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

Abstract

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.

Structural Biology of Influenza Hemagglutinin: An Amaranthine Adventure

“…Over the past decade, structural characterization of bnAbs to the HA have stimulated antiviral design, ranging from small protein binders [142][143][144][145] to peptides [146] to small molecules [147,148]. In addition, the discovery and characterization of bnAbs to HA have reignited aspirations and novel approaches towards a more universal influenza vaccine [149].…”

Potent sialic acid inhibitors that target influenza A virus hemagglutinin

“…Thus, NA inhibitors have limited efficacy against seasonal influenza viruses and emphasize the need to develop alternative anti-IAV therapies. Due to its critical role in the early stage of IAV, HA is a potential target protein for anti-influenza agents 38 – 40 . The HA sequence structure has been extensively explored and roughly divided into two parts: the SA receptor binding site and the fusion domain 25 .…”

Small Molecule Inhibitors of Influenza Virus Entry

“…In addition, two JNJ4796-like inhibitors, GRP-71271 [ 65 ] and IY7640 [ 66 ], have also been reported ( Figure 7 ). Moreover, there are several structurally distinct inhibitor classes, including MBX2329 [ 63 ], LY180299 and other diterpenoid derivatives [ 67 , 68 , 69 ], triperiden derivatives [ 68 , 70 ], GRP-115249 [ 65 ], FA583 [ 64 ], S20 [ 71 ], nylidrin [ 72 ], F0045(S) [ 73 ], and so on ( Figure 8 ).…”

“…Interestingly, the binding sites of JNJ4796, CBS1117 and F0045(S) overlap significantly, localized in a pocket near the fusion peptide ( Figure 12 a) [ 48 , 73 , 96 ]. JNJ4796 is the biggest compound, containing five rings occupying a large region of the target pocket, nonetheless, the binding mode of CBS1117 and F0045(S) are strikingly similar to the binding modes of the B, C, and D rings of JNJ4796 ( Figure 12 b) [ 48 , 73 , 96 ]. Notably, the B-ring of CBS1117 reacts with HA similarly to both the C- and D-ring of JNJ4796 [ 96 ].…”