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

Abstract

Enfuvirtide (ENF) is a clinically used peptide drug for the treatment of HIV infections, but its poor pharmacokinetic profile (T1/2 = 1.5 h in rats) and low aqueous solubility make the therapy expensive and inconvenience. In this study, we present a simple and practical strategy to address these problems by conjugating ENF with polyethylene glycol (PEG). Site-specific attachment of a 2 kDa PEG at the N-terminus of ENF resulted in an ENF-PEG (EP) conjugate with high solubility (≥3 mg/mL) and long half-life in rats (T1/2 = 16.1 h). This conjugate showed similar antiviral activity to ENF against various primary HIV-1 isolates (EC50 = 6-91 nM). Mechanistic studies suggested the sources of the antiviral potency. The conjugate bound to a functional domain of the HIV gp41 protein in a helical conformation with high affinity (Kd = 307 nM), thereby inhibiting the gp41-mediated fusion of viral and host-cell membranes. As PEG conjugation has advanced many bioactive proteins and peptides into clinical applications, the EP conjugate described here represents a potential new treatment for HIV infections that may address the unmet medical needs associated with the current ENF therapy.

Keywords: Conjugation; Enfuvirtide; Fusion inhibitor; Human immunodeficiency virus; Pharmacokinetics; Poly(ethylene glycol).

Graphical abstract

Fig. 1

Synthesis (A) and characterization (B) of EP conjugates (PEG2k-ENF, PEG5k-ENF, ENF-PEG2k, and ENF-PEG5k). All of the peptide sequences were protected by N-terminal acetylation and C-terminal amidation. (A) Reaction conditions: phosphate buffer (pH 7.2), rt, 10 min, 90% for PEG2k-ENF, 85% for ENF-PEG2k, 82% for PEG5k-ENF, and 82% for ENF-PEG5k. (B) MALDI−TOF mass spectrum and HPLC chromatogram (inset) of PEG2k-ENF. All of the other conjugates were characterized in a similar manner. HPLC chromatographic conditions: Phenomenex Synergi Hydro-RP C18 column (4 μm, 10 × 250 mm), 40 → 70% CH₃CN/H₂O linear gradient over 11 min at 2.5 mL/min, UV detection at 220 nm.

Fig. 2

SPR sensorgrams for the binding of ENF and EP conjugates to N46. Peptide N46 was immobilized on the surface of a Biacore sensor chip. Solutions containing different concentrations of ENF or EP conjugates in running buffer (PBS + 0.05% v/v Tween 20, pH 7.4) were flowed over the surface of the chip. The binding curves of ENF and the conjugates at 1 μM are shown in this figure. The dose-dependent sensorgrams are shown in the Supplementary data (Fig. S1). The binding kinetic parameters (Table S1) were calculated using Biacore analysis software. The sequence of N46 is as follows: TLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL.

Fig. 3

CD spectra of ENF and EP conjugates in PBS buffers (pH 7.2) at 20 °C. The α-helicities are calculated relative to an ellipticity value of −33,000° cm²d/mol (at 222 nm) that is assumed to correspond to a helical content of 100% , , (for details, see Table S2). (A) In the absence of peptide N46, both ENF and the conjugates adopted a random coil structure. (B) In the presence of equimolar amounts of N46, the ENF−N46 complex showed a much lower α-helicity than N46, whereas all of the EP conjugate−N46 complexes were fully α-helical, with their ellipticities below −42,000° cm²d/mol (at 222 nm).

Fig. 4

Pharmacokinetic analysis in Sprague–Dawley rats. ENF and PEG2k-ENF were subcutaneously administered at 1.7 μmol/kg (in physiological saline). Four animals per group were used. (A) Pharmacokinetic curves. The concentrations of ENF and the conjugate in plasma were determined using a known HPLC analysis , . Error bars indicate the SD. (B) Pharmacokinetic parameters. Values were calculated using the non-compartmental analysis (NCA,WinNonlin, version 6.2, Pharsight Cary, NC) and expressed as the mean values ± SD. T_1/2 (elimination half-life or terminal half-life) refers to the time required for the concentration of remaining drug in plasma to be reduced by 50% after the pseudo-equilibrium of drug distribution in the body has been reached.