Mechanism of HIV-1 Resistance to Short-Peptide Fusion Inhibitors Targeting the Gp41 Pocket

Abstract

The deep hydrophobic pocket on the N trimer of HIV-1 gp41 has been considered an ideal drug target. On the basis of the M-T hook structure, we recently developed short-peptide-based HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the pocket site and possess highly potent antiviral activity. In this study, we focused on investigating their resistance pathways and mechanisms by escape HIV-1 mutants to SC22EK, a template peptide for MTSC22 and HP23. Two substitutions, E49K and N126K, located, respectively, at the N- and C-heptad repeat regions of gp41, were identified as conferring high resistance to the inhibitors targeting the pocket and cross-resistance to enfuvirtide (T20) and sifuvirtide (SFT). The underlying mechanisms of SC22EK-induced resistance include the following: (i) significantly reduced binding affinity of the inhibitors, (ii) dramatically enhanced interaction of the viral six-helix bundle, and (iii)severely damaged functionality of the viral Env complex. Our data have provided important information for the structure-function relationship of gp41 and the structure-activity relationship of viral fusion inhibitors.

Importance: Enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but the problem of resistance significantly limits its use, calling for new strategies or concepts to develop next-generation drugs. On the basis of the M-T hook structure, short-peptide HIV-1 fusion inhibitors specifically targeting the gp41 pocket site exhibit high binding and antiviral activities. Here, we investigated the molecular pathway of HIV-1 resistance to the short inhibitors by selecting and mapping the escape mutants. The key substitutions for resistance and the underlying mechanisms have been finely characterized. The data provide important information for the structure-function relationship of gp41 and its inhibitors and will definitely help our future development of novel drugs that block gp41-dependent fusion.

FIG 1

Schematic illustration of HIV-1 gp41 and peptide fusion inhibitors. The gp41 numbering of HIV-1_HXB2 is used. FP, fusion peptide; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; TM, transmembrane domain. The sequences corresponding to the T20-resistant site are highlighted in bold, the sequences corresponding to the NHR pocket region are shown in blue, and the sequences corresponding to the CHR pocket-binding domain (PBD) are shown in red. The position and sequence of the M-T hook structure are shown in green. The peptide inhibitors and their sequences are listed, while the E49K substitution in the N peptide N36 and the N126K substitution in the C peptide C34 are highlighted in green.

FIG 2

Alignment of the amino acid sequence of NHR and CHR of HIV-1_NL4-3 and its selected mutants. The positions of selected substitutions are in bold, and numbering is according to that of HIV-1_HXB2 gp41. The pocket-forming sequence in NHR and the pocket-binding domain in CHR are underlined. WT, wild type.

FIG 3

Relative infectivity of HIV-1_NL4-3 and its mutant viruses. The wild-type HIV-1_NL4-3 and mutant pseudoviruses were normalized to a fixed amount by p24 antigen, and viral infectivity was tested in TZM-bl cells using a single-cycle infection assay. The luciferase activity was measured and corrected for background. The luciferase activity of HIV-1_NL4-3 was treated as 100%, and the relative infectivity of other mutant viruses was calculated accordingly. Data were derived from the results of three independent experiments and are expressed as means ± standard deviations.

FIG 4

Binding stability of short-peptide inhibitors determined by CD spectroscopy. The α-helicity and thermostability of 6-HBs formed by SC22EK (A and B), MTSC22 (C and D), or HP23 (E and F) with the N peptide N36 or its E49K mutant were measured. The T_m value was defined as the midpoint of the thermal unfolding transition. The final concentration of each peptide in PBS is 10 μM. The experiments were repeated at least three times, and representative data are shown.

FIG 5

Binding stability of C34, SFT, and a C34 mutant determined by CD spectroscopy. The α-helicity and thermostability of 6-HBs formed by C34 (A and B) or SFT (C and D) with N36 or N36_E49K were measured. The T_m value was defined as the midpoint of the thermal unfolding transition. The final concentration of each peptide in PBS is 10 μM. The binding stability of C34 carrying an N126K mutation (C34_N126K) with N36 or N36_E49K was similarly determined by CD spectroscopy (E and F). The experiments were repeated at least three times, and representative data are shown.

FIG 6

Effects of the E49K and N126K substitutions on the conformation of 6-HB structure. The reactivity of the 6-HBs formed by N36 and C34 or their mutants with conformation-dependent MAbs NC-1 (A) and 17C8 (B) was tested by ELISA. The peptide mixture was coated on the plate wells at 10 μg/ml, and the final concentration of a tested MAb was 5 μg/ml. Data were derived from three independent experiments and are expressed as means ± standard deviations. OD₄₅₀, optical density at 450 nm.

FIG 7

Modeling of the E49K-mediated electrostatic interactions on the 6-HB by the program PyMOL. (A) Structural analysis of the E49K substitution-mediated electrostatic repulsion to the short-peptide inhibitors (Protein Data Base identification [ID] code 3VU5), in which a C-terminal lysine at position 20 (K20) of SC22EK may form a salt bridge interaction with glutamic acid at position 49 (E49) of the viral NHR helix. Thus, the E49K substitution would introduce an electrostatic repulsion to the inhibitors. (B) Axial view of the salt bridge between E49 and K20 in the 6-HB. (C) 6-HB structure of N36 and C34 (Protein Data Base ID code 1AIK). Position 20 of peptide C34 is a native glutamic acid (E20) corresponding to the viral CHR sequence. Thus, the E49K substitution might introduce an electrostatic attraction between viral NHR and CHR helices. (D) 6-HB structure of N36 and SFT (Protein Data Base ID code 3VIE). Similarly, position 21 of the SFT inhibitor carries a glutamic acid (E21), similar to the C34 and viral sequence; thus, the E49K substitution would enhance the binding affinity of SFT via an introduced salt bridge interaction.

FIG 8

Binding stability of truncated short-peptide inhibitors determined by CD spectroscopy. The thermostability of 6-HBs formed by MTSC21 (A), MTSC19 (B), HP22 (C), or HP23E (D) with N36 and N36_E49K was measured. The T_m value was defined as the midpoint of the thermal unfolding transition. The final concentration of each peptide in PBS is 10 μM. The experiments were repeated at least three times, and representative data are shown.