Discovery of Pentacyclic Triterpenoids as ... - ACS Publications

Subscriber access provided by University Libraries | University of Notre Dame

Article

Discovery of Pentacyclic Triterpenoids as Potential Entry Inhibitors of Influenza Viruses Maorong Yu, Longlong Si, Yufei Wang, Yiming Wu, Fei Yu, Pingxuan Jiao, Yongying Shi, Han Wang, Sulong Xiao, Ge Fu, Ke Tian, Yi-Tao Wang, Zhihong Guo, Xin-Shan Ye, Li-He Zhang, and Demin Zhou J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5014067 • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 17, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Discovery of Pentacyclic Triterpenoids as Potential Entry Inhibitors of Influenza Viruses Maorong Yu*,†,§, Longlong Si†,§, Yufei Wang†, Yiming Wu†, Fei Yu†,¤, Pingxuan Jiao†, Yongying Shi†, Han Wang†, Sulong Xiao†, Ge Fu†, Ke Tian#, Yitao Wang ± , Zhihong Guo¶, Xinshan Ye†, Lihe Zhang† and Demin Zhou*, †,§

†

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

#

Stanley Manne Children's Research Institute, Northwestern University, Chicago, Illinois, USA

±

State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao

¶

Department of Chemistry and Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

¤

Medical Faculty of Kunming University of Science and Technology, Kunming 650500, China

Address correspondence to: Professor Demin Zhou or Dr. Maorong Yu School of Pharmaceutical Sciences Peking University #38 Xueyuan Road, Beijing 100191, China Tel: 86-10-8280-5857 Fax: 86-10-8280-5519

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Email: [email protected]; [email protected]

§ These authors contributed equally to this paper


Page 2 of 60

Page 3 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Entry inhibitors are of particular importance in current efforts to develop a new generation of anti-influenza virus drugs. Here we report certain pentacyclic triterpenes exhibiting conserved structure features and with in vitro anti-influenza virus activity comparable to and even higher than that of oseltamivir. Mechanistic studies indicated that these lead triterpenoids bind tightly to the viral envelope hemagglutinin (HA), disrupting the interaction of HA with the sialic acid receptor and thus the attachment of viruses to host cells. Docking studies suggest that the binding pocket within HA for sialic acid receptor potentially acts as a targeting domain, and this is supported by structure-activity data, sialic acid competition studies and broad anti-influenza spectrum as well as less induction of drug resistance. Our study might establish the importance of triterpenoids for development of entry inhibitors of influenza viruses.

Keywords: Pentacyclic triterpenes; Influenza; Entry inhibitor; Hemagglutinin; Sialic acid pocket



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Influenza A virus, a member of the Orthomyxoviridae family, is a major human pathogen that causes annual epidemics and occasional pandemics. Such RNA viruses are negative-sense, single-stranded and roughly spherical and segmented with seven or eight fragments, each one encoding one or two genes. Influenza A viruses are categorized into different subtypes based on two large glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are on the surface of the viral envelope. HA, a lectin, mediates attachment of the virus to target cells via the sialic acid receptor and subsequently fusion of the viral envelope with host membrane, releasing the viral genome into the target cells and initiating infection. NA is an enzyme that cleaves the sialic acid residue tethering the progeny virus and detaches it from infected cells, thus accomplishing one round of infection and virus propagation.1 Currently, 18 types of HA and 11 types of NA have been identified among different influenza viruses.2,3 In the past 100 years, influenza epidemics and pandemics, including H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, and H5N1 in 2009 caused a serious impact on global morbidity, mortality and economy,4-10 and in 2014, influenza A (H7N9) is causing increasing morbidity and mortality in China.11 Currently, two classes of anti-influenza drugs have been developed for interruption of specific processes in influenza infection. Amantadine and rimantadine target the M2 protein which is an ion channel allowing protons to move through the viral envelope to uncoat viral RNA, and thus block the release of viral RNA into the cytoplasm.12 Oseltamivir (Tamiflu) and zanamivir (Relenza), on the other hand, target neuraminidase (NA) protein inhibiting its enzymatic activity


Page 4 of 60

Page 5 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and causing the tethered progeny virus to be unable to escape from its host cells.13,14 However, the emergence of drug-resistant influenza viruses has limited the use of those drugs,15-17 making the identification of novel anti-influenza drugs an urgent task. Influenza virus entry represents a favorable target for drug discovery, since inhibition of the first step of virus infection should result in an efficient block to virus propagation.18, 19 Possible approaches to this include targeting the sialic acid receptor-binding HA protein or disrupting the interactions between the viral and cellular proteins required for entry.18,20 An advantage of the latter strategy is that resistance is less likely to occur since many viruses use similar entry routes.21 Pentacyclic triterpenes are secondary plant metabolites found in different plant organs, with a few species containing up to 30% of their dry weigh.22 It has been suggested that the defense activities of these triterpenes stem from their ability to prevent various pathogen and herbivore infections in the host.22,23 Betulinic acid (BA), a lupane-type triterpene, and oleanolic acid (OA) have been confirmed in many studies to display inhibitory activity against HIV entry and maturation and the triterpene derivative bevirimat (PA-457) is currently in clinical trials.22 Recently, we found that echinocystic acid (EA), an oleanane-type triterpene, and its derivatives exhibit inhibitory activity against HCV entry with IC50 at nanomolar levels.22-25 Here we report certain pentacyclic triterpenoids with conserved structure features displaying in vitro anti-influenza virus activity comparable or higher than that of oseltamivir. Mechanistic studies indicated that these triterpenes bind tightly to hemagglutinin (HA) protein, thus disrupting the interaction of HA with the sialic acid receptor. Furthermore, docking studies suggest that the sialic



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

acid receptor-binding pocket within HA is the domain targeted. This is consistent with structure-activity studies, sialic acid competition assays and the observations of broad anti-influenza spectrum and less induction of drug resistance. This study may establish the importance of triterpenoids as leads for the development of potential entry inhibitors of influenza viruses. RESULTS AND DISCUSSION Discovery of lead triterpenoids. In view of their significant inhibitory effect on both HIV and HCV viruses,22-25 we sought to learn whether such pentacyclic triterpenoids are also active against influenza viruses. The cytopathic effect (CPE) reduction assay and the CellTiter-Glo assay were utilized in parallel to screen a mini-library of EA-glycoconjugates [Supporting Information (SI) Figure 1A]. The CellTiter-Glo screening, an assay monitoring cell proliferation, was utilized to screen and exclude compounds with significant toxicity toward MDCK cells (SI Figure 1B). CPE screening, an assay for measuring the damage to host cells during virus invasion, was utilized to screen and identify compounds which display a reduction of the cytopathic effect on influenza A/WSN/33 virus. We found that Q8, an EA-galactose conjugate (Figure 1A), significantly reduces the viral CPE in MDCK cells (SI Figure 1C). The reduction of the CPE was confirmed by direct microscopic observation which detected far less CPE than in the DMSO control (Figure. 1B). In addition, this triterpenoid exerts a well-defined dose-dependent response against the A/WSN/33 virus based on plague formation (Figure 1C). This is an alternative assay for evaluation of potency, with EC50 calculated to be around 5 µM, almost two-fold lower than that of oseltamivir. No


Page 6 of 60

Page 7 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protective effect was observed with other EA-glycoconjugates including glucose, lactose, maltose and cellobiose (SI Figure 1C). Optimization of lead compound. To explore the structure requirements for maintenance of high anti-influenza activity, a systemic study of Q8 was carried out involving chemical modifications to the aglycone, glycoside and linker moieties in the compound. Some of the synthetic analogs and commercially available related compounds along with their activities are shown in Figure 2. The CPE assay indicated that Y2, an analog of Q8 in which the ester link is replaced by an amide bond, has potency almost identical to that of Q8, indicating the compatibility of ester and amide linkers for anti-A/WSN/33 virus. However, replacement of the galactose moiety in Y2 by mannose, yielding Y16, diminished the potency, as did substitution of the galactose in Q8 by glucose (Q2). This revealed the critical nature of the galactose moiety for the anti-influenza virus activity. This requirement was also found in other active triterpeneglycoconjugates such OA (Y3 and Y4) and ursolic acid (Y5 and Y6), in which galactose is tethered with different aglycones. Replacement of the galactose moiety in these conjugates by mannose (Y7, Y8 and Y9) substantially decreased the anti-influenza activity. Furthermore, it was found that acetylation of the galactose moiety enhances anti-influenza virus activity when the aglycone is OA or ursolic acid (Y3 v. Y4 and Y5 v. Y6). Such acetyl-mediated promotion is also observed in OA-mannose conjugates (Y9 vs. Y10) and consequently it is thought that the introduced acetyl group might be directly involved in anti-influenza virus activity.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

We next explored the aglycone moiety by replacement of EA with OA or with ursolic acid (UA). OA is an analog of EA, but lacking the C-16 hydroxyl group. Such a small change substantially enhances the anti-influenza virus activity (Y4 vs. Y2). The enhancement is further obvious in the acetylated counterparts - Y3 is almost twice as potent as Y1. UA is an analog of OA with the C-20 methyl group shifted to C-19. This scaffold change has no effect on anti-influenza potency when the glycoside is galactose (Y5 v. Y3 and Y6 v. Y4), and has little effect when the glycoside is mannose (Y7 v. Y9 and Y8 v. Y10). Clearly, OA and UA, as the main elements of the aglycone, are both superior to EA in maintenance of higher anti-influenza activity. Further exploration of the EA moiety, which compared to OA or UA has one additional chemically modifiable site, indicated that oxidation of the 3-OH and the 16-OH had less effect on potency when the glycoside is an acetylated galactose (Y11 v. Y1). This suggests that the anti-influenza potency is relatively tolerant to modifications in the aglycone moiety. This was also observed in OA-acetylated galactose in which oxidation of the 3-OH has a substantial effect on activity (Y13 v. Y3). However, simultaneous de-acetylation of the glycoside moiety and oxidation of the 3-OH of the aglycone (Y12, Y14) resulted in diminished activity, suggesting that the acetyl and 3-OH groups are both involved in the anti-influenza activity, and at least one of these moieties is required to maintain substantial activity. In this study, Y3 and Y5, the conjugates of an acetylated galactose moiety with OA and UA aglycones, are the most potent inhibitors against influenza A/WSN/33 virus with EC50 values of 14.2 and 15.1 µM, respectively, in the CPE reduction assay.


Page 8 of 60

Page 9 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Mechanistic exploration. Having identified these structure requirements of triterpenoids for maintaining high anti-influenza activity, we then explored the potential mechanisms by which the influenza infection can be inhibited. The life cycle of influenza virus is around 8-10 h, and is divided into three steps (Figure 3A): virus entry (0-2 h), viral genome replication and translation (2-8 h), and progeny virion release (8-10 h).26 To identify which step(s) the triterpenoids target, we performed a time-of-addition study27 using Y3 as a representative. The expression levels of influenza NP protein in infected MDCK cells, which reflects the extent of viral infection, were measured at five time intervals, 0-10 h, 0-2 h, 2-5 h, 5-8 h and 8-10 h (Figure 3A). We found the NP level at the interval 0-10 h (covering the whole life cycle) and 0-2 h (covering the entry step) was reduced around 90% and 80%, respectively, as compared with the DMSO control. In contrast, no antiviral activity was detected for the remaining three intervals (2-5, 5-8 and 8-10 h) (Figure 3A). These data indicate that Y3 is only effective at the early stage (0-2 h) of the viral lifecycle, presumably at the attachment or the fusion of the virus with the host cell. No inhibitory effect was observed for the remaining steps, i.e. viral genome replication/translation and virion assembly/release. The targeting of the early stage of virus lifecycle was also verified by tracking virus infection. Inoculation of influenza viruses at low temperature with host cells is known to permit only virus attachment but not sequential internalization, which is associated with a finite energy requirement. 28-31

As expected, using green staining, it was found that significant NP protein accumulates on the

plasma membrane when A549 cells are inoculated at 4 oC with influenza A/WSN/33 viruses (MOI



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 60

= 0.5). Addition of Y3 at 1 µM in the culture medium led to far less NP on the cellular membrane and no NP detectable at concentrations ≥10 µM (Figure 3B). Clearly, Y3 is able to block the attachment of influenza viruses to the host cells. In a parallel experiment performed at 37 oC, we found that great populations of host cells were infected with NP protein significantly accumulating in the nucleus (Figure 3C and SI Figure 2A). As expected, addition of Y3 significantly reduces the populations of NP-positive cells but the nuclear intensity of NP protein in certain infected cells was unaffected (Figure 3C and SI Figure 2A). This implies that, once virus enters into the cells, Y3 no longer affects the remaining viral processing. The anti-influenza behavior of Y3 is significantly different from that of ribavirin, which targets viral RNA replication and thus leads to a reduction in the production of NP protein (SI Figure 2A). It is also different from that of OSV, which targets viral release and has no effect on either the populations of infected cells or the nuclear intensity of NP protein (SI Figure 2A). Taken together, these data supported the proposition that Y3 interferes with the early stages (attachment) but not with the later stages of the viral lifecycle. We then designed three experiments, conducted in parallel, with a view to clarifying whether the host cell or the influenza virus was targeted. In the first experiment, namely co-treatment, host cells were inoculated with influenza viruses in the presence of Y3 at 4 oC for 1 h, then washed to remove unbound viruses and compounds, and continuously cultured at 37 oC for 36 hr. In the second experiment, namely pre-treatment of cells, cells were incubated with Y3 at 4 oC for 30 minutes and then washed to remove the unbound compounds. The pretreated cells were exposed to viruses for 1 hr, then washed to remove unbound viruses, and continuously cultured at 37 oC for 36


Page 11 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hr. In the third experiment, namely pre-treatment of the virus, viruses were incubated with Y3 at 4 o

C for 30 minutes and then washed to remove the unbound compounds by ultrafiltration. The host

cells were inoculated with the pretreated viruses for 1 h, then washed to remove unbound viruses, and continuously cultured at 37 oC for 36 h. The co-treatment in which Y3 at a concentration of 50 µM exerts an inhibition rate of 43%, compares with pre-treatment of the cells which had almost no detectable effect while the pre-treatment of virus led to an inhibition rate of 85% (Figure 3D). We concluded that Y3 targets the influenza virus particles but not host cells. Such a mechanism was further supported by the observations of significant morphology changes in the influenza viruses but not in the host cells (Figure 3E). Target identification. After establishing that the virus particles and the early stage of viral life cycle (attachment) were the points of interference, we designed experiments to identify the potential target. A hemagglutination inhibition (HI) assay was first performed to evaluate the potential involvement of the envelope HA protein with Y3. This assay is commonly used to measure flu-specific antibody levels in blood serum. Antibodies at a sufficient concentration, will interfere with the virus attachment to red blood cells and thus inhibit hemagglutination.32,33 We found that Y3 shows the same capability as anti-HA antibody in effectively inhibiting influenza virus-induced aggregation of chicken erythrocytes, and the inhibitory effect is dose-dependent (Figure 4A). This suggests that Y3 and anti-HA antibody have the same target, HA, and thus blocks the interactions of viruses with target cells.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 60

We then characterized the affinity between HA protein and the lead compounds (Q8, Y3, and Y4) by a surface plasmon resonance (SPR) assay. We found that all these compounds bind tightly to the HA immobilized on the CM5 chip, and at concentrations of 3.125-50 µM exerted dose-dependent responses. The calculated KD values for Q8, Y3 and Y4 from the SPR assay were 14.0, 6.0 and 6.8 µM, respectively (Figure 4B and SI Figure 2B). We also found that two negative compounds, OA, the aglycone in Y3 and Y4 and EA, the aglycone in Q8, also bound to HA protein with KD values of 32 and 26 µM respectively (Figure 4B). Such affinities are supposedly traceable to their hydrophobic scaffolds that lead to non-specific binding. It may be noted that OA and EA, despite their much higher hydrophobicities, have much lower affinities than their glycoconjugates. In a control experiment in which NA protein was immobilized on the same chip, no specific affinity was detected for these triterpenoids. The high affinity between HA and the lead compounds suggests that HA is the potential target. Interestingly, high affinities were also observed between lead compounds and HA proteins from other influenza strains including H3N2, H5N1, H7N9 and Flu B virus (SI Table 1). Having identified HA as the potential target, we explored whether Y3-HA binding had any effect on the interaction of HA with its sialic acid receptor, the critical step for attachment of influenza viruses to host cells for initiation of virus infection. According to the SPR assay in which the sialic acid receptor was immobilized on an SA chip, we found HA protein at concentrations of 0.625-20 µg/ml binds tightly to the sialic acid receptor in a dose-dependent manner and KD = 70 nM (Figure 4C), consistent with a previous report34. When Y3 and curcumin, an entry inhibitor


Page 13 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


targeting the HA1 domain,35,36 are added in separate assays, the HA-sialic acid receptor affinity is reduced significantly with KD values of 844 and 507 nM, respectively. In contrast, addition of arbidol, an anti-influenza drug targeting the HA2 domain,35,37 has almost no effect on the affinity (KD 81 nM). Clearly, Y3 has the same capability as curcumin, potentially targeting the same HA1 domain and blocking the binding of HA to its sialic acid receptor. It seems likely that targeting the HA2 domain has no effect on HA-sialic acid receptor interaction. The improved KD value delivered by Y3 suggests that it is more potent than curcumin in its interruption of the HA-sialic acid receptor interaction. Blind docking calculation. In view of HA (HA1) as the potential target and the HA-sialic acid receptor interaction as the potential interruption event, we performed blind docking calculations to investigate such possibilities. The AutoDock program (version 4.2) was employed and the docked conformations of HA-Y3 were determined based on the minimum free energy analyses. According to the computer-aided docking data, Y3 occupied the binding pocket for sialic acid receptor (Figure 5) with an estimated binding energy of -9.44 kcal/mol and an inhibition constant (Ki) = 121.02 nM. The corresponding data for the sialic acid receptor were -6.68 kcal/mol and 12.68 µM, and this suggests that Y3 binds to HA protein more tightly than the sialic acid receptor. One of the influenza virus receptors is the terminal sialic acid of cellular glycoproteins, which bind the shallow depression located at the top of HA1 domain. For all subtypes of influenza viruses, the HA protein is composed of highly conserved HA1 and HA2 domains, which are essential for sialic acid receptor binding and viral envelope fusion respectively.38 A 3Å resolution



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 60

X-ray study of HA complexes with sialyllactose indicates that several hydrogen bonds are formed between sialic acid and the conserved site- and main-chain polar atoms of HA1. In particular, the conserved serine, S136 forms a hydrogen bond with the sialic carboxylate, which is also hydrogen bonded with the amide carbonyl of peptide bond 137. H183 and E190 form hydrogen bonds with the 9-hydroxyl group, and Y98 forms hydrogen bonds with the 8-hydroxyl group (Figure 5). The 5-acetamido nitrogen of sialic acid forms a hydrogen bond with the carbonyl peptide bond 135, and the methyl group of this substituent is in van der Waals contact with L194. The computer-aided docking data indicated that the 3-OH of the OA moiety forms a hydrogen bond with both R133 and the carbonyl group of residue K156, two residues within the HA1 conserved domain associated with sialic acid binding. The amide of peptide bond A137, a critical residue for hydrogen bonding to the carboxylate of sialic acid, forms a hydrogen bond with the 2-estercarbonyl group of the galactose, which is also hydrogen bonded to the amide of residue Q226, also a critical residue involved in sialic acid binding. A fifth hydrogen bond is formed between S145 of HA1 and the 3-carbonyl group of the galactose of Y3. In addition to these hydrogen bonds, multiple hydrophobic interactions were observed between Y3 and its binding pocket, namely, residues V155, H183, S193, P185, Y95, P186, D190, W53, T136, V135 and L194. Among these, H183 and L194 are critical for sialic acid binding via hydrogen bonds. Most of the remaining interactions are within the 190 helix (residues 190 to 198), the 130 loop (residues 135 to 138) and the 220 loop (residues 221 to 228), which are all important for sialic acid binding. These docking data are consistent with results from SAR studies, in particular the requirement for acetyl


Page 15 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


groups in the galactose residue and the 3-hydroxyl group in the OA moiety. Removal of acetyl groups from Y3 significantly reduces the antiviral activity; no activity was detected upon further oxidation of OA 3’-OH. This is reflected in the docked data for the negative compound Y14, wherein the 3-OH and the acetyl groups in lead Y3 has been changed, with an estimated binding energy of -7.13 kcal/mol and an inhibition constant (Ki) = 5.94 µM. Consequently, occupancy of the binding pocket for sialic acid receptor might account for the molecular basis by which Y3 blocks the HA-sialic acid receptor interactions and thus the attachment of viruses to the host cells. Broad anti-influenza spectrum. Given Y3 occupying the pocket for sialic acid receptor, we inquired whether lead triterpenoids such as Y3 exert broad antiviral spectrum and induce less drug resistance. Three potent compounds, Q8, Y3 and Y4, were evaluated against five clinical isolates of influenza A and B viruses (Table 1). Among these, two belong to H1N1 subtype including one oseltamivir-resistant strain (LN/1109), and two belong to H3N2 subtype including one amantadine-resistant strain (HN/1222). The EC50 values of these compounds along with positive controls, oseltamivir, amantadine and ribavirin, were determined and are listed in Table 1. We found Y3, the most potent compound against A/WSN/33 strain (H1N1), exerted remarkable activity with EC50 values in the range of 2.72-7.41 µM, almost similar to ribavirin (RBV), which is an off-label drug targeting many different RNA viruses.39 Furthermore, we found Y3 was effective for all tested strains including the oseltamivir-resistant LN/1109 strain, the amantadine-resistant HN/1222 strain and even the influenza B virus (B/SZ/155) with EC50 values of 6.58, 3.18 and 2.80 µM, respectively. Such a broad anti-influenza spectrum is significantly different from that of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 60

oseltamivir, which is ineffective (EC50 > 200 µM) against PR/8 (H1N1) and LN/1109 (H1N1), much less effective (EC50 > 90 µM) against B/SZ/155 viruses, and amantadine, which is ineffective (EC50 > 200 µM) against HN/1222 (H3N2) and less effective (EC50 > 80 µM) against B/SZ/155. The lead triterpenoids Q8 and Y4 are moderately effective against all test strains and Q8 is somewhat better than Y4 (Table 1). We then explored the propensity of Y3 to induce viral resistance, an increasing problem in clinical use of current anti-influenza virus drugs. Based on the CPE assay, the EC50 of Y3 and amantadine against A/WSN/33 strain (H1N1) are 14.2 µM and 7.0 µM, respectively. Therefore, 100 µM of Y3 and 50 µM of amantadine40 (7-times the EC50) were separately added to a culture medium to perform a multi-passaging experiment. Judging by CPE plaque formation, a remarkable viral resistance is induced by amantadine, suggesting a low-level replication is allowed giving the progeny virus a chance to adapt to the selective pressure. A significant contrast is presented in the parallel experiment where Y3 was substituted for amantadine. It was found that very little plaque formed, suggesting Y3 was still efficient in inhibition of influenza A/WSN/33 virus propagation even after a sixth passage (Figure 6B). The broad anti-influenza virus spectrum and the diminished tendency of induction of drug resistance suggested that Y3 targets a similar entry route essential for many viruses, potentially the pocket for sialic acid receptor. The possibility of Y3 occupancy in the pocket of the sialic acid receptor was supported by a competitive affinity assay. According to the SPR assay, the KD values for arbidol and curcumin, two control compounds, and free sialic acid to HA protein, which was immobilized on a CM5


Page 17 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chip, are 46, 21 and 248 µM, respectively. When arbidol (100 µM), curcumin (100 µM), sialic acid (500 µM) and Y3 (50 µM) flowed separately across the HA-chip surface, the SPR responses were 55.8, 83.6, 14.5 and 43.5 RU respectively (Figure 6A). When arbidol and Y3 were co-added as analytes, an additive response was observed with the SPR value of 90.2 RU. This is not surprising since arbidol and Y3 are thought to bind to HA2 and H1 domains, respectively (Figure 6A). However, co-addition of curcumin and Y3 led to an antagonistic effect (90.3 RU). This is also understandable because it has been suggested that curcumin binds to the region constituting the sialic acid (SA) anchoring residues on the HA1 domain.35,41 Similarly, when free sialic acid and Y3 were co-added as analytes, an antagonistic effect was observed with the SPR value of 51.9 RU, even lower than that of Y3 (Figure 6A). This indicated that sialic acid and Y3, unlike arbidol-Y3 pair, cannot simultaneously bind to HA protein, i.e. they compete with one another during binding to HA protein. We concluded that consistent with the docking data, Y3 and sialic acid might bind in the same domain within HA1 protein. In addition, we performed a polykaryon assay to test whether Y3 targets the HA2 domain. HA is a trimeric glycoprotein containing two polypeptide chains HA1 and HA2.38 The latter chain is responsible for virion internalization via pH-dependent endocytosis. As shown in Figure 6C, a tryptic digestion assay indicates that overexpression of HA protein in Hela cells leads to significant polykaryon formation upon lowering the pH from 7.0 to 4.9, and such a phenotype is independent of the absence or presence of Y3 (50 µM). These data suggested that Y3 does not target the viral fusion step and thus HA2 can be excluded as the potential target. Therefore, the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 60

following tentative mechanism for Y3-mediated anti-influenza activity is proposed: the inhibitor binds tightly to the HA protein, potentially occupying the conserved pocket for sialic acid receptor within HA1 and disrupting its interaction with the sialic acid receptor, thus blocking the attachment of viruses to host cells (Figure 7). Intranasal-treatment of influenza infection with Y3. To evaluate the efficacy of Y3 against influenza virus in vivo, Balb/C mice intranasally infected with 5×LD50 of virus were inoculated intranasally with Y3 twice daily for 5 days, starting one day before infection. The dose chosen (0.25 mg/20g/d and 0.5 mg/20g/d) was due to the limited solubility of Y3. At such doses, no toxicity in terms of body weight and overall health status was observed upon administration for five consecutive days (SI Figure 4). Infected mice treated with zanamivir were used as a positive control. The mouse survival and body weight were monitored daily for 13 days. As shown in Figure 8A, loss of body weight accompanied with other influenza symptoms, such as ruffled fur and reduced activity, was developed in the PBS solvent treated mice 4 days post-infection. On the contrary, significantly less body weight loss was observed upon Y3 administration. The protective effect of Y3 on mice body weight loss, though less effective than that provided by zanamivir, was improved in a dose-dependent manner and such improvement further increased when the Y3 was properly formulated. Moreover, there was significant inhibition of the mortality for infected mice treated with Y3 compared with the untreated mice: 80% (n=5) of the control mice died 10 days post-infection while no Y3-treated mice died even 13 days post-infection (Figure 8B). Taken together, our data suggest that Y3 can protect mice from influenza virus infection in vivo.


Page 19 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSIONS Here we report certain pentacyclic triterpenoids displaying in vitro anti-influenza virus activity comparable and even higher than that of oseltamivir. A systemic exploration of the lead compounds clarifies the structure requirements for maintenance of high activity with oleanolic acid and acetyl galactose moieties as the optimized aglycone and glycoside. These compounds exert broad spectrum activity even against oseltamivir- and amantadine-resistant viruses with diminished induction of viral resistance. The molecular basis of their broad spectrum activity is potentially due to their high affinity for HA protein, which blocks the HA-sialic acid receptor interaction and thus the attachment of influenza viruses to cells. Docking studies suggest that these compounds occupy the conserved pocket for sialic acid receptor, consistent with the SAR data. This study may establish the importance of triterpenes as a new class of lead compounds for the development of potential entry inhibitors of influenza viruses.

EXPERIMENTAL SECTION

Materials. Madin-Darby canine kidney (MDCK) cells, human embryonic kidney 293T (HEK293T) cells, human lung adenocarcinoma A549 cells and HeLa cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, Linz Austria) at 37 °C under 5% CO2. The following viruses were used in this study: pseudotyped vesicular stomatitis virus (VSV), GFP-expressing adenovirus (Ad5-eGFP), encephalomyocarditis virus (EMCV), influenza



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 60

A/WSN/33 (H1N1), A/Puerto Rico/8/34 (H1N1), A/LiaoNing-ZhenXing/1109/2010 (H1N1), A/JiangXi-DongHu/312/2006

(H3N2),

A/HuNan-ZhuHui/1222/2010

(H3N2),

and

B/ShenZhen/155/2005. Mouse anti-NP monoclonal antibody and rabbit anti-GAPDH polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA. USA). Rabbit monoclonal antibodies to influenza virus HA (H1N1) were from Sino Biological Inc. (Beijing, China). The goat anti-mouse IgG conjugated to FITC secondary antibody was from Zhongshan Golden Bridge Biotechnology (Beijing, China). The anti-rabbit IgG and anti-mouse IgG coupled the horseradish peroxidase (HRP) were from Sigma-Aldrich (St. Louis, MO, USA). The HA or NA gene was amplified from influenza A virus A/WSN/33 (H1N1) cDNA by PCR and then inserted between the KpnI and EcoRI or XhoI sites of the pcDNA4/TO vector (Invitrogen, Carlsbad, CA). Ribavirin (RBV) with 98% purity (Sigma-Aldrich, St Louis, MI, USA), Amantadine with 98 % purity (Sigma-Aldrich, St Louis, MI, USA) and OSV with 98 % purity (Hoffmann-La Roche Ltd., Basel, Switzerland) were used as reference compounds in CPE reduction assay and immune-fluorescence microscopy, respectively. All the compounds were dissolved in DMSO to 5 mM as a stock solution. Q8 was synthesized as described previously.22 Purity of all biologically evaluated compounds was assessed to be > 95% by HPLC (Agilent 1260) with detection at 215 nm wavelength.


Page 21 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chemistry. High-resolution mass spectra (HRMS) were obtained with an APEX IV FT_MS (7.0 T) spectrometer (Bruker) in positive ESI mode. NMR spectra were recorded on a Bruker DRX 400 spectrometer at ambient temperature. 1H NMR chemical shifts were referenced to the internal standard TMS (δH = 0.00) or the solvent signal (δH = 3.31 for the central line of MeOD). 13C NMR chemical shifts are referenced to the solvent signal (δC = 77.00 for the central line of CDCl3, δC = 49.00 for the central line of MeOD). Reactions were monitored by thin-layer chromatography (TLC) on a pre-coated silica gel 60 F254 plate (layer thickness 0.2 mm; E. Merck, Darmstadt, Germany) and detected by staining with a yellow solution containing Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24·4H2O (24.0 g) in 6% H2SO4 (500 mL), followed by heating. Flash column chromatography was performed on silica gel 60 (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd.). And the General procedures for purity determination by HPLC are as following: An Agilent 1260 HPLC system (Agilent Technologies, Palo Alto, CA, USA) comprised a quaternary solvent delivery system, an on-line degasser, an auto-sampler, a column temperature controller and DAD detector coupled with an analytical workstation. The column configuration was an Agilent Zorbax SB C18 reserved phase column (5µm, 250 mm×4.6 mm) coupled with an Agilent Zorbax SB C18 guard column (5µm, 10 mm×4.6 mm). The compounds were dissolved in methanol and injection volume was10 µl. Detection wavelength was set at 215 nm, the flow rate was 1.0 ml min−1 and the column temperature was maintained at 30 ºC. The mobile phase was gradient elution which was mixed with solvent A (water) and B (acetonitrile). For triterpene-glycoconjugates with acetylation of the glycoside and aglycone moiety (EA, OA, UA), the gradient program was as follows: 0–40



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 60

min 75% B. For triterpene-glycoconjugates without acetylation of the glycoside, the gradient program was as follows: 0–40 min 45% B. The above gradient program would result in the retention time of between 10 and 40 minute.

General

procedure

A

for

the

synthesis

of

triterpene

glycoconjugates.

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 1.2 eq.) was added to 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosylamine (1 eq.) and corresponding triterpenes (1.2 eq.) with stirring in dry THF . The mixture was stirred at RT for 1 h till the mixture became clear, and then the solution was refluxed overnight. After completion, as determined by TLC, the solvent was evaporated, the residue was suspended with EtOAc (20 mL) and washed with H2O (10 mL × 3), brine and dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography.

General procedure B for the O-deacetylation of triterpene glycoconjugates. NaOMe was added to the O-acetylated compound stirred in MeOH. The mixture was stirred at RT until completion, as determined by TLC and then the reaction mixture was neutralized with HCl (1 M). Water was added and the resulting suspension was filtered. The crude product was purified by column chromatography. Compound Y1. Prepared from EA (200 mg, 0.42 mmol) and 2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosylamine (122 mg, 0.35 mmol) according to general procedure A. The residue was purified by column chromatography (petroleum ether/EtOAc, 2/1 v/v) to give Y1 as a white solid (216 mg, 77%). 1H NMR (400 MHz, CDCl3): δ0.74, 0.79, 0.84, 0.91, 0.93, 1.00, 1.34, 1.99, 2.02,


Page 23 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.03, 2.13 (11 × CH3), 0.74-2.37 (m, other aliphatic ring protons), 2.77-2.80 (m, 1H), 3.23 (dd, 1H, J = 4.2, 10.6 Hz), 3.96-4.09 (m, 3H), 4.23 (brs, 1H), 5.01-5.16 (m, 3H), 5.41 (d, 1H, J = 3.0 Hz), 5.564 (brs, 1H), 7.10 (d, 1H, J = 9.2 Hz). 13C NMR (100 MHz, CDCl3): δ15.5, 15.6, 17.2, 18.1, 20.5, 20.5, 20.6, 23.3, 25.1, 26.7, 27.1, 28.0, 28.6, 30.0, 32.3, 32.5, 34.7, 35.2, 36.8, 38.6, 38.7, 39.7, 40.7, 41.8, 46.6, 46.7, 49.4, 55.2, 60.6, 66.9, 68.2, 70.8, 71.6, 74.6, 78.3, 78.7, 123.8, 142.7, 169.7, 169.9, 170.2, 170.8, 178.5. ESI-HRMS (m/z) [M+H]+ calcd for C44H68NO12, 802.4736, found 802.4755. Compound Y2. Prepared from Y1 (50 mg, 0.06 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y2 as a white solid (35 mg, 89%). 1H NMR (400 MHz, MeOD): δ0.78, 0.86, 0.90, 0.95, 0.96, 0.98, 1.34 (7 × CH3), 0.74-1.94 (m, other aliphatic ring protons), 2.20 (t, 1H, J = 13.4 Hz), 3.04 (dd, 1H, J = 3.6, 14.0 Hz), 3.15 (dd, 1H, J = 4.9, 11.2 Hz), 3.49-3.56 (m, 3H), 3.65-3.67 (m, 2H), 3.87-3.88 (m, 1H), 4.25-4.27 (m, 1H), 4.79 (d, 1H, J = 8.8 Hz), 5.45 (t, 1H, J = 3.1 Hz). 13C NMR (100 MHz, MeOD): δ16.2, 16.3, 18.2, 19.5, 24.5, 26.3, 27.5, 27.9, 28.7, 29.6, 30.9, 33.1, 34.0, 35.8, 36.3, 38.1, 39.8, 40.0, 41.0, 42.2, 43.0, 47.8, 48.4, 50.8, 56.9, 62.4, 70.4, 71.6, 75.0, 75.8, 78.1, 79.7, 81.9, 123.9, 144.6, 181.1. ESI-HRMS (m/z) [M+H]+ calcd for C36H60NO8, 634.4313, found 634.4305. Compound Y3. Prepared from OA (200 mg, 0.44 mmol) and 2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosylamine (127 mg, 0.37 mmol) according to general procedure A. The residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v) to give Y3 as a white solid (148 mg, 51%). 1H NMR (400 MHz, CDCl3): δ 0.79, 0.80, 0.90, 0.91, 0.92, 0.99, 1.16, 2.00, 2.02,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 60

2.05, 2.14 (11 × CH3), 0.72-2.17 (m, other aliphatic ring protons), 2.55 (dd, 1H, J = 3.4, 13.4 Hz), 3.21 (dd,1H, J = 3.6, 10.4 Hz), 3.97-4.13 (m, 3H), 5.03-5.18 (m, 3H), 5.41-5.43 (m, 1H), 5.50 (brs, 1H), 6.68 (d, 1H, J = 9.0 Hz).13C NMR (100 MHz, CDCl3): δ 15.3, 15.5, 17.0, 18.2, 20.4, 20.5, 20.5, 20.7, 23.1, 23.4, 23.9, 25.4, 27.0, 27.1, 28.0, 30.5, 32.3, 32.4, 32.8, 34.0, 36.8, 38.4, 38.6, 39.2, 41.2, 41.9, 46.3, 46.5, 47.4, 55.0, 60.6, 67.0, 68.2, 70.7, 71.6, 78.5, 78.7, 123.4, 143.6, 169.7, 169.9, 170.2, 171.0, 178.9. ESI-HRMS (m/z) [M+H]+ calcd for C44H68NO11, 786.4787, found 786.4784. Compound Y4. Prepared from Y3 (50 mg, 0.06 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y4 as a white solid (33 mg, 85%). 1H NMR (400 MHz, MeOD): δ 0.75, 0.80, 0.90, 0.91, 0.92, 0.96, 1.15 (7 × CH3), 0.71-2.10 (m, other aliphatic ring protons), 2.83 (dd, 1H, J = 2.6, 12.9 Hz), 3.14 (dd, 1H, J = 5.0, 11.3 Hz), 3.48-3.55 (m, 3H), 3.62-3.71 (m, 2H), 3.89 (brs, 1H), 4.80 (d, 1H, 8.0 Hz), 5.31 (brs, 1H). 13C NMR (100 MHz, MeOD): δ 15.8, 16.1, 17.7, 19.2, 23.9, 24.0, 24.3, 26.3, 27.5, 28.1, 28.6, 31.3, 33.4, 33.6, 34.9, 37.8, 39.5, 40.3, 42.0, 42.7, 47.3, 47.3, 48.7, 56.3, 62.2, 70.0, 71.0, 75.4, 77.7, 79.4, 81.5, 123.6, 144.9, 181.1. ESI-HRMS (m/z) [M+H]+ calcd for C36H60NO7, 618.4364, found 618.4361. Compound Y5. Prepared from UA (100 mg, 0.22 mmol) and 2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosylamine (63 mg, 0.18 mmol) according to general procedure A. The residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v) to give Y5 as a white solid (100 mg, 71%). 1H NMR (400 MHz, CDCl3): δ 0.78, 0.80, 0.88, 0.93, 0.95, 0.99, 1.09, 2.00, 2.02,


Page 25 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.05, 2.14 (11 × CH3), 0.71-2.16 (m, other aliphatic ring protons), 3.21 (dd, 1H, J = 5.0, 10.6 Hz), 3.97-4.09 (m, 3 H), 5.02-5.16 (m, 3H), 5.41-5.42 (m, 2H), 6.61 (d, 1H, J = 8.8 Hz). 13C NMR (100 MHz, CDCl3): δ 15.4, 15.5, 17.0, 17.0, 18.1, 20.4, 20.5, 20.5, 20.7, 21.0, 23.0, 23.3, 24.8, 27.0, 27.7, 28.0, 30.7, 32.9, 36.8, 37.0, 38.6 (2C), 38.9, 39.4, 39.5, 42.2, 47.4, 47.8, 53.1, 55.1, 60.7, 67.0, 68.3, 70.7, 71.7, 78.6, 78.8, 126.2, 138.3, 169.7, 170.0, 170.2, 171.0, 178.6. ESI-HRMS (m/z) [M+H]+ calcd for C44H68NO11, 786.4787, found 786.4783. Compound Y6. Prepared from Y5 (60 mg, 0.08 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y6 as a white solid (41 mg, 88%). 1H NMR (400 MHz, MeOD): δ0.78, 0.85, 0.91, 0.96, 0.97, 1.12 (7 × CH3), 0.73-2.23 (m, other aliphatic ring protons), 3.15 (dd, 1H, J = 4.8, 11.0 Hz), 3.49-3.66 (m, 5H), 3.89-3.90 (m, 1H), 4.77-4.82 (m, 1H), 5.31 (t, 1H, J = 3.4 Hz), 7.44 (d, 1H, J = 8.6). 13C NMR (100 MHz, MeOD): δ16.1, 16.4, 17.7, 18.1, 19.5, 21.6, 24.0, 24.4, 25.3, 27.9, 28.8, 29.0, 32.0, 34.3, 38.1, 38.3, 39.8, 40.1, 40.2, 40.8, 41.0, 43.3, 49.1, 54.0, 56.7, 62.2, 70.2, 71.3, 75.8, 77.9, 79.7, 81.9, 82.0, 127.3, 139.7, 181.4. ESI-HRMS (m/z) [M+H]+ calcd for C36H60NO7, 618.4364, found 618.4356. Compound Y7. Prepared from UA (120 mg, 0.26 mmol) and 2,3,4,6-tetra-O-acetyl-β-Dmannopyranosylamine (76 mg, 0.22 mmol) according to general procedure A. The residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v) to give Y7 as a white solid (109 mg, 63%). 1H NMR (400 MHz, CDCl3): δ 0.78, 0.81, 0.85, 0.93, 0.94, 0.99, 1.10, 1.98, 2.04, 2.07, 2.24 (11 × CH3), 0.70-2.20 (m, other aliphatic ring protons), 3.22 (dd, 1H, J = 4.5, 10.4 Hz),



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 60

3.70-3.74 (m, 1H), 4.03 (dd, 1H, J = 2.4, 12.2 Hz), 4.21-4.25 (m, 1H), 5.09 (dd, 1H, J = 3.3, 10.1 Hz), 5.12-5.24 (m, 1H), 5.46-5.49 (m, 2H), 5.30-5.31 (m, 1H), 5.56 (d, 1H, J = 9.3 Hz). 13C NMR (100 MHz, CDCl3): δ15.6, 17.1, 17.1, 18.2, 20.5, 20.7, 20.7, 21.1, 23.0, 23.4, 25.1, 27.1, 27.7, 28.1, 30.8, 32.9, 36.8, 37.0, 38.7, 38.8, 39.4, 39.5, 39.6, 42.6, 47.5, 48.2, 53.9, 55.1, 62.5, 65.5, 70.3, 71.6, 73.7, 76.0, 77.0, 78.9, 125.9, 139.6, 169.7, 169.8, 170.0, 170.6, 178.2. ESI-HRMS (m/z) [M+H]+ calcd for C44H68NO11, 786.4787, found 786.4787. Compound Y8. Prepared from Y7 (60 mg, 0.08mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y8 as a white solid (43 mg, 92%). 1H NMR (400 MHz, MeOD): δ 0.78, 0.88, 0.91, 0.97, 0.98, 1.14 (7 × CH3), 0.74-2.17 (m, other aliphatic ring protons), 3.16 (dd, 1H, J = 4.7, 11.0 Hz), 3.23-3.27 (m, 1H), 3.49-3.58 (m, 2H), 3.65 (dd, 1H, J = 5.5, 11.6 Hz), 3.72 (dd, 1H, J = 1.1, 3.0 Hz), 3.80 (dd, 1H, J = 2.3, 11.6 Hz), 5.07-5.10 (m, 1H), 5.37 (brs, 1H), 7.36 (d, 1H, J = 8.8 Hz). 13C NMR (100 MHz, MeOD): δ16.2, 16.4, 17.7, 18.2, 19.4, 21.6, 23.8, 24.4, 25.8, 27.9, 28.8, 29.0, 31.9, 34.3, 38.1, 38.4, 39.8, 40.1, 40.3, 40.9, 41.0, 43.5, 49.1, 49.3, 54.7, 56.7, 63.1, 68.1, 72.3, 75.6, 79.2, 79.7 (2C), 128.1, 139.4, 180.7. ESI-HRMS (m/z) [M+H]+ calcd for C36H60NO7, 618.4364, found 618.4358. Compound Y9. Prepared from OA (150 mg, 0.33 mmol) and 2,3,4,6-tetra-O-acetyl-β-Dmannopyranosylamine(92 mg, 0.27 mmol) according to general procedure A. The residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v) to give Y9 as a white solid (107 mg, 41%). 1H NMR (400 MHz, CDCl3): δ0.78, 0.79, 0.87, 0.90, 0.92, 0.99, 1.16, 1.98, 2.04, 2.07, 2.23 (11 × CH3), 0.71-2.13 (m, other aliphatic ring protons), 2.45 (dd, 1H, J = 3.7, 13.2 Hz),


Page 27 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.21 (dd, 1H, J = 4.1, 10.4 Hz), 3.71-3.75 (m, 1H), 4.05 (dd, 1H, J = 2.4, 12.2 Hz), 4.22 (dd, 1H, J = 4.9, 12.3 Hz), 5.10 (dd, 1H, J = 3.3, 10.1 Hz), 5.19-5.24 (m, 1H), 5.31-5.34 (m, 2H), 5.51 (d, 1H, J = 9.3 Hz), 6.54 (d, 1H, J = 9.3 Hz). 13C NMR (100 MHz, CDCl3): δ 15.3, 15.5, 16.9, 18.2, 20.4, 20.6 (3C), 23.2, 23.5, 24.0, 25.4, 27.0 (2C), 28.0, 30.5, 32.3, 32.3, 32.8, 33.9, 36.8, 38.4, 38.6, 39.2, 41.9, 42.1, 46.5, 46.6, 47.4, 55.0, 62.4, 65.5, 70.1, 71.4, 73.6, 76.0, 78.7, 122.8, 144.6, 169.7, 169.7, 169.9, 170.5, 178.0. ESI-HRMS (m/z) [M+H]+ calcd for C44H68NO11, 786.4787, found 786.4784. Compound Y10. Prepared from Y9 (50 mg, 0.06 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y10 as a white solid (33 mg, 85%). 1H NMR (400 MHz, MeOD): δ 0.78, 0.85, 0.92, 0.94, 0.95, 0.98, 1.19 (s, 7 × CH3), 0.74-2.17 (m, other aliphatic ring protons), 2.83 (dd, 1H, J = 3.6, 13.4 Hz), 3.15 (dd, 1H, J = 5.0, 11.4 Hz), 3.25-3.28 (m, 1H), 3.50-3.57 (m, 2H), 3.64 (dd, 1H, J = 5.6, 11.7 Hz), 3.74 (brd, 1H, J = 1.4 Hz), 3.81 (dd, 1H, J = 2.2, 11.7 Hz), 5.11-5.13 (m, 1H), 5.39 (brt, 1H), 7.40 (d, 1H, J = 8.8 Hz). 13C NMR (100 MHz, MeOD): δ 16.0, 16.3, 18.1, 19.5, 23.9, 24.6 (2C), 26.3, 27.9, 28.5, 28.8, 31.6, 33.5, 33.9, 34.0, 35.1, 38.1, 39.8, 39.9, 40.8, 43.0, 43.1, 47.6, 47.9, 49.1, 56.7, 63.1, 68.1, 72.3, 75.6, 79.2, 79.7, 79.7, 124.7, 144.7, 180.7. ESI-HRMS (m/z) [M+H]+ calcd for C36H60NO7, 618.4364, found 618.4357. Compound Y11. Dess-Martin Periodinane (191 mg, 0.45 mmol) was added to a solution of Y1 (100 mg, 0.12 mmol) in 8 mL of CH2Cl2. The reaction was stirred at RT for 30 min after completion (TLC), and then excess saturated aqueous Na2S2O3 was added to the reaction mixture. The mixture was extracted with CH2Cl2 (3×10 mL). The combined organic layer was washed with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 60

saturated aqueous NaHCO3, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether/EtOAc, 4/1 v/v) to afford Y11 as a white solid (90 mg, 91%).42 1H NMR (400 MHz, CDCl3): δ 0.82, 0.90, 0.93, 1.05, 1.06, 1.09, 1.22, 2.00, 2.03, 2.04, 2.16 (11 × CH3), 1.25-2.41 (m, other aliphatic ring protons), 2.52-2.59 (m, 1H), 2.63 (d, 1H, J = 15.6 Hz), 3.22 (dd, 1H, J = 3.8, 13.7 Hz), 3.97-4.00 (m, 1H), 4.06-4.08 (m, 2H), 5.09-5.10 (m, 3H), 5.42 (brs, 1H), 5.68 (brs, 1H), 6.76 (d, 1H, J = 7.8 Hz).13C NMR (100 MHz, CDCl3): δ 14.9, 17.0, 19.3, 20.4, 20.5 (2C), 20.7, 21.3, 23.0, 23.4, 26.3, 27.1, 28.6, 30.3, 31.9, 32.6, 33.9, 34.9, 36.6, 38.9, 39.6, 44.4, 45.6, 45.9, 46.0, 46.5, 47.3, 55.2, 59.9, 60.7, 67.0, 67.9, 70.7, 71.9, 78.7, 124.5, 140.1, 169.6, 169.9, 170.2, 170.7, 172.3, 210.3, 216.9.ESI-HRMS (m/z) [M+H]+ calcd for C44H64NO12, 798.4423, found 798.4420.

Compound Y12. Prepared from Y11 (50 mg, 0.06 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y12 as a white solid (32 mg, 81%).1H NMR (400 MHz, MeOD): δ 0.87, 0.95, 0.97, 1.05, 1.08, 1.09, 1.20 (7 × CH3), 1.15-2.27 (m, other aliphatic ring protons), 2.36-2.42 (m, 1H), 2.54-2.63 (m, 1H), 2.98 (d, 1H, J = 14.7 Hz), 3.43 (dd, 1H, J = 3.9, 14.1 Hz), 3.47-3.58 (m, 3H), 3.66-3.67 (d, 2H, J = 6.2 Hz), 3.88-3.89 (m, 1H), 4.82 (d, 1H, J = 8.8 Hz), 5.56 (t, 1H, J = 3.5 Hz).13C NMR (100 MHz, MeOD): δ 15.5, 17.8, 20.6, 21.9, 23.7, 24.6, 27.0, 27.7, 28.6, 31.4, 33.2, 33.3, 35.0, 36.1, 37.9, 40.1, 41.1, 46.7 (2C), 47.1, 48.0, 48.5, 48.9, 56.4, 61.1, 62.2, 70.3, 71.1, 75.9, 78.2, 81.8, 125.2, 142.0, 175.1, 212.7, 220.2. ESI-HRMS (m/z) [M+H]+ calcd for C36H56NO8, 630.4000, found 630.3995. Compound Y13. Dess-Martin Periodinane (127 mg, 0.3 mmol) was added to a solution of Y3


Page 29 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(80 mg, 0.1 mmol) in 8 mL of CH2Cl2. The reaction was stirred at RT for 30 min after completion (TLC), and then excess saturated aqueous Na2S2O3 was added to the reaction mixture. The mixture was extracted with CH2Cl2 (3×10 mL). The combined organic layer was washed with saturated aqueous NaHCO3, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether/EtOAc, 5/1 v/v) to afford Y13 as a white solid (69 mg, 88%). 1H NMR (400 MHz, CDCl3): δ 0.85, 0.90, 0.91, 1.05, 1.06, 1.09, 1.17, 2.00, 2.03, 2.06, 2.14 (11 × CH3), 0.72-2.58 (m, other aliphatic ring protons), 3.97-4.06 (m, 3H), 5.03-5.19 (m, 3H), 5.42-5.43 (m, 1H), 5.52 (brs, 1H), 6.66 (d, 1H, J = 9.0 Hz).13C NMR (100 MHz, CDCl3): δ15.0, 16.9, 19.5, 20.5, 20.6, 20.8, 21.5, 23.2, 23.6, 24.0, 25.4, 26.3, 27.2, 30.6, 32.1, 32.5, 32.8, 34.1, 36.6, 39.2, 39.3, 41.4, 42.1, 46.5, 46.8, 47.4, 55.3, 60.7, 67.1, 68.3, 70.7, 71.8, 78.6, 123.1, 143.8, 169.8, 170.0, 170.3, 171.1, 178.8, 217.6. ESI-HRMS (m/z) [M+H]+ calcd for C44H66NO11, 784.4630, found 784.4615. Compound Y14. Prepared from Y13 (40 mg, 0.05 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y14 as a white solid (28 mg, 89%). 1H NMR (400 MHz, MeOD): δ 0.88, 0.91, 0.94, 1.04, 1.07, 1.18 (7 × CH3), 1.16-2.17 (m, other aliphatic ring protons), 2.34-2.40 (m, 1H), 2.53-2.62 (m, 1H), 2.90 (dd, 1H, J = 3.9, 13.7 Hz), 3.50-3.60 (m, 3H), 3.66-3.67 (m, 2H), 3.89-3.90 (m, 1H), 4.81-4.86 (m, 1H), 5.32 (t, 1H, 3.4 Hz) 7.61 (d, 1H, J = 8.6 Hz). 13C NMR (100 MHz, MeOD): δ15.6, 17.8, 20.7, 21.9, 24.0, 24.1, 24.6, 26.3, 27.0, 28.5, 31.6, 33.4, 33.6, 33.8, 35.1, 35.2, 37.9, 40.3, 40.7, 42.3, 43.1, 47.6, 47.7, 48.2, 48.5, 49.0, 56.6, 62.4, 70.4, 71.2, 75.9, 78.1, 82.0, 123.5, 145.4, 181.4, 220.5.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 60

ESI-HRMS (m/z) [M+H]+ calcd for C36H58NO7, 616.4208, found 616.4203. Compound Y15. Prepared from EA (100 mg, 0.21 mmol) and 2,3,4,6-tetra-O-acetyl-β-Dmannopyranosylamine (61 mg, 0.18 mmol) according to general procedure A. The residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v) to give Y15 as a white solid (89 mg, 63%). 1H NMR (400 MHz, CDCl3): δ 0.78, 0.82, 0.90, 0.92, 0.93, 0.99, 1.32, 1.98, 2.04, 2.07, 2.23 (11 × CH3), 0.72-2.17 (m, other aliphatic ring protons), 2.79 (dd, 1H, J = 3.1, 13.7 Hz), 3.22 (dd, 1H, J = 4.2, 10.6 Hz), 3.69-3.74 (m, 1H), 4.04 (dd, 1H, J = 2.2, 12.2 Hz), 4.21-4.26 (m, 2H), 5.10 (dd, 1H, J = 3.3, 10.1), 5.18-5.23 (m, 1H), 5.34 (d, 1H, J = 2.9 Hz), 5.46-5.50 (m, 2H), 7.05 (d, 1H, J = 9.3 Hz). 13C NMR (100 MHz, CDCl3): δ15.5, 15.7, 17.2, 18.1, 20.5, 20.6, 20.7, 23.4, 25.5, 26.7, 27.1, 27.9, 28.0, 29.9, 32.4, 32.6, 34.4, 35.4, 36.8, 38.6, 38.7, 39.7, 41.3, 42.1, 46.4, 46.8, 49.7, 55.2, 62.4, 65.5, 70.0, 71.4, 73.8, 74.5, 76.3, 78.8, 123.2, 143.1, 169.7, 169.8, 170.1, 170.5, 177.4. ESI-HRMS (m/z) [M+H]+ calcd for C44H68NO12, 802.4728, found 802.4736. Compound Y16. Prepared from Y15 (50 mg, 0.06 mmol) according to general procedure B. The residue was purified by column chromatography (CH2Cl2/MeOH, 10/1 v/v) to afford Y16 as a white solid (34 mg, 87%). 1H NMR (400 MHz, MeOD): δ 0.78, 0.90, 0.95, 0.96, 0.98, 1.38 (7 × CH3), 0.75, 2.04 (m, other aliphatic ring protons), 2.27 (t, 1H, J = 13.4 Hz), 2.87 (dd, 1H, J = 3.5, 13.9 Hz), 3.16 (dd, 1H, J = 4.8, 11.0 Hz), 3.23-3.27 (m, 1H), 3.50-3.52 (m, 2H), 3.72 (brs, 1H), 3.81 (dd, 1H, J = 2.2, 11.7 Hz), 4.24 (brs, 1H), 5.07-5.08 (m, 1H), 5.52 (brs, 1H), 7.75 (d, 1H, J = 10.9 Hz). 13C NMR (100 MHz, MeOD): δ16.3, 18.2, 19.4, 24.5, 26.0, 27.4, 27.9, 28.7, 31.0, 33.1, 34.1, 35.9, 36.3, 38.1, 39.9, 40.1, 41.1, 43.1, 43.2, 47.9, 48.4, 51.0, 56.8, 63.2, 68.2, 72.1, 75.3,


Page 31 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


75.6, 79.2, 79.2, 79.7, 124.9, 144.2, 180.9. ESI-HRMS (m/z) [M+H]+ calcd for C36H60NO8, 634.4313, found 634.4312. Cytopathic effect (CPE) reduction assay. The assay was performed as described by Noah et al.43 with some modifications. MDCK cells were seeded into 96-well plates, incubated overnight and infected with influenza virus (MOI=0.1) suspended in DMEM supplemented with 1% FBS, containing test compound and 2 µg/mL TPCK-treated trypsin, with a final DMSO concentration of 1% in each well. After 40 h of incubation, CellTiter-Glo reagent (Promega Corp., Madison, WI, USA) was added and the plates were read using a plate reader (Tecan Infinite M2000 PROTM; Tecan Group Ltd., Mannedorf, Switzerland). Cytotoxicity test. Cells grown in 96-well plates overnight were cultured in 1% FBS with increasing amounts of the test compounds for 40 h. Cytotoxicity was assessed with the CellTiter-Glo assay described as above. Plaque assay. Confluent cultures of MDCK cells in 12-well plates were infected with 100 plaque forming units (PFU)/well of influenza virus WSN, without or with increasing amounts of the test compounds, for 1 h at 37 °C. Cell monolayers were washed with PBS and overlaid with DMEM containing 0.6% low-melting-point agarose and 2 µg/mL TPCK-treated trypsin in the presence or absence of the test compounds, and incubated at 37 °C. Visible plaques were counted 3 days p.i., and the virus titers were determined. Time-of-addition experiment. MDCK cells were seeded into 6-well plates at 5×105 cells per well 24 hours prior to infection and incubated at 37 °C under 5% CO2. Then MDCK cells were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 60

infected with WSN virus at an MOI of 1. Compound was added at 0-10, 0-2, 2-5, 5-8, or 8-10 h post-infection. The cell lysates were harvested and applied to Western blotting for NP analysis. Attachment assay. The virus was mixed with DMEM containing the compound, followed by incubation for 30 min at 37 °C and then cooled for 1 h at 4 °C. Working on ice, medium was removed from precooled A549 cells, and replaced by the virus mixtures. The cells and virus mixtures were incubated for 1 h on ice, which allowed for virus attachment but prevented fusion. Working on ice, the cells were washed three times with chilled PBS. The cells were fixed with chilled 4% paraformaldehyde for 30 min at 4 °C, followed by three manual wash with chilled PBST. Samples were blocked with 10% goat serum in PBST for 1 h at 37 °C, followed by immunofluorescence assay using anti-NP antibodies for detection of attached virus. Immunofluorescence microscopy. Cells were rinsed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS (PBST). Anti-NP antibody was used to detect NP. Preparation and morphology observation of influenza virus particles. The influenza A/WSN/33 viruses were prepared by reverse genetics and cultured in MDCK cells. The supernatant was harvested and clarified (1000×g, 15 min, 4 °C). The clarified supernatant was concentrated by ultracentrifugation (105×g, 2 h, 4 °C, in a Ti40 rotor). Then the virus precipitation was resuspended in 0.5 ml NTE buffer (100 mM NaCl, 10mM Tris-Cl (pH 7.4), 1 mM EDTA) and purified over a 20-60% sucrose gradient (105×g, 2 h, 4 °C, in a SW40 rotor). The banded viruses were collected, diluted with NTE buffer, pelleted (105×g, 2 h, 4 °C, in a SW40 rotor), and


Page 33 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


resuspended in approximately 1 ml of NTE buffer. The purified viruses were incubated with 50 µM Y3 or DMSO 2 h at 4 °C. The morphology of viruses was observed under transmission electron microscope (TEM) by negative straining. Hemagglutination inhibition (HI) assay. Compound from a three-fold serial dilution in saline was mixed with an equal volume of influenza virus (2 HA units) in the V-bottomed 96-well microplate. Subsequently, 50 µL of freshly prepared chicken red blood cells (cRBC) (1% v/v in saline) were added to each well. The mixture was incubated for 30 min at RT before observing cRBC aggregation on the plate. Surface plasmon resonance (SPR). Interactions between the influenza HA and the compounds were analyzed using the Biacore T200 system (GE Healthcare, Uppsala, Sweden) at 25 °C. Recombinant influenza HA (Sino Biological Inc., Beijing, China) was immobilized on a sensor chip (CM5) using an amine coupling kit (GE Healthcare, Buckinghamshire, UK). Final HA immobilized levels were typically ~16000 RU. Subsequently, compounds were injected as analytes at various concentrations, and using PBS-P (10 mM phosphate buffer with 2.7 mM KCl and 137 mM NaCl, 0.05% Surfactant P20, pH 4.5) as running buffer. For binding studies, analytes were applied at corresponding concentrations in running buffer at a flow rate of 30 µL/min with a contact time of 60 s and a dissociation time of 60 s. Chip platforms were washed with running buffer and 50% DMSO. Data were analyzed with the Biacore evaluation software (T200 Version 1.0) by curve fitting using a 1:1 binding model. To explored whether Y3-HA binding had any effect on interactions between HA and its sialic



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 60

acid receptor, the sialic acid receptor, the α-2,6 glycans (6’ S-Di-LN: Neu5Aca2-6[Galb14GlcNAcb1-3]2b-SpNH-LC-LC-Biotin), kindly provided by the CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China and Scripps Research Institute, Department of Molecular Biology, La Jolla, CA, USA, was immobilized on a SA sensor chip with 320 response units. HA protein at 20, 10, 5, 2.5, 1.25, 0.625, or 0 µg/ml flowed through the chip in absence/presence of Y3 or other compounds at a flow rate of 10 µL/min with a contact time of 120 s and a dissociation time of 600 s. The sialic acid receptor-SA chip was regenerated by glycine (pH2.5). The data were analyzed with Biacore evaluation software (T200 Version 1.0) by curve fitting using a 1:1 binding model. Docking simulation. Compound Q8 and Y3 were docked into the HA protein using AutoDock 4.2.44 The structural template of influenza HA (Protein Data Bank: 1RVT

45

) was

obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). To carry out blind docking experiments, grids of points covering the whole HA head and partial stem were generated with ADT.46 Box size: 126 × 126 × 126 points with a standard space of 0.375 Å. Docking simulations of the compound were carried out using the Lamarckian genetic algorithm and through a protocol with a number of 50 GA runs, an initial population of 300 randomly placed individuals, a maximum number of 25 million energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80. The resulting conformations that differed by less than 2.0 Å in positional root-mean-square deviation (rmsd) were clustered together. Other parameters were set as default. All the relevant torsion angles were treated as rotatable during the docking process, thus allowing


Page 35 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a search of the conformational space. After running autogrid and autodock, the possible poses of compounds in HA were obtained. Virus Resistance. MDCK cells were infected with influenza A/WSN/33 (MOI=0.01) and treated with 100 µM Y3, 50 µM amantadine (Sigma), or left untreated for 24 h.40 The post-infection supernatants were taken and employed for infection in the next round of investigation. This procedure was repeated six times. After each passage, supernatants were assayed for progeny virus yields by plaque assay. Virus yields of mock-treated cells were arbitrarily set as 100%. Polykaryon assay. The polykaryon assay was performed as previously described47 with some modifications. Briefly, HeLa cells were transfected with the pCDNA4/TO-HA plasmid (1.6 µg DNA per well). At 30 h post-transfection, the HA was cleaved by incubation with TPCK-treated trypsin for 15 min at 37 °C. After pre-incubated with test compound for 15 min at 37 °C, the cells were incubated with a pH4.9 buffer containing the corresponding concentration of test compound. After exactly 15 min of incubation at 37 °C, the cells were rinsed, and medium containing 10% FBS was added, followed by 3 h of incubation at 37 °C. Finally, the cells were fixed with 4% paraformaldehyde, stained with crystal violet (Sigma-Aldrich, St Louis, MI, USA), and examined by microscopy. General procedure for in vivo antiviral experiments. The in vivo experiments were performed according to previously reported procedures48-50 with some modifications. Female seven to eight-week-old BALB/c mice were anesthetized by inhalation of ether and were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 60

inoculated intranasally with 5×LD50 of A/WSN/33 influenza virus in 50 µl PBS solution. Drug administration was done by intranasal inoculation in anesthetized mice to deliver the drug topically to the lungs. In our prophylaxis and treatment courses, drug was administered 24 h before infection, 4 h before infection, during infection, 4 h after infection, and then twice daily for 5 days beginning 24 h after infection. Zanamivir was used as a positive control. Daily assessments for weight and mortality were made on all mice. Production and infection of virus-like particles (VLPs). For generating pseudotyped influenza A virus (IAVpp) or vesicular stomatitis virus (VSVpp), 293T cells were cotransfected with plasmids encoding influenza A/WSN/33 virus HA and NA, or vesicular stomatitis virus G protein (VSV-G), and the envelope protein (Env) and Vpr deficient HIV vector carrying a luciferase reporter gene inserted into the Nef position. At 48 h post-transfection, cell culture supernatants were harvested, passed through 0.45-µm pore-size filters, aliquoted, and stored at -80°C. For infection experiments, the cell monolayers were inoculated with diluted IAVpp or VSVpp in the presence or absence of test compounds. Luciferase activity in cell lysates was measured 72 h after infection using the Bright-Glo Reagent (Promega Corp., Madison, WI, USA).

AUTHOR INFORMATION Corresponding Author *Phone: +86-10-82805857. Fax: +86-10-82805857. Email: [email protected]. Author Contributions


Page 37 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We gratefully acknowledge the antiviral broad spectrum activity tested by Professor Jiandong Jiang, Professor Yuhuan Li and Mrs. Linlin Ma from Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College. This work was supported by the National Basic Research Program of China (973 Program; Grant No. 2010CB12300) and the National Natural Science Foundation of China (Grants Nos. 813611680027, 81101239, 20932001, 91029711, 20852001, 81373271, and 81202975) and Science and Technology Development Fund of Macao (074/2012/A3, 077/2011/A3). Maorong Yu was supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences. ABBREVIATIONS USED EA, echinocystic acid; RBV, ribavirin; GL, glycyrrhizin; OSV, oseltamivir; PFU, plaque-forming unit;

EDC,

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride;

NHS,

N-hydroxysuccinimide; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone

REFERENCES: 1.

Suzuki, Y. Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses. Biol. Pharm. Bull. 2005, 28, 399-408.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.

Page 38 of 60

Tong, S.; Li, Y.; Rivailler, P.; Conrardy, C.; Castillo, D. A.; Chen, L. M.; Recuenco, S.; Ellison, J. A.; Davis, C. T.; York, I. A.; Turmelle, A. S.; Moran, D.; Rogers, S.; Shi, M.; Tao, Y.; Weil, M. R.; Tang, K.; Rowe, L. A.; Sammons, S.; Xu, X.; Frace, M.; Lindblade, K. A.; Cox, N. J.; Anderson, L. J.; Rupprecht, C. E.; Donis, R. O. A distinct lineage of influenza A virus from bats. Proc. Natl. Acad. Sci. U S A 2012, 109, 4269-4274.

3.

Tong, S.; Zhu, X.; Li, Y.; Shi, M.; Zhang, J.; Bourgeois, M.; Yang, H.; Chen, X.; Recuenco, S.; Gomez, J.; Chen, L. M.; Johnson, A.; Tao, Y.; Dreyfus, C.; Yu, W.; McBride, R.; Carney, P. J.; Gilbert, A. T.; Chang, J.; Guo, Z.; Davis, C. T.; Paulson, J. C.; Stevens, J.; Rupprecht, C. E.; Holmes, E. C.; Wilson, I. A.; Donis, R. O. New world bats harbor diverse influenza A viruses. PLoS Pathog. 2013, 9, e1003657.

4.

De Clercq, E. Antiviral agents active against influenza A viruses. Nat. Rev. Drug. Discov. 2006, 5, 1015-1025.

5.

Webby, R. J.; Webster, R. G. Are we ready for pandemic influenza? Science 2003, 302, 1519-1522.

6.

Kilbourne, E. D. Influenza pandemics of the 20th century. Emerg. Infect. Dis. 2006, 12, 9-14.

7.

Yuen, K. Y.; Wong, S. S. Human infection by avian influenza A H5N1. Hong Kong Med. J. 2005, 11, 189-199.

8.

Buchy, P.; Mardy, S.; Vong, S.; Toyoda, T.; Aubin, J. T.; Miller, M.; Touch, S.; Sovann, L.; Dufourcq, J. B.; Richner, B.; Tu, P. V.; Tien, N. T.; Lim, W.; Peiris, J. S.; Van der Werf, S. Influenza A/H5N1 virus infection in humans in Cambodia. J. Clin. Virol. 2007, 39, 164-168.


Page 39 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9.

Michaelis, M.; Doerr, H. W.; Cinatl, J. J. Of chickens and men: avian influenza in humans. Curr. Mol. Med. 2009, 9, 131-151.

10. Yeh, J. Y.; Coumar, M. S.; Horng, J. T.; Shiao, H. Y.; Kuo, F. M.; Lee, H. L.; Chen, I. C.; Chang, C. W.; Tang, W. F.; Tseng, S. N.; Chen, C. J.; Shih, S. R.; Hsu, J. T.; Liao, C. C.; Chao, Y. S.; Hsieh, H. P. Anti-influenza drug discovery: structure-activity relationship and mechanistic insight into novel angelicin derivatives. J. Med. Chem. 2010, 53, 1519-1533. 11. Khazeni, N.; Hutton, D. W.; Collins, C. I.; Garber, A. M.; Owens, D. K. Health and Economic Benefits of Early Vaccination and Nonpharmaceutical Interventions for a Human Influenza A (H7N9) Pandemic: A Modeling Study. Ann. Intern. Med. 2014, 160, 684-694. 12. Bouvier, N. M.; Palese, P. The biology of influenza viruses. Vaccine 2008, 26 Suppl 4, D49-53. 13. von Itzstein, M.; Wu, W. Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Van Phan, T.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Et, A. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 1993, 363, 418-423. 14. Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.; Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R. C. Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J. Am. Chem. Soc. 1997, 119, 681-690. 15. Bright, R. A.; Medina, M. J.; Xu, X.; Perez-Oronoz, G.; Wallis, T. R.; Davis, X. M.; Povinelli,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 60

L.; Cox, N. J.; Klimov, A. I. Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 2005, 366, 1175-1181. 16. Deyde, V. M.; Xu, X.; Bright, R. A.; Shaw, M.; Smith, C. B.; Zhang, Y.; Shu, Y.; Gubareva, L. V.; Cox, N. J.; Klimov, A. I. Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J. Infect. Dis. 2007, 196, 249-257. 17. Moscona, A. Global transmission of oseltamivir-resistant influenza. N. Engl. J. Med. 2009, 360, 953-956. 18. Vanderlinden, E.; Naesens, L. Emerging antiviral strategies to interfere with influenza virus entry. Med. Res. Rev. 2014, 34, 301-339. 19. Song, G.; Yang, S.; Zhang, W.; Cao, Y.; Wang, P.; Ding, N.; Zhang, Z.; Guo, Y.; Li, Y. Discovery of the first series of small molecule H5N1 entry inhibitors. J. Med. Chem. 2009, 52, 7368-7371. 20. Matsubara, T.; Sumi, M.; Kubota, H.; Taki, T.; Okahata, Y.; Sato, T. Inhibition of influenza virus infections by sialylgalactose-binding peptides selected from a phage library. J. Med. Chem. 2009, 52, 4247-4256. 21. Dimitrov, D. S. Virus entry: molecular mechanisms and biomedical applications. Nat. Rev. Microbiol. 2004, 2, 109-122. 22. Yu, F.; Wang, Q.; Zhang, Z.; Peng, Y.; Qiu, Y.; Shi, Y.; Zheng, Y.; Xiao, S.; Wang, H.; Huang, X.; Zhu, L.; Chen, K.; Zhao, C.; Zhang, C.; Yu, M.; Sun, D.; Zhang, L.; Zhou, D.


Page 41 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Development of oleanane-type triterpenes as a new class of HCV entry inhibitors. J. Med. Chem. 2013, 56, 4300-4319. 23. Wang, H.; Wang, Q.; Xiao, S. L.; Yu, F.; Ye, M.; Zheng, Y. X.; Zhao, C. K.; Sun, D. A.; Zhang, L. H.; Zhou, D. M. Elucidation of the pharmacophore of echinocystic acid, a new lead for blocking HCV entry. Eur. J. Med. Chem. 2013, 64, 160-168. 24. Yu, F.; Peng, Y.; Wang, Q.; Shi, Y.; Si, L.; Wang, H.; Zheng, Y.; Lee, E.; Xiao, S.; Yu, M.; Li, Y.; Zhang, C.; Tang, H.; Wang, C.; Zhang, L.; Zhou, D. Development of bivalent oleanane-type triterpenes as potent HCV entry inhibitors. Eur. J. Med. Chem. 2014, 77, 258-268. 25. Xiao, S.; Wang, Q.; Si, L.; Shi, Y.; Wang, H.; Yu, F.; Zhang, Y.; Li, Y.; Zheng, Y.; Zhang, C.; Wang, C.; Zhang, L.; Zhou, D. Synthesis and anti-HCV entry activity studies of beta-cyclodextrin-pentacyclic triterpene conjugates. ChemMedChem 2014, 9, 1060-1070. 26. Shapiro, G. I.; Gurney, T. J.; Krug, R. M. Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs. J. Virol. 1987, 61, 764-773. 27. Furuta, Y.; Takahashi, K.; Kuno-Maekawa, M.; Sangawa, H.; Uehara, S.; Kozaki, K.; Nomura, N.; Egawa, H.; Shiraki, K. Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 2005, 49, 981-986. 28. Hahon, N.; Booth, J. A.; Eckert, H. L. Cell attachment and penetration by influenza virus. Infect. Immun. 1973, 7, 341-351.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 60

29. Haywood, A. M.; Boyer, B. P. Time and temperature dependence of influenza virus membrane fusion at neutral pH. J. Gen. Virol. 1986, 67 ( Pt 12), 2813-2817. 30. Jones, J. C.; Turpin, E. A.; Bultmann, H.; Brandt, C. R.; Schultz-Cherry, S. Inhibition of influenza virus infection by a novel antiviral peptide that targets viral attachment to cells. J. Virol. 2006, 80, 11960-11967. 31. Hoffmann, J.; Schneider, C.; Heinbockel, L.; Brandenburg, K.; Reimer, R.; Gabriel, G. A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment. Antiviral Res. 2014, 104, 23-33. 32. Pedersen, J. C. Hemagglutination-inhibition test for avian influenza virus subtype identification and the detection and quantitation of serum antibodies to the avian influenza virus. Methods Mol. Biol. 2008, 436, 53-66. 33. Kumakura, S.; Shibata, H.; Isobe, T.; Hirose, M.; Ohe, M.; Nishimura, N.; Onoda, K.; Nagai, A.; Yamaguchi, S. Comparison of hemagglutination inhibition assay and enzyme immunoassay for determination of mumps and rubella immune status in health care personnel. J. Clin. Lab. Anal. 2013, 27, 418-421. 34. Suenaga, E.; Mizuno, H.; Penmetcha, K. K. Monitoring influenza hemagglutinin and glycan interactions using surface plasmon resonance. Biosens Bioelectron 2012, 32, 195-201. 35. Yang, J.; Li, M.; Shen, X.; Liu, S. Influenza A virus entry inhibitors targeting the hemagglutinin. Viruses 2013, 5, 352-373. 36. Ou, J. L.; Mizushina, Y.; Wang, S. Y.; Chuang, D. Y.; Nadar, M.; Hsu, W. L.


Page 43 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Structure-activity relationship analysis of curcumin analogues on anti-influenza virus activity. FEBS J 2013, 280, 5829-5840. 37. Leneva, I. A.; Russell, R. J.; Boriskin, Y. S.; Hay, A. J. Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol. Antiviral Res. 2009, 81, 132-140. 38. Skehel, J. J.; Wiley, D. C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 2000, 69, 531-569. 39. Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.; Robins, R. K. Broad-spectrum

antiviral

activity

of

Virazole:

1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide. Science 1972, 177, 705-706. 40. Kumar, N.; Sharma, N. R.; Ly, H.; Parslow, T. G.; Liang, Y. Receptor tyrosine kinase inhibitors that block replication of influenza a and other viruses. Antimicrob. Agents Chemother. 2011, 55, 5553-5559. 41. Ou, J. L.; Mizushina, Y.; Wang, S. Y.; Chuang, D. Y.; Nadar, M.; Hsu, W. L. Structure-activity relationship analysis of curcumin analogues on anti-influenza virus activity. FEBS J 2013, 280, 5829-40. 42. Vedejs, E.; Piotrowski, D. W.; Tucci, F. C. Oxazolium-derived azomethine ylides. External oxazole activation and internal dipole trapping in the synthesis of an aziridinomitosene. J. Org. Chem. 2000, 65, 5498-5505. 43. Noah, J. W.; Severson, W.; Noah, D. L.; Rasmussen, L.; White, E. L.; Jonsson, C. B. A



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 60

cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals. Antiviral Res. 2007, 73, 50-59. 44. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785-2791. 45. Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.; Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley, D. C.; Skehel, J. J. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 2004, 303, 1838-1842. 46. Sanner, M. F. Python: a programming language for software integration and development. J. Mol. Graph Model 1999, 17, 57-61. 47. Vanderlinden, E.; Goktas, F.; Cesur, Z.; Froeyen, M.; Reed, M. L.; Russell, C. J.; Cesur, N.; Naesens, L. Novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin. J. Virol. 2010, 84, 4277-4288. 48. Ryan, D. M.; Ticehurst, J.; Dempsey, M. H.; Penn, C. R. Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase). Antimicrob. Agents Chemother. 1994, 38, 2270-2275. 49. Gubareva, L. V.; McCullers, J. A.; Bethell, R. C.; Webster, R. G. Characterization of influenza A/HongKong/156/97 (H5N1) virus in a mouse model and protective effect of


Page 45 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


zanamivir on H5N1 infection in mice. J. Infect. Dis. 1998, 178, 1592-1596. 50. Nicol, M. Q.; Ligertwood, Y.; Bacon, M. N.; Dutia, B. M.; Nash, A. A. A novel family of peptides with potent activity against influenza A viruses. J. Gen. Virol. 2012, 93, 980-986.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 60

Figure legends Figure 1. Discover anti-influenza virus lead compound Q8. (A) The structure of EA-galactose conjugate (Q8). (B) Validation of the protection of MDCK cells from influenza A/WSN/33 virus by Q8. (C) The Q8 dose-dependent reduction of viral plaque formation. The calculated EC50 is around 5 µM, almost two-fold lower than that of oseltamivir (OSV). Glycyrrhizic acid (GL) acts as negative control. Figure 2. Explore the structure-activity relationship of triterpene-glycoconjugates against influenza A/WSN/33 virus. The structure requirements for maintaining high anti-influenza virus activity were explored by systematic modifications at the constitutive aglycone, glycoside and linker. The potency of each compound was tested at the concentration 50 µM by cytopathic effect reduction assay. The inhibition rate of the mock-treated cells was arbitrarily set as 0%. Figure 3. Explore the mechanism of the lead compound as an inhibitor of influenza viruses. (A) Design time-of-addition experiments to identify which step of life cycle Y3 targets, i.e. virus entry (0-2 hr), viral genome replication and translation (2-8 hr), or progeny virion release (8-10 hr). Detect the expression levels of influenza NP protein in infected MDCK cells at five time intervals, 0-10 h, 0-2 h, 2-5 h, 5-8 h and 8-10 h. Only at two intervals, 0-10 h (covering the whole life cycle) and 0-2 h (covering the entry step), NP expression was significantly reduced (around 90% and 80%, respectively) as compared with DMSO control. (B) Track infected cells stained with anti-NP antibody (green) and DAPI (blue) under a microscope to identify the mode of action of Y3. A549 cells was inoculated with influenza A/WSN/33 viruses (MOI=0.5) at 4oC in the presence of Y3 at


Page 47 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentration 0-100 µM. The attachment of NP protein on cellular membrane was prevented upon addition of Y3. (C) The different effect of Y3 on the populations of infected cells and nuclear accumulation of the NP protein. Inoculation of A549 cells with A/WSN/33 viruses under 37oC rather than 4oC caused a great population of infected cells and significant nuclear accumulation of the NP protein. Addition of Y3 led to significant reduction of NP-positive cell populations but not the nuclear intensity of NP protein in certain infected cells. (D) Three experiments, namely co-treatment, pre-treated cell and pre-treated virus, were designed to clarify whether the host cell or the influenza virus was targeted. (E) The effects of Y3 on the morphology of host cells and influenza virus particles. Figure 4. Identify HA as the potential target of lead compound. (A) Comparisons of the behaviors of Y3 vs. anti-HA antibody in inhibition of influenza virus-induced aggregation of chicken erythrocytes. Y3 exerted identical capability as anti-HA antibody in inhibition of hemagglutination in a dose-dependent manner. (B) Characterization of the affinity between lead compounds (Q8 and Y3) and HA protein, which was immobilized on a CM5 sensor chip, based on the surface Plasmon resonance assay. The aglycone of the lead compounds including EA and OA as well as the negative compound ribavirin (RBV) were also tested. Their KD values were labeled in the corresponding curves. (C) The effect of Y3-HA binding on interactions between HA and its sialic acid receptor. HA protein flew across the sialic acid receptor-chip surface in absence/presence of the lead compound (20 µM Y3, 100 µM Arbidol or 20 µM Curcumin), and lead KD at 70, 844, 81 and 507 nM. Curcumin was taken as positive control, while Arbidol was



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 60

taken as negative control. Figure 5. Structural representative of Y3 binding within HA protein (Protein Data Bank: 1RVT) according to blind docking calculation. The final docked conformations of Y3 bound HA were determined based on the minimum free energy. Y3 was suggested to occupy the conserved binding pocket for sialic acid. The complex of HA with sialyllactose (LSTc) as a reference was derived from a 3-Å-resolution X-ray structure, in which a group of hydrogen bonds are formed between sialic acid and the conserved site-chain or main-chain polar atoms of HA. By comparison, the computer-aided docking data indicated that the 3-hydroxyl group of the oleanolic acid of Y3 forms hydrogen bond with both the guanidine of residue Arg133 and the carbonyl group of residue Lys156, two residues with the conserved domain for sialic acid binding. The amide of peptide bond Ala137, one critical residue for hydrogen bonding to the carboxylate of sialic acid, forms a hydrogen bond with the 2-carbonyl group of the β-galactose, which is also hydrogen bonded to the amide of residue Gln226, also one critical residue involved in sialic acid binding. The 5th hydrogen bond is formed between Ser145 of HA and the 3-carbonyl group of the β-galactose of Y3. In addition, multiple hydrophobic interactions were observed between Y3 and its binding pocket, namely, residues Val155, His183, Ser193, Pro185, Tyr95, Pro186, Asp190, Trp53, Thr136, Val135, and Leu194. Among them, His183 and Leu194 are critical for sialic acid binding via hydrogen bonds. The estimated binding energies for Y3-HA and sialic acid were -9.44 and -6.68 kcal/mol with inhibition constants (Ki) of 121.02 nM and12.68 µM. Figure 6. (A) A competitive affinity assay was designed to verify the possibility of Y3 occupancy


Page 49 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


at the pocket for the sialic acid receptor. Arbidol (100 µM), curcumin (50 µM), sialic acid (500 µM) and Y3 (50 µM) flew separately across the HA-chip surface and showed a SPR response. When arbidol or curcumin was co-added with Y3 as the analyte, an additive response was observed. However, when free sialic acid and Y3 were co-added as the analyte, rather than an additive but antagonistic effect was observed with the SPR value as barely 51.9 RU, even lower than that of Y3. (B) Test the tendency of induction of viral resistance by continuous Y3 treatment. Microscopy observations of plaque formation at the 1st, 4th and 6th passage of a multi-passaging experiment treated either by Y3 or amantadine. Quantitative analysis of the relative yield of progeny virus by plaque assay at each round of total six rounds propagation. MDCK cells were infected with influenza A/WSN/33 (MOI=0.01) and treated with Y3, amantadine, or left untreated. At 24h post-infection supernatants were taken and employed for infection in the next round of investigation. Virus yields of mock-treated cells were arbitrarily set as 100%. (C) The effect of Y3 on HA-mediated polykaryon formation. Overexpression of HA protein in Hela cells led to significant polykaryon formation upon lowering pH from 7.0 to 4.9 irrespective of the absence or presence of Y3. Figure 7. A tentative mechanism for Y3-mediated anti-influenza infection: Y3 binds to influenza HA protein, disrupts its interaction with sialic acid receptor and thus the attachment of influenza viruses to host cells. Figure 8. Evaluation of the efficacy of Y3 against influenza infection via intranasal administration. Balb/C mice, five in each group, were inoculated with 5LD50 of virus and intranasally



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 60

administrated with Y3 at dose 0, 0.25 and 0.5mg/20g/d twice daily for 5 days, starting one day before infection. The body weights (A) and survival (B) of infected mice were monitored daily for 10 and 13 days, respectively. Infected mice treated with zanamivir were used as positive control.


Page 51 of 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment



Page 52 of 60

Page 53 of 60





Page 54 of 60

Page 55 of 60





Page 56 of 60

Page 57 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7




Page 58 of 60

Page 59 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Comparisons of the anti-IAV activity of lead compounds with that of other antiviral therapeutics CC50 (µM)a

Compound

EC50 (µM)b

PR/8 (H1N1) c

JX/312

HN/1222 (H3N2) B/SZ/155

(H3N2)

Y3

>200

7.41 ± 0.05

6.58 ± 1.17

2.72 ± 0.35

3.18 ± 0.08

2.80 ± 0.74

Y4

>200

9.33 ± 0.60

33.25 ± 0.64

66.67 ± 0.17

26.25 ± 3.01

43.88 ± 19.38

Q8

>200

17.50 ± 0.43

17.80 ± 12.57

16.89 ± 5.38

25.59 ± 9.14

36.21 ± 7.21

7.41 ± 0.21

0.44 ± 0.14

8.58 ± 1.65

>200

>83.24

Amantadine >200

a

LN/1109 (H1N1)

OSV

>200

>200

>200

2.06 ± 0.58

8.75 ± 4.41

91.07 ± 34.51

RBV

>200

4.02 ± 1.27

5.75 ± 0.35

4.19 ± 0.35

3.56 ± 0.24

1.32 ± 0.60

CC50, the concentration required to reduced normal, non-infected cell viability by 50%. Values

represent the mean of duplicate samples from three independent experiments. b

EC50, the concentration required

to reduced inhibition of viral infection-induced

cytopathogenicity by 50%. Values represent the mean of duplicate samples from three independent experiments. c

PR/8 (H1N1), A/Puerto Rico/8/34 (H1N1); LN/1109 (H1N1), A/LiaoNing-ZhenXing/1109/2010

(H1N1);

JX/312

(H3N2),

A/JiangXi-DongHu/312/2006

(H3N2);

A/HuNan-ZhuHui/ 1222/2010 (H3N2); B/SZ/155, B/ShenZhen/155/2005.


HN/1222

(H3N2),


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents graphic


Page 60 of 60

Discovery of Pentacyclic Triterpenoids as ... - ACS Publications

Recommend Documents