Small-Molecule Inhibitors of Dengue-Virus Entry

Abstract

Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal “stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC90s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a “pocket-open" state, which has lost affinity for the stem peptide and cannot support the final “zipping up" of the stem.

The Bioactive Lipid 4-Hydroxyphenyl Retinamide Inhibits Flavivirus Replication

“…Vero cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The HEK293T, BHK21, Huh7.5, C6/36 cells were grown as previously described (7)(8)(9).…”

“…The virus sources are noted in parentheses: DENV-2 strain NGC (L. Gehrke), Kunjin virus (M. Diamond), Modoc virus (ATCC VR-1260), vesicular stomatitis virus (VSV) (S. Whelan), and poliovirus 1 (PV1) (ATCC VR-1562). Infections of Vero cells with these viruses were performed as previously described for DENV-2 (7,9), and viral titers were determined by a plaque formation assay. Hepatitis C virus (HCV) Japanese fulminant hepatitis strain 1 (JFH1) was produced from linearized pJFH-1 plasmid (T. Wakita).…”

Glycoprotein E of the Japanese encephalitis virus forms virus-like particles and induces syncytia when expressed by a baculovirus

“…As there is no effective clinical therapeutic intervention in flaviviral infection, the identification of novel inhibitors to target the virus life cycle is urgently required (Geiss et al, 2009;Shi, 2002). The E protein of flaviviruses mediates interactions between the virus and host cells during the initial stages of infection; hence it is widely targeted for the development of antiviral molecules, including inhibitory peptides (Bai et al, 2007;Costin et al, 2010;Hrobowski et al, 2005), therapeutic antibodies (Thompson et al, 2009) and small molecules (Kampmann et al, 2009;Schmidt et al, 2012). Here, we have established a new approach for antiviral screening targeted to JEV E, and in particular to its role in the attachment and fusion processes.…”

A Small-Molecule Inhibitor of Hepatitis C Virus Infectivity

“…7C) suggests that HCV II-1 either locks the viral envelope in its prefusion state or promotes an envelope conformation change that is incapable of fusion. Along these lines, it has been observed that the Dengue2 entry inhibitor 1662G07 blocks viral fusion by stabilizing an intermediate conformation of the Dengue2 viral envelope that cannot complete the fusion-promoting conformational change (37). Further studies will be necessary to elucidate how HCV II-1 prevents HCV particles from fusing with the endosome whether through a direct interaction with E2 or other means.…”