dengue infection causes various clinical symptoms ranging from mild fever to severe hemorrhagic fever and dengue shock syndrome [ 13 ] .
the virus uses host cell ribosomes to translate its genomic rna to full - length precursor polyprotein .
subsequently , the host cell furin and dengue ns2b - ns3 serine protease ( ns2b - ns3pro ) cleave viral polyprotein at various regions to produce structural and nonstructural viral proteins [ 46 ] .
the ns3 protein is one of the viral non - structural proteins that possess enzymatic activities .
the n - terminal of this protein contains 180 amino acid residues that represent ns3 protease [ 7 , 8 ] , while c - terminal region contains amino acid residues that represent rna helicase and rna - stimulated ntpase [ 9 , 10 ] .
the activity of ns2b - ns3pro depends on the interaction with its cofactor ns2b to form a ns2b - ns3pro complex .
it has been found that the disruption of ns2b - ns3pro functions inhibits viral replication .
therefore , ns2b - ns3pro is considered as a potential target for the design of antiviral drugs . at present , a legitimate vaccine or treatment to prevent or to cure this disease is unavailable .
these facts emphasize the need for a better understanding of the mechanism of viral infection and propagation in the host cell to combat this disease .
recently , computational studies indicated that cationic cyclic peptides have potential inhibition towards dengue ns2b - ns3pro [ 15 , 16 ] .
protegrin-1 ( pg-1 , rggrlcycrrrfcvcvgr ) is an eighteen amino acids cationic cyclic peptide with high content of basic residues and two disulphide bonds .
the peptide is originally isolated from porcine white blood cells and considered as an antibiotic agent against a broad range of microorganisms [ 17 , 18 ] .
the formation of two disulphide bonds between cysteine residues endows pg-1 with a stabile -hairpin secondary structure that is crucial for antimicrobial activity [ 19 , 20 ] .
therefore , removal of these two disulphide bonds is found to noticeably reduce the antimicrobial activity of pg-1 .
the peptide is able to penetrate and disrupt the cell membrane by homodimerization [ 22 , 23 ] .
the mechanism of this activity depends on the secondary structure and the cationic nature of pg-1 molecules which are essential to generate pores in the cell membrane of microorganism [ 2427 ] . in this study ,
our objective was to examine the efficacy of pg-1 as cationic cyclic peptide to inhibit dengue serine protease and subsequently reduce viral replication in host cells .
the linear peptides were prepared by automated peptide synthesis using symphony parallel synthesizer ( protein technologies , tucson , az , usa ) by standard solid - phase peptide synthesis .
the lyophilized peptide was dissolved in 20% dmso solution in a round bottom flask and stirred on a magnetic stirrer to produce a peptide concentration of 1.1  mm .
the formation of the first disulphide bond was completed within 24  hrs . the solution was then lyophilized to proceed for second disulfide formation .
the lyophilized peptide with the first disulphide bond was dissolved in acetic acid water ( 4  :  1 ) so that the peptide concentration was 1.2  mm and iodine ( 10 equivalents ) was added in one portion .
the reactions were stirred at 25c for 60  min and then quenched by diluting with water and extracting with ccl4 to remove excess iodine .
purification of crude cyclised peptide was accomplished by rp - hplc ( agilent 1200 series ) .
the identity of the purified peptide with 98% purity was confirmed by lc - ms ( shimadzu lc / ms 2020 , single quad ) . to produce single chain protease nsb2- ( g4-t - g4)-ns3
, the ns2b fragments were amplified individually by pcr using the primer pairs ns2b - f ( 5-atactgaggatcc gccgatttggaactg-3 ) and ns2blinker - r ( acctactaggtacctcctccacccagtgtctgttcttc ) .
the ns2b - ns3 was amplified by ns3linker - f ( 5-atctataggtaccggcggtggaggtgctggagtattgtgg-3 ) and ns3-r ( 5-agcataagcttaagcttcaattttct-3 ) .
the linker sequence was added to ns2blinker - r and ns3linker - f primers which included the site for kpni restriction enzyme ( all restriction sites are underlined ) .
the pcr product of ns2b fragment was digested with bamhi and kpni while ns2b fragment was digested with kpni and hindiii .
purified fragments were cloned into pqe30 plasmid downstream of 6his tag . the escherichia coli x -
the recombinant e. coli was inoculated in luria - bertani liquid medium ( 1% tryptone , 1% nacl , 0.5% yeast extract , w / v , ph 7.0 ) supplemented with 100  mg / l ampicillin and cultured overnight at 37c . in brief , 10  ml of overnight grown culture was added to 1000  ml of medium and incubated with shaking at 37c until the optical density at 600  nm reached 0.5 .
subsequently , isopropylthio--d - galactoside ( iptg ) was then added to a final concentration of 0.5  mm and the bacteria were cultured for an additional 5  hrs at 37c in a shaking incubator to induce protein expression .
finally , bacterial cells were harvested by centrifugation at 4000  rpm for 15  min at 4c .  
the recombinant ns3pro was produced as soluble proteins and , therefore , the purification had been performed by his gravitrap flow precharged ni sepharose 6 fast column ( amersham biosciences , usa ) according to the manufacturer 's instructions . in brief , the column was normalized with phosphate buffer ( 20  mm sodium phosphate buffer and 500  mm nacl , ph 7.4 ) .
the sample was loaded into the column and the column was washed with binding buffer ( phosphate buffer containing 20  mm imidazole , ph 7.4 ) .
the recombinant protein was eluted with elution buffer ( phosphate buffer containing 200  mm imidazole , ph 7.4 ) .
the bioassay used in this study was modified from the method published by kiat and coworkers .
these reaction mixtures consisted of 100  m fluorogenic peptide substrate ( boc - gly - arg - arg - mca ) , 2  m ns2b - ns3pro complex , with or without pg-1 of varying concentrations , buffered at ph 8.5 with 200  mm tris - hcl .
the pg-1 was initially prepared in tris - hcl buffer and assayed at five different concentrations .
subsequently , the substrate was added and the mixture was further incubated at the same temperature for 30 minutes .
triplicates were performed for all measurements and the readings were taken using tecan infinite m200 pro fluorescence spectrophotometer .
substrate cleavage was optimized at the emission at 440  nm upon excitation at 350  nm .
the readings were then used for calculating km values of peptide substrate and ic50 values of peptide inhibitors using nonlinear regression models in graphpad prism 5.01 software .  
the mk2 cell lines were seeded at 1  10 cells per well in triplicate at optimal conditions ( 37c , 5% co2 in humidified incubator ) in 96 well plates .
pg-1 was diluted to serial concentrations 2.5 , 12.5 , 25 , 50 , 100 and 200  m with dmem media supplemented with 2% fbs .
the cell culture was analyzed at 24 , 48 and 72 hours using nonradioactive cell proliferation assay ( promega , usa ) according to the manufacture protocol .
the mk2 cell lines were grown in a 24-well tissue culture plate ( 1  10 cells / well ) , incubated 24 hrs under optimal conditions ( 37c and 5% co2 ) .
denv-2 was added to the wells ( moi of 2 ) followed by incubation for 1  hr with gentle shaking every 10  min for optimal virus to cell contact .
the virus supernatant was removed , and the cells were washed twice with fresh serum free dmem media to remove residual virus . new complete dmem media containing 2.5 , 7.5 and 12.5  m of pg-1 were added and the cultures were incubated for 24 , 48 and 72  hrs .
afterwards , cellular supernatants were collected and stored at 80c for viral quantification by real - time pcr .   for quantification of denv-2 copies ,
the standard curve was generated by 10-fold serial dilution of known copies of denv-2 rna .
viral rna was extracted from culture supernatant using qiamp viral rna minikit ( qiagen , germany ) according to the manufacturer 's instructions . a fragment located at the 5utr region of the virus genome
one - step rt - pcr using sybr green master kit ( qiagen , germany ) was used to conduct absolute quantification using abi7300 machine from applied biosystems ( foster city , ca ) .
the pcr programme included 1 cycle of 50c for 2  min , 1 cycle of 95c for 10  min , and 40 cycles of 95c for 15 sec and 60c for 1  min .
results were analyzed using sequence detection software version 1.3 ( applied biosystems , foster city , ca , usa ) .
all the assays were done in triplicates and the statistical analyses were performed using graphpad prism version 5.01 ( graphpad software , san diego , ca ) .
the recombinant ns2b - ns3pro was produced as a soluble protein in e. coli and purified by nickel column ( figure 1(a ) ) .
further purification was applied using gel affinity chromatography to achieve more than 95% of enzyme purity ( figure 1(b ) ) .
the activity of purified enzyme had been assessed at 37c by catalyzing the fluorogenic peptide substrate t - butyloxycarbonylglycyl - l - arginyl - l - arginyl - l-4-methylcoumaryl-7-amide ( boc - gly - arg - arg - mca ) .
the pg-1 peptide was added to the protease reaction at different concentrations and the inhibition profile was plotted as shown in figure 2 .  
it was observed that the inhibition potential increased with pg-1 concentration and the highest inhibition ( 95.7% ) with low concentrations of pg-1 was at 40  m .
the kinetic assay study indicated that pg-1 competitively inhibited ns2b - ns3pro activity ( alpha value 3.14 ) with ki value 5.85  m ( table 1 ) .
intriguingly , the maximum enzyme velocity decreased threefold when pg-1 concentration was 20  m ( figure 3 ) .
previous studies have shown that various types of natural and chemical compounds were able to inhibit dengue ns2b - ns3pro activity .
for example , some of plant natural compounds , such as chalcones , have shown good inhibition potential against the dengue protease ( ki value 2125  m ) .
the synthesized peptidic -keto - amide compound inhibited dengue ns2b - ns3pro with ki value of 47  m .
most recent study indicated that the retrotripeptides with an arylcyanoacrylamide group as n - terminal cap exhibited high inhibition potential against dengue protease with ki value of 4.6  m .
most recent study showed that the inhibition potential of some chemical compounds towards ns2b - ns3pro measured by ic50 was 15.4 , 20.4 , and 27.0  m .
cytotoxicity and compound stability can be considered as major limitations of the practical application of protease inhibitors . in this study , to test pg-1 toxicity , mk2 cell lines were incubated with increasing concentrations of pg-1 for 24 , 48 , and 72 hrs .
the pg-1 peptide showed toxic effect against mk2 cell lines at concentrations greater than 12.5  m ( figure 4 ) .
other studies indicated that pg-1 was also toxic to 293a cell lines ( human embryonic kidney cells ) at 50  g / ml ( 25  m )   and more than 50% of human red blood cells lyses were observed at 80  g / ml ( 40  m ) .
therefore , three concentrations at nontoxic range were used to test pg-1 stability and ability to reduce dengue viral replication in mk2 cell lines .
the results showed that the viral copy number significantly ( p < 0.001 ) reduced with increasing concentrations of pg-1 ( figure 5(a ) ) .
furthermore , the highest inhibition percentage was observed when the pg-1 concentration was 12.5  m at 24 , 48 and 72  hrs ( figure 5(b ) ) .
however , the low concentrations exhibited less inhibition percentage at 48 and 72  hrs as compared with 24  hrs , indicating that the pg-1 stability declined with longer incubation in culture media ( figures 5(a ) and 5(b ) ) .
similarly , it has been showed that at low doses ( 4  g / ml ) , pg-1 has significant in vitro antimicrobial activity with low in vivo toxicity ( up to 8  mg / kg i.v .
this may be accounted by its short half - life in vivo as its level in mice plasma that was injected with 4  mg / ml i.v
although pg-1 showed significant inhibition profile towards dengue virus in this study and to human immunodeficiency virus 1 ( hiv1 ) in another study , the peptide instability should be considered as a major concern .
the results in this study may give a clear picture that would then help in engineering new sequence of peptides to retain the antiviral activity against dengue while increasing its stability and eliminating the toxic characteristics of pg-1 .
 dengue diseases have an economic as well as social burden worldwide . in this study , 
 the antiviral activity of protegrin-1 ( pg-1 , rggrlcycrrrfcvcvgr ) peptide towards dengue ns2b - ns3pro and viral replication in rhesus monkey kidney ( mk2 ) cells was investigated . 
 the peptide pg-1 was synthesized by solid - phase peptide synthesis , and disulphide bonds formation followed by peptide purification was confirmed by lc - ms and rphplc . 
 dengue ns2b - ns3pro was produced as a single - chain recombinant protein in e. coli . 
 the ns2b - ns3pro assay was carried out by measuring the florescence emission of catalyzed substrate . 
 real - time pcr was used to evaluate the inhibition potential of pg-1 towards dengue serotype-2 ( denv-2 ) replication in mk2 cells . 
 the results showed that pg-1 inhibited dengue ns2b - ns3pro at ic50 of 11.7  m . 
 the graded concentrations of pg-1 at nontoxic range were able to reduce viral replication significantly ( p < 0.001 ) at 24 , 48 , and 72  hrs after viral infection . 
 however , the percentage of inhibition was significantly ( p < 0.01 ) higher at 24  hrs compared to 48 and 72  hrs . 
 these data show promising therapeutic potential of pg-1 against dengue infection , hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors . 