Dengue virus (DENV) causes ~96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue 1,2 . There are no antivirals available to prevent or treat dengue. We describe a highly potent DENV inhibitor (JNJ-A07) that exerts nano-to picomolar activity against a panel of 21 clinical isolates, representing the natural genetic diversity of known geno-and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus unveiling an entirely novel mechanism of antiviral action. JNJ-A07 has an excellent pharmacokinetic profile that results in outstanding efficacy against DENV infection in mouse infection models. Delaying start of treatment until peak viremia results in a rapid and significant reduction in viral load. An analogue is currently in further development. MAIN TEXTDengue is currently considered one of the top10 global health threats 1 . Annually, an estimated 96 million develop dengue disease 2 , which is likely an underestimation [3][4][5] . The incidence has increased ~30-fold over the past 50 years. The virus is endemic in 128 countries in (sub-)tropical regions, with an estimated 3.9 billion people at risk of infection. A recent study predicts an increase to 6.1 billion people at risk by 2080 6 . The upsurge is driven by factors such as rapid urbanization and the sustained spread of the mosquito vectors [6][7][8] . DENV has four serotypes (further classified into genotypes), which are increasingly co-circulating in endemic regions. A second infection with a different serotype increases the risk of severe dengue 9,10 . The vaccine Dengvaxia ® , which is approved in a number of countries for those aged ≥9 years, is only recommended for those with previous dengue exposure 11,12,13 . There are no antivirals for the prevention or treatment of dengue; the development of pan-serotype DENV inhibitors has proven challenging 14,15 .

Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.Integration of retroviral DNA is an essential step in the life cycle of human immunodeficiency virus (HIV) (29). The integration process is facilitated by the viral integrase (IN) enzyme which catalyzes the insertion of the viral DNA into the host genome in a multistep process involving viral and host proteins. HIV IN recognizes and binds specific sequences in the long terminal repeats (LTRs) of the viral retrotranscribed DNA in the cytoplasm. After DNA binding, IN cleaves GT dinucleotides from the 3Ј termini of the linear cDNA in a process called 3Ј processing (2). The processed viral DNA, as part of the preintegration complex, is then translocated into the nucleus, where IN inserts the viral DNA into the host chromosome by a process called strand transfer (2,12,13). There are few cellular enzymes with comparable function to HIV integrase (24), apart from the V(D)J polynucleotide recombinase RAG1 (34). Therefore, the IN enzyme has been considered an attractive target for antiretroviral therapy for the last decade (27, 36). Recent progress has resulted in two IN inhibitors (5, 30), with one drug in late-stage clinical trials and one currently approved for use in treatment-experienced patients (18). For all currently targeted retroviral proteins, inhibition with antiretroviral drugs has led to emergence of resistance in treated patients, often leading to treatment failure and requiring changes in the composition of the highly active antiretroviral therapy (HAART) drug regimen (6). Nevertheless, the emergence of new classes of drugs will enable new combinations of inhibitors to be used and will offer more treatment options to HIV-infected pati...

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.cocrystal structure | respiratory syncytial virus | TMC353121 | viral fusion T o allow the deposition of their nucleic acid genome into a host cell, and to initiate their replication cycle, enveloped viruses have evolved complex membrane fusion machinery that includes a fusion protein (1, 2). Based on structural similarity, the viral fusion proteins from different viruses have been grouped into three distinct classes: I, II, and III (3, 4). Prototypic trimeric class I fusion proteins include HIV-1 gp41, influenza hemagglutinin and the fusion proteins from paramyxoviruses. The fusion protein (F) of respiratory syncytial virus (RSV), a paramyxovirus belonging to the pneumovirinae subfamily, assembles into a homotrimer that is cleaved at two proximal furin cleavage sites during biosynthesis, priming the protein for membrane fusion. Proteolytic cleavage of the fusion protein precursor (F 0 ) yields two polypeptides, F 1 and F 2 , joined by a disulfide bridge (Fig. 1). F 1 consists of an N-terminal hydrophobic fusion peptide, followed by a first heptad-repeat (HR1), an intervening globular domain, and a second heptadrepeat (HR2), which itself is N-terminal to the viral transmembrane and cytoplasmic regions (3). Once fusion is triggered, dramatic refolding of the prefusion conformation of the viral fusion protein occurs. Functional and structural studies have provided evidence that a folding intermediate is formed that contains a coiled-coil structure of three HR1 heptad repeats (5-8). This intermediate allows the fusion peptide to be inserted into the plasma membrane of a target cell. In the final stage of membrane fusion, the HR1-CTC structure irreversibly refolds into a 6HB complex with three HR2 heptad-repeats, resulting in membrane merger and stable fusion pore formation (5-14). In many viruses that rely on class I fusion proteins, the central HR1 trimeric coiled-coil (HR1-CTC) contains a hydrophobic pocket in each of its three grooves that has been proposed as a potential drug binding site (9, 10).The therapeutic value of inhibiting 6HB formation was establ...

In quite a few diseases, drug resistance due to target variability poses a serious problem in pharmacotherapy. This is certainly true for HIV, and hence, it is often unknown which drug is best to use or to develop against an individual HIV strain. In this work we applied ‘proteochemometric’ modeling of HIV Non-Nucleoside Reverse Transcriptase (NNRTI) inhibitors to support preclinical development by predicting compound performance on multiple mutants in the lead selection stage. Proteochemometric models are based on both small molecule and target properties and can thus capture multi-target activity relationships simultaneously, the targets in this case being a set of 14 HIV Reverse Transcriptase (RT) mutants. We validated our model by experimentally confirming model predictions for 317 untested compound – mutant pairs, with a prediction error comparable with assay variability (RMSE 0.62). Furthermore, dependent on the similarity of a new mutant to the training set, we could predict with high accuracy which compound will be most effective on a sequence with a previously unknown genotype. Hence, our models allow the evaluation of compound performance on untested sequences and the selection of the most promising leads for further preclinical research. The modeling concept is likely to be applicable also to other target families with genetic variability like other viruses or bacteria, or with similar orthologs like GPCRs.

Emergence of resistance to raltegravir reduces its treatment efficacy in HIV-1-infected patients. To delineate the effect of resistance mutations on viral susceptibility to integrase inhibitors, in vitro resistance selections with raltegravir and with MK-2048, an integrase inhibitor with a second-generation-like resistance profile, were performed. Mutation Q148R arose in four out of six raltegravir-selected resistant viruses. In addition, mutations Q148K and N155H were selected. In the same time frame, no mutations were selected with MK-2048. Q148H/K/R and N155H conferred resistance to raltegravir, but only minor changes in susceptibility to MK-2048. V54I, a previously unreported mutation, selected with raltegravir, was identified as a possible compensation mutation. Mechanisms by which N155H, Q148H/K/R, Y143R and E92Q confer resistance are proposed based on a structural model of integrase. These data improve the understanding of resistance against raltegravir and cross-resistance to MK-2048 and other integrase inhibitors, which will aid in the discovery of second-generation integrase inhibitors.