Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region

Influenza A viruses pose a serious pandemic risk, while
generation
of efficient vaccines against seasonal variants remains challenging.
There is thus a pressing need for new treatment options. We report
here a set of macrocyclic peptides that inhibit influenza A virus
infection at low nanomolar concentrations by binding to hemagglutinin,
selected using ultrahigh-throughput screening of a diverse peptide
library. The peptides are active against both H1 and H5 variants,
with no detectable cytotoxicity. Despite the high sequence diversity
across hits, all tested peptides were found to bind to the same region
in the hemagglutinin stem by HDX-MS epitope mapping. A mutation in
this region identified in an escape variant confirmed the binding
site. This stands in contrast to the immunodominance of the head region
for antibody binding and suggests that macrocyclic peptides from in
vitro display may be well suited for finding new druggable sites not
revealed by antibodies. Functional analysis indicates that these peptides
stabilize the prefusion conformation of the protein and thereby prevent
virus–cell fusion. High-throughput screening of macrocyclic
peptides is thus shown here to be a powerful method for the discovery
of novel broadly acting viral fusion inhibitors with therapeutic potential.