The Discovery and Characterization of Rigid Amphipathic Fusion Inhibitors (RAFIS), a Novel Class of Broad-Spectrum Antiviral Compounds

Abstract

Antiviral drugs targeting viral proteins or their interactions with cellular proteins lead to rapid selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, virion envelopes must locally convert to negative curvature to form hourglass-shaped hemifusion stalks. Unlike cellular vesicles, virion envelopes fuse to the outer leaflets of cell membranes without lipid redistribution between leaflets, and using only the energy released by glycoprotein binding and rearrangement. Enrichment in inverted-cone shaped lysophospholipids in the outer leaflet of vesicles disfavors negative curvature, inhibiting fusion. Such lipids, however, are toxic and not pharmacologically useful. My hypothesis is that pharmacologically useful small amphipathic molecules of appropriate shape (hydrophilic region of larger crosssection than the hydrophobic one) can intercalate within virion lipids, inhibiting the formation of the negative curvature required for fusion of all enveloped viruses. Herein, I report the discovery and characterization of the novel antiviral mechanisms of a novel family of rigid amphipathic nucleosides. One such compound, dUY11, disfavors the formation of the required negative curvature. Exposure to dUY11 inhibits the infectivity of several otherwise unrelated enveloped viruses (IC50, 50 nM), including hepatitis C and genital herpes virus. dUY11 has no major cytotoxic/static effects (SI>1,800). dUY11 does not inhibit viral DNA replication, but inhibits infectivity produced by cells treated after infection, even with acyclovir or phosphonoacetic acid resistant strains. The targets of dUY11 are not encoded in the viral genomes. Consistently, resistance to dUY11 has not been detected. This thesis serves as proof-of-concept that small molecule fusion inhibitors targeting envelope bilayer curvature are viable antiviral strategies. These strategies may help in overcoming the limitations of clinical antivirals, including narrow specificity and rapid resistance selection. dUY11 inhibits infection and acts extracellularly, highly desirable properties for microbicides. dUY11 has therapeutic potential, too, targeting and inhibiting functions conserved among all otherwise unrelated enveloped viruses. The novel antiviral mechanisms of action presented herein may impact future development of prophylactic and therapeutic antivirals.