Crystal Structure of Refolding Fusion Core of Lassa Virus GP2 and Design of Lassa Virus Fusion Inhibitors

ABSTRACTSC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.IMPORTANCET20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

Download Full-text

Discovery of (aza)indole derivatives as novel respiratory syncytial virus fusion inhibitors

MedChemComm ◽

10.1039/c9md00178f ◽

2019 ◽

Vol 10 (6) ◽

pp. 970-973

Author(s):

Xiufang Zheng ◽

Chungen Liang ◽

Lisha Wang ◽

Kun Miao ◽

Baoxia Wang ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Indole Derivatives ◽

New Class ◽

Fusion Inhibitors ◽

Virus Fusion ◽

Syncytial Virus

A new class of (aza)indole derivatives have been identified as potent RSV fusion inhibitors.

Download Full-text

Discovery of Piperazinylquinoline Derivatives as Novel Respiratory Syncytial Virus Fusion Inhibitors

ACS Medicinal Chemistry Letters ◽

10.1021/acsmedchemlett.5b00234 ◽

2016 ◽

Vol 7 (6) ◽

pp. 558-562 ◽

Cited By ~ 18

Author(s):

Xiufang Zheng ◽

Lisha Wang ◽

Baoxia Wang ◽

Kun Miao ◽

Kunlun Xiang ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Fusion Inhibitors ◽

Virus Fusion ◽

Syncytial Virus

Download Full-text

Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein

Journal of Virology ◽

10.1128/jvi.00597-16 ◽

2016 ◽

Vol 90 (15) ◽

pp. 6799-6807 ◽

Cited By ~ 15

Author(s):

Sundaresh Shankar ◽

Landon R. Whitby ◽

Hedi E. Casquilho-Gray ◽

Joanne York ◽

Dale L. Boger ◽

...

Keyword(s):

Small Molecule ◽

Envelope Glycoprotein ◽

Small Molecule Inhibitors ◽

Hemorrhagic Fever ◽

Lassa Virus ◽

Ph Sensing ◽

Antiviral Therapies ◽

Hemorrhagic Fevers ◽

Fusion Inhibitors ◽

Stable Signal

ABSTRACTArenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever.IMPORTANCEHemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

Download Full-text