SC29EK, a peptide fusion inhibitor with enhanced alpha-helicity, inhibits replication of human immunodeficiency virus type 1 mutants resistant to enfuvirtide

Abstract

Peptides derived from the alpha-helical domains of human immunodeficiency virus (HIV) type 1 (HIV-1) gp41 inhibit HIV-1 fusion to the cell membrane. Enfuvirtide (T-20) is a peptide-based drug that targets the step of HIV fusion, and as such, it effectively suppresses the replication of HIV-1 strains that are either wild type or resistant to multiple reverse transcriptase and/or protease inhibitors. However, HIV-1 variants with T-20 resistance have emerged; therefore, the development of new and potent inhibitors is urgently needed. We have developed a novel HIV fusion inhibitor, SC34EK, which is a gp41-derived 34-amino-acid peptide with glutamate (E) and lysine (K) substitutions on its solvent-accessible site that stabilize its alpha-helicity. Importantly, SC34EK effectively inhibits the replication of T-20-resistant HIV-1 strains as well as wild-type HIV-1. In this report, we introduce SC29EK, a 29-amino-acid peptide that is a shorter variant of SC34EK. SC29EK blocked the replication of T-20-resistant HIV-1 strains and maintained antiviral activity even in the presence of high serum concentrations (up to 50%). Circular dichroism analysis revealed that the alpha-helicity of SC29EK was well maintained, while that of the parental peptide, C29, which showed moderate and reduced inhibition of wild-type and T-20-resistant HIV-1 strains, was lower. Our results show that the alpha-helicity in a peptide-based fusion inhibitor is a key factor for activity and enables the design of short peptide inhibitors with improved pharmacological properties.

FIG. 1.

(A) Schematic diagram of HIV-1 gp41 and sequences of C-HR-derived peptides. FP, fusion peptide; TM, transmembrane domain. The residues at each position in the helical turns are denoted in italics. *Z, an artificial amino acid, norleucine, instead of methionine, to avoid oxidation of the side chain of methionine; †, possible electrostatic interactions indicated by lines and correlating amino acids. (B) One heptad helical turn.

FIG. 2.

Analysis of N36 and various C-HR-derived peptides complexes by CD spectroscopy. CD spectra for N36- and C-HR-derived peptide complexes (A) and mutated N36-C29 or SC29EK complexes (B). (C) Temperature-dependent transitions of the dissociation degree of N36 and various C-HR-derived peptide complexes. (D) T_ms of complexes of various N-HR peptides and C-HR peptides. NA, not available. (E) Relation between EC₅₀s of C-HR-derived peptides and T_ms of N36 and various C-HR-derived peptide complexes. The strength of the correlation between EC₅₀s and T_ms is increased (r² = 0.8002) when the data for SC22EK are excluded.

FIG. 3.

Effect of serum components on antiviral activity. Antiviral activities in the presence of serum at various concentrations (5, 10, 20, and 50%) were determined by the MAGI assay. (A) Black dots indicate EC₅₀s (nM), each of which was determined three times independently (*, P = 0.01 by Student's t test). Antiviral activities of T-20 (B) and SC29EK (C) in human HIV-seronegative sera (hs1 and hs2) were assessed by counting the number of blue cells. Bars indicate the percentages of blue cell counts in PBS, FCS, and human serum compared with the count obtained with no antiviral agents (control, for which the value was 100%). Error bars represent the standard deviation of each mean.