Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic ␤-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4= to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease. Dengue virus (DENV) is a member of the flavivirus genus, which includes several viruses that are important human pathogens, including yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). The four serotypes of DENV are estimated to cause 50 to 100 million human infections worldwide every year in tropical and subtropical regions (16). There are currently no clinically approved vaccines or therapeutics for DENV. Understanding the molecular details of DENV infection is essential for vaccine and antiviral development.Flaviviruses contain a single strand of positive-sense RNA that encodes three structural and seven nonstructural (NS) proteins that are translated as a single polypeptide chain. NS3 contains two functions, an N-terminal serine protease and a C-terminal RNA helicase. The catalytic triad (His51, Asp75, and Ser135) is within the NS3 protease domain, but a region of NS2B is also required for catalytic activity (15). NS2B contains three predicted transmembrane helices, ensuring that the NS2B-NS3 protease is bound to the endoplasmic reticulum. The viral polyprotein is cleaved into the individual proteins by a combination of host proteases and the viral NS2B-NS3 protease (6, 30). The proteolytic activity of the viral protease is essential for viral replication, making it an excellent antiviral target (18).Ligand-free structures of flavivirus proteases have been solved for DENV-1 and -2 and a WNV active-site mutant (1, 7, 13). In addition, the structure of Mur...

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Background: The NS5 protein from dengue virus comprises a methyltransferase and a polymerase domain connected by a linker region. Results: Linker residues enhance polymerase activity and thermostability. Conclusion: A crystal structure of the dengue virus polymerase reveals that linker residues contribute to protein stability. Significance: These results should accelerate the development of antivirals against dengue virus, a major human pathogen.

We performed a fragment screen on the dengue virus serotype 3 RNA-dependent RNA polymerase using x-ray crystallography. A screen of 1,400 fragments in pools of eight identified a single hit that bound in a novel pocket in the protein. This pocket is located in the polymerase palm subdomain and conserved across the four serotypes of dengue virus. The compound binds to the polymerase in solution as evidenced by surface plasmon resonance and isothermal titration calorimetry analyses. Related compounds where a phenyl is replaced by a thiophene show higher affinity binding, indicating the potential for rational design. Importantly, inhibition of enzyme activity correlated with the binding affinity, showing that the pocket is functionally important for polymerase activity. This fragment is an excellent starting point for optimization through rational structurebased design.Dengue virus (DENV) 3 is the most widespread mosquitoborne viral infection. Disease symptoms of DENV-infected patients range from a mild fever to severe plasma leakage and hemorrhagic shock (1). There are four serotypes of DENV (DENV-1 to -4) that concurrently circulate around the world in tropical and subtropical regions. The vast majority of clinical cases are not reported, and there are estimated to be approximately 390 million human cases of dengue worldwide per year (2). There is currently no licensed vaccine or antiviral to treat DENV infection, underlining the urgency for the development of safe therapeutics (3). DENV is a member of the Flavivirus genus that also includes other viruses that are pathogenic to humans such as West Nile virus (WNV), yellow fever virus, Japanese encephalitis virus (JEV), and tick-borne encephalitis virus.One of the most attractive antiviral targets is the DENV RNA-dependent RNA polymerase (RdRp) because (i) viral polymerases are clinically proven therapeutic targets and (ii) the RdRp is the most conserved viral protein among the four serotypes of DENV so that the likelihood of a single compound with pan-serotype activity is higher than compounds targeting other viral proteins. The DENV RdRp activity resides in the C-terminal two-thirds of the viral nonstructural protein 5 (NS5) (reviewed in Ref. 4), whereas the N-terminal one-third of DENV NS5 encodes a methyltransferase (5).Crystal structures of the RdRp domain and full-length NS5 have been determined (6, 7). The overall architecture of the polymerase resembles a right hand with fingers, palm, and thumb subdomains. DENV RdRp catalyzes de novo initiation, as well as elongation. Like other RdRps that perform de novo RNA synthesis, DENV RdRp has a fully encircled active site (8). These polymerases undergo a conformational change from a closed to an open conformation during the transition from de novo initiation to elongation. During this process, the initiation loop (also known as the priming loop) is thought to move out of the active site, in a manner similar to the hepatitis C virus polymerase (9).A number of in vitro enzyme assays have been developed for DENV Rd...

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

Flavivirus nonstructural protein 5 (NS5) contains an N-terminal methyltransferase (MTase) domain and a C-terminal polymerase (RNA-dependent RNA polymerase [RdRp]) domain fused through a 9-amino-acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: the NS5 proteins from Japanese encephalitis virus (JEV) and Zika virus (ZIKV) adopt one conformation, while the NS5 protein from dengue virus serotype 3 (DENV3) adopts another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures. Replacement of the DENV2 NS5 linker with DENV1, DENV3, DENV4, JEV, and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, while the JEV and ZIKV NS5 linkers abolished replication. Thus, the JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design. IMPORTANCE Flaviviruses include important human pathogens, such as dengue virus and Zika virus. NS5 is a nonstructural protein essential for flavivirus RNA replication with dual MTase and RdRp enzyme activities and thus constitutes a major drug target. Insights into NS5 structure, dynamics, and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling that of NS5 from JEV and ZIKV. Replacement of the DENV2 NS5 linker with the JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic flavivirus NS5 linkers impede DENV2 NS5 protein-protein interactions that are essential for virus replication.

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

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