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.