NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models

2021 ◽  
Vol 13 (579) ◽  
pp. eabb2181
Author(s):  
Stephanie A. Moquin ◽  
Oliver Simon ◽  
Ratna Karuna ◽  
Suresh B. Lakshminarayana ◽  
Fumiaki Yokokawa ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Pharmacokinetic Studies ◽  
Resistance Mutations ◽  
Binding Studies ◽  
Log Reduction ◽  
Elimination Half ◽  
Preclinical Animal Models

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

scholarly journals Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1393
Author(s):  
Thanyaporn Dechtawewat ◽  
Sittiruk Roytrakul ◽  
Yodying Yingchutrakul ◽  
Sawanya Charoenlappanit ◽  
Bunpote Siridechadilok ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
Multifunctional Protein ◽  
Nonstructural Protein 1 ◽  
Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

scholarly journals Characterization and pharmacokinetic parameters of recombinant soluble interleukin-4 receptor

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2396-2403 ◽  
Author(s):  
CA Jacobs ◽  
DH Lynch ◽  
ER Roux ◽  
R Miller ◽  
B Davis ◽  
...  
Keyword(s):  
Half Life ◽  
Integral Membrane Protein ◽  
Interleukin 4 ◽  
Soluble Form ◽  
Pharmacokinetic Parameters ◽  
Pharmacokinetic Studies ◽  
Cdna Clones ◽  
Elimination Half ◽  
Interleukin 4 Receptor

Abstract The interleukin-4 receptor (IL-4R) is expressed as a 140-Kd membrane glycoprotein that binds IL-4 with high affinity. Recently, cDNA clones for the murine IL-4R have been isolated. One clone encodes an integral membrane protein, while another encodes a protein in which translation is terminated before the transmembrane region, thus producing a soluble form of the IL-4R (sIL-4R). HeLa cell clones overexpressing sIL-4R were isolated using a novel filter-overlay and 125I-IL-4 ligand binding technique. Quantitative analysis demonstrated that the kinetics and affinity of IL-4 binding to the recombinant sIL-4R were similar to the native membrane-bound IL-4R. As low doses of sIL-4R specifically inhibited IL-4-induced proliferative responses in vitro, sIL-4R biodistribution and elimination parameters were evaluated to assess the pharmacokinetic potential of sIL-4R as a therapeutic agent. Pharmacokinetic studies demonstrated that radiolabeled sIL-4R had a distribution half-life of 9 minutes and an elimination half-life of 2.3 hours following intravenous (IV) administration. When administered by intraperitoneal or subcutaneous (SC) injection, the elimination half- lives were prolonged to 4.2 hours and 6.2 hours, respectively. Although the initial blood level of sIL-4R was reduced if administered by SC injection, the bioavailability was comparable with IV administration. The main sites of sIL-4R elimination were the liver and kidney.

scholarly journals Illustrating and homology modeling the proteins of the Zika virus

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 275 ◽  
Author(s):  
Sean Ekins ◽  
John Liebler ◽  
Bruno J. Neves ◽  
Warren G. Lewis ◽  
Megan Coffee ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Crystal Structures ◽  
Zika Virus ◽  
Antiviral Drug ◽  
Homology Model ◽  
Model Quality ◽  
Glycoprotein E ◽  
Starting Point ◽  
Homology Models

The Zika virus (ZIKV) is a flavivirus of the familyFlaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it eitherin vitroorin vivo. ZIKV therefore epitomizes a neglected disease. Several suggested steps have been proposed which could be taken to initiate ZIKV antiviral drug discovery using both high throughput screens as well as structure-based design based on homology models for the key proteins. We now describe preliminary homology models created for NS5, FtsJ, NS4B, NS4A, HELICc, DEXDc, peptidase S7, NS2B, NS2A, NS1, E stem, glycoprotein M, propeptide, capsid and glycoprotein E using SWISS-MODEL. Eleven out of 15 models pass our model quality criteria for their further use. While a ZIKV glycoprotein E homology model was initially described in the immature conformation as a trimer, we now describe the mature dimer conformer which allowed the construction of an illustration of the complete virion. By comparing illustrations of ZIKV based on this new homology model and the dengue virus crystal structure we propose potential differences that could be exploited for antiviral and vaccine design. The prediction of sites for glycosylation on this protein may also be useful in this regard. While we await a cryo-EM structure of ZIKV and eventual crystal structures of the individual proteins, these homology models provide the community with a starting point for structure-based design of drugs and vaccines as well as a for computational virtual screening.

scholarly journals Chloroquine Inhibits Dengue Virus Type 2 Replication in Vero Cells but Not in C6/36 Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Kleber Juvenal Silva Farias ◽  
Paula Renata Lima Machado ◽  
Benedito Antônio Lopes da Fonseca
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Mammalian Cells ◽  
Plaque Assay ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Significant Difference ◽  
Dengue Virus Type 2

Dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. Since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of Vero and C6/36 cells infected with dengue virus type 2 (DENV-2). Real-time RT-PCR and plaque assays were used to quantify the DENV-2 load in infected Vero and C6/36 cells after chloroquine treatment. Our results showed that a dose of 50 μg/ml of chloroquine was not toxic to the cells and induced a statistically significant inhibition of virus production in infected Vero cells when compared to untreated cells. In C6/36 cells, chloroquine does not induce a statistically significant difference in viral replication when compared to untreated cells, showing that this virus uses an unlikely pathway of penetration in these cells, and results were also confirmed by the plaque assay (PFU). These data suggest that the inhibition of virus infection induced by chloroquine is due to interference with acidic vesicles in mammalian cells.

scholarly journals Inhibition of Dengue Virus by an Ester Prodrug of an Adenosine Analog

2010 ◽  
Vol 54 (8) ◽  
pp. 3255-3261 ◽  
Author(s):  
Yen-Liang Chen ◽  
Zheng Yin ◽  
Suresh B. Lakshminarayana ◽  
Min Qing ◽  
Wouter Schul ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Antiviral Therapy ◽  
Yellow Fever Virus ◽  
Pharmacokinetic Analysis ◽  
Pharmacokinetic Studies ◽  
Hydroxyl Groups ◽  
Viral Pathogen ◽  
Oral Dosing

ABSTRACT Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2′-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3′- and 5′-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3′,5′-O-diisobutyryl-2′-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.

scholarly journals Ficus septicaplant extracts for treating Dengue virusin vitro

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3448 ◽  
Author(s):  
Nan-Chieh Huang ◽  
Wan-Ting Hung ◽  
Wei-Lun Tsai ◽  
Feng-Yi Lai ◽  
You-Sheng Lin ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Viruses ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Immunofluorescence Assay ◽  
Aichi Virus ◽  
Specific Therapy ◽  
Enveloped Virus ◽  
Positive Strand Rna

Dengue virus types 1-4 (DENV-1-4) are positive-strand RNA viruses with an envelope that belongs to theFlaviviridae. DENV infection threatens human health worldwide. However, other than supportive treatments, no specific therapy is available for the infection. In order to discover novel medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plantsin vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves and stem fromFicus septicaBurm. f. had a promising anti-DENV-1 and DENV-2 effect. However, infection with the non-envelopepicornavirus, Aichi virus, was not inhibited by treatment withF. septicaextracts.F. septicamay be a candidate antiviral drug against an enveloped virus such as DENV.

scholarly journals Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

2020 ◽  
Author(s):  
Sisi Kang ◽  
Mei Yang ◽  
Zhongsi Hong ◽  
Liping Zhang ◽  
Zhaoxia Huang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Structural Information ◽  
Antiviral Drug ◽  
Binding Domain ◽  
Binding Studies ◽  
Terminal Domain ◽  
Rna Binding Domain

AbstractThe outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

scholarly journals Illustrating and homology modeling the proteins of the Zika virus

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 275 ◽  
Author(s):  
Sean Ekins ◽  
John Liebler ◽  
Bruno J. Neves ◽  
Warren G. Lewis ◽  
Megan Coffee ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Crystal Structures ◽  
Zika Virus ◽  
Antiviral Drug ◽  
Homology Model ◽  
Glycoprotein E ◽  
Starting Point ◽  
Homology Models

The Zika virus (ZIKV) is a flavivirus of the familyFlaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it eitherin vitroorin vivo. ZIKV therefore epitomizes a neglected disease. Several suggested steps have been proposed which could be taken to initiate ZIKV antiviral drug discovery using both high throughput screens as well as structure-based design based on homology models for the key proteins. We now describe preliminary homology models created for NS5, FtsJ, NS4B, NS4A, HELICc, DEXDc, peptidase S7, NS2B, NS2A, NS1, E stem, glycoprotein M, propeptide, capsid and glycoprotein E using SWISS-MODEL. Eleven out of 15 models pass our criteria for selection. While a ZIKV glycoprotein E homology model was initially described in the immature conformation as a trimer, we now describe the mature dimer conformer which allowed the construction of an illustration of the complete virion. By comparing illustrations of ZIKV based on this new homology model and the dengue virus crystal structure we propose potential differences that could be exploited for antiviral and vaccine design. The prediction of sites for glycosylation on this protein may also be useful in this regard. While we await a cryo-EM structure of ZIKV and eventual crystal structures of the individual proteins, these homology models provide the community with a starting point for structure-based design of drugs and vaccines as well as a for computational virtual screening.

scholarly journals Elvitegravir: A New HIV Integrase Inhibitor

2009 ◽  
Vol 20 (2) ◽  
pp. 79-85 ◽  
Author(s):  
Kazuya Shimura ◽  
Eiichi N Kodama
Keyword(s):  
Antiviral Drug ◽  
Integrase Inhibitor ◽  
Phase Iii ◽  
Hiv Integrase ◽  
Integrase Inhibitors ◽  
Resistance Mutations ◽  
Hiv 1

Integration is a distinctive and essential process in the HIV infection cycle and thus represents an attractive antiviral drug target. Integrase inhibitors combined with other classes of drug might contribute to long-lasting suppression of HIV type-1 (HIV-1) replication for many patients. Of the numerous potential integrase inhibitor leads that have been reported, few have reached clinical trials and only one, raltegravir, has been approved (in late 2007) for the treatment of HIV-1-infected patients. Another integrase inhibitor, elvitegravir, is currently showing promise in Phase III clinical studies. Once-daily administration of elvitegravir has a comparable antiviral activity to twice-daily of raltegravir in HIV-1-infected patients. Here, we highlight the salient features of elvitegravir: its chemical structure compared with representative integrase inhibitors, mechanism of action, in vitro and in vivo activity against HIV and other retroviruses, and the effect of integrase polymorphisms and resistance mutations on its anti-HIV activity.

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