Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the β-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “Wing” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication. Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human pathogens. The only licensed vaccine has limited efficacy and an antiviral therapy is not available. The multifunctional non-structural protein 1 (NS1) of DENV is secreted from infected cells, counteracts antiviral immune response and contributes to the pathogenesis of DENV infection. In addition, NS1 is essential for the viral replication cycle but the underlying mechanism is unknown. Here we determined the viral interactome of NS1 and identified a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with NS4A and NS4B. This interaction is required for RNA replication. Additionally, we identified a domain in NS1 important for efficient secretion of this protein. Finally, we demonstrate that NS1 is required for the biogenesis of the membranous DENV replication organelle. This function does not require RNA replication and is independent from NS1 interaction with NS4A-2K-4B. Our results provide new insights into the role of NS1 in DENV RNA replication and establish a genetic map of residues in NS1 required for the diverse functions of this protein. These results should aid in the design of antiviral strategies targeting NS1, with the aim to suppress viral replication as well as severe disease manifestations.

Introduction of novel and diverse functional groups in drug discovery is always seen with hesitancy until good activity and low toxicity characteristics are proven. The introduction of fluorine in drug-like compounds is now a well-accepted strategy in medicinal chemistry. However, polyfluoroalkyl groups, with the exception of trifluoromethyl substituents, are not well explored yet. Our aim is to show to the readers how polyfluorinated groups can be beneficial to the properties of pharmaceutically active compounds by highlighting the structure-activity relationship (SAR) studies that led to the selection of polyfluorinated moieties as key structural features. Despite the fact that the use of higher polyfluoroalkyl/aryl moieties is still in its infancy, we believe that they will soon acquire the same importance of their lower parents.

Prostate cancer (PC) is one of the major causes of male death worldwide and the development of new and more potent anti-PC compounds is a constant requirement. Among the current treatments, (R)-bicalutamide and enzalutamide are non-steroidal androgen receptor antagonist drugs approved also in the case of castration-resistant forms. Both these drugs present a moderate antiproliferative activity and their use is limited due to the development of resistant mutants of their biological target. Insertion of fluorinated and perfluorinated groups in biologically active compounds is a current trend in medicinal chemistry, applied to improve their efficacy and stability profiles. As a means to obtain such effects, different modifications with perfluoro groups were rationally designed on the bicalutamide and enzalutamide structures, leading to the synthesis of a series of new antiproliferative compounds. Several new analogues displayed improved in vitro activity towards four different prostate cancer cell lines, while maintaining full AR antagonism and therefore representing promising leads for further development. Furthermore, a series of molecular modelling studies were performed on the AR antagonist conformation, providing useful insights on potential protein-ligand interactions.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca²⁺- and phosphatidylserine (PS)–dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein β2GP1 (β2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX–derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.