Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

A pan-serotype dengue virus inhibitor targeting the NS3-NS4B interaction.

Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue1,2. There are no antiviral agents available to prevent or treat dengue. Here, we describe a highly potent dengue virus inhibitor (JNJ-A07) that exerts nanomolar to picomolar activity against a panel of 21 clinical isolates that represent the natural genetic diversity of known genotypes 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 revealing a previously undescribed mechanism of antiviral action. JNJ-A07 has a favourable pharmacokinetic profile that results in outstanding efficacy against dengue virus infection in mouse infection models. Delaying start of treatment until peak viraemia results in a rapid and significant reduction in viral load. An analogue is currently in further development.

Modelling the impact of JNJ-1802, a first-in-class dengue inhibitor blocking the NS3-NS4B interaction, on in-vitro DENV-2 dynamics.

Dengue virus (DENV) is a public health challenge across the tropics and subtropics. Currently, there is no licensed prophylactic or antiviral treatment for dengue. The novel DENV inhibitor JNJ-1802 can significantly reduce viral load in mice and non-human primates. Here, using a mechanistic viral kinetic model calibrated against viral RNA data from experimental in-vitro infection studies, we assess the in-vitro inhibitory effect of JNJ-1802 by characterising infection dynamics of two DENV-2 strains in the absence and presence of different JNJ-1802 concentrations. Viral RNA suppression to below the limit of detection was achieved at concentrations of >1.6 nM, with a median concentration exhibiting 50% of maximal inhibitory effect (IC50) of 1.23x10-02 nM and 1.28x10-02 nM for the DENV-2/RL and DENV-2/16681 strains, respectively. This work provides important insight into the in-vitro inhibitory effect of JNJ-1802 and presents a first step towards a modelling framework to support characterization of viral kinetics and drug effect across different host systems.

Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein.

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.

From Oxetane to Thietane: Extending the Antiviral Spectrum of 2'-Spirocyclic Uridines by Substituting Oxygen with Sulfur.

In continuation of our efforts of finding novel nucleoside inhibitors for the treatment of viral diseases, we initiated a discovery research program aimed at identifying novel nucleos(t)ide inhibitors for emerging diseases like Dengue and Chikungunya. Based on the previously reported 2'-spiro-oxetane uridine derivatives active against Hepatitis C Virus (HCV), we envisaged its sulfur analogue as an interesting congener both from a synthetic as well as biological point of view. Surprisingly, we found the 2'-spirothietane uridine derivatives not only to be active against HCV and Dengue virus (DENV), viruses belonging to the flavivirus family, but also to demonstrate activity against alphaviruses like Chikungunya virus (CHIKV) and Sindbis virus (SINV).

Resistance mutations in human immunodeficiency virus type 1 integrase selected with elvitegravir confer reduced susceptibility to a wide range of integrase inhibitors.

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.

Characterization of a dengue NS4B inhibitor originating from an HCV small molecule library.

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.

A fluorescence-based high-throughput screening assay to identify HIV-1 inhibitors.

Highly active antiretroviral therapy (HAART) dramatically increases the long-term survival rates of human immunodeficiency virus type 1 (HIV-1) infected patients. Yet, poor adherence to therapy, adverse effects and the occurrence of resistant viruses can compromise the efficacy of HAART regiments. Therefore, there remains a clear unmet medical need for novel drugs and treatment options. In this chapter, we describe an HIV-1 antiviral high-throughput screening assay based on an HIV-1 permissive T lymphoblastoid MT4 cell line, stably transfected with a construct carrying an HIV-1 long terminal repeat promoter driving the expression of a reporter gene (enhanced green fluorescent protein). This assay runs in a 384-well format and enables the identification of HIV-1 inhibitors during a high-throughput screening campaign. In parallel, a cytotoxicity assay is performed to evaluate the compound-related in vitro toxicity.

An automated time-of-drug-addition assay to routinely determine the mode of action of HIV-1 inhibitors.

Cell-based high-throughput screening campaigns are widely used to identify novel antiviral compounds, for example, against human immunodeficiency virus type 1 (HIV-1). Typically, these assays enable identification of compounds that potentially target any viral or cellular factor involved in the viral replication cycle. Unraveling the mechanism of action of these active compounds is an important step to facilitate further drug development. Time-of-addition (TOA) assays are an elegant tool to achieve this goal by comparing the TOA profile of novel compounds with those of well-studied reference compounds. Downscaling to a 384-well format and automation significantly increase the capacity of the TOA assay, enabling compound handling around the clock. Mechanical liquid dispensing with optimized time points for compound addition ensures robustness (Z'>0.8) and maximal resolution in profiling novel antiviral compounds. The presented methodology has been optimized for routine use and allows for fully automated high-throughput screening to support the process in search for novel inhibitors of HIV-1.

Which compound to select in lead optimization? Prospectively validated proteochemometric models guide preclinical development.

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.

Primary mutations selected in vitro with raltegravir confer large fold changes in susceptibility to first-generation integrase inhibitors, but minor fold changes to inhibitors with second-generation resistance profiles.

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.