REVIEW ARTICLE Progress for dengue virus diseases Towards the NS2B–NS3pro inhibition for a therapeutic-based approach Sonia Melino and Maurizio Paci Department of Chemical Science and Technology, University of Rome ‘Tor Vergata’, Italy Keywords dengue hemorrhagic fever; dengue virus; NS3; protease inhibitors; vaccines; viral diseases; viral serine protease Correspondence S Melino, Dipartimento di Scienze e ` Tecnologie Chimiche, Universita di Roma ‘Tor Vergata’ via della Ricerca Scientifica, 00133 Rome, Italy Fax: +39 0672594328 Tel: +39 0672594449 E-mail: melinos@uniroma2.it (Received 22 January 2007, revised 16 March 2007, accepted 17 April 2007) doi:10.1111/j.1742-4658.2007.05831.x Transmitted by the Aedes aegypti mosquito, the dengue virus is the etiological agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, and, as such, is a significant factor in the high death rate found in most tropical and subtropical areas of the world Dengue diseases are not only a health burden to developing countries, but pose an emerging problem worldwide The immunopathological mechanisms appear to include a complex series of immune responses A rapid increase in the levels of cytokines and chemical mediators during dengue disease plays a key role in inducing plasma leakage, shock and hemorrhagic manifestations Currently, there are no vaccines available against dengue virus, although several tetravalent live-attenuated dengue vaccines are in clinical phases I or II, and prevention through vaccination has become a major priority on the agendas of the World Health Organization and of national ministries of health and military organizations An alternative to vaccines is found in therapeutic-based approaches Understanding the molecular mechanisms of viral replication has led to the development of potential drugs, and new molecular viral targets for therapy are emerging The NS3 protease domain of the NS3 protein is responsible for processing the viral polyprotein and its inhibition is one of the principal aims of pharmacological therapy This review is an overview of the progress made against dengue virus; in particular, it examines the unique properties – structural and functional – of the NS3 protease for the treatment of dengue virus infections by the inhibition of viral polyprotein processing One hundred million cases of dengue fever (DF) are estimated by the World Health Organization to occur yearly, together with between 250 000 and 500 000 cases of dengue hemorrhagic fever (DHF) Extensive plasma leakage in various serous cavities of the body, including the pleura, the pericardium and the peritoneal cavities, may result in profound shock, the so-called dengue shock syndrome (DSS) The case ⁄ fatality rate of DHF in most countries is about 5%, although appropriate symptomatic treatment has been successful in reducing the mortality of DHF to less than 1% Most fatalities occur among children and young adults DF and DHF are primarily diseases of tropical and subtropical areas, but represent a typical example of a global disease The transmission of dengue virus (DENv) has Abbreviations ADE, antibody-dependent enhancement; DENv, Dengue virus; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; E protein, glycoprotein E; ER, endoplasmic reticulum; HCV, hepatitis C virus; NS, nonstructural; NS3pro, NS3 protease domain; NTPase, nucleotide three phosphate hydrolase; protein C, nucleocapsid protein C; prM, protein M 2986 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS S Melino and M Paci increased considerably in recent years as a result of the expansion of the Aedes aegypti mosquito to different geographic areas, and DHF has spread from South East Asia to the Western Pacific and the Americas A substantial number of people travelling to endemic regions are also infected each year In the last year, DHF has again been on the increase in India and in several Asian countries because of seasonal factors Dengue is one of the most important mosquito-borne viral diseases affecting humans; its global distribution is comparable to that of malaria and an estimated 2.5 billion people live in areas at risk from epidemic transmission (Fig 1) In 1906, Bancroft published the first evidence implicating the mosquito A aegypti as the vector of DENv [1] DENv was originally classified as an arthropod-borne animal virus (arbovirus) The arboviruses comprise infection agents that are biologically transmitted between susceptible hosts by hematophagous arthropods and are classified in different virus families according to viral genes, virion structure and the viral replication cycle DENv belongs to the Flavivirus genus of the family Flaviviridae that are members of the positive-stranded virus supergroup [2] Progress for dengue virus diseases The normal cycle of DENv infection is considered to be human–mosquito–human Feeding on an infected and viremic human enables the female mosquito to transmit the virus after an incubation period of 8–10 days, during which DENv infection, replication and dissemination results in the infection of salivary glands The mosquito is able to transmit DENv for its entire life The DENv is transmitted to a person when an infected mosquito introduces anticoagulant substances, present in its saliva to prevent the recipient’s blood clotting, during feeding Four antigenically distinct members of the DENv serotype complex have been identified (DEN-1, DEN2, DEN-3 and DEN-4), and these are considered as four distinct species belonging to the mosquito-borne cluster clade IX of Flavivirus [2] The corresponding viruses of the four serotypes are genetically closely related to one another All DENv serotypes can cause DF, a mild self-limiting acute febrile illness, but 1–5% of patients with DF may experience more complicated and severe diseases, such as DHF and DSS [3,4] Infection with one of these four DENv serotypes provides immunity to only that serotype for life; therefore, persons living in a dengue-endemic area can have more Fig Map of the countries of the world at risk of dengue virus infections in 2006, according to the World Health Organization (http:// gamapserver.who.int/mapLibrary/) Figure reproduced by permission of the World Health Organization FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2987 Progress for dengue virus diseases S Melino and M Paci than one dengue infection during their lifetime Sequential heterotypic infection has been shown to increase virus replication, and thus the probability of developing DHF, by a process known as antibodydependent enhancement (ADE) [5–7] However, there are still cases of DHF and DSS that cannot be adequately explained by ADE, an example being in the confirmed cases of primary infection [8] Accurate knowledge of the viral life cycle is essential in order to highlight potential targets for antiviral therapy and to obtain key information for the rational design of antiviral drugs Virus structure and replicative cycle The structure of the DENv is relatively simple The virions are spherical particles 40–50 nm in diameter, containing three structural proteins: the nucleocapsid protein C (C; 12–14 kDa); protein M (prM; an kDa nonglycosylated membrane protein); and the glycoprotein E (E; 51–59 kDa), which is the major envelope protein present as a homodimer The DENv genome is a single-stranded positive-sense RNA that is encapsidated by protein C in an icosahedral structure The genomic RNA presents a single long ORF encoding the three structural proteins (C, prM and E) and seven nonstructural (NS1–5) proteins (Fig 2) It is translated as a single polyprotein, which is cleaved by proteases of viral and host origin Outside the ORF, there are the 5¢- and 3¢-UTRs, which have secondary structure and are crucial in the initiation and regulation of translation, replication and virion assembly [9–11] Genome organization The first step in the viral infection process is the binding to a cell-surface receptor This step is mediated by E protein, identified as a viral attachment protein for DENv, which leads to virus penetration into the host cell [12–17] prM also seems to have an essential role in the control of the fusion activity of E protein and is necessary for the correct folding of E protein by acting as a chaperone-like protein [18–21] Following entry and fusion, the translation of the genomic RNA into a polyprotein is the first event in DENv-infected cells A small polypeptide is synthesized, the RNA– ribosomes–nascent protein complex docks at the endoplasmic reticulum (ER), and translation and processing of the viral polyprotein continue in association with the ER Processing of the polyprotein is performed by cellular and viral proteases Cleavage at NS1–NS2A occurs soon after synthesis by a still-unknown host protease of the ER [22], while cleavages at C–prM, prM–E, E–NS1 and NS4A–NS4B junctions are performed by a host-cell signal peptidase resident in the ER, and the cleavages at NS2A–NS2B, NS2B–NS3, NS3–NS4A and NS4B–NS5 junctions (see Fig 2) are performed by the viral serine protease, NS3 [23] NS proteins are involved in different functions of the replicative cycle NS1 glycoprotein (42–50 kDa) is present on the cell surface Its colocalization with doublestranded RNA, together with other evidence, suggests that intracellular NS1 protein plays a role in the replication of viral RNA [24,25] NS2A, NS2B, NS4A and NS4B are small hydrophobic proteins that are associated with the membranes In particular, NS2B is associated with the NS3 protease to form an active serine protease complex [26] NS3 is implicated in the ORF 5’ UTR 3’ UTR CAP Polyprotein and processing Structural proteins C prM E Non structural proteins NS1 NS2A NS2B NS3 NS4A NS4B NS5 Furin cleavage NS2B-NS3 protease cleavage Signal peptidases cleavage Unidentified protease in ER 2988 Fig Organization of the dengue virus (DENv) RNA genome and scheme of the proteolytic processing of the DENv polyprotein FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS S Melino and M Paci Progress for dengue virus diseases Table Vaccines against dengue fever by the Initiative for Vaccine Research, World Health Organization, updated February 2006 Type of vaccine Live attenuated tetravalent vaccine Live attenuated 3¢-NCR Live attenuated two-dose vaccine Live attenuated DENv-2 and DENv-2 ⁄ 1,2 ⁄ 3, ⁄ chimeric vaccines Live chimeric virus tetravalent vaccine dengue ⁄ yellow fever vaccine Tetravalent chimeric, F mutant Tetravalent E-NS1 fusion protein subunit DNA and recombinant modified vaccinia Ankara Pharmaceutical company or research group Status of development Sanofi-Pasteur ⁄ Mahidol National Institute of Health GlaxoSmithKline Center for Disease Control and Prevention Acambis ⁄ Sanofi-Pasteur Phase II (pediatric) Phase I (as monovalent) PhaseII Preclinical US Food and Drug Administration Hawaii Biotech National Institute of Health Preclinical Preclinical Preclinical polyprotein processing and RNA replication The NS3 protein (69 kDa) is a multifunctional protein with an N-terminal protease domain (NS3pro) (1–180), an RNA triphosphatase, an RNA helicase and an RNAstimulated NTPase domain in the C-terminal region [26,27] The protease and NTPase enzymatic functions share an overlapping region between residues 160 and 180 of the NS3 protein [28] The RNA triphosphatase may contribute to RNA capping [29], whereas the NTP ⁄ helicase activity may separate nascent RNA strands from the template [30] The NS3 viral protease is absolutely essential (along with the viral-encoded cofactor NS2B) for viral replication In addition to the cleavages at the protein junctions in the polyprotein described above, the viral serine protease is also responsible for internal cleavages in the C, NS2A, NS3 and NS4A proteins, the significance of which is not yet known (see Fig 2) [31–33] The most conserved flavivirus protein is NS5 It is characterized by a methyltransferase motif in the N-terminal domain and by an RNA-dependent RNA polymerase located at its C-terminal domain [34,35] After processing of the viral proteins, most of the NS proteins associate with the 3¢-UTR of viral RNA to form a replication complex for RNA synthesis [2] The association of protein C with genomic RNA on the cytosolic face of the ER membrane is the initial step of virion assembly The particles are transported through the secretory pathway to the cell surface for release Dengue vaccine: a possible solution to DENv infection In the absence of effective antiviral drugs, vaccination offers a good chance for decreasing the incidence of these diseases, live virus vaccines being the most prom- Phase I (adults) ising and cost-effective (Table 1) However, currently, no approved vaccines are available, and various strategies have been used to develop dengue vaccines [36– 38] Vaccine development has been complicated by the potential risk of vaccination resulting in the ADE of future heterotypic infection [36,39] Different strategies for the development of dengue vaccines include live attenuated and inactivated viruses, recombinant subunits, protein expression in Escherichia coli, recombinant baculoviruses, recombinant poxviruses, chimeric viruses derived from infectious cDNA clones of DENv, and naked DNA vaccines In preclinical evaluation using no-human primates, chimeric tetravalent vaccines have been demonstrated to produce high levels of neutralizing antibody and viremia protection against all serotypes after a single dose, and clinical trials are in progress [37,38,40] Another type of dengue vaccine is the DNA vaccine, which represents a promising gene-based vaccine strategy considered suitable for developing a dengue tetravalent vaccine [41,42] Several flavivirus DNA vaccines, including those against dengue, have already been developed [43–46] Recently, a new dengue tetravalent DNA vaccine against DENv-3 and DENv-4, based on a prM ⁄ E strategy and combined with two previously constructed DNA vaccines against DENv-1 and DENv-2, has been constructed [47] Molecular biology techniques have facilitated the development of recombinant subunit vaccines Several structural (E and prM) and nonstructural proteins stimulate immunity, and the nonstructural proteins NS1 and NS3 are the dominant sources of cross-reactive CD4+ and CD8+ cytotoxic T-lymphocyte epitopes [48–51] Passive immunizations using monoclonal antibodies, and active immunization studies using purified proteins, provided evidence that these proteins are important for inducing protective immunity [52–59] A synergistic increase in neutralizing antibody titers by FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2989 Progress for dengue virus diseases S Melino and M Paci simultaneous immunization with the DNA and protein vaccine has been demonstrated [60–62] Recently, a capsid protein of DEN-2 virus has also been used in order to obtain statistically significant protection against the infective homologous virus This suggests that effective protection against the four serotypes might be attainable only by immunization with the four corresponding capsids, or with one of them including the immunodominant cytotoxic epitopes of the others [63] The major pharmaceutical companies are currently developing a treatment against the disease A tetravalent live attenuated vaccine was developed at the Walter Reed Army Institute of Research, Silver Spring, Maryland, licensed to GlaxoSmithKline [36]; this is the first two-dose vaccine to show a 100% immune response against all four virus subtypes that cause the disease The vaccine is expected to enter Phase III in 2007 and be commercially available thereafter, if its efficacy and safety is proven Therapeutic approaches – NS3 protease inhibition as a response to DENv Viral inhibitors have been widely studied in in vitro systems as supportive medical care and for symptomatic treatment; they represent an important aid for patients and for improving survival in severe forms of disease Antiviral therapeutic strategies involve virusbinding blocking to prevent intracellular virus multiplication and maturation Based on the putative receptor role of heparan sulphate for DENv, inhibition of virus binding and entry has been obtained using polyanionic compounds such as heparin [64,65], sulphated polysaccarides extracted from algae [66,67] or polyoxometallates [68,69], and these have been recently described as inhibitors of DENv-2 multiplication in Vero cells Acetylsalicylate and its metabolite sodium salicylate specifically inhibit DENv-2 and Japanese encephalitis virus replication [70] A specific p38 mitogen-activated protein kinase inhibitor seems to be involved in the mechanism of salicylates in suppressing the flavivirus infection Recently, in fact, the inhibition of virus replication through the prevention of virus-associated apoptosis of infected cells represents a new potential pharmacological target for the control of flavivirus infection [71,72] Inhibitors of viral replication have also been studied, for example, ribavirin, and interferon-a, -b and -c [73–75] In recent years, the RNAdependent RNA polymerase and the methyltransferase activity of NS5 protein have been studied as specific viral targets for chemotherapeutic strategies in order to prevent RNA strand elongation and RNA capping, 2990 respectively [76] Recently, nitric oxide has been shown to suppress DENv RNA and protein accumulation in infected cells [77,78] The target of nitric oxide action in viral RNA synthesis has been investigated and the selective inhibitory effect on the de novo synthesis of RNA via the inhibition of RNA-dependent RNA polymerase activity has been identified [79] The serine protease domain of NS3 protein plays a central role in the replicative cycle of DENv [80] Like other viral proteases, the DENv NS3 protease represents an attractive therapeutic target for the development of novel antiviral agents Studies over the past 20 years have shown that many viruses encode one or more proteases [81,82] that catalyze the processing of viral polyprotein or maturational processing of precapsids and which are required for the production of infectious virions The discovery and development of inhibitors of the viral protease activity assumed clinical relevance, as has been demonstrated in cases involving the treatment of patients with acquired immunodeficiency syndrome (AIDS) or hepatitis C virus (HCV) [83–87] Studies on the viral protease significantly increase our understanding of the life cycle of viruses, the mechanism of proteolytic processing and the regulation of cellular processes A recurring theme from structural and sequence analyses is the remarkable compactness of these enzymes In addition, most contain no disulfide bridges, in contrast to many classical cellular proteases, and, moreover, cofactors such as metal ions or peptides are frequently required to stabilize the viral protease [88–90] Most viral proteases have little sequence homology with cellular proteins, even when they share the same backbone fold These characteristics lead to a very different substrate specificity of the viral proteases with very important implications for the design and development of their efficient inhibitors, while undesirable cross reactivity against cellular enzymes can be minimized In the DENv life cycle, proteases from the host (furin and secretase) and from the virus (NS3 protease) are required to process the polyprotein precursor into the individual functional proteins [91], and it has also been observed that inactivating mutations of the DENv NS3 protease (NS3pro) cleavage sites in the polyprotein precursor abolish viral infectivity [92,93] This suggests that NS3pro is a promising drug target for flaviviral inhibitors Structural and functional studies on NS3 protease NS3 viral protease is a trypsin-like protease, which, together with the NS2B cofactor, is essential for the FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS S Melino and M Paci virus replication The importance of this protease activity in viral viability is underscored by the finding that mutations abolishing the activity, when they are introduced in the context of an infectious cDNA clone, eliminate virus recovery [94] The N-terminal 184 amino acid-long domain of the NS3 multifunctional protein (69 kDa) is the serine protease (NS3pro) with a functional catalytic triad (His51, Asp75 and Ser135 in DEN-2) The importance of these catalytic triad residues in the mechanism of homologous flaviviral NS3 serine proteases was established by site-directed mutagenesis of these residues, which abolished protease activity Serine proteases are the best studied of the four classes (serine, aspartic, metallo and cysteine) of proteases [95] The basic mechanism consists of a charge relay system that transfers the negative charge on the buried carboxyl via the histidine to the serine The transfer of the Ser Oc proton to the histidine converts the serine into a strong nucleophile for the attack on the peptidyl carbonyl of the substrate The substrate is oriented by the binding of the amino acid side chain of the P1 residue in the S1 pocket [96], a hydrogen bond between the backbone NH of the P1 residue and two hydrogen bonds between the carbonyl oxygen of the scissible bond and two backbone NH groups of the enzyme (oxyanion binding hole) The reaction is carried on through a tetrahedral transition state with an acylenzyme intermediate The DENv NS3 protease is also commonly designated as being a member of the flavivirin enzyme family (EC 3.4.21.91 and S07.001 Peptidase MEROPS peptidase database http://merops.sanger.ac.uk), which comprise the NS2B–NS3 endoproteases of the Flavivirus genus [97,98] The presence of a small activating cofactor protein is a prerequisite for the optimal catalytic activity of the flaviviral proteases with natural polyprotein substrates [99,100] The DENv NS3 protease requires the presence of the nonstructural NS2B protein for its activity [26] The NS2B–NS3 conjugate has been shown to cleave the precursor polypotein at NS2A ⁄ NS2B, NS2B ⁄ NS3, NS3 ⁄ NS4A and NS4B ⁄ NS5 junctions, as well as at internal sites within C, NS2A, NS3 and NS4A [23,101,102] The NS2B protein is composed of seven domains, which can be separated on the basis of their relative hydrophobicity (domains I–VII) [103] The hydrophobic core residues, belonging to domain IV (G69–E80), were proposed to interact with NS3pro This domain is flanked by two hydrophilic stretches (domains III and V) Studies using mutant plasmids transfected into cells have shown that the fragment of 40 residues of the NS2B encompassing domains III to V, is the Progress for dengue virus diseases minimal region necessary for inducing the protease activity of NS3pro [104,105] Moreover, the NS2B– NS3 association, demonstrated by co-immunoprecipitation experiments, is also mediated by this hydrophilic region [104] Comparing the kinetic properties of NS3 and NS2B–NS3, it has been suggested that NS2B generates additional specific interactions with the P2 and P3 residues of the substrates [106] Currently, the molecular details of the mechanism by which the NS2B cofactor stimulates the activity of the protease are not yet known The analogy with the HCV protease has offered some structural and mechanistic explanations for the activation of this flaviviral protease by its cofactor However, unlike the HCV NS3 protein analog, it seems that in the case of the DEN-2 NS3pro the cofactor activity cannot be supplied in trans with a small peptide derived from the cofactor NS2B [105] Other serine proteases (subtilisin, a-lytic protease) are also known to require a pro-region, such as NS2B, for inducing a productive folding leading to the active form In these cases, once the protein is folded, the necessary pro-region does not remain bound to the active enzyme The results obtained regarding the NS2B–NS3pro complex indicate that NS2B also functions as a molecular chaperone in assisting the folding of NS3pro to the active conformation [105,107] A new construct of the recombinant form of the NS3pro fused to a 40-residue cofactor and corresponding to the hydrophilic part of NS2B by a glycine linker was engineered and expressed in E coli, and demonstrated activity against hexapeptide substrates modified as chromogenic paranitroanilide derivates [105] Expression of the construct CF40GlyNS3pro (the amino acid sequence is shown in Fig 3) resulted in substantially high yields of the soluble and active recombinant protein, which was significantly more active than the refolded NS3pro and CF40NS3pro (lacking the Gly linker) In fact, although the DENv NS3 protease exhibits NS2B-independent activity with small substrates such as N-a-benzoyl-l-arginine-p-nitroanilide, the activity towards peptide substrates is stimulated significantly in the presence of the NS2B protein [26,106] Recently, it has been proposed that the Fx3F motif is the common structural element involved in cofactor binding to the protease [108] This motif consists of two bulky hydrophobic residues separated by three unspecified residues; it has been speculated that additional residues, located outside this sequence motif, would contribute to the stringent specificity of the protease for the corresponding polyprotein substrate [108] A mutagenesis study with the DENv NS2B cofactor has revealed that substitution of the F residues (corres- FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2991 Progress for dengue virus diseases S Melino and M Paci Fig Sequence alignment between DENv-2 NS2B–NS3pro (2FOM pdb) and West Nile virus NS2B–NS3pro (2FP7 pdb) obtained using the T-COFFEE program, version 1.41 (http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/ tcoffee_cgi/index.cgi) [135] The catalytic residues are in bold and the numbers refer to the DENv-2 NS2B–NS3pro sequence ponding to residues Leu75 and Ile79) with alanine results in a decrease of the NS2B–NS3pro autoprocessing to approximately 55 and 75% of the wild-type value [109] By contrast, the replacement of W61 located outside this sequential motif yielded a catalytically inactive enzyme [109] Moreover, in agreement with these results, the W61 residue is also present in the CFNS3d protein complex, which is an active form of the enzyme obtained by limited proteolysis of the CF40GlyNS3pro [107] All these results suggest a pivotal function for this invariant residue in protease activation Other experiments, examining the role of NS2B cofactors, indicate that in addition to activating proteolytic activity, NS2B is necessary for promoting membrane association of the NS3 complex [110], and cryoimmunoelectron microscopy studies have suggested that functional NS2B–NS3 proteolytic activity may be compartmentalized into specific membranous structures [111] This finding suggests that the protease activity may be affected by the membrane environment; in fact, the CF40GlyNS3pro activity in vitro was increased by the presence of zwitterionic and nonionic detergents at low concentrations [105] Structural biological studies The initial structural study was performed by Brinkworth et al [103], using a sequence homology approach of NS3 protease with HCV NS3 protease, which has been widely studied and whose structure has been resolved by X-ray and NMR spectroscopy [112–114] By molecular modelling, a number of insights concerning the cofactor interaction and substrate specificity were obtained The model, by analogy with HCV NS4A, predicted that the NS2B peptide encompassing residues Gly72– Gly83 could be sufficient to function as a peptide cofactor in vitro [103] Moreover, the model suggested a substrate specificity in the P1 position for the basic 2992 Lys or Arg residues because of the presence of an acidic Asp129 residue, present in the active cleft at six residues before the catalytic Ser135 and conserved in all the flavivirus sequences Other interactions between DEN2 NS3pro and the substrate have been predicted by this model, such as a possible H-bond between the Asp75 and the P2 residues and a hydrophobic interaction between the P1¢ residue and the Val52 or Tyr41 residues The resolution of the crystal structure of ˚ NS3pro [115] at 2.1 A has been reported (Fig 4A) This structure differs significantly from that of HCV NS3pro, resembling more that of HCV NS3pro in complex with the cofactor NS4A and, in particular, the first tract of 30 residues of the protein appears with a different conformation In particular, the structure obtained shows a rather limited extension cleft region between the two domains of DEN-2 NS3pro and it was not useful in elucidating the specific interactions with the substrate beyond P2 and P2¢ For these reasons, the structure obtained in the absence of the cofactor could not be used to design specific inhibitors, although it represented an important starting point for the determination of the DEN-2 NS2B–NS3pro complex structure The X-ray structure of the complex NS3pro with a Bowman Birk inhibitor [116] has been compared with the results reported for the DEN-2 NS3pro Differences are particularly pronounced for the stretch of residues 127–136, including the catalytic Ser135 residue, and lead to a different orientation of Asp129, which has electrostatic interactions with the P1 Arg residue However, these observations may not have physiological implications considering that the regulatory component, NS2B, was not present in the complex The structural NMR studies on CF40GlyNS3pro show the presence of a substantially flexible or unfolded region of the protein that is responsible for the aggregation at high concentrations and makes the determination of the solution structure very unlikely [107] FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS S Melino and M Paci Progress for dengue virus diseases A N-Term C-Term Asp 75 His 51 Ser 135 B NS2B 43-96 N-Term C-Term Fig Ribbon representations of the NS3 protease (NS3pro) (1BEF pdb) (A) and the NS2B–NS3pro (2FOM pdb) (B) structures The NS2B 43–96 fragment [108,110] is shown in blue In red are the side chains of the catalytic residues His51, Asp75 and Ser135 ˚ Recently, the crystallographic structure (at 1.5 A resolution) of the active form of the NS2B–NS3pro protein, including the 47-residue core region of NS2B via a glycine linker (such as CF40GlyNS3pro), has become available (2FOM pdb; Fig 4B) [117] Overall, the structure is topologically close to that reported previously (six b-strands in two b-barrels with the catalytic triad located at the cleft between the two barrels) Nevertheless, it presents relevant differences in the secondary and tertiary structure that are important for definition of the structural and functional roles of the NS2B cofactor However, the X-ray structure does not appear to be structurally well defined in some regions This suggests that these regions may adopt multiple conformations when passing from the solution to the crystal state, as has also been observed in the NMR experiments Differences have also been found in the length and location of secondary structural elements, which assume great importance for the solubility and proteolytic activity of this protein form As observed for the complex of HCV NS4A–NS3pro [112,113], NS2B contributes a b-strand (residues 51–57) to the stability of the N-terminal b-barrel of NS3 in contrast to the previously reported prediction by homology modelling, where the interacting fragment of the NS2B was the 70–81 tract [103] On the other hand, the expression of a truncated NS2B–NS3pro form, including only the 40–66 residues of NS2B, gives a soluble but catalytically inactive form of the enzyme [117] This suggests that the region 40– 66 of the cofactor is important in the folding of the protein and that the C-terminal part of the cofactor, which is absent in the truncated form, directly interacts with the substrate-binding site On the other hand, the soluble form of the CFNS3d protein complex, obtained by limited proteolysis of CF40Gly–NS3pro, which conserves 52% of the proteolytic activity, consists of the D6–E179 region of NS3pro and the NS2B fragment D50–E80 The 1H-15N heteronuclear single quantum coherence spectrum of the uniformly labelled 15 N-CFNS3d shows a good cross-peak dispersion, indicating a stable folded state of the protein [107] All data confirm that the NS2B fragment D50–E80 has a strong interaction with NS3pro and is also able to promote in trans the activity of the enzyme when correctly folded This finding indicates that this cofactor region has an important role in the conformational stability of the active site In the crystal structure, the electron density beyond the NS2B residue 76 is discontinuous, revealing that this region may adopt several conformations probably as a consequence of its great flexibility in solution No evidence of direct interactions of NS2B with the active site are found in this structure, giving no structural explanation of its absolute requirement by NS3pro for activity On the contrary, the crystallographic structure of the homologous NS2B–NS3pro protein of West Nile virus has shown direct interactions of the C-terminal part of NS2B with the active site of the NS3pro The C-terminal part of NS2B wraps around NS3pro and, in particular, the Arg78–Leu87 residues form a b-strand in NS2B, which links the N-terminal tract of NS3pro The structure of the West Nile virus NS2B– NS3 pro-inhibitor complex has also elucidated (2FP7 pdb) [117], the details of the S1 pocket, formed by Gly151, Tyr161, Tyr150, Asp129 and the backbone FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2993 Progress for dengue virus diseases S Melino and M Paci residues of Tyr130–Thr132 Asp129 is located at the bottom of the pocket, stabilizing the positively charged side chain of P1 arginine It has also been reported that in this case the S2 pocket is dominated by the negative electrostatic charge originating from the NS2B residues Asp82, Gly83 and Asn84 that are close to the positively charged guanidinium group of the P2 arginine [117] These findings could also explain the importance of a basic residue at P2 for the DEN-2 NS2B–NS3pro protein, and are in agreement with the observed loss of binding when the P2 arginine is replaced by an alanine residue The importance of this contribution is in the observation that the formation of the active protease differs substantially from those observed with other cofactor-activated viral proteases, such as HCV NS4A–NS3pro [113,114] The structures currently available explain well the huge increase in activity of flaviviral NS3pro in the presence of NS2B, and may be useful in the development of drugs to treat the flaviviral diseases Substrate specificity of NS2B–NS3pro The first step towards designing an inhibitor of the viral protease is to identify substrate specificity The selectivity of the proteases for particular substrates results from the presence of specific binding sites on the enzyme for amino acid side chains of the substrate(s) The virus-encoded proteases display an unusual degree of selectivity for their natural polyprotein substrates and only very few cases are known where the viral enzyme reacts with protein substrates derived from the host cell [118,119] In the case of viral proteases, the identification of a high turnover substrate is usually difficult [120] because the kinetic parameters of synthetic peptides based on the natural cleavage sites are generally unfavorable [121] The NS3 protease in the absence of the cofactor reacts with small model substrates for serine proteases, such as N-a-benzoyl-l-arginine-p-nitroanilide, and activity of the NS3 protease towards the substrate is higher than that of the NS2–NS3 complex [106] This suggests that substrate recognition in the complex requires additional interactions, extending beyond the P1 site, for optimal activity Other studies have indicated that NS2B–NS3pro requires the presence of Lys ⁄ Arg and Arg, respectively, at the P2 and P1 positions, for achieving substrate proteolysis, and that the cleavage motifs have features in common with the physiological cleavage sites [122,123] The best substrate identified, using synthetic combinatorial libraries of peptides and single substrate kinetics, is the fluorogenic 2994 peptide Bz-nKRR-acmc, which shows, for DEN-2 protease, an apparent Km value of 12 ± lm, a kcat of 1.4 ± 0.1Ỉs)1 and a catalytic efficiency, expressed as kcat ⁄ Km, of 112 100 ± 18 500Ỉm)1Ỉs)1 [122] The substrate pocket of the NS3 proteases from the four serotypes consists of a number of highly conserved residues within the S1–S4 region The enzymes of the four serotypes appear to share very similar substrate specificities, which implies that it is possible to develop a single inhibitory agent targeting all four dengue NS3 proteases [122] Basic or aliphatic residues at P3 and P4, and small or polar residues at P1¢ (Ser > Gly > Ala), are required [123,124] Moreover, the P3 and P4 positions also contribute significantly to ground state binding, providing additional evidence for enzyme–substrate interactions that extend beyond S2 to S2¢ [122] The introduction of an arginine residue at P3 results in an almost four-fold increase in kcat ⁄ Km, and the introduction of an arginine residue at P3 and P4 in the capsid protein-derived tetrabasic sequence RRRR results in a 30-fold increase in kcat ⁄ Km A higher degree of selectivity for serine at the P3¢ position is needed, whereas selection of residues at the P2¢, and especially at the P4¢ positions seems to be relatively unrestrained [123] Recently, the specifics of substrate recognition by NS3pro from DENv have been mapped using a library of the 9-mer peptides to the cleavable sequences with the general P4–P3–P2–P1– P1¢–P2¢–P3¢–P4¢–Gly structure [124] The N terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag, respectively The amino acid sequences of the peptides corresponding to the junction regions efficiently cleaved by the DENv protease are shown in Table In addition, other potential sites of the NS2B–NS3pro that are efficiently cleaved have been identified, also on the basis of the high homology with the West Nile virus NS3 protease [124] These sites are in the NS3pro ⁄ helicase protein and correspond to the sequences 1659RKKRRLTIM1666, 1674KTKRYLPA1681 and 1930AQRRGRIG1937 [117] A library obtained by randomization of the P1¢ and P2¢ positions of the peptide 2522GKRGGAK2529 with different amino acids has been used to demonstrate that the NS2B–NS3pro can accommodate, in these positions, a number of the amino acid residues, including the bulky hydrophobic Trp, Phe and Tyr, but does not tolerate the presence of the negatively charged Asp and Glu residues [124] In contrast, the homologous proteases from West Nile virus and from Japanese encephalitis virus prefer small (like Gly) or polar amino acid residues in the P1¢ and P2¢ positions Noteworthy, the West Nile virus protease processes substrates with a P2 FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS S Melino and M Paci Progress for dengue virus diseases Table The amino acid sequences of the cleavage sites of the NS2B–NS3pro protease in the precursor polyprotein flDenotes the scissile bond, and the P1 residues are shown in bold Lys more efficiently than those with a P2 Arg, in contrast to the four serotypes of dengue protease [122], which are more active against substrates with Arg instead of Lys at P2 Recent work shows that the cofactor residue at NS2B-84 is associated with a preference for Lys or Arg at the substrate P2 position In particular, the presence of an Asn or an Asp residue at NS2B-84 leads to a preference for a Lys residue at P2 of the native substrate, while the substitution with Ser, Thr or Glu at NS2B-84 leads to a preference for an Arg residue at the P2 position [125] Thus, the finding that DENv proteases exhibit a preference for Arg at the P2 position could be explained by the presence of Ser or Thr at NS2B-84 [125] On the basis of these recent studies, the DENv enzyme seems to adopt a restricted specificity to process the natural cleavage sites of the polyprotein precursor, but this specificity is less stringent than the homologous viral proteases Inhibition of NS3 protease, a therapeutic target In a first step towards design of an inhibitor for the DENv NS3 serine protease, the standard inhibitors of serine proteases have been assayed The serine protease inhibitor, aprotinin, has been shown to inhibit the four CF40GlyNS3pro proteases with high affinity (Ki ¼ 79, 25, 88, 6.4 pm for DEN 1–4 CF40GlyNS3pro proteases, respectively), whereas other serine protease inhibitors show a low ability in inhibiting the viral protease [105,122] Similarly to the HCV NS3 protease, the existence of a high-affinity binding site in the nonprime region of the enzyme offers the possibility of developing effective inhibitors against the DENv protease by combinatorial optimization of the cleavage sites For this reason, small-molecule inhibitors based upon the peptide substrates have been synthesized as inhibitors of NS3pro (Table 3) N-terminal cleavage site peptides, corresponding to the P6–P1 region of the Table Representative competitive inhibitors of the dengue NS2B–NS3pro serine protease Type of compound Compound Ki value (lM) Reference a-keto-amide Amide Ac-FAAGRR-a keto–SL-CONH2 Ac-FAAGRR-CONH2 Ac-RTSKKR-CONH2 Ac-KKR-CONH2 Ac-FAAGRR-CHO Bz-Nle-Lys-Arg-Arg-H Bz-Ala-Lys-Arg-Arg-H Bz-Phe-Lys-Arg-Arg-H Bz-Nle-Lys-Arg-Phe-H Bz-Nle-Lys-Arg-Trp-H Bz-Nle-Phe-Arg-Arg-H Bz-D-Nle-Lys-Arg-Arg-H Bz-Lys-Arg-Arg-H Bz-Arg-Arg-H Bz-Nle-Lys-Arg-Arg-(p-guanidinyl)Phe-H Bz-Nle-Lys-Arg-Arg-CF3 Bz-Nle-Lys-Arg-Arg-B(OH)2 Panduratin A 4-Hydroxypanduratin A 47 25.87 12.14 22.31 16 5.8 5.3 6.8 15.9 7.5 15.8 9.4 1.5 12.0 2.8 0.85 0.043 25 21 [105] [126] [126] [126] [105] [131] [131] [131] [131] [131] [131] [131] [131] [131] [131] [130] [130] [133] [133] Aldehyde Trifluoromethyl ketone Boronic acid Cyclohexenyl chalcone derivative FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS 2995 Progress for dengue virus diseases S Melino and M Paci polyprotein, were found to act as competitive inhibitors of the enzyme, with Ki values ranging from 67 to 12 lm The NS2A ⁄ NS2B cleavage site, RTSKKR, is the peptide with the lowest Ki value However, in contrast to HCV NS3 protease, the cleavage products and their analogs not appreciably inhibit this protease In fact, the peptides corresponding to the P1¢–P5¢ region of the polyprotein cleavage sites not show any inhibitory effect on enzymatic activity, even at mm concentration [126] Peptidic a-keto amide inhibitors have been well characterized as reversible competitive inhibitors for other serine proteases, including HCV NS3 protease [127– 129] Similarly, in the case of DENv NS3 protease, some peptidic a-ketoamide inhibitors have been synthesized [105,130] in order to determine their inhibitory efficacy Except for the a-ketoamide peptide Ac-FAAGRR-aketo-SL-CONH2 [105], which showed a Ki value of 47 lm, the other synthesized a-ketoamide peptides showed no effect up to 500 lm concentration [130] Substrate-based peptide aldehydes have also been synthesized, resulting in low micromolar inhibitors with a Ki value up to 1.5 lm (see Table 2) [105,131] A systematic structure–activity relationship study has revealed that the P2 Arg residue is more important for enzyme interactions than P1 Arg and that the dipeptide aldehyde inhibitors have a low micromolar activity [131] Furthermore, the replacement of P1 arginine with Phe and Trp does not induce a loss of the inhibitory activity [131] In the published structure of DEN-2 NS3pro-MbBBI (1DF9), obtained in the absence of the NS2B cofactor, the P1 Arg residue has electrostatic interactions with Y150 and S163 and forms two H-bonding interactions with D129 [116,132] Thus, when P1 Arg is replaced with Phe or Trp, the hydrogen bonds with Y150 and S163 are lost, but p–p interactions between P1 Phe ⁄ Trp and Y150 are possible and stabilize inhibitor binding [131] Potent inhibitors have been identified by incorporating trifluoromethyl ketone and boronic acid onto the substrate peptide [130] Tetrapeptide boronic acid proved to be the most potent inhibitor of DENv NS3 protease, having a Ki of 43 nm, whereas the trifluoromethyl ketone occupies an intermediate position between that of peptide aldehyde and peptide boronic acid [131] Recent studies have shown that some natural compounds, such as chalcones isolated from Boesenbergia rotunda (a common spice belonging to a member of the ginger family), are able to inhibit the DENv NS3 protease In particular, the cyclohexenyl chalcone derivates, 4-hydroxypanduratin A and panduratin A, show good competitive inhibitory activities against DEN-2 virus NS3 protease, having apparent Ki values of 21 2996 and 25 lm, respectively [133] Although several inhibitors of DENv proteases have been tested, selective viral protease inhibition has not been obtained to date and inhibitors for clinical trials are not yet available Conclusions Albeit there are still no specific vaccines or chemotherapy regimes for the prevention and treatment of DF and DHF, the understanding and the biochemical characterization of the life cycle of DENv have made substantial progress over the past few years, and all the life cycle stages represent potential targets for antiviral drug discovery The DENv NS3 protein, as in other viral pathologies, is a valid molecular target for the development of antiviral compounds Inhibitors against the DENv protease will be of great benefit in the design of drugs to combat other related flaviviruses, as well as Japanese encephalitis virus and West Nile virus In vitro inhibitors against NS2B–NS3pro protease for the four serotypes are now available However, the fact that the protease activity in vitro of the NS2B– NS3pro (or CF40GlyNS3) is present only at alkaline pH, suggests that in the physiological environment of the host cell, further interactions with an unknown activator or post-translational modifications are required for optimal activity This situation could be similar to that of kallikrein Recently, in fact, glycosoaminoglycans or kosmotropic salts have been found to induce a relevant increase of human kallikrein activity [134] In the presence of these compounds, the optimum pH of this secreted serine-type protease shifted towards lower values (from pH towards pH 7.5) [134] The interaction between the NS2B cofactor and NS3pro seems to be important not only for the correct fold of the protease but also for the correct interaction with the substrate The binding site of the NS2B cofactor could be examined for a view to inhibitor development, and competitive inhibitors of the binding site might be able to inhibit the correct fold of the protease At present, no inhibitors of the cofactor–protease binding are available, although the resolution of the crystallographic structure and the production of mutants can help to develop specific inhibitors of the binding to the cofactor Some regions of the NS2B–NS3pro structure are not yet well defined, and their resolution will be important for the complete understanding of the structure–function correlations, such as the resolution of the structure of its complex with an inhibitor On the other hand, the NTPase ⁄ helicase region of the NS3 protein, and the surface of NS3 protein with FEBS Journal 274 (2007) 2986–3002 ª 2007 The Authors Journal compilation ª 2007 FEBS S Melino and M Paci the NS5 replicase, could represent alternative drug targets Thus, the use of a pharmacological therapy using combinations of different inhibitors, similarly to other viral therapies, could minimize the development of rapid resistance In conclusion, considerable effort has recently been made towards inhibiting the viral replication of DENv Much remains to be performed to achieve results suitable for experimentation in clinical trials and to produce a drug for blocking the DENv spread To date, funding for a co-ordinated strategy against dengue has been disappointing This is probably attributable to the spread of dengue diseases in the world’s poorest countries Our hope therefore is that the major international funding agencies will now seriously consider increasing their commitment to combat these diseases The need is all the more urgent now that climate change has made the spread of DENv to Western 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Pacific and the Americas A substantial number of people travelling to endemic regions are also infected each year In the last year, DHF has again been on the increase in India and in several Asian... Melino and M Paci Progress for dengue virus diseases Table Vaccines against dengue fever by the Initiative for Vaccine Research, World Health Organization, updated February 2006 Type of vaccine... Compound Ki value (lM) Reference a- keto-amide Amide Ac-FAAGRR -a keto–SL-CONH2 Ac-FAAGRR-CONH2 Ac-RTSKKR-CONH2 Ac-KKR-CONH2 Ac-FAAGRR-CHO Bz-Nle-Lys-Arg-Arg-H Bz-Ala-Lys-Arg-Arg-H Bz-Phe-Lys-Arg-Arg-H