influenza remains a serious health threat 
 and a future pandemic risk , as evidenced by recent outbreaks of h5n1 
 and h1n1 . these influenza type a strains , which cause acute 
 upper respiratory tract infections with 
 high morbidity and mortality , possess three viral - coat proteins : hemagglutinin 
 ( ha ) , neuraminidase ( na ) , and m2 .
ha and na are glycoproteins that 
 recognize terminal sialic - acid ( sa ) residues on host - cell surface 
 receptors , and m2 is a proton channel critical for virus assembly 
 and replication . upon attachment via sa 
 binding , ha mediates viral entry into the cell . following viral replication , 
 na facilitates the liberation of new virions from the cellular surface 
 by cleaving the (26)- or (23)-ketosidic 
 linkages that connect terminal sa residues to cell - surface glycoproteins .
na is conserved in all wild - type influenza viruses , and its inhibition 
 halts viral propagation by interfering with effective shedding .
consequently , 
 it is an attractive target for anti - influenza drug design . who recommends stockpiling na inhibitors such as zanamivir ( relenza , glaxosmithkline ) 
 and oseltamivir ( tamiflu , roche ) , which have 
 recently replaced older drugs like rimantadine and amantadine .
however , the threat of an h1n1 flu pandemic , the sudden emergence of oseltamivir - resistant h1n1 , and the emergence of potentially pathogenic 
 h3n2 and h5n1 strains warrant ongoing efforts to identify novel anti - influenza 
 compounds .
consequently , many researchers have expended considerable 
 effort in the pursuit of antiviral small molecules via bioinformatics 
 studies , hit - and - lead discovery approaches , and analogue synthesis .
natural compounds constitute an indispensable source of potential 
 inhibitors from which these studies can draw .
for example , the plant flavonoids isoscutellarein and isoscutellarein-8-methyl 
 ether , derived from the leaves and roots of scutellaria baicalensis , have been known to inhibit h1n1 na since 1995 . in total , about 160 similar plant compounds have been isolated 
 and tested to date ( reviewed in ref ( 27 ) ) .
inspired by the wealth and diversity 
 of malaysian tropical plants , we recently developed the natural product 
 discovery ( nadi ) repository ( http://www.nadi-discovery.com/ ) as an online resource .
this resource 
 consists of several databases , including nadi - meps , which describes 
 the ethnopharmacological use of various malaysian plants , and nadi - chem , 
 which stores the three - dimensional structures of over 3000 compounds 
 from more than 250 malaysian plants for use in computational - biology 
 studies .
virtual screening has shown great promise in sorting 
 through large libraries of potential bioactive molecules like those 
 in the nadi repository .
for example , in 2008 , rollinger et al . demonstrated 
 how natural - product / ethnopharmacological - guided virtual screening 
 can be used to select a particular class of natural products for subsequent 
 study .
compounds they isolated from the gum resin asafetida yielded 
 four experimentally confirmed inhibitors that were subsequently shown 
 to inhibit human rhinovirus in the low micromolar range .
two of these 
 compounds were present among their original virtual hits . here , we report a similar virtual screen 
 of the nadi compound database aimed at influenza drug discovery .
although 
 the nadi provides useful ethnopharmacological information about each 
 plant , we aimed to blindly predict the activity of each compound in silico without regard for traditional regional medical 
 practices .
the top predicted inhibitors principally came from five 
 distinct plants : garcinia mangostana , eurycoma 
 longifolia , tabernaemontana divaricata , brucea javanica , and momordica charantia .
these plants were subjected to in vitro evaluation 
 in order to characterize h5n1 na inhibition .
all the plant extracts 
 demonstrated some inhibitory activity ; g. mangostana extracts yielded the highest percent inhibition ( 82.95% at 250 g / ml ) .
fractions obtained from the extracts of these plants were subsequently 
 tested for na activity , ultimately leading to the isolation of 12 
 compounds from g. mangostana ( 4 ) , m. charantia ( 2 ) , b. javanica ( 1 ) , t. divaricata ( 1 ) , and e. longifolia ( 4 ) 
 that also inhibited h5n1 neuraminidase .
of these 12 compounds , four 
 had already been identified as hits in our initial nadi virtual screen .
we are hopeful that the virtual - screening methodology described 
 here will become an increasingly effective tool for rapidly identifying 
 bioactive plants for additional experimental study .
the three - dimensional structures of 3000 nadi and 2000 
 nci compounds were obtained from www.nadi-discovery.com and dtp.nci.nih.gov , respectively .
compounds that did 
 not satisfy lipinski s rule of five for drug - likeness were 
 discarded .
an additional 58 known inhibitors 
 of neuraminidase a with ki or ic50 values less than or equal to 25 nm were identified using the bindingdb 
 database .
structures of these compounds were generated 
 using schrdinger s ligprep module ( 2011 ) . in brief , 
 protonation states were assigned at ph 7.0 using epik . for each compound , ligprep identified all tautomeric and one stereoisomeric 
 state . where appropriate , one low - energy ring conformation was determined 
 per ligand .
both the small 
 molecules and the h5n1 na receptor were prepared for docking using 
 the autodocktools ( adt ) software package .
the receptor was prepared from an x - ray crystal structure of h5n1 
 neuraminidase ( pdb i d : 2hu4 ) .
hydrogen atoms were 
 added to all ligands and na models using adt , and ligand rotatable 
 bonds were assigned via autotors .
grid boxes comprised 
 of 60  60  60 points spaced 0.375  apart and centered 
 on the na active site were calculated for the following atom types : 
 c , a ( aromatic c ) , n , o , s , h , f , cl , br , i , p , and e ( electrostatic ) 
 using autogrid3 .
the parameters of the lamarckian 
 genetic algorithm ( lga ) were as follows : population size of 50 , elitism 
 of 1 , mutation rate of 0.02 , crossover rate of 0.80 , local search 
 rate of 0.06 , 250  000 energy evaluations , and 100 search runs . 

the final docked conformations were clustered using a cluster tolerance 
 of 1.0  root - mean - square deviation ( rmsd ) .
the ligand pose with 
 the lowest predicted free energy of binding , chosen from the most 
 populated cluster , was used in subsequent analysis .
solvents used for extraction 
 and column chromatographic analysis were of analytical grade ( ar ) , 
 and solvents used in the liquid chromatographic analysis were of hplc 
 grade .
22-(4-methylumbelliferyl)-a - d - n - acetylneuraminic 
 acid sodium salt hydrate ] , mes [ 2-(n - morpholino ) 
 ethanesulfonic acid ] , and dana [ 2,3-didehydro-2-deoxy - n - acetylneuraminic 
 acid ] were obtained from sigma ( malaysia ) .
melting 
 points were determined using a smp20 melting point apparatus ( stuart ) 
 and a differential scanning calorimeter ( perkinelmer ) .
ftir spectra 
 were recorded using an ir prestige-21 instrument ( shimadzu ) and a 
 nicolet 6700 ftir - atr spectrometer ( thermo scientific ) available at 
 the centre for drug research , universiti sains malaysia .
uv spectra 
 were recorded using a u-2000 spectrophotometer ( hitachi , japan ) and 
 a uv 1601pc spectrophotometer ( shimadzu ) .
ms spectra were acquired 
 using a 1100 series lc - msd trap g2445l ( agilent technologies , palo 
 alto , usa ) .
h and c nmr spectra were 
 both recorded at 500 mhz using an avance iii hd spectrometer ( bruker ) . 

semipolar compounds were dissolved in deuterated chloroform ( cdcl3 ) , and polar compounds were dissolved in 99% cd3od .
a centrifugal tlc ( cyclograph , analtech inc . ; newark , de , usa ) 
 was used to separate fractions .
sample purification was achieved using 
 a pu-980 hplc system ( jasco ; tokyo , japan ) equipped with a solvent 
 delivery pump , a 7725i sample injector ( rheodyne ; cotati , ca , usa ) 
 with a 250 l sample loop , a uv-975 detector ( jasco ; tokyo , 
 japan ) , and a d-2500 chromato - integrator ( hitachi ; tokyo , japan ) . 

mass spectra were measured on a 1100 series lc - msd trap g2445 vl ( agilent 
 technologies , palo alto , california , usa ) .
the hplc system was used 
 to check for peak purity . a microplate reader ( turner - biosystem , usa ) 

the 
 leaves of t. divaricata were collected around penang , 
 oven - dried ( 45 c ) , and powdered
. the seeds of b. javanica were obtained from solo , east java , indonesia , and were likewise 
 dried and ground into powder .
e. longifolia plant 
 and root samples were identified and purchased in perak , malaysia , 
 by the pharmaceutical company hovid berhad ( ipoh ) .
voucher specimens 
 for all the plant materials with the exception of e. longifolia were deposited in the herbarium of the school of biological sciences , 
 universiti sains malaysia ( no .
11301 , 11302 , 1298 , and 1299 for g. mangostana , m. charantia , t. 
 divaricata , and b. javanica , respectively ) . 
 an e. longifolia voucher specimen ( no .
a description of the extraction , fractionation , and isolation of 
 specific compounds can be found in the supporting 
 information , together with experimentally measured purities , 
 melting points , and ir / nmr spectra .
munana ( sigma , m8639 ) in 32.5 mm mes ( sigma , 
 m8250 ) buffer ( ph 6.5 ) served as the substrate , and neuraminidase 
 from viral h5n1 ( sinobio ) in mes buffer served as the enzyme .
the 
 chemical compounds , plant extracts , and fractions were dissolved in 
 2.5% dmso and diluted to various concentrations ranging from 0.488 
 g / ml to 250  g / ml in mes buffer . for the assay , neuraminidase 
 ( 25 l ) was added to 25 l of sample solution mixed with 
 a buffer in 96-well microplates .
after 1 h , 100 ml of stop solution 
 was added to each well , and 4-methylumbelliferone was immediately 
 quantified fluorometrically using a modulus microplate reader ( turner 
 biosystem , usa ) . the excitation and emission wavelengths were set 
 at 365 and 450 nm , respectively .
the percentage of na inhibition over a range of compound 
 concentrations was determined by fitting experimental data to the 
 logistic graph .
dana and oseltamivir carboxylate were used as standard 
 inhibitors ( positive controls ) in the bioassay .
the detergent assay 
 was adapted from the work of feng and shoichet , by adding 0.01% triton - x-100 to the mes buffer .
prior 
 to performing a virtual screen of the nadi database using autodock 
 3.05 , we first validated our docking 
 procedure .
an oseltamivir molecule was extracted from a crystallographic 
 na structure ( pdb i d : 2hu4(42 ) ) and redocked into the 
 same binding pocket .
the docked ligand pose was similar to the crystallographic 
 pose ( rmsd = 0.84  , figure 1 ) , demonstrating 
 that the autodock docking parameters used are amenable to this system .
superimposition 
 of the docked and crystallographic oseltamivir poses ( green and blue , 
 respectively ) .
we next verified that our virtual - screening technique was 
 capable of identifying known na inhibitors .
the compounds of the nci 
 diversity set 1 ( presumed decoys / negatives ) , together with 58 known 
 neuraminidase a inhibitors with ki or 
 ic50 values less than or equal to 25 nm ( positives ) , were 
 docked using the same protocol described above .
the area under the 
 roc curve generated from this initial benchmark screen was 0.99 per 
 the trapezoidal rule ( figure 2 ) , suggesting 
 that our screening methodology is highly predictive .
receiver operator curve 
 generated from the initial benchmark screen of the nci diversity set 
 i ( presumed decoys ) and known potent na inhibitors .
the area under 
 the curve is 0.99 , suggesting the screening methodology is highly 
 predictive .
satisfied that our virtual - screening protocol could be effectively 
 used to identify na inhibitors , we next performed a virtual screen 
 of 3000 nadi compounds .
the top 100 compounds with the best 
 docking scores ( table s1 ) were derived 
 from a total of 31 local malaysian plants .
g. mangostana , e. longifolia , t. divaricata , m. charantica , and b. javanica possessed 
 the greatest number of predicted h5n1 inhibitors ( table s2 ) and so were selected for further experimental evaluation .
methanol ( meoh ) extracts of these five plants ( figure 3 ) , as well as their fractions ( table 1 and figure s1 ) , were tested for 
 h5n1 na inhibition . with the exception of e. longifolia , all plants showed marked na inhibition .
g. mangostana , m. charantia , t. divaricata , b. javanica , and e. longifolia had 82.95% , 
 72.61% , 62.79% , 55.80% , and 29.62% na inhibition at 250 g / ml , 
 respectively .
in general , isolated fractions of these plant extracts 
 ( table 1 ) also inhibited neuraminidase .
fraction 
 f3 from g. mangostana had 84.49% inhibition , fractions 
 f3 and f4 from m. charantia had > 70% inhibition , 
 all three b. javanica fractions had > 50% inhibition , 
 and t. divaricata had only modest inhibition ( 12 
 to 36% ) . interestingly , though the inhibition of the whole meoh e. longifolia extract was negligible , fraction f4 from this 
 species did demonstrate reasonable inhibition ( 56.47% ) .
percent h5n1 
 na inhibition of five plant meoh extracts ( 2500.488 g / ml ) . 

the calculated ic50 for the plant extracts are g mangostana = 50.93  0.02 g / ml , m 
 charantia = 79.43  0.06
g / ml , b. javanica = 184.93  0.04 g / ml , t. divaricata = 164.81  0.03 g / ml . efforts 
 were next undertaken to isolate specific inhibitory compounds from 
 the extracts of these five plants .
within the time frame of the experiment , 
 we isolated 12 compounds from the fractions of g. mangostana ( 4 ) , m. charantia ( 2 ) , t. divaricata ( 1 ) , b. javanica ( 1 ) , and e. longifolia ( 4 ) ( table 2 and figure 
 s2 ) .
of these 12 compounds , five compounds showed > 50% inhibition 
 at 250 g / ml , with ic50 values ranging from 89.71 
 to 275.45 m .
four of the 12 compounds were present in the original 
 virtual screen , including gartanin , with an ic50 value 
 of 126.64  0.13 m ( table 2 ) .
an 
 additional four compounds ( rubraxanthone , kuguacin j , 13,21-dihyroeurycomanone , 
 and 13,21-epoxyeurycomanone ) were absent from the nadi database 
 at the time of screening and so could not have been identified in silico .
in contrast , -mangostin , garcinone c , 
 eurycamanol , and daucosterol failed to rank among the top 100 hits 
 of the virtual screen ( table s1 ) .
compounds marked with asterisks were 
 subjected to additional testing to rule out nonspecific inhibition 
 by self aggregation .
momordicin i and kuguachin j from m. charantia showed 15.58% and 21.42% na inhibition , respectively .
m ) , far better than any of 
 the fractions or extracts from this species .
as expected , the compounds 
 from e. longifolia exhibited low inhibition , never 
 exceeding the 34.50% inhibition of 13 , 21-epoxyeurycomanone .
all four compounds from g. mangostana demonstrated 
 > 80% inhibition at 250 g / ml , with ic50 values 
 of 91.95  0.09 , 89.71  0.08 , 95.49  0.08 , and 126.64 
  0.13 m for -mangostin , rubraxanthone , garcinone 
 c , and gartanin , respectively .
-mangostin and gartanin have 
 been previously isolated from g. mangostana pericarps 
 and have been shown to inhibit c. perfringens neuraminidase 
 with ic50 values of 12.2  1.2 and 2.9  0.3 
 m , respectively .
the presence 
 of -mangostin , gartanin , and garcinone c in asian mangosteen 
 pericarps has been well documented , and lian and 
 rahmani communicated the presence of rubraxanthone in the malaysian 
 mangosteen at a recent conference .
to 
 our knowledge , however , rubraxanthone has not been previously identified 
 as a neuraminidase inhibitor .
it is interesting to note that a series of xanthone derivatives 
 from c. triscuspidata has also been shown to inhibit 
 bacterial neuraminidase at the nanomolar level . among the 12 nadi compounds tested , compounds from g. mangostana showed the highest inhibition rates and were 
 therefore selected for additional testing to rule out inhibition by 
 nonspecific aggregation . according to feng and shoichet
, most aggregators show a greater than 50% decrease 
 in inhibition in the presence of detergent .
the tested compounds demonstrated 
 a 1015% decrease at 250 g / ml , far less than 50% .
furthermore , 
 the dose response curve of self - aggregating compounds is typically 
 markedly less steep in the presence of detergent .
in contrast , the steepness of the dose response curves 
 of our compounds was unaffected by detergent ( figure s3 ) .
garcinone c is representative of these xanthone analogues and had 
 a consistent binding pose when docked using two distinct programs 
 ( autodock 3.05 and schrdinger s glide/2011 , data not shown ) .
we therefore focused on this compound in order 
 to better understand the potential binding modes of these analogues . 

the autodock pose of garcinone c is shown in figure 4a and b. potential interactions between the docked compound 
 and the na receptor were identified using binana , poseview , visual molecular dynamics , and visual inspection .
garcinone c may form 
 hydrogen bonds with the y347 , r118 , y406 , d151 , and q276 side chains 
 ( figure 4a ) , as well as extensive cation- 
 interactions with r371 , r292 , and r152 ( figure 4b ) .
the crystallographic pose of oseltamivir , a potent approved drug , 
 is shown for reference ( figure 4c ) . predicted pose 
 of garcinone c , docked into the neuraminidase active site .
( b ) predicted cation- interactions between 
 r371 , r292 , r152 , and the xanthone moiety .
( c ) the crystallographic 
 pose of oseltamivir , a potent inhibitor , shown for reference ( pdb 
 i d : 2hu4 ) .
for example , the oseltamivir 
 acetamide carbonyl oxygen atom forms a hydrogen bond with the r152 
 side chain .
if the docked pose of garcinone c is correct , a 2-methylbutan-2-ol 
 moiety is positioned at the same location ; the corresponding -mangostin 
 and rubraxanthone moieties are entirely hydrophobic .
the careful addition 
 of a hydrogen - bond acceptor at this location may improve potency .
furthermore , all approved neuraminidase inhibitors take advantage 
 of strong electrostatic interactions between negatively charged ligand 
 carboxylate groups and positively charged na arginine residues , most 
 notably r371 .
the novel xanthone inhibitors are also predicted to 
 form interactions with active - site arginine side chains via cation 
 interactions .
while these interactions are substantial , adding negatively 
 charged moieties to the xanthones at the appropriate locations might 
 lead to even stronger electrostatic interactions . finally , we 
 note that , like other na inhibitors , the xanthone compounds are not 
 predicted to bind in the 150-loop cavity .
their predicted binding poses are adjacent , however , and moieties 
 could potentially be added to the xanthone scaffold that extend into 
 this highly flexible subpocket .
a number of recent medicinal - chemistry studies have applied virtual 
 screening to natural - product databases .
for example , a recent virtual 
 screen of 57 natural plant metabolites identified hesperidin and narirutin 
 ( isolated constituents of citrus junos ) , as well 
 as proanthocyanidin , ursolic acid , sitosterol , tangeretrin , abbssinone , 
 nobiletin , and tannic acid , as potential h1n1 neuraminidase inhibitors .
this work was not validated by bioassay , but 
 hesperidin is known to inhibit influenza growth . encouraged by this previous success , we aimed to demonstrate 
 that virtual screening can be used to select specific plants from 
 among the rich biodiversity of south east asia for further in vitro pharmacological studies . despite time and resource 
 restraints ,
virtual screening proved effective at identifying active 
 botanical / nadi compounds , even when larger - scale efforts were impossible . to demonstrate that our 
 virtual - screening protocol could effectively identify na inhibitors , 
 we first docked known na inhibitors and presumed decoys from the nci 
 diversity set 1 .
given the promising results 
 of our initial nci benchmark virtual screen , we next applied the same 
 docking protocol to the compounds of the natural product database 
 ( nadi ) .
the docking results prompted us to experimentally test five 
 tropical plants for na inhibition , as described in the results section .
remarkably , none of these five plants were 
 mentioned in a recent review of 3000 references describing natural - product 
 influenza inhibition , attesting to the 
 novelty of our discovery .
as far as we are aware , with the exception 
 of g. mangostana(49 ) this 
 is the first time these plants have been reported to have antineuraminidase 
 activity . despite our success identifying true inhibitors , it 
 is curious that the correlation between predicted free energies and 
 experimentally measured potencies was poor . as warren et al
. demonstrated 
 in their critical assessment of nine docking tools , this is a general problem independent of the docking program 
 used .
one of the advantages of the method herein described is that 
 it does not rely on a single docking score .
docking scores were used 
 only to guide the selection of appropriate plants , each containing multiple predicted na inhibitors , for further experimental 
 examination .
we identified five tropical 
 plants that show inhibitory activity against h5n1 neuraminidase : g. mangostana , e. longifolia , t. 
 divaricata , b. javanica , and m. 
 charantia .
parts of these plants  leaves , roots , and 
 fruits  were subjected to various extraction methods , chromatographed , 
 and further fractionated for biological testing .
fractions and extracts 
 of the five plants exhibited good to moderate anti - h5n1 neuraminidase 
 activity .
g. mangostana had the highest inhibition 
 ( 82.95% at 250 g / ml ) .
rubraxanthone , -mangostin , and garcinone 
 c , with ic50 values ranging from 89.71 to 95.49 m , 
 were particularly notable .
while we are hopeful that further 
 optimization of these compounds will yield more potent analogs , we 
 acknowledge that currently approved na - inhibiting therapeutics are 
 much stronger binders .
the primary purpose of this study is to present 
 a novel computational method for prioritizing biologically active 
 plants for subsequent pharmacological study .
the work described here 
 affords a glimpse into the potential of both virtual screening and 
 the nadi compound library , which contains a growing number of malaysian 
 natural - plant products .
we expect that similar virtual - screening approaches 
 will serve as useful tools to guide the future identification of additional 
 bioactive , medicinal plants .
 recent 
 outbreaks of highly pathogenic and occasional drug - resistant influenza 
 strains have highlighted the need to develop novel anti - influenza 
 therapeutics . here , we report computational and experimental efforts 
 to identify influenza neuraminidase inhibitors from among the 3000 
 natural compounds in the malaysian - plants natural - product ( nadi ) database . 
 
 these 3000 compounds were first docked into the neuraminidase active 
 site . 
 the five plants with the largest number of top predicted ligands 
 were selected for experimental evaluation . 
 twelve specific compounds 
 isolated from these five plants were shown to inhibit neuraminidase , 
 including two compounds with ic50 values less than 92 m . 
 
 furthermore , four of the 12 isolated compounds had also been identified 
 in the top 100 compounds from the virtual screen . 
 together , these 
 results suggest an effective new approach for identifying bioactive 
 plant species that will further the identification of new pharmacologically 
 active compounds from diverse natural - product resources . 
Introduction
Methods
Results
Discussion
Conclusion