Inhibition of Dengue Virus Protease by Eugeniin, Isobiflorin, and

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Article Cite This: ACS Omega 2019, 4, 1525−1533

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Inhibition of Dengue Virus Protease by Eugeniin, Isobiflorin, and Biflorin Isolated from the Flower Buds of Syzygium aromaticum (Cloves) Hafiza Nosheen Saleem,† Farwa Batool,† Hafiz Javed Mansoor,† Syed Shahzad-ul-Hussan,*,‡ and Muhammad Saeed*,† Department of Chemistry and Chemical Engineering, Syed Babar Ali School of Science and Engineering, and ‡Department of Biology, Syed Babar Ali School of Science and Engineering, Lahore University of Management Sciences, 54792 Lahore, Pakistan

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†

S Supporting Information *

ABSTRACT: Dengue virus (DENV) infections are rampant in tropical and subtropical regions of the world with millions of people at risk. There is still no specific antiviral treatment available against these infections. Amongst the different potential therapeutic targets, DENV protease is considered an important target because of its crucial role in the viral replication cycle. We are reporting here a potent DENV protease inhibitor, eugeniin (3), which has been isolated from cloves, along with two other weaker inhibitors, isobiflorin (1) and biflorin (2). In this study, the IC50 values of 3 against the proteases of DENV serotype-2 and -3 were found to be 94.7 nM and 7.5 μM, respectively. Mechanistically, the compounds 1−3 exhibited a competitive type of inhibition, which were further substantiated by computational docking and saturation transfer difference (STD) NMR spectroscopy. Atomic-level details of the binding of these molecules at the active site of the protease suggested extensive interactions mediated by a network of hydrogen bonds and hydrophobic contacts. With further evaluation, these inhibitors are highly promising in the context of antiviral therapeutics development against DENV.

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medicinal chemistry and drug discovery.11 Therefore, development of direct-acting antiviral (DAA) drugs for the treatment of DENV infections can also be an attractive area of research for anti-dengue drug discovery. For discovering new DAA drugs, various viral factors have been proposed as drug targets.12,13 The positive-sense singlestranded RNA genome of DENV contains a single open reading frame that is translated inside the host cell into a long, intertwined polyprotein chain at the site of rough endoplasmic reticulum (ER). The polyprotein is cleaved into seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and three structural [capsid (C), membrane (M), and envelope (E)] proteins, which are subsequently packaged into a new virion. To accomplish this post-translational processing, viral (NS3) and host proteases (signalase and furin) play a central role. Normally, the loops of the polyprotein protruding into the ER lumen are cleaved by the host proteases; however, for the cleavages of the cytoplasmic loops, a viral protease is required.14 Analogous to the other pathogenic members of the Flavivirus genus, the proteolytic activity of the DENV protease

INTRODUCTION The past few decades have witnessed a sudden rise in flaviviral infections in tropical and subtropical regions, with a potential of penetrating into remote areas because of international travel and trade. Particularly, the infections caused by dengue virus (DENV) or Zika virus have raised major public health concerns.1−4 Epidemiological data suggest that DENV infections affect approximately 400 million people each year globally.4 Clinical symptoms of DENV infection range from a mild self-limited fibril illness to fatal dengue hemorrhagic fever and dengue shock syndrome.5 In the recent epidemic outbreaks, four serotypes of DENV (DENV1-4) have been detected along with the emergence of a fifth one in Southeast Asia.6 To date, no specific medicine is available for the treatment of DENV infections. Although, recently Sanofi-Pasteur developed a tetravalent dengue vaccine (CYD-TDV, Dengvaxia) that is licensed for clinical use in several endemic countries,7,8 yet its effectiveness in general population has been questioned.9,10 Availability of treatment regimens and preventive vaccines are equally important to control these infections. In the recent history, hepatitis C virus (HCV) infections are successfully cured by targeting the virus-specific factors directly, and this strategy has revolutionized the area of © 2019 American Chemical Society

Received: October 18, 2018 Accepted: January 2, 2019 Published: January 17, 2019 1525

DOI: 10.1021/acsomega.8b02861 ACS Omega 2019, 4, 1525−1533

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27.8 ± 2.4 μM and (1.4 ± 0.8) × 10−1/s, respectively, for DENV2 NS2BNS3pro, and 50.3 ± 4.2 μM and (7.4 ± 2.4) × 10−2/s for DENV3 NS2BNS3pro, respectively. The enzymatic efficiency, Kcat/KM, was calculated to be (5.0 ± 1.0) × 103 M−1s−1 for DENV2 NS2BNS3pro and (1.5 ± 0.5) × 103 M−1s−1 for DENV3 NS2BNS3pro. Preparation and Screening of the Extracts of Plant Materials. Selected plant materials (Supporting Information, Table S1) were extracted in ethanol. After evaporation of the solvent, a gummy material was obtained. The concentrated stock solutions of extracts were prepared by dissolving 10 mg of the gummy material in DMSO (1 mL) and used for initial screening at a concentration of 200 μg/mL to ascertain percentage inhibition of DENV2 NS2BNS3pro. Almost all the tested extracts showed significant background fluorescence because of their chemical constituents. However, the increase in the fluorescence due to free AMC appeared linear within the (AMC) concentration range used for these experiments (data not shown). Thus, the net rate of change of fluorescence (ΔRFU/s) within the first 5−10 min was used in the calculation of percentage inhibition of the activity in the presence of the plant extracts. As shown in Figure 1, most of

has been traced within the first 185 residues of the N-terminal domain of the NS3 protein (NS3pro). For the activation and formation of the catalytic site of the NS3pro, another nonstructural protein NS2B is required.15,16 Together, the complex NS2BNS3pro is responsible for the cleavage of the polyprotein at the junctions of NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5.14 Thus, because of its pivotal role in the replication of the virus, DENV NS2BNS3pro can be exploited as an important drug target for discovering DAA drugs. Previous efforts for discovering inhibitors of DENV NS2BNS3pro have resulted in the identification of several peptidic, peptidomimetic, and small organic molecules.17 However, to the best of our knowledge, no inhibitor has reached to the clinical trial, probably due to unfavorable pharmacokinetics and pharmacodynamic properties and/or lower target affinity.14 Natural products can offer an attractive source of new medicines because of their diverse structural and chemical properties. Secondary metabolites, isolated from natural products, have shown properties of specifically recognizing viral components without off-target effects that result in specific antiviral activities.18−20 Recently, antiviral activities from several herbal medicines and natural products against pathogenic viruses such as DENV, hepatitis B virus (HBV), HCV, herpes simplex virus, human immunodeficiency virus, and so forth have been reviewed.21 Therefore, to identify new natural product-based “hits”, we tested DENV protease inhibitory activity of plant materials with a known history of utilization in the traditional medicines against viral and febrile diseases.22−33 In this study, ethanol extracts of 14 plant materials were prepared and screened against recombinant DENV proteases from serotype-2 (DENV2). The initial screening resulted in the identification of the Syzygium aromaticum (cloves) extract with very strong inhibition of the DENV2 protease. Bioactive fractions of S. aromaticum were further subjected to the bioassay-guided isolation, which eventually led to the identification of previously known 5,7-dihydroxy-2-methylchromone-8C-β-D-glucopyranoside [isobiflorin (1)],34 5,7dihydroxy-2-methylchromone-6C-β-D-glucopyranoside [biflorin (2)],34 and eugeniin (3)19,35,36 as the active chemical constituents responsible for the inhibition. The purified compounds also show inhibition of the protease of serotype3 (DENV3 NS2BNS3pro). This article reports the IC50 values, the mechanism of inhibition, and the atomic-level details of the interactions of these inhibitors with DENV protease determined by computational docking and saturation transfer difference (STD)−NMR experiments.

Figure 1. Percentage of inhibition of DENV2 NS2BNS3pro by ethanol extracts of different plant materials. The plant extracts at a concentration of 200 μg/mL were added in the reaction mixture containing 100 mM Tris−HCl, pH 9.0, 20 mM NaCl, 40% glycerol, 2 mM CHAPS, 50 μM substrate, and 100 nM protease. The reaction was monitored for 30 min, and the net change of fluorescence because of the release of the AMC fluorophore in the presence of the extract was compared with the change of fluorescence in the absence of the extract. *The activity of proteases in the absence of the extract is used as a negative control. **Aprotinin (5 μM) was used as a positive control. Each bar represents a mean value of three replicates.

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the tested extracts demonstrated around 50% inhibition of DENV2 protease. Ethanol extracts of Caesalpinia crista and Citrullus colocynthis demonstrated minimum inhibition, that is, 90%) inhibition of the protease at 200 μg/mL concentration. For the positive control, a well-known serine protease inhibitor, aprotinin was used, which showed around 100% inhibition at 5 μM concentration. The protease-catalyzed reaction in the absence of any test compound or inhibitor was used as a negative control. The extracts showing at least 50% inhibition at 200 μg/mL were further subjected to determine the inhibitory dose response by varying concentrations between 1000 and 1 μg/ mL (Supporting Information, Table S1). The IC50 value of the

RESULTS AND DISCUSSION Expression, Purification, and Characterization of DENV2 and DENV3 NS2BNS3pro Enzymes. Recombinant NS2BNS3pro of DENV2 and DENV3 were expressed and purified for in vitro enzyme assays, by using the conventional methodology as described elsewhere.37−39 The enzymatic activity was measured by using a fluorogenic tetrapeptide substrate, Bz-Nle-Lys-Arg-Arg-AMC. Upon catalytic action of the protease on the substrate, 7-amino-4-methylcoumarine (AMC) was released and the enhanced fluorescence was then measured. Rate of enhancement of fluorescence was correlated to the rate of the reaction. The apparent KM and Kcat values determined for the recombinant proteases were found to be 1526


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crude ethanol extract of S. aromaticum was found to be 15.3 ± 5.8 μg/mL for DENV2 protease. The potential of the crude ethanol extracts of S. aromaticum to inhibit the DENV2 protease led us to further investigate for the identification of chemical constituents responsible for this activity. Identification of Eugeniin (3), Isobiflorin (1), and Biflorin (2) from the S. Aromaticum Extract. Ethanol extract of S. aromaticum was fractionated in different organic solvents including hexane, dichloromethane, and ethyl acetate, and subsequently, the inhibitory activity of every fraction was determined against DENV2 NS2BNS3pro. Except hexane fraction, all other fractions showed over 90% inhibition at 200 μg/mL concentrations. These fractions were then subjected to the measurement of IC50 values. The IC50 values of the ethyl acetate and aqueous fractions were around 12- and 22-fold lower than that of the parent ethanol extract. This indicated that the protease inhibition activity of S. aromaticum was due to its polar constituents (Table 1).

The purified compounds were structurally characterized by using 1H and 13C NMR spectroscopy, and the compounds related to the peak 1, 2, and 4 were identified as previously reported isobiflorin (1), biflorin (2), and eugeniin (3), respectively (Figure 2).34,35,40,41 IC50 Values of the Purified Compounds. The IC50 values of the purified 1−3 were determined against NS2BNS3pro of both serotype-2 and -3 of DENV after optimizing the concentration range of each compound to be used in enzymatic assay and subsequently fitting the data into a dose-response model. Eugeniin (3) was identified as the most potent among the three tested compounds with an IC50 value of 94.7 ± 2.5 nM against DENV2 protease. Isobiflorin (1) and biflorin (2) on the other hand showed moderate inhibitions with an apparent IC50 value of 58.9 ± 1.3 and 89.6 ± 4.4 μM, respectively (Figure 3A). IC50 values of the three compounds were also measured against DENV3 NS2BNS3pro. In this regard, concentrations of 1 and 2 were varied from 1 mM to 7.8 μM in 2-fold serial dilutions (eight concentrations), and concentrations of 3 from 100 μM to 45.7 nM in 3-fold serial dilutions (eight concentrations) after optimizing the concentration range. Compound 3 found out to be a better DENV3 protease inhibitor than 1 and 2: the apparent IC50 values of 1, 2, and 3 were determined as 219 ± 1.4, 337 ± 1.2, and 7.53 ± 1.6 μM, respectively (Figure 3B). However, inhibition of DENV3 protease by compound 3 was around 80-fold weaker as compared to DENV2. The lower inhibitory activity of 3 for DENV3 protease can be attributed to two mutations in the active site, as will be later explained in the Supporting Information (Figure S5). Interestingly, compounds 1 and its isomer 2 showed similar IC50 against proteases of both DENV genotypes, showing that the position of the glucose moiety does not affect the inhibitory properties of these compounds. Type of Protease Inhibition. Previously discovered small molecule inhibitors of dengue protease are found to bind to the active site,42 orthosteric site,43 or allosteric site44−46 of the enzyme. To determine whether the observed inhibition of the proteases by the compounds 1−3 is due to binding at the active site or at the orthosteric/allosteric site, we conducted enzyme kinetic analyses by monitoring the enzyme activity and inhibition at varying concentrations of the inhibitor and the substrate (60−4.6 μM). The data obtained were fitted to the linear Lineweaver−Burk Model (Supporting Information) that showed an increase in the KM value in response to increasing inhibitor concentration (Figure 4, Supporting Information, Figures S3 and S4). Also, the trend lines in each case passed through almost the same y-intercept, indicating no change in Vmax of both proteases in response to inhibitor concentrations. The enzyme kinetic data, therefore, showed a competitive type

Table 1. Percent Inhibition and IC50 Values of Solvent Fractions against DENV2 NS2BNS3pro fraction ethanol extract (parent) hexane fraction dichloromethane fraction ethyl acetate fraction aqueous fraction aprotinin (control, 5 μM)

% inhibitiona

IC50 (μg/mL)

± ± ± ± ± ±

15.3 ± 5.8 NDb 12.7 ± 2.5 1.3 ± 0.6 0.7 ± 0.1 0.5 ± 0.1

93.5 32.0 93.0 99.1 99.3 99.9

3.5 3.5 7.9 5.4 3.7 0.7

A single concentration of 200 μg/mL for the extract/fraction was tested. The given values are mean of three replicates. bNot determined. a

High-pressure liquid chromatography (HPLC) analysis of the aqueous fraction yielded a very complex chromatogram with overlapped peaks (data not shown). Therefore, on a semipreparative C18 column, 9 fractions of 75 mL each were blindly collected, freeze-dried, and tested for protease inhibition. The fraction eluted between 35 and 45% methanol in water demonstrated the highest inhibitory activity. In subsequent HPLC separation of this fraction by using the gradient system # 2 (Supporting Information), four major peaks and many minor peaks were observed. Therefore, repeated HPLC purifications were conducted to isolate the major peaks at retention times of 12.7 min (peak 1), 13.5 min (peak 2), 20.5 (peak 3), and 23.0 min (peak 4) (Supporting Information, Figure S1). Peak 3 showed no protease inhibition and was discarded. The solvent from the collected fractions was finally evaporated to yield 2.0, 5.6, and 15.2 mg amorphous solids from the peak 1, 2, and 4, respectively.

Figure 2. Chemical structures of isobiflorin (1), biflorin (2), and eugeniin (3) isolated from the aqueous fraction of S. aromaticum. 1527


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Figure 3. Determination of IC50 values of isobiflorin (1) (○), biflorin (2) (×), and eugeniin (3) (△) for DENV2 NS2BNS3pro (A) and DENV3 NS2BNS3pro (B). Concentrations of 1−3 were optimized and serial dilutions covering ∼0 to ∼100% enzyme inhibition were used. Eugeniin (3) demonstrated the lowest IC50 values against both proteases. Each data point is a mean value of three replicates.

Figure 4. Mechanism of inhibition demonstrated by eugeniin (3). Lineweaver−Burk plot showing competitive inhibition of (A) DENV2- and (B) DENV3 NS2BNS3pro. Initial rates of the enzymatic reactions were measured at varying concentrations of the substrate (60−4.6 μM) in the absence (×) and in the presence of 75 nM (○) and 150 nM (△) of eugeniin (3) in A, and in the absence (×) and in the presence of 5 μM (○) and 10 μM (△) of eugeniin (3) in B.

In the STD spectrum of 3, extraordinary signal enhancement, of almost equal intensity, for all of the aromatic protons was observed (Figure 5A). This suggests extensive interactions of the ligand involving all five aromatic rings in the formation of the contact surface with the protein. However, protons of the carbohydrate ring did not show any significant STD effect suggesting that the sugar moiety is not directly involved in mediating ligand−protein interactions. Similarly, the STD spectra of both of 2 and 1 (Figure 5B,C) showed the main STD effects from aromatic protons and almost no effect from sugar protons. This suggests that the two rings of the chromone skeleton, that exists almost perpendicular to the pyranose ring, are primarily involved in mediating interactions of these molecules with the protein. The methyl group at position c also did not show any significant STD enhancement indicating that the methyl group is possibly oriented away from the interface. Molecular Docking. To further illustrate the binding interactions of these compounds with DENV NS2BNS3 protease, docking simulation was performed using Autodock Vina.49 In the docking simulation, the cocrystallized structure of DENV3 protease (PDB ID: 3U1I) and energy minimized structures of 1−3 were used. Because the active sites of both DENV2 and DENV3 proteases are conserved with minor

of inhibition of both proteases by the compounds 1−3, that is, the inhibitor and the substrate compete for the same binding site. The Ki values of 3 was determined to be 125.2 nM for DENV2 protease, and 7.1 μM for DENV3 protease. The isomeric 1 and 2 showed likewise competitive inhibitions against both proteases (Supporting Information, Figures S3 and S4). STD−NMR Spectroscopy and Molecular Docking. To characterize atomic-level interactions of these inhibitors with the protease, we performed STD−NMR experiments. STD is a ligand-based NMR technique that provides information about the protons of a ligand in close contact with a protein during binding and can depict binding orientation of the ligand.47,48 The test compounds were initially dissolved in DMSO-d6 to prepare 30 mM stock solutions. Each sample was then prepared in a buffer consisting of 20 mM sodium phosphate buffer (pH 6.8), 30 μM DENV2 NS2BNS3pro, 1.5 mM compound, 50 mM sodium chloride, and 5% DMSO-d6. Onedimensional STD−NMR spectra were recorded by selectively irradiating protein resonances at −1.0 ppm and off-resonance frequency at 40 ppm using a train of 50 ms Gaussian-shaped radio frequency pulses separated by 1 ms delays at 45 db as the optimized power level. To suppress residual water signals, a binomial 3−9−19 pulse sequence was applied. 1528


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Figure 6. Molecular docking assessments of compounds 1−3 with DENV3 protease. Structures of NS3 and NS2B are colored gray and light blue, respectively, in the surface-displayed presentations. (A) Subsites S1−S4, defined on the basis of orientation of the tetrapeptide substrate residues, and P1−P4 in the substrate binding sites are approximately encircled in red. Optimized structures of 1 (B), 2 (C), and 3 (D) docked at the substrate binding site of the DENV3 NS2BNS3pro cocrystal structure (PDB ID: 3U1I) with a top docking score of −6.8, −7.2, and −10.2 kcal/mol, respectively, as determined by the AutoDock Vina program.

of Gly-133 and Ser-135. Furthermore, the residues Pro-132, Tyr-161, Tyr-150, and Val-155, lined with the S1 cavity, exhibit nonpolar van der Waals interaction and/or π−π interactions with HHDP (Figure 7). These interactions explain the strong STD enhancement observed for the aromatic Hd and H e protons (100 and 90% STD enhancement, respectively) (Figure 5A). Similarly, the three gallate functionalities at 1-, 2-, and 3-positions of glucose exhibit Hbonding through their hydroxyl groups, and van der Waals interactions (Table 2) with several residues of S2 and S3 subsites, explaining over 90% STD effects for the aromatic Ha, Hb, and Hc protons (Figure 5A). The sugar moiety despite present in the middle of the binding cleft parallel to the protein surface does not exhibit any direct interaction with the protein. It remains slightly distant from the protein surface acting as a scaffold for aromatic rings that make remarkable interactions with the protein. The docked structure of 3 seems to completely cover the catalytic triad (His-51, Asp-75, and Ser135) which provides the possible basis of its potent protease inhibitory activity. Docking of isobiflorin (1) shows the top scoring pose (docking score = −6.8 kcal/mol) nested at the S2 subsite of the substrate binding site (Figure 6B), demonstrating hydrogen bonding interactions of one of the phenolic hydroxyl groups with Asp-75 at a distance of 2.95 Å and of the sugar hydroxyl groups with the −NεH and −NH2 groups of Arg-54 at a distance of ∼2.70 and 3.24 Å, respectively (Supporting Information, Figure S6). 3-OH and 6-OH of the sugar moiety also show hydrogen bonding interactions with Trp-50 (2.96 Å) and Gly residues of the Gly4SerGly4 linker (2.98 Å). In the docked structure, the aromatic Ha and Hb atoms of 1 occupy the positions close to Asp-75 and His-51 corroborating the

Figure 5. Characterization of binding interactions by STD−NMR. STD (blue) and reference (red) spectra of 1.5 mM 3 (A), 2 (B), and 1 (C) in the presence of 30 μM DENV2 NS2BNS3pro. Aromatic protons and their corresponding peaks are labeled with single-digit alphabets, whereas sugar ring protons and their corresponding peaks are indicated with numbers. Signals from residual water, DMSO-d6, and acetone are also shown along with few peaks of impurities between 2 and 3.5 ppm. STD enhancement of each signal was calculated in percent of the strongest STD effect.

variations, and no crystal structure of the catalytically active closed conformation is available for the DENV2 protease, we used the crystal structure of DENV3 protease (3U1I) for these studies. Docking results demonstrated that the compounds 1− 3 were well accommodated at the flat, solvent-exposed and highly polar substrate binding site of DENV3 NS2BNS3 protease (Figure 6B−D). The docked models of 1−3 were evaluated on the basis of the docking score (Table 2). The docked structure of 3 (docking score = −10.2 kcal/ mol), the most active compound, shows extensive interactions with the residues of the subsites S1, S2, and S3 of the substrate-binding pocket of the protease (Figure 6D).50 The hexahydroxydiphenoyl (HHDP) moiety of the compound occupies a position deep inside the S1 cavity and exhibits H bond interactions with Asp-129, Phe-130, and the −NH group 1529


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Table 2. Docking Score and Binding Interaction of the Isolated Compounds with the Catalytic Site Residues of DENV NS2B/ NS3pro compound

docking score (kcal/mol)

isobiflorin (1) biflorin (2) eugeniin (3)

−6.8 to −6.3 (10 models) −7.2 to −6.7 (10 models) −10.2 to −9.7 (10 models)

interacting residues (H-bonding) Trp-50, Arg-54, Asp-75, Gly-linker Met-84, Ile-86, Asn-152, Gly-153, Tyr-161 Asp-75, Asp-81, Met-84, Asp-129, Phe-130, Gly-133, Ser-135

other close contact residues (within 4 Å space) His-51, Val-72, Asp-81, Asn-152 Thr-83, Arg-85, Val-154, Val-155 His-51, Arg-54, Pro-132, Tyr-150, Val-154, Val-155, Tyr-161

observed for it. Other notable residues within the 4 Å distance are shown in Table 2.

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CONCLUSIONS In conclusion, we have identified three compounds 1−3 from the aqueous fraction of S. aromaticum (cloves), which inhibits recombinantly expressed proteases of DENV2 and DENV3. These compounds appear to bind to the catalytic site of the protease and inhibit its enzyme activity with IC50 values in nanomolar to micromolar range. Atomic-level details of the binding interactions of these compounds with the proteases were described by STD−NMR spectroscopy and molecular docking simulations. Because the activity of DENV protease is essential for the maturation of nascent viral polyprotein, eugeniin (3) with IC50 values in nanomolar range exhibits high potential to inhibit dengue replication. Nevertheless, the effectiveness of these compounds to inhibit DENV replication in cell culture-based assay needs to be evaluated in the followup study. S. aromaticum (cloves) is a flavoring spice widely used as a food ingredient. Therefore, these compounds from cloves, expected to exhibit minimum or no cytotoxic effects, a characteristic of promising therapeutic agents.

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EXPERIMENTAL SECTION General. HPLC-based analyses were performed on the Alliance e2695 separation module (waters) connected with a photodiode array detector (2998, waters) by using a Discovery C18 (25 cm × 4 mm, 5 μm; Supelco) column. Semipreparative HPLC purifications were conducted on an Agilent 1100 series system connected with a G1314A variable wavelength detector, by using a Discovery C18 (25 cm × 10 mm, 5 μm; Supelco) column. DENV NS2BNS3 bioassays were performed on the EnSpire multimode plate reader (PerkinElmer). NMR spectra were acquired on a Bruker AVANCE New 600 MHz spectrometer equipped with a triple resonance z-gradient probe. All NMR data were processed and analyzed with TopSpin 4.0 software. Characterization of DENV NS2BNS3 Bioassays. Preparation and purification of DENV2 and DENV3 NS2BNS3pro were accomplished by using the standard procedures reported elsewhere.37,38,51 Protease activity of the purified DENV2 and DENV3 NS2BNS3pro was assessed in an opaque 96-well plate containing 100 μL reaction mixture/well. The reaction was conducted in a buffer containing 50 mM Tris, pH 9.0, 10 mM NaCl, 25% glycerol, 1 mM CHAPS, and 50 μM tetrapeptide substrate, Bz-Nle-Lys-Arg-Arg-AMC. The reaction was initiated by the addition of the 100 nM DENV2 or DENV3 NS2BNS3pro enzyme, and the release of fluorogenic AMC was measured at 37 °C after every 60 s interval by using a spectrofluorometer (Molecular Devices) for a total time of 30 min. The excitation and emission wavelengths were set at 380 and 460 nm, respectively. For the determination of Michaelis− Menten parameters (KM, Vmax, Kcat), the concentration of DENV NS2BNS3pro was fixed at 100 nM, while that of the

Figure 7. LigPlot analysis of the DENV NS2BNS3pro-eugeniin (3) complex. The bonds in 3 are shown in blue and those in protein residues are shown in orange. Hydrogen bonding interactions are shown as green dashed lines, and van der Waals interactions are shown as red-spiked half circles. Residue numbering is based on individual chains (A = NS2B and B = NS3 chain).

STD signal enhancement of these protons, that is, 98 and 100%, respectively, in the STD−NMR spectrum. The methyl group of 1 in the docked pose points away from the protease catalytic site and thus shows only a weak (8%) signal enhancement. Other residues of the catalytic site surrounded by 1 within 4 Å distance are given in the Table 2. The docked structure of isomeric biflorine (2) (docking score = −7.2 kcal/mol) occupies the position between S4 and S3 subsites (Figure 6C), exhibiting interactions with NS3pro as well as the cofactor NS2B. The chromone nucleus rests on Val-154 and Val-155 of subsite S4, which renders several hydrogen bond interactions of the hydroxyl groups of the sugar moiety and phenolic ring with residues Asn-152 (3.02 Å), Tyr161 (2.84 Å), and backbone CO/NH of Ile-86 (3.20 Å), Gly-153 (2.63 and 2.69 Å), and Met-84 (2.51 and 2.72 Å) (Supporting Information, Figure S7). Positioning 2 this way would place the aromatic Ha proton about 3.4 Å away from Val-155, and the aromatic Hb proton about 4.3 Å away from Ile-86, thus causing STD enhancement of about 98 and 100%, respectively, for these H-atoms. Because, the methyl group of the chromone moiety was protruding away from protein, and toward the solvent, only a weak STD signal (10%) was 1530


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400 to 0.05 μg/mL concentrations, were prepared in DMSO and bioassays were conducted as above. HPLC Analyses and Purification of the Active Chemical Constituents. The lyophilized aqueous fraction of S. aromaticum was dissolved in a mixture of methanol/water (3:1), and a 20 μL injection was analyzed by using a C18 column (4.6 × 250 mm, Supelco) on analytical HPLC. At a flow rate of 1 mL/min, the mobile phase was changed from initial 100% H2O to 100% MeOH in 30 min linear gradient, while monitoring the eluates at 277 nm. For purification, a manual injection of 1 mL extract was separated on a semipreparative C18 column (10 × 250 mm, Supelco) eluted with a flow rate of 3.0 mL/min. The dual solvent gradient was linearly changed from 100% H2O at the start to 50% MeOH in 30 min and then to 100% MeOH in the next 10 min (semipreparative gradient system #1, Supporting Information). Eluates were blindly collected for every 5 min intervals, lyophilized, and tested for the biological activity. The active fractions were rechromatographed repeatedly using the same column, but with a second mobile phase gradient system (#2, Supporting Information). Almost pure peaks were collected in the second system and occasionally needed a second round of purification by the same system #2. Identical peaks were combined, lyophilized, and characterized by spectroscopy techniques (Supporting Information, Figures S8 and S9 for 1 H NMR spectra of 3). For the 50% inhibitory concentration (IC50) of 1−3, DENV2/DENV3 NS2BNS3pro bioassays were conducted as above in the presence of different concentrations of the compounds (0.032−500 μM), dissolved in DMSO. The final concentration of DMSO in each reaction mixture was less than 5%. At least eight data points were obtained for inhibitor concentrations in serial dilution. IC50 values were calculated by plotting data using GraphPad Prism v.7 software. Type of Inhibition. The activity of DENV2/DENV3 NS2BNS3pro was monitored in the same way as described before, with varying concentrations of both inhibitors and the substrate (4.6−60 μM). For isobiflorin (1), two concentrations of 40 and 80 μM were used for DENV2 protease. For biflorin (2), two concentrations of 75 and 150 μM were used for DENV2 protease. For eugeniin (3), two concentrations of 75 and 150 nM were used for DENV2 protease, and 5 and 10 μM for DENV3 protease. The reactions were started after addition of the 100 nM enzyme in all reactions, and the change of fluorescence per s(ΔRFU/s) was measured in the first 5−10 min. The data were fit to the Lineweaver−Burk equation (Supporting Information) in order to determine the mechanism and kinetic parameters for each compound by using GraphPad Prism v.7 software. Computational Assessment of Binding Interactions. The 3D structure of DENV NS2BNS3pro cocrystallized with a covalently linked substrate, Bz-NLE-LYS-ARG-Arg-H, was downloaded from the Protein Data Bank website (PDB ID: 3U1I). The water molecules and heterogroups were removed from the structure by using PyMOL software. The input pdbqt files were prepared by using AutoDock Tool (ADT), bundled with the MGLTools package (version 1.5.7). All hydrogens were added in the editing of the protein structure, and nonpolar hydrogens were merged. Structures of 1−3 were drawn by the ChemDraw software package, saved in MDL Molfile format, and then converted into 3D structures by importing into Avogadro software. The energy was minimized by applying UFF force field. The energy minimized structures

substrate varied between 100 μM and 45 nM by using eight 3fold serial dilutions. The progression curve was fit in a nonlinear regression by using Graphpad Prism. Preparation of Plant Extract. The specific parts of plants (stem, bark, legumes, or seeds, see Table S1) were purchased from local vendors in the dry form and were ground to fine powder by using a mortar and pestle and/or by an electric blender. In each case, approximately 10 g powdered plant material was suspended in 30−40 mL of ethanol and kept at room temperature under dark for 5−7 days. The dark browncolored extracts were separated from the solid material by centrifugation, and the supernatants were decanted into roundbottomed flasks. The solvent was removed by using a rotary evaporator in each case to obtain a dark gummy material (ethanol extract). Stock solutions of each plant extract were prepared by dissolving 10 mg of the ethanol extract in 1 mL of DMSO and stored at −20 °C until further use. Inhibition of DENV NS2B-NS3pro by Plant Extracts. The DENV NS2BNS3pro activity inhibition assays were conducted in an opaque 96-well plate containing 100 μL total reaction mixture. The reaction was conducted in a buffer containing 50 mM Tris, pH 9.0, 10 mM NaCl, 25% glycerol, 50 μM Bz-Nle-Lys-Arg-Arg-AMC, and 1 mM CHAPS in the presence of 200 μg/mL plant extracts. The concentration of DMSO in each reaction mixture was less than 5%. The reaction in triplicates was initiated by addition of 100 nM DENV2 NS2B-NS3pro by a multichannel pipet, and release of fluorogenic AMC was measured after 60 s interval by using a spectrofluorometer for a maximum 30 min reaction time. A solvent (DMSO) control was run for each assay plate by measuring activity of DENV NS2BNS3pro in the absence of the plant extract (DMSO control). The effect of background fluorescence was measured, preparing a calibration curve by spiking known concentrations of free AMC in the same volume of the reaction mixtures containing the buffer, enzyme, and plant extract, and comparing the slope of this curve with that obtained by free AMC concentrations in reaction mixtures containing the buffer, enzyme, no plant extract, and no substrate. For the positive control, aprotinin was used in 5 μM concentration. All experiments were conducted in at least in triplicates. Initial velocities (rates) of reactions were measured, and the percentage (%) inhibition was then calculated by using the formula: Ä ÉÑ ijÅÅÅ v yz ÑÑ exp ÑÑ jjÅÅ z ÑÑ × 100zzz % inhibition = 100 − jjÅÅ jjÅÅ vpro − v bg ÑÑ zz ÑÖ kÅÇ { where vexp is the rate of fluorescence increase in the presence of the plant extract or the tested compound; vpro and vbg are the rates of fluorescence change in the presence and absence of the protease, respectively, when no plant extract/compound was added in the reaction mixture. Solvent Fractionation of S. aromaticum and Measurement of IC50. The ethanol extract of S. aromaticum flower buds (1 kg), prepared as described earlier was suspended in 10% aqueous ethanol and sequentially extracted with hexane (three times), chloroform (three times), and ethyl acetate (three times). The solvent fractions and the remaining aqueous fraction were evaporated to dryness. The dried fractions (20 mg each) were dissolved in 1 mL of DMSO to prepare stock solution, and percentage inhibition of the solvent extracts was measured as described above. For the measurement of IC50 values, 3-fold serial dilutions of the test extract, ranging from 1531


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Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease. N. Engl. J. Med. 2015, 373, 1195−1206. (8) Capeding, M. R.; Tran, N. H.; Hadinegoro, S. R. S.; Ismail, H. I. H. M.; Chotpitayasunondh, T.; Chua, M. N.; Luong, C. Q.; Rusmil, K.; Wirawan, D. N.; Nallusamy, R.; Pitisuttithum, P.; Thisyakorn, U.; Yoon, I.-K.; van der Vliet, D.; Langevin, E.; Laot, T.; Hutagalung, Y.; Frago, C.; Boaz, M.; Wartel, T. A.; Tornieporth, N. G.; Saville, M.; Bouckenooghe, A. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014, 384, 1358− 1365. (9) Dans, A. L.; Dans, L. F.; Lansang, M. A. D.; Silvestre, M. A. A.; Guyatt, G. H. Controversy and debate on dengue vaccine series-paper 1: review of a licensed dengue vaccine: inappropriate subgroup analyses and selective reporting may cause harm in mass vaccination programs. J. Clin. Epidemiol. 2018, 95, 137−139. (10) Larson, H. J.; Hartigan-Go, K.; de Figueiredo, A. Vaccine confidence plummets in the Philippines following dengue vaccine scare: why it matters to pandemic preparedness. Hum. Vaccines Immunother. 2018, 1−3. (11) Lam, B.; Henry, L.; Younossi, Z. Sofosbuvir (Sovaldi) for the treatment of hepatitis C. Expert Rev. Clin. Pharmacol. 2014, 7, 555− 566. (12) Behnam, M. A. M.; Nitsche, C.; Boldescu, V.; Klein, C. D. The medicinal chemistry of dengue virus. J. Med. Chem. 2016, 59, 5622− 5649. (13) Qadir, A.; Riaz, M.; Saeed, M.; Shahzad-ul-Hussan, S. Potential targets for therapeutic intervention and structure based vaccine design against Zika virus. Eur. J. Med. Chem. 2018, 156, 444−460. (14) Nitsche, C.; Holloway, S.; Schirmeister, T.; Klein, C. D. Biochemistry and medicinal chemistry of the dengue virus protease. Chem. Rev. 2014, 114, 11348−11381. (15) Yusof, R.; Clum, S.; Wetzel, M.; Murthy, H. M. K.; Padmanabhan, R. Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J. Biol. Chem. 2000, 275, 9963−9969. (16) Cahour, A.; Falgout, B.; Lai, C. J. Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease. J Virol. 1992, 66, 1535−42. (17) Nitsche, C. Strategies towards protease inhibitors for emerging flaviviruses. Adv. Exp. Med. Biol. 2018, 1062, 175−186. (18) Hussein, G.; Miyashiro, H.; Nakamura, N.; Hattori, M.; Kakiuchi, N.; Shimotohno, K. Inhibitory effects of sudanese medicinal plant extracts on hepatitis C virus (HCV) protease. Phytother. Res. 2000, 14, 510−516. (19) Kurokawa, M.; Hozumi, T.; Basnet, P.; Nakano, M.; Kadota, S.; Namba, T.; Kawana, T.; Shiraki, K. Purification and characterization of eugeniin as an anti-herpesvirus compound from Geum japonicum and Syzygium aromaticum. J. Pharmacol. Exp. Therapeut. 1998, 284, 728−35. (20) Rothan, H. A.; Zulqarnain, M.; Ammar, Y. A.; Tan, E. C.; Rahman, N. A.; Yusof, R. Screening of antiviral activities in medicinal plants extracts against dengue virus using dengue NS2B-NS3 protease assay. Trop. Biomed. 2014, 31, 286−96. (21) Lin, L.-T.; Hsu, W.-C.; Lin, C.-C. Antiviral natural products and herbal medicines. J. Tradit. Complement. Med. 2014, 4, 24−35. (22) Panchabhai, T. S.; Kulkarni, U. P.; Rege, N. N. Validation of therapeutic claims of Tinospora cordifolia: a review. Phytother. Res. 2008, 22, 425−441. (23) Kean, J. D.; Downey, L. A.; Stough, C. A systematic review of the Ayurvedic medicinal herb Bacopa monnieri in child and adolescent populations. Complement. Ther. Med. 2016, 29, 56−62. (24) Triantafyllidi, A.; Xanthos, T.; Papalois, A.; Triantafillidis, J. K. Herbal and plant therapy in patients with inflammatory bowel disease. Ann Gastroenterol. 2015, 28, 210−220. (25) Nikolić, M. M.; Jovanović, K. K.; Marković, T. L.; Marković, D. L.; Gligorijević, N. N.; Radulović, S. S.; Kostić, M.; Glamočlija, J. M.;

were saved as pdb format and then optimized by ADT as described above.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02861. Chemicals and materials used in this study; expression, purification, and characterization of dengue proteases; preparation of plant extracts; HPLC gradient systems; preparation of protease structure for docking; and selected plant material and IC50 values of crude ethanol extracts (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.S.-u.-H.). *E-mail: [email protected] (M.S.). ORCID

Muhammad Saeed: 0000-0002-9229-838X Author Contributions

This study was conceived and the experiments were designed by M.S. Bioassays, inhibition kinetic studies, and HPLC analyses/purifications were performed by H.N.S. Preparation of the plant extracts and stock solutions were performed by F.B. and H.J.M. STD NMR spectroscopy data were collected, analyzed, and written by S.S.-u.-H. The article was written mainly by HNS with the assistance of F.B., H.J.M., and proofread by M.S. and S.S.-u.-H. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Higher Education Commission (HEC) of Pakistan (grant no. NRPU#5912) and faculty initiative funding (FIF) from LUMS.

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REFERENCES

(1) Kaptein, S. J.; Neyts, J. Towards antiviral therapies for treating dengue virus infections. Curr. Opin. Pharmacol. 2016, 30, 1−7. (2) Gulland, A. Burden of dengue fever is higher than previously thought. BMJ 2013, 347, f6280. (3) Wilder-Smith, A.; Gubler, D. J.; Weaver, S. C.; Monath, T. P.; Heymann, D. L.; Scott, T. W. Epidemic arboviral diseases: priorities for research and public health. Lancet Infect. Dis. 2017, 17, e101− e106. (4) Bhatt, S.; Gething, P. W.; Brady, O. J.; Messina, J. P.; Farlow, A. W.; Moyes, C. L.; Drake, J. M.; Brownstein, J. S.; Hoen, A. G.; Sankoh, O.; Myers, M. F.; George, D. B.; Jaenisch, T.; Wint, G. R. W.; Simmons, C. P.; Scott, T. W.; Farrar, J. J.; Hay, S. I. The global distribution and burden of dengue. Nature 2013, 496, 504−507. (5) Monath, T. P. Dengue: the risk to developed and developing countries. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2395−2400. (6) Mustafa, M. S.; Rasotgi, V.; Jain, S.; Gupta, V. Discovery of fifth serotype of dengue virus (DENV-5): A new public health dilemma in dengue control. Med. J. Armed Forces India 2015, 71, 67−70. (7) Hadinegoro, S. R.; Arredondo-García, J. L.; Capeding, M. R.; Deseda, C.; Chotpitayasunondh, T.; Dietze, R.; Ismail, H. I. H. M.; Reynales, H.; Limkittikul, K.; Rivera-Medina, D. M.; Tran, H. N.; Bouckenooghe, A.; Chansinghakul, D.; Cortés, M.; Fanouillere, K.; Forrat, R.; Frago, C.; Gailhardou, S.; Jackson, N.; Noriega, F.; Plennevaux, E.; Wartel, T. A.; Zambrano, B.; Saville, M. Efficacy and 1532


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Article

Soković, M. D. Antimicrobial synergism and cytotoxic properties of Citrus limon L., Piper nigrum L. and Melaleuca alternifolia (Maiden and Betche) Cheel essential oils. J. Pharm. Pharmacol. 2017, 69, 1606−1614. (26) Boskabady, M. H.; Alitaneh, S.; Alavinezhad, A. Carum copticum L.: a herbal medicine with various pharmacological effects. BioMed Res. Int. 2014, 2014, 569087. (27) Ghasemian, M.; Owlia, S.; Owlia, M. B. Review of antiinflammatory herbal medicines. Adv. Pharmacol. Sci. 2016, 2016, 9130979. (28) Kalauni, S. K.; Awale, S.; Tezuka, Y.; Banskota, A. H.; Linn, T. Z.; Asih, P. B. S.; Syafruddin, D.; Kadota, S. Antimalarial activity of cassane- and norcassane-type diterpenes from Caesalpinia crista and their structure-activity relationship. Biol. Pharm. Bull. 2006, 29, 1050− 1052. (29) Dugoua, J.-J.; Seely, D.; Perri, D.; Cooley, K.; Forelli, T.; Mills, E.; Koren, G. From type 2 diabetes to antioxidant activity: a systematic review of the safety and efficacy of common and cassia cinnamon barkThis article is one of a selection of papers published in this special issue (part 1 of 2) on the Safety and Efficacy of Natural Health Products. Can. J. Physiol. Pharmacol. 2007, 85, 837−847. (30) Liu, Q.; Meng, X.; Li, Y.; Zhao, C.-N.; Tang, G.-Y.; Li, H.-B. Antibacterial and Antifungal Activities of Spices. Int. J. Mol. Sci. 2017, 18, 1283−1344. (31) Elmaci, I.; Altinoz, M. A. Thymoquinone: An edible redoxactive quinone for the pharmacotherapy of neurodegenerative conditions and glial brain tumors. A short review. Biomed. Pharmacother. 2016, 83, 635−640. (32) Aggarwal, B. B.; Prasad, S.; Reuter, S.; Kannappan, R.; Yadev, V. R.; Park, B.; Kim, J. H.; Gupta, S. C.; Phromnoi, K.; Sundaram, C.; Prasad, S.; Chaturvedi, M. M.; Sung, B. Identification of novel antiinflammatory agents from Ayurvedic medicine for prevention of chronic diseases: ″reverse pharmacology″ and ″bedside to bench″ approach. Curr. Drug Targets 2011, 12, 1595−1653. (33) Al-Zuhair, H.; el-Sayeh, B.; Ameen, H. A.; al-Shoora, H. Pharmacological studies of cardamom oil in animals. Pharmacol. Res. 1996, 34, 79−82. (34) Zhang, Y.; Chen, Y. Isobiflorin, a chromone C-glucoside from cloves (Eugenia Caryophyllata). Phytochemistry 1997, 45, 401−403. (35) Nonaka, G.-I.; Harada, M.; Nishioka, I. Eugeniin, a new ellagitannin from Cloves. Chem. Pharm. Bull. 1980, 28, 685−687. (36) Takechi, M.; Tanaka, Y. Purification and characterization of antiviral substance from the bud of Syzygium aromatica. Planta Med. 1981, 42, 69−74. (37) Li, J.; Lim, S. P.; Beer, D.; Patel, V.; Wen, D.; Tumanut, C.; Tully, D. C.; Williams, J. A.; Jiricek, J.; Priestle, J. P.; Harris, J. L.; Vasudevan, S. G. Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries. J. Biol. Chem. 2005, 280, 28766− 28774. (38) Lin, K.-H.; Ali, A.; Rusere, L.; Soumana, D. I.; Yilmaz, N. K.; Schiffer, C. A. Dengue virus NS2B/NS3 protease inhibitors exploiting the prime side. J. Virol. 2017, 91, e00045−00017. (39) Leung, D.; Schroder, K.; White, H.; Fang, N.-X.; Stoermer, M. J.; Abbenante, G.; Martin, J. L.; Young, P. R.; Fairlie, D. P. Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors. J. Biol. Chem. 2001, 276, 45762−45771. (40) Tanaka, T.; Orii, Y.; Nonaka, G.-i.; Nishioka, I. Tannins and related compounds. CXXIII Chromone, acetophenone, and phenylproponoid glucosides and their gallol and/or hexahydroxydiphenoyl esters from the leaves of Syzysium aromaticum Merr. et Perry. Chem. Pharm. Bull. 1993, 41, 1232−1237. (41) Lee, S.-H.; Tanaka, T.; Nonaka, G.-i.; Nishioka, I. Hydrolysable tannins from euphorbia thymifolia. Phytochemistry 1990, 29, 3621− 3625. (42) Weng, Z.; Shao, X.; Graf, D.; Wang, C.; Klein, C. D.; Wang, J.; Zhou, G.-C. Identification of fused bicyclic derivatives of pyrrolidine

and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors. Eur. J. Med. Chem. 2017, 125, 751−759. (43) Li, Z.; Sakamuru, S.; Huang, R.; Brecher, M.; Koetzner, C. A.; Zhang, J.; Chen, H.; Qin, C.-f.; Zhang, Q.-Y.; Zhou, J.; Kramer, L. D.; Xia, M.; Li, H. Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease. Antiviral Res. 2018, 150, 217−225. (44) Padmapriya, P.; Fathima, S. G.; Ramanathan, G.; Yuvaraj, V.; Sheriff, K. A.; Kaveri, K.; Gunasekaran, P.; Sivagnanam, U. T.; Thennarasu, S. Development of antiviral inhibitor against dengue 2 targeting NS3 protein: In vitro and in silico significant studies. Acta Trop. 2018, 188, 1−8. (45) Beesetti, H.; Tyagi, P.; Medapi, B.; Krishna, V. S.; Sriram, D.; Khanna, N.; Swaminathan, S. A quinoline compound inhibits the replication of dengue virus serotypes 1-4 in Vero cells. Antiviral Ther. 2018, 23, 385−394. (46) Brecher, M.; Li, Z.; Liu, B.; Zhang, J.; Koetzner, C. A.; Alifarag, A.; Jones, S. A.; Lin, Q.; Kramer, L. D.; Li, H. A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease. PLoS Pathog. 2017, 13, e1006411. (47) Mayer, M.; Meyer, B. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J. Am. Chem. Soc. 2001, 123, 6108− 6117. (48) Bewley, C. A.; Shahzad-ul-Hussan, S. Characterizing carbohydrate-protein interactions by nuclear magnetic resonance spectroscopy. Biopolymers 2013, 99, 796−806. (49) Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455− 461. (50) Noble, C. G.; Seh, C. C.; Chao, A. T.; Shi, P. Y. Ligand-bound structures of the dengue virus protease reveal the active conformation. J. Virol. 2011, 86, 438−446. (51) Yang, C.-C.; Hsieh, Y.-C.; Lee, S.-J.; Wu, S.-H.; Liao, C.-L.; Tsao, C.-H.; Chao, Y.-S.; Chern, J.-H.; Wu, C.-P.; Yueh, A. Novel dengue virus-specific NS2B/NS3 protease inhibitor, BP2109, discovered by a high-throughput screening assay. Antimicrob. Agents Chemother. 2010, 55, 229−238.

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Inhibition of Dengue Virus Protease by Eugeniin, Isobiflorin, and

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