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Sialidase inhibitory activity of diarylnonanoid and neolignan compounds extracted from the seeds of Myristica fragrans
Bioorganic & Medicinal Chemistry Letters 27 (2017) 3060–3064
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Sialidase inhibitory activity of diarylnonanoid and neolignan compounds extracted from the seeds of Myristica fragrans Ji-Young Park a,c, Su Hwan Lim a,b,c, Bo Ram Kim a, Hyung Jae Jeong a, Hyung-Jun Kwon a, Gyu-Yong Song b, Young Bae Ryu a,⇑, Woo Song Lee a,⇑ a b
Natural Product Materials Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejon 305-764, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 February 2017 Revised 11 May 2017 Accepted 17 May 2017 Available online 18 May 2017 Keywords: Sialidase NanA NanB Myristica fragrans Malabaricone C
a b s t r a c t Sialidases are key virulence factors that remove sialic acid from host cell surface glycans, thus unmasking receptors to facilitate bacterial adherence and colonization. In this study, we report the isolation and characterization of novel inhibitors of the Streptococcus pneumoniae sialidases NanA, NanB, and NanC from Myristica fragrans seeds. Of the isolated compounds (1–12), malabaricone C showed the most pneumococcal sialidases inhibition (IC50 of 0.3 lM for NanA, 3.6 lM for NanB, and 2.9 lM for NanC). These results suggested that malabaricone C and neolignans could be potential agents for combating S. pneumoniae infection agents. Ó 2017 Elsevier Ltd. All rights reserved.
Pathogenic bacteria resistant to existing drugs occasionally result in fatal infectious diseases. The need to combat these superbugs mandates the development of new drugs capable of overcoming antibacterial resistance. The gram-positive bacterium Streptococcus pneumoniae is a major cause of bacterial meningitis, which is the most frequent cause of otitis media and sepsis in children and the primary cause of community-acquired and hospitalacquired pneumonia in adults.1,2 Several virulence factors may contribute to bacterial colonization and infection. Sialidases from pathogenic bacteria are a key virulence factor, as they remove sialic acid from host cell surface glycans, unmasking certain receptors to facilitate bacterial adherence and colonization.2–4 All S. pneumoniae clinical isolates investigated to date have prominent sialidases activities which have been found to be involved in sepsis and septic shock.5,6 Moreover, analysis of pathogens with sialiase deletion demonstrated that sialidase acts in the initial stages of pulmonary infection by targeting glycoconjugates and biofilm production.7 Three distinct sialidases, NanA, NanB and NanC, are encoded by the S. pneumoniae genomes and possess a carbohydrate-binding module (CMB). A study of neuraminidase genes in clinical pneumococcal isolates identified NanA, NanB and NanC to be present in
⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Bae Ryu), [email protected] (W. Song Lee). c Both authors contributed equally to the work. http://dx.doi.org/10.1016/j.bmcl.2017.05.055 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
100%, 96% and 51% of these strains, respectively, with NanC being more prevalent in the cerebrospinal fluid.8 Gene-knockout studies in mouse models have shown that NanA and B are essential for S. pneumoniae infection.9,10 In an attempt to develop drugs against pneumococcal infectious diseases using naturally derived sialidase inhibitors, we have synthesized and expressed the full-length genes for the sialidases NanA, NanB, and NanC in Escherichia coli (E. coli). The three genes encoding NanA (Fig. S3, GenBank accession no. COT45929.1, PDB: 2VVZ), NanB (Fig. S4, GenBank accession no. U43526.1, PDB: 2VW0) and NanC (Fig. S5, GenBank accession no. AE005672.3) of S. pneumoniae were synthesized. (Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany). The synthesized genes were inserted into the cloning sites of a pET151/d-TOPO vector (Invitrogen, Carlsbad, CA) containing a 6x His-tag at the C-terminus. S. pneumoniae sialidases were expressed and purified from BL21 (DE3) E. coli (HIT, Real biotech Co., Taipei, Taiwan). The purified sialidases were detected at approximately 56.6, 77.7, and 82.4 kDa with greater than 90% purity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified sialidases had specific activities (Km values) of 43.9, 51.6, and 188.7 lM, respectively, using 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (MUNANA) as substrate (Fig. S1). Since sialidase inhibitors are substrate analogs (Neu5Ac2en) typically exhibit competitive kinetics,11–13 it is interesting to explore natural non-substrate mimics such as chalcones,
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flavonoids, and xanthones.14–16 Previously, we identified and characterized a naturally-derived sialidase inhibitor diplacone from the fruit of Paulownia tomentosa, which showed potent sialidase (Clostridium perfringens, NanI) inhibition.15 In this study, we report that nutmeg constituents can target the S. pneumoniae sialidases NanA, NanB, and NanC. Nutmeg is the seed of Myristica fragrans, and has been used as a spice in sweet and savoury preparations, as well as for its medicinal properties. Studies have reported that nutmeg exhibits a broad range of pharmacological properties, including anti-inflammatory, antibacterial, antioxidant, antiangiogenic, and anticarcinogenic effects.17–22 Neolignans and malabaricones are the major active components of M. fragrans and show various bioactivities. However, despite several reports of anti-bacterial activities, none have evaluated and characterized the sialidase inhibition capabilities of M. fragrans components. During our search for natural inhibitors of S. pneumoniae sialidases, we found that an M. fragrans ethanol extract exhibited sialidase inhibitory activity (Nan A: 62.2%, NanB: 70.5% and NanC: 75.1%) at 30 lg/ml (Table S1). M. fragrans (9.6 kg) were ground and extracted with ethanol for 1 week at room temperature. The ethanol extract was evaporated until completely dry (520 g). Ethanolic extracts of M. fragrans were fractionated using polar solvents to perform a comparative analysis of their constituents by HPLC. The ethyl acetate fraction was found to be present in high quantities (Fig. S2). The extracts were suspended with water and extracted again with hexane and ethyl acetate to obtained hexane (330 g), ethyl acetate layer (101 g), and water layers (89 g). The hexane layer was subjected to a column over silica gel (10 40 cm) and eluted with gradient elution using hexane/ethyl acetate (100:1 ? 1:2) mixtures to obtain fractions 1–10. Fr. 2 (68.4 g) was chromatographed via a silica gel column using hexane/ethyl acetate (50:1 ? 1:1) to give compound 1 (17.8 g). Fr. 4 HO
(21.8 g) was purified by column chromatography using hexane/ ethyl acetate (50:1 ? 10:1) to obtain compound 2 (837.8 mg). Fr. 9 (22.1 g) was separated using a silica gel column and eluted with gradient elution using hexane/ethyl acetate (50:1 ? 10:1). Subfractions were chromatographed on an RP C-18 column with 70% acetonitrile to yield compound 3 (235.2 mg), compound 4 (153.2 mg) and compound 6 (48.3 mg). Fr.6 and Fr.7 were chromatographed on a RP C-18 column and eluted with 70% acetonitrile to yield compound 7 (51.6 mg), compound 10 (19.3 mg), compound 11 (8.5 mg), and compound 12 (8.6 mg). The ethyl acetate layer was chromatographed over a silica gel column using MPLC with a gradient solvent of hexane/ethyl acetate to yield ten subfractions based on TLC profile. The EA-Fr. 2 was applied to a C18 column using 70% acetonitrile to obtain compound 5 (28.6 mg). EA-Fr. 8 (2.9 g) was chromatographed over a Sephadex LH-20 column using methanol as the eluting solvent to give seven subfractions. The subfraction EA-Fr. 8–7 (659.3 mg) was repeatedly purified using a Sephadex LH-20 column and recycling HPLC to obtain compound 8 (47.4 mg) and compound 9 (113.6 mg). The isolated compounds (1–12) were six phenyl propens (1–6), four neolignans (7–10), and two diarylnonanoids (11 and 12), and identified as 3,5-dihydroxyestragole (1), methoxyeugenol (2), myristicin (3), myrislignan (4), myrislignanometin E (5), maceneolignan H (6), licarin A (7), licarin B (8), 50 -methoxylicarin B (9), verrucosin (10), malabaricone B (11), and malabaricone C (12). These compounds confirmed though comparison of their spectroscopic data with those in previous studies (Fig. 1).23–26 The inhibition activity and selectivity of the twelve isolated compounds against the S. pneumoniae sialidases NanA, NanB, and NanC were investigated.27 All compounds were first tested at a maximal concentration (200 lM), following which results from progressive two-fold serial dilutions were used for IC50 determination. NanA and NanB have been implicated in
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11 Fig. 1. Chemical structures of isolated compounds from M. fragrans.
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Table 1 Inhibitory effects of isolated compounds (1–12) on S. pneumoniae NanA. Compounds
IC50 (lM)a
Inhibition mode (Ki, lM)b
1 2 3 4 5 6 7 8 9 10 11 12
2.0 ± 0.2 6.9 ± 1.8 196.9 ± 37.4 25.6 ± 1.6 1.6 ± 0.9 294.5 ± 28.2 7.9 ± 2.3 1.5 ± 0.4 44.2 ± 4.7 37.2 ± 5.0 0.4 ± 0.01 0.3 ± 0.02
Noncompetitive (4.9 ± 0.3) Noncompetitive (4.2 ± 0.2) NTc Noncompetitive (15.1 ± 0.6) Noncompetitive (3.0 ± 0.1) NT Noncompetitive (9.4 ± 0.8) Noncompetitive (1.3 ± 0.04) Noncompetitive (47.5 ± 3.5) Noncompetitive (21.9 ± 0.6) Competitive (0.5 ± 0.03) Competitive (0.1 ± 0.01)
a All compounds were examined in at least of three experiments; IC50 (50% inhibitory concentration) values for the compounds represent the concentration causing 50% enzyme activity loss. b Inhibition constant. c Not tested.
S. pneumoniae-mediated pathogenesis, and are considered to be valid drug targets. Since many studies have confirmed that NanA and NanB can be inhibited by weak sialidase inhibitors such as Neu5Ac2en and the b-amino-sulfonic acid family, the focus of this study was to identify a naturally-derived inhibitor of greater potency. First, we evaluated the inhibitory effect of compounds 1–12 on S. pneumoniae sialidase hydrolytic activity and confirmed using DANA (Neu5Ac2en) as a positive sontol. The IC50 value of DANA with respect to S. pneumoniae sialidases inhibition were 4.8 ± 1.1 lM (for Nan A) and 45.1 ± 2.5 lM (for Nan B). All the isolated compounds except 3 and 6 exhibited a dose-dependent inhibitory effect on all three S. pneumoniae sialidases (Fig. S12). As shown in Table 1, NanA IC50 values (not including compounds 3 and 6) ranged from 0.3 to 44.2 lM, indicating significant inhibition. However, inhibitory activity was slightly affected by subtle structural changes. Of the isolated compounds, the diarylnonanoid derivatives malabaricone B (11, IC50 = 0.4 ± 0.01 lM) and malabaricone C (12, IC50 = 0.3 ± 0.02 lM) showed highly inhibitory activity than isolated component from M. fragrans in this study. Additionally, the number of free hydroxyl groups influenced the inhibition potency of phenyl propen derivatives (1–6); of the monomeric phenyl propenes (1–3), 3,5-dihydroxy analog 1 (IC50 = 2.0 ± 0.2 lM) > 4-hydroxy analog 2 (IC50 = 6.9 ± 1.8 lM) hydroxyl group substituted with [3, 4] dioxole analog 3 (IC50 = 196.9 ± 37.4 lM);
in the dimeric derivatives (4–6), phenylprop-2-en-1-ol analog 5 showed the most potent inhibition and 25- to 200-fold more activity than did the dimerin phenyl propene 4 (IC50 = 25.6 ± 1.6 lM) and 6 (IC50 > 200 lM). Interestingly, the neolignan with dioxole moiety, licarin B (8), displayed significant activity against NanA (IC50 = 1.5 ± 0.4 lM). The IC50 values of neolignans (7–10) for NanA inhibition ranged between 1.5 and 44.2 lM (Table 1). Second, all NanA-tested compounds were evaluated for NanB inhibitory activity. Within the sequence encompassed by the structure of NanB reported, NanB shares 26.5% sequence identity with NanA, and 56.2% sequence identity with NanC. NanB is an intramolecular trans-sialidases with specificity towards a2-3 linked sialic acid substrate, and differs in its substrate specificity from the other pneumococcal neuraminidase, NanA. These activity of NanB plays an important role in bacterial nutrition during pneumococcal infection of the respiratory tract and sepsis.12 The inhibition assay results of NanB are summarized in Table 2. Among the examined compounds, malabaricone C was also the most potent NanB inhibitor (IC50 = 3.6 ± 1.3 lM). For the phenyl propenes, the number of hydroxyl groups and the replacement of prop-2-en-1ol resulted in marginal increase in inhibitory activity [1 (IC50 = 27.1 ± 1.6 lM) and 5 (IC50 = 20.9 ± 2.6 lM)]. As noted previously, the replacement of the 4,5-dihydroxy group by a dioxole group (8, IC50 = 14.0 ± 1.6 lM) led to stronger inhibition compared to that reported for 7. Compounds 1–12 were then evaluated for their effect on NanC, which shares 27.9% sequence identity with NanA and 56.2% sequence identity with NanB, and is an unusual sialidase in that its primary reaction product is Neu5Ac2en. NanC has been found to be a market for hemolytic uremic syndrome (HUS), a deadly complication of invasive pneumococcal disease.29 The IC50 values of compounds 1–12 for NanC are summarized in Table 2. Although pneumococcal sialidase are different structural homologs, malabaricone C again displayed the most potent inhibition, with an IC50 value of 2.9 ± 0.5 lM. NanA and NanB discussed active chemotype campaigns showed similar trends to NanC inhibition, but with more moderate potency overall [2 (33.6 lM) vs 1 (60.5 lM), 5 (24.6 lM) vs 4 (103.2 lM), 8 (30.7 lM) vs 7 (112.9 lM)]. On the basis of these observations, we have successfully obtained inhibitors of S. pneumoniae sialidases from natural products. And we investigated the kinetic mechanisms of inhibitors with IC50 values of 50 lM or less. Enzyme kinetics were measured with a series of substrate concentrations and various inhibitor concentrations. We found that M. fragrans-derived sialidase inhibitors could be divided into two classes based on their mode of inhibition.
Table 2 Inhibitory effects of isolated compounds (1–12) on S. pneumoniae NanB and NanC. Compounds
1 2 3 4 5 6 7 8 9 10 11 12
S. pneumoniae Nan B
S. pneumoniae Nan C
IC50 (lM)a
Inhibition modeb (Ki, lM)
IC50 (lM)
Inhibition mode (Ki, lM)
27.1 ± 1.6 77.6 ± 39.0 NAd 229.3 ± 5.6 20.9 ± 2.6 NA 95.8 ± 19.0 14.0 ± 1.6 92.1 ± 35.3 331.2 ± 22.3 5.7 ± 1.5 3.6 ± 1.3
Noncompetitive NTc NT NT Noncompetitive NT NT Noncompetitive NT NT Noncompetitive Noncompetitive
60.5 ± 15.6 33.6 ± 7.0 NA 103.2 ± 9.8 24.6 ± 0.7 NA 112.9 ± 19.9 30.7 ± 5.6 317.3 ± 7.4 273.8 ± 49.6 14.3 ± 3.2 2.9 ± 0.5
Noncompetitive Noncompetitive NT NT Noncompetitive NT NT Noncompetitive NT NT Noncompetitive Noncompetitive
(74.0 ± 4.3)
(39.2 ± 2.4)
(13.5 ± 1.1)
(5.6 ± 1.7) (3.0 ± 0.2)
(91.8 ± 8.0) (30.2 ± 1.4)
(37.8 ± 2.4)
(12.1 ± 0.6)
(5.8 ± 0.2) (2.1 ± 0.05)
a All compounds were examined in at least three experiments; IC50 (50% inhibitory concentration) values for the compounds represent the concentration causing 50% enzyme activity loss. b Inhibition constant. c Not tested. d No activity.
J.-Y. Park et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3060–3064
A 0.18
In this study, we identified for the first time sialidase inhibitor from the seeds of M. fragrans, with one of the 12 isolated compounds, 12, showing exemplary potency. By extending our study to parent compounds similar in chemical structure to the isolated compounds, we were able to uncover some key aspects of sialidase function. These results not only provide further insight into the pathogenic mechanisms of S. pneumoniae, infection but also suggest that isolated phenyl propene and diarylnonanoid compounds could be potentially used in future molecular therapies for bacterial infectious disease. Further in vivo evaluation of malabaricone C (12) will be reported in due course.
0.16 1/Rate (RFU/sec)
0.14 0.12 0.10 0.08 0.06 0.04 0.02 -2
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2 4 [8], ( M)
6
8
Conflict of interest The authors declare no conflict of interest.
B 0.14
Acknowledgements
1/Rate (RFU/sec)
0.12
This research was supported by the KRIBB Research Initiative Program and Fishing Commercialization Technology Development Program through KIMST funded by the Ministry of Oceans and Fisheries, South Korea, J.-.Y. Park and H.-.J. Kwon were supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1C1B2015753).
0.10 0.08 0.06 0.04 0.02
A. Supplementary data -1
0
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3
4
[11], ( M)
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References
1/Rate (RFU/sec)
0.05 0.04 0.03 0.02 0.01 -0.4 -0.2 0.0
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05. 055.
0.2 0.4 0.6 [12] ( M)
0.8
1.0
Fig. 2. Graphical determination of the inhibition type for compounds 8, 11 and 12. (A–C) Dixon plots for the inhibitory activity of compounds 8, 11 and 12, respectively, against S. pneumoniae NanA hydrolysis activity in the presence of different substrate concentrations (12.5 lM, d; 25 lM, s; and 50 lM, .).
The isolated compounds, aside from the diarylnonanoids (11 and 12), exhibited noncompetitive inhibition characteristics against NanA. As shown in Fig. 2, the kinetic plots showed that compound 8 had a noncompetitive mechanism because increasing substrate concentrations resulted in a family of lines with different slopes, but a common x-axis intercept. Importantly, the potent NanA inhibitors 11 and 12, the diarylnonanoids, showed a competitive inhibitory mode of action, as the Lineweaver-Burk plots for 1/V versus 1/[S] resulted in a family of straight lines with the same y-axis intercept for NanA inhibition (Fig. 2). Most compounds present noncompetitive inhibition for NanB and NanC (listed in Table 2, Fig. S14 and S15). Despite having similar molecular sizes and functionalities, the isolated compounds inhibited pneumonial sialidases by bothe competitive and noncompetitive inhibition mechanisms.
1. Luotonen J, Herva E, Karma P, Timonen M, Leinonen M, Makela PH. Scand J Infect Dis. 1981;13:177–183. 2. Mitchell TJ. Res Microbiol. 2000;151:413–419. 3. Brittan JL, Buckeridge TJ, Finn A, Kadioglu A, Jenkinson HF. Mol Oral Microbiol. 2012;27:270–283. 4. Bogaert D, De Groot R, Hermans PW. Lancet Infect Dis. 2004;4:144–154. 5. Paulson JC, Kawasaki N. Nat Biotechnol. 2011;29:406–407. 6. Chen GY, Chen X, King S, et al. Nat Biotechnol. 2011;29:428–435. 7. Parker D, Soong G, Planet P, Brower J, Ratner AJ, Prince A. Infect Immun. 2009;77:3722–3730. 8. Pettigrew MM, Fennie KP, York MP, Daniels J, Ghaffar F. Infect Immun. 2006;74:3360–3365. 9. Manco S, Hernon F, Yesilkaya H, Paton JC, Andrew PW, Kadioglu A. Infect Immun. 2006;74:4014–4020. 10. Kadioglu A, Weiser JN, Paton JC, Andrew PW. Nat Rev Microbiol. 2008;6:288–301. 11. Xu G, Potter JA, Russell RJ, Oggioni MR, Andrew PW, Taylor GL. J Mol Biol. 2008;384:436–449. 12. Gut H, King SJ, Walsh MA. FEBS Lett. 2008;582:3348–3352. 13. Xu G, Kiefel MJ, Wilson JC, Andrew PW, Oggioni MR, Taylor GL. J Am Chem Soc. 2011;133:1718–1721. 14. Park J-Y, Jeong HJ, Kim YM, et al. Bioorg Med Chem Lett. 2011;21:5602–5604. 15. Lee Y, Ryu YB, Youn HS, et al. Acta Crystallogr D Biol Crystallogr. 2014;70:1357–1365. 16. Ryu YB, Curtis-Long MJ, Lee JW, et al. Bioorg Med Chem. 2009;17:2744–2750. 17. Shafiei Z, Shuhairi NN, Md Fazly Shah Yap N, Harry Sibungkil CA, Latip J. Evid Based Complement Alternat Med eCAM. 2012;2012:825362. 18. Narasimhan B, Dhake AS. J Med Food. 2006;9:395–399. 19. Cao GY, Xu W, Yang XW, Gonzalez FJ, Li F. Food Chem. 2015;173:231–237. 20. Chung JY, Choo JH, Lee MH, Hwang JK. Phytomedicine. 2006;13:261–266. 21. Shinohara C, Mori S, Ando T, Tsuji T. Biosci Biotechnol Biochem. 1999;63:1475–1477. 22. Patro BS, Bauri AK, Mishra S, Chattopadhyay S. J Agric Food Chem. 2005;53:6912–6918. 23. Valente VMM, Jham GN, Jardim CM, Dhingra OD, Ghiviriga I. J Food Res. 2015;4:51–57. 24. Du SS, Yang K, Wang CF, et al. Chem Biodivers. 2014;11:1449–1456. 25. Li F, Yang XW. Phytochemistry. 2008;69:765–771. 26. Isogai A, Murakoshi S, Suzuki A, Tamura S. Agr Biol Chem. 1973;37:1479–1486. 27. Procedure of S. pneumoniae sialidases inhibition assay:As described, the inhibitory effects of compounds on S. pneumoniae sialidases were measured
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using a FRET method. In this assay, the 4-methylumbelliferyl-a-D-Nacetylneuraminic acid (Sigma Chemical Co., St. Louis, MO, USA) was used as a substrate, and the enzyme activity was determined by measuring the increase in fluorescence by continuously monitoring the reactions at 450/40 nm with excitation at 365 nm using a SpectraMax M2e Multimode Reader (Molecular Devices Co., USA). The IC50 values of the isolated compounds were measured in a reaction mixture containing each enzyme (final concentration of NanA, 2.2 nM; NanB, 17.8 nM; NanC, 22.9 nM), the test compounds (from 0 to 200 lM), and 50 lM of substrate in 20 mM Sodium phosphate buffer (pH 7.5, containing 300 mM NaCl). To determine the enzyme activity, the experimental data was fit to a logistic curve with Eq. (1), a time-drive protocol was used and the initial velocity was recorded over a range of concentrations, and the data were analyzed using a nonlinear regression program [Sigma Plot (SPCC Inc., Chicago, IL)].
Inhibition activity ð%Þ ¼ 100 ½ðS S0Þ=ðC C0Þ 100
ð1Þ
where C is the fluorescence of the control (enzyme, buffer, and substrate) after 60 min of incubation, C0 is the fluorescence of the control at 0 min, S is the fluorescence of the tested samples (enzyme, sample solution, and substrate) after
incubation, and S0 is the fluorescence of the tested samples at 0 min.The inhibition mechanism was determined, and the apparent inhibition constants (Ki) for the respective sialidases (NanA, NanB and NanC) were performed on the test compounds, for which the IC50 values were below 50 lM. The test compounds were studied at three different concentrations that were chosen based on the IC50 values obtained with each sialidases (approximately 1/2IC50, IC50, 2IC50). The concentrations of marker substrates were chosen (approximately 1/4 Km, 1/2 Km, Km) with regard to their Michaelis-Menten kinetics. The Ki values were calculated by nonlinear regression analysis by fitting different models of enzyme inhibition to the kinetic data using SigmaPlot Enzyme Kinetics Module 1.3 (SPSS Inc., Chicago, IL). The inhibition mechanism of the compounds was determined by comparing the statistical results, including the Akaike’s information criterion values, of different inhibition models and selecting the one with the best fit.28 28. Li X-Q, Andersson TB, Ahlstr¨om M, Weidolf L. Drug Metab Dispos. 2004;32:821–827. 29. Janapatla RP, Hsu MH, Hsieh YC, Lee HY, Lin TY, Chiu CH. Clin Microbiol Infect. 2013;19:480–486.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Sialidase inhibitory activity of diarylnonanoid and neolignan compounds extracted from the seeds of Myristica fragrans Ji-Young Park a,c, Su Hwan Lim a,b,c, Bo Ram Kim a, Hyung Jae Jeong a, Hyung-Jun Kwon a, Gyu-Yong Song b, Young Bae Ryu a,⇑, Woo Song Lee a,⇑ a b
Natural Product Materials Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejon 305-764, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 February 2017 Revised 11 May 2017 Accepted 17 May 2017 Available online 18 May 2017 Keywords: Sialidase NanA NanB Myristica fragrans Malabaricone C
a b s t r a c t Sialidases are key virulence factors that remove sialic acid from host cell surface glycans, thus unmasking receptors to facilitate bacterial adherence and colonization. In this study, we report the isolation and characterization of novel inhibitors of the Streptococcus pneumoniae sialidases NanA, NanB, and NanC from Myristica fragrans seeds. Of the isolated compounds (1–12), malabaricone C showed the most pneumococcal sialidases inhibition (IC50 of 0.3 lM for NanA, 3.6 lM for NanB, and 2.9 lM for NanC). These results suggested that malabaricone C and neolignans could be potential agents for combating S. pneumoniae infection agents. Ó 2017 Elsevier Ltd. All rights reserved.
Pathogenic bacteria resistant to existing drugs occasionally result in fatal infectious diseases. The need to combat these superbugs mandates the development of new drugs capable of overcoming antibacterial resistance. The gram-positive bacterium Streptococcus pneumoniae is a major cause of bacterial meningitis, which is the most frequent cause of otitis media and sepsis in children and the primary cause of community-acquired and hospitalacquired pneumonia in adults.1,2 Several virulence factors may contribute to bacterial colonization and infection. Sialidases from pathogenic bacteria are a key virulence factor, as they remove sialic acid from host cell surface glycans, unmasking certain receptors to facilitate bacterial adherence and colonization.2–4 All S. pneumoniae clinical isolates investigated to date have prominent sialidases activities which have been found to be involved in sepsis and septic shock.5,6 Moreover, analysis of pathogens with sialiase deletion demonstrated that sialidase acts in the initial stages of pulmonary infection by targeting glycoconjugates and biofilm production.7 Three distinct sialidases, NanA, NanB and NanC, are encoded by the S. pneumoniae genomes and possess a carbohydrate-binding module (CMB). A study of neuraminidase genes in clinical pneumococcal isolates identified NanA, NanB and NanC to be present in
⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Bae Ryu), [email protected] (W. Song Lee). c Both authors contributed equally to the work. http://dx.doi.org/10.1016/j.bmcl.2017.05.055 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
100%, 96% and 51% of these strains, respectively, with NanC being more prevalent in the cerebrospinal fluid.8 Gene-knockout studies in mouse models have shown that NanA and B are essential for S. pneumoniae infection.9,10 In an attempt to develop drugs against pneumococcal infectious diseases using naturally derived sialidase inhibitors, we have synthesized and expressed the full-length genes for the sialidases NanA, NanB, and NanC in Escherichia coli (E. coli). The three genes encoding NanA (Fig. S3, GenBank accession no. COT45929.1, PDB: 2VVZ), NanB (Fig. S4, GenBank accession no. U43526.1, PDB: 2VW0) and NanC (Fig. S5, GenBank accession no. AE005672.3) of S. pneumoniae were synthesized. (Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany). The synthesized genes were inserted into the cloning sites of a pET151/d-TOPO vector (Invitrogen, Carlsbad, CA) containing a 6x His-tag at the C-terminus. S. pneumoniae sialidases were expressed and purified from BL21 (DE3) E. coli (HIT, Real biotech Co., Taipei, Taiwan). The purified sialidases were detected at approximately 56.6, 77.7, and 82.4 kDa with greater than 90% purity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified sialidases had specific activities (Km values) of 43.9, 51.6, and 188.7 lM, respectively, using 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (MUNANA) as substrate (Fig. S1). Since sialidase inhibitors are substrate analogs (Neu5Ac2en) typically exhibit competitive kinetics,11–13 it is interesting to explore natural non-substrate mimics such as chalcones,
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flavonoids, and xanthones.14–16 Previously, we identified and characterized a naturally-derived sialidase inhibitor diplacone from the fruit of Paulownia tomentosa, which showed potent sialidase (Clostridium perfringens, NanI) inhibition.15 In this study, we report that nutmeg constituents can target the S. pneumoniae sialidases NanA, NanB, and NanC. Nutmeg is the seed of Myristica fragrans, and has been used as a spice in sweet and savoury preparations, as well as for its medicinal properties. Studies have reported that nutmeg exhibits a broad range of pharmacological properties, including anti-inflammatory, antibacterial, antioxidant, antiangiogenic, and anticarcinogenic effects.17–22 Neolignans and malabaricones are the major active components of M. fragrans and show various bioactivities. However, despite several reports of anti-bacterial activities, none have evaluated and characterized the sialidase inhibition capabilities of M. fragrans components. During our search for natural inhibitors of S. pneumoniae sialidases, we found that an M. fragrans ethanol extract exhibited sialidase inhibitory activity (Nan A: 62.2%, NanB: 70.5% and NanC: 75.1%) at 30 lg/ml (Table S1). M. fragrans (9.6 kg) were ground and extracted with ethanol for 1 week at room temperature. The ethanol extract was evaporated until completely dry (520 g). Ethanolic extracts of M. fragrans were fractionated using polar solvents to perform a comparative analysis of their constituents by HPLC. The ethyl acetate fraction was found to be present in high quantities (Fig. S2). The extracts were suspended with water and extracted again with hexane and ethyl acetate to obtained hexane (330 g), ethyl acetate layer (101 g), and water layers (89 g). The hexane layer was subjected to a column over silica gel (10 40 cm) and eluted with gradient elution using hexane/ethyl acetate (100:1 ? 1:2) mixtures to obtain fractions 1–10. Fr. 2 (68.4 g) was chromatographed via a silica gel column using hexane/ethyl acetate (50:1 ? 1:1) to give compound 1 (17.8 g). Fr. 4 HO
(21.8 g) was purified by column chromatography using hexane/ ethyl acetate (50:1 ? 10:1) to obtain compound 2 (837.8 mg). Fr. 9 (22.1 g) was separated using a silica gel column and eluted with gradient elution using hexane/ethyl acetate (50:1 ? 10:1). Subfractions were chromatographed on an RP C-18 column with 70% acetonitrile to yield compound 3 (235.2 mg), compound 4 (153.2 mg) and compound 6 (48.3 mg). Fr.6 and Fr.7 were chromatographed on a RP C-18 column and eluted with 70% acetonitrile to yield compound 7 (51.6 mg), compound 10 (19.3 mg), compound 11 (8.5 mg), and compound 12 (8.6 mg). The ethyl acetate layer was chromatographed over a silica gel column using MPLC with a gradient solvent of hexane/ethyl acetate to yield ten subfractions based on TLC profile. The EA-Fr. 2 was applied to a C18 column using 70% acetonitrile to obtain compound 5 (28.6 mg). EA-Fr. 8 (2.9 g) was chromatographed over a Sephadex LH-20 column using methanol as the eluting solvent to give seven subfractions. The subfraction EA-Fr. 8–7 (659.3 mg) was repeatedly purified using a Sephadex LH-20 column and recycling HPLC to obtain compound 8 (47.4 mg) and compound 9 (113.6 mg). The isolated compounds (1–12) were six phenyl propens (1–6), four neolignans (7–10), and two diarylnonanoids (11 and 12), and identified as 3,5-dihydroxyestragole (1), methoxyeugenol (2), myristicin (3), myrislignan (4), myrislignanometin E (5), maceneolignan H (6), licarin A (7), licarin B (8), 50 -methoxylicarin B (9), verrucosin (10), malabaricone B (11), and malabaricone C (12). These compounds confirmed though comparison of their spectroscopic data with those in previous studies (Fig. 1).23–26 The inhibition activity and selectivity of the twelve isolated compounds against the S. pneumoniae sialidases NanA, NanB, and NanC were investigated.27 All compounds were first tested at a maximal concentration (200 lM), following which results from progressive two-fold serial dilutions were used for IC50 determination. NanA and NanB have been implicated in
MeO
MeO
O O
HO OH
OMe
OMe
3
2
1
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OH OMe
O
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OMe
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O
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OMe OMe
8
9 O
OH HO
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O
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MeO
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HO
HO
11 Fig. 1. Chemical structures of isolated compounds from M. fragrans.
HO 12
OH
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Table 1 Inhibitory effects of isolated compounds (1–12) on S. pneumoniae NanA. Compounds
IC50 (lM)a
Inhibition mode (Ki, lM)b
1 2 3 4 5 6 7 8 9 10 11 12
2.0 ± 0.2 6.9 ± 1.8 196.9 ± 37.4 25.6 ± 1.6 1.6 ± 0.9 294.5 ± 28.2 7.9 ± 2.3 1.5 ± 0.4 44.2 ± 4.7 37.2 ± 5.0 0.4 ± 0.01 0.3 ± 0.02
Noncompetitive (4.9 ± 0.3) Noncompetitive (4.2 ± 0.2) NTc Noncompetitive (15.1 ± 0.6) Noncompetitive (3.0 ± 0.1) NT Noncompetitive (9.4 ± 0.8) Noncompetitive (1.3 ± 0.04) Noncompetitive (47.5 ± 3.5) Noncompetitive (21.9 ± 0.6) Competitive (0.5 ± 0.03) Competitive (0.1 ± 0.01)
a All compounds were examined in at least of three experiments; IC50 (50% inhibitory concentration) values for the compounds represent the concentration causing 50% enzyme activity loss. b Inhibition constant. c Not tested.
S. pneumoniae-mediated pathogenesis, and are considered to be valid drug targets. Since many studies have confirmed that NanA and NanB can be inhibited by weak sialidase inhibitors such as Neu5Ac2en and the b-amino-sulfonic acid family, the focus of this study was to identify a naturally-derived inhibitor of greater potency. First, we evaluated the inhibitory effect of compounds 1–12 on S. pneumoniae sialidase hydrolytic activity and confirmed using DANA (Neu5Ac2en) as a positive sontol. The IC50 value of DANA with respect to S. pneumoniae sialidases inhibition were 4.8 ± 1.1 lM (for Nan A) and 45.1 ± 2.5 lM (for Nan B). All the isolated compounds except 3 and 6 exhibited a dose-dependent inhibitory effect on all three S. pneumoniae sialidases (Fig. S12). As shown in Table 1, NanA IC50 values (not including compounds 3 and 6) ranged from 0.3 to 44.2 lM, indicating significant inhibition. However, inhibitory activity was slightly affected by subtle structural changes. Of the isolated compounds, the diarylnonanoid derivatives malabaricone B (11, IC50 = 0.4 ± 0.01 lM) and malabaricone C (12, IC50 = 0.3 ± 0.02 lM) showed highly inhibitory activity than isolated component from M. fragrans in this study. Additionally, the number of free hydroxyl groups influenced the inhibition potency of phenyl propen derivatives (1–6); of the monomeric phenyl propenes (1–3), 3,5-dihydroxy analog 1 (IC50 = 2.0 ± 0.2 lM) > 4-hydroxy analog 2 (IC50 = 6.9 ± 1.8 lM) hydroxyl group substituted with [3, 4] dioxole analog 3 (IC50 = 196.9 ± 37.4 lM);
in the dimeric derivatives (4–6), phenylprop-2-en-1-ol analog 5 showed the most potent inhibition and 25- to 200-fold more activity than did the dimerin phenyl propene 4 (IC50 = 25.6 ± 1.6 lM) and 6 (IC50 > 200 lM). Interestingly, the neolignan with dioxole moiety, licarin B (8), displayed significant activity against NanA (IC50 = 1.5 ± 0.4 lM). The IC50 values of neolignans (7–10) for NanA inhibition ranged between 1.5 and 44.2 lM (Table 1). Second, all NanA-tested compounds were evaluated for NanB inhibitory activity. Within the sequence encompassed by the structure of NanB reported, NanB shares 26.5% sequence identity with NanA, and 56.2% sequence identity with NanC. NanB is an intramolecular trans-sialidases with specificity towards a2-3 linked sialic acid substrate, and differs in its substrate specificity from the other pneumococcal neuraminidase, NanA. These activity of NanB plays an important role in bacterial nutrition during pneumococcal infection of the respiratory tract and sepsis.12 The inhibition assay results of NanB are summarized in Table 2. Among the examined compounds, malabaricone C was also the most potent NanB inhibitor (IC50 = 3.6 ± 1.3 lM). For the phenyl propenes, the number of hydroxyl groups and the replacement of prop-2-en-1ol resulted in marginal increase in inhibitory activity [1 (IC50 = 27.1 ± 1.6 lM) and 5 (IC50 = 20.9 ± 2.6 lM)]. As noted previously, the replacement of the 4,5-dihydroxy group by a dioxole group (8, IC50 = 14.0 ± 1.6 lM) led to stronger inhibition compared to that reported for 7. Compounds 1–12 were then evaluated for their effect on NanC, which shares 27.9% sequence identity with NanA and 56.2% sequence identity with NanB, and is an unusual sialidase in that its primary reaction product is Neu5Ac2en. NanC has been found to be a market for hemolytic uremic syndrome (HUS), a deadly complication of invasive pneumococcal disease.29 The IC50 values of compounds 1–12 for NanC are summarized in Table 2. Although pneumococcal sialidase are different structural homologs, malabaricone C again displayed the most potent inhibition, with an IC50 value of 2.9 ± 0.5 lM. NanA and NanB discussed active chemotype campaigns showed similar trends to NanC inhibition, but with more moderate potency overall [2 (33.6 lM) vs 1 (60.5 lM), 5 (24.6 lM) vs 4 (103.2 lM), 8 (30.7 lM) vs 7 (112.9 lM)]. On the basis of these observations, we have successfully obtained inhibitors of S. pneumoniae sialidases from natural products. And we investigated the kinetic mechanisms of inhibitors with IC50 values of 50 lM or less. Enzyme kinetics were measured with a series of substrate concentrations and various inhibitor concentrations. We found that M. fragrans-derived sialidase inhibitors could be divided into two classes based on their mode of inhibition.
Table 2 Inhibitory effects of isolated compounds (1–12) on S. pneumoniae NanB and NanC. Compounds
1 2 3 4 5 6 7 8 9 10 11 12
S. pneumoniae Nan B
S. pneumoniae Nan C
IC50 (lM)a
Inhibition modeb (Ki, lM)
IC50 (lM)
Inhibition mode (Ki, lM)
27.1 ± 1.6 77.6 ± 39.0 NAd 229.3 ± 5.6 20.9 ± 2.6 NA 95.8 ± 19.0 14.0 ± 1.6 92.1 ± 35.3 331.2 ± 22.3 5.7 ± 1.5 3.6 ± 1.3
Noncompetitive NTc NT NT Noncompetitive NT NT Noncompetitive NT NT Noncompetitive Noncompetitive
60.5 ± 15.6 33.6 ± 7.0 NA 103.2 ± 9.8 24.6 ± 0.7 NA 112.9 ± 19.9 30.7 ± 5.6 317.3 ± 7.4 273.8 ± 49.6 14.3 ± 3.2 2.9 ± 0.5
Noncompetitive Noncompetitive NT NT Noncompetitive NT NT Noncompetitive NT NT Noncompetitive Noncompetitive
(74.0 ± 4.3)
(39.2 ± 2.4)
(13.5 ± 1.1)
(5.6 ± 1.7) (3.0 ± 0.2)
(91.8 ± 8.0) (30.2 ± 1.4)
(37.8 ± 2.4)
(12.1 ± 0.6)
(5.8 ± 0.2) (2.1 ± 0.05)
a All compounds were examined in at least three experiments; IC50 (50% inhibitory concentration) values for the compounds represent the concentration causing 50% enzyme activity loss. b Inhibition constant. c Not tested. d No activity.
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A 0.18
In this study, we identified for the first time sialidase inhibitor from the seeds of M. fragrans, with one of the 12 isolated compounds, 12, showing exemplary potency. By extending our study to parent compounds similar in chemical structure to the isolated compounds, we were able to uncover some key aspects of sialidase function. These results not only provide further insight into the pathogenic mechanisms of S. pneumoniae, infection but also suggest that isolated phenyl propene and diarylnonanoid compounds could be potentially used in future molecular therapies for bacterial infectious disease. Further in vivo evaluation of malabaricone C (12) will be reported in due course.
0.16 1/Rate (RFU/sec)
0.14 0.12 0.10 0.08 0.06 0.04 0.02 -2
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0
2 4 [8], ( M)
6
8
Conflict of interest The authors declare no conflict of interest.
B 0.14
Acknowledgements
1/Rate (RFU/sec)
0.12
This research was supported by the KRIBB Research Initiative Program and Fishing Commercialization Technology Development Program through KIMST funded by the Ministry of Oceans and Fisheries, South Korea, J.-.Y. Park and H.-.J. Kwon were supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1C1B2015753).
0.10 0.08 0.06 0.04 0.02
A. Supplementary data -1
0
1
2
3
4
[11], ( M)
C
0.06
References
1/Rate (RFU/sec)
0.05 0.04 0.03 0.02 0.01 -0.4 -0.2 0.0
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05. 055.
0.2 0.4 0.6 [12] ( M)
0.8
1.0
Fig. 2. Graphical determination of the inhibition type for compounds 8, 11 and 12. (A–C) Dixon plots for the inhibitory activity of compounds 8, 11 and 12, respectively, against S. pneumoniae NanA hydrolysis activity in the presence of different substrate concentrations (12.5 lM, d; 25 lM, s; and 50 lM, .).
The isolated compounds, aside from the diarylnonanoids (11 and 12), exhibited noncompetitive inhibition characteristics against NanA. As shown in Fig. 2, the kinetic plots showed that compound 8 had a noncompetitive mechanism because increasing substrate concentrations resulted in a family of lines with different slopes, but a common x-axis intercept. Importantly, the potent NanA inhibitors 11 and 12, the diarylnonanoids, showed a competitive inhibitory mode of action, as the Lineweaver-Burk plots for 1/V versus 1/[S] resulted in a family of straight lines with the same y-axis intercept for NanA inhibition (Fig. 2). Most compounds present noncompetitive inhibition for NanB and NanC (listed in Table 2, Fig. S14 and S15). Despite having similar molecular sizes and functionalities, the isolated compounds inhibited pneumonial sialidases by bothe competitive and noncompetitive inhibition mechanisms.
1. Luotonen J, Herva E, Karma P, Timonen M, Leinonen M, Makela PH. Scand J Infect Dis. 1981;13:177–183. 2. Mitchell TJ. Res Microbiol. 2000;151:413–419. 3. Brittan JL, Buckeridge TJ, Finn A, Kadioglu A, Jenkinson HF. Mol Oral Microbiol. 2012;27:270–283. 4. Bogaert D, De Groot R, Hermans PW. Lancet Infect Dis. 2004;4:144–154. 5. Paulson JC, Kawasaki N. Nat Biotechnol. 2011;29:406–407. 6. Chen GY, Chen X, King S, et al. Nat Biotechnol. 2011;29:428–435. 7. Parker D, Soong G, Planet P, Brower J, Ratner AJ, Prince A. Infect Immun. 2009;77:3722–3730. 8. Pettigrew MM, Fennie KP, York MP, Daniels J, Ghaffar F. Infect Immun. 2006;74:3360–3365. 9. Manco S, Hernon F, Yesilkaya H, Paton JC, Andrew PW, Kadioglu A. Infect Immun. 2006;74:4014–4020. 10. Kadioglu A, Weiser JN, Paton JC, Andrew PW. Nat Rev Microbiol. 2008;6:288–301. 11. Xu G, Potter JA, Russell RJ, Oggioni MR, Andrew PW, Taylor GL. J Mol Biol. 2008;384:436–449. 12. Gut H, King SJ, Walsh MA. FEBS Lett. 2008;582:3348–3352. 13. Xu G, Kiefel MJ, Wilson JC, Andrew PW, Oggioni MR, Taylor GL. J Am Chem Soc. 2011;133:1718–1721. 14. Park J-Y, Jeong HJ, Kim YM, et al. Bioorg Med Chem Lett. 2011;21:5602–5604. 15. Lee Y, Ryu YB, Youn HS, et al. Acta Crystallogr D Biol Crystallogr. 2014;70:1357–1365. 16. Ryu YB, Curtis-Long MJ, Lee JW, et al. Bioorg Med Chem. 2009;17:2744–2750. 17. Shafiei Z, Shuhairi NN, Md Fazly Shah Yap N, Harry Sibungkil CA, Latip J. Evid Based Complement Alternat Med eCAM. 2012;2012:825362. 18. Narasimhan B, Dhake AS. J Med Food. 2006;9:395–399. 19. Cao GY, Xu W, Yang XW, Gonzalez FJ, Li F. Food Chem. 2015;173:231–237. 20. Chung JY, Choo JH, Lee MH, Hwang JK. Phytomedicine. 2006;13:261–266. 21. Shinohara C, Mori S, Ando T, Tsuji T. Biosci Biotechnol Biochem. 1999;63:1475–1477. 22. Patro BS, Bauri AK, Mishra S, Chattopadhyay S. J Agric Food Chem. 2005;53:6912–6918. 23. Valente VMM, Jham GN, Jardim CM, Dhingra OD, Ghiviriga I. J Food Res. 2015;4:51–57. 24. Du SS, Yang K, Wang CF, et al. Chem Biodivers. 2014;11:1449–1456. 25. Li F, Yang XW. Phytochemistry. 2008;69:765–771. 26. Isogai A, Murakoshi S, Suzuki A, Tamura S. Agr Biol Chem. 1973;37:1479–1486. 27. Procedure of S. pneumoniae sialidases inhibition assay:As described, the inhibitory effects of compounds on S. pneumoniae sialidases were measured
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using a FRET method. In this assay, the 4-methylumbelliferyl-a-D-Nacetylneuraminic acid (Sigma Chemical Co., St. Louis, MO, USA) was used as a substrate, and the enzyme activity was determined by measuring the increase in fluorescence by continuously monitoring the reactions at 450/40 nm with excitation at 365 nm using a SpectraMax M2e Multimode Reader (Molecular Devices Co., USA). The IC50 values of the isolated compounds were measured in a reaction mixture containing each enzyme (final concentration of NanA, 2.2 nM; NanB, 17.8 nM; NanC, 22.9 nM), the test compounds (from 0 to 200 lM), and 50 lM of substrate in 20 mM Sodium phosphate buffer (pH 7.5, containing 300 mM NaCl). To determine the enzyme activity, the experimental data was fit to a logistic curve with Eq. (1), a time-drive protocol was used and the initial velocity was recorded over a range of concentrations, and the data were analyzed using a nonlinear regression program [Sigma Plot (SPCC Inc., Chicago, IL)].
Inhibition activity ð%Þ ¼ 100 ½ðS S0Þ=ðC C0Þ 100
ð1Þ
where C is the fluorescence of the control (enzyme, buffer, and substrate) after 60 min of incubation, C0 is the fluorescence of the control at 0 min, S is the fluorescence of the tested samples (enzyme, sample solution, and substrate) after
incubation, and S0 is the fluorescence of the tested samples at 0 min.The inhibition mechanism was determined, and the apparent inhibition constants (Ki) for the respective sialidases (NanA, NanB and NanC) were performed on the test compounds, for which the IC50 values were below 50 lM. The test compounds were studied at three different concentrations that were chosen based on the IC50 values obtained with each sialidases (approximately 1/2IC50, IC50, 2IC50). The concentrations of marker substrates were chosen (approximately 1/4 Km, 1/2 Km, Km) with regard to their Michaelis-Menten kinetics. The Ki values were calculated by nonlinear regression analysis by fitting different models of enzyme inhibition to the kinetic data using SigmaPlot Enzyme Kinetics Module 1.3 (SPSS Inc., Chicago, IL). The inhibition mechanism of the compounds was determined by comparing the statistical results, including the Akaike’s information criterion values, of different inhibition models and selecting the one with the best fit.28 28. Li X-Q, Andersson TB, Ahlstr¨om M, Weidolf L. Drug Metab Dispos. 2004;32:821–827. 29. Janapatla RP, Hsu MH, Hsieh YC, Lee HY, Lin TY, Chiu CH. Clin Microbiol Infect. 2013;19:480–486.