The key finding in this paper is the feasibility of creating synthetic θ-defensin analogs with equal or greater activity against IAV than their natural counterparts. The analogs comprised two distinct subgroups, Hapivirins and Diprovirins. Some members of each group had neutralizing activity comparable to that of wild type defensins. The Hapivirins resembled primate retrocyclins in having an intramolecular ladder of three evenly spaced disulfide bonds. Strikingly, changing a single amino acid in the hairpin loop of HpVs resulted in marked changes in antiviral activity. Several HpVs had 50% neutralizing activity at concentrations of ~500ng/ml. Several DpVs had similar activity since they reduced viral infectivity to <20% of control at 3 μg/ml and DpV1632 had a 50% neutralizing dose <0.4 μg/ml (). In our prior studies, the most active θ-defensins RC2 and RC101 showed 50% neutralizing activity of ~1.25 μg/ml. Hence, based on neutralizing activity alone the HpVs and DpVs are worthy of further investigation.
We also tested a limited set of the more potent antiviral peptides for antibacterial and antifungal activity. Of interest, DpVs 13 and 16 also caused strong inhibition of growth of S. aureus
and C. albicans
, greatly exceeding the activity of HNPs. Both peptides also showed similar inhibition of C. albicans
, and greater inhibition of S. aureus
, than RC1. In contrast, HpV17 had modest antibacterial and antifungal activity indicating that antiviral and antibacterial (or antifungal activity) are not consistently correlated. The broad spectrum antimicrobial activity of the DpVs is another attractive feature of this group of peptides. S. aureus
superinfection is a catastrophic consequence of IAV infection and hence a peptide capable of inhibiting both IAV and S. aureus
could be valuable (26
Structure-function analysis of the HpV and DpV variants tested provides insights into the molecular features that are most important for antiviral activity. While cationic charge has been considered the most important determinant of defensin-mediated antimicrobial activity, our findings suggest that increasing hydrophobicity can have a marked impact on antiviral activity. The feature of HpVs conferring the best antiviral activity was increased “non-aromatic type” hydrophobicity in the loop of the molecule (position 7) delivered by L-cyclohexylalanine (Cha) or (S)-octahydroindole-2-carboxylic acid (Oic) -which can be considered a constrained analog of Cha. Because HpV11 and HpV17 were among the most active HpVs, their side chains may represent optimal hydrophobicity as well as spatial arrangements for maximal antiviral effect, especially considering that fairly similar substitutions did not confer similar effects (see ).
DpVs 1–21 were designed to simplify the β-hairpin structure of HpVs, which is imparted and rigidified by a “tri-disulfide ladder”. The β-hairpin structure of DpVs was imparted by incorporating the –(D)Pro–(L)Pro– moiety described by Robinson and co-workers (25
). In this group of analogues, the most effective substitutions were L-isoleucine (DpV13) and L-leucine (DpV16), with isoleucine being superior to leucine. Substitution with L-valine also led to increased activity compared to alanine or polar amino acids although this increase was modest compared to the L-isoleucine or L-leucine substitutions. Of interest, the D-isomers of leucine, valine or alanine all had lower activity than the L-isomers.
Given the strong activity of the L-leucine variant (DpV16) this peptide became the starting point for a series of additional peptides having substitutions at positions 1, 5, and 12. In this case hydrophobic substitutions were not clearly advantageous, although Tle, Cpg, Chg, Tyr analogues showed considerable activity. Increased activity was seen with more polar substitutions: i.e., threonine, serine and arginine. Since the parental compound (DpV16) has limited solubility in the aqueous solutions, further increase in hydrophobicity may be detrimental, while polar residues are beneficial.
Retaining the intact sequence of DpV16 but reducing cysteines 2 and 13 or adding PEG5
, methylsulfonyl attachments or Aoa-PEG5
to the molecule gave the greatest increase in activity. The fact that addition of PEG5
or methylsulfonyl group to the peptide significantly increased activity may be useful, since these attachments may also prolong the half life of the peptide in physiological conditions. The presence of the aminooxyacetic acid is particularly beneficial. Its effects may be partially explained by the presence of modified hydroxylamine group. Hydroxylamine was shown to inactivate IAV (29
) by cleaving fatty acids from viral haemagglutinin, lowering fusogenic and hemolytic activity. Although the concentration of hydroxylamine in the aforementioned experiments was much higher (1 M), perhaps DpV1632 augments its intrinsic antiviral activity by delivering the Aoa moiety to sites that are conducive to allow fatty acid cleavage. Replacing the cysteines with various polar or charged amino acids generally resulted in loss of activity.
HNPs were reported to exert effects on epithelial cells that inhibit replication of IAV (5
). In the present experiments, IAV was generally pre-incubated with defensins before infection of epithelial cells. To ascertain if the activity of HpV and DpV resulted from events before or after viral internalization, we compared pre-incubating IAV with the peptide, to introducing the peptides after the virus had interacted with cells for 15 or 45 minutes. Surprisingly, delaying peptide addition for as little as 15 minutes markedly diminished neutralizing activity. This suggests that under our experimental conditions the HpVs and DpVs exerted their antiviral effects by interacting with the virus itself. We believe that the ability of collectins or antimicrobial peptides to induce viral aggregation is an important correlate of antiviral activity. Viral aggregation can reduce particle numbers and promote clearance of virus from the airway through mucociliary action or uptake by phagocytes. We studied IAV aggregation mainly with peptides that showed strong activity in the neutralization assays. In general, highly neutralizing HpVs and DpVs also had strong viral aggregating activity, so these properties may be closely related. Note that the aggregating activity of the most potent peptides tested in this paper (e.g., HpV11 or HpV17) greatly exceeded that of retrocyclin RC2, which had the strongest aggregating activity among the θ-defensins tested in our prior studies. Some of the HpVs and DpVs also caused aggregation of bacteria or zymosan. The ability of the peptides to induce aggregation suggests that they may oligomerize as has been reported for some defensins and retrocyclins (31
). We have found that oligomerization is important for the antiviral activity of collectins (32
HNPs and retrocyclins have been reported to have pro- and anti-inflammatory effects in the lung and to promote viral or bacterial uptake by phagocytes (4
). The opsonizing activity of defensins may relate to the ability of these defensins to induce viral or bacterial aggregation (28
). HpVs and DpVs showed opsonizing activity for IAV comparable to that shown by HNPs and RCs. Significantly, peptides that displayed strong aggregating activity, such as HpV17, also had the strongest opsonizing activity. It is notable that the HpVs and DpVs were also capable of inhibiting IAV induced TNFα responses in monocytes. Production of TNFα and other strong pro-inflammatory cytokines may be deleterious during IAV infection (33
) so this effect could be beneficial during severe IAV infection in vivo
Overall, our findings suggest that developing synthetic θ-defensin analogs as potential therapeutics for IAV is promising and should be pursued further by in vivo
studies. One concern might be local inflammatory effects of the peptides is administered through the respiratory tract; however, recent reports indicate that retrocyclins applied in this manner have significant protective activity against serious respiratory pathogens including SARS and avian influenza (H5N1) in mice (35
). Since retrocyclins are also highly effective against other viruses, including HIV and HSV, it will be important to determine if the HpVs and DpVs are similarly active against these other viruses. If so, the small size and simple structures of DpVs and HpVs could make them excellent candidates for use as topical microbicides as well as potential systemic therapeutics. Of note, HpVs and DpVs appeared to have additive neutralizing activity when combined with SP-D, quite unlike the competitive effects seen when HNP1 and SP-D were combined. These findings suggest that HpVs or DpVs would not interfere with functional activities of SP-D if used to enhance lung immunity in vivo.