GS-5806 Inhibits Pre- to Postfusion Conformational Changes of the Respiratory Syncytial Virus Fusion Protein

Abstract

b GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity. R espiratory syncytial virus (RSV) is an enveloped, singlestranded, negative-sense RNA virus that belongs to the Pneumovirinae subfamily of Paramyxoviridae (1). RSV infects the respiratory tract of infants, young children, and immunocompromised adults, causing severe disease (2-11). GS-5806 is a small-molecule inhibitor of RSV replication that is active against a diverse collection of RSV A and RSV B clinical isolates, with a mean 50% effective concentration (EC 50 ) of 0.43 nM (12, 13). GS-5806 blocks RSV fusion (F) protein-mediated cell-cell fusion, and mutations that confer drug resistance map to the RSV F gene, suggesting that the target of GS-5806 is the RSV F protein. Viral-cell membrane coalescence mediated by paramyxovirus fusion proteins involves several proteins, such as an attachment protein, cell surface receptors, and other cellular components that trigger conformational changes in the fusion protein that catalyze fusion of the two membranes (14-16). In vitro, triggering of preto postfusion conformational changes of RSV F proteins can be achieved by lowering the ionic strength of the buffer or by increasing the temperature (17-19). The conformational changes expose the buried hydrophobic fusion peptides, which interact with fusion peptides of neighboring molecules to form rosette-like structures (see Fig. S2, top panel, in the supplemental material). These macromolecular structures are distinct and easily observed by electron microscopy (EM). The conformational changes can also be triggered in the presence of liposomes prepared in low-ionic-strength buffer. The RSV F protein triggered in the presence of liposomes inserts into the lipid bilayer presumably mediated by the fusion peptides. To evaluate the effects of GS-5806 on the pre-to postfusion conformational changes of RSV F, we expressed the extracellular domain of RSV F protein (⌬TM-RSV F) in HEK293 cells. The protein was stored in high-ionic-strength buffer (500 mM NaCl, 250 mM imidazole, 20 mM Tris, pH 8.0) to keep it in pretriggered conformation. On exposure to low-ionic-strength buffer (10 mM HEPES, pH 8.0) (see Fig. S1 in the supplemental material), ⌬TM-RSV F formed rosettes or inserted into liposomes that were easily observable by EM (see Fig. S2, bottom panel, in the supplemental material). These experiments were used to measure the effect of GS-5806 on the conformational changes of ⌬TM-RSV F protein (18).⌬TM-RSV F protein was triggered in the presence of GS-5806 (5-fold molar excess over protein), an inactive analog of GS-5806 (GSC-1), or 0.1% dimethyl sulfoxide (DMS...

Molecular mechanism of respiratory syncytial virus fusion inhibitors

“…Furthermore, our proposed mechanism is consistent with that recently proposed for GS-5806 (ref. 30), suggesting that all RSV fusion inhibitors act as antagonists of F triggering.…”

GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process

“…While GS-5806 was inactive in an RSV replicon assay that measures viral RNA transcription and replication functions, it effectively inhibited cell-to-cell fusion (syncytia) mediated by RSV F protein. Evaluation of liposomes containing pretriggered RSV F protein revealed that GS-5806 inhibits the pre-to postfusion conformational change within the F protein necessary for viral fusion with the host cell membrane (41,42). Finally, drug susceptibility determinants mapped to the RSV F gene, indicating that the fusion protein is the likely target of GS-5806.…”

Respiratory syncytial virus entry and how to block it

“…Crystallographic experiments later revealed that the small molecules bind inside the central cavity of the prefusion conformation and interact with residues in the fusion peptide and the second heptad repeat 141 . Functional studies with soluble F proteins 142 and membrane-bound F proteins 141 demonstrated that the small molecules function as antagonists that prevent The fusion (F) protein can interact with one or more attachment factors to bind the virus to the host cell, a process that is greatly enhanced by the viral attachment protein (not shown). The F protein may also interact with a functional receptor that induces refolding of the prefusion conformation to the prehairpin intermediate.…”