J Med Chem. 2014 Dec 11;57(23):10058-71.

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

 

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

Email: deminzhou@bjmu.edu.cn; maorongyu6@126.com

  • These authors contributed equally to this paper

 

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.

PMID: 25383779

 

Supplement:

Influenza A virus, a member of the Orthomyxoviridae family, is a major human pathogen that causes annual epidemics and occasional pandemics. 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. Oseltamivir (Tamiflu) and zanamivir (Relenza), on the other hand, target neuraminidase (NA) protein inhibiting its enzymatic activity and causing the tethered progeny virus to be unable to escape from its host cells. However, the emergence of drug-resistant influenza viruses has limited the use of those drugs, 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. 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. An advantage of the latter strategy is that resistance is less likely to occur since many viruses use similar entry routes.1

 Figure 1

Figure 1. The structure of Y3.

Pentacyclic triterpenes are secondary plant metabolites found in different plant organs, with a few species containing up to 30% of their dry weigh. 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. 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. 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.2,3 In view of their significant inhibitory effect on both HIV and HCV viruses, we sought to learn whether such pentacyclic triterpenoids are also active against influenza viruses.1

A mini-library of triterpene-glycoconjugates was screened for their anti-influenza virus activity. We found that Y3, a conjugate of an acetylated galactose moiety with OA aglycones, significantly reduces the viral CPE in MDCK cells by direct microscopic observation. Its anti-influenza virus activity was confirmed by plague formation reduction assay in which Y3 exerts a well-defined dose-dependent response against the A/WSN/33 virus with EC50 calculated to be around 5 μM, almost two-fold lower than that of oseltamivir.

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: virus entry (0-2 h), viral genome replication and translation (2-8 h), and progeny virion release (8-10 h). We found 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. Further research indicated that Y3 interferes with the attachment of viruses to host cells but not with the later stages of the viral lifecycle.

After establishing that the early stage of viral life cycle (attachment) were the points of interference, we designed experiments to identify the potential target. We first clarified whether the potential target was on the host cell or the influenza virus. As described in the paper featured at this site, Y3 targets the influenza virus particles but not host cells.1 Due to the important role of hemagglutinin (HA) on the envelope of influenza viruses during the viral entry process, a series of assays were designed to explore possibility of HA as the target of Y3. We discover that Y3 could tightly bind to HA protein and disrupt the interaction between HA and sialic acid receptor. More interestingly, the docking calculation and related evidences indicated that Y3 occupied the binding pocket on the HA for sialic acid receptor with an estimated binding energy of -9.44 kcal/mol and an inhibition constant (Ki) = 121.02 nM.

Therefore, a 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 HA and disrupting its interaction with the sialic acid receptor, thus blocking the attachment of viruses to host cells.

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.

Figure 2

Figure 2. A tentative mechanism model for Y3-mediated anti-influenza activity. Left: Y3 binds to influenza HA protein, disrupts its interaction with sialic acid receptor and thus the attachment of influenza viruses to host cells. Right: Structural representative of Y3 binding within HA protein (Protein Data Bank: 1RVT) according to blind docking calculation.

 

References:

  1. Yu, M.; Si, L.; Wang, Y.; Wu, Y.; Yu, F.; Jiao, P.; Shi, Y.; Wang, H.; Xiao, S.; Fu, G.; Tian, K.; Wang, Y.; Guo, Z.; Ye, X.; Zhang, L.; Zhou, D. Discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses. J Med Chem 2014, 57, 10058-10071.
  2. 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. Development of oleanane-type triterpenes as a new class of HCV entry inhibitors. J Med Chem 2013, 56, 4300-4319.
  3. 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.

 

Acknowledgement:

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.

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann