NS 3 and NS 5 Proteins : Important Targets for Anti-Dengue Drug Design

A dengue é uma doença negligenciada responsável pelo óbito de 22.000 pacientes por ano em áreas onde a doença é endêmica. Embora grandes esforços tenham sido investidos em pesquisas na busca de tratamentos para a dengue, não há ainda nenhuma vacina ou antiviral disponível no mercado para tratamento desta patologia. Entre as estratégias que vêm sendo utilizadas para a busca e desenvolvimento de antivirais contra a dengue, a inibição de enzimas é aquela que tem se mostrado a mais promissora. O presente artigo de revisão descreve os recentes avanços no que tange à utilização de enzimas do vírus da dengue como alvos para o desenho de antivirais.


Introduction
Dengue fever and dengue hemorrhagic fever are the most rapidly spreading mosquito-borne diseases in the world today, with an observed 30-fold increase of reported cases over the last 50 years. 1The World Health Organization estimates that, globally, 2.5 billion people are at risk, and annually, 50-100 million people become infected, of which approximately 0.5 million require hospitalization 2 and 22,000 deaths occur each year in areas where it is endemic. 3he disease has four viral serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), and its spectrum ranges from asymptomatic infection to dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), and may lead the patient to death. 4All four serotypes of dengue virus are transmitted to humans by the Aedes aegypti and Aedes albopictus mosquitoes. 5e cost of dengue in the Western Hemisphere alone is estimated to be $ 2.1 billion per year. 61][12] The optimal dengue drugs encompass a good safety profile, resolve symptoms rapidly and reduce risks of severity, and achieve comparable inhibition of all four DENV serotypes. 13he antiviral agents for dengue can inhibit (i) viral entry; (ii) the viral protein NS4B; (iii) the viral capsid; (iv) the viral protease; (v) the viral helicase; (vi) the viral methyltransferase; (vii) the viral polymerase; and (viii) the host target.Inhibition of viral enzymes has been proven to be the most successful of antiviral approaches. 8,13,14onsidering this fact, the aim of this review is to summarize major progress towards drugs that inhibit viral enzymes.

Viral Structure
The dengue virus particle is about 50 nm in diameter.The 10,723-nucleotide RNA genome encodes an uninterrupted open reading frame (ORF), directing the synthesis of a polyprotein precursor in the order NH 2 -C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH, where C is the capsid protein, M is the membrane-associated protein, E is the envelope protein, and NS1 through NS5 are nonstructural proteins. 15he capsid protein (C) has about 12 and 14 kDa, and has several residues of basic amino acids such as lysine and arginine, which allow these to interact with the viral genome for construction of the nucleocapsid.The prM protein is a glycoprotein presenting 18.1-19.1 kDa which cleavages the N-terminal region, originating the M protein.The cleavage process is associated with the virus maturation, and prM and M are intracellular and extra cellular proteins, respectively.The M protein with 8.2-8.5 kDa is one of the two proteins that form the viral envelope, and it is involved in the penetration of the virus of the host cell. 16he glycoprotein E functions on the virus and in the host cells causing cell tropism, a membrane fusion cell, resulting in the neutralization of antibodies and hemaglutination. 17his glycoprotein has three distinct domains bound by S-S (sulfur-sulfur) connection: I, II, and III domains have 495 amino acids and a molecular weight of 56-57 kDa. 18,19he prM protein forms an intracellular heterodimer, that stabilizes the E protein. 16PrM is cleaved during the exocytose in virion liberation.Such a process originates the M protein, 19 possessing a new conformation, which is necessary for the right disposition of E. This fact suggests a chaperone-like function of the M protein. 16he NS1 protein has approximately 45 kDa and exists in multiple forms (monomer, dimer, and hexamer) in different compartments of insect cells and dengue virus-infected mammals.The NS1 protein is found in a membraneassociated or soluble form that is secreted by the infected cells, and therefore can be found in cell culture mediums or serums from infected patients. 20,21The functions of the NS1 dengue virus infections have not been clearly elucidated. 21S2A is a 22-kDa hydrophobic protein that was previously shown to be important for viral replication and pathogenesis.NS2A participates in viral RNA synthesis, 22 viral assembly, 23 virus-induced membrane formation, 23 contributes to the production of NS1, 24 and inhibits interferon α/β response. 25he NS2B is a co-factor for the NS3 protein.NS3 is a multi-functional enzyme, acting as a protease for polyprotein processing, an RNA (ribonucleic acid) triphosphatase for capping nascent viral RNA, and a helicase for unwinding the double-stranded replicative form of RNA. 26NS3 and NS5 (vide infra) are sufficiently conserved within the four serotypes.
The flavivirus proteases, including NS2B-NS3 protease, are essential for viral replication and infectivity.Chambers and collaborators show that yellow fever virus genetically modified to contain only inactive NS2B-NS3 protease is unable to infect target cells. 27Similarly, Rothan and collaborators show that the treatment of cells with a peptide that inhibits NS2B-NS3 protease decreases dengue virus infection by 80%. 28he NS4A protein has been implicated in substantial re-arrangements of internal membranes, permitting facile virus RNA synthesis and assembly; however, the mechanism by which this occurs is unknown. 29,30NS4B consisting of 248 amino acids has been shown to be part of the viral replication complex and is also implicated in dengue virus pathogenicity. 24,31NS4B, as one of the dengue virus proteins with RNA interference (RNAi) suppression activity, is independent of the interferon inhibition functions of NS4B. 32S5 is the largest (104 kDa) and the most conserved protein in DENV.It is also a bifunctional enzyme with a methyltransferase domain (MTase; residues 1-296) at its N-terminal end and a RNA-dependent RNA polymerase (RdRp; residues 320-900) 33 at its C-terminal end. 34pecifically, residues 320-368 are strictly conserved among the flaviviruses.These residues are also implicated to interact with NS3. 35,36

NS2B/NS3 Protease and NS3 Helicase
The bipartite NS3 protease-NS3 helicase is an enzyme that is central to flavivirus replication and polyprotein processing.Dissecting the structural and functional properties of this protein in its full-length state is therefore key to improving our understanding of the flavivirus life cycle and informing the design of effective antiviral drugs.It remains unclear why NS3 harbors several catalytic activities within one polypeptide chain.However, the conservation of this arrangement across the Flaviviridae genus suggests some functional relevance. 37S3 protease is a trypsin-like serine protease shown to harbor a classic serine protease catalytic triad comprised of residues histidine 51 (His51), aspartic acid 75 (Asp75), and serine 135 (Ser135). 38The N-terminal one-third of the dengue virus NS3 protease (NS3pro) is required for protease activity, and the C-terminal two-thirds are associated with the enzymatic functions of a nucleoside triphosphatase and RNA helicase. 39Falgout and collaborators demonstrated that the activating NS2B cofactor is a prerequisite for Vol. 25, No. 10, 2014   catalytic activity of the NS3 protease. 40The two-component NS2B-NS3pro protease represents a more structurally relevant target than NS3pro alone for functional studies and drug discovery research.However, the mechanism by which NS2B contributes to high activation of NS3pro remains poorly understood.Elucidation of the prerequisite role of NS2B will pave the way for discovering and designing new drugs against dengue diseases. 41he viral protease is responsible for cleavage at a number of sites, including NS2A-NS2B, NS2B-NS3, NS3-NS4A, and NS4B-NS5; it also cleaves just upstream of the signal sequences at the C-prM and NS4A-NS4B junctions, within NS2A, and within NS3 itself. 42he fact that NS2B-NS3pro recognizes only sites that contain two cationic residues, whereas trypsin recognizes sites containing a single cationic residue, has necessitated the development of new classes of inhibitors for targeting the active site. 43The structural boundaries originally annotated within the polyprotein suggested that the NS3 domain encoded the functional protease.Later studies showed that expression of NS3 alone did not lead to production of an active protease; however, including a portion of NS2B with NS3 led to full proteolytic activity. 44iral proteases are proven antiviral targets, as evidenced by the clinical availability of ten human immunodeficiency virus 1 (HIV-1) protease inhibitors 45 and the hepatitis C virus (HCV) protease inhibitors. 44Thus, it is plausible that a protease inhibitor for dengue virus would be efficacious in the clinic.However, experience with HIV-1 and HCV indicates that there are certain drawbacks associated with protease inhibitors, and these are important considerations in developing dengue virus protease inhibitors. 13he helicase domain of NS3 (NS3Hel, residues 180-618) has seven structural motifs reminiscent of superfamily 2 helicases. 46It has three subdomains with significant sequence identity and structural similarity to other flavivirus helicases. 47,48Subdomains I and II are also structurally similar to the corresponding domains in the hepatitis C virus, suggesting a common functional mechanism. 49he combined activities of a polynucleotide-stimulated helicase and nucleoside triphosphatase (NTPase) in the C-terminal domain are required for melting secondary structures prior to initiation of RNA synthesis and for the unwinding of RNA duplexes, either to separate double stranded RNA (dsRNA) intermediates formed during viral RNA synthesis or as a translocase that can remove proteins bound to viral RNA. 50Mutational analysis has shown that the adenosine triphosphatase (ATPase), helicase and NTPase activities of DENV NS3 share a common active site. 51Dengue viruses with impaired helicase activities lose the ability to replicate, demonstrating the importance of the NS3 helicase domain in the viral life cycle 52 and that inhibitors or modulators for these enzymes are potentially of interest as therapeutic agents. 53n the HCV NS3 protein, such interdependence of different domains for their respective activities has been extensively characterized, and a strong interdependence in the enzymatic activities of the protease and helicase domains of HCV NS3 protein has been demonstrated.Previous studies have suggested a significant regulatory role of the protease domain on flavivirus NS3 helicase activity, [48][49][50][51][52][53] but, recently, a study showed that the NS3 protease domain had no significant effect on the ATPase and helicase activities of DENV NS3. 54he important role of NS3 for the replication of the dengue virus makes this protein an interesting site for viral inhibiting.

NS5 Methyltransferase and NS5 Polymerase
The NS5 is about 900 amino acids long and comprises a methyltransferase domain at its N terminus and an RNA-dependent RNA polymerase domain at its C-terminal end.][57][58] The domain RNA-dependent RNA polymerase (RdRp) is responsible for the replication of the positive-strand RNA genome in an asymmetric and semi-conservative process in which the antigenome is only present in a double-stranded RNA replication intermediate. 59In DENV infections, the NS5 protein is primarily localized within the nucleus.However, not all flavivirus RdRps localize to the nucleus.The rationalization for a viral RdRp localizing to the nucleus when its actual enzymatic functions in the virus life cycle are required in the cytoplasm is currently unknown but is actively being investigated. 60However, these observations do suggest that, apart from its enzymatic functions, NS5 may also engage in virus-host interactions and actively interact with the host environment. 61iral MTases are involved in the mRNA capping process, transferring a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the N7 atom of the cap guanine and onto the 2'OH group of the ribose moiety of the first RNA nucleotide.In the genus Flavivirus, both (guanine-N7)-methyltransferase (N7MTase) and (nucleoside-2'-O-)-methyltransferase (2'OMTase) activities have been associated with the N-terminal domain of the viral NS5 protein. 33,62,63he linkage between the two NS5 domains does not seem to be relevant to the functions of these two domains, since the RdRp activity is not altered by the presence of the glucosyltransferases (GTase) and MTase domain, and the GTase and MTase activities are not affected by the RdRp domain. 64,65he interdependence of the NS3 and NS5 activities, suggested by the sequence of reactions during genome replication and capping, was shown by experiments in which NS5 stimulates NS3's NTPase and RNA 5'triphosphatase (5'-RTPase) activities, [66][67][68] and where NS3 stimulates NS5's GTase activity. 64he enzymes codified by NS5 show they have important functions in the replication of the virus, playing a key role and suggesting NS5 as a potential antiviral target.

Dengue Inhibitors
The best characterized DENV nonstructural proteins are NS3 and NS5, which are multifunctional proteins presenting several enzymatic activities.These proteins are the most conserved ones in all four dengue virus serotypes.Therefore, NS3 and NS5 are considered attractive targets for the search and development of dengue antiviral chemotherapeutics. 36It is important to mention that inhibition of virus enzymes is one of the most successful approaches for drug antiviral discovery. 13In this section, several compounds that have been described in the literature as promising dengue antiviral drugs will be presented.
Conotoxins are a mixture of peptide neurotoxins produced by cone snails to paralyze prey and be used for defense.Crude venom extracts from five different Conus species were tested for serotype 2 DENV protease inhibitory activity, 72 being that the extract from Conus marmoreus was the only effective kind.High performance liquid chromatography (HPLC) fractioning of C. marmoreus extract afforded, among others, conotoxin MrIA, 73 which was found to significantly inhibit serotype 2 NS2B-NS3 protease activity (inhibitory constant, K i = 2.2 µmol L -1 ).The activity of this conotoxin was attributed to a disulfide bond-mediated loop.Inspired by this structural motif, several cyclic peptides containing a disulfide bond were synthesized.Peptide 8 was the most effective, presenting K i = 2.2 ± 0.2 µmol L -1 .
Studies conducted with tetrapeptides identified compound 9 to possess a boronic acid functionality as the tetrapetide with the highest affinity (K i = 43 nmol L -1 ). 74,75he various peptides investigated so far exhibit inhibitory activities in biochemical protease assays.However, this is not the case in cell-based viral infection assays.This fact is attributed to their poor permeability and stability.One important fact is that cyclization of the peptides results in improved stability and cell permeability.Screening of small-molecular-weight compounds is another important approach that has been utilized in the search for dengue antiviral drugs.Figure 2 depicts the structure of some lead compounds that have been identified in these screening campaigns.
In screening of DENV protease inhibitors a problem found is the occurrence of false positives.A selective high-throughput bioassay, based on tryptophan fluorescence, was described to overcome this problem. 76This methodology identified phtalazine as DENV protease inhibitor.In addition, the assays were capable to discriminate between specific and non-specific analogues concerning DENV protease activitiy. 76It was demonstrated that specific analogues quenched DENV protease fluorescence and showed variation in IC 0 (inhibitory concentration) values.In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves.
A screening of approximately 12,000 small-molecularweight compounds resulted in the selection of guanidine derivatives, such as 11 (Figure 2), as competitive inhibitors of pan-dengue and West Nile virus NS3 protease inhibitors with IC 50 values in the 1-70 µmol L -1 range.The compounds displayed low cytotoxicity.Although from in vitro assays the derivatives showed protease activity, antiviral activity against DENV-2 on Vero cells was not detectable. 77 docking investigation screening of a small set of compounds that dock into the P1 pocket and the catalytic site of the DENV-2 NS3 protease domain apo-structure led to the discovery of the compounds ADRP0006 (12) and ARDP0009 (13).78 The 50% effective concentration (EC 50 ), 50% cytotoxic concentration (CC 50 ), and selective index (SI) were determined for both compounds (compound 12: EC 50 = 4.2 ± 1.9 µmol L -1 , CC 50 = 69 ± 4 µmol L -1 , SI = 16.6;compound 13: EC 50 = 35.8± 8 µmol L -1 , CC 50 > 300 µmol L -1 , SI > 8.1). Inaddition to inhibiting protease activity in vitro, both compounds were also capable of inhibiting viral replication in cell culture experiments.78 Twenty three analogues of compounds 12 were purchased for testing against DENV-2 NS2B-NS3 protease.Due to solubility problems, only ten analogues were selected for a preliminary protease screening.Among these compounds, four displayed protease inhibitory activities comparable to ADRP 0006 (12), while other four analogues (structures 14-17, Figure 2) presented superior activity (∼2 to ∼60-fold higher).79 Steuer and co-workers investigated the biological effects of α-ketoamides and α-ketoamides derivatives on DENV virus protease.80 All the compounds evaluated presented moderate activities in the in vitro DENV protease assays.However, compound 18, which presented the highest inhibitory activity (39.1%), displayed a remarkably inhibited DENV replication in a cell-culture assay in a dose-dependent manner, achieving a more than 1000-fold reduction of virus titers at non-cytotoxic concentrations. In this study, they described ivermectin (19), selamactin (20), methylbenzethonium chloride (21), tyrothricin (22) and alexidine hydrochloride (23) (Figure 3) as a lead for DENV-2 NS3-NS2B protease inhibitors.The lowest determined K i value was 12 ± 1.2 µmol L -1 , associated with compound 23.It is also important to mention that compounds 18-23 were also effective on the related NS3 protease of West Nile virus.81 The exploitation of aminobenzamide, quinoline, and benzotiazole scaffolds has led to the discovery of compounds that can be explored in dengue antiviral discovery (Figure 4).
Compound 24, an amidobenzamide, 82 is active against dengue and West Nile virus proteases, presenting K i values, respectively, equal to 8.77 and 5.55 µmol L -1 .
Investigations by Lai and colleagues disclosed benzoathiazole derivatives 26 and 27 as DENV protease inhibitors. 84,85Chemical 26 presents an IC 50 value equal to 0.91 ± 0.05 µmol L -1 , and a K i value of 2.36 ± 0.13 µmol L -1 .Dengue and West Nile virus NS2B/NS3 proteases are inhibited by compound 27 (IC 50 values were found to be 3.75 ± 0.06 and 4.22 ± 0.07 µmol L -1 , respectively).Kinetics studies support a competitive mode of inhibition by this compound.

Compounds Inhibiting Helicase
The in silico study was conducted with a selected region of the ssRNA (single stranded RNA) site in the crystal structure of the NS3 helicase domain of the Kunjin virus, an Australian variant of the West Nile Virus, and identified ivermectin (19) (Figure 3), as a DENV helicase inhibitor (IC 50 of 500±70 nmol L -1 ).Kinetic studies proved that the mechanism of inhibition is uncompetitive.Moreover, it was demonstrated that ivermectin (19) is capable of inhibiting in vitro dengue, West Nile Virus, and yellow fewer virus replication. 87gure 3. Structures of ivermectin (19), selamectin (20), benzothonium chloride (21), tyrothricin (22), and alexidine hydrochloride (23).
Recently, a high-throughput screening of a 200,000 compounds library identified the benzoxazole ST-610 (29) (Figure 5) as a dengue helicase inhibitor. 87The compound inhibited all four dengue serotypes in cell culture.In addition, ST-610 is non-cytotoxic and non-mutagenic.Finally, this benzoxazole showed marginal effects in the DENV AG-129 mouse model, with < 10-fold viremia reduction. 88

NS5 Methyltransferase and NS5 Polymerase Inhibitors
A series of small-molecular weight compounds possessing the general structure 30 (Figure 6) are compounds that could block dengue virus methyltransferase. 89,90The investigators tested twelve compounds for its inhibitory activity against DENV-3 methyl transferase (for both N7 and 2'-O methyl transferase activities).Among the evaluated compounds, the chlorinated derivative 31 was the most active, presenting K i = 0.82 ± 0.06 for N7-methylation and K i = 0.17 ± 0.02 for 2'-O methylation.
Figure 6 depicts the structures of DENV RdRp inhibitors.They can be divided into two major categories, namely nucleoside and non-nucleoside inhibitors.
Compounds 32 and 33 are nucleoside inhibitors.NITD-008 (32) corresponds to an adenosine analogue and presents high potency against DENV replication.In vivo experiments with DENV-infected mice showed that peak viremia was suppressed and completely prevented death in a lethal mouse model.Moreover, no adverse effects were noticed when rats were orally treated with this nucleoside.However, severe side-effects were observed in both rats and dogs after 2 weeks of application.As a consequence, further development of compound 32 was ended. 91,92alapiravir (33) is another example of nucleoside originally developed by Hoffmann-La Roche for treatment of hepatitis C virus.However, this clinical development was finished because of side effects on patients. 93This nucleoside was reconsidered for a phase II trial for DENV.The nucleoside presented low efficiency at different doses applied to two groups of adult dengue patients.The reasons for this failure remain unclear. 12he non-nucleoside 34 (Figure 6) was identified after a high-throughput screening of about 1.8 million compounds.This N-sulphonylanthranilic acid derivative  inhibits DENV-4 RdRp activity in a dose-responsive manner, with IC 50 of 113 µmol L -1 .Using a replicon assay, it was noticed that compound 34 also inhibits DENV-2 replication (EC 50 of 100 µmol L -1 ).It was also demonstrated that this compound binds selectively, though weakly, to DENV RdRp.The related compound 35 also inhibited DENV-2 RdRp activity more efficiently with an IC 50 of 7.2 µmol L -1 . 94,95

Conclusions
Two general strategies can be pursued for any antiviral therapy.The first is to inhibit viral targets.The second is to inhibit host targets.Inhibition of targets in the host continues to be risky since it is not yet known what all the mechanisms of infection and disease development in the host are, particularly in cases of dengue hemorrhagic fever.Another point is that the action of the drug should be well known to minimize side effects.
In this case, inhibition of viral targets is a good way.The best characterized DENV proteins are nonstructural proteins NS3 and NS5, which are multifunctional proteins presenting several enzymatic activities.These proteins are the most conserved ones in all four dengue virus serotypes.It is a good point to create an efficient drug that acts in all four serotypes.
Even with forthcoming advances, a drug for clinical trials has not yet been obtained.A greater understanding of the viral replicative cycle in the host and the variations that occur in different serotypes will be necessary for the choice and improvement of a suitable drug.
Although challenging, recent research has generated optimistic results and has encouraged researchers to develop an effective therapy against the dengue virus in the near future.
André S. de Oliveira is a Biologist, MSc in Cellular and Structural Biology by Federal University of Viçosa (2011) and is a PhD student in Cellular and Structural Biology at the same University.He is currently a Professor at the Federal Institute of Education, Science and Technology in northern MinasGerais.Acts mainly in immunology and working on the following topics: dengue vaccine and heterologous protein expression.