Hoang Duc Manh¹, Tran Minh Ngoc¹, Do Thi Ha¹, Nguyen Minh Khoi¹,

Le Viet Dung¹, Mai Kien Dinh², Jong Seog Ahn^3,*,

¹Institute of Medicinal Material, Vietnam; ²Vietnam Institute of Seas and Islands

³Korea Research Institute of Bioscience and Biotechnology (KRIBB);

*Corresponding author: jsahn@kribb.re.kr

(Received September, 15^th, 2015)

Summary

Protein Tyrosine Phosphatase 1B Inhibitory Effect of Oxotomaymycin Devivatives from Actinomycetes sp. AN1827

Protein tyrosine phosphatase 1B (PTP1B) is considered as a potential therapeutic target for the treatment of diabetes and obesity. Bioassay-guided fractionation of the chloroform-soluble fraction of Actinomycetes sp. AN1827, using an in vitro PTP1B inhibitory assay led to the identification of oxotomaymycin (1) and oxotomaymycin methyl ether (2). This study suggested that the oxotomaymycin derivatives from Actinomycetes sp. might be a considerable source of PTP1B inhibitor. This study would be benefit for the development of anti-diabetes or anti-obesity agents.

Keywords: Actinomycetes, Diabetes, Protein tyrosine phosphatase-1B (PTP1B), Oxotomaymycin.

The increased incidence of type 2 diabetes mellitus (T2DM) and obesity in the population is an alarming rate world-wide, was previously observed primarily in adult populations, associated with a sedentary lifestyle, but has now also become a medical problem in children [1,2]. The relationship between obesity and T2DM has a polygenetic component and is associated with insulin resistance [3]. Insulin resistance is evident in many tissues that are important for glucose homeostasis, including muscle, liver and - more recently - in fat and at the level of the central nervous system. Metabolic insulin signal transduction occurs through activation of the insulin receptor (IR), including autophosphorylation of tyrosine (Tyr) residues in the insulin-receptor activation loop [4]. Several protein tyrosine phosphatases (PTPs), comprising receptor protein tyrosine phosphatase (rPTP-α), leukocyte antigen-related tyrosine phosphatase (LAR), SH2-domain-containing phosphotyrosine phosphatase (SHP2) and protein tyrosine phosphatase 1B (PTP1B) have been implicated in the dephosphorylation of the IR [5]. There is substantial evidence supporting PTP1B as the critical PTP controlling insulin signaling pathway. PTP1B seems to be a key regulator of insulin-receptor activity that acts at the insulin receptor and at downstream signaling insulin receptor substrate proteins [6]. Therefore, it has been suggested that compounds that reduce PTP1B activity or expression levels could not only be used for the treatment of type 2 diabetes but also obesity. In our efforts to discover anti-diabetes agents from microorganisms, we found that an EtOAc-soluble extract of Actynomycetes sp. AN1827 inhibited PTP1B activity (75% inhibition at 30 mg/ml). The present report describes the isolation and characterization of compounds from the EtOAc extract of Actynomycetes sp. AN1827 broth and their inhibitory activity against PTP1B enzyme.

2. Experiment

2.1 Actinomycetes material

Strain AN1827 was collected from Bongsalli, Boseongup, Boseong-gun, Chungnam province, Korea in August, 2008. Cultural characteristics and physiological properties of isolates were studied by using media described by Shirling and Gottlieb [7]. The organism grew on Bennett agar for 6 days at 28°C to form colonies with diameter of 25-36 mm. The sample was deposited at the Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB).

2.2. General experimental procedures

A slant culture of AN1827 grown on Bennett agar was inoculated into 1000 mL Erlenmeyer flasks containing 200 mL of a Bennett broth. The flasks were shaken on rotary shaker for 2 days at 28°C. Two hundred milliliters of the seed culture was transferred into 8 liters of a production medium (Bennet’s broth) in a 16-liter jar fermentor. The fermentation was carried out at 28°C for 7 days. The filtered culture broth was extracted with n-hexane, EtOAc, BuOH. Bioactivity-guided fractionation of the EtOAc-soluble fraction shown highest activity and the resulting organic extract was subjected to C₁₈ functionalized silica gel flash column chromatography, eluting with a stepwise gradient consisting of MeOH in H₂O (20% to 100% MeOH with 10% increment for each step; 500 mL each). The fraction eluted at 30% and 40% MeOH showed stronger activity while compared with other eluted fractions (data not shown). The fraction eluted with 40% MeOH was subjected to preparative HPLC (RS Tech Optima Pak^Ò C₁₈ column (10 × 250 mm, 10 mm particle size), CH₃CN/H₂O 29:71, 12.5-13.8 min) to give oxotomaymycin (1, 5.4 mg). The fraction eluted with 30% MeOH was subjected to preparative HPLC (RS Tech Optima Pak^Ò C₁₈ column (10 × 250 mm, 10 mm particle size, CH₃CN/H₂O 29:71, 30.0-31.5 min) to give oxotomaymycin methyl ether (2, 4.2 mg).

2.3. Protein tyrosine phosphatase 1B inhibitory activity assay

The PTP1B (human, recombinant) was purchased from BIOMOL^® International LP (USA) and the enzyme activity was measured using p-nitrophenyl phosphate (pNPP) as a substrate. Each 96-well (final volume: 200 μL) were added 2 mM pNPP and PTP1B (0.05–0.1 μg) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT) with or without test compounds (1-2). After incubation at 37°C in 30 minutes, the reaction was then terminated with 10N NaOH. The quantity of p-nitro phenol produced was estimated via measurements of absorbance at 405 nm. The non-enzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of the PTP1B enzyme.

3. Result and discustion

Bioactivity-guided fractionation of the n-hexane, EtOAc and BuOH soluble fractions of Actinomycetes sp. using an in vitro PTP1B inhibitory assay, the EtOAc soluble fraction showed higher activity with 75% inhibition at 30 μg/mL comparison with n-hexane and BuOH solution fractions (less than 50% inhibition at 30 μg/mL), respectively. The EtOAc soluble fraction (1.1g) was subjected to C₁₈ functionalized silica gel flash column chromatography and using HPLC to purified 1 and 2 as described in the general experimental procedures.

Compound 1 was obtained as crystal. The IR spectrum of 1 showed bands in the range between 3300 and 3400 cm^-1 due to hydroxyl and amide (NH) group and two bands at 1685 and 1610 cm^-1 due to amide carbonyl group. The ¹³C NMR spectra of 1 has showed 15 carbons and mass spectra shown 289.4 [M+H]⁺ and a molecular formula is C₁₅H₁₆O₄N₂. The ¹H and also the ¹³C NMR spectra showed the amide carbonyl carbon (δ_C 167.9 and 172.1), the oxygen-bearing methane carbon (δ_C 56.7) and the nitrogen-bearing carbon (δ_C 63.1), N-CH₂ (δ_C 52.6). These suggested that compound 1 indicated resonances typical of tomaymycin which was isolated from Streptomyces achromogenes var. Tomaymycetes [8]. Based on those data and in comparison with literature [8], compound 1 was identified as oxotomaymycin (Figure 1).

Table 1. ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) data of compounds 1 and 2 (recorded in CD₃OD)

In order to screen for PTP1B inhibitors of the isolated compounds from Actinomycetes sp. AN1827, all the isolated compounds were assayed for their inhibitory activity against PTP1B, and the results are presented in Table 2. Compounds 1 and 2 exhibited significantly inhibitory effects against PTP1B with IC₅₀ values of 25.1 ± 1.1 and 31.3 ± 0.6 mM, respectively. The known PTP1B inhibitors, RK-682 (IC₅₀ = 5.0 ± 0.5 mM) and ursolic acid (IC₅₀ = 3.9 ± 0.3 mM) [9] were used as positive controls in this assay.

Table 2. Inhibitory activity of compounds 1–2 against PTP1B

^a IC₅₀ values were determined by regression analyses and expressed as mean ± SD of three replicates.

^b Positive control ⁹

PTP1B is known to have several binding sites, such as those electrostatic, hydrophobic, and hydrogen-bond binding, and to have several N-terminals that are capable of binding to an acidic site. As with the insulin-signaling pathway, the leptin-signaling pathway can be attenuated by PTPs and compelling evidences implies the involvement of PTP1B in this process [10,11]. Therefore, is has been suggested that compounds that reduce PTP1B activity or expression levels could be used for treating both type 2 diabetes and obesity. Tomaymycin exhibited a strong inactivating effect against various bacteriophages and animal viruses but otomaymycin did not show such activity [8]. In our results, oxotomaymycin (1, IC₅₀ = 21.5 ± 1.1 mM) showed somewhat stronger than oxotomaymycin methyl ether (2, IC₅₀ = 31.3 ± 0.6 mM). Although the structure-activity relationship of these compounds were not thoroughly investigated, due to the presence of methoxyl group at C-7 position in both isolated compounds, the difference in the methoxyl group at C-8 in 2 instead for hydroxyl group at 1 was responsible for the change of activity. This result was attributed to the increased polarity of the compounds induced by the presence of hydroxyl groups, which may have decreased the affinity for a hydrophobic site of the enzyme.

4. Conclusion

This study for the first time determined that an oxotomaymycin derivative is inhibiting PTP1B enzyme. According to these results, it is suggest that the metabolites from Actinomycetes sp. AN1827 might be considerable source to treatment of diabetes and obesity. Therefore, further investigation and optimization of these derivatives might enable the preparation of new PTP1B inhibitors potentially useful in the treatment of type 2 diabetes and obesity.