Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

Abstract

We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxymethyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC 50 ) and cytotoxic concentration (CC 50 ) values of 0.7 M and >100 M, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14 C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.The family Flaviviridae consists of three genera, Flavivirus, Pestivirus, and Hepacivirus. Many viruses from the genus Flavivirus are arthropod-borne and cause significant human diseases. The four serotypes of dengue virus (DENV) alone pose a health threat to 2.5 billion people worldwide, leading to 50 to 100 million human infections and 500,000 cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) each year (11). Besides DENV, other flaviviruses, such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV), also cause frequent outbreaks throughout the world. No clinically approved antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are available only for YFV, JEV, and TBEV. The development of a successful DENV vaccine has been challenging due to the facts that (i) the vaccine should simultaneously induce a long-lasting protection against all four DENV serotypes and (ii) an incompletely immunized individual may be sensitized to the lifethreatening DHF or DSS. The challenge associated with the development of a vaccine underlines the importance of antiviral development for DENV and other flaviviruses.The flavivirus genome is a single-strand RNA of plus-sense polarity. The single open reading frame from the 11-kb viral genome encodes three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Nonstructural proteins are responsible for viral replication. Of those, NS3 and NS5 have enzymatic activities. The N-terminal domain of NS3 (with NS2B as a cofactor) functions as a serine protease, and the C-terminal domain acts as an RNA helicase, an RNA triphosphatase, and an NTPase (10,34,35). The N-...

Antiviral screening identifies adenosine analogs targeting the endogenous dsRNALeishmaniaRNA virus 1 (LRV1) pathogenicity factor

“…Common features of the two selective anti-LRV1 compounds include the 2′-C methyl and the adenine base moieties, although 2′ C-methyl G and C were inactive against both Leishmania and LRV1. A similar pattern was observed for dengue virus RDRP inhibitors, where only adenosine analogs demonstrated antiviral activity (51). Following uptake, in Leishmania most purine nucleosides are metabolized to nucleobases, the major exception being adenosine, which is phosphorylated directly by adenosine kinase (31).…”

Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence

“…A small-molecule adenosine nucleotide analog was reported previously to completely protect S221-infected mice for up to 11 days, while untreated mice succumbed to primary infection by day 4 (11,74), in a mortality pattern similar to that reported here. In two other studies, one using a PPMO targeting the 5Ј-terminal region of the genome and/or the CS1 region (roughly equivalent in targeting to the DC-1 and DC-6 siRNAs here) (60) and the other using a heparin sulfate mimetic (39), the average survival time of AG129 mice receiving a lethal challenge of mouse brain-adapted DENV-2 strain New Guinea C was extended for about a week.…”

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

“…Derivatives of these ribonucleotide analogs may serve as potential antiviral molecules to combat ZIKV infection. It has been shown before that 2=-C-ethynyl-7-deaza-ATP is also a potent inhibitor of HCV and dengue virus RdRps, and its antiviral activity has been extensively studied (16,18,24).…”

“…Significant nucleoside-based drug discovery and development have been undertaken and are ongoing to combat viral infections caused by Flaviviridae members, such as HCV and dengue virus (13)(14)(15)(16)(17)(18)(19). Sofosbuvir, an FDA-approved ribonucleotide analog targeting HCV NS5b polymerase, has been shown to be a very effective and safe drug for the treatment of HCV infection, and these findings provided a proof of concept for the use of ribonucleotide analogs as effective antiviral drugs against Flaviviridae (20,21).…”