A Cyclic Phosphoramidate Prodrug of 2′-Deoxy-2′-Fluoro-2′-C-Methylguanosine for the Treatment of Dengue Virus Infection

ABSTRACT Monophosphate prodrug analogs of 2′-deoxy-2′-fluoro-2′-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue virus activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are among the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2′-deoxy-2′-fluoro-2′-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate, prodrug 17, demonstrating well-balanced anti-dengue virus cellular activity and in vitro stability profiles. We further determined the PBMC concentration of active triphosphate needed to inhibit virus replication by 50% (TP50). Compound 17 was assessed in an AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reductions at 100 and 300 mg/kg twice a day (BID), respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs exceeded the TP50 value, demonstrating TP50 as the target exposure for efficacy. In dogs, oral administration of compound 17 resulted in high PBMC triphosphate levels, exceeding the TP50 at 10 mg/kg. Unfortunately, 2-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that “no observed adverse effect level” (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of compound 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue virus infection.

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A Cyclic Phosphoramidate Prodrug of 2’-deoxy-2’-fluoro-2’-C-methylguanosine for the Treatment of Dengue Infection

10.1101/2020.04.13.040287 ◽

2020 ◽

Author(s):

Ratna Karuna ◽

Fumiaki Yokokawa ◽

Keshi Wang ◽

Jin Zhang ◽

Haoying Xu ◽

...

Keyword(s):

Oral Administration ◽

Mononuclear Cells ◽

Pulmonary Inflammation ◽

Dengue Infection ◽

Hcv Rna ◽

Peripheral Blood Mononuclear ◽

Cellular Assays ◽

Adverse Effect Level ◽

Effect Level

ABSTRACTMonophosphate prodrug analogs of 2’-deoxy-2’-fluoro-2’-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are one of the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2’-deoxy-2’-fluoro-2’-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate prodrug 17 demonstrating a well-balanced anti-dengue cellular activity and in vitro stability profiles. In dogs, oral administration of 17 resulted in high PBMC triphosphate level, exceeding TP50 (the intracellular triphosphate concentration at which 50% of virus replication is inhibited) at 10 mg/kg. Compound 17 demonstrated 1.6- and 2.2 log viremia reduction in the dengue mouse model at 100 and 300 mg/kg twice daily, respectively. At 100 mg/kg twice daily, the terminal triphosphate concentration in PBMCs reached above TP50, defining for the first time the minimum efficacious dose for a nucleos(t)ide prodrug. In the two-week dog toxicity studies at 30 to 300 mg/kg/day, no observed adverse effect level (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue infection.

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Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection

Journal of Virology ◽

10.1128/jvi.72.5.3999-4004.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 3999-4004 ◽

Cited By ~ 75

Author(s):

Anuja Mathew ◽

Ichiro Kurane ◽

Sharone Green ◽

Henry A. F. Stephens ◽

David W. Vaughn ◽

...

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

T Lymphocyte ◽

Mononuclear Cells ◽

Cytotoxic T Lymphocyte ◽

Nonstructural Proteins ◽

Dengue Virus Infection ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Lymphocyte Responses

ABSTRACT We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.

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Activation of Peripheral Blood Mononuclear Cells by Dengue Virus Infection Depotentiates Balapiravir

Journal of Virology ◽

10.1128/jvi.02841-13 ◽

2013 ◽

Vol 88 (3) ◽

pp. 1740-1747 ◽

Cited By ~ 38

Author(s):

Y.-L. Chen ◽

N. Abdul Ghafar ◽

R. Karuna ◽

Y. Fu ◽

S. P. Lim ◽

...

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Dengue Virus Infection ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

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Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection

Journal of Virology ◽

10.1128/jvi.01096-16 ◽

2016 ◽

Vol 90 (24) ◽

pp. 11122-11131 ◽

Cited By ~ 9

Author(s):

Meihui Xu ◽

Roland Züst ◽

Ying Xiu Toh ◽

Jennifer M. Pfaff ◽

Kristen M. Kahle ◽

...

Keyword(s):

Virus Infection ◽

Antibody Response ◽

Dengue Virus ◽

Secondary Infection ◽

Immune Memory ◽

Protective Capacity ◽

Dengue Virus Infection ◽

Immune Correlates

ABSTRACT Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo . IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive “recall” or “anamnestic” response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

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