Cyanovirin-N (CV-N) has been shown to reveal broad neutralizing activity against human immunodeficiency virus (HIV) and to specifically bind Manα(1→2)Manα units exposed on various
glycoproteins of enveloped viruses, such as
influenza hemagglutinin (HA) and Ebola
glycoprotein. Chemically synthesized dimannosylated HA
peptides bound domain-swapped and dimeric CV-N with either four
disulfide-bonds (
Cys-Cys), or three
Cys-Cys bonds and an intact fold of the high-affinity binding site at an equilibrium dissociation constant K D of 10 μM.
Cys-Cys mutagenesis with ion-pairing
amino-acids glutamic acid and
arginine was calculated by in silico structure-based
protein design and allowed for recognizing dimannose and dimannosylated
peptide binding to low-affinity binding sites (K D ≈ 11 μM for one C58-C73 bond, and binding to dimannosylated
peptide). In comparison, binding to HA was achieved based on one ion-pairing C58E-C73R substitution at K D = 275 nM, and K D = 5 μM for two C58E-C73R substitutions. We were utilizing a
triazole bioisostere linkage to form the respective mannosylated-derivative on the HA
peptide sequence of residues
glutamine,
glycine, and
glutamic acid. Thus, mono- and dimannosylated
peptides with N-terminal
cysteine facilitated site-specific interactions with HA
peptides, mimicking a naturally found N-linked glycosylation site on the HA head domain.