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 .

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...

A preceding paper (Bonfanti et al. J. Med Chem. 2007, 50, 4572-4584) reported the optimization of the pharmacokinetic profile of substituted benzimidazoles by reducing their tissue retention. However, the modifications that were necessary to achieve this goal also led to a significant drop in anti-RSV activity. This paper describes a molecular modeling study followed by a lead optimization program that led to the recovery of the initial potent antiviral activity and the selection of TMC353121 as a clinical candidate.

We previously reported the discovery of substituted benzimidazole fusion inhibitors with nanomolar activity against respiratory syncytial virus (Andries, K.; et al. Antiviral Res. 2003, 60, 209-219). A lead compound of the series was selected for preclinical evaluation. This drug candidate, JNJ-2408068 (formerly R170591, 1), showed long tissue retention times in several species (rat, dog, and monkey), creating cause for concern. We herein describe the optimization program to develop compounds with improved properties in terms of tissue retention. We have identified the aminoethyl-piperidine moiety as being responsible for the long tissue retention time of 1. We have investigated the replacement or the modification of this group, and we suggest that the pKa of this part of the molecules influences both the antiviral activity and the pharmacokinetic profile. We were able to identify new respiratory syncytial virus inhibitors with shorter half-lives in lung tissue.