current approaches in antiviral drug discovery against the

Send Orders for Reprints to [email protected]

3428 Current Pharmaceutical Design, 2014, 20, 3428-3444

1381-6128/14 $58.00+.00 © 2014 Bentham Science Publishers

Current Approaches in Antiviral Drug Discovery Against the Flaviviridae Family

Aida Baharuddin1, Asfarina Amir Hassan3, Gan Chye Sheng1, Shah Bakhtiar Nasir2, Shatrah Othman1, Rohana Yusof1, Rozana Othman3 and Noorsaadah Abdul Rahman2*

1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia;

2Deparment of

Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; 3Department of Pharmacy, Faculty of Medi-

cine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abstract: Viruses belonging to the Flaviviridae family primarily spread through arthropod vectors, and are the major causes of illness and death around the globe. The Flaviviridae family consists of 3 genera which include the Flavivirus genus (type species, yellow fever virus) as the largest genus, the Hepacivirus (type species, hepatitis C virus) and the Pestivirus (type species, bovine virus diarrhea). The flaviviruses (Flavivirus genus) are small RNA viruses transmitted by mosquitoes and ticks that take over host cell machinery in order to propagate. However, hepaciviruses and pestiviruses are not antropod-borne. Despite the extensive research and public health concern as-sociated with flavivirus diseases, to date, there is no specific treatment available for any flavivirus infections, though commercially avail-able vaccines for yellow fever, Japanese encephalitis and tick-born encephalitis exist. Due to the global threat of viral pandemics, there is an urgent need for new drugs. In many countries, patients with severe cases of flavivirus infections are treated only by supportive care, which includes intravenous fluids, hospitalization, respiratory support, and prevention of secondary infections. This review discusses the strategies used towards the discovery of antiviral drugs, focusing on rational drug design against Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Yellow Fever virus (YFV) and Hepatitis C virus (HCV). Only modified peptidic, non-peptidic, natural compounds and fragment-based inhibitors (typically of mass less than 300 Da) against structural and non-structural pro-teins are discussed.

Keywords: Dengue virus (DENV), West Nile virus (WNV),Japanese encephalitis virus (JEV), Yellow Fever virus (YFV), Hepatitis C virus (HCV), drug discovery, antiviral.

1. INTRODUCTION

The genus Flavivirus is a group of aboviruses that belongs to the Flaviviridae family. It consists of over 70 viruses mostly trans-mitted by mosquitoes or ticks [1]. West Nile virus (WNV), Japa-nese encephalitis virus (JEV), Dengue virus (DENV), Tick-borne encephalitis virus (TBEV), Yellow fever virus (YFV), Murray Val-ley encephalitis virus (MVLEV) and St Louis encephalitis virus (SLEV) are considered important global flavivirus pathogens which are responsible for human and animal diseases and deaths. Most of these viruses cause severe hemorrhaging fever while others cause neuroinvasive diseases ranging from mild febrile illness to fatal encephalitis. DENV, JEV, YFV and TBEV are the most dangerous flaviviruses that have mortality rates up to 30% [2]. The viruses are endemic in many areas of the world and thousands of lives are lost during recurring outbreaks. Although extensive researches have been conducted, currently there is no specific treatment available for any flavivirus infection, though commercially available vac-cines for yellow fever, Japanese encephalitis and tick-born encepha-litis exist [3]. Available license drug such as ribavirin is known to suppress the replication of some flaviviruses in vitro, however, in vivo studies have been limited to few rodent models [4]. Further-more, the management of mosquito and tick-borne populations has shown to be problematic [5]. In addition, due to the globalization and gradual climate changes, the viruses are emerging in new geo-graphic areas and populations, thus there is an urgent need to find new drugs to fight these emerging threats [3]. For example, in the USA, the first case of WNV was reported in New York in the year 1999. Since then, more than 30,000 clinical cases of WNV and neuroinvasive diseases were reported by the Centers for Disease

*Address correspondence to this author at the Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; Tel: +603-7967 4254; Fax: +603-7967 4193; E-mail: [email protected]

Control and Prevention (CDC), with the largest outbreaks in the summer of 2002, 2003 and 2012 (ref). Currently, the most impor-tant mosquito borne disease is the dengue fever with an estimated 50 million cases of febrile illness each year, and an increasing number of cases for hemorrhagic fever [6].

The flavivirus genome is a positive single-stranded RNA with a size of ~11 kb, which includes the 5’ and 3’ noncoding regions of approximately 100 to 600 nucleotides, respectively (Fig. 1). In the infected cells, the messenger RNA (mRNA) of the RNA genome encodes three structural proteins (C, capsid protein; Pre M, precur-sor membrane protein; and envelope E protein) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3 [protease, heli-case], NS4A, NS4B and NS5 [methyltransferase, RNA-dependent RNA polymerase]) that form the apparatus involved in viral synthe-sis and replication [7, 8]. Virus-encoded serine protease, NS2B-NS3, and host-encoded proteases, including signalase and furin, are responsible for the processing of the three structural and seven NS proteins encoded by the viral mRNA [9]. Currently, researchers are targeting the structural and non-structural viral proteins for drug development [10-14]. According to previous knowledge of drugs that are now in clinical trials, RNA dependent RNA polymerase (RdRp) has shown to be the most promising target. This enzyme plays a critical role in viral replication by forming part of a mul-timeric complex, and since there is no host cellular protein equiva-lent to flavivirus RdRp, no toxicity issues exist [14]. Like anti-HIV protease inhibitors that are already in the market for clinical use, flavivirus protease is another attractive target for drug development. However, since cellular serine protease such as furin also recog-nises two basic amino acid residues at P1 and P2 positions [15], its toxicity issues remain to be determined. Another attractive choice for antiviral therapeutics is the virus entry inhibitors. For example, virus entry inhibitors have been developed and approved for treat-ment of HIV due to the emergence of drug resistance and cellular toxicity towards inhibitors of DNA and RNA polymerases and pro-teases [16-18]. Another novel target for flaviviruses is the NS3

Current Approaches in Antiviral Drug Discovery Current Pharmaceutical Design, 2014, Vol. 20, No. 21 3429

RNA helicasethat forms a multimeric complex with NS5, which is required for viral replication, and apparently needed for unwinding the double-stranded RNA intermediate [12]. In addition to these approaches where the virus-coded function is blocked, another ap-proach is to block the cellular functions needed for the viral multi-plication. In the later approach, knowledge of the virus-host protein interactions is fundamental.

Development of drugs through trial-and-error is time-consuming and may be costly. With the advancement of science in drug discovery, scientists are focusing more on structure-based rational drug design techniques that link NMR and crystallography, computational advances in docking and virtual screening, and high-throughput screening (HTS) with combinatorial chemistry to in-crease the speed and efficiency of drug discovery. On another note, structure-based drug design may improve initial discovery, but it has yet to significantly improve the effectiveness or reduce the cost of clinical trials. In rational drug design, the process is more stream-lined and requires detail knowledge of the target of the drug, as well as the drug itself. It usually involves the study of the three-dimensional structure of a drug target, followed by the search of a

compound that can interact with the target. By aiming at well-characterized specific target of the virus, it is hoped that the se-lected compound would be specific and potent in its activity, de-sirably with less side effects. Rational drug design requires sound knowledge of chemistry as well as biology because the bioactivities of drugs depend on the chemical interactions between the drugs and their targets. Two possible rational approaches that could be used in the design of antiviral compounds are by modification of the known antiviral drugs or inhibitors, and design of new antiviral compounds directed at specific steps in the replication cycle of particular vi-ruses. Several other strategies for discovery of chemotherapeutic agents for flaviviruses include reverse genetic system-based screen-ing, macromolecular inhibitors like antibodies and interferon, nu-cleic acid-based agents (for example antisense oligomers and siRNA) [19] and vaccine development [20]. However, in this re-view article, only strategies for discovery of modified peptidic, non-peptidic, natural compounds and fragment-based inhibitors (typi-cally of mass less than 300 Da) against DENV, WNV, JEV, YFV and HCV structural and non-structural proteins are discussed.

Fig. (1). Flavivirus genome and polyprotein organization. The flavivirus genome consists of a single-stranded 11 kb positive-sense RNA genome with a single open reading frame that codes for structural and non-structural protein regions, including the 5’ and 3’ untranslated region (UTR) and the 5’-CAP. The open reading frame encodes an immature polyprotein precursor (~ 3400 amino acids) that is co- and post-translationally cleaved into three structural proteins and seven non-structural proteins. Sites of polyprotein cleavage mediated by NS2B-NS3, cellular signalase and furin and unknown protease are shown. Putative functions of these proteins during infection are described. The most characterized non-structural proteins with multiple enzymes activities, which are required for viral replications are NS3 (70 kDa) and NS5 (104 kDa). Mutations that affect the activity of NS3 and NS5 impaired viral replication [186]. NS1 protein (46 kDa) is needed for viral replication and is likely to be involved in negative-strand synthesis by an unknown mechanism. NS2A (22 kDa) is a small hydropho-bic transmembrane protein that plays a role in generation of virus-induced membranes during virus assembly [7]. NS4A (16 kDa) is an integral membrane protein that induces membrane rearrangements to form viral replication complex [187, 188]. NS4B protein (27 kDa) inhibits the type I interferon response of host cells [189], and interaction with NS3 protein may modulate viral replication [190]. Reproduced from Pastorino et al [191] with permission.

3430 Current Pharmaceutical Design, 2014, Vol. 20, No. 21 Baharuddin et al.

2. DENGUE VIRUS

DENV exists in four different serotypes and each of these sero-types shares similar genome organization and replication strategies. All four serotypes result in identical syndromes to the human body. Among the four serotypes, DENV type 2 is the most prevalent. Currently there are several X-ray structures of apo and inhibitor bound NS2B-NS3pro deposited in the Protein Data Bank as shown in Table 1. The protease protein structure is similar to a chymotryp-sin-like fold with two six-stranded -barrels. The catalytic triad (His51-Asp75-Ser135) is located at the cleft between the two -barrels, which represents the binding site and is shown to be rather shallow with small binding pocket (Fig. 2). The crystal structures of apo NS2B-NS3 provides an intriguing insight into the interactions of the protease and helical domains, while the availability of a pro-tein structure with a bound inhibitor is critical for structure-based design. DENV drug discovery process has been initially hampered due to the lack of sufficient DENV NS2B-NS3 co-crystal structures indicating that NS2B co-factor appropriately interacts with the ac-tive site. Fortunately, suitable ligand-inhibitor complexes for drug design are currently available from DENV protease (Table 1). Re-cently, Deng et al [21] reported a compound, obtained through virtual screening, to be active against NS2B-NS3 protease. This compound was selected as the lead structure after considering its biological activity, structural variability and synthetic accessibility. In fact, modification on the functional group on R1-R4 substituents in this compound has resulted in four new inhibitors with moderate in vitro inhibition activity. Their findings indicated that this com-pound inhibited DENV NS2B-NS3 protease in the cleavage of sub-strate with IC50 value of 13.12 ± 1.03 μM. Therefore, it was sug-gested that this compound was a good candidate inhibitor worthy of structure modification and optimization. Schuller [22] found a few tripeptide aldehyde inhibitors that were successfully synthesized, and their inhibitory effects against DENV type 2 protease were tested. Results revealed that the tripeptide aldehyde phenylacetyl-KRR-H, showed lower micromolar inhibition for DENV type 2 protease compared to reference peptide inhibitor, benzoyl-nKRR-H. Nevertheless, the peptide inhibitor gave poor inhibition towards DENV NS3 protease due to the marked structural flexibility of the protein especially towards its co-factor NS2B. On the other hand, docking study of phenylacetyl-KRR-H to a homology model of DENV type 2 NS3 protease showed that the tripeptide aldehyde was successfully docked to a closed-form of the protease, with the

cap groups aligned well and potentially engaged in the - interac-tions with Tyr161. Noble et al [23] reported the crystal structure of DENV type 3 NS3 protease, covalently bound to a peptide as well as to aprotinin, a small protein basic pancreatic trypsin inhibitor. Unlike aprotinin, which bound to the DENV type 3 NS3 protease in an open conformation, the peptide bound to DENV type 3 NS3 protease induced the target molecule to adopt close conformation. Basically, DENV type 3 NS3 protease forms a closed conformation in which the hydrophilic -hairpin region of NS2B wraps around the NS3 protease core in the presence of the peptide (Fig. 2B). On the other hand, aprotinin high affinity for DENV protease is gained from multiple interactions, with most of its inhibitory activity comes from active site steric hindrance.

The development of new retro di- and tripeptide hybrids that did not require highly electrophilic warheads (serine-trap) such as aldehydes, keto-amides, and boronic acids to combine with macro-cyclic fragments to achieve affinities in the low micromolar range have been described by Nitsche et al [24]. Retro peptides were found to give the best activities for both DENV and WNV prote-ases. Retro and retro-inverse peptide sequences of target are usually used for protein inhibition study and to understand the intermolecu-lar recognition. For retro peptides, only the C- and N-termini of a given peptide sequence are interchangable. And as for retro-inverse peptides, the streocenters are also interchangable. An inversion of the sequence from R-Arg-Lys-NH2 to the non-retro sequence R-Lys-Arg-NH2 resulted in loss of DENV activity and invariant activ-ity for WNV protease. The DENV protease was found to be more sensitive to molecular changes in retro-peptide inhibitors compared to WNV protease. This study summarized that retro peptides were the best choice for creation of potent and selective DENV protease inhibitors, and were superior to natural sequences or retroinverse peptides.

A guanidinilated 2,5-dideoxystreptamine was identified as a competitive inhibitor of NS3 protease for all four DENV serotypes and WNV with IC50 values in the range of 1-70 μM [25]. This com-pound was discovered through screening of about ~12 000 com-pound library using FRET 96-well plate based assay. The concen-tration of the tested compound was fixed at 25 μM. Hits were iden-tified if the compounds inhibited the proteolytic activity more than 40% and were characterized as follows: determination of the IC50 against both DENV (all four serotypes) and WNV NS2B-NS3 pro-

Table 1. X-ray crystal structures available in PDBa for DENV protease.

DENV serotypes PDBa Resolution

(Å)

Comment Reference

DENV1 3L6P 2.20 NS2B-NS3prob [175]

DENV1 3LKW 2.00 NS2B-NS3prob

(active site mutant)

[175]

DENV1 2WZQ 2.80 NS2B-NS3 full length [176]

DENV2 2FOM 1.50 NS2B-NS3prob [126]

DENV3 3U1I 2.30 NS2B-NS3prob complexed with peptide

[BEZ][NLE]KR[OAR]

[177]

DENV3 3U1J 1.80 NS2B-NS3pro complexed with aprotinin [177]

DENV4 2VBC 3.15 NS2B-NS3 full length [178]

DENV4 2WHX 2.20 NS2B-NS3 full length [176]

aProtein data bank (PDB) bNS2-NS3protease (NS2-NS3pro)


teases, identification of potential promiscuous behavior and the mechanism of inhibition. Generally, inhibition of DENV type 4 NS2B-NS3 protease activities is reduced by 2 to 5 fold compared to the other serotypes. The highly positively charged nature of the compound suggested that they acted as substrate mimetics and in-teracted with the highly negatively charged non-prime portion of the substrate-binding pocket. Inhibition kinetics study by colorimet-ric pNA assay demonstrates competitive behavior for DENV type 2 and WNV NS2B-NS3 proteases. The compounds only interacted with the non-prime side and catalytic machinery within the active site, indicating that the compounds exclusively bind only to the non-prime side. The Vero cell cytotoxicity was assessed using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bro-mide)-based viability assay and the viral growth was measured by ELISA detection of virally produced envelope protein. The com-pound at 100 M showed low cytotoxicity nor measurable reduc-

tion of DENV type 2 growths in Vero cells, which could be due to their highly cationic nature.

Knehans et al [26] demonstrated the use of in silico fragment-based drug design approach to identify small molecule inhibitors against DENV NS2B-NS3 protease. This approach was believed to be less time consuming compared to the normal high-throughput docking. Instead of docking millions of compounds, only a small set of compounds is required to be docked. The DENV protease model was then subjected to fragment-based drug design approach. Molecular fragments were then generated by retrosynthetically cleaving 14 million compounds of the ZINC screening database. Fragments that displayed high binding affinity towards DENV pro-tease binding site were then identified by high throughput docking with Autodock Vina. Two sets of high-scoring fragments were later chosen based on the S1 and S2 binding pocket scores. High-scoring fragments were then linked to generate a potential DENV protease

Fig. (2). Comparison of the dengue NS2B-NS3 protease [175], and the dengue NS2B-NS3 protease complex with a peptide [177]. Catalytic residues His51, Asp75 and Ser131 are colored in red, the NS2B in green, the NS3pro in cyan and the peptide, [BEZ][NLE]KR[OAR] in yellow. Surface diagram representa-tion of (A) NS2B-NS3pro and (B) NS2B-NS3pro complex with a peptide of [BEZ][NLE]KR[OAR]. Significant conformational changes were observed for the NS2B-NS3pro complex with a peptide in which the hydrophilic -hairpin region of NS2B (green) wrapped around the NS3 protease core in the presence of the peptide as shown in figure 2B. (C) Superposition of NS2B-NS3 (cyan) and NS2B-NS3 complex with a peptide of [BEZ][NLE]KR[OAR] (blue) catalytic sites demonstrated that this region was similarly structured in the two proteins. Catalytic residues His51, Asp75 and Ser131 are colored cyan and blue in the NS2B-NS3 and NS2B-NS3 complex with peptide structures, respectively.


inhibitor. Molecular docking has predicted the binding mode of this potential inhibitor and found that this compound interacted with Asp129 of the S1 pocket via hydrogen bonding and electrostatic interactions. A total of 23 compounds were then tested experimen-tally and only two compounds were discovered to inhibit dengue protease (IC50=7.7 μM and 37.9 μM, respectively). Both com-pounds exhibited almost similar IC50 values in protease assay and may act as covalent inhibitors. The ester group may also react with the nucleophilic groups outside the active site, opening the possibil-ity for unspecific or allosteric binding. However, enzyme kinetic analysis of both compounds indicated a competitive mode of inhibi-tion, as expected by active site directed inhibitors. In this analysis, the first and second compound exhibited an inhibition constant Ki of 2.0 μM and 31.1 μM for DENV type 2, respectively.

Frimayanti et al [27] used fragment-based approach based on natural product extracts (4-hydroxypanduratin A and panduratin A) and synthesized compound, 246 DA. Our previous work indicated that 4-hydroxypanduratin A and panduratin A, isolated from finger root (Boesenbergia rotunda) exhibited inhibitory activities against DENV type 2 NS3pro with Ki value of 21 and 25 μM, respectively. These components of finger root were known to be competitively inhibiting the protease activity [28]. Using fragment-based ap-proach, the ligands were divided into separate portions or frag-ments: hydrophilic, hydrogen bonding and van der Waals frag-ments, followed with docking of each fragment into the active site of DENV type 2 NS2B-NS3pro (PDB code 2FOM) using the MOE software package (Chemical Computing Group). All fragments were minimized before docking. CHARMM27 force field was used for docking purposes. Fragments that formed critical interactions with the binding site of the serine protease NS3 would serve as a starting point, and by linking each fragment, new ligands were con-structed. The new ligands were further docked against NS3pro in a similar manner. The predicted Ki of the fragments and the new ligands were significantly lower when compared to the initial docked ligands, thus indicating tighter binding. The new ligands were able to bind well into the NS3pro active site, suggesting a greater potential as competitive dengue inhibitors.Indeed, as was reported, fragment-based drug design have several advantages such as it is a very fast method, and a large variety of molecules could be generated due to the possibility of enormous combinations of frag-ments to be connected [29].

Major conformational changes and well-defined molecular structures of pre- and post-fusion E-protein present several targets for the design of inhibitors (Fig. 3). The crystal structure of the E-protein indicates a ligand-binding pocket that is filled with a deter-gent molecule, -octyl- -D-glucoside ( -OG) [30]. This finding led to a number of docking studies to identify and optimize possible inhibitors targeting at this position of the E protein [31-33]. One such computational study used a docking program to screen 135,000 compounds for inhibition of E protein via the -OG pocket [34]. Several inhibitors obtained from the docking study displayed micromolar inhibition against DENV, YFV and Kunjin virus. A compound named A5 directly inhibited E protein-mediated fusion as shown by the cell-based fusion assays. Further study and optimization of these compounds could lead to the discovery of potent inhibitors. Besides targeting the -OG pocket, small peptides have been designed against the E protein stem region. The C-terminus of the stem protein is essential for membrane fusion and is adjacent to the membrane-bound anchor region [35, 36]. The conserved sequence-specific binding interactions of the post-fusion DENV E were exploited for the design of peptides inhibitors. The E protein sequence of the stem region is conserved among all the four DENV serotypes and other flaviviruses. Compared to the inhibitors targeting at the -OG pocket, these inhibitors were unique in that they inhibited the virus following entry into the cell, rather than prevention of entry itself. It was speculated that these peptides were

Fig. (3). Conformational changes of the flavivirus envelope (E) glycopro-tein. The elongated E ectodomain has three distinct, structurally defined domains (DI colored in red, DII in yellow, and DIII in blue) that are joined by flexible hinges. The fusion loop is shown in purple. The green colored regions represent the -OG. (A) The pre-fusion E glycoprotein head-to-tail homodimer arrangement looking towards the center of the virus and (B) from the side after 90 °C rotation around its long axis. One monomer is shown as a ribbon diagram with its three domains mostly adopting the -sheet folds. Surface shaded volume represented the other monomer. The DI (red)-DIII (blue) of one of the E protein provides a hydrophobic pocket where the fusion loop (purple) from the other E molecule is buried. (B) The side views indicated that each E ectodomain monomer elongates parallel to the viral lipid envelope as the -helical stem region (light green) and a two-helix transmembrane anchor (light blue). The -helical stem region is half buried in the outer lipid layer. The C-terminus of the E ectodomain is con-nected to the transmembrane helices via the -helical stem. Two antiparallel transmembrane helices are the M protein represented by the light orange diagram is buried under E protein layer. Only one stem-anchor region is shown for one E monomer. (C) The E-glycoprotein homotrimer in the post-fusion arrangement shown from the top and (D) from the side after a 90° rotation. (D) The E monomers are oriented parallel to each other with their peptides fusion (purple) exposed on one end of the trimer. DIII (blue) show major conformational changes where its move to the side of DI (red) and towards DII (yellow). The stem region (dashed in light green) is predicted to bind in the hydrophobic groove created by the interface of two neighboring DIIs (yellow) of the E trimer that extend towards the fusion loop (purple). Reproduced from Kaufmann and Rossmann [181] with permission.


able to bind to the virion in a non-specific manner as they were taken up by the cells. In the late endosome, the virus is exposed to the low pH causing conformational rearrangements, thus inducing the tight binding of the peptides, and inhibition of membrane fusion [37-39]. A similar study done by Costin et al [39] used the pre-fusion DENV type 2 E protein and computer modeling techniques to discover peptide inhibitors. Rough outer surface morphology was observed on particles treated with the peptide inhibitors, compared to the untreated smooth outer surface of mature DENV, which indi-cates that the E proteins were likely rearranged. The virion structure was grossly affected as confirmed by the treated virion structures that were no longer icosahedral [39]. Other studies showed that these inhibitors were likely to inhibit the interaction of the trans-membrane regions and the fusion loop [40].

3. WEST NILE VIRUS – NEEDS AN INTRO PARAGRAPH JUST LIKE DV AND JE.

West Nile virus (WNV) infections first appeared in the United States in 1999 (Centers for Disease Control and Prevention, June 2013). This virus causes epidemics of febrile illness, meningitis, encephalitis, and flaccid paralysis. Infected mosquitoes spread the virus that causes it. WNV has a spherical, enveloped capsid with a diameter of ~50 nm and contains single-stranded RNA. The virus has 2 genetic lineages; lineage 1 strains are found in North Amer-ica, Europe, Africa, Asia and Australia; lineage 2 strains have been isolated only in sub-Saharan Africa and Madagascar [41].

Due to the well-established antihelicase activities of numerous DNA-interacting agents, several NTPase/helicase inhibitors that act by interacting with a DNA substrate have been developed against WNV. In vitro studies showed that the antiviral agent ribavirin (1-

-D-ribofuranosyl-1H-1,2,4-triazole-3 carboxamide), a prodrug which resembles purine RNA nucleotides was active against WNV, and had been considered as a specific treatment for WNV diseases [42-44]. It was known to exhibit several antiviral properties such as inhibition of inosine monophosphate dehydrogenase (IMPDH) that is required for de novo guanine synthesis, which in turn is needed for viral replication [45]. In addition to the fact that thein vitro study indicated that only high concentrations of ribavirin could inhibit replication of WNV, its clinical efficacy was also restricted because of the lack of controlled clinical trial. Furthermore, ri-bavirin lacked the ability to cross the blood-brain barrier [46]. Other nucleoside inhibitor like cyclopentenylcytosine, mycophenolic acid, 6-azauridine acetate, pyrazofurin, 2-thio-azauridine have been re-ported to have antiviral activity against WNV [44]. Among these inhibitors, mycophenolic acid was the most promising inhibitors since it exhibited potency at therapeutic concentration and inhibited other flaviviruses [47]. The inhibitory mechanism of mycophenolic acid was by directly binding to IMPDH [48] thus decreasing guanosine level to a point where viral RNA synthesis was inter-rupted [49]. A patent described mycophenolic acid as a carbarmate

prodrug, which was metabolized in the liver to produce an active moiety of mycophenolic acid but with undesirable pharmacological properties [50]. However, this prodrug could provide a lead for flavivirus inhibition and could be improved by chemical modifica-tion. Drugs like ribavirin and mycophenolate are nucleoside inhibi-tors that were developed against the host factors. Recent in vitro and in vivo study indicated that mycophenolic acid exhibited poten-tial therapeutic activity against JEV with an IC50 of 3.1 μg/ml, and a therapeutic index of 16 [51]. Borowski et al showed that imi-dazo[4,5-d]pyridine-4,7(5H, 6H)-dione inhibited the helicase activ-ity of WNV NS3 with an IC50 of 30 μM. This compound also inhib-ited WNV replication[52]. Nucleoside analog inhibitors developed for HCV, 2’-C-methyl-adenosine and 2’-C-methyl-guanosine also showed inhibition towards WNV with EC50 of 5.1 μM and 30 μM, respectively [53].

To date, there are three crystal structures of WNV NS2B-NS3pro in complex with inhibitors and one apo crystal structure of WNV NS2-NS3pro (Table 2). Like DENV protease, the catalytic triad (His51-Asp75-Ser135) is located at the cleft between the two

-barrels with arginine preferably located at the non-prime site P1 position of the protease active site, while arginine or lysine at the P2 position, thus highlighting the importance of electrostatic inter-actions with the negative S1 and S2 pockets [54]. Subsequently, Stoermer et al reported potent and small molecule inhibitors, cati-onic tripeptides with non-peptidic at the N-terminus and aldehyde at the C-terminus for WNV protease [55]. One of the compounds with Ki of 9 nM was stable in serum (more than 90% intact after 3 hrs at 37°C), cell permeable and exhibits antiviral activity (IC50=1.6 μM) without cytotoxicity (IC50>400 μM). Recently, Shüller et al synthe-sized and analyzed a series of 17 tripeptides with the sequence X-KRR-H and X-KKR-H, where X is a varying N-terminal cap group [56]. The nature of the cap groups was selected to cover a large range of physicochemical properties such as length, polarity, flexi-bility and bulkiness. Bulk syntheses of tripeptide aldehydes were done according to standard solution-phase chemistry [57]. The pep-tides showed strong inhibition against WNV NS3 protease com-pared to DENV type 2 protease where the IC50 values were in sub-micromolar and micromolar ranges, respectively.

A non-peptidic inhibitor (4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl)-methylsulfanyl-methanimidamide was identi-fied to be a lead compound against WNV NS3 protease from auto-mated fragment-based docking study [58]. The compound showed a good ratio of binding affinity versus molecular weight, with ligand efficiency of 0.33 kcal/mol per non-hydrogen atom. This compound was discovered through screening of approximately 12,000 com-pounds from the iResearch database (ChemNavigator Inc. 2006) using Filter v2.0.1 (OpenEye Scientific Software, Inc). Selections of compounds were based on the structural information of the S1 to S3 pockets of the active site which contained 19 hydrogen bond acceptors. The binding free energy was calculated using the LIECE

Table 2. X-ray crystal structures available in PDBa for WNV protease.

PDB Resolution

(Å)

Comment Reference

2FP7 1.68 NS2B-NS3prob complexed with Bz-NKRR-H [126]

2GGV 1.80 NS2B-NS3prob [179]

2IJO

2.30

NS2B-NS3prob complexed with aprotinin/BPTI [179]

3E90 2.40 NS2B-NS3prob complexed with Naph-KK-H [180]

aProtein data bank (PDB) bNS2-NS3protease (NS2-NS3pro)


(linear interaction energy model with continuum electrostatic solva-tion) approach [59] and the compound libraries were prepared using the CHARMM (Chemistry at HARvard Macromolecular Mechan-ics) program [60]. Based on the most favorable LIECE energies and the most intermolecular hydrogen bond formation, 651 compounds were selected for visual inspection. Only 22 compounds were cho-sen for 15N-HSQC NMR spectral experiment to determine the binding mode and inhibitor binding specificity into the active site of NS3 protease. Two other experimental techniques, namely the en-zymatic assay and tryptophan fluorescence quenching assay were performed to determine the inhibitory activities. Su et al [61] de-scribed in detail the usage of NMR spectroscopy to determine the binding mode of low molecular mass inhibitors of the WNV NS2B-NS3pro.

A novel ligand with potential inhibitory activity against the NS3 protease of WNV, as well as for DENV, JEV, Murray Valley encephalitis virus (MVEV), and Usutu virus (USUV), has been identified via in silico techniques[62]. In this study, homology models for WNV, DENV, JEV, and USUV were constructed using MODELLER 9v7 based on the available NS3 protein structure of MVEV in the PDB (PDBid 2WV9) as template. The protease cata-lytic triad consisting of His51, Asp75, and Ser175 were determined based on a previous study [63]. The lead compound or the ‘seed molecule’ was revealed based on the previous available inhibitor studies for NS3 protein [64]. Following the ‘Lipinski’s Rule of Five’, fragment 1H-1,2,4-triazole was identified as the ‘seed mole-cule’ and docked into the MVEV NS3 protein. The resulting ‘seed molecule’ conformation was used for building 100 novel ligands for the inhibitory site in NS3 protein using the Ligbuilder v1.2 software (http: //mdl.ipc.pku.edu.cn/drug_design/work/ligbuilder.html). From virtual screening, only 10 out of 100 ligands were selected based on the maximum binding affinity. Finally, out of the10 ligands, only one was used for docking analysis with WNV, DENV, JEV, MVEV and USUV proteases. The overall free energy of bind-ing ( G bind) of the docked protease-ligand complexes ranged from -5.9 to -6.7 kcal/mol. The negative and low values of G bind indicated strong favorable binding interactions between the prote-ases and the ligand (in its most favorable conformation). The se-lected ligand could be considered as a potential broad inhibitor against NS3 protein of flavivirus family since the NS3 protein of WNV, DENV, JEV, MVEV and USUV exhibit similar structural features.

A series of meta and para aminobenzamide scaffold derivatives were synthesized, followed by in vitro screening against WNV and DENV proteases [65]. The Ki values of the most potent compound, namely 7n, against WNV and DENV proteases were 5.55 and 8.77 μM, respectively. According to the kinetic data, compound 7n ex-hibited competitive binding mode against both proteases. The com-petitive binding mode was further supported by the predicted con-formation of compound 7n docked at the active site. This article provides an example on how iterative medicinal chemis-try/structure-activity relationship studies and in vitro screening might develop new chemical scaffolds to yield potent inhibitors. A cinnamyl moiety combined with the ketoamide electrophiles has been identified to be potent inhibitors for WNV protease as well as for DENV protease [66]. Sufficient potency and selectivity were achieved with this covalent mode of inhibition. In another study [67], the promising cinnamyl moiety was combined with an alterna-tive electrophile, the nitrile group, resulting in 3-aryl-2-cyano-acyrylamide formation. A total of 86 analogs were synthesized based on the 3-aryl-2-cyanoacrylamide scaffolds to study the struc-ture-activity relationships in detail. The most potent inhibitor for WNV protease was the para-hydroxy substituted analog with Ki value of 44.6 μM. This analog also inhibited DENV protease with Ki value of 35.7 μM. The discovery of chimeric peptide inhibitors, which inhibited the activation of the NS3 protease of a virus from the Flaviviridae family has been described in a patent invented by

Glay et al [68]. However, these chimeric peptides were not capable of penetrating the target cells, thus, did not inhibit the viral infec-tion in cell lines. The inhibition of the viral infection was achieved by combining the chimeric peptides with a cell-penetrating seg-ment.

The present WNV NS2B-NS3 inhibitors are mainly peptide-based that target directly at the active site of NS3pro domain with Ki values in a nanomolar range [56, 69]. However, the in vivo prac-tical use of these peptide inhibitors is limited. Several modestly successful high-throughput screening (HTS) assays to identify al-losteric inhibitor against WNV NS2B-NS3pro have been devel-oped. Johnston et al [70] identified a noncompetitive inhibitor of WNV protease, a common 5-amino-1-(phenyl)sulfonyl-pyrazol-3-yl scaffold, by HTS of a small library (screened 65,000 compounds) through the National Institutes of Health (NIH) Molecular Libraries Initiative (MLI). These allosteric inhibitor interacted with the NS2B-binding cavity in the NS3pro domain, and the interaction intervened with the productive conformation of the active NS2B-NS3pro proteins complex [71]. The discovery of allosteric inhibi-tors could be a more superior drug discovery strategy since the active site of the human and viral serine protease is largely con-served.

Shiryaev et al [72] applied a focused structure-based approach to identify de novo allosteric small molecule against NS2B-NS3pro using virtual ligand screening (VLS) technology. In this study, about 275,000 molecules, openly available from NCI compound library of the Developmental Therapeutics Program NCI/NIH (http: //dtp.nci.nih.gov) have been used for high throughput in silico docking. The crystal structure of NS2B-NS3pro protein (PDB code 2IJO) was used in the docking simulations with the NS2B removed from the protein complex. A binary space partitioning tree-like structure was applied in order to increase the VLS performance. The Q-MOL L.L.C., San Diego, CA molecular modeling package with the Optimized Potential for Liquid Simulations (OPLS) force field [73] was used for protein-ligand complex optimization. Exten-sive experimental in vitro and cell based tests were conducted which led to the identification of three most promising novel inhibi-tory scaffolds. In vitro experiments indicated that these inhibitory scaffolds exhibited nanomolar level of inhibitions towards WNV NS2B-NS3pro. Furthermore no cross-reactivity was observed when these inhibitors were tested against human furin, and they moder-ately inhibited the highly homologous DENV NS2B-NS3pro. Cell based experiments confirmed the specificity of the inhibitors since flaviviral replication was inhibited. Recent study by Samanta et al [74] discovered a non-peptidic zwitterion-type inhibitor, a 9,10-dihydro-3H,4aH-1,3,9,10a-tetraazaphenanthren-4-one scaffold via screening a small library of 110 compounds with various scaffolds. The compounds were screened at 100 μM to filter potential inhibi-tors in the preliminary WNV NS2B-NS3 protease inhibition assay. Focused optimized libraries of 69 compounds were synthesis based on 9,10-dihydro-3H,4aH-1,3, 9,10a-tetraazaphenanthren-4-one scaffold and thus led to the discovery of a novel uncompetitive inhibitor 1a40 with IC50 of 5.41 ± 0.45 μM. The binding mode of compound1a40 was identified using molecular docking simulation with the AutoDock4 program.

A recent report by Jia et al [75] showed palmatine, a chemical compound from Coptis chinensis Franch could specifically inhibit WNV NS2B-NS3 protease. Palmatine was demonstrated to non-competetitively inhibit protease activity with IC50 of 96 μM. No detectable cytotoxicity was detected (EC50=3.6 μM and CC50 [a 50% cytotoxicity concentration]=1,031 μM). Dengue virus and yellow fever virus were also suppressed by palmatine in a dose dependent manner.

The envelope glycoprotein (E) of flaviviruses belongs to class II and comprises mainly of beta sheets. Since viral fusion proteins mediate cell entry by undergoing a series of conformational changes, peptides that mimic portions of these beta sheets might


inhibit structural rearrangements of the fusion proteins, thus prevent viral infection. Hrobowski et al [76] used physiochemical algo-rithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) [77] in combination with known structural data to iden-tify potential peptide inhibitors of WNV as well as DENV infectiv-ity that targeted the viral E protein. The peptides were designed based on the known secondary structures of several subdomains of E protein, which included the fusion peptide domain, a portion of sub-domain IIb, the pre-anchor stem region following domain III, and the transmembrane domain. The selected peptides were synthe-sized via solid-phase conventional N- -9-flurenylmethyl-oxy-carbonyl chemistry (Genemed Synthesis, San Francisco, CA), puri-fied by reverse-phase high performance liquid chromatography and confirmed by amino acid analysis and electrospray masspectrome-try. Unlike the inhibition of flavivirus by neutralizing antibodies that appeared to be involved in receptor blocking by binding to domain III, peptides designed based on domain IIb and the pre-anchor stem region showed to be involved in structural rearrange-ments during fusion, rather than making direct contact with cellular receptors. The viral entry inhibitions by the peptides were con-firmed by viral inhibition assays with IC50 in the 10 μM range. Cell viability assayed with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay indicated that these inhibitory peptides were noncytotoxic and acted in a sequence specific man-ner.

4. JAPANESE ENCEPHALITIS AND YELLOW FEVER VI-RUSES

Japanese encephalitis (JE) causing acute infection and inflam-mation of the brain is mainly a pediatric disease with 30,000-50,000 cases reported annually [78]. Around half of the JE survivors have severe neurological sequel with JE causing high fatality rate of 30%. Despite the catastrophes it causes, JE has remained a tropical disease uncommon in the West [79, 80]. Like other flavivirus infec-tions, currently there is no antiviral therapy to treat the disease and to date, the main strategies to control JE infections are by preven-tive methods such as vaccination and mosquito control [81-83]. Although there are five genotypes (I-V) of the envelope gene (E), the current approved vaccines are designed only for genotype III virus [84]. Although commercial vaccines are available which have reducedthe number of JE cases, about 55,000 (81%) of the total cases still exist in the areas even with well-established JE vaccina-tion program [85]. The drawback of the current available JE vac-cines, although they are relatively safe and effective, is that multi-ple doses are required [86, 87]. In addition, the delivery cost to the poor communities still remains a challenge[88]. Thus, at present antiviral therapy remains a useful alternative when the vaccination system does not cover the whole population at risk of JEV infec-tion.

Unlike DENV and WNV proteases, which are extensively char-acterized as potential drug targets [89, 90], JEV protease is com-paratively less studied with regards to its structure-activity relation-ship. To date, there is no x-ray structure available for JEV protease (NS3) complexed with NS2B. Currently, only one x-ray crystal structure of the catalytic domain of JEV NS3 helicase/nucleoside triosphatase is available at a resolution of 1.8 Å [91]. The crystal structure of JEV NS3 consists of three domains, with an asymmet-ric distribution of charges on its surface, and has a tunnel large enough to fit single-stranded RNA. The NTP-binding pocket is made of three motifs; namely motif I (Walker A), II (Walker B) and VI. Based on mutagenesis study, all residues in Walker A motif (Gly199, Lys200 and Thr201), polar residues within the NTP-binding pocket (Gln457, Arg461 and Arg464), and also Arg458 (outside of the pocket in motif VI) were important for NTPase and helicase activities in viral replication. Consequently, NS3 heli-case/NTPase seems to be a promising antiviral drug target.

Borowski et al [92] reported that the helicase activities could be inhibited by the peptides containing Arg-rich conserved motif (mo-tif VI) of the NS3 helicase/ NTPase. The inhibition of the viral enzyme activity may result from blockade of the intramolecular interactions of the NTPase/helicase domains. The JEV peptides (1959 to 1975 amino acids) that were homologous to the 17 pep-tides located in motif IV within domain 2 of HCV NS3 heli-case/NTPase (1484 to1500 amino acids) were synthesized and evaluated for NS3 helicase/NTPase inhibition activity. Shortened versions of the 17 peptides (5-, 8-, 11-, 14-peptide) were synthe-sized and tested for inhibition of the unwinding activity of the en-zyme. JEV peptides moderately inhibited JEV helicase activity with an IC50 of 196 ± 9.4 μM. Kinetic analysis showed that the binding of the peptides did not interfere with the NTPase activity of the enzyme, but could be optimized and serve as lead compounds to reduce virus propagation.

Recently, Kaczor and Matosiuk [93] used structure-based vir-tual screening to identify novel inhibitors against NS3 heli-case/NTPase. This study reported, for the first time, new potential competitive JEV NS3 helicase/NTPase inhibitors that are structur-ally different from nucleosides and their analogs. A natural ligand, ATP, and two known JEV NS3 helicase/NTPase inhibitors, namely compounds 1 and 2[94] were docked into the binding pocket using the flexible docking method of Surflex [95] incorporated in SY-BYL8.0. Ligand-enzyme complex obtained for ATP and inhibitors (compounds 1 and 2) of NS3 helicase/NTPase were further opti-mized with Yamber3 force field energy in YASARA Dynamics [96]. This step was performed for optimization of the residue con-formations located at the binding pocket, thus provided the final enzyme structure that was possible for use in virtual screening. The reported mutagenesis study and literature data regarding the mechanism of ATP hydrolysis by helicase/NTPase [97] agreed with the obtained binding mode of ATP interactions with the enzyme. The optimized ligand-enzyme complexes were then utilized to gen-erate structure-based pharmacophore model, using Interaction Gen-eration module of DISCOVERY STUDIO 2.1, which contained 15 hydrogen bond donors, 3 hydrogen acceptors and no lipophilic moieties. The pharmacophore model was verified positively as it recognized the known inhibitors 1-2 as hits and sensitive to false positive as none of the noncompetitive inhibitors 3-4 and inactive compounds 5-7 was interacting with the ATP-binding site in the virtual screening of a database of 10,000 ZINC drug-like com-pounds. In the subsequent screening, 15 hits with the highest scores were selected from about 1,161,000 lead-like compounds freely available from ZINC database [98]. The screening was performed using the Screen Library module of DISCOVERY STUDIO 2.1, based on consensus scoring procedure, which combined the screen-ing and docking results [99]. Based on the calculated Preadmet server (preadmet.bmdrc.org), most of the compounds were simu-lated to not cross the blood-brain barrier easily, except for 5 com-pounds which could be good candidates for further ADMET opti-mization. Nevertheless, the antiviral activities of these compounds warrant experimental validation. Despite this, reliability of the re-ported computational results were enhanced by three factors: first, a refined crystal structure of the catalytic domain of JEV NS3 heli-case/NTPase was used to construct the pharmacophore model; sec-ond, residues contributing to the ATP-binding site were discovered in the mutational studies; and third, usage of the consensus screen-ing procedure improved the hit list.

Currently there are two available structure of envelope protein domain III of JEV solved by NMR (PDB code 1PJW) [100] and x-ray crystallography (PDB code 3P54) [101]. Li et al [102] showed that the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII exhibited antiviral activities against JEV (NJ 2008 strain). The EDIII protein at a concentration of 25 μg/ml and loop3 peptide at a concentration of 10 ± 1.4 μM exhib-ited the ability to 50% inhibit JEV infection in BHK-21 cells. The


EDIII of JEV consists of six antiparallel strands namely 1, 2, 3, 4, 5, and 6, which correspond to strand A, B, C, D, E, F and G

as in WNV and YFV. The antiviral activity of EDIII domain was determined by using plaque assay, quantitative PCR and western blot at 48 hours post JEV infection (multiplicity of infec-tion/MOI=0.02). According to the NMR structure, four of the ex-posed peptide loops before or between the -barrel loops of EDIII, namely loop1 (amino acids 302-309), loop2 (amino acids 329-337), loop3 (amino acids 362-370) and loop4 (amino acids 385-391), were chosen for antiviral screening against JEV. The idea of syn-thesizing these exposed peptide loops located on the surface of EDIII was based on two information: that the EDIII protein can inhibit virus entry as shown in DENV type 1 and 2 as well as in WNV [103-105], and several studies showed that some peptides located on the envelope protein exhibited the ability to inhibit virus infection as in DENV type 2 and WNV [106-108]. Even at 200 μM, the loop peptides did not exhibit significant cytotoxicity in BHK-21 cells. In order to address whether loop3 inhibition was sequence-specific, a scrambled loop3 was synthesized. A plaque assay showed that the scrambled loop3 did not inhibit JEV infection. The binding of virus after incubation with peptide was detected with ELISA (Enzyme-linked immunosorbent assay). Significant de-crease in virus attachment in the different concentration of the pep-tide loop3 was detected especially at 100 μM (with viral load of p < 0.02). However no significant changes in the JEV titer was ob-served when the peptide loop3 was added after virus infection, demonstrating that peptide loop3 exerted its inhibitory ability via interfering with virus attachment. In vivo study showed that, when administered before lethal JEV challenge, peptide loop3 could sig-nificantly increase the survival rate in mice. This study indicated that other peptides of the envelope protein might also be important for the post-binding process of JEV entry since JEV DEIII and the loop3 peptide could not inhibit JEV infection completely in BHK-21 and in mice. Nevertheless due to the conservation between enve-lope protein domain III and the loop3 peptides, the inhibitory loop3 could serve as a lead to develop new therapeutics for the JEV of different genoypes or other flavivirus infections.

Yellow fever virus (YFV) is one of the most lethal and feared diseases. It originates from Africa and spread to the western hemi-sphere during the slave trade era, with the first epidemic reported in 1648 in the Yucatan [109]. However for unknown reason, yellow fever has not spread to Asia yet. The illness caused by YFV ranges from a self-limited febrile illness to severe liver disease with bleed-ing. Like other flaviviruses, in most cases, patients with the most clinically severe cases of YF are treated by supportive care. As for JEV, vaccines too have been an integral part in fighting YFV. The first attenuated vaccine against YFV namely 17D strain was dis-covered by Theiler and coworker. This work was awarded the No-bel Prize in 1951. The development of live-attenuated 17D vaccines in the 1930s was a turning point in the history of the disease. The 17D vaccine has been considered to be one of the safest and most effective live virus vaccines ever developed [110, 111]. Around the same time, another life-attenuated vaccine was designed, called the French neutrophic vaccine (FNV) and was used widely in the 1960s in francophone Africa, and led to the disappearance of the disease [110]. However recent re-emergence has been reported in South America and Africa and has brought this virus back to the public attention [112]. This could be most likely due to the decline of vac-cination, unreachable populations, lack of mosquito control and increased transportation of people and goods. According to Khasnatinov et al [113], currently there is a major gap of knowledge of how to manage and treat sick patients infected with JEV or manage the serious vaccine-associated adverse events. To date, no effective specific antiviral therapy for yellow fever has been identified. So there is a clear need for safe and effective drugs to treat patients in each steps of the disease progression. Currently there are eleven JEV protein structures (apo and ligand) consist of envelope (E), NS3 helicase, and NS5 methyltransferase available in Protein Data Bank solved by NMR and X-ray crystallography [114-

Bank solved by NMR and X-ray crystallography [114-116]. The ligand structure of helicase consists of complex with ADP (PDB code 1YMF) and ligand structure of methyltransferase consists of complexes with S-adenosyl-L-homocysteine (PDB code 3EVA, 3EVB), GTP (PDB code 3EVD), GpppA (PDB code 3EVE), and Me7-GpppA (PDB code 3EVF).

Although NS3 protease is an attractive potential target in search for inhibitors in JEV,DENV and WNV, this is not the case for YFV. Since DENV, WNV, and YFV NS2B-NS3 proteases share similar cleavage recognition sites [117, 118], Löhr et al [119] pos-tulated that peptidic inhibitors designed against DENV [120, 121] and WNV [25, 69] could potentially work on the YFV protease. A construct of YFV protease namely YFV CF40-gly-NS3pro190 was designed with 47 core amino acids of NS2B [122] and the N-terminal 190 amino acids NS3 protease domain via a linker Gly4-Ser-Gly4. Active site-titration with the competitive, tight-binding inhibitor aprotinin (Ki=17.6±0.56 nM) and curve fitting based on the Morrison equation [123] indicates that the total amount of ac-tive protein in the purified was 100%. Proteolytic activity was tested with two different substrates; the tripeptide Boc-GRR-AMC [124, 125] and the tetrapeptide Bz-nKRR-AMC [126]. As in DENV [124], the enzyme showed nearly 10-fold higher affinity and proc-essed it for 34-fold greater efficiency for tetrapeptide Bz-nKRR-AMC substrate while the catalytic efficiency for Boc-GRR-AMC was comparable to the work done by Bessaud et al [125]. The in-creased catalytic efficiency in YFV and DENV for tetrapeptide Bz-nKRR-AMC could be due to the additional P4 residue of norleucine in the tetrapeptide sequence. Based on this result, further YFV pro-tease enzyme characterization and inhibitor studies were explored using tetrapeptide Bz-nKRR-AMC substrate. The Ki value of sub-strate-based peptide Bz-nKRR-H for DENV is 5.8 μM [120, 121] and 2.1 μM for WNV [69]. Thirteen peptide-based inhibitors were synthesized according to the aldehyde derivative of the substrate-based peptide Bz-nKRR-H. Evaluation of the importance of the P4-P3-P2-P1 positions was carried out. The potency of Bz-nKRR-H against YFV was nearly 15-fold more potent than DENV and 5-fold better than WNV. According to the enzyme-inhibitor constant val-ues, replacement with electrophilic boronic acid group improved the potency to almost 8-fold while complete loss and significant loss of activities were observed when P1 (Arg) and P2 (Arg) were replaced with either Ala, Phe, or Lys. Replacement of P3 (Lys) and P4 (Nle) with either Ala or Phe and shortening to tri- and dipeptides did not significantly affect the inhibitory activity. These results showed that the major determinants of the peptidic binding were S1 and S2 subsites. In order to understand the binding mode of the inhibitors, a homology model approach was taken where the crystal structure of WNV NS2B-NS3 protease in complex with Bz-nKRR-H (PDB code 2FP7) was used as the template. Prime 1.2 (Schrödinger) was used for performing the homology modeling, and the docking of individual peptidic aldehydes was done using SYBYL 6.9 and further optimized with MacroModel 9.0. The YFV homolog model agreed with the kinetic analysis data where the S1 and S2 pockets were the dominant interaction sites for the peptidic inhibitor which resembled the WNV crystal structure [126]. As in YFV protease inhibition study, WNV protease shows that the re-placement of the Arg with Lys in P2 position was tolerated but not in P1 [69, 126]. However this was not the case for DENV protease where Lys replacement in P1 and P2 positions were not tolerated [121], suggesting that the DENV S1 pocket might be different. The knowledge obtained from this paper indicates that it may be possi-ble to design pan-flavivirus drugs against NS2B-NS3 protease and the peptidic inhibitors can be used as a starting point for rational drug design.

Recently Mastrangelo group [127] discovered that anti-helminthic drug ivermectin as a highly potent inhibitor of YFV replication (sub-nanomolar EC50 values). This drug also selectively inhibited cell culture of JEV, TBEV and DENV type 2 (in sub-


micromolar EC50values). Furthermore in vitro analysis showed that ivermectin inhibited the YFV, WNV and DENV type 2 helicase domains at sub-micromolar concentrations. Ivermectin has been licensed for more than 20 years for the treatment of parasitic infec-tions in man. The idea was to target specifically against the RNA binding and unwinding mechanisms mediated by NS3 helicase [128, 129] and not against the ATP-binding pocket. This was due to the ATP-mimetic molecules designed as inhibitors against NS3 helicase may create adverse effects on the host cell since ATP is the key nucleotide of host cell metabolism. The group used insilico docking to search for possible inhibitors targeting at ssRNA access site of NS3 helicase. A modelled of WNV NS3 helicase complex with RNA was built by superimposition of the WNV NS3 helicase (PDB code 2QEQ) [129] with the DNA-bound bacterial helicase PcrA (PDB code 3PJR) [130] since when this work was initiated, no DENV or YFV helicase in complex with RNA structure was available. The modeled of WNV NS3 helicase complex with RNA was used to locate the putative ssRNA access within the WNV NS3 helicase domain. Later, a crystal structure of DENV in complex with ssRNA was solved (PDB code 2JLU, 2JLW) [131] and con-firmed the model-based prediction. The predicted putative ssRNA binding site was then explored for inhibitors binding via in silico docking low molecular weight of 1280 commercially compounds from the virtual Library of Pharmaceutically Active Compounds (LOPAC) accessed from Sigma-Aldrich. The docking was per-formed with the AutoDock4 sofware package. Three compounds with free binding energy ( G) between -11.5 to -9.5 kcal/mol for WNV NS3 helicase were identified from the docking search were ivermectin, ouabain (a poisonous cardiac glycoside) and paromo-mycin sulphate (an aminoglycoside antibiotic). These commercially available drugs were obtained from Sigma-Aldrich and chosen for further biochemical evaluation. Two different types of helicase assays were performed in order to show that the selected drugs inhibit the NS3 helicase unwinding activity were; an assay using radiolabelled double-stranded RNA substrate in the presence of Mg2+ and ATP [132] and FRET-based helicase assay [133]. The ATPase activity of NS3 helicase was not affected by ivermectin at a concentration of 1 μM as indicated by NS3 ATPase assay, showing that this compound did not restrict the helicase ATP binding site. The ivermectin was predicted to bind at a distance of ~25 Å from the ATP binding site. The NS5 RdRp activity assay showed that ivermectin at concentration of 1 μM did not inhibit NS5 RdRp ac-tivity of RdRp domains and the NS5 full-length proteins from DENV and WNV. Furthermore, thermofluorometric assays indi-cated that the inhibition of helicase activity dids not result from ligand-induced protein aggregation or denaturation.Specificity of ivermectin for the flaviruses helicase (WNV, DENV, JEV, TBEV) was confirmed since activity assays of HCV helicase was not inhib-ited by ivermectin (up to 100 μM). HCV was chosen for compari-son since the virus belongs to the family Flaviviridae and is the closest to flaviruses helicase. Due to the failure in obtaining crystal structure of the protein-ligand complex, in silico approached was used to simulate the conformations of ivermectin inside the ssRNA access site in order to characterize the part of protein involved in ivermectin binding. Based on virtual docking, two amino acid namely Thr408-Asp409 of DENV, Thr409-Asp410 of WNV and Thr413-Asp414 were found to frequently interacts with ivermectin. Mutation of Thr to Ile and Asp to Glu preserved the double-stranded RNA unwinding activity but were not inhibited by iver-mectin. This result indicated that ivermectin closely interact with these residues thus support the predicted structural analysis of ivermectin bind at the single-stranded RNA access site. Kinetic assays showed that the mechanism of invermectin is via noncom-petitive inhibition by binding to NS3 helicase/RNA complex to inhibit the unwinding activity. According to a time-of-drug-addition assay, ivermectin is only active during the first 14 hours after virus enters the cells. This meant that the compound isonly effective in certain phase of the viral life cycle where the NS3 helicase is func-

tionally active [134]. However other mechanisms besides helicase inhibition may contribute to the action of ivermection inhibition in cell culture since the EC50 value in YFV cell culture is 0.5 nM while the IC50 value of NS3 helicase of YFV is 120 nM. Since ivermectin has been used in man for the treatment of a variety of parasitic disease for more than 20 years, the potential of ivermectin to go through clinical trials for the treatment of life-threatening flaviviruses may not require much effort. Currently, ivermectin has been patented for the treatment, prevention or amelioration of flavivirus infections [135].

5. HEPATITIS C VIRUS

HCV infection exists throughout the world, and believed to be transmitted only by blood. Until year 1990/91 where anti-HCV screening tests for blood donors were introduced in Europe and the United States, HCV remain the major cause of transfusion-associated hepatitis [136]. Acute infection is generally asympto-matic thus the incidence of HCV on a global scale is not well known [137]. To date there are no vaccines against HCV. Accord-ing to World Health Organization (WHO) there are more than 170 million chronic carriers who are at risk of developing liver cancer or/and liver cirrhosis with about 3% of the world’s population in-fected with HCV [138]. HCV is currently classified into six major genotypes [139-144], which differ from each other by more than 30% at the nucleotides level. Although HCV share some similari-ties with other members of theFlavivirus genus, it does exhibit dif-ferences in the genome organization as shown in (Fig. 4) where the HCV 5’UTR is not capped. The HCV NS5B RdRp was extensively studied in the hope to find a novel drug to treat HCV since this protein possesses pivotal role in the synthesis and replication of viral RNA [143]. Several inhibitors of the NS5B RdRp have been developed and currently are in preclinical and clinical development (for example TMC649128, R-7128, PSI-7977, PSI-938, INX-189, IDX184) [145]. Another traditional antiviral target besides NS5B is the NS3 protease. Despite the catalytic site being a shallow and largely hydrophobic groove as observed in DENV and WNV NS3 protease thus making it a difficult target, several compounds inhibi-tors of the NS3 protease of HCV have either been approved for use in humans (for example telaprevir (VX-950) and boceprevir (SCH503034)) or currently in preclinical and clinical trials (such as BI12202, MK-7009, TMC435, ITMN-191) [146]. Current standard of care for HCV-infected patients consists of combined therapy of PEG IFN- and ribavirin. However this therapy has its drawback as we previously described in the WNV section. The crystal structure of HCV NS5B has right hand architecture with fingers, thumb and palm domains. The active site is located within the palm encircled by the fingers and thumb polymerase subdomains [147, 148]. A spherical hydrophobic pocket-allosteric binding site proximal to the active site has been identified, which comprises of Pro197, Arg200, Leu384, Met414 and Tyr448 [148].

In a study by Patel et al [149], 3D QSAR (quantitative struc-ture-activity relationship) was used to interrogate the inhibitory activity of benzimidazol and its derivatives toward HCV NS5B RdRp. They revealed that for NS5B inhibitors to have substantial inhibitory activity, it requires hydrogen bond donor and receptor groups at the 5-position of the indole ring, and an R substituent at the chiral carbon.This conclusion was made based on the results generated form the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of 67 benzimidazole derivatives as HCV NS5 inhibitor.In this respect, acquisition of a high-resolution NS5B protein structure into the structure-activity analysis (SAR), followed by NMR and modeling studies established the improved structure configuration of ben-zimidazol 5-carboxamide derivatives. The incorporation of the - -disubstituted amino acid at the designated site of the derivatives enhanced the inhibition of the enzymatic activity up to IC50 = 40 nM [150]. However, despite their potencies, further development of these derivatives as potential drug candidates were hampered by the


high EC50 values [151]. This has resulted in the replacement of the benzimidole core of truncated carboxylic acid residues with a greater lipophilic N-methyindole isoesters. This replacement dem-onstrated significant improvement in the in vitrostudy with an EC50 value less then 100 nM [152].

HCV NS3-NS4A is another attractive drug target since it is also essential for the replication of HCV. The crystal structure of HCV NS3-NS4A possesses a catalytic triad consisting of a protease do-main and a helicase domain that are segregated and connected by a single strand [153]. A competitive HCV NS3-NS4A serine protease inhibitor, VX-950 (telaprevir) developed by Vertex and Johnson & Johnson had been recently released into the clinical practice [154]. Telaprevir is a potent peptidomimetic inhibitor of HCV serine pro-tease withan IC50 of 280 nM. It functions by forming a reversible covalent enzyme-inhibitor complex with the viral protease, espe-cially with the genotype 1a HCV, at a steady-state inhibition con-stant (Ki) of 7 nM. Noteworthy, Li and co-workers [155] discovered new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors, which has a smaller molecular weight thanthe first gen-eration drug such as telaprevir. A systematic SAR analyses was performed by exploring various linkers and substitutions around the benzoxaborole moiety as well as the P2* groups in order to achieve better in vivo absorption and bioavailability. The inhibitory activi-ties of the synthesized compounds were evaluated by FRET NS3-NS4A protease domain assays, while in vitro assay was determined by acquiring 1a and 1b HCV replicon. Two compounds were identi-fied with low nanomolar potency. In the FRET NS3-NS4A protease

assay, compound 5g and 17 exhibited IC50 of 1.7 nM and 3.1 nM, respectively. The linker-truncated compound 5j exhibited improved in vivo absorption by 4.9% and oral bioavailability by 4.3%. These findings suggested that further reduction of molecular weight and polar surface area in benzoxaborole-based compounds could pro-vide an effective strategy towards the discovery of drug-like boron-containing HCV protease inhibitiors.

In order to overcome the rapid emergence of the HCV variance that showed resistance towards the first generation HCV protease inhibitors [156], researchers haddeveloped two strategies: screening compound with the clinical HCV protease mutants, and generating improved method in assisting the search for novel HCV inhibitors [157-159]. A new virtual screening method was recently published providing another alternative approach in conducting structure-based drug design [157]. In this method, a new induced-fit docking system termed GENIUS was introduced. As a result, 97 out of 166,206 compounds were identified against HCV NS3-NS4A pro-tease (PDB: 1DXW).Subsequent protease inhibition and cell-based assays identified two potential compounds with similar potency of 10 M and EC50 values of 13 and 23 M. Compound 10, that ex-hibited a potency of 10 M with an IC50 value of 8.58 M and an EC50 value of 12 M was identified as a lead compound for future synthetic development. This compound, unlike the first generation compounds that were on clinical trials [154], lacks an asymmetric carbon and does not have a macrocyclic structure. Novel urea-based HCV protease inhibitors against protease resistant mutants of HCV have been discovered via collaborative effort of GlaxoSmithKline

Fig. (4). HCV genome organization and translation. The HCV genome consists of a positive single-stranded RNA genome (9.6 kb) with a single open reading frame codes for structural and non-structural proteins including the 5’ untranslated region (UTR) with four highly structured domains (I, II, III and IV) and the internal ribosom entry site (IRES), and the 3’ UTR consists of stable stem-loop structures and an internal poly(U)/polyprimidine tract. The single open reading frame codes for polyprotein of slightly more than 3000 amino acids depending on the HCV genotype. The polyprotein enzymatic processing of the 10 HCV proteins are shown. Cleavage site for host-cell signal peptidase are represented by the diamond shape (colored in purple), autocatalytic cleavage by the NS2-NS3 zinc-dependent metalloproteinase of the NS2-NS3 junction is represented by the triangle shape (colored in yellow) and NS3-NS4 serine proteinase com-plex cleavage sites are represented by the clear triangle shape [182-185].


and Ancor Pharmaceuticals [159]. The macrocyclic urea2, a by-product in the synthesis of benzoxaborole 1[191], which showed inhibitory activity against HCV NS3-NS4A offered substantial opportunity for lead compound optimization. Thus, by setting mac-rocyclic urea 2 as a lead compound, 24 urea analogues (namely compounds 3-26) were synthesized and evaluated in panel laborato-ries against a variety of clinical HCV protease mutants Ala156Ser, Ala156Thr, Ala156Val, Asp168Ala, Asp168Val, and Arg155Lys. Several compounds, for examplecompound 12, demonstrated higher potency in HCV replicon assay with an IC50of 0.01 nM, as compared to those leading inhibitors such as ciluprevir, teleprevir and danoprevir that hadIC50> 1.99 nM. In addition, following oral administration in vivo, compound 12exhibited a longhalf-lifeof 8.1 hour in the rat’s liver [159].

Kota and co-workers [161] reported that short peptides derived from the HCV core structural protein possessed antiviral properties. These inhibitors prevented viral core dimerization,thus perturbing viral release. This rational design was based on the idea that pro-tein-protein interactions could be inhibited by peptides derived from one of the interacting partneror by small molecules [162]. The NIH AIDS Research and Reference Program (htt: //www.aidsreagent.org) provided 14 synthetic peptides designed to span the first 109 residues of the HCV core structural protein of the HCV H77 1a genotype. The peptides consisting of 18 residues, with 11 residues apart, were resynthesized with 95% purity or more as verified by HPLC. These peptides were then tested for their inhibi-tory activities using in-house developed Amplified Luminescent Proximity Homogeneous Assay (ALPHA). As a result, two pep-tides demonstrated appreciable inhibition of core dimerization, namely SL173 that showed 68% inhibition and SL174 with 63% inhibition. SL175 peptide, identical to SL174 but with deleted 3 amino acid residues at the C terminal, showed a 50% inhibition activity with an IC50of 21.9 M. A florescence polarization assay revealed that SL175 peptide interactedwith the hydrophilic core protein region, comprising 106 residues with a Kdof 1.9 M. When measured by surface plasmon resonance, peptide SL175 was shown to bind to core protein of 169 residues with a Kd of 7.2 M [160]. However, these three peptides did not inhibit viral replication, but blocked the release of infectious virus when added to HCV-infected cells. Nevertheless these core-derived peptides would be useful as a tool to study the role of core dimerization in the virus life cycle and interaction of core protein with cellular protein such as Dicer [163]. These core-peptides could also act as peptidomimetic drugs against HCV infection with improved affinity and serum stability.

A short peptide derived from the N-terminus of host claudin-1 (CLDN1) protein, which inhibited virus entry at the post binding stage had been identified [64]. This approach was feasible due to the sufficient knowledge of the HCV entry-processing field. It is known that HCV uses at least four cellular membrane proteins for entry: CD81, scavenger receptor B1 (SR-B1), claudin-1 (CLDN1) and occluding (OCLN) [165-168]. The rational design was based on the postulation that HCV entry might be blocked due to the competing peptides derived from host entry factors located on the cell surface and incoming virion. A library consisting of 121 over-lapping 18-mer peptides with 13 amino acids offset spanning the entire protein sequences of human CD81, SR-B1, CLDN1 and OCLN were designed f to inhibit HCV pseudoviral particle (HCVpp) infection of Huh7.5.1 cells. Due to solubility problem, thirty-two peptides were abandoned. Out of 89 peptides, only 2 peptides derived from the first transmembrane region of CLDN1,namely CL58 and CL59, were identified to inhibit HCVpp entry by more than 80% at a concentration of 50 μM. CL58 was more potent in inhibiting HCV entry compared to CL59. To deter-mine the maximal inhibition of CL58, eight additional 18-mer pep-tides (namely CL58.1 until CL58. 8) were synthesized with differ-ent length and sequence. The 18-mer peptides were synthesized

with a-3-amino acid offset, thus extended further into the first transmembrane and extracellular loop region of CLDN1. However none of these peptides showed inhibition to the extent as that shown by the parental CL58. In the following optimization, the length of the peptides were altered by adding or deleting residues from the CL58 C terminus. Shortening the peptide by 2, 4, or 6 amino acids caused the IC50 to drastically increase, while extending the peptide by 2, 4, or 6 amino acids only slightly increased the IC50. Slightly lower IC50was observed with a D-isomer of CL58, thus parental CL58 length and sequence seemed to be the optimum for inhibiting HCV entry. In order to understand how CL58 interacted with HCV envelope (E1 and E2) proteins, a coimmunopreciptation experi-ments using Flag-tagged CL58 were performed. According to the coimmunoprecipitation experiment, CL58 exerted its effect by in-teracting with HCV glycoproteins, since the E1 and E2 envelope proteins of HCV and HCVpp co-precipitated with CL58 tagged with Flag peptide at the C terminus. However, it was unclear how CL58 mediated the inhibition. Cell culture experiments indicated that CL58 did not affect the cellular distribution of CLDN1 and was not cytotoxic even at a concentration 100-fold higher from its IC50as determined using MTT assay.These finding indicated that a new class of inhibitors that blocked virus-host interactions could be developed. Furthermore the strategy described in this study may offer advantages since the inhibition target is the cellular protein thus reduce the likelihood of developing resistance.

An exciting study was performed to possibly overcome the peptide instability using self-assembling nanotubes. Montero and coworkers [169] presented a detailed study on using peptide nano-tubes to effectively block the entry of HCV into target cells. Origi-nally such inhibitors were discovered against bacterial membranes and adenovirus [170-172]. A cyclic D,L- -peptide library was screened for anti-HCV activity and nine promising amphiphilic peptides were identified. The peptides exerted the anti-HCV activi-ties through self-assembly into inhibitory nanotubes which acted after entry and before protein synthesis. The inhibitory nanotubes also controlled the spread of the virus in culture and were likely to interact with a ‘specialized cellular membrane’ to inhibit either membrane fusion or pH control. More studies are needed to deter-mine exactly how these peptides are inhibiting HCV and how to use them against DENV and related flaviruses.

High-throughput screening of a library of more than 1 million small molecules against HCVppwith genotype 1b clinical isolate 432-4 on modified hepatic cell line had led to the discovery of a group of compounds, which inhibited viral entry with the best IC50 of 0.016 M [173]. Synthetic chemistry efforts were used to inter-rogate the SAR of the triazine series (EI-2 - EI-12) using N2-(4-nitrophenyl)-N4-pentyl-6-(2,2,2-trifluoroethoxy)-1,3,5-triazine-2,4-diamine as a lead compound, and were assessed against HCVpp 1a and 1b. As a result, it was found that the para- and meta-methyl analogs delivered a 4-fold greater potency when compared to the nitro analogs. Introduction of para- substituents that closely mim-icked the polarity and shape of the nitro analogs afforded sub-100 nM EC50 values in both HCVpp subtypes. A patent recently in-vented by Gary et al [174] described the isolation of peptide fusion inhibitors for flavivirus infection. The idea of this invention was based on the knowledge that in order to deliver the viral genome into the cell cytoplasm, the virion envelope of flaviviruses and membranes of the target cell had to fuse. It was hoped that by em-ploying peptides or peptide derivatives that blocked the fusion, the entry of the viral genome into the target cell could be blocked.The peptides or peptide derivatives as fusion inhibitors for flavivirus family are identical (at least 70% identity) to the amino acid se-quences of HCV fusion inhibitory peptides. A non-peptide drug can be developed, once an effective inhibitor is identified. The discov-ery of these peptides could serve as lead target for development of peptide drugs to treat flavivirus infections.


6. CONCLUSION

The helicase (NS3), protease (NS2B/NS3) and polymerase (NS5) are enzymes that are traditionally targeted for antiviral drug discovery. This is largely because loss of these specific proteins has proven lethal for virus replication, thus drugs targeted at these en-zymes may reduce viral load. According to the discussed antiviral discovery against flaviviruses (DENV, WNV, JEV, YFV), the complex of NS3 proteases with inhibitors are mostly characterized for DENV, while allosteric inhibitors against NS3 protease have been identified and characterized mainly for WNV. The NS3 heli-case inhibitors have been immensely studied especially in JEV and YFV. Inhibitors developed for NS5 polymerases are mostly charac-terized and studied for HCV where several NS5 polymerases inhibi-tors (nucleoside and non-nucleoside polymerase) have reached preclinical and clinical phases of drug development, and others are commercially available. Limited research publications on small molecule inhibitors against JEV and YFV NS3 serine proteases and NS5 polymerases compared to the other flaviruses may probably be due to the decline in the infection caused by these viruses because of the available vaccines. However, current increase in the JEV and YFV infections in the area where vaccination are available creates a demand to find new antiviral drugs. Due to the existing mutation in the presence of the inhibitors of both NS3 (protease/helicase) and NS5 polymerase, new viral targets are constantly in demand. Cur-rently, the E protein is one of the attractive novel targets for design-ing entry inhibitors for flaviruses. The development of inhibitors against E protein becomes feasible due to the availability of E pro-tein X-ray crystal structures and sufficient information of its func-tion. The E protein as a drug target provides several strategies for designing potent peptide inhibitors for entry and also endosomal escape via inhibiting membrane fusion. The discovery of nanotube inhibitors that target the cellular membrane as shown in HCV pro-vides new strategies for developing fusion inhibitors for other flaviviruses.Another promising strategy for the discovery of new inhibitors against flaviviruses is by targeting the host proteinsthat interact with the flavivirus structural and nonstructural proteins. However, the drawback of targeting the host proteins is that it could provide serious side effects to human. On the other hand, targeting cellular proteins will overcome the viral mutations as seen in clini-cal development of NS3 protease and NS5 polymerase of HCV. Advances in computational techniques and HTS have increased the speed of flaviviral drug development in terms of function and struc-ture determination. In order to use rational drug design approach, sufficient information of the viral protein functions is essential.The possibility to precisely target specific viral processes in the virus life cycle is higher if greater understanding of the viral protein func-tions is acquired. This development is sometimes hampered by the difficulty in getting X-ray structures to guide the design of better inhibitors because it is often difficult to design inhibitors based on structure–guided principles without knowing the actual structure. Fortunately other complementary techniques such as NMR as well as cryo-electron microscopy might provide some clues about the structure as well as the function. Besides, traditionally drug targets such as NS3 protease/helicase and NS5 polymerase, and novel tar-gets like NS1, NS2A and NS4A/B may pose as new targets for inhibitor design, since currently, no approaches have been at-tempted on these proteins.

7. OUTLOOK

The field of flavivirus antiviral development is clearly moving forward as indicated by new options discussed in many studies. It is quite interesting to see the study of known drug-like anti-helminthic drug, ivermectin, that was originally developed for the treatment of parasitic infections in man, proved effective against the YFV, WNV and DENV type 2 helicase domain, as well as inhibited cell culture of YFV, WNV and DENV type 2.Similarly the discovery of self-assembling nanotubes that originally inhibited bacteria had proven to have antiviral activities against HCV, thus may lead to the under-

standing of the nature of the membranes of the viral lifecycle. Fur-thermore the rapidly expanding list of Food and Drug Administra-tion (FDA)-approved HCV inhibitors that mostly targets the NS3 proteinase and NS5B polymerase is now validated by other mem-bers of the Flaviviridae family. Although the journey from com-pound discovery to marketable drug is a long and expensive proc-ess, novel drug discovery is clearly beneficial and must be encour-aged. Therefore, further studies should focus on novel drug devel-opment as well as with existing drug with novel uses. Many antivi-ral compounds showed great success in vitro but either not tested or failed in vivo, thus more in vivo studies will greatly benefit drug design process. For example, many of the literature studies de-scribed only the potency of the E protein inhibitors of the -OG, but few continued the study in cell culture luciferase assays and none have been tested in vivo. Proper animal models of infection for DENV is more representative of the genuine DENV infection com-pared to cell culture. The goal of antiviral research is to control the human infections thus compounds discovered are desperately needed to move beyond the bench and into the clinical trials. Indeed the future bears hope for a breakthrough in flaviviral drug discovery with the advance technologies of biomedical at hand. However it is the responsibility of the government of different countries and WHO to extend the fruit of this research so it can be delivered to the large populations at an affordable cost.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS

The authors would like acknowledge the University of Malaya Research Grant (UMRG RP002/2012) and the Ministry of Educa-tion Research Grant (FRGS FP019/2013A).

REFERENCES

[1] Burke DS. Monath TP. Flaviviruses. In Fields Virology, 4th ed, Edited by Knipe DM. Howley PM. Philadelphia, PA: Lippincott Williams & Wilkins 2001.

[2] Pugachev KV, Guirakhoo F, Trent DW, Monath TP. Traditional and novelapproaches to flavivirus vaccines. Int J Parasitol 2003; 33: 567-82.

[3] Mackenzie JS, Gubler DJ, Petersen LR. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 2004; 10: S98-S109.

[4] Leyssen P, De Clercq E, Neyts J. Molecular strategies to inhibit the replication of RNA viruses. Antiviral Res 2008; 78: 9-25.

[5] Hombach J, Barrett ADT, Cardosa MJ, et al. Review on flavivirus vaccine development. Proceedings of a meeting jointly organized by the World Health Organization and the Thai Ministry of Public Health, 26-27 April 2004, Bangkok, Thailand. Vaccine 2005; 23: 2689-95.

[6] Guzman MG, Kouri G. Dengue: an update. Lancet Infect Dis 2002; 2: 33-42.

[7] Harris E, Holden KL, Edgil D, et al. Molecular Biology of flavivi-ruses. New Treatment Strategies for Dengue and Other Flaviviral Diseases: Novartis Foundation Symposium 277. John Wiley & Sons 2006.

[8] Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspec-tive of the flavivirus life cycle. Nat Rev Microbiol 2006; 3: 13-22.

[9] Leung JY, Pijlman GP, Kondratieva N, et al. Role of nonstructural protein NS2A in flavivirus assembly. J Virol 2008; 82: 4731-41.

[10] Rice CM, Lenches EM, Reddy SR, et al. Rice Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 1985; 229: 726-33.

[11] Perera R, Khaliq M, Kuhn RJ. Closing the door on flaviviruses: Entry as a target for antiviral drug design. Antiviral Res 2008; 80: 11-22.

[12] Lescar J, Dahai L, Xu T, et al. Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from Dengue virus as a target. Antiviral Res 2008; 80: 94-101.


[13] Dong H, Zhang B, Shi PY. Flavivirus methyltransferase: a novel antiviral target. Antiviral Res 2008; 80: 1-10.

[14] Malet H, Massé N, Selisko B, et al. The flavivirus polymerase as a target for drug discovery. Antiviral Res 2008; 80: 23-35.

[15] Thomas G. Furin at the cutting edge: from protein traffic to em-bryogenesis and disease Nat Rev Mol Cell Biol 2002; 10: 753-66.

[16] Bridges S. Basic science priorities for therapeutics research. Res Initiat Treat Action 2003; 8: 25-27.

[17] DeMarco SJ, Henze H, Lederer A, et al. Discovery of novel, highly potent and selective beta-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles. Bioorg Med Chem 2006; 14: 8396-404.

[18] Daelemens D, Lu R, De Clercq E, et al. Characterization of a repli-cation-competent, integrase-defective human immunodeficiency vi-rus (HIV)/simian virus 40 chimera as a powerful tool for the dis-covery and validation of HIV integrase inhibitors. J Virol 2007; 81: 4381-5.

[19] Deas TS, Binduga-Gajewska I, Tilgner M, et al. Inhibition of flavivirus infections by antisense oligomers specifically suppress-ing viral translation and RNA replication. J Virol 2005; 79: 4599-609.

[20] Ray D, Shi PY. Recent Advances in Flavivirus Antiviral Drug Discovery and Vaccine Development. Recent Patents on Anti-Infective. Drug Discov 2006; 1: 45-55.

[21] Deng J, Li N, Hongchuan L, et al. Discovery of novel small mole-cule inhibitors of dengue viral NS2B-NS3 protease using virtual screening and scaffold hopping. J of Med Chem 2012; 55: 6278-93.

[22] Schuller A, Yin Z, Brian Chia CS, et al. Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease. Antiviral Res 2011; 92: 96-101.

[23] Noble CG, Seh CC, Chao AT, et al. Ligand-Bound Structures of the Dengue Virus Protease Reveal the Active Conformation. J of Virology 2012; 86: 438-46.

[24] Nitsche C, Behnam MAM, Steuer C, et al. Retro peptidehybrids as selective inhibitors of the dengue virus NS2B-NS3 protease, Anti-viral Res 2012; 94: 72-79.

[25] Cregar-Hernandez L, Jiao GS, Johnson AT, et al. Small molecule pan-Dengue and West Nile Virus NS3 protease inhibitors. Antivir Chem Chemother 2011; 21: 209-18.

[26] Knehans T, Schuller A, Doan DN, et al. Structure guided fragment-based in silico drug design of dengue protease inhibitors. J Comput Aided Mol Des 2011; 25: 263-74.

[27] Frimayanti N, Zain SM, Lee VS, et al. Fragment-based molecular design of new competitive dengue Den2 Ns2b/Ns3 inhibits from the components of fingerroot (Boesenbergia rotunda). In Silico Biol 2011-2012; 11: 29-37.

[28] Tan SK, Pippen R, Yusof R, et al. Inhibitory activity of cylohex-enyl chalcone derivatives and favanoids of fingerroot, Boesenber-gia rotunda (l), towards dengue-2 virus ns3 protease. Bioorg Med Chem Lett 2006; 16: 3337-40.

[29] Kubinyi H. 3d qsar in drug design: Theory, method and applica-tions. Springer: 1993; Vol. 1.

[30] Modis Y, Ogata S, Clements D, et al. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA 2003; 12: 6986-91.

[31] Poh MK, Yip A, Zhang S, et al. A small molecule fusion inhibitor of dengue virus. Antiviral Res 2009; 84: 260-6.

[32] Wang QY, Patel SJ, Vangrevelinghe E, et al. A small-molecule dengue virus entry inhibibitor. Antimicrob Agents Chemother 2009; 5: 1823-31.

[33] Yennamalli R, Subbarao N, Kampmann T, et al. Identification of novel target sites and an inhibitor of the dengue virus E protein. J Comput Aided Mol Des 2009; 23: 333-41.

[34] Kampmann T, Yennamalli R, Campbell P, et al. In silico screening of small molecule libraries using the dengue virus envelope E pro-tein has identified compounds with antiviral activity against multi-ple flaviviruses. Antiviral Res 2009; 84: 234-41.

[35] Zhang W, Chipman PR, Corver J, et al. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 2003; 10: 907-12.

[36] Allison SL, Stiasny K, Stadler K, et al. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein. Eur J Virol 1999; 73: 5605-12.

[37] Schmidt AG, Yang PL, Harrison SC, et al. Peptide inhibitors of dengue-virus entry target a late-stage fusion intermediate.PLoS

Pathog 2010; 6: e1000851. Available at doi: 10.1371/journal.ppat.1000851.

[38] Schmidt AG, Yang PL, Harrison SC, et al. Peptide inhibitors of flavivirus entry derived from the E protein stem. J Virol 2010; 84: 12549-54.

[39] Costin JM, Jenwitheesuk E, Lok SM, et al. Structural optimization and de novo design of dengue virus entry inhibitory peptides. PLoS Negl Trop Dis 2010; 4: e721. Available at doi: 10.1371/journal.pntd.0000721.

[40] Nicholson CO, Costin JM, Rowe DK, et al. Viral entry inhibitors block dengue antibody-dependent enhancement in vitro. Antiviral Res 2011; 89: 71-4.

[41] Hayes EB, Komar N, Nasci RS, et al. Epideniology and transmis-sion dynamics of West Nile Virus Diseases. Emerg.Infect. Dis Aug 2005; 11(8): 1167-73

[42] Jordan I, Briese T, Fischer N, Lau JYN, Lipkin WI. Ribavirin in-hibits West Nile virus replication and cytopathic effect in neural cells. J Infect Dis 2000; 182: 1214-7.

[43] Anderson JF, Raha JJ. Efficacy of interferon -2b and ribavirin against West Nile Virus in vitro. Emerg Infect Dis 2002; 8: 107-8.

[44] Morrey JD, Smee DF, Sidwell RW, et al. Identification of active antiviral compounds against a New York isolate of West Nile virus. Antiviral Res 2002; 55: 107-16.

[45] Leyssen P, Balzarini J, De Clercq E, et al. The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase. J of Virol 2005; 79: 1943-7.

[46] Murray KO, Walker C, Gould E. The virology, epidemiology, and clinical impact of West Nile virus: a decade of advancements in re-search since its introduction into the Western Hemisphere. Epide-miol Infect 2011; 139: 810-7.

[47] Leyssen P, Balzarini J, De Clercq E, et al. The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase. J Virol 2005; 79: 1943-7.

[48] Sintchak MD, Fleming MA, Futer O, et al. Structure and mecha-nism of inosine monophosphate dehydrogenase in complex with the immunosuppressant mycophenolic acid. Cell 1996; 85: 921-30.

[49] Diamond MS, Zachariah M, Harris E. Mycophenolic acid inhibits Dengue virus infection by preventing replication of viral RNA. Vi-rology 2002; 304: 211-21.

[50] Stamos DP, Inventor; Vertex Pharmaceutical, Inc., assignee. Pro-drugs of carbamate inhibitors of IMPDH. United States patent US20046825224. 2004 Nov.

[51] Sebastian L, Madhusudana SN, Ravi V, et al. Mycophenolic acid inhibits replication of Japanese encephalitis virus. Chemotherapy 2011; 57: 56-61.

[52] Borowski P, Lang M, Haag A, et al. Characterization of imi-dazo[4,5-d] pyridazine nucleosides as modulators of unwinding re-action mediated by West Nile virus nucleoside triphospha-tase/helicase: evidence for activity on the level of substrate and/or enzyme. Antimicrob Agents Chemother 2002; 46: 1231-9.

[53] Migliaccio G, Tomassini JE, Carroll SS, et al. Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J Biol Chem 2003; 278: 49164-70.

[54] Chappell KJ, Stoermer MJ, Fairlie DP, et al. West Nile virus NS3 protease: insights into substrate binding and processing through combined modelling, protease mutagenesis and kinetic studies. J Biol Chem 2006; 281: 38448-58.

[55] Stoermer MJ, Chappell KJ, Liebscher S, et al. Potent cationic in-hibitors of West Nile virus NS2B/NS3 protease with serum stabil-ity, cell permeability and antiviral activity. J Med Chem 2008; 51: 5714-21.

[56] Shüller A, Yin Z, Chia CSB, et al. Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease. Antiviral Res 2011; 92: 96-101.

[57] Moulin A, Martinez J, Fehrentz JA. Synthesis of peptide aldehydes. J Pept Sci 2007; 13: 1-15.

[58] Ekonomiuk D, Su XC, Ozawa K, et al. Discovery of a non-peptidic inhibitor of West Nile Virus NS3 Protease by high-throughput docking. PLoS Neglected Tropical Diseases 2009; 3: 1-9.

[59] Aqvist J, Medina C, Samuelsson JE. A new method for predicting binding affinity in computer-aided drug design. Protein Eng 1994; 7: 385-91.


[60] Brooks BR, Bruccoleri RE, Olafson BD, et al. CHARMM: a pro-gram for macromolecular energy, minimization, and dynamics cal-culations. J Comput Chem 1983; 4: 187-217.

[61] Su XC, Ozawa K, Qi R, et al. NMR analysis of the Dynamic Ex-change of the NS2B cofactor between open and closed conforma-tions of the west nile virus NS2B-NS3 protease. PLoS Negl Trop Dis 2009; 3: e561. Available at doi: 10.1371/journal.pntd.0000561.

[62] Jitendra S, Vinay R. Structure based drug designing of a novel anti-flaviviral for nonstructural 3 protein. Bioinformation 2011; 6: 57-60.

[63] Tambunan US, Alamudi S. Designing cyclic peptide inhibitor of dengue virus NS3-NS2B protease by using molecular docking ap-proach. Bioinformation 2010; 5: 250-4.

[64] Cregar-Hernandez L, Jiao GS, Johnson AT, et al. Small molecule pan-Dengue and West Nile Virus NS3 protease Inhibitors. Antivir Chem Chemother 2011; 21: 209-18.

[65] Aravapalli S, Lai H, Teramoto T, et al. Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaf-fold. Bioorg Med Chem 2012; 21: 4140-8.

[66] Steuer C, Gege C, Fischl W, et al. Synthesis and biological evalua-tion of a-ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture. Bioorg Med Chem 2011; 19: 4067-74.

[67] Nitsche C, Steuer C, Klein CD. Arylcyanoacrylamides as inhibitors of the Dengue and West Nile virus proteases. Bioorg Med Chem 2011; 19: 7318-37.

[68] Glay SC, Vivian JG, Miguel MDA, et al. inventors; Centro de ingenieria genetica y biotecnologia (Ciudad de la habana, Cuba), assignee. Chimerical peptidic molecules with antiviral properties against the virus of the flaviviridae family. United States patent US 20100273702. 2009 Dec.

[69] Knox JE, Ma NL, Yin Z, et al. Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors. J Med Chem 2006; 49: 6585-90.

[70] Johnston PA, Phillips J, Shun TY, et al. HTS identifies novel and specific uncompetitive inhibitors of the two-component NS2B-NS3 proteinase of West Nile virus. Assay Drug Dev Technol 2007; 5: 737-50.

[71] Sidique S, Shiryaev SA, Ratnikov BI, et al. Structure- activity relationship and improved hydrolytic stability of pyrazole deriva-tives that are allosteric inhibitors of West Nile virus NS2B-NS3 proteinase. Bioorg Med Chem Lett 2009; 19: 5773-7.

[72] Shiryaev SA, Cheltsov AV, Gawlik K, et al. Virtual Ligand Screening of the National Cancer Institute (NCI) Compound Li-brary Leads to the Allosteric Inhibitory Scaffolds of the West Nile Virus NS3 Proteinase. Assay Drug Dev Technol 2011; 9: 69-78.

[73] Jorgensen WL, Rives JT. The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin. J Am Chem Soc 1988; 110: 1657-66.

[74] Samanta S, Cui T, Lam Y. Discovery, synthesis, and in vitro evaluation of West Nile virus protease inhibitors based on the 9,10-dihydro-3H,4aH-1,3,9,10a-tetraazaphenanthren-4-one scaffold. Chem Med Chem 2012; 7: 1 210-6.

[75] Jia F, Zou G, Fan J, Yuan Z. Identification of palmatine as an in-hibitor of West Nile virus. Arch Virol 2010; 155: 1325-9.

[76] Hrobowski YM, Garry RF, Michael SF. Peptide inhibitors of den-gue virus and West Nile virus infectivity. Virol J 2005; 2: 1-10.

[77] White SH, Snider C, Jaysinghe S, et al. Membrane ProteinExplorer version 2.2a. http: //blancobiomoluciedu/mpex/ 2003.

[78] CDC. Japanese encephalitis among three U.S. Travelers Returning from Asia, 2003-2008. MMWR Morb Mortal Wkly Rep 2009; 58: 737-40.

[79] Ghosh D, Basu A. Japanese Encephalitis-A Pathological and Clini-cal Perspective. PLoS Negl Trop Dis 2009; 3: e437. Available at doi: 10.1371/journal.pntd. 0000437.

[80] Erlanger TE, Weiss S, Keiser J, et al. Past, present and future of Japanese encephalitis. Emerg Infect Dis 2012; 15: 1-7.

[81] Beasley D, Lewthwaite P, Solomon T. Current use and develop-ment of vaccines for Japanese encephalitis. Exp Opinion Biological Therap 2006; 8: 95-106.

[82] Solomon T. Recent advances in Japanese encephalitits. J Neuro Virol 2003; 9: 274-83.

[83] Solomon T. New vaccines for Japanese encephalitis. Lancet Neurol 2008; 7: 116-8.

[84] Solomon T, Ni H, Beasley DW, et al. Origin and evolution of Japanese encephalitis virus in southeast Asia. J Virol 2003; 77: 3091-8.

[85] Food and Drug Administration. Product approval information [package insert]. JE-Vax (Japanese encephalitis virus vaccine inac-tivated). Research Foundation for Microbial Diseases of Osaka University, Osaka, Japan. Available at http: //www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm179149.htm.

[86] Campbell GL, Hills SL, Fischer M, et al. Estimated global inci-dence of Japanese encephalitis: a systematic review. Bulletin of the World Health Organization BLT 2011; 10.085233: 1-22.

[87] Solomon T. Control of Japanese encephalitis–within our grasp?N Engl J Med 2006; 355: 869-71.

[88] World Health Organization. Global Advisory Committee on Vac-cine Safety.Wkly Epidemiol Rec 2008; 83: 37-44.

[89] Phong MY, Moreland NJ, Lim SP, et al. Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target. Biosci Rep 2011; 31: art: BSR20100142. doi: 10.1042/BSR2010014

[90] ] Bessaud M, Pastorino BAM, Peyrefitte CN, et al. Functional characterization of the NS2B/NS3 protease complex from seven vi-ruses belonging to different groups inside the genus Flavivirus. Vi-rus Research 2006; 120: 79-90.

[91] Yamashita T, Unno H, Mori Y, et al. Crystal structure of the cata-lytic domain of Japanese encephalitis virus NS3 heli-case/nucleoside triphosphatase at a resolution of 1.8 A. Virology 2008; 373: 426-36.

[92] Borowski P, Heising MV, Miranda IB, et al. Viral NS3 helicase activity is inhibited by peptides reproducing the Arg-rich conserved motif of the enzyme (motif VI). Biochme Pharmacol 2008; 76: 28-38

[93] Kaczor A, Matosiuk D. Structure-based virtual screening for novel inhibitors of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase. FEMS Immunol Med Microbiol 2010; 58: 91-101.

[94] Zhang N, Chen HM, Koch V et al. Ring-expanded (‘fat’) nucleo-side and nucleotide analogues exhibit potent in vitro activity against flaviviridae NTPases/helicases, including those of the West Nile virus, hepatitis C virus, and Japanese encephalitis virus. J Med Chem 2003; 46: 4149-64.

[95] Jain AN. Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine. J Med Chem 2003; 46: 499-511

[96] Krieger E & Vriend G. Models@Home – distributed computing in bioinformatics using a screensaver based approach. Bioinformatics 2002; 18: 315-8.

[97] Frick DN, Lam AM. Understanding helicases as a means of virus control. Curr Pharm Design 2006; 12: 1315-38.

[98] Irwin JJ & Shoichet BK. ZINC – a free database of commercially available compounds for virtual screening. J Chem Inf Model 2005; 45: 177-82.

[99] Feher M. Consensus scoring for protein–ligand interactions. Drug Discov Today 2006; 11: 421-8.

[100] Wu KP, Wu CW, Tsao YP, et al. Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein. J Biol Chem 2003; 278: 46007-13.

[101] Luca VC, Abimansour J, Nelson CA, et al. Crystal structure of the Japanese encephalitis virus envelope protein. J Virol 2012; 86: 2337-46.

[102] Li C, Li-ying Zhang LY, Sun MX, et al. Inhibition of Japanese encephalitis virus entry into the cells by the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII. An-tiviral Res 2012; 94: 179-83.

[103] Chu J, Rajamanonmani R, Li J, et al. Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glyco-protein. J Gen Virol 2005; 86: 405-12.

[104] Bai F, Town T, Pradhan D, et al. Antiviral peptides targeting the West Nile Virus envelope protein. J Virol 2007; 81: 2047-55.

[105] Zhang S, Bovshik EI, Maillard R, et al. Role of BC loop residues in structure, function and antigenicity of the West Nile virus envelope protein receptor-binding domain III. Virology 2010; 403: 85-91.

[106] Hung JJ, Hsieh MT, Young MJ, et al. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells. J Virol 2004; 78: 378-88.


[107] Schmidt AG, Yang PL, Harrison SC. Peptide inhibitors of flavivi-rus entry derived from the E protein stem. J Virol 2010; 84: 12549-54.

[108] Smit JM, Moesker B, Rodenhuis-Zybert I, et al. Flavivirus Cell Entry and Membrane Fusion. Viruses 2011; 3: 160-71.

[109] Carter HR. Yellow Fever: An Epidemiological and Historical Study of Its Place of Origin. Baltimore, Maryland: Williams & Wilkins 1931.

[110] Monath TP. Yellow fever: Victor, Victoria? conqueror, conquest? Epidemics and research in the last forty years and prospects for the future. Am J Trop Med Hyg 1991; 45: 1-43.

[111] Barrett AD, Higgs S. Yellow fever: a disease that has yet to be conquered. Annu Rev Entomol 2007; 52: 209-29.

[112] Gubler DJ. The changing epidemiology of yellow fever and den-gue, 1900 to 2003: full circle? Comp Immunol Microbiol Infect Dis 2004; 27: 319-30.

[113] Khasnatinov MA, Ustanikova K, Frolova TV, et al. Non-Hemagglutinating Flaviviruses: Molecular Mechanisms for the Emergence of New Strains via Adaptation to European Ticks. PLoS ONE 2009; 4: e7295. Available at doi: 10.1371/journal.pone.0007295.

[114] Volk DE, Gandham SH, May FJ, et al. Yellow fever envelope protein domain III NMR structure (S288-K398). Virology 2009; 394: 12-8.

[115] Thompson AA, Geiss BJ, Peersen OB, et al. Analysis of flavivirus NS5 methyltransferase cap binding. J Mol Biol 2009; 385: 1643-754.

[116] Wu J, Bera AK, Kuhn RJ, et al. Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and prote-olytic processing. J Virol 2005; 79: 10268-77.

[117] Chambers TJ, Hahn CS, Galler R, et al. Flavivirus genome organi-zation, expression, and replication. Annu Rev Microbiol 1990; 44: 649-88.

[118] Falgout B, Pethel M, Zhang YM et al. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of den-gue virus nonstructural proteins. J Virol 1991; 65: 2467-75.

[119] Löhr K, Knox JE, Phong WY, et al. Yellow fever virus NS3 prote-ase: peptide-inhibition studies. J of Gen Virol 2007; 88: 2223-7.

[120] Yin Z, Patel SJ, Wang WL, et al. Peptide inhibitors of dengue virus NS3 protease. 1. Warhead. Bioorg Med Chem Lett 2006; 16: 36-9.

[121] Yin Z, Patel SJ, Wang WL, et al. Peptide inhibitors of dengue virus NS3 protease. 2. SAR study of tetrapeptide aldehyde inhibitors. Bioorg Med Chem Lett 2006; 16: 40-3.

[122] Chambers TJ, Grakoui A, Rice CM. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 protein-ase domain and NS2B are required for cleavages at dibasic sites. J Virol 1991; 65: 6042-50.

[123] Copeland RA. Enzymes: a Practical Introduction to Structure, Mechanism and Data Analysis, 2nd ed. New York: Wiley 2000.

[124] Yusof R, Clum S, Wetzel M, et al. Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J Biol Chem 2000; 275: 9963-9.

[125] Bessaud M, Pastorino BAM, Peyrefitte CN, et al. Functional char-acterization of the N2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus. Virus Res 2006; 120: 79-90.

[126] Erbel P, Shiering N, D’Arcy A, et al. Structural basis for the activa-tion of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 2006; 13: 372-273.

[127] Mastrangelo E, Pezzullo M, Burghgrave TD, et al. Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug. J Antimicrob Chemother 2012; 67: 1884-94.

[128] Luo D, Xu T, Watson RP, et al. Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein. EMBO J 2008; 27: 3209-19.

[129] Mastrangelo E, Milani M, Bollati M, et al. Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein. J Mol Biol 2007; 372: 444-55.

[130] Velankar SS, Soultanas P, Dillingham MS, et al. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 1999; 97: 75-84.

[131] Luo DH, Xu T, Watson RP, et al. Dengue virus 4 NS3 helicase in complex with ssRNA. EMBO J 2008; 27: 10278-88.

[132] Wu J, Bera AK, Kuhn RJ, et al. Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and prote-olytic processing. J Virol 2005; 79: 10268-77.

[133] Boguszewska-Chachulska AM, Krawczyk M, Stankiewicz A, et al. Direct fluorometric measurement of hepatitis C virus helicase ac-tivity. FEBS Lett 2004; 567: 253-8.

[134] Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspec-tive of the flavivirus life cycle. Nat Rev Microbiol 2005; 3: 13-22

[135] Milani De Mayo De Mari et al. inventor; Consiglio Nazionale Delle Richerche, Katholieke Universiteit Leuven-K.U. Leuven R&D, Aix-Marseille Universite, assignee.Avermectins and Milbe-mycins for the treatment prevention or amelioration of flavivirus infections. United States patent US 20120208778 A1. 2012 Aug.

[136] van der Poel, CL. Hepatitis C virus and blood transfusion: past and present risks. Hepatology 1999; 31: 101-6.

[137] World Health Organization (WHO). Hepatitis C. Weekly Epidemi-ological Record 1997; 72: 65-9.

[138] World Health Organization (WHO).Global surveillance and control of hepatitis C. Report of a WHO Consultation organized in collabo-ration with the Viral Hepatitis Prevention Board, Antwerp, Bel-gium. J Viral Hepat1999; 6: 35-47.

[139] Hoofnagle JH. Course and outcome of hepatitis C. Hepatology 2002; 36: S21-29.

[140] Liang TJ, Rehermann B, Seeff LB, et al. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med 2000: 132; 296-305.

[141] Kuo G, Choo QL, Alter HJ, et al.An assay for circulating antibod-ies to a major etiologic virus of human non-a, non-B-Hepatitis.Science 1989; 244: 362-4.

[142] Barazani Y, Hiatt JR, Tong MJ, et al. Chronic viral hepatitis and hepatocellular carcinoma. World J Surg 2007; 31: 1243-8.

[143] Das D, Hong J, Chen SH, et al. Recent advances in drug discovery of benzothiadiazine and related analogs as HCV NS5B polymerase inhibitors. Bioorg Med Chem 2011; 19: 4690-703.

[144] Rehman S, Ashfaq UA, Javed T. Antiviral drugs against hepatitis C virus. Genet Vaccines Ther 2011; 9: 1-10.

[145] Yang PL, Gao M, Lin K, et al. Anti-HCV drugs in the pipe-line.Curr Opin Virol 2011; 1: 607-16.

[146] Manns MP, Foster GR, Rockstroh JK, et al. The way forward in HCV treatment-finding the right path. Nat Rev Drug Discovery 2008; 6: 991-1000.

[147] Lesburg CA, Cable MB, Ferrari E, et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 1999; 6: 937-43.

[148] Bressanelli S, Tomei L, Roussel A, et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci USA 1999; 96: 13034-9.

[149] Patel PD, Patel MR, Kaushik-Basu N, et al. 3D QSAR and molecu-lar docking studies of benzimidazole derivatives as hepatitis C vi-rus NS5B polymerase inhibitors. J Chem Inf Model 2008; 48: 42-55.

[150] LaPlante SR, Gillard JR, Jakalian A, et al. Importance of ligand bioactive conformation in the discovery of potent indole-diamide inhibitors of the hepatitis C virus NS5B. J Am Chem Soc 2010; 132: 15204-12.

[151] McKercher G, Beaulieu PL, Lamarre D, et al. Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate. Nucl Acids Res 2004; 32: 422-31.

[152] Beaulieu PL, Gillard J, Jolicoeur E, et al. From benzimidazole to indole-5-carboxamide Thumb Pocket I inhibitors of HCV NS5B polymerase. Part 1: Indole C-2 SAR and discovery of diamide de-rivatives with nanomolar potency in cell-based subgenomic repli-cons. Bioorg Med Chem Lett 2011; 21: 3658-63.

[153] Yao N, Reichert P, Taremi SS, et al. Molecular views of viral poly protein processing revealed by the crystal structure of the hepatitis C virus bifuctional protease-helicase. Structure 1999; 7: 1353-63.

[154] Perni RB, Almquist SJ, Bryan RA, et al. Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepati-tis C virus NS3-4A serine protease. Antimicrob. Agents Chemother 2006; 50: 899-909.

[155] Li X, Zhang S, Zhang YK, et al. Synthesis and SAR of acyclic HCV NS3 protease inhibitors with novel P4-benzoxzborole moie-ties. Bioorg Med Chem Lett 2011; 21: 2048-54.

[156] Rong L, Dahari H, Ribeiro RM, et al. Rapid emergence of protease inhibitor resistance in hepatitis C virus. Sci Transl Med 2010; 2: 30-2.


[157] Takaya D, Yamashita A, Kamijo K, et al. A new method for in-duced fit docking (GENIUS) and its application to virtual screening of novel HCV NS3-4A protease inhibitors.Bioorg Med Chem 2011; 19: 6892-904.

[158] Kazmierski WM, Hamatake R, Duan M, et al. Discovery of novel urea-based hepatitis C protease inhibitors with high potency against protease-inhibitor-resistant mutants. J Med Chem 2012; 55: 3021-6.

[159] Barbato G, Cicero DO, Cordier F, et al. Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protein do-main. EMBO J 2000; 19: 1195-206.

[160] Ding CZ, Zhang YK, Li X, et al. Synthesis and biological evalua-tions of P4-benzoxaborole-substituted macrocyclic inhibitors of HCV NS3 protease. Bioorg Med Chem Lett 2010; 20: 7317-22.

[161] Kota S, Coito C, Mousseau G, et al. Peptide inhibitors of hepatitis C virus core oligomerization and virus production. J Genl Virol 2009; 90: 1319-28.

[162] Wells JA, McClendon, CL. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 2007; 450: 1001-9.

[163] Che W, Zhang Z, Chen J, et al. HCV core protein interacts with Dicer to antagonize RNA silencing. Virus Res 2008; 133: 250-8.

[164] Si Y, Liu S, Liu X, Jacobs JL, et al. A human claudin-1-derived peptide inhibits hepatitis C virus entry. Hepatology 2012; 56: 507-15.

[165] Scarselli E, Ansuini H, Cerino R, et al. The human scavenger receptor class B type I is a novel candidate receptor for the hepati-tis C virus. EMBO J 2002; 21: 5017-25.

[166] Evans MJ, von Hahn T, Tscherne DM, et al. Claudin-1 is a hepati-tis C virus co-receptor required for a late step in entry. Nature 2007; 446: 801-5.

[167] Liu S, Yang W, Shen L, et al. Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated dur-ing infection to prevent superinfection. J Virol 2009; 83: 2011-4.

[168] Ploss A, Evans MJ, Gaysinskaya VA, et al. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 2009; 457: 882-6.

[169] Montero A, Gastaminaza P, Law M, et al. Self-assembling peptide nanotubes with antiviral activity against hepatitis C virus. Chem Biol 2011; 18: 1453-62.

[170] Dartois V, Sanchez-Quesada J, Cabezas E, et al. Systemic antibac-terial activity of novel synthetic cyclic peptides. Antimicrob Agents Chemother 2005; 49: 3302-10.

[171] Fernandez-Lopez S, Kim HS, Choi EC, et al. Antibacterial agents based on the cyclic D,L-alpha-peptide architecture. Nature 2001; 412: 452-5.

[172] Horne WS, Wiethoff CM, Cui C, et al. Antiviral cyclic D,L-alpha-peptides: targeting a general biochemical pathway in virus infec-tions. Bioorg Med Chem 2005; 13: 5145-53.

[173] Bildick CJ, Wichroski MJ, Pendri A, et al.A novel small molecule inhibitor of hepatitis C virus entry. PLoS Pathog 2010; 6: e1001086. Available at doi: 10.1371/journal.ppat.1001086.

[174] Gary RF, Dash S, Coy DH, et al, inventor; The Administrators of the Tulane Educational Fund.The Rockefeller Univer-sity.Assignee.The Flavivirus fusion inhibitors. United States patent US815336. 2012 April.

[175] Chandramouli S, Joseph JS, Daudenarde S, et al. Serotype-specific structural differences in the protease-cofactor complexes of the dengue virus family. J Virol 2010; 84: 3059-67.

[176] Luo D, Wei N, Doan DN, et al. Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications. J Biol Chem 2010; 285: 18817-27.

[177] Noble CG, Seh CC, Chao AT, et al. Ligand-bound structures of the dengue virus protease reveal the active conformation. J Virol 2011; 86: 438-46.

[178] Luo DH, Xu T, Hunke C, et al. Crystal structure of the NS3 prote-ase-helicase from dengue virus. J Virol 2008; 82: 173-83.

[179] Aleshin AE, Shiryaev SA, Strongin AY, et al. Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold. Protein Sci 2007; 16: 795-806.

[180] Robin G, Chappell K, Stoermer MJ, et al. Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conforma-tion. J Mol Biol 2009; 385: 1568-77.

[181] Kaufmann B, Rossmann MG. Molecular mechanisms involved in the early steps of flavivirus cell entry. Microbes Infect 2011; 13: 1-9.

[182] Lindenbach BD, Rice CM. Flaviviridae: the viruses and their repli-cation. In Fields Virology, 4th ed. Edited by Knipe DM and How-ley PM. Lippincott Williams & Wilkins 2001.

[183] Hijikata M, Mizushima H, Akagi T, et al. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 1993; 67: 4665-75.

[184] Popescu C-I, Callens N, Trinel D, et al. NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly. PLoS Pathog 2011; 7: e1001278. Available at doi: 10.1371/journal.ppat.1001278.

[185] Chevaliez S, Pawlotsky JM. HCV Genome and Life Cycle. In: Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006. Chapter 1. Available from: http: //www.ncbi.nlm.nih.gov/books/NBK1630/.

[186] Matusan AE, Pryor MJ, Davidson AD, et al. Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase mo-tifs: effects on enzyme activity and virus replication. J Virol 2001; 75: 9633-43.

[187] Miller S, Kastner S, Krijnse-Locker J, et al. The non- structural protein 4A of dengue virus is an integral membrane protein induc-ing membrane alterations in a 2K-regulated manner. J Biol Chem 2007; 282: 8873-82.

[188] Roosendaal J, Westaway EG, Khromykh A, et al. Regulated cleav-ages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein. J Virol 2006; 80: 4623-32.

[189] Munoz-Jordan JL, Laurent-Rolle M, Ashour J, et al. Inhibition of alpha/beta interferon signaling by the NS4B protein of flaviviruses. J Virol 2005; 79: 8004-13.

[190] Umareddy I, Chao A, Sampath A, et al. Dengue virus NS4B inter-acts with NS3 and dissociates it from single-stranded RNA. J Gen Virol 2006; 87: 2605-14.

[191] Pastorino B, Nougairéde A, Wurtz N, et al. Role of host cell factors in flavivirus infection: Implications for pathogenesis and develop-ment of antiviral drugs. Antiviral Res 2010; 87: 281-94.

Received: June 30, 2013 Accepted: August 27, 2013

current approaches in antiviral drug discovery against the

Documents