Site-Specific PEGylation of HR2 Peptides: Effects of PEG Conjugation Position and Chain Length on HIV-1 Membrane Fusion Inhibition and Proteolytic Degradation

Abstract

Peptides derived from the HR1 or HR2 regions of the HIV-1 envelope glycoprotein gp41 have been shown to be effective inhibitors to prevent virus−host cell membrane fusion. These peptide drugs, however, suffer from relatively short plasma half-lives and are susceptible to enzymatic degradation. Modification of peptides/proteins with poly-(ethylene glycol) (PEG) is a well-established strategy to overcome these limitations. This manuscript presents the results of a systematic study on the influence of the site of PEGylation of HR2-derived peptides, as well as of PEG molecular weight on the biological activity and proteolytic stability of these conjugates. Investigation of the fusion inhibitory efficacy of the conjugates in a model cell−cell based assay revealed a loss in activity for the PEGylated peptides as compared to the wild-type HR2-derived peptide. The loss of activity, however, can be minimized by controlling the site of PEGylation, more specifically, by introducing the PEG chain at one of the more central positions along the non-interacting αhelical surface of the peptides. The proteolytic stability of the PEG−peptide conjugates was assessed in a trypsin-based model assay, which revealed an up to 3.4-fold increase in degradation half-life that may help to compensate for the lower inhibitory efficacy of the PEG−peptide conjugates as compared to the wild-type peptide. The results of this study emphasize the power of site-specific PEGylation to improve the stability of peptide/protein drugs while minimizing adverse effects on biological activity.

Site-Specific Polymer Attachment to HR2 Peptide Fusion Inhibitors against HIV-1 Decreases Binding Association Rates and Dissociation Rates Rather Than Binding Affinity

“…While these polymers may not provide prolonged renal retention, conjugation of these relatively low molar mass polymers have shown to provide up to 3.4-fold increase in degradation half-life when incubated with the protease trypsin. 19 Furthermore, conjugation of similar low molar mass polymers as described in this manuscript would provide valuable insights that would not easily accessible through conjugation of high molar mass polymers.…”

“…The negative controls (S20C peptide – polymer conjugates) exhibited no infection inhibition over the investigated concentration range (Figure S9) and confirmed previous observations made with syncytia assays. 19,20 …”

“…35,36 In terms of the interaction studied here, the peptide – polymer conjugate must first diffuse up to 5-Helix before it can bind. As HR2 peptides are random coils (unordered) in solution, 19,28 the binding step involves zipping up of the HR2 helix as well as displacement of water and any local structural rearrangments of 5-Helix. Likewise, dissociation reverses these conformational transitions before the peptide – polymer conjugate can diffuse away.…”

Polymeric Anti‐HIV Therapeutics

“…Through a judicious choice of PEGylation along the peptide chain on amino acid residues that are on the periphery of the heptad repeat as opposed to the N-and C-peptide termini, the loss in efficacy upon PEGylation was further reduced to only 3-fold over the unmodified HR2-derived peptide (1.5 AE 0.1 Â 10 À9 M). [101] Dimeric fusion inhibitors (32), consisting of a short PEG-7 linker between the N-termini of two HR2 derived peptides, have exhibited a four-fold increase in efficacy over the unimeric HR2 derived peptide. [102] Similar dimeric peptide compounds have also shown to be effective fusion inhibitors against strains of HIV-1 that are resistant against the unmodified, unimeric T-20 peptide.…”

N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides

“…PEGylation of peptide substrates has also been used to increase resistance, though this often requires 10–40 kDa branched PEG units, and can result in changes in the properties of the peptide and sterics can interfere with binding. 25 Previous work in our lab has shown that appending small cyclic peptides to the N-terminus of a linear peptide can extend its lifetime in cytosolic environment, taking advantage of the size of the catalytic cleft as well as the N-terminus recognition. 14 However, cyclization can be synthetically challenging or low yielding, diminishing broad utility.…”