c-Src protein kinase inhibitors block assembly and maturation of dengue virus

Abstract

Dengue virus is a mosquito-borne flavivirus that represents an important emerging infectious disease and is an international health concern. Currently, there is no vaccine or effective antiviral therapy to prevent or to treat dengue virus infection. The slow progress in developing antiviral agents might be alleviated by the availability of efficient high-throughput anti-dengue virus screening assays. In this study, we report an immunofluorescence imagebased assay suitable for identification of small molecule inhibitors of dengue virus infection and replication. Using this assay, we have discovered that inhibitors of the c-Src protein kinase exhibit a potent inhibitory effect on dengue virus (serotypes 1-4) and murine flavivirus Modoc. Mechanism of action studies demonstrated that the c-Src protein kinase inhibitor dasatinib prevents the assembly of dengue virions within the virus-induced membranous replication complex. These results demonstrate that this cell-based screen may provide a powerful means to identify new potential targets for anti-dengue drug development while simultaneously providing pharmacological probes to investigate dengue virus-host cell interactions at the biochemical level. gen that causes dengue fever (DF) and a severe lifethreatening illness, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (1). DENV is a small, enveloped, positive-stranded RNA virus that belongs to the Flavivirus genus of the Flaviviridae family. Four distinct serotypes (DENV1 to -4) of dengue viruses are transmitted to humans through the bites of the mosquito species, Aedes aegypti and Aedes albopictus (2). It has been estimated that Ϸ50-100 million cases of DF, and Ϸ250,000-500,000 cases of DHF occur every year (3). Furthermore, 2.5 billion of people are at risk for infection in subtropical and tropical regions of the world (4) in the absence of effective intervention. The intracellular life cycle of DENV begins with receptor-mediated endocytosis of the virus into cells, followed by fusion of the viral envelope protein with the late endosomal membrane, which results in the release of the viral genome into the cytoplasm for replication. Replication of the viral RNA genome occurs within membrane-bound complexes formed from the endoplasmic reticulum membrane. Subsequently, virus particles are assembled and released via the host cell secretory machinery (5). Although replication of DENV involves complex interaction between viral proteins and cellular factors, many of these interactions remain unidentified and uncharacterized. Small molecules that specifically target different steps in the viral replication cycle could potentially be used as ''tool compounds'' to facilitate biochemical characterization of these host-virus interactions and might also be used to identify pharmacological intervention points for treatment of DENV infection. Although extensive studies have been carried out over the years to understand the pathogenicity of DENV infection, little progress has been made in the development of specific anti-DE...

Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis

“…Glucosidase is responsible for the proper folding and glycosylation of the flaviviral prM, E, and NS1 (11). Host kinases have been shown to be involved in DENV and WNV assembly and secretion (8,19). The cholesterol biosynthesis pathway has been linked to DENV entry and replication as well as to the host immune response (29,42).…”

The Small Molecules AZD0530 and Dasatinib Inhibit Dengue Virus RNA Replication via Fyn Kinase

“…Previous results from a screen of small-molecule kinase inhibitors had identified several compounds that impair DV infection in mammalian cells (18). Among those, the dual Src and Abl kinase inhibitors AZD0530 and dasatinib (Fig.…”

“…Among those, the dual Src and Abl kinase inhibitors AZD0530 and dasatinib (Fig. 1A) were found to have antiviral activity at a postentry stage of the viral life cycle (18). We quantified the anti-DV2 activity and the cytotoxicity of both drugs in Huh7 cells ( Fig.…”

“…Since the inhibitor collection we screened included several inhibitors that have been U.S. Food and Drug Administration (FDA)-approved or that are in late stages of clinical development, we were additionally motivated by the opportunity to identify tool compounds that could be used to validate specific kinases as anti-DV targets in vitro and in vivo. AZD0530 and dasatinib, two pharmacologically validated compounds known to be potent inhibitors of Src and Abl kinases, were found to potently inhibit DV in cell culture (18). Here, we report our efforts to better understand the target and mechanism of action underlying the anti-DV activity of these compounds.…”

“…These studies have typically used biochemical screening assays that focus on one specific aspect of the viral infectious cycle, such as, for instance, viral entry (16), RNA polymerase activity (17), or RNA capping. We had previously developed a microscopy-based assay to permit identification of small molecules that inhibit any aspect of the DV infectious cycle (18).…”