Flavivirus NS5 protein encodes methyltransferase and RNAdependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.The genus Flavivirus from the family Flaviviridae contains more than 70 viruses, many of which are important human pathogens causing major public health threats worldwide. Dengue virus (DENV) 2 is a mosquito-borne flavivirus responsible for 50 -100 million human infections and ϳ20,000 deaths each year (1). Besides DENV, West Nile virus (WNV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus also cause significant human diseases (1). No antiviral therapy is currently available for treatment of flavivirus infections. Therefore, development of antiviral therapy is urgently needed for flaviviruses.The flavivirus genome is a single strand, plus-sense RNA of about 11 kb in length. The genomic RNA contains a 5Ј-untranslated region (UTR), a single open reading frame, and a 3Ј-UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins (2). Three structural proteins (capsid, pre-membrane, and envelope) are primarily involved in virus particle formation, and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are mainly responsible for viral replication (2). Among these, NS3 and NS5 possess enzymatic activities and have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5Ј-RNA triphosphatase, and nucleoside triphosphatase (3-5). NS5 is the largest and the most conserved viral protein. The N-terminal part of NS5 is a methyltransferase that methylates the N-7 and 2Ј-O positions of the viral RNA cap structure (6 -9);...

Obesity and type 2 diabetes are chronic metabolic diseases, and those affected could benefit from the use of a-amylase inhibitors to manage starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3, isolated from an Apocynaceae plant show promise for further development as orally active a-amylase inhibitors. These linear peptides retain the stability known for cystine-knot peptides in the presence of harsh treatment. They are resistant to heat treatment and endopeptidase and exopeptidase degradation, which is characteristic of cyclic cystine-knot peptides. Our NMR and crystallography analysis also showed that wrightides, which are currently the smallest proteinaceous a-amylase inhibitors reported, contain the backbone-twisting cis-proline, which is preceded by a nonaromatic residue rather than a conventional aromatic residue. The modeled structure and a molecular dynamics study of Wr-AI1 in complex with yellow mealworm a-amylase suggested that, despite having a similar structure and cystine-knot fold, the knottin-type a-amylase inhibitors may bind to insect a-amylase via a different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine-knot a-amylase inhibitors and their biosynthesis in plants follow a secretory protein synthesis pathway. Together, our findings provide insights for the use of the pseudocyclic a-amylase inhibitors as useful leads for the development of orally active peptidyl bioactives, as well as an alternative scaffold for cyclic peptides for engineering metabolically stable human a-amylase inhibitors.

cWe report a highly reproducible method to crystallize the RNA-dependent RNA polymerase (RdRp) domain of dengue virus serotype 3 (DENV-3), allowing structure refinement to a 1.79-Å resolution and revealing amino acids not seen previously. We also present a DENV-3 polymerase/inhibitor cocrystal structure at a 2.1-Å resolution. The inhibitor binds to the RdRp as a dimer and causes conformational changes in the protein. The improved crystallization conditions and new structural information should accelerate structure-based drug discovery. Many flaviviruses are significant human pathogens. However, no antiviral therapy is currently available for the treatment of flavivirus infections. The flavivirus RNA-dependent RNA polymerase (RdRp), located at the C-terminal two-thirds of nonstructural protein 5 (NS5), is an attractive target for antiviral development (1-3). Like other polymerases, the flavivirus RdRp adopts a right-hand configuration composed of the fingers, palm, and thumb subdomains (4-6). Unfortunately, because of their flexibility, several segments of the protein are missing in the current flavivirus RdRp unliganded crystal structures, including several loops at the interface between the fingers and thumb subdomains (5, 6). Moreover, crystals of the dengue virus serotype 3 (DENV-3) RdRp were obtained at 4°C following a tedious dehydration procedure (7), making high-throughput structure-based drug discovery very inconvenient. It is therefore critical to develop a robust crystallization protocol to allow structural determination of DENV RdRp in complex with small-molecule inhibitors. A simplified crystallization protocol will be invaluable for structurebased rational design of inhibitors of DENV RdRp.Here we report such a procedure to grow crystals of DENV-3 RdRp at the temperature of 18°C that routinely diffract to a resolution higher than 2.0 Å. This allowed an improved refinement of the original RdRp structure (PDB code 2J7U [6]), revealing several amino acids hitherto not seen. Using these improved conditions for crystallization, we present the first cocrystal structure of a flavivirus RdRp with an inhibitor. This cocrystal structure shows that the inhibitor induces major conformational changes in the DENV-3 RdRp.An improved protein purification protocol for the reproducible crystallization of DENV-3 RdRp. The previous conditions used to grow DENV-3 RdRp crystals required a complicated dehydration protocol through gradual transfer of the crystals into increasing concentrations of polyethylene glycol (PEG) (7). This method was difficult to routinely reproduce because crystals were obtained only after a few weeks at 4°C. To improve crystallization, we established a new purification protocol of the DENV-3 RdRp that avoided the use of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), EDTA, or ␤-mercaptoethanol. Instead, we included Tris (2-carboxyethyl)-phosphine (TCEP; Thermo Scientific) in the final RdRp solution to prevent protein oxidation. The addition of TCEP minimizes protein precipita...

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC (50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions. b s t r a c tIn drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC 50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other proteinligand interactions. Ó 2009 Elsevier Inc. All rights reserved.High-throughput screening (HTS) 2 is a major method in drug discovery for new small lead molecules. In high-throughput enzyme inhibition assays, more than a million compounds are usua...

Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. IMPORTANCEThe reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans-complementation enhances our understanding of NS4B in flavivirus replication. Many flaviviruses are significant human pathogens, including dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). The four serotypes of DENV represent the most prevalent mosquito-borne viral pathogen for humans. DENV causes about 390 million human infections annually, leading to 96 million cases with manifest symptoms (1). No clinically approved vaccine or antiviral is currently available for DENV. A pilot study of the most advanced DENV vaccine candidate reported an overall efficacy of only 30.2% in a recent clinical trial (2). More efforts are needed to understand the biology of DENV and other flaviviruses to enable vaccine development.The genomes of flaviviruses are single-stranded, positive-sense RNAs of 11 kb in length. The genomic RNA harbors a long open reading frame (ORF) flanked by the 5=-and 3=-untranslated regions (UTRs). The ORF encodes a polyprotein which...