Electrostatically constrained α-helical peptide inhibits replication of HIV-1 resistant to enfuvirtide

Abstract

Alpha-helical peptides, such as T-20 (enfuvirtide) and C34, derived from the gp41 carboxyl-terminal heptad repeat (C-HR) of HIV-1, inhibit membrane fusion of HIV-1 and the target cells. Although T-20 effectively suppresses the replication of multi-drug resistant HIV variants both in vitro and in vivo, prolonged therapy with T-20 induces emergence of T-20 resistant variants. In order to suppress the emergence of such resistant variants, we introduced charged and hydrophilic amino acids, glutamic acid (E) and lysine (K), at the solvent accessible site of C34. In particular, the modified peptide, SC34EK, demonstrates remarkably potent inhibition of membrane fusion by the resistant HIV-1 variants as well as wild-type viruses. The activity was specific to HIV-1 and little influenced by serum components. We found a strong correlation between the anti-HIV-1 activities of these peptides and the thermostabilities of the 6-helix bundles that are formed with these peptides. We also obtained the crystal structure of SC34EK in complex with a 36 amino acid sequence (N36) comprising the amino-terminal heptad repeat of HIV-1. The EK substitutions in the sequence of SC34EK were directed toward the solvent and generated an electrostatic potential, which may result in enhanced alpha-helicity of the peptide inhibitor. The 6-helix bundle complex of SC34EK with N36 appears to be structurally similar to that of C34 and N36. Our approach to enhancing alpha-helicity of the peptide inhibitor may enable future design of highly effective and specific HIV-1 inhibitors.

Design of Peptide-based Inhibitors for Human Immunodeficiency Virus Type 1 Strains Resistant to T-20

“…Moreover, this strategy should not result in more adverse effect than those that might be obtained during use of the original peptide or oligonucleotide reagents. Recently we (6,30,31) and others (5) reported that hydrophilic amino FIGURE 4. Replication kinetics of HIV-1 S138X variants (X, any natural amino acid).…”

“…acid substitutions stabilized the ␣-helix of C-HR peptides and increased their binding affinity to N-HR, thus providing potent anti-HIV activity. This property may be one of the key attributes of the recently developed potent peptide inhibitors, SC34EK (6,30), T-20EK (31), or T-2429 (5), that have been reported to efficiently inhibit T-20 resistant variants. However, the S138A substitution on T-20 in the present study had little effect on the random coil structure, as judged by CD (data not shown), indicating that T-20 S138A increases its binding affinity not by simply enhancing the ␣-helicity of this region (5,6).…”

“…T-2635 is another efficient peptide fusion inhibitor that was recently developed and is also modified extensively (19 substitutions in 38 amino acids) (5). Also, SC34EK is an electrostatically constrained peptide that also suppresses replication of T-20-resistant variants, and it required 12 substitutions in the original C34 inhibitor (6,30). Hence, it is possible to improve the potency of existing peptide inhibitors through intense modeling and iterative testing in in vitro studies that could lead to the design and synthesis of improved peptide drugs.…”

Resistance Profiles of Novel Electrostatically Constrained HIV-1 Fusion Inhibitors

“…1) (17). SC34EK, which has unidirectional EK motifs, demonstrated a 5-fold enhanced activity compared with the original C34 (15,17). We demonstrated that the ␣-helical structure was stabilized by electrostatic in-* This work was supported, in whole or in part, by National Institutes of Health Grants AI076119, AI074389, and AI079801 (to S. G. S.), in part by a grant for Promotion of AIDS Research from the Ministry of Health and Welfare of Japan (to E. N. K. and M. M.), a grant for Research on Health Science Focusing on Drug Innovation from the Japan Health Science Foundation (to E. N. K. and M. M.), and a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to E. N. K.).…”

“…These inhibitors include tifuvirtide (T-1249) (12), T-2410 (13), and sifuvirtide (14), which are able to suppress T-20-resistant variants. We have developed electrostatically constrained fusion inhibitors, SC34 and SC34EK, which inhibit replication of T-20-resistant HIV-1 (15). SC34 was designed to be more soluble and have enhanced ␣-helicity, by engineering electrostatic interactions between glutamic acid and lysine substitutions at i and iϩ4 positions in the solvent-interacting site (EK motif) (16) of the parental C-HRderived C34 peptide ( Fig.…”