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Inhibition of the NS2B-NS3 – Towards a Causative Therapy for Dengue Virus Diseases

Gerd Katzenmeier#

Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon No. 4 Rd., Nakornpathom 73170, Thailand

Abstract The high impact of diseases caused by dengue viruses on global health is now reflected in an increased interest in the identification of drug targets and the rationale-based development of antiviral inhibitors which are suitable for a causative treatment of severe forms of dengue virus infections – dengue haemorrhagic fever and dengue shock syndrome. A promising target for the design of specific inhibitors is the dengue virus NS3 protease which – in the complex with the small activator NS2B – catalyses processing of the viral polyprotein at a number of sites in the nonstructural region. The NS3 protease is an indispensable component of the viral replication machinery and inhibition of this protein offers the prospect of eventually preventing dengue viruses from replication and maturation. After nearly a decade of mainly genetic analysis of flaviviral replication, recent studies have contributed substantial biochemical information on polyprotein processing including the 3-dimensional structure of the dengue virus NS3 protease domain, the mechanism of co-factor-dependent activation and sensitive in vitro assays which are needed for studies on substrate specificity and the development of high-throughput assays for inhibitor screening. This review discusses recent biochemical findings which are relevant to the design of potential inhibitors directed against the dengue virus NS3 protease. Keywords: Dengue virus, NS2B/NS3, polyprotein, protease, inhibitor, treatment.

Viral polyprotein processing nucleotides and encodes a single polyprotein precursor of 3,391 Dengue viruses, members of the Flaviviridae residues[3]. Individual viral proteins are family, possess single-stranded, positive arranged in the order C-prM-E-NS1-NS2A- sense RNA genomes and generate mature NS2B-NS3-NS4A-NS4B-NS5. Proteolytic viral proteins by co- and post-translational cleavages in the N-terminal region of the proteolytic processing of a polyprotein viral polyprotein are mediated by a host precursor catalysed by host cell and virus- and yields three structural encoded [for review see refs. 1, 2 proteins C, prM and E, which constitute the and references herein]. The genomic RNA virion[4]. Before the virion exits the cell, prM of dengue virus serotype 2 contains 10,723 is cleaved by a cellular -type

# E-mail: [email protected]; Tel.: (662)-800-3624-1237; Fax: (662)-441-9906

58 Dengue Bulletin – Vol 28, 2004 Inhibition of NS2B/NS3 Protease prohormone convertase in the post-Golgi Cleavage sites recognized by the acidic compartment to yield the M protein[5]. NS2B/NS3 protease consist of ‘dibasic’ Cleavages at the NS1/NS2A and residues Lys-Arg, Arg-Arg and Arg-Lys at the NS4A/NS4B junctions are catalysed by a (nonprime) P1 and P2 positions of the signalase bound to the membranes of the cleavage site sequence followed by short ER[6,7]. Proteolytic cleavages in the chain residues such as Gly, Ala and Ser at nonstructural region of the polyprotein are the (prime) P1’ position. The “non- mediated by a heterodimeric complex of canonical” NS2B/NS3 site contains a Gln NS3 with the activator protein NS2B which residue at the P2 position (Figure 1). catalyses in cis (intramolecular) cleavages at the NS2A/NS2B and NS2B/NS3 sites and in The NS3 protease domain trans (intermolecular) cleavages at the NS3/NS4A and NS4B/NS5 polyprotein The existence of a -like serine junctions[8-10]. Additional cleavages protease domain in the N-terminal region of mediated by the NS3 protease within the C, the flaviviral NS3 proteins was originally NS2A, NS4A and within a conserved C- predicted by sequence comparisons between [14] terminal portion of NS3 itself have been cellular and virus-encoded proteases . The described in the literature[11-13]. NS2B-NS3 of the Flavivirus genus which at present comprises at least 68 known members, are now commonly Figure 1. The 3-dimensional structure of designated as (EC 3.4.21.91)[15,16]. the dengue virus NS3 protease The dengue virus 69 kDa NS3 protein is a (shown here is a superimposition of a multifunctional protein with a serine ribbon-presentation of the Ca trace on a protease domain located within the N- space-filling surface model. Residues of terminal 167 amino acid residues[17] and the (His51, Asp75, Ser135) activities of a nucleoside triphosphatase are shown as sticks. The figure was (NTPase) and RNA helicase in the C- [18] generated by Deepview Swiss-Pdb Viewer) terminal moiety . A catalytic triad consisting of residues His51, Asp75 and Ser135 was identified by site-directed mutagenesis experiments and replacement of the catalytic serine by alanine resulted in [19] Ser135 an enzymatically inactive NS3 protein . His51 The NS3 protease is an essential component for maturation of the virus and viable virus was never recovered from infectious cDNA Asp75 clones carrying mutations in the NS3 sequence which abolished protease activity[20]. Interaction of the helicase portion of NS3 with the viral RNA- dependent RNA polymerase NS5 may promote the association of the viral replicase complex to the membranes of the ER[21].

Dengue Bulletin – Vol 28, 2004 59 Inhibition of NS2B/NS3 Protease

Figure 2. Schematic of dengue virus polyprotein processing (shown here are sites on the flavivirus polyprotein cleaved by host-encoded proteases and the virus-encoded two-component protease NS2B-NS3)

C prM E NS1 NS2ANS2BNS3 NS4ANS4BNS5

NS2B/NS3 protease Signal peptidases Furin protease Unidentified protease

The 3-dimensional structure of the N- natural polyprotein substrates. Although the terminal 185 residues of the dengue virus dengue virus NS3 protease exhibits NS2B- NS3 protease domain (NS3pro) was independent activity with model substrates resolved at a resolution of 2.1 Å[22]. The for serine proteases, enzymatic cleavage of overall folding of the protein resembles the dibasic is markedly enhanced with 6-stranded b-barrel conformation typical for the NS2B-NS3 co-complex and the -like serine proteases. presence of the NS2B activation sequence is Interestingly, the structure of the dengue indispensable for the cleavage of virus NS3 protease is closer to that of the polyprotein substrates in vitro[24]. The initial hepatitis C virus NS3/NS4A co-complex characterization of the co-factor than to the unliganded HCV NS3 protease, requirement for the dengue virus NS3 an observation which is suggestive of major protease had revealed that the minimal structural differences in the co-factor- region necessary for protease activation was dependent activation mechanism of the two located in a 40-residue hydrophilic segment proteases[23]. The substrate of of NS2B[25]. The hydrophobic flanking NS3pro is relatively shallow and contourless regions of the 14 kDa NS2B protein are and specific -substrate interactions likely to be involved in targeting the were not predicted to extend beyond the protease complex to the membranes of the P2 and P2’ positions of the substrate ER where genome replication occurs. Fusion in the absence of the NS2B co- of the NS2B core sequence to the NS3 factor[22] (Figure 2). protease domain yielded a catalytically active NS2B(H)-NS3p protein, which, upon expression in E. coli and subsequent The NS2B co-factor refolding, displayed autoproteolytic processing at the NS2B/NS3 site conducive The presence of a small activating protein or [24] co-factor is a prerequisite for optimal activity to the formation of a non-covalent adduct . Incorporation of a flexible nonamer linker, of the flaviviral NS3 proteases with their Gly4-Ser-Gly4, between the NS2B core

60 Dengue Bulletin – Vol 28, 2004 Inhibition of NS2B/NS3 Protease segment and the protease domain resulted reaction with the synthetic substrate peptide in a cleavage-resistent protease with GRR-AMC. optimized enzymatic activity against For the HCV NS3-NS4A protease hexapeptide substrates representing native complex, large structural rearrangements polyprotein cleavage junctions[26]. A leading to a catalytic triad, which is recombinant construct representing the full- conformationally optimized for proton length NS2B co-factor linked to the NS3 shuttle during catalysis, were observed as a protease domain was enzymatically active result of co-factor binding[30]. No 3- with peptide substrates derived from the dimensional structure is available for the polyprotein; however, this protein was dengue virus NS2B-NS3 co-complex and completely resistant to proteolytic self- the precise mechanism of co-factor- cleavage[27,28]. dependent activation is not fully elucidated An essential requirement for the correct as yet. In particular, it is an open question as association of the co-factor with the to whether binding of the substrate protease is the presence of hydrophobic contributes to the formation of an ‘induced residues which act as ‘anchor’ for the fit’ conformation as observed with the HCV protease – co-factor interaction. Recently, NS3 protease[31,32]. based on mutagenesis experiments and sequence comparisons of known flaviviral Substrate specificity co-factors, the “Fx3F” - motif was proposed as the common structural element involved So far, only very limited efforts have been [29] in co-factor binding to the protease . The undertaken to analyse the precise substrate “Fx3F” - motif is comprised of two bulky requirements and determinants of cleavage hydrophobic residues separated by three efficiency for the dengue virus NS3 protease. unspecified residues and it was This is surprising in the light of the fact that hypothesized that additional residues development of inhibitors against serine located at the N-terminus of the activation proteases usually starts with optimal peptide sequence would contribute to the stringent substrates derived from the nonprime side specificity of the protease for the wherein the scissile amide bond is replaced polyprotein substrate. A mutagenesis study by an electrophile which reacts with the with the dengue virus NS2B co-factor had catalytic serine residue. revealed that substitutions of the “F” - residues (Leu75 and Ile79 in NS2B of The NS3 protease reacts with small dengue virus type 2) by alanine resulted in model substrates for serine proteases such as preponderant effects on the catalytic activity N-a-benzoyl-L-arginine-p-nitroanilide (BAPA) of the NS3 protease rather than on substrate and activity of the unliganded NS3 protease binding [P. Niyomrattanakit, unpublished towards this substrate is higher than that of data]. A single residue in the N-terminal the NS2B-NS3 co-complex[24], a finding region of NS2B, Trp62, was critical for which suggests that substrate recognition in protease activation and replacement of this the complex requires additional interactions residue yielded a NS3 protease which was extending beyond the P1 side for optimal catalytically inactive in autoproteolysis and activity. A number of fluorogenic tripeptides containing dibasic residues at the P1 and P2

Dengue Bulletin – Vol 28, 2004 61 Inhibition of NS2B/NS3 Protease positions are cleaved by NS2B(H)-NS3pro 10-fold better than the best commercially protease and the best substrate identified in available substrate tested so far, GRR-AMC these experiments was GRR-AMC, which [J. Wikberg, unpublished data]. In the near had a Km value of 180 mM, a kcat value of future, these investigations will likely lead to 0.031 s-1 and a catalytic efficiency expressed the identification of NS3 substrates with -1 -1 [24] as kcat / Km of 172 s M . optimized sequence length and improved binding affinities, which can be applied as Chromogenic p-nitroanilide peptides sensitive probes for enzyme activity in high- representing hexameric sequences of the throughput inhibitor screenings. native polyprotein cleavage junctions were tested in a photometric assay and the most efficiently cleaved substrate derived from Perspectives for inhibitor the NS4B/NS5 site (Ac-TTSTRR-pNA) had a -1 development Km of 346 mM, kcat of 0.095 s and a kcat / Km of 275 s-1 M-1 [26]. Principally, every step of viral Recently, we have shown by a HPLC- morphogenesis, from cell-entry, uncoating, based assay with fluorometric detection that replication and assembly of new virus the NS2B-NS3pro protease incorporating a particles, is a potential target for antiviral full-length NS2B co-factor could cleave N- inhibitors. However, the molecular events in terminally dansylated 12mer peptides the infectious cycle of flaviviruses such as mimicking native polyprotein junctions in dengue virus are characterized only to a the absence of microsomal membranes[28]. very limited extent, making the design of However, this protein was completely specific inhibitors an adventurous task. In inactive in autocleavage and the efficiency contrast, proteases and their inhibitors have of this recombinant protease with the been intensively studied because of their peptide substrate was markedly reduced potential for the development of selective when compared to constructs incorporating antiviral compounds. Although tremendous the 40-residue NS2B core sequence, likely progress in the field is indicated by the due to structural distortions induced by the design and clinical use of antiviral drugs flanking regions of NS2B and inefficient against HIV (AIDS) and hepatitis C virus activation of the protease. proteases, the potential of dengue and related flaviviral proteases for inhibitor Currently, a detailed study on substrate discovery is largely unexploited. In response specificity of the dengue virus NS3 protease to the global problem of the dengue virus is in progress, which uses combinatorial epidemics, considerable efforts are now libraries of internally quenched fluorogenic being devoted to the development of drugs peptides labelled with the aminobenzoyl / which will eventually be suitable for a p-nitrotyrosine reporter pair. For a substrate chemotherapeutic intervention in acute peptide based on the NS3/NS4A cleavage dengue diseases, not only by academic site, Abz-AAGRK?SLTLY(NO2)R-NH2 (? institutions but also by pharmaceutical denotes the cleavage site), a Km value of 141 companies (for example see -1 mM, a kcat of 0.18 s and a kcat / Km of 1262 http://www.nitd.novartis.com). s-1 M-1 was found, which is approximately

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In a first step towards a rational inhibitor suggest the existence of a high-affinity design for the dengue virus NS3 serine binding site in the non-prime region of the protease, inhibition by synthetic peptides enzyme and offer the prospect of mimicking uncleavable transition state developing effective inhibitors against the isosteres of the P6-P2’ residues of the native dengue virus protease by combinatorial polyprotein sites, was demonstrated[26]. The optimization based on the structure(s) of peptides with an a-keto amide in place of native polyprotein cleavage site peptides. the scissile amide bond acted as competitive However, in general, peptide-based inhibitors of the NS3 protease with Ki - inhibitors exhibit poor pharmacokinetic values in the micromolar range. properties and usually the conversion of Replacement of the P1’-P2’ residues by a these structures into less “peptide-like” carboxyl-terminal aldehyde in the compounds (“peptidomimetics”) is required NS3/NS4A-derived peptide (Ac-FAAGRR- to generate drug-like entities. CHO) yielded a competitive inhibitor with a Inhibitor discovery for the dengue virus K of 16 mM. For the dengue virus protease, i NS3 protease is currently limited by the lack the hexapeptides displayed K - values which i of a 3-dimensional structure for the NS2B- were only 2- to 6-fold lower than the K - m NS3 co-complex; however, it can be value for the corresponding substrate, a expected that crystallographic studies and feature which discriminates dengue virus NMR-experiments will provide more insight NS3 from the HCV protease, where product into the structure and catalytic mechanism of inhibitors had binding affinities which were the enzyme in the near future. In addition, one order of magnitude lower than those for powerful computer modelling approaches the substrates[33,34]. exist which may help to obtain information Product inhibition of the HCV NS3 required for a rational drug design even in protease by cleavage-site derived peptides the absence of crystallographic structures. led to the discovery of very potent inhibitors Proteochemometric modelling (PCM) is of this enzyme with IC – values in the 50 currently explored as a tool to analyse the nanomolar range by cyclic optimization of structural and physicochemical properties the inhibitor structures[33,34]. Recently, we which are necessary for the interaction of have shown that peptides representing non- potential inhibitors with the dengue virus prime-side residues of the dengue virus NS3 NS3 protease target structure[35]. Preliminary protease act as competitive inhibitors of the data obtained by this approach suggest that enzyme, whereas prime-side peptides the proteochemometric models are valid appeared to have negligible effects on and useful for the accelerated design of enzyme inhibition at concentrations >1.0 novel inhibitors. This approach does not mM. (S. Chanprapaph, unpublished data). Ki only circumvent the traditional erratic dug - values for hexapeptides derived from all 4 discovery process with its high attrition rates, dengue virus cleavage sites were in the low but also allows to incorporate potential micromolar range and the best inhibitor was resistance of the target and the development based on the NS2A/NS2B site (Ac-RTSKKR- of ‘drug resistance – resistant’ compounds as m CONH2) and gave a Ki of 12 M. In analogy an initial consideration in the design process. to the HCV NS3 protease, these findings The existence of large conformational

Dengue Bulletin – Vol 28, 2004 63 Inhibition of NS2B/NS3 Protease ensembles is particularly challenging in the biomedical targets in the dengue virus case of rapidly mutating viral , polyprotein would even make a therapy where a design against a moveable target feasible, which uses combinations of would require large sets of corresponding different inhibitors and therefore could inhibitors. In the future, these problems are minimize the risk of rapid resistance likely to be addressed by ‘shotgun development. approaches’ to the structure of enzyme- It can also be expected that progress for inhibitor complexes and the identification of inhibitors against the dengue virus protease hot spots of ‘interaction flexibility’ by the will be of large benefit for drug design use of fast, high-resolution methods such as against related human pathogens of the NMR. Detailed accounts on this strategy are flavivirus complex such as virus, given in Reference 36. Japanese encephalitis virus and West Nile virus. Conclusions However, in order to bring an effective anti-dengue drug from the ‘bench to the Substantial progress has been made over the past few years in the biochemical bedside’, several questions and limitations need to be addressed. These include the characterization of the dengue virus NS2B- evaluation of prognostic markers for disease NS3 two-component protease. The data, severity, the pathobiochemistry of dengue which are available now, make the dengue haemorrhagic fever and shock syndrome, virus NS3 protease a valid molecular target the problem of selectivity against for the development of antiviral compounds. pharmacologically relevant human proteases A large repertoire of powerful methods for such as furin and the risk of adverse effects. inhibitor development and evaluation exists Potential complications may also arise from which includes state-of-the-art technology in the presence of four related dengue organic synthesis and computer-aided serotypes in the case that their NS3 molecular design. Although there is no proteases show marked differences in their suitable animal model available for dengue inhibition profiles. Intensive efforts and virus diseases, initial screening of potential sustained multidisciplinary research is antiviral compounds would be facilitated by required in the future to cope with the well-established insect and mammalian cell challenging task of a causative treatment for culture systems which are useful to monitor dengue virus diseases. the effects of anti-NS3 inhibitors on the propagation of the virus. Acknowledgements Moreover, alternative drug targets which are present on the dengue virus NS3 We thank Dr J. Wikberg, Department of protein can be exploited for inhibitor Pharmaceutical Biosciences, Uppsala development. These include the binding site University, Sweden, for intensive discussions of the NS2B co-factor to the NS3 protease, and access to data prior to publication. This the NS3 NTPase / helicase portion and the work was supported by grants from the interaction surface of NS3 with the NS5 Thailand Research Fund (TRF). replicase. The presence of multiple

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