Glycosylated Enfuvirtide: A Long-Lasting Glycopeptide with Potent Anti-HIV Activity

Abstract

Many peptide-based therapeutics have short circulatory half-lives. We report here that the pharmacokinetics of an anti-HIV peptide drug enfuvirtide (ENF) can be dramatically improved by a chemical glycosylation approach. A set of glycosylated ENFs with varying glycosylation sites and glycan structures were synthesized. Among these, a sialic acid-introduced peptide (SL-ENF) demonstrated a 15-fold extended half-life in rats relative to ENF (T1/2: 23.1 vs 1.5 h), and its antiviral potency was comparable to that of ENF (EC50: 2 vs 3 nM). SL-ENF bound to a functional fragment of the HIV fusogenic protein gp41 and formed complexes with high affinity and α-helicity, revealing the mechanism behind its potent antiviral activity. Because it is widely accepted in biology that glycosylation protects proteins from denaturation and proteases, our approach may be useful for the development of novel protein and peptide drugs with enhanced pharmaceutical properties.

Enfuvirtide−PEG conjugate: A potent HIV fusion inhibitor with improved pharmacokinetic properties

“…Notably, the EC 50 value of PEG2k-ENF (4 nM) was comparable to that of ENF (3 nM). The chemical modification at the C-terminus of ENF has an adverse impact on its antiviral activity [11,12,20], because the lipophilic residues (WNWF) in this region are required for the anchoring of the peptide into the membrane interface where the fusion occurs [28,29]. Our data were therefore in agreement with these earlier findings and revealed that the attachment of PEG2k to the N-terminus of ENF did not lead to a significant decrease in its antiviral efficacy.…”

“…Following our established glycosylation strategy [20], we investigated the site-specific addition of PEG to the terminus of ENF using a thiol-maleimide coupling reaction ( Fig. 1).…”

“…All peptides used in this study were synthesized using a solidphase Fmoc method at Beijing Scilight Biotechnology LLC (Beijing, China), purified by HPLC (purity >95%), and characterized by MALDIÀTOF MS. The sources of HIV-1 SF33 and TZM-bl, as well as the methods used for their preservation and passage have been described previously [20,41]. The clinical isolates of HIV-1 subtypes B 0 , BC, and AE were obtained from treatment-naive patients and stored at our laboratory in Chinese Center for Disease Control and Prevention [46,47].…”

“…As part of our ongoing research towards the development of persistent inhibitors against HIV fusion, we previously developed a series of glycosylated ENFs with long in vivo half-lives that completely retained their antiviral activities [20]. In a similar manner to PEGylation, the glycosylation of peptides and proteins leads to an increase in their molecular size and steric bulk, which can have a significant impact on their biophysical and physiological properties.…”

“…Based on our previous experience of the glycosylation of ENF [20], and the fact that PEGylation has advanced many bioactive proteins and peptides into clinical applications [23], we sought to prepare the ENFÀPEG (EP) conjugate as a simple alternative. By controlling the modification site of ENF and the molecular weight (MW) of PEG, we successfully synthesized an EP conjugate with a remarkably improved pharmacokinetic profile.…”