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

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.In the early 1990s, a number of highly potent anti-human immunodeficiency virus type 1 (HIV-1) peptides derived from the C-heptad repeat (CHR) domain of the HIV-1 envelope glycoprotein (Env) transmembrane subunit gp41 were discovered (21, 22, 35, 59, 61). Biophysical and biochemical analyses suggest that the CHR peptides inhibit HIV-1 Env-mediated membrane fusion by interacting with the viral gp41 N-heptad repeat (NHR) domain to form heterologous trimer-of-heterodimer complexes, thus blocking gp41 six-helix bundle (6-HB) core formation, a critical step in virus-cell fusion (4, 5, 31, 52, 57).T20 (generic name, enfuvirtide; brand name, Fuzeon), a 36-mer CHR peptide (amino acids aa] 638 to 673) containing a heptad repeat (HR) sequence-binding domain (HBD) and a tryptophan-rich domain (TRD) (Fig. ​(Fig.1)1) (30, 61), was licensed by the U.S. FDA as a first-generation HIV fusion inhibitor. T20 is very effective in inhibiting infection by HIV-1, especially the strains resistant to current antiretroviral therapies (24). However, many patients are now failing to respond to T20 because the viruses have developed T20 resistance (34, 51, 56, 62).Open in a separate windowFIG. 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).T1249, a second-generation HIV fusion inhibitor, is a 39-mer peptide consisting of a pocket-binding domain (PBD), an HBD, and a TRD (Fig. ​(Fig.1).1). T1249 was shown to have a longer half-life than T20 in primates (7) and greater anti-HIV-1 potency than T20 in clinical studies and to be active against some T20-resistant HIV-1 variants (7, 14, 27, 38). However, the clinical development of T1249 was discontinued due to formulation difficulties (37).T1144, a third-generation HIV fusion inhibitor, is a 38-mer peptide containing a PBD and an HBD (Fig. ​(Fig.1).1). T1144 was designed by modifying the amino acid sequence of T651 (peptide C38; aa 626 to 673) to increase α-helicity and 6-HB stability and to improve pharmacokinetic properties (10). T1144 and its analog peptides are effective against viruses that are resistant to T20 (11).Sifuvirtide, a new generation of HIV fusion inhibitor, is a 34-mer peptide analogue of C34 containing a PBD and an HBD. Our previous studies have shown that sifuvirtide is more effective than T20 against both primary and laboratory-adapted HIV-1 strains. Pharmacokinetic studies of sifuvirtide demonstrated longer decay half-lives than T20 (19). Sifuvirtide is under phase II clinical trial (www.fusogen.com). Most recently, we found that the combination of sifuvirtide with T20 resulted in potent synergistic effect against T20-sensitive and -resistant HIV-1 strains (43). These findings encouraged us to test whether combining T20 with T1249 and/or T1144 would also have synergistic anti-HIV-1 activity since next-generation HIV fusion inhibitors, like C34 and sifuvirtide, also contain a PBD that can interact with pocket-forming sequence in the gp41 NHR. In this study, we were also motivated to address the mechanism(s) underlying a synergic effect. Once this effect is confirmed, a novel combination therapy could be designed for the treatment of HIV/AIDS patients who have failed to respond to T20 or other antiretroviral drugs.