ReviewStrategies for development of dengue virus inhibitors
Introduction
Family Flaviviridae consist of three genera, Flavivirus, Pestivirus, and Hepacivirus. The genus Flavivirus consists of more than 70 viruses, many of which are arthropod-borne and cause human diseases including fevers, encephalitis, and hemorrhagic fevers (Gubler et al., 2007). Pathogenic flaviviruses include the four serotypes of dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). No clinically approved antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are available only for YFV, JEV, and TBEV. It is therefore a priority of public health to develop therapeutics for flavivirus infections.
Flaviviruses are small enveloped viruses about 50 nm in diameter, containing a single positive sense RNA that is approximately 11 kb in length. The viral genome encodes three structural proteins (capsid [C], premembrane [PrM], and envelope [E] proteins) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5; Fig. 1). The structural proteins form the viral particle. The non-structural proteins participate in the replication of the RNA genome, virion assembly (Kummerer and Rice, 2002, Liu et al., 2003), and invasion of innate immune response (Guo et al., 2005, Liu et al., 2005, Munoz-Jordan et al., 2003, Munoz-Jordan et al., 2005). Of these, only NS3 and NS5 have known enzymatic activities, making them ideal antiviral targets, since the enzymatic activity can be used to develop an assay for HTS campaigns. The N-terminal domain of NS3, together with NS2B, contains a serine protease activity; the C-terminal domain functions as an RNA helicase, an RNA triphosphatase, and an NTPase (Falgout et al., 1993, Wengler and Wengler, 1991, Wengler and Wengler, 1993). The N-terminal domain of NS5 contains a methyltransferase activity; the C-terminal domain serves as an RNA-dependent RNA polymerase (RdRp) (Ackermann and Padmanabhan, 2001, Egloff et al., 2002, Ray et al., 2006, Tan et al., 1996). Other non-structural proteins are required for RNA replication, among which NS2A, NS2B, NS4A, and NS4B are transmembrane proteins that form the scaffold for the viral replication complex (Lindenbach and Rice, 1997, Miller et al., 2006, Miller et al., 2007). Although the exact topology of the replication complex remains to be determined, the non-structural proteins without known enzymatic activity are valid antiviral targets.
Many aspects of the flavivirus replication and pathogenesis have been recently reviewed (Dong et al., 2008, Kroschewski et al., 2008, Malet et al., 2008, Perera et al., 2008, Xu et al., 2005). Here we concentrate on antiviral approaches and methodologies, using DENV as an example.
Section snippets
Protease NS2B/NS3
Viral proteases are a proven antiviral targets. There are currently nine HIV-1 protease inhibitors in clinical use (Menéndez-Arias, 2010) and a few HCV protease inhibitors in various stages of clinical trials (Soriano et al., 2008). DENV protease domain consists of the first 170 amino acids of NS3, with an amino acid homology of >50% among various members of the genus Flavivirus (Valle and Falgout, 1998). Like other flaviviruses, DENV protease is a serine protease with a catalytic triad (His51,
Host target-based approach
Flaviviruses utilize host proteins for their entry, translation, polyprotein cleavage, replication, and assembly. The host proteins are potential antiviral targets, provided that they are druggable, and that their inhibition is not toxic for the host (Table 1).
Structure-based rational design
The use of high-resolution protein crystallography for structure-based design is a powerful approach to increase the affinity and potency of lead antiviral compounds. Confirmed hits from screening can be either co-crystallized with the protein target or soaked into pre-formed apo-protein crystals to determine the exact position and manner of binding. This information can then be used in conjunction with docking and molecular modeling to design compounds that would be predicted to have a higher
Replication-based HTS approach
A number of cell-based assays have been developed to screen for antiviral compounds. Compared with the target-based approach, viral replication-based assays cover multiple steps and targets involved in a viral infection cycle.
Concluding remarks
The major goal of anti-DENV therapy is to prevent patients with DF (mild disease form) from development of DHF and DSS (severe disease form). Prospective studies of clinically characterized patients indicated that plasma levels of viremia and viral NS1 were 10- to 100-fold higher in patients with DHF/DSS than in those with DF (Gubler et al., 1981, Libraty et al., 2002). The rationale for anti-DENV therapy is to develop inhibitors that can suppress viremia by ≥10-fold in vivo. Both host and
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