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

Abstract

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen

“…Interestingly, the observed trend of decreased affinities for other group 1 HAs is similar to the diminished affinities previously reported for stem-targeting molecule JNJ4796 (22). Future HTS efforts will focus on these individual HA subtypes to try to identify common small-molecule motifs that can be optimized in parallel across group 1 HAs.…”

“…These C-ring interactions of F0045(S) are strikingly similar to the interactions made by the D-ring of small molecule JNJ4796 in the JNJ4796-H1 HA complex (PDB ID 6CF7) ( Fig. 4C) (22). Nonpolar interactions of F0045(S) include contacts with the sidechains of Thr 318 , Val 40 from HA1 and Trp 21 , Thr 41 , IIe 45 , IIe 48 and Val 52 from HA2.…”

“…2B, S2). We next employed an established trypsin susceptibility assay to assess if F0045 protected H1/PR8 from low pH induced conformational changes, akin to the endosomal membrane fusion event (20)(21)(22). Briefly, H1/PR8 HA is susceptible to trypsin digestion in the low pH post-fusion state, but stem-binding small molecules (i.e., Umifenovir, JNJ4796) or bnAbs (i.e., FI6v3, CR9114) prevent the HA from undergoing these conformation rearrangements on lowering of the environmental pH ( Fig.…”

“…The P7 specificity for group 1 is due in part to glycosylation of Asn38 in group 2 HAs as well as substitution of Thr49 (group 1) for a larger Asn (group 2) that interferes with binding. In another collaboration with Janssen, we also disclosed the small molecule JNJ4796, an HA stem-targeted inhibitor that is orally active in mice (22). JNJ4796 can neutralize a broad spectrum of human pandemic, seasonal, and emerging group 1 influenza A viruses and has promise as a therapeutic option with a complementary mechanism-of-action to existing antiviral drugs for the treatment of influenza.…”

“…The primary issue with the identification of HA-targeted small molecules is the lack of robust, rapid, and cost-effective high-throughput screening (HTS) assays (22)(23)(24). Identification of new antiviral small molecules have been attempted with both phenotypic and ELISA assay formats, including a dual myxovirus reporter assay (23) and an AlphaLISA assay (amplified luminescent proximity homogeneous assay) (22).…”