A small-molecule fusion inhibitor of influenza virus is orally active in mice

INTRODUCTION Annual influenza epidemics and episodic pandemics continue to cause widespread illness and mortality. Strategies to prevent and treat acute influenza infection have remained limited to seasonal influenza vaccination and a small arsenal of antiviral drugs. Thus, there is an urgent need for additional prophylactic and therapeutic options, including new targets and mechanisms of action, to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and the emergence of antiviral drug resistance. RATIONALE The recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the highly conserved hemagglutinin (HA) stem as a promising target for development of universal vaccines and complementary therapeutics. Even though this spurred several bnAbs to be evaluated as passive immunotherapy in clinical trials, antibodies are large and complex molecules that are generally unsuited for oral delivery. We therefore set out to utilize the structural details of the molecular interactions and mechanisms of HA stem bnAbs to identify an orally active small molecule that mimics bnAb functionality. Influenza A viruses can be separated in group 1 and group 2 on the basis of their HA subtype (H1 to H18), and anti-stem bnAbs usually bind to group 1 or to group 2 viruses, but a few can target both. RESULTS We screened a diverse chemical library for compounds that selectively target the group 1 HA epitope of bnAb CR6261 through a binding assay that detects displacement of a CR6261-based designed small protein. Benzylpiperazines were identified as a major hit class, with JNJ7918 being the most promising candidate. Consistent with its binding to the functional HA stem epitope, this compound also neutralized influenza infection in vitro. Key chemical modifications were subsequently introduced to optimize binding and neutralization potency, as well as properties dictating metabolic stability and oral bioavailability, to finally afford JNJ4796. This lead compound binds and neutralizes a broad spectrum of influenza A group 1 viruses in vitro and protects mice against lethal and sublethal influenza challenge after oral administration. The compound also effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Like bnAb CR6261, the mechanism of action of JNJ4796 was demonstrated to be based on inhibition of the pH-sensitive conformational change of HA that triggers fusion of the viral and endosomal membranes and release of the viral genome into the host cell. Cocrystal structures with H1 and H5 HAs reveal that JNJ4796 recapitulates the original CR6261-HA hotspot interactions and provide detailed and valuable information on the minimal epitope in the HA1-HA2 fusion region of the stem for an antiviral small molecule to neutralize influenza A group 1 viruses. CONCLUSION We identified an orally active small molecule against influenza A HA that mimics the binding and functionality of the broadly neutralizing antibody CR6261. The small molecule targets the conserved HA stem region, acts as a fusion inhibitor by inhibiting conformational changes that lead to the postfusion HA structure, and neutralizes a broad spectrum of human pandemic, seasonal, and emerging group 1 influenza A viruses. Thus, the compound holds promise as an urgently sought-after therapeutic option offering a complementary mechanism of action to existing antiviral drugs for the treatment of influenza virus infection, and that should further aid development of universal therapeutics that prevent entry of influenza virus in host cells.