Combinations of the first and next generations of human immunodeficiency virus (HIV) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtide- sensitive and -resistant HIV type 1 strains

Abstract

T20 (generic name, enfuvirtide; brand name, Fuzeon) is a first-generation human immunodeficiency virus (HIV) fusion inhibitor approved for salvage therapy of HIV-infected patients refractory to current antiretroviral drugs. However, its clinical use is limited because of rapid emergence of T20-resistant viruses in T20-treated patients. Therefore, T1249 and T1144 are being developed as the second- and third-generation HIV fusion inhibitors, respectively, with improved efficacy and drug resistance profiles. Here, we found that combinations of T20 with T1249 and/or T1144 resulted in exceptionally potent synergism (combination index, <0.01) against HIV-1-mediated membrane fusion by 2 to 3 orders of magnitude in dose reduction. Highly potent synergistic antiviral efficacy was also achieved against infection by laboratory-adapted and primary HIV-1 strains, including T20-resistant variants. The mechanism underlying the synergistic effect could be attributed to the fact that T20, T1249, and T1144 all contain different functional domains and have different primary binding sites in gp41. As such, they may work cooperatively to inhibit gp41 six-helix bundle core formation, thereby suppressing virus-cell fusion. Therefore, these findings strongly imply that, rather than replacing T20, combining it with HIV fusion inhibitors of different generations might produce synergistic activity against both T20-sensitive and -resistant HIV-1 strains, suggesting a new therapeutic strategy for the treatment of HIV-1 infection/AIDS.

FIG. 1.

Functional domains of HIV fusion inhibitors and the interaction model. (A) Schematic view of the HIV-1_HXB2 gp41 molecule and sequences of the first-, second-, and third-generation HIV fusion inhibitors. FP, fusion peptide; TM, transmembrane domain; CP, cytoplasmic domain. (B) Interaction between the NHR and CHR peptides. The dashed lines between the NHR and CHR domains indicate the interaction between the residues located at the e and g and a and d positions in the NHR and CHR, respectively. The PBD, HBD, and TRD in the CHR peptides are shown in blue, light blue, and orange, respectively. The HR sequence, the region of aa 36 to 45 (determinant for T20 resistance and the primary binding site for T20), and the pocket-forming sequence in the NHR are shown in red, purple, and green, respectively. The interaction between the PBD and pocket-forming sequence is critical for stabilization of the 6-HB (3).

FIG. 2.

Synergistic effect of combinations of T20 with T1249 and/or T1144 and T1144 analogues T267227 and C38 on inhibition of HIV-1-mediated cell-cell fusion as determined by a dye transfer assay (21, 33). Ratios are as follows: T20-T1249, 4:1 (A); T20-T1144, 4:1 (B); T20-T1249-T1144, 4:1:1 (C); T20-T267227, 5:1 (D); T20-C38, 10:1 (E); and T1144-T2672227, 1:1 (F). Each sample was tested in quadruplicate, the experiment was repeated twice, and a representative set of data is shown.

FIG. 3.

Synergistic effect of combinations of T20 with T1249 and/or T1144 on inhibition of HIV-1 IIIB infection. Ratios are as follows: T20-T1249, 4:1 (A); T20-T1144, 16:1 (B); and T20-T1249-T1144, 16:4:1 (C). Each sample was tested in triplicate, the experiment was repeated twice, and a representative set of data is shown.

FIG. 4.

Analysis of the interaction between N46 and the CHR peptide(s) by CD spectroscopy. All peptides and their complexes were measured at 8 μM in PBS. For peptide(s) underlined or not underlined data are presented as the experimental spectra (underlined) and calculated theoretical noninteracting spectra (not underlined), as previously described (28, 40). Combinations are indicated on the panels.

FIG. 5.

Synergistic effect on inhibition of 6-HB formation resulting from combinations of T20 with T1249 and/or T1144 as measured by ELISA (45). Ratios are as follows: T20-T1249, 16:1 (A); T20-T1144, 16:1 (B); T20-T1249-T1144, 16:1:1 (C). Each sample was tested in triplicate. The results shown are a representative set of data from two independent experiments.

FIG. 6.

Putative models of action of different generations of HIV fusion inhibitors, T20, T1249, and T1144, which contain distinct functional domains. T1144 binds to the HR sequence and pocket-forming sequence in the NHR domain via its PBD and HBD, respectively, to form a stable heterologous 6-HB. T20 interacts with HR sequence in the NHR and lipid membranes through its HBD and TRD, respectively (1, 29, 30, 49, 58). T1249 may function either like T1144 through the PBD and HBD (model I) or like T20 via the HBD and TRD (model II). Binding of T20 to one of the three grooves on the NHR trimer may prolong the exposure of the fusion intermediate, which may promote T1249 or T1144 to interact with the unoccupied grooves, resulting in a synergistic effect against gp41-mediated membrane fusion.