Influenza neuraminidase: A druggable target for natural products

Contents

• 1 Introduction

• 2 Influenza NA – an established drug target for combating influenza
  • 2.1 Influenza NA function
  • 2.2 Influenza NA classification
  • 2.3 Influenza NA structure
  • 2.4  Catalytic mechanism
  • 2.5 Approved NAIs
  • 2.6 Mechanisms conferring drug resistances in influenza
  • 2.7 Similarities and differences of influenza NA with NA of other biological systems

• 3 Experimental evaluation of NA inhibition
  • 3.1 Methods used in discovery and functional analysis of first generation NAIs
  • 3.2 Advantages and disadvantages of methods currently applied to identify novel NAIs

• 4 Inhibition of influenza NA by natural compounds
  • 4.1 Evaluation of published data
  • 4.2 Compound classes and sources of natural NAIs
  • 4.3 Phenolic compounds as NAIs
  • 4.4  Isoprenoids – a compound class showing no effect on NA
  • 4.5 Extracts with promising NA inhibitory activity

• 5 Strategies for the discovery of NA inhibiting natural compounds
  • 5.1  In vitro screening of extracts–Bioassay-guided fractionation and isolation
  • 5.2 Exploitation of empirical knowledge
  • 5.3 Exploiting influenza NA inhibition by computer-guided approaches

• 6 Conclusions and future perspectives

• References

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Abstract

Covering: 2000 to 2011

The imminent threat of influenza pandemics and repeatedly reported emergence of new drug-resistant influenza virus strains demonstrate the urgent need for developing innovative and effective antiviral agents for prevention and treatment. At present, influenza neuraminidase (NA), a key enzyme in viral replication, spread, and pathogenesis, is considered to be one of the most promising targets for combating influenza. Despite the substantial medical potential of NA inhibitors (NAIs), only three of these drugs are currently on the market (zanamivir, oseltamivir, and peramivir). Moreover, sudden changes in NAI susceptibility revealed the urgent need in the discovery/identification of novel inhibitors. Nature offers an abundance of biosynthesized compounds comprising chemical scaffolds of high diversity, which present an infinite pool of chemical entities for target-oriented drug discovery in the battle against this highly contagious pathogen. This review illuminates the increasing research efforts of the past decade (2000–2011), focusing on the structure, function and druggability of influenza NA, as well as its inhibition by natural products. Following a critical discussion of publications describing some 150 secondary plant metabolites tested for their inhibitory potential against influenza NA, the impact of three different strategies to identify and develop novel NAIs is presented: (i) bioactivity screening of herbal extracts, (ii) exploitation of empirical knowledge, and (iii) computational approaches. This work addresses the latest developments in theoretical and experimental research on properties of NA that are and will be driving anti-influenza drug development now and in the near future.

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Ulrike Grienke

Ulrike Grienke studied pharmacy at the University of Innsbruck, Austria (2001–2008), where she already started with her phytochemical investigations on traditional Chinese medicinal plants and evaluation of computational methods (neural networks) for exploring the chemical space relevant for acetylcholinesterase inhibitors. In 2008, she received a PhD scholarship granted by the Austrian Science Fund (FWF), which enabled her to continue her studies on bioactive natural products under the supervision of Prof. Judith M. Rollinger. She has published four research papers, gave four talks at international conferences and is about to finalize her PhD studies.

Michaela Schmidtke

Michaela Schmidtke studied biology at the University of Chişinău, Moldova (1980–1985). After receiving her PhD in Antiviral Research from the Faculty of Biology at the Friedrich-Schiller-University, Jena, Germany (1992), she had post-doctorial training at the Institute of Immunology, University Marburg with the focus on coxsackievirus B3-immune cell interactions. Thereafter, she was a postdoctoral fellow at the Department of Virology and Antiviral Therapy, Jena University Hospital, Germany ending up with the habilitation thesis and the venia legendi in Virology. Her research interests focus on the development of novel anti-enteroviral and anti-influenza virus inhibitors communicated in more than 70 papers and patents.

Susanne von Grafenstein

Susanne von Grafenstein studied pharmacy at the Ludwig-Maximilians-University, Munich, Germany (2004–2008) and registered as pharmacist in 2009. During her training, she participated in several virtual screening projects at the University of Innsbruck, Austria. There, she joined the research group of Prof. Klaus R. Liedl, starting her PhD studies in 2010. At present her research is focused on the investigation of molecular dynamics and identification of new inhibitors in a project on influenza neuraminidase.

Johannes Kirchmair

Johannes Kirchmair studied pharmacy at the University of Innsbruck (1999–2004) and received his PhD degree under the guidance of Prof. Thierry Langer (2007), specializing in virtual screening. He was application scientist at Inte:Ligand GmbH, Vienna, Austria, assistant lecturer at the University of Innsbruck and post-doctoral research fellow with Prof. Klaus R. Liedl and Prof. Klaus-Jürgen Schleifer (BASF SE, Ludwigshafen, Germany). Since 2010 he is working as research assistant with Prof. Robert C. Glen at the Unilever Centre for Molecular Science Informatics, University of Cambridge, United Kingdom. His work is communicated in 35 peer-reviewed papers and book chapters.

Klaus R. Liedl

Klaus R. Liedl is head of the Department of Theoretical Chemistry at the Faculty of Chemistry and Pharmacy, University of Innsbruck. After he finished his chemistry and mathematics studies (1989, 1992), he studied theoretical physics and obtained a PhD in chemistry (1995). In 2006 he finished his doctorate in juridical science focusing on intellectual property rights complementing his chemical research as proven by his more than 130 publications in the field. His main research interests are drug development and the understanding and prediction of thermodynamics in the course of biomolecular binding processes and reactions.

Judith M. Rollinger

Judith M. Rollinger is Professor of Pharmacognosy at the University of Innsbruck, Austria, member of the Center for Molecular Biosciences Innsbruck, and elected member of the GA Board of Directors (2011–2013). After studying Pharmacy, she received her PhD in 1999 and the venia legendi in Pharmacognosy (2007) at the University of Innsbruck. Her research interests include developing new approaches, e.g. integrating computational techniques and ethno-pharmacological knowhow in natural product research, for rationalized discovery of natural leads targeting neurodegenerative diseases, viral infections and inflammation. Publications (60) resulting from her research appear in highly ranked international journals, as book contributions, and patents.

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1 Introduction

Belonging to the Orthomyxoviridae, influenza A and B are responsible for an acute contagious respiratory infection commonly known as “flu”. The disease appears in waves of seasonal flu characterized by fever, cough, sore throat and myalgia, with the ability to develop severe life-threatening conditions, such as pneumonia.¹ Moreover, epidemics as well as pandemics reoccur periodically. Considered a worst-case scenario, the influenza pandemic of 1918 (known as the Spanish Flu) took the lives of an estimated 20 to 40 million people worldwide.^2–4

Influenza neuraminidase (NA) has been established as a primary drug target for the prophylaxis and treatment of influenza infections, which are still a major threat to human health due to high morbidity and mortality rates, especially when affecting risk groups like children, the old, and people with chronic diseases.⁵ In contrast to the first approved anti-influenza drugs, the ion channel blockers amantadine (Symmetrel^®, approved 1966) and rimantadine (Flumadine^®, approved 1993), NA inhibitors (NAIs) are well tolerated and act against influenza A as well as B viruses.^6,7 After the introduction of the first NAIs in 1999, interest in the development of drugs targeting this viral enzyme wound down for several years – the market was well served. But, with the outbreak of A/H5N1, the avian influenza (also known as “bird flu”), concerns about a major pandemic started to emerge. The person-to-person transmission of A/H5N1 is limited, but in case these viruses developed efficient human-to-human transmission, estimates show that such a variant may cause a death toll exceeding the devastating Spanish Flu.⁸ Moreover, in the season of 2007/2008 a naturally oseltamivir-resistant H1N1 mutant emerged suddenly and spread worldwide.⁹ Awareness rose further with the flu pandemic of 2009 caused by a new A/H1N1 strain (swine-origin influenza H1N1 virus; S–OIV), which was characterized by a high transmission rate but low virulence.¹⁰ Though this pandemic developed less critically than anticipated these events highlight the urgent need for novel antiviral therapeutics.^11,12

Moreover, with today's very narrow drug portfolio for the prophylaxis and treatment of flu, public health is threatened by the imminent risk of a sudden emerge of drug-resistant viral strains.^13–16 Such sudden changes in NAI susceptibility were observed in influenza A viruses circulating worldwide in the last 3 years.^17,18

Therefore, twelve years after the launch of Relenza^® (zanamivir (1)) and Tamiflu^® (oseltamivir (2)) – the two commonly applied/prescribed NAIs worldwide – the search for novel lead structures in the fight against this aggressive pathogen is again an area of intense research.^19,20 Emphasis on more sophisticated strategies is needed to overcome shortcomings and drawbacks of currently available drugs and leads, such as side effects and emerging drug resistance.

Nature provides an abundance of structurally diverse chemical scaffolds that represent a rich source for lead structures in drug development.^21,22 In the past century various compounds isolated from natural sources have been extensively studied and tested for their anti-viral and anti-influenza activity.^23–25 This review focuses on natural products specifically targeting the viral surface protein NA, as do currently available first-line drugs stockpiled worldwide in the case of a pandemic. The survey provides an overview on reported activities as well as an outlook on future prospects.

2 Influenza NA – an established drug target for combating influenza

2.1 Influenza NA function

Influenza NA, a glycoside hydrolase (EC 3.2.1.18) and hemagglutinin (HA) are antigenic glycoproteins on the surface of influenza virions. NA and HA both recognize carbohydrate structures and bind to terminal sialic acid (3) units on the surface of the host cell.

Binding of HA to its receptor 3 initiates viral entry into the host cell. NA also binds 3 and hydrolytically cleaves the α-(2,3) and α-(2,6) glycosidic linkage of terminal 3. Thereby, NA reduces the number of receptor binding sites for HA on host cells as well as on progeny viruses. This allows the mature virus to detach from the host cell during release and prevents self aggregation mediated by HA.^26–28 As a consequence, NA plays a critical role for virus spread in infected tissues and enables virus transmission during infection. Due to the antagonistic functions of HA and NA, a balanced interplay between the activity of both viral envelope glycoproteins is essential for efficient entry and release of influenza viruses.²⁹ Additionally, NA's function is related to the mobility of influenza virus within the respiratory tract. NA cleaves the glycan structure of mucus and allows the virus to reach host cells.^30–32

2.2 Influenza NA classification

Nine NA subtypes were classified by antigenic characteristics. While all nine subtypes are found in avian hosts, only N1 and N2 subtypes were found in influenza virus strains causing human infections. According to their phylogenetic relationship and structure the nine subtypes split into two families: group-1 includes N1, N4, N5, and N8, whereas group-2 comprises N2, N3, N6, N7, and N9.³³ The most prominent structural feature of group-1 NAs is the formation of a pocket located at the 150-loop. Contrary to other structurally determined N1 NAs, the one of the 2009 H1N1 virus lacks the 150-cavity.^34,35

2.3 Influenza NA structure

The structure of an NA entity on the viral surface is composed of four identical subunits forming a homotetramer of around 240 kDa. The tetramer has a mushroom-like shape, where the head is composed of four catalytic domains (Fig. 1A) and the stalk is formed by the extended N-terminal sequences of the four subunits. The stalk amino sequence can vary in length and glycosylation for different subtypes. However, a membrane anchoring helix is conserved for each monomer. In contrast to the stalk region, the head domains – isolated by proteolytic cleavage – are accessible to X-ray crystallography and therefore have been thoroughly investigated within the past 25 years.^17,30,36,37 In NA crystals the subunits assemble around a four-fold axis. The typical β-sheet dominated six-blade propeller fold of each subunit was first observed in 1983 by Varghese et al.³⁶ for N2 and is conserved for other subtypes (N9,^38,39 N4,³³ N6,³³ N8,³³ N1;^33,34,40,41 Influenza B NA^42,43). In the centre of each subunit conserved residues form the catalytic active site (Fig. 1B).^36,37,44

Fig. 1 A) NA as tetramer with the inhibitor DANA (4) (blue) bound to the four active sites. The top right NA chain is coloured according to residue number. Glycosylation is indicated in gold. B) Active site of NA subtype N9 (PDB code 1f8b) with 4 (blue).

2.4 Catalytic mechanism

The proposed catalytic mechanism is derived from the binding mode of the endogenous NA ligand 3 to influenza B NA.⁴² It was further explored by biochemical investigations as well as computational studies on influenza A NA.^45–47 Three conserved arginine residues forming a sub-pocket stabilize the orientation of the carboxylate group of 3. In consequence, they direct the pyranoside ring into a boat conformation. Thus, formation of an oxocarbocation intermediate is favoured by this arrangement. The regeneration of the free 3 by addition of a water molecule results in the β-anomer, which subsequently transforms to its favourable α-anomer by mutarotation.^45,46 A conserved aspartate is supposed to activate the water molecule and orients it in the optimal position. The cationic transition is proven by deuterium-labelled kinetic experiments.⁴⁵ Reduced analogues of 3, DANA (2-deoxy-2,3-didehydro-N-acetylneuraminic acid) (4) and FANA (2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid) (5), show micromolar inhibitory activity.⁴⁸

The conformation of the reduced pyranoside ring is similar to the transition state, and therefore the compounds match the binding pocket without undergoing ring deformation (Fig. 1B).^46,49,50 The binding of 4 and 5 was crucial for elucidation of the catalytic mechanism. Additionally, they were important as lead compounds for the structure-based development of NAIs.^46,47

2.5 Approved NAIs

NAIs show broad spectrum activity against influenza A and B.⁵¹ Commonly reported side effects of these drugs cover nausea, vomiting, dizziness, sinusitis, runny or stuffy nose, cough, diarrhea, and headache.¹

The close correspondence of highly-conserved residues of the active sites of all NAs of influenza A and B viruses renders this enzyme the most promising drug-target for the development of novel lead compounds.^42,52,53 Derived from 3, three NAIs have been brought to the market for the prevention and treatment of influenza virus infection. In 1999, zanamivir (Relenza^®) was approved by the FDA as inhalative NAI. Currently, a Phase III study of intravenous zanamivirversus oral oseltamivir in adults and adolescents hospitalized with influenza is ongoing.⁵⁴

Furthermore, the ethyl ester derivative oseltamivir (Tamiflu^®) was developed for improved bioavailability, making it the first approved orally absorbed NAI.⁵⁵Oseltamivir acts as a prodrug, which is turned into the active carboxylate metabolite by hepatic esterases.⁵⁶ Tamiflu^® was stockpiled for treatment of pandemic flu in many countries.⁵⁷

More recently, several cyclopentane and pyrrolidine derivatives have been developed as novel inhibitors of NA. Among those, peramivir (6) (Rapiacta^®), an intravenously applied NAI, was first launched in Japan for the treatment of hospitalized and pediatric patients. In the U.S., 6 is currently unapproved but was tolerated under a temporary emergency use authorization by the FDA between October 2009 and June 2010 for hospitalized patients with known or suspected H1N1 influenza infection.^58,59 All NAIs need to be administered no later than 36–48 h after manifestation of the symptoms in order to be effective.⁶⁰

Also dimeric and diversely decorated analogues of zanamivir and oseltamivir have been reported. They in part exhibit enhanced efficacy in prolonging antiviral effects in vivo and are superior in inhibiting virus replication.^61–63 Moderate effort has been made so far in designing novel disruptors with a decreased vulnerability toward resistance-conferring mutations, e.g. by selecting residues for ligand anchoring genetically stable due to their contributions to substrate binding.⁶⁴

2.6 Mechanisms conferring drug resistances in influenza

Changes in proteins of approved anti-influenza targets, i.e. the M2 channel protein as well as the NA, can confer resistance and restrict prevention and treatment of influenza. Ion channel blockers, as well as NAI-resistant influenza viruses, can emerge and spread suddenly.^65–70 Based on structural specifics and replication strategies, influenza viruses may evolve by two different ways (i) genetic reassortment, which describes the combination of genome fragments from different virus strains, and (ii) mutations leading to single amino acid exchanges. Reassortment events can be related with pandemic outbreaks in humans, as the immune defence is facing a virus strain with new antigenic characteristics resulting in inadequate immune response. As a result, drug susceptibility can be affected. For example, the H1N1 influenza A strain that emerged 2009 in humans and caused a pandemic showed a new genomic arrangement. While the NA gene and the gene for the matrix protein originate in Eurasian swine strains, the other gene segments have clustered in a previous triple reassortment (human, classical swine, and avian) in a North American porcine lineage of influenza A.⁷¹ The M gene segment from Eurasian swine influenza A viruses conferred resistance to ion channel blockers and the NA gene segment from those viruses enables efficient NAI treatment.¹⁸ In addition to genetic reassortment, influenza viruses show a high genetic variability within the different gene segments due to point mutations introduced by the viral polymerase that lacks a proof reading capacity. In contrast, the active site of the viral NA was shown to be highly conserved. However, spontaneously emerging point mutations within the inhibitor-binding site of NA were shown to confer oseltamivir and/or zanamivir resistance, e.g. mutations of E119, D198, R152, H274, and R292.^51,72 Most of the reported mutations concern active site residues that are in direct contact with the natural substrate upon binding. These residues are highly conserved in the different subtypes and mutations of these residues are often associated with reduced biochemical activity, expression or transmission rates.^73,74 A clinically significant case of an in vivo mutation (R152K) causing zanamivir-resistance in influenza B NA was reported in 1998;⁷⁵ other isolated cases of drug resistance were identified occasionally.⁵¹

Due to the highly conserved structure of the active site of the viral NA the susceptibility towards NAIs in clinical isolates did not change during the first years of their application.^9,17 The situation, however, suddenly changed for the worse when oseltamivir-resistant H1N1 viruses spontaneously emerged and spread globally in the flu season of 2007/2008.^6,9,76Oseltamivir-resistance was related to the H274Y mutation. Initially described in a clinical study of oseltamivir, the mutation remained unnoticed at first.⁷⁷ It occurs at a residue without direct contact with the inhibitor. Results from comparative crystal structure analysis revealed a conformational shift and electrostatic effect induced by the H274Y amino acid exchange.⁷⁸ The bulkier tyrosine induces a conformational shift of E276. The charged carbonic acid group of E276 is directed to a position directly facing the hydrophobic pentyloxy moiety of oseltamivir. While this mutation knocks out the activity of oseltamivir, zanamivir is still active in the mutant due to its hydrophilic glycerol moiety interacting with E276. The world-wide spread of the resistance indicates that strains carrying this mutation show a good transferability. This is in contrast to preliminary studies of the H274Y mutation.^79,80 Bioinformatics analyses of sequence data from extensive surveillance indicate that the circulating oseltamivir-resistant strain does not only show an isolated mutation (H274Y), but is rather accompanied by additional mutations. The point mutation D344N was deemed to be essential for spreading oseltamivir-resistant strains,⁷⁸ while another study showed H274Y strains spreading without D344N mutation.⁸¹ Bloom et al. combined sequence analysis with experimental comparison of NA expression and NA activity. They observed that V234M and R222Q can restore the expression level of a H274Y NA to the wild type level.⁸²

A misbalance in NA/HA activity was also observed as a viral mechanism to overcome NA inhibition in cell culture-based assays. For example, influenza strains treated with NAIs were observed to exhibit mutations in HA e.g. additional glycosylation sites reducing HA affinity to sialic acid. As a result, those strains do not depend on NA activity for virus release and showed no susceptibility to NAIs in MDCK cells.^31,83,84 The relevance of these findings for flu treatment is unknown.

Taken together, the sudden emergence and worldwide spread of NAI-resistant influenza strains may occur despite a highly conserved antiviral target. Based on these prospective findings, identification of novel NAIs breaking resistance is an urgent demand to ensure treatment of future influenza infections.

2.7 Similarities and differences of influenza NA with NA of other biological systems

The enzyme NA is not only specific to viruses but has also been reported as surface protein of other respiratory pathogens, including bacteria e.g. Hemophilus influenzae, Streptococcus pneumoniae, and Pseudomonas aeruginosa.^85–87 These bacterial NAs can cleave α-(2,3)-linked 3 from glycoconjugates.²⁶ Their function in pathogenesis is under discussion.^27–29,87 The crystal structures of S. pneumoniae and P. aeruginosaNA have been reported.⁸⁸ The structures revealed similarities with other bacterial NAs e.g. from Salmonella typhimurium, Vibrio cholerae, and Clostridium perfringens, including their interaction with the NAIs 4 and 5 or even striking differences, respectively.^89–93 Interestingly, some research groups apply in vitro assays with bacterial NA (e.g. from C. perfringens and V. cholerae) to identify anti-viral agents.^94–96 They use commercially available fluorescence (FL) and chemiluminescence (CL) based test systems that work with both viral and bacterial NAs because the substrates used are recognized by viral as well as bacterial NAs.⁹⁷ Unfortunately, data comparing the two test setups, which would be helpful regarding their validity and significance, is scarcely available. Taking this into account, IC₅₀ values obtained by assays using bacterial NA must be regarded with caution if compared with data obtained by assays using viral NA.

In addition to viral and bacterial NAs, there are also mammalian enzymes involved in sialic acid metabolism, including endogenous NA, sialyltransferases, and CMP-synthase. Human NAs (huNAs) can be classified into four groups: intra-lysosomal (NEU1),^98,99 cytosolic (NEU2),^100,101plasma membrane-bound (NEU3),^102,103 and lysosomal or associated with mitochondria (NEU4).¹⁰⁴ In some recent studies the putative inhibitory effects of influenza NAIs on related endogenous enzymes have been evaluated.^105–107 Though their sequence of amino acids is differing, the structural arrangement of the mammalian NAs is similar to the viral enzymes.¹⁰⁸ Inhibition of huNAs is discussed as a reason for side effects of antiviral agents. In vitro studies with established NAIs did not show inhibitory effect on huNAs, which could explain neurological side effects.¹⁰⁵ Nevertheless, inhibitors can be accommodated in the sialic acid binding pocket of huNA and corresponding crystal structures are available. This structural information on huNA can be helpful in the development and structural optimization of novel more specific NAIs.¹⁰⁷ To avoid cross targeting, amino acids without equivalents in huNA should be preferred as interaction reference point for influenza NAIs.

3 Experimental evaluation of NA inhibition

3.1 Methods used in discovery and functional analysis of first generation NAIs

The first synthetic inhibitors of influenza NA were discovered without the structure of the active site and the detailed cleavage mechanism known. However, function and ligand/substrate of this viral enzyme were already determined. This knowledge allowed on the one hand the establishment of first generation NA inhibitory assays with the phenyl-α-ketoside of N-acetylneuraminic acid or fetuein as substrates to measure enzyme activity and to search for inhibitors.^109,110 On the other hand specific synthetic NAIs, 4 and 5 were discovered by using these NA inhibitory assays.^110,111 The NA-specific competitive mechanism of inhibition was demonstrated by measuring the inhibition constant K_i. Cell culture-based assays e.g. virus elution from erythrocytes, plaque size reduction, and comparison of inhibition of single step growth cycle and multicycle replication helped to confirm the antiviral activity of 4 and 5.^48,110–113Electron microscopy showed that virions grown in the presence of 5 form aggregates near the cell surface.¹¹² The observed virus aggregation proved the hypothesis that 5 directly interacts with the influenza NA and by this manner blocks the enzymatic removal of sialic acid from the virus envelope. As a result, sialylated particles are produced containing the receptor sites for HA of other virus particles. Formation of virus aggregates results in reduction of hemaglutination and infectivity titer. Furthermore, results from these fundamental early experiments have indicated differences between NA of subtype N1 and N2 for the first time.¹¹¹ The concentration of 5 required for inhibition of N2 was 50–200 times higher than that for N1 influenza A viruses. This implies structural differences that have to be taken into account in the search for new NAIs.

About 20 years after the discovery of first specific NAIs, the crystal structure of NA of subtype N2 was solved.^114,115 This fuelled the application of computational methods and structure-based design resulting in the development of the highly active N-acetyl-α-D-neuraminic acid (NANA) (8) derivative zanamivir.¹¹⁶ Shortly afterwards, oseltamivir, a second highly active NAI was discovered.⁵⁵ The biological activity of both compounds was demonstrated in NA inhibitory assays and cell culture-based experiments.

Until now, NA-inhibitory assays, cell culture-based, and structure-based design are used to discover and characterize novel NAIs. However, the methods were adapted in part to automated microtests to save time and material, and to avoid subjective evaluation. They were also refined and standardized.

3.2 Advantages and disadvantages of methods currently applied to identify novel NAIs

Today, functional NA inhibition assays are accepted to be the method of choice in NAI susceptibility surveillance.

They are highly standardized, approved in primary screening for novel NAIs for a long time,^{110,111,117–119} and more predictive of in vivo susceptibility than cell culture-based assays.¹²⁰ State of the art functional NA enzyme inhibition assays are either based on fluorescence (FL) or chemiluminescence (CL).^116,121,122 The substrate for FL assays, 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA) (7), is cleaved by NA to release 8and the fluorescent compound 4-methylumbelliferone (MU) (9) (Chart 1A).

Chart 1 A) Fluorescent NA inhibition assay: MUNANA (7) cleaved by NA to give 8 and 4-methylumbelliferone (9). B) Chemiluminescent NA inhibition assay: NA-Star (10) cleaved by NA to give 11 and 12.

The second assay uses the chemiluminescent substrate sodium (2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3.3.1.1^3,7]decan}-4-yl-5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosid)onate (NA-Star) (10) available as component of NA-Star kit (Tropix, Applied Biosystems, Darmstadt, Germany) (Chart 1B). In the latter case the substrate is enzymatically hydrolyzed to give 11and the respective chemiluminescent product 12. The quantity of FL or CL is negatively proportional to the inhibitory activity of the measured sample. Parameters showing influence on the reliability of generated results include buffer, pH, and substrate concentration in the applied test system.

Comparative studies with both functional inhibition assays were performed with zanamivir and/or oseltamivir. Buxton et al. showed that the use of substrate 10 results in a 67-fold reduction in detection limit in comparison with the fluorogenic substrate 7 from 200 pM to 3 pM NA.¹²¹ The IC₅₀ values of zanamivir determined with a panel of influenza A and B viruses were nearly similar in the two NA assays. However, according to two more comprehensive reports on zanamivir and oseltamivir susceptibility of seasonal viruses using standardised commercially kits available now, it was noted that IC₅₀s may vary for the same virus if the NA inhibitory assay was done using substrate 7 and 10.^123,124 In both studies, the CL assay results in 2- to 12-times lower IC₅₀ values than the FL assay. The results from NAI susceptibility surveillance studies with FL- as well as CL-based NA-inhibitory assays indicate that virus type, subtype, and strain as well as the NAIs tested have an influence on 50% inhibitory concentrations.^{69,121,123–125} Based on the proven reproducibility, ease of automation, time required for the assay, and greater sensitivity, the CL assay was selected for NAI susceptibility testing of influenza virus isolates circulation globally. It seems to be also the best choice for antiviral testing.

Cell culture-based assays need to be performed to determine the cytotoxicity of identified NAIs. They can also be applied for confirmation of anti-influenza activity of NAIs but with some limitations. For example, plaque reduction assay that has been traditionally applied for studying the anti-influenza activity of compounds can be used.^{18,111,113,116,126} Although laborious and time consuming, the drug susceptibility interpretation is generally very reliable. However, the experience from NAI susceptibility testing showed that reduction of plaque number does not necessarily reflect the susceptibility of a given viral NA to the respective inhibitor or the antiviral potential of NAIin vivo.^{77,113,116,120,127}Reduction of plaque number was dependent on the cell line and virus strain used.^127,128 Alternatively, plaque size reduction was used as a study parameter because a somewhat better correlation between NA inhibition and antiviral activity was found when the influence of NAIs on plaque size was studied.^{77,84,127,129} Other procedures currently used for the routine antiviral or virus susceptibility screening include different dye uptake assays.^130–132 According to our own experience they work in anti-influenza NAI screening after careful validation under multicycle replication condition. The antiviral activity of NAIs was also confirmed by focus reduction assays^18,133 or analyzing virus yield reduction.^128,129,131 Using a low multiplicity of infections for most but not all influenza A viruses, a good correlation between enzyme inhibition and virus yield reduction can be observed. Results from assays that facilitate studies on prevention of virus spread in infected cell monolayers under multicycle replication conditions can further underline the antiviral activity of this inhibitor class. Such assays include, for instance, immunohistochemical staining of influenza nucleoprotein^84,128 or electron microscopy.^51,112,127 In summary, all these culture-based antiviral assays can help to provide evidence for NAI activity against infectious virus. But, the interpretation of results is complicated by several factors unique to NAIs.¹³⁴ In particular, the validity of results depends on a balanced function of HA and NA. It can be affected negatively by the receptor expression pattern of test cells^133,135 as well as changes in the binding efficiency of HA to sialic acid receptors e.g. by additional glycosylation.^84,136 As a result, viruses become partially independent from NA function for release and can spread from cell-to-cell, also in the presence of NAI. This explains the observed influence of the virus strain and cell line used on the inhibitory effect in all cell culture-based assays. Therefore, a careful evaluation of cell culture-based assays with approved NAIs considering the impact of test virus and cell line is necessary for successful application of those assays in antiviral research.

Today, NA-directed in silico screening and computational docking studies represent a highly useful method to find new hit compounds from available structure collections and to characterize the putative binding mode of NAIs, respectively.^19,137–139 Moreover, knowledge on the dynamics of NA opens new possibilities for the development of next generation NAIs, as summarised below. Potentially active molecules identified or designed by computational approaches can be purchased or synthesised and subsequently evaluated in biological assays. Quantitative structure–activity relationship (QSAR) data gained from experiments can be used to find novel compounds with higher activity and/or compatibility. Increasing knowledge of NAI resistance and mutations enables the consideration of resistant viruses in computational enzyme- as well as cell culture-based approaches. The close interplay between modern computational studies and functional inhibition assays allows for fast discovery and characterization of highly active, resistance-breaking inhibitors of NA.

4 Inhibition of influenza NA by natural compounds

4.1 Evaluation of published data

In the past decade many research teams have demonstrated renewed interest in investigating substances with regard to their NA inhibiting potential. A number of chemically diverse compounds have been identified endowed with this activity. 2634 references (without duplicates) were collected from SciFinder¹⁴⁰ (accessed 2011/05/05) using the research topic “influenza neuraminidase” constrained to the publication years (2000–2010) and the document types journal, letter, review, and patent. This answer set was further refined by either the keyword “natural product” (33 ref.) or “compound” (269 ref.). Each category was then analyzed in respect to the frequency between the years 2000 and 2010. Fig. 2 reveals that beside the overall upward trend, the number of literature reports dealing with natural products targeting influenza NA has tremendously increased since 2009.

Fig. 2 Comparison of number of publications dealing with influenza NA related to the keywords “natural product” (blue) and “compound” (grey) between the years 2000 and 2010.

This tendency might be correlated with the emergence of the H1N1 flu pandemic in 2009, which underlines the urgent need for novel antiviral therapeutics.

In the present survey natural products with publicly accessible information concerning their NA inhibiting potential are reviewed. For this purpose SciFinder¹⁴⁰ (accessed 2011/01/18) was used to explore the research topic “influenza neuraminidase”. Search results were constrained to the publication years 2000 till present, and to the document types journal, letter, review, and patent (2761 ref. without duplicates). For further analysis, this set was organized by the subject category “organisms” (1639 ref.). Plant species or keywords related to natural products were then selected manually (21 ref.). In an additional query approach, not to neglect insufficiently tagged literature, further research topics such as “influenza sialidase”, “influenza neuraminidase inhibitor”, and “influenza sialidase inhibitor” were explored. Furthermore, primarily obtained search results were refined by using chemical structure classes as keywords. In a final step, false positive records, such as IC₅₀ values obtained by the use of bacterial instead of viral NA assays, were sorted out.

For a clear presentation of the pool of information gained, an in-depth evaluation was performed considering only reported results of NA inhibition assays of pure compounds. This procedure was accompanied by difficulties, because not all research groups use the same viral strain or isolate, assay type, positive control or concentration unit. Table 1 provides a compilation of essential information on natural compounds tested for NA inhibition. Comprised data is ordered alphabetically by compound structure class and aims at providing insight not only into influenza NA inhibiting compounds and applied assay types, but also the distribution of NA-inhibiting secondary metabolites between plant species and families, respectively. In this context it needs to be emphasized that apart from natural compounds with significant NA inhibiting effects, also those showing only weak or no activity on the respective target have been taken into account. We decided to include reported negative test results due to their potential value for future work, viz. (i) to avoid a retesting of inactive compounds, (ii) to validate hypotheses (e.g. computational models), and (iii) to enable the establishment of SARs.

For the sake of clarity, a set of three distinct influenza viral subtypes, i.e.H1N1, H3N2, and H9N2, were selected to provide an overview on reported NA inhibiting activities, without going further into detail by specifying the viral strain and isolate, respectively. In case recombinant H1N1 (rvH1N1) has been used, the corresponding IC₅₀ values are marked with the letter b (^b) in Table 1. If compounds have been tested on several strains or isolates of one viral subtype, an activity range is given. For standardization reasons, IC₅₀ values originally published in μg mL⁻¹ or ng mL⁻¹ have been converted to μM or nM and are marked with the letter a (^a). References to primary literature are provided.

4.2 Compound classes and sources of natural NAIs

Published data from NAIs from synthetic chemistry and natural sources have roughly been classified by Zhang et al. in 2010.¹⁴¹ The survey of our literature search focusing on natural products (Table 1) allowed categorizing influenza NA inhibiting natural compounds in detail. Due to already mentioned difficulties, i.e. heterogeneous test systems, a threshold between effective and ineffective NAIs needed to be drawn. In this review, compounds with IC₅₀ values >100 μM obtained by FL assays and >50 μM obtained by CL assays were considered inactive. Fig. 3 provides the distribution by percentage of chemical classes of reported NA inhibiting natural compounds. Among the most prominent scaffolds are flavonoids and (oligo)stilbenes. Other chemical classes investigated for their potential on influenza NA are not present in this diagram, e.g.monoterpenes and triterpenes, because they could not reach the set threshold.

Fig. 3 Percentage of natural product compound classes showing inhibition of influenza NA (publication years: 2000 until present).

4.3 Phenolic compounds as NAIs

Natural product research dealing with influenza NAIs focuses mainly on the biological activity of substituted phenyl-benzopyranes, so called flavonoids, which are widespread in the plant kingdom. In particular, species belonging to the Fabaceae family have been intensively studied.^142–145Flavonoid constituents are categorized into ten major sub-groups, i.e.aurones, biflavonoids, catechins, chalcones, flavanones, flavanonols, flavans, flavones, flavonoles, and isoflavones.

In the context of druglikeness or druggability, flavonoids represent an interesting compound class of low-molecular weight chemical entities accompanied with mostly well-know bioavailability and innocuousness.¹⁴⁶ Various research groups working with these polyphenolic compounds studied their SARs, allowing the establishment of an NA activity order according to the involved functional groups and scaffolds. Liu et al. investigated 25 commonly occurring flavonoids and record the following order of NA inhibiting potency: aurones > flavon(ol)es > isoflavones > flavanon(ol)es and flavan(ol)es.¹⁴² Efforts to categorize NA inhibitory activity measured by a standard FL assay resulted in the classification of three groups: (i) good (IC₅₀ < 40 μM), (ii) moderate (IC₅₀ 40–80 μM), and (iii) weak (IC₅₀ > 80 μM) inhibitors.¹⁴² Thus, essential functional groups for good NA inhibiting effects of flavonoids are hydroxyl groups in position 4′ and 7, an oxo group in position 4, and a double bond between position 2 and 3. Concerning the aurone subtypes, essential functionalities are a hydroxyl group in position 6, an oxo group in position 3, and a double bond between position 2 and the phenylidene moiety.¹⁴² The presence of a bulky sugar moiety distinctly reduces the NA inhibitory potential due to an assumed sterical hindrance.¹⁴² Recently, these findings were incorporated in a theoretical computational approach by Mercader et al. to predict NA inhibiting activity based on quantitative structure–activity relationships (QSAR) exploiting already reported activities of flavonoids including relevant 2D and 3D descriptors.¹⁴⁷

Although Liu and co-workers ranked substances belonging to the structure class of aurones (IC₅₀ between 22.0 and 72.0 μM; H1N1) as most potent NAIs among flavonoid derivatives,¹⁴² no further studies on the NA inhibiting properties of these compounds have been published to the knowledge of the authors.

Biflavonoids like ginkgetin (13) with an IC₅₀ of 97.09 μM (H1N1), isolated from Ginkgo biloba and Cephalotaxus harringtonia, hinokiflavone (14) (IC₅₀ 77.63 μM; H1N1), and 4′-O-methylochnaflavone (15) (IC₅₀ 2.01–3.54 μM; H1N1) have successfully been identified as potential NAIs.^19,147,148

Catechin (16) and its derivatives epicatechin (17) and gallocatechin (18) were found to exert no or only weak NA inhibition.^142,149

Fourteen C-methylated flavonoids (chalcones, flavanones, isoflavones, and one flavanonol) from Cleistocalyx operculatus were found to have NA inhibiting properties, with (E)-4,2′,4′-trihydroxy-6′-methoxy-3′,5′-dimethylchalcone (19) (IC₅₀ 3.31–20.45 μM) and 2,2′,4′-trihydroxy-6′-methoxy-3′,5′-dimethylchalcone (20) (IC₅₀ 2.55–28.12 μM) showing the strongest effects against NA from the novel influenza H1N1 (WT) and oseltamivir-resistant H1N1 (H274Y mutant), respectively.¹⁵⁰

Ryu et al. reported the isolation and NA inhibition evaluation of 18 polyphenols from the methanol extract of the roots of Glycyrrhiza uralensis.¹⁴³ The investigated compounds include chalcones, (prenylated) flavonoids, (prenylated) coumarins and one phenylbenzofuran. Among the chalcones, isoliquiritigenin (21) exhibited the most potent inhibitory activity, with an IC₅₀ value of 9.0 μM towards recombinant influenza virus A (rvH1N1).^143,151 Most favourable effects against rvH1N1 were demonstrated for the coumestan glycyrol (22) (IC₅₀ 3.1 μM), which is characterized by a condensed furan ring in contrast to classical coumarins lacking this feature. In addition, the presence of a free hydroxyl group in position 7 and an isoprene residue in position 6 seem to be essential functionalities for these scaffolds.¹⁴³

Flavones, such as apigenin (23), apiin (24), and luteolin (25), have also proven significant activities against influenza NA with IC₅₀ values between 25.92 and 33.71 μM (H1N1).¹⁵² Jeong and co-workers investigated five flavonols isolated from the roots of Rhodiola rosea and determined their NA inhibiting properties.¹⁵³ For in-depth SARs they compared the activities to a further nine available flavonoids. Regarding the inhibitory effects towards rvH1N1 quercetin (26), gossypetin (27), and herbacetin (28) exhibited the most promising activities (IC₅₀ 2.2, 2.6, and 8.8 μM).

Within the sub-group of reviewed isoflavones, only daidzein (29) and genistein (30) displayed mentionable inhibitory activities against NA with IC₅₀ values of 37.1 and 77.1, respectively (H1N1).¹⁴²

Recently, (oligo)stilbenes isolated from Vitis amurensis and Gnetum pendulum were reported to exert anti-viral activities by targeting influenza NA. Among the most potent constituents were (+)-viniferol C (31) (IC₅₀ 8.94–61.16 μM; H1N1) and gnetupendin B (32) (IC₅₀ 13.14 μM; H1N1).^154,155

In addition, various compounds not belonging to the mentioned chemical classes or sub-groups have been investigated on their NA-inhibiting properties. One example is represented by vomifoliol (33), a 2-cyclohexen-1-one derivative, isolated from Chaenomeles speciosa showing an IC₅₀ of 10.39 μM (H1N1).¹⁵⁶

4.4 Isoprenoids – a compound class showing no effect on NA

As discussed before, reports on compounds with experimentally determined inactivity on influenza NA have also been regarded as valuable input for future research efforts. Three monoterpenes from the essential oil of Melaleuca alternifolia (Myrtaceae) and five triterpenes occurring in Chaenomeles speciosa (Rosaceae) were evaluated for their inhibitory potential on NA. All tested compounds of this structural class did not exhibit any inhibitory activity on NA.^156,157

4.5 Extracts with promising NA inhibitory activity

Publications dealing with promising NA inhibiting plant extracts have appeared rather infrequently during the last decade. The majority of research groups seem to preferentially elucidate bioactivity and mechanism of action of isolated pure compounds instead of extracts, allowing for publication in higher-ranked journals. Another aspect might be that extracts representing multi-component mixtures are rather tested in general antiviral assays, such as viral replication tests, than in more specific assays directly evaluating the NA activity. Nevertheless, eastern medicines, such as, especially, the traditional Chinese medicine (TCM), describe well-defined anti-influenza preparations composed of one or more herbs instead of individual compounds. In most cases the active principle(s) and mechanism of action is unknown or not fully understood. A review published in 2006 by Wang et al. deals in detail with TCM-derived anti-influenza agents from plants.¹⁵⁸ This issue was also the subject of a recently published review by Geet al. describing the importance of four TCM anti-flu prescriptions in the fight against the 2009 H1N1 pandemic in China.¹⁵⁹ In a very recent study, water extracts from 439 traditional Chinese medicinal plants were investigated for their NA inhibiting activities in vitro. Among the five extracts analyzed in vivo using a mouse model, the extract of Melia toosendan significantly increased the survival rate of H1N1 viral infected mice.¹⁶⁰

5 Strategies for the discovery of NA inhibiting natural compounds

Researchers in the field of phytochemistry are faced with a nearly unmanageable quantity of structurally diverse natural products as only the estimated number of plant species on earth is about 400 000.¹⁶¹ Given such issues the search for novel innovative natural leads resembles looking for a needle in a haystack. In the very recent analysis of Newman et al., approved drugs for all kind of diseases were statistically evaluated in respect to what structures led to the design of the compounds subsequently approved and what origins they have.¹⁶² In the case of antiviral agents, 80% of 46 entities registered in the last 29 years (1981–2010), can be classified as natural product-botanicals, synthetic but natural product mimics, natural product pharmacophores, or a combination of the latter two.¹⁶²Oseltamivir is one of the success stories of anti-influenza drug synthesis having its roots in nature: the abundant plant constituents, quinic acid (34) and shikimic acid (35), are used as starting materials for the development of oseltamivir.⁵⁵ Both natural compounds are intermediates in the biosynthesis of essential aromatic amino acids and also precursors to lignans and phenols.^163,164

They accumulate to some extent, primarily in gymnosperms and woody dicotyledons. The most important source of 35 has been found in the genus Illicium, where it was first isolated from I. religiosum in 1885.¹⁶⁵ Star anise (I. verum) turned out as very rich source of 35, and was accordingly used in large amounts to yield this much valued precursor for the semi-synthesis of oseltamivir.¹⁶⁶

In order to rationalize the access to novel natural NA inhibiting leads, which otherwise would be dependent on serendipity, three different strategies have been applied so far: (i) in vitro screening of natural compounds or extracts followed by bioassay-guided fractionation and isolation, (ii) strategies based on the exploitation of empirical knowledge, and (iii) computational approaches. Also the combination of heuristic approaches (e.g. ligand or structure based) with empirical ones might be favourable.

5.1 In vitro screening of extracts–Bioassay-guided fractionation and isolation

Random selection of natural material followed by an unbiased in vitro screening allows first activity categorizations of multi-component mixtures.^167,168 Although the identity of the individual compounds responsible for the measured effect cannot be elucidated in this way, promising natural starting material can be selected for further processing. Extracts displaying desirable activities are in most cases subjected to a bioassay-guided fractionation with the goal to identify the active principle(s). In the course of this process the individual bioactive components responsible for the given effects can ideally be traced down to a molecular level, allowing structure elucidation.¹⁶⁸ This strategy represents one of the classical lead finding methods especially applied by academic research groups. At a first glance this approach is more straightforward than laborious isolation and structure elucidation of single components without knowledge of proven bioactivity of the mother sample. However, for this strategy experience is of pivotal importance to avoid pitfalls, e.g. assay-disturbing constituents.

A respective screening strategy was applied by Lee and co-workers who investigated crude extracts from 260 plant species on their inhibiting potential towards NA of C. perfringens.⁹⁶ These results provide highly valuable data for further selection and processing of promising plant material e.g. in the course of bioassay-guided fractionations.

Many other research groups successfully identified novel NAIs based on such large scale anti-influenza screening campaigns; e.g. Dao et al. discovered 14 bioactive C-methylated flavonoids (Table 1) from Cleistocalix operculatus buds by using an anti-influenza screening approach.¹⁵⁰

In the course of a project by the Chinese Academy of Medical Sciences more than 10 000 plants were tested.¹⁵² Among them a pronounced NA inhibiting effect was observed for the herb extract of Elsholtzia rugulosa, which prompted Liu et al. to further investigate this plant and isolate constituents responsible for its activity, e.g.23 (H3N2; IC₅₀ 28.90 μM) and 25 (H3N2; IC₅₀ 32.63 μM) (Table 1).¹⁵²

5.2 Exploitation of empirical knowledge

This procedure takes advantage of ethnopharmacological hints about beneficial anti-influenza effects, mainly of herbal remedies as well as reports on anti-influenza or anti-viral multi-component mixtures or even individual compounds of not yet identified mechanism of action.

Concerning toxicity and bioavailability, naturally derived remedies and their ingredients used by humans for decades, centuries or even millennia are supposed to be safer and more efficacious than substances missing this background.¹⁶⁷ In particular, preparations with ethnomedical background might be favourable compared to synthetic compounds evoking from combinatorial chemistry.¹⁶⁷ Natural compounds occupy an even larger and more complex chemical space compared to synthetic compounds.¹⁶⁹

Some extracts (e.g. from Agrimonia pilosa,¹⁷⁰Echinacea purpurea,¹⁷¹ and Prunus mume¹⁷²) or multi-component mixtures (e.g.polyphenol fractions from Punica granatum¹⁷³ and secoiridoid glucosides from Ligustrum lucidum¹⁷⁴) have already shown a significant reduction of virus-induced cytopathic effects and general anti-viral or anti-influenza activity. These phenomenological effects often observed in folk medicine give valuable hints for tracing activity on a target specific level exploring a possible impact on influenza NA. Concerning viral infections, a great number of preparations containing natural materials have shown bioactivity with often complementary and overlapping antiviral mechanisms of action.^24,25 In most cases therapy with these simply prepared multi-component mixtures of complex chemical composition is intrinsically tied to traditional medicines, i.e.TCM, Ayurveda or indigenous medicines.¹⁶⁷ Many of the contained secondary metabolites, possibly active principles, have never been examined chemically or biochemically using modern medical knowledge. Phytochemical and pharmacological work performed with ethnomedical anti-viral agents mainly from plants revealed a variety of active metabolites from different chemical classes, e.g.stilbenoids from Gnetum pendulum,¹⁵⁵C-methylated flavonoids from Cleistocalix operculatus,¹⁵⁰ and polyphenols from Glycyrrhiza uralensis.¹⁴³

Successful identification of novel bioactive leads from the follow-up of phenomenological effects or ethnopharmacological hints is based on the selection of the correct starting material, its medicinal preparation, the kind of application, and the pharmacological profile.¹⁷⁵ This strategy, however, is not a guarantee to discover constituents acting on the supposed target. Reported positive effects in flu treatment, regardless if obtained from clinical trials or empirical knowledge handed down to the present, can only give clues for further research efforts. One might not know if therapy is successful due to NA inhibition or if improvement of health conditions is simply triggered by a stimulation of the immune system. Furthermore, other anti-influenza targets, e.g. the non-structural NS1A protein, the nucleoprotein, and the viral polymerase should not be disregarded.¹⁷⁶

5.3 Exploiting influenza NA inhibition by computer-guided approaches

Computational strategies to identify novel NAIs can be classified in ligand-based, structure-based or combined approaches, according to the data and methods employed.¹⁷⁷ Given the accessibility of influenza NA to X-ray crystallography, structure-based methods have been playing a major role in the development of NAIs.¹⁷⁸Zanamivir, the first approved inhibitor of influenza NA resulted from a highly successful optimization campaign during which affinity to the target was increased by 5000-fold, driven by computational analyses using the computer software GRID.¹⁷⁹

Molecular mechanics (MM) calculations based on demanding molecular dynamics (MD) simulations were applied to estimate the binding energy of potential optimisations during the development of zanamivir.⁴⁶ However, ligand-based methods have also been employed and successfully identified, e.g., NSC 404789 (36), a uronic acid derivative as an inhibitor of influenza NA (IC₅₀ 1.8 μM).¹⁸⁰

Protein–ligand docking is one of the most popular structure-based in silico methods. It is particularly useful for identifying bioactive compounds and for deriving the potential binding mode of small organic molecules to their target. Using a docking-based virtual screening approach, NSC 89853 (37) was recently identified as a bulky NAI for N1. Docking studies suggest that this compound requires an open-form binding site to be accommodated.¹⁸¹

Employing MD simulations of influenza NA, Amaro et al. observed extended conformational shifts of the 150- and the 430-loop.^182,183,184 It appears that the binding site of influenza NA can open to a much larger extent than anticipated from prior X-ray structure analyses, which can be systematically explored for the development of more potent, chemically diverse inhibitors. In this context, computational analyses of multiple target conformations of H5N1 NA revealed novel protein hot spots proximate to the sialic acid binding region that could serve as potent anchor points for small organic inhibitors.¹⁸⁵

In general, docking programs consider the target structure to be rigid. Some algorithms allow including a certain degree of target flexibility during the docking process; however, far-reaching conformational arrangements are not sampled by most of the conventional docking algorithms. Ensemble-based docking has turned out to be a particularly powerful approach to address highly flexible targets in virtual screening. Thereby, a collection of representative protein conformations (as observed during the MD simulations or in X-ray crystallographic experiments) is generated and, subsequently, virtual screening is performed on each of these structures. Promising candidate molecules are then selected from all individual hit lists in order to cover a large proportion of the biomolecule's phase space. In the case of influenza NA, it has become increasingly popular to sample target flexibility employing MD simulations to generate a number of representative protein conformations (conformational ensemble) for docking in order to obtain superior performance.¹⁸⁶

Inspired by the observations of a highly flexible 150-loop, which has been initially observed in X-ray structures, several novel scaffolds for inhibitors targeting extended conformations of the influenza NA have previously been reported.¹³⁷ The fact that the loop region was found to be more flexible in the apo form of group-1 compared to group-2 NAs, renders it an interesting structural characteristic that could be exploited for rational design of novel NAIs.³³ Rudrawar et al. reported the first experimental observation of derivatives of 3 stabilising the 150-loop of group-1 influenza NA in its open conformation,¹⁸⁷ a proof of concept for the rational design of NAIs selectively targeting group-1 NAs.

Among novel scaffolds that were identified by taking these aspects into account, initial in vitro testing confirmed the predicted activity of several of them, e.g. NSC 117079 (38).¹⁸⁸NA conformations derived from MD simulations were also used to deduce analogues of zanamivir and oseltamivir with predicted superior activities.¹⁸⁹

Recently, we have identified novel lead structures of NAIs from plants.¹¹⁸ Based on promising literature data on the inhibiting potential of a methanol extract of Alpinia katsumadai (Zingiberaceae) towards NA from C. perfringens,⁹⁶ the seed extract of this plant was phytochemically investigated to track down the active principle(s). Six constituents were isolated, with four diarylheptanoids showing in vitro influenza NA inhibitory activity in a CL based enzyme inhibition assay within the low micromolar range against human influenza virus A/PR/8/34 of subtype H1N1. Katsumadain A (39) (IC₅₀ 1.05 ± 0.42 μM) was identified as the most promising compound (Table 1). This bulky diarylheptanoid, characterized by a monocyclic α-pyrone moiety and an additional phenyl group also inhibited the NA of four H1N1 swine influenza viruses with IC₅₀ values between 0.59 and 1.64 μM, whereas the NA of an oseltamivir-resistant human H1N1 isolate was only weakly affected.

In addition to 39, four active compounds (40–43) comprising simple diarylheptanoid skeletons with two peripheral phenyl groups connected via a heptyl chain were used for SAR analyses.

The potential binding mode of 39 was resolved using protein–ligand docking. 39 is unlikely to bind to influenza NA in a target conformation observed in X-ray experiments due to its large molecular volume. MD simulations were performed to derive conformations of influenza NA. A representative frame of a well-populated cluster of the MD trajectory could be identified that allowed the establishment of favourable interaction patterns between the ligand and the protein. Overall a very good surface complementarity was observed. In particular, the extended conformation of the 430- and the 245-loop appeared to be essential for the accommodation of the ligand.

The proposed binding mode of 39 was taken as a starting point for shape-focused screening to identify novel active molecules from the NCI database (Developmental Therapeutics Program, DTP, National Cancer Institute, U.S.) and all five selected compounds showed activities in the very low micromolar range.¹⁹ Four of them, i.e.quercetin 5,7,3′,4′-tetramethyl ether (44), artocarpin (45), 15, and 1-(5-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)-2-phenyl-ethanone (46) are flavonoid derivatives (Table 1). 45, the most active compound identified, inhibits oseltamivir-sensitive H1N1 strains with an IC₅₀ range of 0.18–0.30 μM.¹⁹ Interestingly, this compound also exhibits significant activity on an oseltamivir-resistant H1N1 strain (IC₅₀ 0.55 μM).¹⁹Fig. 4 depicts a docking pose of 45 and the suggested binding interactions with amino acid residues from the NA protein backbone.

Fig. 4 Artocarpin (45) (carbons lavender, oxygens red) docked to a representative frame of the MD simulation of influenza NA. The docking pose suggests the formation of hydrogen bonding interactions with several Arg and Glu residues present in the binding site. Hydrophobic contacts are formed with Arg224 and Tyr406.

6 Conclusions and future perspectives

The threat of influenza pandemics claiming utterly devastating death tolls and the small number of effective drugs to fight this pathogen, including the emergence of resistances, are the main driving forces behind the search for innovative NAIs.

Within the past three years, scientific communities all over the world have shown renewed interest in the search for novel anti-viral agents from plant origin. Natural compounds are widely recognized as privileged structures, trimmed by evolutionary processes to interact with macromolecular targets. However, working with them requires expertise and might be paved with challenges, viz. legal access to correctly identified starting material, analytics, isolation from multi-component mixtures (extracts), purification, structure determination and chemical complexity, just to name some aspects. Natural product research is accordingly highly multifaceted and interdisciplinary, and asks for target-oriented strategies exploiting their bioactivities. This might be of relevance for outsourcing (parts of) lead discovery campaigns focusing on natural products from industry to academic groups, as happened in the past decades. Concerning antiviral research and in particular anti-influenza agents, input from ethnopharmacological knowhow and phenomenological proofs about antiviral effects are highly valuable hints for the selection of promising natural materials. In combination with heuristic approaches based on previous structural knowledge about the target's binding site and its ligands, optimal starting points are available to further rationalize the efficient search for improved NAIs. This survey aims at bundling these data by summarising the recently reported discoveries on influenza NA and the mechanism of resistance to common NAIs together with available information on natural products tested for their influenza NA-inhibiting potential (∼150 compounds). It is anticipated that the recent findings of the enzyme flexibility and thereby extended binding pocket of the influenza NA, as well as the progress in computational methods exploiting these findings will fuel natural product research activities for effective and innovative drugs to fight influenza.

Table 1 Natural compounds with measured NA inhibitory potential available in the public domain (activity of positive controls is given in brackets)^cdefg

Compound name	No.	Natural source	Family	NA inhibition IC50 [μM]			Assay type	Ref.
				H1N1	H3N2	H9N2
a For clarity purposes IC50 values originally published in μg mL^−1 or ng mL^−1 have been converted to μM or nM, respectively. b rvH1N1 = recombinant H1N1. c n. g.: not given. d Os: oseltamivir. e Os ac: oseltamivir acid. f Neu5Ac2en: 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. g MA: 8-methoxyapigenin.
Coumarins
glycyrin	47	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	10% at 200^b μM	n. g.	n. g.	FL	143
				(Os: 1.59^b nM)
licopyranocoumarin	48	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	10% at 200^b μM	n. g.	n. g.	FL	143
				(Os: 1.59^b nM)
Coumestanes
glycyrol	22	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	3.1^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
isoglycyrol	49	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	92.4^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
Diarylheptanoids
(3S)-1,7-diphenyl-(6E)-6-hepten-3-ol	40	Alpinia katsumadai Hayata	Zingiberaceae	4.13 (Os: 0.13 nM)	n. g.	n. g.	CL	118
(3S,5S)-trans-3,5-dihydroxy-1,7-diphenyl-1-heptene	41	Alpinia katsumadai Hayata	Zingiberaceae	29.75 (Os: 0.13 nM)	n. g.	n. g.	CL	118
(E,E)-5-hydroxy-1,7-diphenyl-4,6-heptadien-3-one	42	Alpinia katsumadai Hayata	Zingiberaceae	4.67 (Os: 0.13 nM)	n. g.	n. g.	CL	118
alnustone	43	Alpinia katsumadai Hayata	Zingiberaceae	6.10 (Os: 0.13 nM)	n. g.	n. g.	CL	118
katsumadain A	39	Alpinia katsumadai Hayata	Zingiberaceae	0.59–1.05 (Os: 0.06–0.13 nM)	n. g.	n. g.	CL	118
Flavonoids and related structure types
Aurones
(2E)-4,6-dihydroxy-2-[(4-hydroxyphenyl) methylene]-3(2H)-benzofuranone	50	n. g.	n. g.	25.6 (Os ac: 15 nM)	22.3 (Os ac: 3.2 nM)	n. g.	FL	142,147
(2E)-6-hydroxy-2-(phenylmethylene)-3(2H)-benzofuranone	51	n. g.	n. g.	72.0 (Os ac: 15 nM)	73.3 (Os ac: 3.2 nM)	n. g.	FL	142,147
(2E)-6-hydroxy-2-[(4-hydroxyphenyl)methylene]-3(2H)-benzofuranone	52	n. g.	n. g.	22.0 (Os ac: 15 nM)	22.1 (Os ac: 3.2 nM)	n. g.	FL	142,147
sulfuretin	53	n. g.	n. g.	29.6 (Os ac: 15 nM)	27.7 (Os ac: 3.2 nM)	n. g.	FL	142,147
Biflavonoids
4′-O-methylochnaflavone	15	Ochna sp.; Lonicera japonica Thunb.	Ochnaceae; Caprifoliaceae	2.01–3.54 (Os: 0.12–0.23 nM)	n. g.	n. g.	CL	19
ginkgetin	13	Ginkgo biloba L.; Cephalotaxus harringtonia Knight ex J.Forbes	Ginkgoaceae; Cephalotaxaceae	97.09^a (MA: 32.57^a μM)	17.26^a (MA: 29.81^a μM)	n. g.	n. g.	147,148
hinokiflavone	14	n. g.	n. g.	77.63^a (Os: 1.66 nM)	n. g.	n. g.	n. g.	147
Catechins
catechin	16	n. g.	n. g.	>100 (Os ac: 15 nM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142
epicatechin	17	Gouania obtusifolia Vent. ex Brongn.	Rhamnaceae	>100 (Os ac: 15 nM); 5.49 (Neu5Ac2en: 44.47 μM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142,149
gallocatechin	18	Gouania obtusifolia Vent. ex Brongn.	Rhamnaceae	10.22 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
Chalcones
(E)-4,2′,4′-trihydroxy-6′-methoxy-3′,5′-dimethylchalcone	19	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	3.31–20.45 (Os: 12.29–95.22 nM)	n. g.	18.57 (Os: 10.34 nM)	FL	150
2,2′,4′-trihydroxy-6′-methoxy-3′,5′-dimethylchalcone	20	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	2.55–28.12 (Os: 12.29–95.22 nM)	n. g.	23.12 (Os: 10.34 nM)	FL	150
2′,4′-dihydroxy-3′-methyl-6′-methoxychalcone	54	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	26.02–93.77 (Os: 12.29–95.22 nM)	n. g.	66.48 (Os: 10.34 nM)	FL	150
2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone	55	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	5.03–35.23 (Os: 12.29–95.22 nM)	n. g.	21.74 (Os: 10.34 nM)	FL	150
2′-methoxyisoliquiritigenin	56	Glycyrrhiza uralensis Fisch. ex DC.; Caesalpinia sappan L.	Fabaceae	24.3^b (Os: 1.59^b nM); 54.02^a (Os ac: 3.52^a nM)	64.38^a (Os ac: 1.65^a nM)	n. g.	FL	143,144
5-prenylbutein	57	Glycyrrhiza inflata Batalin; Erythrina addisoniae Hutch. & Dalziel	Fabaceae	76.01^a (Os: 16.42^a - 127.21^a nM); 64.43^a (Os: 127.21^a nM)	n. g.	60.14^a (Os: 15.81^a nM); 104.30^a (Os: 15.81^a nM)	FL	145,151
abyssinone VI	58	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	70.23^a (Os: 127.21^a nM)	n. g.	65.23^a (Os: 15.81^a nM)	FL	145
echinatin	59	Glycyrrhiza inflata Batalin	Fabaceae	8.18^a - 21.46^a (Os: 16.42^a - 127.21^a nM)	n. g.	21.09^a (Os: 15.81^a nM)	FL
isoliquiritigenin	21	Glycyrrhiza inflata Batalin; Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	13.35^a - 32.82^a (Os: 16.42^a - 127.21^a nM); 9.0^b (Os: 1.59^b nM)	n. g.	37.81^a (Os: 15.81^a nM)	FL	143,151
isoliquiritin	60	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	124.0^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
isoliquiritin apioside	61	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	12.9^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
kanzonol C	62	Glycyrrhiza inflata Batalin	Fabaceae	192.06^a (Os: 16.42^a - 127.21^a nM)	n. g.	134.93^a (Os: 15.81^a nM)	FL
licoagrochalcone A	63	Glycyrrhiza inflata Batalin; Erythrina addisoniae Hutch. & Dalziel	Fabaceae	159.05^a (Os: 16.42^a - 127.21^a nM); 66.31^a (Os: 127.21^a nM)	n. g.	175.48^a (Os: 15.81^a nM); 61.75^a (Os: 15.81^a nM)	FL	145,151
licochalcone A	64	Glycyrrhiza inflata Batalin	Fabaceae	12.41^a - 56.41^a (Os: 16.42^a - 127.21^a nM)	n. g.	53.13^a (Os: 15.81^a nM)	FL
licochalcone D	65	Glycyrrhiza inflata Batalin	Fabaceae	80.76^a (Os: 16.42^a - 127.21^a nM)	n. g.	99.35^a (Os: 15.81^a nM)	FL
licochalcone F	66	Glycyrrhiza inflata Batalin	Fabaceae	106.32^a (Os: 16.42^a - 127.21^a nM)	n. g.	118.82^a (Os: 15.81^a nM)	FL
sappanchalcone	67	Caesalpinia sappan L.	Fabaceae	49.95^a (Os ac: 3.52^a nM)	74.05^a (Os ac: 1.65^a nM)	n. g.	FL	144
Flavanones
(2S)-2,7-dihydroxy-5-methoxy-6,8-dimethylflavanone	68	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	282.58 (Os: 95.22 nM)	n. g.	284.78 (Os: 10.34 nM)	FL	150
(2S)-5,7-dihydroxy-6,8-dimethylflavanone	69	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	333.59 (Os: 95.22 nM)	n. g.	319.12 (Os: 10.34 nM)	FL	150
(2S)-5-hydroxy-7-methoxy-6,8-dimethylflavanone	70	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	>500 (Os: 95.22 nM)	n. g.	>500 (Os: 10.34 nM)	FL	150
(2S)-6-formyl-8-methyl-7-O-methylpinocembrin	71	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	268.59 (Os: 95.22 nM)	n. g.	256.25 (Os: 10.34 nM)	FL	150
(2S)-7-hydroxy-5-methoxy-8-methylflavanone	72	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	>500 (Os: 95.22 nM)	n. g.	>500 (Os: 10.34 nM)	FL	150
(2S)-8-formyl-5-hydroxy-7-methoxy-6-methylflavanone	73	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	289.94 (Os: 95.22 nM)	n. g.	298.91 (Os: 10.34 nM)	FL	150
(2S)-8-methylpinocembrin	74	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	>500 (Os: 95.22 nM)	n. g.	>500 (Os: 10.34 nM)	FL	150
(S)-naringenin	75	n. g.	n. g.	>100 (Os ac: 15 nM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142
hesperidin	76	n. g.	n. g.	>100 (Os ac: 15 nM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142
liquiritigenin	77	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	46.8^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
liquiritin	78	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	82.3^b (Os: 1.59^b nM); >100 (Os ac: 15 nM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142,143
liquiritin apioside	79	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	18.2^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
Flavanonoles
(2S,3S)-2,3-trans-5,7-dihydroxy-6,8-dimethyldihydroflavonol	80	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	374.67 (Os: 95.22 nM)	n. g.	367.87 (Os: 10.34 nM)	FL	150
engeletin	81	Gouania obtusifolia Vent. ex Brongn.	Rhamnaceae	5.44 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
Flavans
7,4′-di-O-galloyltricetinflavan	82	n. g.	n. g.	49.14^a (Os: 1.66 nM)	n. g.	n. g.	FL	147
7-O-galloyltricetinflavan	83	n. g.	n. g.	35.49^a (Os: 1.66 nM)	n. g.	n. g.	FL	147
Flavones
8-methoxyapigenin	84	n. g.	n. g.	43.80^a (Os: 1.66 nM)	n. g.	n. g.	FL	147
apigenin	23	Elsholtzia rugulosa Hemsl.	Lamiaceae	31.63^a (Os ac: 16.53^a nM); 33.4^b(Os: 1.59^b nM)	28.90^a (Os ac: 3.52^a nM)	n. g.	FL	142,147,152,153
apiin	24	Elsholtzia rugulosa Hemsl.	Lamiaceae	25.92^a (Os ac: 16.53^a nM)	44.89^a (Os ac: 3.52^a nM)	n. g.	FL	152
artocarpin	45	Artocarpus heterophyllus Lam. & other A. species	Moraceae	0.18–0.30 (Os: 0.12–0.23 nM)	n. g.	n. g.	CL	19
cosmosiin	85	n. g.	n. g.	46.9^b (Os: 1.59^b nM)	n. g.	n. g.		153
chrysin	86	n. g.	n. g.	45.7 (Os ac: 15 nM)	33.36 (Os ac: 3.2 nM)	n. g.	FL	142,147
dinatin	87	n. g.	n. g.	46.3 (Os ac: 15 nM)	26.0 (Os ac: 3.2 nM)	n. g.	FL	142,147
galuteolin	88	Elsholtzia rugulosa Hemsl.	Lamiaceae	47.35^a (Os ac: 16.53^a nM)	52.23^a (Os ac: 3.52^a nM)	n. g.	FL	142,147,152
luteolin	25	Elsholtzia rugulosa Hemsl.	Lamiaceae	33.68^a (Os ac: 16.53^a nM); 11.0^b(Os: 1.59^b nM)	32.63^a (Os ac: 3.52^a nM)	n. g.	FL	142,147,152,153
luteolin 3′-glucuronyl acid methyl ester	89	Elsholtzia rugulosa Hemsl.	Lamiaceae	44.42^a (Os ac: 16.53^a nM)	50.97^a (Os ac: 3.52^a nM)	n. g.	FL	152
scutellarin	90	n. g.	n. g.	50.6 (Os ac: 15 nM)	47.3 (Os ac: 3.2 nM)	n. g.	FL	142,147
vitexin	91	n. g.	n. g.	46.5 (Os ac: 15 nM)	45.1 (Os ac: 3.2 nM)	n. g.	FL	142,147
Flavonoles
astragalin	92	n. g.	n. g.	38.4^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
gossypetin	27	n. g.	n. g.	2.6^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
herbacetin	28	Rhodiola rosea L.	Crassulaceae	8.9^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
isoquercitrin	93	Ziziphus cambodiana Pierre	Rhamnaceae	9.87 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
kaempferol	94	Rhodiola rosea L.	Crassulaceae	11.2^b (Os: 1.59^b nM); 58.6 (Os ac: 15 nM)	38.1 (Os ac: 3.2 nM)	n. g.	FL	142,147,153
kaempferol 3-O-α-L-rhamnoside	95	Gouania obtusifolia Vent. ex Brongn.	Rhamnaceae	5.47 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
kumatakenin	96	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	36.4^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
leucoside	97	n. g.	n. g.	42.21^a (Os: 1.66 nM)	n. g.	n. g.	n. g.	147
licoflavonol	98	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	20.6^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
linocinamarin	99	n. g.	n. g.	44.2^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
myricetin	100	n. g.	n. g.	82.6 (Os ac: 15 nM)	46.2 (Os ac: 3.2 nM)	n. g.	FL	142,147
nicotiflorin	101	n. g.	n. g.	31.7^b (Os: 1.59 nM)	n. g.	n. g.		153
quercetin	26	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	58.4 (Os ac: 15 nM); 2.2^b (Os: 1.59^b nM)	87.6 (Os ac: 3.2 nM)	n. g.	FL	142,147,153,156
quercetin 3-O-β-xylopyranosyl-(12)-O-α-rhamnopyranoside	102	Ziziphus cambodiana Pierre	Rhamnaceae	4.82 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
quercetin 5,7,3′,4′-tetramethyl ether	44	Sambucus nigra L.	Adoxaceae	0.39–1.09 (Os: 0.12–0.23 nM)	n. g.	n. g.	CL	19,190
quercitrin	103	Gouania obtusifolia Vent. ex Brongn.; Ziziphus cambodiana Pierre	Rhamnaceae	7.89 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
rhamnetin	104	Caesalpinia sappan L.	Fabaceae	48.69^a (Os ac: 3.52^a nM)	76.20^a (Os ac: 1.65^a nM)	n. g.	FL	144
rhamnocitrin	105	n. g.	n. g.	51.5 (Os ac: 15 nM)	83.9 (Os ac: 3.2 nM)	n. g.	FL	142,147
rhodiolinin	106	Rhodiola rosea L.	Crassulaceae	10.3^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
rhodionin	107	Rhodiola rosea L.	Crassulaceae	32.2^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
rhodiosin	108	Rhodiola rosea L.	Crassulaceae	56.5^b (Os: 1.59^b nM)	n. g.	n. g.	FL	153
rutin	109	n. g.	n. g.	52.2 (Os ac: 15 nM); 34.4^b (Os: 1.59^b nM)	87.7 (Os ac: 3.2 nM)	n. g.	FL	142,147,153
Isoflavones
7-hydroxy-5-methoxy-6,8-dimethylflavone	110	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	414.32 (Os: 95.22 nM)	n. g.	362.87 (Os: 10.34 nM)	FL	150
7-hydroxy-5-methoxy-6,8-dimethylisoflavone	111	Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry	Myrtaceae	430.51 (Os: 95.22 nM)	n. g.	391.01 (Os: 10.34 nM)	FL	150
corylin	112	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	>115^a (Os: 127.21^a nM)	n. g.	>115^a (Os: 15.81^a nM)	FL	145
daidzein	29	n. g.	n. g.	37.1 (Os ac: 15 nM)	26.6 (Os ac: 3.2 nM)	n. g.	FL	142,147
erysenegalensein M	113	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	>115^a (Os: 127.21^a nM)	n. g.	>115^a (Os: 15.81^a nM)	FL	145
erythraddison A	114	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	>115^a (Os: 127.21^a nM)	n. g.	>115^a (Os: 15.81^a nM)	FL	145
formononetin	115	n. g.	n. g.	>100 (Os ac: 15 nM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142
genistein	30	n. g.	n. g.	77.1 (Os ac: 15 nM)	134.4 (Os ac: 3.2 nM)	n. g.	FL	142,147
glyasperin C	116	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	20% at 200^b μM (Os: 1.59^b nM)	n. g.	n. g.	FL	143
glyasperin D	117	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	20% at 200^b μM (Os: 1.59^b nM)	n. g.	n. g.	FL	143
isowighteone	118	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	>115^a (Os: 127.21^a nM)	n. g.	>115^a (Os: 15.81^a nM)	FL	145
licorisoflavan A	119	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	30% at 200^b μM (Os: 1.59^b nM)	n. g.	n. g.	FL	143
ononin	120	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	30% at 200^b μM (Os: 1.59^b nM)	n. g.	n. g.	FL	143
sophoricoside	121	n. g.	n. g.	>100 (Os ac: 15 nM)	>100 (Os ac: 3.2 nM)	n. g.	FL	142
wighteone	122	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	>115^a (Os: 127.21^a nM)	n. g.	>115^a (Os: 15.81^a nM)	FL	145
(Oligo)stilbenes
(+)-ampelopsin A	123	Vitis amurensis Rupr.	Vitaceae	215.30–261.60 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
(+)-viniferol C	31	Vitis amurensis Rupr.	Vitaceae	8.94–61.16 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
amurensin G	124	Vitis amurensis Rupr.	Vitaceae	38.15–42.47 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
amurensin-K	125	Vitis amurensis Rupr.	Vitaceae	14.43–48.44 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
erythraddison B	126	Erythrina addisoniae Hutch. & Dalziel	Fabaceae	29.30^a (Os: 127.21^a nM)	n. g.	23.94^a (Os: 15.81^a nM)	FL	145
gnetin D	127	Gnetum pendulum C.Y.Cheng	Gnetaceae	30.18^a (Os ac: 3.52^a nM)	38.26^a (Os ac: 1.65^a nM)	n. g.	FL	155
gnetulin	128	Gnetum pendulum C.Y.Cheng	Gnetaceae	36.15^a (Os ac: 3.52^a nM)	41.20^a (Os ac: 1.65^a nM)	n. g.	FL	155
gnetupendin B	32	Gnetum pendulum C.Y.Cheng	Gnetaceae	13.14^a (Os ac: 3.52^a nM)	24.71^a (Os ac: 1.65^a nM)	n. g.	FL	155
isorhapontigenin	129	Gnetum pendulum C.Y.Cheng	Gnetaceae	35.62^a (Os ac: 3.52^a nM)	63.50^a (Os ac: 1.65^a nM)	n. g.	FL	155
napalensinol B	130	Vitis amurensis Rupr.	Vitaceae	30.42–52.30 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
piceid	131	Vitis amurensis Rupr.	Vitaceae	110.25–150.81 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
shegansu B	132	Gnetum pendulum C.Y.Cheng	Gnetaceae	32.07^a (Os ac: 3.52^a nM)	40.43^a (Os ac: 1.65^a nM)	n. g.	FL	155
trans-resveratrol	133	Gnetum pendulum C.Y.Cheng	Gnetaceae	79.74^a (Os ac: 3.52^a nM)	64.84^a (Os ac: 1.65^a nM)	n. g.	FL	155
trans-vitisin B	134	Vitis amurensis Rupr.	Vitaceae	23.89–66.73 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
trans-ε-viniferin	135	Vitis amurensis Rupr.	Vitaceae	88.54–173.79 (Os: 3.89–70.88 nM)	n. g.	n. g.	FL	154
Isoprenoid compounds
Monoterpenes
terpinen-4-ol	136	Melaleuca alternifolia Cheel	Myrtaceae	not effective	n. g.	n. g.	FL	157
terpinolene	137	Melaleuca alternifolia Cheel	Myrtaceae	not effective	n. g.	n. g.	FL	157
α-terpineol	138	Melaleuca alternifolia Cheel	Myrtaceae	not effective	n. g.	n. g.	FL	157
Sesquiterpene
trans,trans-farnesol	139	Alpinia katsumadai Hayata	Zingiberaceae	81.40 (Os: 0.13 nM)	n. g.	n. g.	CL	118
Triterpenes
3-O-acetylursolic acid	140	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
3β-acetoxyurs-11-en-13β,28-olide	141	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
masilinic acid	142	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
oleanolic acid	143	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
speciosaperoxide	144	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
tormentic acid	145	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
ursolic acid	146	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
Phenylpropanoids & analogues
(6S,7E,9R)-6,9-dihydroxy-4,7-megastigmadien-3-one 9-O-[β-D-xylopyranosyl(1→6)-glucopyranoside]	147	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
1-(5-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)-2-phenyl-ethanone	46	n. g.	n. g.	1.33–2.21 (Os: 0.12–0.23 nM)	n. g.	n. g.	CL	19
1,4-dicaffeoylquinic acid	148	Cynara scolymus L.; Arnica montana L.; Echinacea purpurea (L.) Moench	Asteraceae	17.0 (Os: 0.12–0.23 nM)	n. g.	n. g.	CL	19
3,4-dihydroxybenzoic acid	149	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	8.24^a (Os: 0.99^a μM)	n. g.	n. g.	FL	156
gallic acid	150	Mangifera odorata Griff.	Anacardiaceae	>2600 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
iriflophenone 2-O-β-glucoside	151	Mangifera odorata Griff.	Anacardiaceae	>110 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
iriflophenone 3-C-β-glucoside	152	Mangifera odorata Griff.	Anacardiaceae	80.71 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
licocoumarone	153	Glycyrrhiza uralensis Fisch. ex DC.	Fabaceae	27.8^b (Os: 1.59^b nM)	n. g.	n. g.	FL	143
mangiferin	154	Gouania obtusifolia Vent. ex Brongn.; Mangifera odorata Griff.	Rhamnaceae; Anacardiaceae	0.82 (Neu5Ac2en: 44.47 μM)	n. g.	n. g.	FL	149
roseoside	155	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	not effective	n. g.	n. g.	FL	156
vomifoliol	33	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	10.39^a (Os: 0.99 μM)	n. g.	n. g.	FL	156
Miscellaneous
1-[5-(2-formylfuryl)methyl] hydrogen 1-hydroxyethane-1,2-dicarboxylate	156	Prunus mume Siebold & Zucc.	Rosaceae	1640.0^a (Os: 1.50^a nM)	n. g.	n. g.	FL	191
2-hydroxy-2-[(5-formyl-2-furanyl)methyl]-1,2,3-propanetricarboxylic acid ester	157	Prunus mume Siebold & Zucc.	Rosaceae	1620.0^a (Os: 1.50^a nM)	n. g.	n. g.	FL	191
2-hydroxy-4-[(5-formyl-2-furanyl)methyl]-butanedioic acid ester	158	Prunus mume Siebold & Zucc.	Rosaceae	713.0^a (Os: 1.50^a nM)	n. g.	n. g.	FL	191
5-hydroxymethyl-2-formylfuran	159	Prunus mume Siebold & Zucc.	Rosaceae	33400.0^a (Os: 1.50^a nM)	n. g.	n. g.	FL	191
brazilein	160	Caesalpinia sappan L.	Fabaceae	93.22^a (Os ac: 3.52^a nM)	86.54^a (Os ac: 1.65^a nM)	n. g.	FL	144
brazilin	161	Caesalpinia sappan L.	Fabaceae	69.51^a (Os ac: 3.52^a nM)	50.65^a (Os ac: 1.65^a nM)	n. g.	FL	144
methyl 3-hydroxybutanedioic ester	162	Chaenomeles speciosa (Sweet) Nakai	Rosaceae	12.83^a (Os: 0.99^a μM)	n. g.	n. g.	FL	156
mumefural	163	Prunus mume Siebold & Zucc.	Rosaceae	207.0^a (Os: 1.50^a nM)	n. g.	n. g.	FL	191
protosappanin A	164	Caesalpinia sappan L.	Fabaceae	94.40^a (Os ac: 3.52^a nM)	78.97^a (Os ac: 1.65^a nM)	n. g.	FL	144
spinosulfate A	165	unidentified pycnidial fungus	n. g.	0.5 (Neu5Ac2en: 0.9 μM)	n. g.	n. g.	FL	192

---

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