Discovery of JNJ-1802, a First-in-Class Pan-Serotype Dengue Virus NS4B Inhibitor.

Dengue is a global public health threat, with about half of the world's population at risk of contracting this mosquito-borne viral disease. Climate change, urbanization, and global travel accelerate the spread of dengue virus (DENV) to new areas, including southern parts of Europe and the US. Currently, no dengue-specific small-molecule antiviral for prophylaxis or treatment is available. Here, we report the discovery of JNJ-1802 as a potent, pan-serotype DENV inhibitor (EC50's ranging from 0.057 to 11 nM against the four DENV serotypes). The observed oral bioavailability of JNJ-1802 across preclinical species, its low clearance in human hepatocytes, the absence of major in vitro pharmacology safety alerts, and a dose-proportional increase in efficacy against DENV-2 infection in mice were all supportive of its selection as a development candidate against dengue. JNJ-1802 is being progressed in clinical studies for the prevention or treatment of dengue.

Discovery of Acyl-Indole Derivatives as Pan-Serotype Dengue Virus NS4B Inhibitors.

In the absence of any approved dengue-specific treatment, the discovery and development of a novel small-molecule antiviral for the prevention or treatment of dengue are critical. We previously reported the identification of a novel series of 3-acyl-indole derivatives as potent and pan-serotype dengue virus inhibitors. We herein describe our optimization efforts toward preclinical candidates 24a and 28a with improved pan-serotype coverage (EC50's against the four DENV serotypes ranging from 0.0011 to 0.24 µM for 24a and from 0.00060 to 0.084 µM for 28a), chiral stability, and oral bioavailability in preclinical species, as well as showing a dose-proportional increase in efficacy against DENV-2 infection in vivo in mice.

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.

Ovalbumin-containing core-shell implants suitable to obtain a delayed IgG1 antibody response in support of a biphasic pulsatile release profile in mice.

A single-injection vaccine formulation that provides for both a prime and a boost immunization would have various advantages over a multiple-injection regime. For such a vaccine formulation, it is essential that the booster dose is released after a certain, preferably adjustable, lag time. In this study we investigated whether a core-shell based implant, containing ovalbumin as core material and poly(DL-lactic-co-glycolic acid) of various monomer ratios as shell material can be used to obtain such a booster release. An in vitro release study showed that the lag time after which the ovalbumin was released from the core-shell implant increased with increasing lactic to glycolic acid ratio of the polymer and ranged from 3-6 weeks. Fluorescence spectroscopy showed minimal differences between native ovalbumin and ovalbumin from core-shell implants that were incubated until just before the observed in vitro release. In addition, mice immunized with a subcutaneous inserted core-shell implant containing ovalbumin showed an ovalbumin-specific IgG1 antibody response after a lag time of 4 or 6-8 weeks. Moreover, delayed release of ovalbumin caused higher IgG1 antibody titers than conventional subcutaneous vaccination with ovalbumin dissolved in PBS. Collectively, these findings could contribute to the further development of a single-injection vaccine, making multiple injections of the vaccine superfluous.

Discovery of 1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2'-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (JNJ-54257099), a 3'-5'-Cyclic Phosphate Ester Prodrug of 2'-Deoxy-2'-Spirooxetane Uridine Triphosphate Useful for HCV Inhibition.

JNJ-54257099 (9) is a novel cyclic phosphate ester derivative that belongs to the class of 2'-deoxy-2'-spirooxetane uridine nucleotide prodrugs which are known as inhibitors of the HCV NS5B RNA-dependent RNA polymerase (RdRp). In the Huh-7 HCV genotype (GT) 1b replicon-containing cell line 9 is devoid of any anti-HCV activity, an observation attributable to inefficient prodrug metabolism which was found to be CYP3A4-dependent. In contrast, in vitro incubation of 9 in primary human hepatocytes as well as pharmacokinetic evaluation thereof in different preclinical species reveals the formation of substantial levels of 2'-deoxy-2'-spirooxetane uridine triphosphate (8), a potent inhibitor of the HCV NS5B polymerase. Overall, it was found that 9 displays a superior profile compared to its phosphoramidate prodrug analogues (e.g., 4) described previously. Of particular interest is the in vivo dose dependent reduction of HCV RNA observed in HCV infected (GT1a and GT3a) human hepatocyte chimeric mice after 7 days of oral administration of 9.

Discovery and development of simeprevir (TMC435), a HCV NS3/4A protease inhibitor.

Hepatitis C virus is a blood-borne infection and the leading cause of chronic liver disease (including cirrhosis and cancer) and liver transplantation. Since the identification of HCV in 1989, there has been an extensive effort to identify and improve treatment options. An important milestone was reached in 2011 with the approval of the first-generation HCV NS3/4A protease inhibitors. However, new therapies are needed to improve cure rates, shorten treatment duration, and improve tolerability. Here we summarize the extensive medicinal chemistry effort to develop novel P2 cyclopentane macrocyclic inhibitors guided by HCV NS3 protease assays, the cellular replicon system, structure-based design, and a panel of DMPK assays. The selection of compound 29 (simeprevir, TMC435) as clinical candidate was based on its excellent biological, PK, and safety pharmacology profile. Compound 29 has recently been approved for treatment of chronic HCV infection in combination with pegylated interferon-α and ribavirin in Japan, Canada, and USA.

Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

Nucleotide prodrugs of 2'-deoxy-2'-spirooxetane ribonucleosides as novel inhibitors of the HCV NS5B polymerase.

The limited efficacy, in particular against the genotype 1 virus, as well as the variety of side effects associated with the current therapy for hepatitis C virus (HCV) infection necessitates more efficacious drugs. We found that phosphoramidate prodrugs of 2'-deoxy-2'-spirooxetane ribonucleosides form a novel class of HCV NS5B RNA-dependent RNA polymerase inhibitors, displaying EC50 values ranging from 0.2 to >98 µM, measured in the Huh7-replicon cell line, with no apparent cytotoxicity (CC50 > 98.4 µM). Confirming recent findings, the 2'-spirooxetane moiety was identified as a novel structural motif in the field of anti-HCV nucleosides. A convenient synthesis was developed that enabled the synthesis of a broad set of nucleotide prodrugs with varying substitution patterns. Extensive formation of the triphosphate metabolite was observed in both rat and human hepatocyte cultures. In addition, after oral dosing of several phosphoramidate derivatives of compound 21 to rats, substantial hepatic levels of the active triphosphate metabolite were found.

Cell-based antiviral assays for screening and profiling inhibitors against dengue virus.

Dengue, a mosquito-borne virus of the Flaviviridae family, is reemerging as one of the most important human pathogens in tropical and subtropical regions of the world. It is estimated that 2.5 billion people live in areas at risk for transmission of dengue virus (DENV). Furthermore, it causes significant morbidity and mortality with 50-100 million infections per year. Currently, there are no vaccines commercially available and no effective antiviral drugs for treatment of DENV infections. In this chapter, we describe a plaque reduction assay and a cell-based high-throughput antiviral assay for identifying inhibitors against DENV. The latter is a homogeneous high-throughput assay that has been fully validated according to the Food and Drug Administration (FDA) guidelines for assay validation and can be used for screening compound libraries.

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055.

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 µM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.

Pharmacokinetics-pharmacodynamics of a respiratory syncytial virus fusion inhibitor in the cotton rat model.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, young children, elderly persons, and severely immunocompromised patients. Effective postinfection treatments are not widely available, and currently there is no approved vaccine. TMC353121 is a potent RSV fusion inhibitor in vitro, and its ability to reduce viral loads in vivo was demonstrated in cotton rats following prophylactic intravenous administration. Here, the pharmacokinetics of TMC353121 in the cotton rat, which is semipermissive for RSV replication, were further explored to build a pharmacokinetic-pharmacodynamic (PK-PD) model and to estimate the plasma drug levels needed for significant antiviral efficacy. TMC353121 reduced the viral titers in bronchoalveolar lavage fluid in a dose-dependent manner after a single subcutaneous administration and intranasal RSV inoculation 24 h after compound administration. The viral titer reduction and plasma TMC353121 concentration at the time of RSV inoculation were well described using a simple E(max) model with a maximal viral titer reduction (E(max)) of 1.5 log(10). The plasma drug level required to achieve 50% of the E(max) (200 ng/ml) was much higher than the 50% inhibitory concentration observed in vitro in HeLaM cells (0.07 ng/ml). In conclusion, this simple PK-PD approach may be useful in predicting efficacious exposure levels for future RSV inhibitors.

Essentiality of FASII pathway for Staphylococcus aureus.

Recently, Brinster et al. suggested that type II fatty-acid biosynthesis (FASII) is not a suitable antibacterial target for Gram-positive pathogens because they use fatty acids directly from host serum rather than de novo synthesis. Their findings, if confirmed, are relevant for further scientific and financial investments in the development of new drugs targeting FASII. We present here in vitro and in vivo data demonstrating that their observations do not hold for Staphylococcus aureus, a major Gram-positive pathogen causing several human infections. The observed differences among Gram-positive pathogens in FASII reflects heterogeneity either in fatty-acid synthesis or in the capacity for fatty-acid uptake from the environment.

Structure-based design of a benzodiazepine scaffold yields a potent allosteric inhibitor of hepatitis C NS5B RNA polymerase.

HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c (K(D) = 0.79 nM), which has approximately 20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c.

Antiviral suppression vs restoration of RIG-I signaling by hepatitis C protease and polymerase inhibitors.

BACKGROUND & AIMS: Expression of the nonstructural protein (NS)3/4A protease in cells infected with hepatitis C virus (HCV) results in cleavage of the mitochondrial antiviral-signaling protein (MAVS) and disruption of signaling pathways that lead to viral activation of interferon regulatory factor 3 (IRF-3) and synthesis of type 1 interferons (IFN-alpha/beta). High concentrations of inhibitors of NS3/4A reverse this signaling defect, but quantitative analyses of this potential therapeutic effect are lacking. This study quantitatively assessed the rescue of IRF-3 signaling by NS3/4A inhibitors, compared with in vitro antiviral activity. METHODS: Antiviral activities of 2 NS3/4A protease inhibitors (TMC435350 and an analog, TMC380765) and a nonnucleoside polymerase inhibitor (Tib-3) were determined in HCV replicon cells and in cells infected with genotype 1a and 2a viruses. The capacity to rescue IRF-3 activation in these cells was assessed by monitoring IFN-beta promoter activity following challenge with Sendai virus. Inhibitor-induced changes in NS3 and MAVS expression were assessed in immunoblots. RESULTS: Both protease inhibitors were capable of rescuing IFN-beta promoter activation but only at concentrations approximately 100-fold the antiviral 50% effective concentration (EC(50)) for genotype 1 virus. No rescue was observed with the polymerase inhibitor, even at a concentration 600-fold greater than the EC(50). IRF-3 activation did not correlate with reductions in NS3/4A levels or detection of full-length MAVS. Overexpression of the product of NS3/4A cleavage of MAVS did not result in a dominant-negative effect on signaling. CONCLUSIONS: NS3/4A protease inhibitors can restore IRF-3 signaling in HCV-infected cells but only at concentrations far in excess of the antiviral EC(50).