. Natl. Acad. Sci. USA 99, 11357–11362. 119


Potent protein tyrosine phosphatase 1B (PTP1B) inhibiting constituents from Anoectochilus chapaensis and molecular docking studies.

Canthinone alkaloids are novel protein tyrosine phosphatase 1B inhibitors.

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

Brominated polyunsaturated lipids with protein tyrosine phosphatase-1B inhibitory activity from Chinese marine sponge Xestospongia testudinaria.

Geranylated 2-arylbenzofurans from Morus alba var. tatarica and their α-glucosidase and protein tyrosine phosphatase 1B inhibitory activities.

Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw.

Zinc ions modulate protein tyrosine phosphatase 1B activity.

Protein-tyrosine phosphatase 1B substrates and metabolic regulation.

The mechanism of allosteric inhibition of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase-1B modulates pancreatic β-cell mass.

Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects.

Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells.

Effect of Catnip Charcoal on the In Vivo Pharmacokinetics of the Main Alkaloids of Rhizoma Coptidis.

New triterpenoids with protein tyrosine phosphatase 1B inhibition from Cedrela odorata.

New limonoids from Coptidis Rhizoma-Euodiae Fructus couple.

New compounds from acid hydrolyzed products of the fruits of Momordica charantia L. and their inhibitory activity against protein tyrosine phosphatas 1B.

Rhizoma Coptidis: A Potential Cardiovascular Protective Agent.

Pharmacokinetics of two alkaloids after oral administration of rhizoma coptidis extract in normal rats and irritable bowel syndrome rats.

Inhibitory effect of morin on tyrosinase: insights from spectroscopic and molecular docking studies.

Synthesis and evaluation of several oleanolic acid glycoconjugates as protein tyrosine phosphatase 1B inhibitors.

Design and synthesis of paracaseolide A analogues as selective protein tyrosine phosphatase 1B inhibitors.

Gene deletion of protein tyrosine phosphatase 1B protects against sepsis-induced cardiovascular dysfunction and mortality.

Design, synthesis and molecular modelling studies of novel 3-acetamido-4-methyl benzoic acid derivatives as inhibitors of protein tyrosine phosphatase 1B.

Regulation of growth hormone induced JAK2 and mTOR signalling by hepatic protein tyrosine phosphatase 1B.