Covalent stabilization of coiled coils of the HIV gp41 N region yields extremely potent and broad inhibitors of viral infection

Abstract

Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001) Proc. Natl. Acad. Sci. USA 98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17)3, has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17)3 (i) inhibits fusion in a cell-cell fusion assay (IC50 = 260 pM); (ii) is the most potent fusion inhibitor described to date (IC50 = 40-380 pM) in a single-cycle infectivity assay against HIV(HXB2), HIV(NL4-3), and HIV(MN-1); (iii) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and (iv) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17)3 was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17)3 an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.

Fig. 1.

Thermal denaturation curves of IZN17, N17IZ, and (CCIZN17)₃, as monitored by CD at 222 nm in 5 mM Hepes, pH 7.3/150 mM NaCl/2 M guanidinium hydrochloride. The melting temperatures (T_m), are 50.7°C, 61.5°C, and >90°C for N17IZ, IZN17, and (CCIZN17)₃, respectively.

Fig. 2.

Schematic model of the designed IZN17 and (CCIZN17)₃ molecules. The IZN17 helices forming a homotrimeric coiled-coil are represented by cylinders. Each chimeric N peptide consists of an N-terminal designed trimeric coiled-coil (light gray) fused to a portion of the sequence from the N-helix region of gp41 (dark gray), namely N17 (residues 568-584 of HIV_HXB2). The (CCIZN17)₃ helices are covalently stabilized at the N terminus by three interchain disulfides between each pair of cysteines of each peptide chain. Only one of the possible combinations of the three disulfides is drawn.

Fig. 3.

Formation of the covalent trimer (CCIZN17)₃ monitored by HPLC-MS. (a) Overlay of the chromatograms of the precursor CCIZN17 (dashed line) and of the oxidized (CCIZN17)₃ after 18 h of incubation (solid line). (b and c) Hypermass reconstruction of the raw electrospray MS data of monomeric CCIZN17 (observed mass, 5,175.0 Da; calculated mass, 5,175.0 Da) (b) and the covalent trimer (CCIZN17)₃, whose observed mass of 15,520.1 Da (calculated mass, 15,520.2 Da), is in agreement with the formation of three disulfide bonds (c).

Fig. 4.

Inhibition of infectivity of various HIV isolates. The calculated IC₅₀ values for (CCIZN17)_3, IZN17, 5-helix, and DP178 are (respectively) 0.04, 1.3, 9.9, and 1.2 nM (against HIV_HXB2) (a); 0.046, 33.66, 12.0, and 1.7 nM (against HIV_NL4-3)(b); and 0.38, 394.7, 159.4, and 5.5 nM (against HIV_MN-1)(c).

Fig. 5.

Inhibition of HIV fusion in a cell-cell fusion assay. The calculated IC₅₀ values for (CCIZN17)₃ and DP178 are 0.26 nM and 14.5 nM, respectively.