Long-Acting HIV-1 Fusion Inhibitory Peptides and their Mechanisms of Action

Abstract

The clinical application of HIV fusion inhibitor, enfuvirtide (T20), was limited mainly because of its short half-life. Here we designed and synthesized two PEGylated C34 peptides, PEG2kC34 and PEG5kC34, with the PEG chain length of 2 and 5 kDa, respectively, and evaluated their anti-HIV-1 activity and mechanisms of action. We found that these two PEGylated peptides could bind to the HIV-1 peptide N36 to form high affinity complexes with high α-helicity. The peptides PEG2kC34 and PEG5kC34 effectively inhibited HIV-1 Env-mediated cell–cell fusion with an effective concentration for 50% inhibition (EC50) of about 36 nM. They also inhibited infection of the laboratory-adapted HIV-1 strain NL4-3 with EC50 of about 4–5 nM, and against 47 HIV-1 clinical isolates circulating in China with mean EC50 of PEG2kC34 and PEG5kC34 of about 26 nM and 32 nM, respectively. The plasma half-life (t1/2) of PEG2kC34 and PEG5kC34 was 2.6 h and 5.1 h, respectively, and the t1/2 of PEGylated C34 was about 2.4-fold and 4.6-fold longer than C34 (~1.1 h), respectively. These findings suggest that PEGylated C34 with broad-spectrum anti-HIV-1 activity and prolonged half-life can be further developed as a peptide fusion inhibitor-based long-acting anti-HIV drug for clinical use to treat HIV-infected patients who have failed to respond to current anti-retrovirus drugs.

Analysis of insertion mutants in distal α9 portion of C-terminal heptad repeat (CHR) of HIV-1 gp41 subunit

“…To explore the difference between 653+A and 653+Q mutants, corresponding synthetic CHR peptides were used. Although N36 and C34 have been used widely to analyze 6HB formation (27)(28)(29), we were unable to obtain interpretable CD data using these peptides. Because our mutation is an alanine insertion rather than alanine substitution, this may have resulted in a more destabilizing effect on 6HB formation with shorter peptides.…”

Polyethylene Glycol 40-Modified Peptide with High Therapeutic Efficacy in Simian-Human Immunodeficiency Virus-Acutely Infected Rhesus Monkeys

“…We constructed PEG-C34 conjugates using thiol-maleimide Michael reactions ( Fig. 1) (21,35). C34 is a 34-mer peptide derived from the gp41 CHR (residues 628 to 661) and includes Asn637, which forms a conserved glycosylation site in native gp41 (37).…”

“…HIV inhibition assay. The antiviral activities of PEG-NC conjugates were measured as previously described (21,35). Briefly, TZM-b1 cells were incubated on a tissue culture plate (96 well, 1 ϫ 10 4 cells/well) at 37°C overnight, then titrated PEG-NC conjugates were added (100 l in DMEM), followed by 200 50% tissue culture infective doses (TCID 50 ) of HIV-1 NL4-3 .…”

“…Pharmacokinetic analysis. The pharmacokinetic profile of PEG40-NC was studied in 7-week-old Sprague-Dawley rats (n ϭ 4; 180 to 200 g) as described previously (21,24,35). A single subcutaneous injection of PEG40-NC or C34 (1.7 mol/kg in physiological saline) was given to each rat (35).…”

“…C34 is a first-generation HIV-1 fusion inhibitor with more potent anti-HIV-1 activity than T20 (10). Furthermore, we have demonstrated C34 has a different resistance mechanism with T20 and retained high antiviral activity against T20-resistant strains (35). However, the low solubility of C34 undermines its potential use as a clinical drug (36).…”