Printer Friendly

Antiviral Metabolites from Saudi Red Sea Sponge Hyrtios species as Inhibitors of HCV NS3/4A Protease.

Byline: Usama Wahid Hawas

Summary

Indole and AY-carboline alkaloids were isolated and identified from sponge, Hyrtios sp, collected from the Saudi Arabia Red Sea coast. The structure of the isolated compounds was elucidated on the basis of mass spectrometry and detailed NMR spectroscopy. The biological properties of the isolated metabolites were explored for anti-HCV protease as well as antimicrobial activity. The isolated alkaloids showed strong to moderate inhibition of HCV NS3/4A protease with IC50 ranging from 3.94 to 45.98g/ml.

Key words: Red Sea, Hyrtios sp., Indole and AY-carboline, Antimicrobial, HCV protease.

Introduction

The increasing number of people in the world having health problems caused by deadly diseases such as Hepatitis C virus (HCV) is a cause for alarm. The virus is a growing public health problem, affecting an estimated more than 3% of the world's population [1], and causing liver fibrosis, which may lead to cirrhosis and eventually development of liver cancer [2]. No vaccine exists to prevent HCV infection and interferon-alpha, either alone or in combination with the ribavirin is the only licensed therapy. For chronic HCV infection, the therapy is expensive, associated with poor response rates (40-50%), and laden with significant side effects such as depression, psychoses, and extreme fatigue [3].

The recent clinical trials of a specific HCV protease inhibitor (telaprevirin) in combination with peginterferon and ribavirin have shown improved rates of sustained virologic response to as high as 65%, but unfortunately the rate of anemia, nausea, diarrhea, pruritis, and rash has been also increased [4]. The (HCV) viral serine protease and RNA polymerase have shown to be excellent targets for selective anti-HCV chemotherapy. The clinical studies with a limited number of HCV protease and polymerase inhibitors resulted in encouraging more studies [5].

Sponges belong to the genus Hyrtios have proven to be a rich source of bioactive diverse secondary metabolites of different classes. Previous chemical investigations of different Hyrtios sp. revealed the presence of sesterterpenes [6, 7], sesquiterpenes [8, 9], macrolides [10], indole and AY- carboline alkaloids [11-13]. Many of these metabolites displayed interesting biological activities [6-15]. Spongistatins are the most important metabolites of the genus Hyrtios discovered to date, which have been showed powerful anticancer activity [15].

In the course of a program aiming at the isolation of bioactive natural products from marine sponges, Hyrtios sp was collected from Saudi Arabia Red Sea site. Here, we describe the isolation and structural characterization of indole and AY-carboline alkaloids from the EtOAc extract of Hyrtios sp. The isolated pure metabolites were evaluated for their antimicrobial activity, as well as for their inhibitory effect on HCV NS3-NS4A protease using a SensoLyteTM 520 HCV protease assay kit.

Experimental

General Experimental Procedures

NMR experiments were measured on Jeol (500 MHz). The chemical shifts were expressed in d (ppm) using DMSO-d6 as solvent and TMS as internal reference. ESI-MS were recorded on a Waters-Micromass Quattro Premier Triple Quadrupole mass spectrometer. UV-VIS spectra were recorded on a Perkin-Elmer Lambda 25 UV/VIS spectrometer. Rf-values were measured on Polygram SIL F/UV254 (Merck-pre-coated sheets). Size exclusion chromatography was done on Sephadex LH-20 (Pharmacia).

Enzymes and Chemicals

The SensolyteTM 520 HCV protease assay kit Fluorimetric (Lot# AK71145-1020), HCV NS3- NS4A protease, HCV NS3-NS4A protease inhibitor 2 (cat# 25346), and SensolyteTM Green protease assay kit Fluorimetric (Lot# AK71124-1011) were purchased from AnaSpec (San Jose, CA, USA). Soybean trypsin-chymotrypsin inhibitor was purchased from Sigma Aldrich (St. Louis, MO, USA). FalconTM MicrotestTM 384-well 120 l black assay plates, non-sterile, no lid, were purchased from Becton Dickinson (Tokyo, Japan).

Animal Material

The sponge material of Hyrtios sp. was collected by hand using scuba at depths between 20-25 m off Jeddah at the Red Sea, Saudi Arabia, in March 2013. The collected sponge was identified in the Department of Marine Biology, Faculty of Marine Science, King Abdel Aziz University.

Extraction and Isolation

The frozen sponge materials (600 g, wet, wt) were extracted exhaustively with methanol then CH2Cl2 at room temperature. The combined organic extracts were concentrated under reduced pressure and suspended in H2O/MeOH (9:1). The resulted mixture was extracted with n-hexane to give 2.1 g, and then extracted successively with CH2Cl2, EtOAc, and finally n-BuOH. Each of the extracts was concentrated under reduced pressure to give 1.1 g of CH2Cl2 extract, 1.4 g of EtOAc extract, and 2.1 g of n-BuOH extract, respectively.

The EtOAc soluble fraction was subjected to silica gel column chromatography using gradient elution with n-hexane/EtOAc/MeOH mixtures of increasing polarity. Nine fractions were obtained and the soluble fractions were repeatedly chromatographed on preparative silica gel followed by Sephadex LH-20 eluted with MeOH to yield compounds 1 (8 mg), 2 (6 mg), 3 (9 mg), 4 (11 mg), 5 (7 mg), and 6 (5 mg).

5-Hydroxy-1H-indole-3-carboxylic acid (1)

Orange powder. TLC, Rf = 0.32 (butanol: acetic acid: water, 4:1:5, upper phase). 1H NMR (DMSO-d6, 500 MHz 12.08 (1H, s, 3-COOH), 9.48 (1H, br, 1-NH), 8.45 (1H, s, H-2), 7.62 (1H, d, J = 8 Hz; H-6), 7.60 (1H, br; H-4), 7.12 (1H, d, J = 8 Hz; H-7). 13C NMR (DMSO-d6, 125 MHz): 168.6 (3- COOH), 156.7 (C-5), 136.2 (C-2), 134.2 (C- 7a), 128 (C-3a), 119.2 (C-6), 114.1(C-7), 111.6 (C-3), 108.4 (C-4). ESI-MS: m/z 177 [M+H]+.

5-Hydroxy-1H-indole-3-carboxylic acid methyl ester (2)

Orange powder. TLC, Rf = 0.48 (n-butanol: acetic acid: water, 4:1:5 upper phase). 1H NMR (DMSO-d6, 500 MHz): 9.48 (1H, br, 1-NH), 8.45 (1H, s, H-2), 7.60 (1H, m, H-4/6), 7.12 (1H, br, H-7), 3.87 (3H, s, 5-OCH3). 13C NMR (DMSO-d6, 125 MHz): 168.2 (3-COOH), 156.7 (C-5), 136.4 (C-2), 134.2 (C-7a), 128 (C-3a), 119.1 (C-6), 113.1(C-7), 111.6 (C-3), 107.4 (C-4), 55.3 (5-OCH3). EI-MS: m/z 191 [M].

6-Hydroxy-1,2,3,4-tetrahydro-AY-carboline-3- carboxylic acid (3)

Greenish powder. TLC, Rf = 0.36 (n- butanol: acetic acid: water, 4:1:5 upper phase); UV max (MeOH): 228, 271, 355. 1H NMR (DMSO-d6, 500 MHz): 10.62 (1H, s, 1-NH), 7.08 (1H, d, J = 8.5 Hz; H-8), 6.89 (1H, s, H-5), 6.55 (1H, d, J = 8.5 Hz; H-7), 3.42 (2H, m, 1-CH2), 3.01 (1H, m, H-3), 2.88 (2H, m, 4-CH2). 13C NMR (DMSO-d6, 125 MHz): 171.3 (3-COOH), 150.9 (C-6), 131.3 (C- 9a), 128.4 (C-8a), 125 (C-4b), 112.1 (C-8), 111.8 (C- 7), 109 (C-4a), 102.9 (C-5), 55.1 (C-3), 38.6 (C-1), 27.8 (C-4). ESI-MS: m/z 233 [M+H]+.

Hyrtioreticulin A (4)

White powder; [a]25 +18 (c 1.0, MeOH). 1H NMR (DMSO-d6, 500 MHz): 11.37 (1H, s, 3- COOH), 9.13 (1H, br, 1-NH), 8.17 (1H, s, H-13), 7.64 (1H, br, H-12), 7.29 (1H, d, J = 8 Hz; H-8), 6.99 (1H, d, J = 2 Hz; H-5), 6.88 (1H, d, J = 8 Hz; H-7), 4.9 (1H, m, H-1), 3.51 (1H, m, H-3), 3.19/2.45 (2H, m, 10-CH2), 2.99/2.46 (2H, m, 4-CH2). 13C NMR (DMSO-d6, 125 MHz): 167.7 (3-COOH), 158.1 (C- 6), 136.4 (C-13), 132.2 (C-8a), 132.1 (C-11), 129.1 (C-9a), 128.4 (C-4b), 121.2 (C-12), 119.3 (C-7), 111.7 (C-8), 109 (C-4a), 106 (C-5), 53.8 (C-3), 56 (C-1), 30.3 (C-10), 25.2 (C-4). (+) ESI-MS: m/z 313 [M+H]+.

Hyrtioreticulin B (5 )

White powder; [a]25 -39 (c 1.0, MeOH). 1H NMR (DMSO-d6, 500 MHz): 11.37 (1H, s, 3- COOH), 9.11 (1H, br, 1-NH), 8.18 (1H, s; H-13), 7.62 (1H, br, H-12), 7.28 (1H, d, J = 8 Hz; H-8), 6.99 (1H, d, J = 2 Hz; H-5), 6.88 (1H, d, J = 8 Hz; H-7), 4.8 (1H, m, H-1), 3.51 (1H, m, H-3), 3.18/2.44 (2H, m, 10-CH2), 2.99/2.46 (2H, m, 4-CH2). 13C NMR (DMSO-d6, 125 MHz): 167.7 (3-COOH), 158 (C- 6), 136.4 (C-13), 132.2 (C-8a), 132.1 (C-11), 129.1 (C-9a), 128.4 (C-4b), 121.2 (C-12), 119.3 (C-7), 111.7 (C-8), 109 (C-4a), 106 (C-5), 56 (C-3), 63 (C- 1), 28.8 (C-10), 23.5 (C-4). (+) ESI-MS m/z 313 [M+H]+.

Cinnamic acid (6)

White powder. Rf = 0.69 (ethyl acetate:methanol:water 70:5:1). 1H NMR (DMSO-d6, 500 MHz): 6.2 (1H, d, J = 16 Hz, H-8), 7.52 (2H, d, J = 8 Hz m, H-2/6), 7.20 (2H, d, J = 8 Hz, H-3/5), 7.48 (d, J = 16 Hz, H-7); 13C NMR (DMSO-d6, 125 MHz): 168.0 (C-9), 144.9 (C-4), 134.1 (C-7), 126 (C-2/6), 121 (C-1), 116.2 (C-3/5), 115.2 (C-8).

Antimicrobial Assays

The antimicrobial spectrum and activity of the isolated compounds 1-5 from EtOAc extract were determined by the agar plate diffusion assay [16].

10l of the tested sample was then applied to sterile paper disc (Whatman) of 6 mm diameter, placed on the surface of indicator test plate and incubated at the temperature that permitted optimal growth of the test organisms. For determination of antibacterial and antifungal activities, indicator bacteria were grown overnight in LB medium, yeasts were grown in YPG medium (10 g/l yeast extract, 10 g/l peptone, 100 g/l glucose) for 24 h, and fungi were grown in potato dextrose agar (PDA) for 3-4 days. The minimum inhibition concentration (MIC) of the pure compounds was expressed as g/ml by the dilution method.

Assay for Determination of HCV Protease Inhibitory Activity

Samples of 2 l of a compound dissolved in dimethyl sulfoxide (DMSO) were placed in each well of a 384-well microplate, then 8 l of recHCV PR (0.5 g/ml) were added, and the plate was briefly agitated. Finally, 10 l of the freshly prepared substrate [Ac-Asp-Glu-Dap (QXLTM520)-Glu-Glu- Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH2] were added with sequential rotational shaking. The reaction mixture was incubated for 30 min at 37C. The fluorimetric analyses were performed on an automated TECAN GENios plate reader (MAnnedorf, Switzerland) with excitation wavelength at 485 nm and emission at 530 nm. Each compound was tested in triplicate. HCV PR inhibition (%) was calculated using the following equation: % Inhibition = (Fsubstrate Ftest compound) x 100/Fsubstrate where Fsubstrate is the fluorescence of the substrate and enzyme without test compounds, and Ftest compound is the fluorescence of the assay mixture with the added compound.

Results and Discussion

Characterization of Isolated Compounds

A combination of column chromatography on silica gel and Sephadex LH-20 of the EtOAc extract of Hyrtios sp. The EtOAc extract was submitted to a combination of silica gel and Sephadex LH-20 column chromatography (CC) to afford six known metabolites (Fig. 1). Compounds 1- 5 were detected by TLC on silica gel as yellow and dark spots under UV light change to red by spraying with Ehrlich's reagent. These UV absorbing spots were tentatively identified as indole and AY-carboline alkaloids due to their color reactions.

NMR spectroscopy (1D- and 2D-NMR) and mass spectrometry (MS) were extensively used in structural elucidation. The isolated compounds were identified based on the spectral analyses and comparison with literature data. The isolated compounds were identified as two indoles, 5- hydroxy-1H-indole-3-carboxylic acid (1) and 5- hydroxy-1H-indole-3-carboxylic acid methyl ester (2) [17]; three AY-carboline derivatives, 6-hydroxy- 1,2,3,4-tetrahydro-AY-carboline-3-carboxylic acid (3), hyrtioreticulin A (4) and hyrtioreticulin B (5) [18]; and cinnamic acid (6). The compounds 1, 3 and 6 were isolated for the first time from Hyrtios genus.

HCV NS3-NS4A Protease Inhibition

The isolated compounds 1-5 from the ethyl acetate extract were tested for their inhibitory activity against HCV PR using HCV NS3 protease inhibitor 2 as a positive control [19]. The isolated compounds showed potent activity against HCV NS3-NS4A protease with IC50 values from 3.94 to 42.98g/ml (Table-1). Compounds 1 and 2 were strongly inhibitory with IC50 values of 3.49 and 4.25g/ml, respectively, while compounds 3 and 4 were mild inhibitory with an IC50 values of 31.45 and 42.98 g/ml, respectively. Compound 5 was inactive as inhibitor of HCV PR as compared to other alkaloids. These results demonstrated that the HCV PR activity of indole carboxylic acid (1) was decreased through esterfication in compound 2. Furthermore, AY- carboline carboxylic acid (3) is more active than its derivative at C-1 in compounds 4 and 5.

These findings indicated that the inhibitory activity against HCV protease for these compounds would be reinforced by the increasing of free carboxylic group in indole structure and weakened by he blockade of the methylene group in AY-carboline structure.

Table-I: Inhibition of HCV NS3-NS4A protease by compounds 1-5.

###Isolated compounds###HCV protease inhibitory activity (%) (IC50 g/ml)

###Indole carboxylic acid (1)###3.94 1.9 (14.2 M)

###Indole-methyl ester (2)###4.25 1.3 (14.5 M)

###-Carboline carboxylic acid (3)###31.45 1.4 (135.5 M)

###Hyrtioreticulin A (4)###42.98 2.5 (137.8 M)

###Hyrtioreticulin B (5)###greater than 1000

###HCV-I2###1.5 0.5 (1.64 M)

Antimicrobial activity

The isolated alkaloids 1-5 were subjected to agar diffusion assays for antimicrobial activity.

Tested metabolites showed different potent antimicrobial activities with minimum inhibitory micrograms/ml against Gram-positive and Gram- negative bacteria as well as fungi (Table-2). The results were obtained using Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 5141), Escherichia coli (ATCC 10536), and Pseudomonas aeruginosa (NTCC 6750) as test bacterial strains and Candida albicans and Aspergillus niger as fungal indicators, indicated that compounds 1, 2, 3 and 5 exhibited potent activity against B. subtilis with MIC values of 1.9, 1.1, 3.3 and 2.9 g/ml and against P. aeruginosa with MIC values of 1.2, 1.8, 1.3 and 2.5 g/ml, respectively. Compounds 2, 3 and 4 inhibited moderately the growth of Staphylococcus aureus with MIC values of 3.0, 2.8 and 3.4 g/ml, respectively. Candida albicans was highly sensitive to compound 1 with MIC 1.4 g/ml, while Aspergillus niger was moderately sensitive to compound 3 with MIC 3.2 g/ml.

Table-2: Antimicrobial activity of the isolated compounds 1-5

###Microorganism

###MIC (g/ml)

###1###2 3 4 5

###Indicator bacteria

###Bacillus subtilis ATCC6633###1.9###1.1###3.3###6.8###2.9

Staphylococcus aureus ATCC5141###3.8###3.0###2.8###3.4###4.3

###Escherichia coli ATCC10536###4.7###4.0###3.9###5.2###5.0

Pseudomonas aeruginosa NTCC6750###1.2###1.8###1.3###5.9###2.5

###Indicator fungi

###Candida albicans###1.4 5.5 4.6 9.0 5.4

###Aspergillus niger###7.3 6.0 3.2 7.8 7.3

Conclusion

In conclusion, our search for marine-derived bioactive compounds led to the investigation of five indole and AY-carboline alkaloids along with phenolic acid from the ethyl acetate extract of Saudi Red Sea sponge Hyrtios species. The isolated alkaloids were evaluated for their antimicrobial activity and for their inhibitory effect on HCV NS3-NS4A protease. The compounds have powerful antimicrobial activity and exhibited strong to moderate inhibition of HCV NS3/4A protease.

Acknowledgement

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah under grant No (150-005-D1434). The authors, therefore, acknowledge with thank DSR technical and financial support.

References

1. A. Wasley and M. J. Alter, Epidemiology of hepatitis C: geographic differences and temporal trends, Semin. Liver Dis., 20, 1 (2000).

2. M. J. Alter, D. Kruszon-Moran, O. V. Nainan, G. M. McQuillan, and F. Gao, The prevalence of hepatitis C virus infection in the United States, N. Engl. J. Med., 341, 556 (1999).

3. J. G. McHutchison, S. C. Gordon, E. R. Schiff, M. L. Shiffman, W. M. Lee, V. K. Rustgi, Z. D. Goodman, M. H. Ling, S. Cort, and J. K. Albrecht, Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C, N. Engl. J. Med., 339, 1485 (1998).

4. J. G. McHutchison, G. T. Everson, S. C. Gordon, I. M. Jacobson et al., Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N. Engl. J. Med., 360, 1827 (2009).

5. Y. S. Tsantrizos, Peptidomimetic therapeutic agents targeting the protease enzyme of the human immunodeficiency virus and hepatitis C virus. Acc. Chem. Res., 41, 1252 (2008).

6. G. R. Pettit, R. Tan, and Z. A. Cichacz, Antineoplastic agents: Isolation and structure of sesterstatin 6 from the Indian Ocean sponge Hyrtios erecta, J. Nat. Prod., 68, 1253 (2005).

7. D. T. A. Youssef, L. A. Shaala, and S. Emara, Antimycobacterial scalarane-based sesterterpenes from the Red Sea sponge Hyrtios erecta, J. Nat. Prod., 68, 1782 (2005).

8. I. C. Pina, M. L. Sanders, and P. Crews, Puupehenone congeners from an Indo-Pacific Hyrtios sponge, J. Nat. Prod., 66, 2 (2003).

9. M. Salmoun, C. Devijver, D. Daloze, J-C. Braekman, R. Gomez, M. de Kluijver, and R. W. M. van Soest. New sesquiterpenes/quinones from two sponges of the genus Hyrtios, J. Nat. Prod., 63, 452 (2000).

10. M. Kobayashi, S. Aoki, H. Sakai, K. Kawazoe, N. Kihara, T. Sasaki, and I Kitagawa, Altohyrtin A, a potent anti-tumor macrolide from the Okinawan marine sponge Hyrtios altum, Tetrahedron Lett. 34, 2795 (1993).

11. D. T. A. Youssef, Hyrtioerectines AC, cytotoxic alkaloids from the Red Sea sponge Hyrtios erectus. J. Nat. Prod., 68, 1416 (2005).

12. S. Aoki, Y. Ye, K. Higuchi, A. Takashima, Y. Tanaka, I. Kitagawa, and M. Kobayashi, Novel neuronal nitric oxide synthase (nNOS) selective inhibitor, aplysinopsin-type indole alkaloid, from marine sponge Hyrtios erecta, Chem. Pharm. Bull., 49, 1372 (2001).

13. D. T. A. Youssef, L. A. Shaala, and H. Z. Asfour, Bioactive Compounds from the Red Sea Marine Sponge Hyrtios Species. Mar. Drugs, 11, 1061 (2013).

14. W. Gul and M. T. Hamann, Indole alkaloid marine natural products: An established source of cancer drug leads with considerable promise for the control of parasitic, neurological and other diseases. Life Sci., 78, 442 (2005).

15. G. R. Pettit, Z. A. Cichacz, F. Gao, C. L. Herald, M. R. Boyd, J. M. Schmidt, and J. N. A. Hooper, Isolation and structure of spongistatin, J. Org. Chem., 58, 1302 (1993).

16. N. S. Egorov, Antibiotics: A scientific approach, Mirpublishers, Moscow, pp. 170 (1985).

17. P. Sauleau, M-T. Martin, M-E. T. H. Dau, D. T. A. Youssef, and B-K. Marie-Lise, Hyrtiazepine, an Azepino-indole-Type Alkaloid from the Red Sea Marine Sponge Hyrtios erectus, J. Nat. Prod, 69, 1676 (2006).

18. R. Yamanokuchi, K. Imada, M. Miyazaki, H. Kato, T. Watanabe, M. Fujimuro, Y. Saeki, S. Yoshinaga, H. Terasawa, N. Iwasaki, H. Rotinsulu, F Losung, R. E. P. Mangindaan, M. Namikoshi, N. J. de Voogd, H. Yokosawa, and S. Tsukamoto, Hyrtioreticulins AE, indole alkaloids inhibiting the ubiquitin- enzyme, from the marine sponge Hyrtios reticulates. Bioorg. Med. Chem., 20, 4437 (2012).

19. R. S. El Dine, A. R. Abdel Monem, A. M. El- Halawany, M. Hattori, and E. Abdel-Sattar, HCV-NS3/4A Protease Inhibitory Iridoid Glucosides and Dimeric Foliamenthoic Acid Derivatives from Anarrhinum orientale, J. Nat. Prod., 74, 943 (2011).
COPYRIGHT 2015 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Hepatitis C virus
Author:Hawas, Usama Wahid
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Apr 30, 2015
Words:3180
Previous Article:Solid-phase Extraction on Magnetic Multi-walled Carbon Nanotubes Coupled with Flame Atomic Absorption Spectrometry for Determining Lead and Cadmium...
Next Article:Optimization and Modeling of Process Variables of Biodiesel Production from Marula Oil using Response Surface Methodology.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters