Inhibition of VHR Dual-Specificity Protein Tyrosine Phosphatase Activity by Flavonoids Isolated from Scutellaria baicalensis: Structure-Activity Relationships

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Three flavonoids: norwogonin, dihydronorwogonin and baicalein, were isolated from the roots of Scutellaria baicalensis, as potential inhibitors of VHR dual-specificity protein tyrosine phosphatase (DS-PTPase). Norwogonin (IC₅₀ = 1.1μM), dihydronorwogonin (IC₅₀ = 2.9μM) and baicalein (IC₅₀ = 2.4μM) showed potent inhibitory activity toward VHR, but had no inhibitory activity against T-cell protein tyrosine phosphatase or serine/threonine protein phosphatase 1. From comparisons to the inhibitory activities of other similar flavonoids, it could be suggested that the presence of a hydroxy group in the B ring of flavonoids interferes with the inhibitory activity toward VHR DS-PTPase.

Reversible phosphorylation of protein is a regulation mode of cellular processes. Even though earlier studies have focused on the protein phosphorylation induced by protein kinases, it is now clear that protein dephosphorylation plays an equally integral role in the cellular signaling pathways. There are two families of protein phosphatases. Protein serine/threonine phosphatase (PPase) catalyzes the dephosphorylation of phosphoserine and phosphothreonine residues, whereas protein tyrosine phosphatase (PTPase) removes phosphates from phosphotyrosine residues of substrate proteins. Dual-specificity protein tyrosine phosphatase (DS-PTPase) is a new subfamily of PTPase and has catalytic activity on both tyrosine and serine/threonine residues even though PTPase acts just on tyrosine residue. The vaccinia open reading-frame H1-related protein phosphatase (VHR) was the first DS-PTPase identified in humans [1]. VHR has a central regulation role in cell cycle progression and intracellular signaling mediated by the mitogen-activated protein (MAP) kinase [2], [3]. Although there are many known inhibitors of PPase and PTPase, only few compounds were reported as inhibitors of DS-PTPase [4]. So, DS-PTPase-specific inhibitors may provide further insight into the regulatory role of cellular signaling, and might be candidates for therapeutics, such as anticancer and immunomodulating agents.

During the search for inhibitors of VHR, from medicinal plants, three flavonoids: norwogonin (1), dihydronorwogonin (2) and baicalein (3), were isolated as potential inhibitors of VHR from Scutellaria baicalensis (Labiatae). The roots of this plant are widely used as a medicinal herb against respiratory tract infections, chronic bronchitis, scarlet fever, infectious hepatitis and biliary tract infections [5].

The structures of the three isolated compounds (1, 2 and 3), from the roots of Scutellaria baicalensis, and other common flavonoids such as luteolin (4), baicalin (5), quercetin (6), naringenin (7), hesperetin (8), daidzein (9) and genistein (10) are shown in Fig. [1]. Their inhibitory activities toward VHR DS-PTPase, T-cell PTPase and PPase1 were measured, and are summarized in Table [1]. Compared to the positive control, RK-682 (IC₅₀ = 2 μM), as a competitive inhibitor of VHR DS-PTPase [6], [7], compounds 1, 2 and 3 also showed strong VHR DS-PTPase inhibitory activities, with IC₅₀ = 1.1 μM, 2.9 μM and 2.4 μM, respectively, but the other flavonoids showed no inhibitory activities toward VHR DS-PTPase. Moreover, compounds 1, 2 and 3, and all the other flavonoids did not inhibit T-cell PTPase, or PPase1, so these isolated compounds should be proposed to have specific inhibitory activity toward DS-PTPase.

Taking into account the structure of several flavonoids, it could be suggested that the absence of a hydroxy group on the B ring of the flavonoids 1, 2, and 3 is important for their inhibitory activity toward VHR DS-PTPase. In contrast, the presence of a hydroxy group on the B ring such as in flavone (4), flavonol (6), flavanones (7, 8), isoflavones (9, 10) dramatically reduced their inhibitory activity toward VHR DS-PTPase. In addition, compound (5) in which glucuronic acid substituted for the hydroxy group at the C-8 position of the A ring, showed no inhibitory activity toward VHR DS-PTPase, even though a hydroxy group was not present in the B ring.

As a result, the isolated compounds, norwogonin (1), dihydronorwogonin (2), baicalein (3), from the roots of Scutellaria baicalensis, could be specific inhibitors of VHR DS-PTPase. It might be expected that these compounds would be useful in exploring DS-PTPase-mediated signaling pathways in cellular proliferation and differentiation, and their potential usefulness will be further studied.

Materials and Methods

The roots of Scutellaria baicalensis Georgi were purchased from the herbal medicine cooperative association of Daejeon, Korea, in October 1999. A voucher specimen No. 062 was deposited in our laboratory. The authenticity of the plant was confirmed by G. Bai, Professor of Chungnam National University.

Glutationine-S-transferase (GST)-VHR fusion protein (over 98 % purity) was prepared as described by Ishibashi et al. [8] and T-cell protein tyrosine phosphatase was purchased from New England BioLabs (Beverly, USA). Protein phosphatase 1 catalytic subunit (α-isoform), para-nitrophenyl phosphate (p-NPP), naringenin (approx. 95 %), luteolin, quercetin, hesperetin (minimum 95 %), genistein (approx. 98 %), daidzein (minimum 98 %) and baicalin were obtained from Sigma Chemical Co (St. Louis, USA). All other chemicals were the highest pure grade available.

The dried roots of the S. baicalensis (1 kg) were extracted with methanol at room temperature for a week. The methanolic extract (35 g) was extracted with ethyl acetate. The ethyl acetate layer (15 g) was subjected to column chromatography on a silica gel (Merck, 0.040 - 0.063 mm, 200 g) and eluted with ethyl acetate and a gradient of ethyl acetate-methanol (20 : 1→ 1 : 1, v/v). The elution gave 8 fractions (each of 800 ml) and each fraction was monitored by in vitro dephosphorylation activity. Fractions 5 - 6 (acetate-methanol, 5 : 1) with the highest inhibitory activity were combined and evaporated to give 8 g solid material. The active fractions were applied to rechromatography over RP-18 (YMC*GEL, ODS-A, 60 Å, 70/230 mesh, 80 g). Elution with a step gradient of methanol-H₂O (30 % MeOH→ 50 % MeOH→ 70 % MeOH→ 90 % MeOH→ 100 % MeOH) gave 10 fractions (300 ml) and the active fractions 5 - 6 (methanol-H₂O, 7 : 3) were evaporated (5g) and then applied to the Sephadex LH-20 with a chloroform-n-hexane-methanol (2 : 3:1) as an eluent collecting 20 ml fractions. Fraction 6 - 22 with inhibitory activity was purified on an HPLC system (Shimadzu, SPD-6A) with the following conditions: a J'sphere ODS-H80, 150 × 10 mm, S-4 μm column, using methanol-H₂O (7 : 3) as eluent at a flow rate 4 ml/min. The compounds; 1 (19 mg), 2 (22 mg) and 3 (78 mg), were over 95 % purity. The chemical structures of isolated compounds were determined using ¹H-NMR, ¹³C-NMR, HMQC, EI-mass spectrometry and by comparison with published data [9], [10], [11]. Compound 1 was identified as norwogonin, compound 3 as baicalein and compound 2 as the (S)-form dihydronorwogonin [12].

DS-PTPase was assayed with the GST-VHR fusion enzyme, overexpressed in Escherichia coli [4]. The reaction mixture containing GST-VHR fusion enzyme, 10 mM p-NPP and assay buffer (50 mM succinate, 1 mM EDTA, 140 mM NaCl, 0.05 % Tween 20, pH 6.0) was incubated at 30 °C for 1 hour. The reaction was terminated by the addition of 1 N NaOH, and the dephosphorylation activity measured at 410 nm [13]. PTPase activities were measured using a T-cell PTPase from New England BioLabs, Inc. Dephosphorylation activity was determined in a reaction mixture containing the assay buffer (25 mM imidazole, 50 mM NaCl, 2.5 mM Na₂EDTA, 5 mM DTT, pH 7.0), 100 μg/ml BSA and artificial substrate, 10 mM p-NPP. After 1-hour incubation at 37 °C, the reaction was stopped by the addition of 1 N NaOH, and absorbance at 410 nm was measured [14]. The catalytic activity of PPase1, from Sigma Co., was measured at 37 °C using p-NPP as a substrate. Reactions were performed for 30 min in the assay buffer (50 mM Tris-HCl, 0.1 % β-mercaptoethanol, 1 mM Na₂EDTA, 1 mM MnCl₂, 20 mM MgCl₂, pH 7.6). The reaction was stopped by the addition of 1 N NaOH, and the p-NPP hydrolyzed measured by absorbance at 410 nm [15].

Acknowledgements

This research was supported by a grant from the Ministry of Science and Technology in Korea.

References

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• 14 Zander N F, Lorenzen J A, Cool D E, Tonks N K, Daum G, Krebs E G, Fischer E H. Purification and characterization of a human recombinant T-cell protein tyrosine-phosphatase from a baculovirus expression system.  Biochemistry. 1991;  30 6964-70
  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar

Jong Seog Ahn, Ph. D.

Korea Research Institute of Bioscience and Biotechnology (KRIBB)

P.O. Box 115

Yusong

Daejeon 305-600

South Korea

Phone: +82-42-860-4312

Fax: +82-42-860-4595

Email: jsahn@mail.kribb.re.kr

References

• 1 Denu J M, Zhou G, Wu L, Zhao R, Yuvaniyama J, Saper M A, Dixon J E. The purification and characterization of a human dual-specific protein tyrosine phosphatase.  J Biol Chem. 1995;  270 3796-803
  CrossrefPubMedSearch in Google Scholar
• 2 Todd J L, Tanner K G, Denu J M. Extracellular regulated kinase (ERK) 1 and 2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR.  J Biol Chem. 1999;  274 13 271-80
  PubMedSearch in Google Scholar
• 3 Alonso A, Saxena M, Williams S, Mustelin T. Inhibitory role for dual specificity phosphatase VHR in T cell antigen receptor and CD28-induced Erk and Jnk activation.  J Biol Chem. 2001;  276 4766-71
  CrossrefPubMedSearch in Google Scholar
• 4 Sodeoka M, Sampe R, Kojima S, Baba Y, Usui T, Ueda T, Osada H. Synthesis of a tetronic acid library focused on inhibitors of tyrosine and dual-specificity protein phosphatases and its evaluation regarding VHR and cdc25B inhibition.  J Med Chem. 2001;  44 3216-22
  CrossrefPubMedSearch in Google Scholar
• 5 Huang K C, The Pharmacology of Chinese H erbs. Antibacterial, antiviral, and antifungus herbs. CRC Press Tokyo; 1993: 287-91
  Search in Google Scholar
• 6 Hamaguchi T, Sudo T, Osada H. RK-682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle progression at G₁phase.  FEBS Letters. 1995;  372 54-8
  CrossrefPubMedSearch in Google Scholar
• 7 Usui T, Kojima S, Kidokoro S, Ueda K, Osada H, Sodeoka M. Design and synthesis of a dimeric derivative of RK-682 with increased inhibitory activity against VHR, a dual-specificity ERK phosphatase: implications for the molecular mechanism of the inhibition.  Chem & Biol. 2001;  8 1209-20
  PubMedSearch in Google Scholar
• 8 Ishibashi T, Bottaro D P, Chan A, Miki T, Aaronson S A. Expression cloning of a human dual-specificity phosphatase.  Proc Natl Acad Sci USA. 1992;  89 12 170-4
  PubMedSearch in Google Scholar
• 9 Tomimori T, Miyaichi Y, Imoto Y, Kizu H, Tanabe Y. Studies on the constituents of Scutellaria species. II. On the flavonoid constituents of the root of Scutellaria baicalensis Georgi.  Shoyakugaku Zasshi. 1983;  103 607-11
  PubMedSearch in Google Scholar
• 10 Jacupovic J, Zdero C, Grenz M, Tsichritzis F, Lehmann L, Hashemi-Nejad S M, Bohlmann F. Twenty-one acylphloroglucinol derivatives and further constituents from South African Helichrysum species.  Phytochemistry. 1989;  28 1119-31
  CrossrefPubMedSearch in Google Scholar
• 11 Tomimori T, Miyaichi Y, Imoto Y, Kizu H. Studies on the constituents of Scutellaria species. V. On the flavonoid constituents of ”Ban Zhi Lian”, the whole herb of Scutellaria baicalensis rivularis Wall.  Shoyakugaku Zasshi. 1984;  38 249-52
  PubMedSearch in Google Scholar
• 12 Hu B H, Liu Y L. Studies on the structures of new flavonoids from the root of Scutellaria amoena .  Acta Pharmaceutica Sinica. 1989;  24 200-6
  PubMedSearch in Google Scholar
• 13 Zhou G, Denu J M, Wu L, Dixon J E. The catalytic role of Cys¹²⁴ in the dual specificity phosphatase VHR.  J Biol Chem. 1994;  269 28 084-90
  PubMedSearch in Google Scholar
• 14 Zander N F, Lorenzen J A, Cool D E, Tonks N K, Daum G, Krebs E G, Fischer E H. Purification and characterization of a human recombinant T-cell protein tyrosine-phosphatase from a baculovirus expression system.  Biochemistry. 1991;  30 6964-70
  CrossrefPubMedSearch in Google Scholar
• 15 Zhang Z, Bai G, Deans-Zirattu S, Browner M F, Lee E YC. Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli .  J Biol Chem. 1992;  267 1484-90
  PubMedSearch in Google Scholar

Jong Seog Ahn, Ph. D.

Korea Research Institute of Bioscience and Biotechnology (KRIBB)

P.O. Box 115

Yusong

Daejeon 305-600

South Korea

Phone: +82-42-860-4312

Fax: +82-42-860-4595

Email: jsahn@mail.kribb.re.kr