Current Trends and Limitations in Dengue Antiviral Research

Abstract

Dengue is the most prevalent arthropod-borne viral disease worldwide and affects approximately 2.5 billion people living in over 100 countries. Increasing geographic expansion of Aedes aegypti mosquitoes (which transmit the virus) has made dengue a global health concern. There are currently no approved antivirals available to treat dengue, and the only approved vaccine used in some countries is limited to seropositive patients. Treatment of dengue, therefore, remains largely supportive to date; hence, research efforts are being intensified for the development of antivirals. The nonstructural proteins, 3 and 5 (NS3 and NS5), have been the major targets for dengue antiviral development due to their indispensable enzymatic and biological functions in the viral replication process. NS5 is the largest and most conserved nonstructural protein encoded by flaviviruses. Its multifunctionality makes it an attractive target for antiviral development, but research efforts have, this far, not resulted in the successful development of an antiviral targeting NS5. Increase in structural insights into the dengue NS5 protein will accelerate drug discovery efforts focused on NS5 as an antiviral target. In this review, we will give an overview of the current state of therapeutic development, with a focus on NS5 as a therapeutic target against dengue.

Keywords: NS5; antiviral targets; dengue virus; flavivirus; non-nucleoside inhibitors; nucleoside inhibitors; polymerase.

Figure 1

Genome Structure and Organization of the Dengue Virus. The dengue virus is spherical in shape, enveloped, and contains a positive-sense, single-stranded RNA genome. The genome encodes a precursor polyprotein, with an open-reading frame ~11 kb in length. Following viral entry into the host cell, the polyprotein is cleaved into three structural proteins (capsid, pre-membrane, and envelope), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A with a 2k signal peptide at the C-terminal, NS4B, and NS5).

Figure 2

Life Cycle of the Dengue Virus and Antiviral Targets. The viral membrane attaches to host cell receptors (DC-SIGN, CLEC5A, heparan sulfate receptors) and viral entry occurs through receptor-mediated endocytosis. The viral genome is released into the cytoplasm followed by translation of the polyprotein encoding the open reading frame. Polyprotein processing leads to its cleavage into structural and nonstructural (NS) proteins by host proteases and the NS2B-NS3 viral protease. Viral replication occurs on the endoplasmic reticulum by NS proteins followed by viral assembly and trafficking of immature viral particles to the trans-Golgi network (TGN). The acidic environment of the TGN facilitates viral maturation and subsequent release of mature viral particles from the host cell. A list of inhibitors against targets at different stages of the viral cycle are indicated. DC-SIGN, dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin; CLEC5A, human C-type lectin-like molecule.

Figure 3

The Enzymatic Functions of Dengue NS5. NS5 plays a major role in the flaviviral replication complex including RNA synthesis, RNA capping, and RNA methylation. The exact sequence of events is not yet fully understood, but a general scheme (A) is widely accepted. The (+) strand genomic RNA is used by the NS5 RdRp as a template to synthesize a (−) strand RNA which results in a replicative dsRNA intermediate. The (−) strand of the dsRNA intermediate then serves as a template for (+) strand synthesis, after which the dsRNA product is released and used for additional synthesis of (+) strands. Newly synthesized (+) strands are capped and methylated to form (+) strands of genomic RNA. The 5′-RNA capping begins with the triphosphatase activity of the NS3 helicase, where the terminal phosphate at the 5′-end of the (+) strand RNA is removed leading to a diphosphorylated RNA. The NS5 methyltransferase (MTase) caps the RNA through its guanylyl-transferase activity, where guanine monophosphate (GMP) is transferred to the 5′-end of the diphosphorylated (+) strand RNA from GTP. The capped RNA is methylated by the NS5 MTase via two sequential methylations: an initial methylation at the N7 position of the guanine cap, and a subsequent methylation at the 2′O position of the first RNA nucleotide. S-adenosyl-L-methionine (SAM) is utilized as a methyl donor in both methylations and is converted to S-adenosyl-L-homocysteine (SAH) as a by-product of the reactions. A cartoon representation (B) of a crystal structure of the full-length dengue serotype 2 NS5 is shown with the canonical features of the NS5 RdRp. PDB ID: 5ZQK.

Figure 4

Representative Crystal Structures of NS3 and NS5 with Small Molecule Inhibitors. Cartoon representations of the (A) NS2B-NS3 protease, (B,C) NS5 RNA-dependent RNA polymerase (RdRp), and (D,E) NS5 methyltransferase (MTase) crystal structures are shown with inhibitor-bound small molecule (green sticks). (A) Compound 8 is bound to the protease active site of the NS3 protein; (B) RK-0404678 is bound to the RNA binding site of the NS5 RdRp, close to the priming loop; (C) HeE1-2Tyr is bound to the RNA template tunnel, between the fingers subdomain and the priming loop; (D) Ribavirin 5′-triphosphate is bound to the GTP binding site of the NS5 MTase; (E) Sinefungin is bound to the SAM binding site of the NS5 MTase. All structure representations were made in PyMOL.