Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency

Abstract

Peptides derived from the heptad repeat 2 (HR2) region of the HIV fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, enfuvirtide, is used for the treatment of therapy-experienced AIDS patients. The mechanism of action of these peptides is binding to a critical intermediate along the virus-cell fusion pathway, and accordingly, increasing the affinity for the intermediate yields more potent inhibitors. We took a different approach, namely to increase the potency of the HR2 peptide inhibitor C34 by targeting it to the cell compartment where fusion occurs, and we show here that a simple, yet powerful way to accomplish this is attachment of a cholesterol group. C34 derivatized with cholesterol (C34-Chol) shows dramatically increased antiviral potency on a panel of primary isolates, with IC(90) values 15- to 300-fold lower than enfuvirtide and the second-generation inhibitor T1249, making C34-Chol the most potent HIV fusion inhibitor to date. Consistent with its anticipated mechanism of action, the antiviral activity of C34-Chol is unusually persistent: washing target cells after incubation with C34-Chol, but before triggering fusion, increases IC(50) only 7-fold, relative to a 400-fold increase observed for C34. Moreover, derivatization with cholesterol extends the half-life of the peptide in vivo. In the mouse, s.c. administration of 3.5 mg/kg C34-Chol yields a plasma concentration 24 h after injection >300-fold higher than the measured IC(90) values. Because the fusion machinery targeted by C34-Chol is similar in several other enveloped viruses, we believe that these findings may be of general utility.

Fig. 1.

Currently accepted model for HIV fusion and for the mechanism of action of HR2-derived peptide inhibitors. (Top) Binding of the gp120 subunit (green) of the trimeric envelope glycoprotein to the CD4 receptor and the chemokine coreceptor (CXCR4 or CCR5) triggers a conformational change in the unmasked subunit gp41 (gray and violet), where two regions, the N-terminal HR1 (gray), and the C-terminal HR2 (violet) become separated in the so-called “hairpin intermediate,” which bridges the viral and cell membranes. Collapse of the hairpin intermediate into a 6-helix bundle drives viral–cell membrane fusion. (Middle) Schematic representation of gp41, using the same color code for HR1 and HR2. (Bottom) MOA of HR2-derived FI. C34 binds the HR1 coiled-coil and prevents transition to the 6-helix bundle. It is apparent from the figure that C34 must bind to HR1 in antiparallel orientation. The boundaries of the gp41 regions encompassed by C34 and T20 are indicated (FP, fusion peptide; PTM, proximal to the transmembrane region; TM, transmembrane region; CP, cytoplasmic region).

Fig. 2.

Pharmacokinetics of C34-Chol in mice. C34-Acm or C34-Chol (3.5 mg/kg) in 10 mM glycine buffer containing 10% ethanol was administered s.c. to C57BL/6 mice, and the concentration of peptide was monitored with time. The calculated pharmacokinetic parameters are given in Table S1.