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 highthroughput screening to identify molecules with low µM to nM 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 HA trimer against approximately 72,000 compounds. The crystal structure of H1/Puerto Rico/8/1934 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 2 discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities. Summary

Protective neutralizing epitopes in SARS‐CoV‐2

“…Overall, these concerted conformational changes bring the cell and viral membranes into close proximity that ultimately leads to membrane fusion, which is critical for releasing the viral genome into the target cell. Inhibitors that interrupt the transition from prefusion to postfusion forms prevent infection by several viruses, including influenza virus 102,103 and SARS‐CoV‐2 101,104,105 . More details regarding spike proteins of coronaviruses and their cell entry mechanisms can be found in several excellent reviews 3,18,76,99,106,107,108,109,110,111 …”

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 ].…”

Repurposing of Drugs Against Mutated Strain of Eurasian Avian Like H1N1 (EA H1N1) Swine Flu Virus, Genotype 4(G4) Virus

“…Haemagglutinin is a protein responsible for viral entry to the host, thus, determining pathogenicity and mediating membrane fusion.It has a great role in antigenic drift and shift as well and is assumed to be an important target for drug discovery .The HA protein has now already been established as a validated drug target against influenza virus. However, no any FDA-approved therapeutics have been succeeded in specifically blocking the receptor binding or the fusion machinery to prevent the functions of HA (Yao et al, 2020) ..PA is RNA dependent RNA polymerase of influenza virus that functions as endonuclease. ( Homology modeling was employed to prepare the three dimensional structure of the respective target proteins using SwissModel online tool.…”