Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25-100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.

Lasso peptides belong to the class of ribosomally synthesized and post-translationally modified peptides. Their common distinguishing feature is an N-terminal macrolactam ring that is threaded by the C-terminal tail. This lasso fold is maintained through steric interactions. The isolation and characterization of xanthomonins I-III, the first lasso peptides featuring macrolactam rings consisting of only seven amino acids, is now presented. The crystal structure of xanthomonin I and the NMR structure of xanthomonin II were also determined. A total of 25 variants of xanthomonin II were generated to probe different aspects of the biosynthesis, stability, and fold maintenance. These mutational studies reveal the limits such a small ring imposes on the threading and show that every plug amino acid larger than serine is able to maintain a heat-stable lasso fold in the xanthomonin II scaffold.

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC 50 s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC 50 s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Dengue viruses (DENVs) are enveloped positive-strand RNA viruses and belong to the family Flaviviridae. DENV is the most important arthropod-borne viral infection. Over one-third of the world population lives in areas of DENV endemicity, and an estimated 390 million infections occur every year. In addition, the number of countries having experienced DENV epidemics has risen from 9 in 1970 to more than 100 today (1, 2). Furthermore, the number of diagnosed infections across America, Southeast Asia, and the Western Pacific nearly doubled from 1.2 million in 2008 to over 2.3 million in 2010 (2). Four different DENV serotypes have been identified so far. Recently, evidence for an additional subtype has been presented (3). Serotypes 1 to 4 are now prevalent in Asia, Africa, and America, and the regions where dengue is endemic are still increasing (4-6), with dengue endangering even Europe and the United States due to vector spread. DENV infections can be associated with dengue fever, but up to 88% of the infections remain inapparent (7). These nonpersistent infected patients serve besides persistently infected mosquitoes as a virus reservoir. Severe DENV infections and especially reinfections may lead to dengue hemorrhagic fever and dengue shock syndrome, with lethality up to 5% (2,8,9). There is neither a vaccination nor a specific treatment for DENV infections.The DENV genome contains a single open reading frame, which encodes the structural proteins capsid, membrane precursor (prM), and envelope and the nonstructural proteins NS1, NS2, NS3, NS4, and NS5 (10). Cellular proteases and the viral serine protease (PR) are responsible for cleaving the viral precursor polyprotein into functional proteins. The DENV PR consists of the amino-terminal domain of the NS3 protein and requires NS2B, a 14-kDa protein, as a cofactor to form a stable complex. This heterodimeric PR cleaves at the capsid-prM, NS2A/NS2...