SC29EK, a Peptide Fusion Inhibitor with Enhanced α-Helicity, Inhibits Replication of Human Immunodeficiency Virus Type 1 Mutants Resistant to Enfuvirtide

Abstract

Peptides derived from the α-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 α-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 α-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 α-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.

Broad Antiviral Activity and Crystal Structure of HIV-1 Fusion Inhibitor Sifuvirtide

“…Pseudoviruscontaining culture supernatants were harvested 48 h after transfection, filtered by 0.45-m pore size, and stored at Ϫ80°C in 1-ml aliquots until use. The 50% tissue culture infectious dose (TCID 50 ) of a single thawed aliquot of each pseudovirus batch was determined in TZM-bl cells. The antiviral activity of SFT or T20 was determined using TZM-bl cells as described previously.…”

“…The mixture was added to TZM-bl cells (10 4 /well) and incubated at 37°C for 48 h. Luciferase activity was measured using luciferase assay reagents (Promega, Madison, WI) and a luminescence counter (Promega) according to the manufacturer's instructions. The percent inhibition by the peptides and 50% inhibitory concentration (IC 50 ) values were calculated as described previously (27,28).…”

“…The antiviral activity of SFT or T20 was determined using TZM-bl cells as described previously. Briefly, the peptides were prepared with 10 series of dilutions in a 3-fold stepwise manner, mixed with 100 TCID 50 viruses, and incubated for 1 h at room temperature. The mixture was added to TZM-bl cells (10 4 /well) and incubated at 37°C for 48 h. Luciferase activity was measured using luciferase assay reagents (Promega, Madison, WI) and a luminescence counter (Promega) according to the manufacturer's instructions.…”

“…The mutant viruses were generated by transient transfection of NL4-3 plasmids into 293T cells. The virus stocks were harvested 48 h post-transfection and quantified for TCID 50 . Inhibition of the peptides (SFT and T20) on the NL4-3 mutants was performed as described for pseudoviruses.…”

“…The results from the single cycle infection assays are presented in Table 2. Compared with T20, SFT displayed more potent inhibitory activity, with mean IC 50 (17)(18)(19)(20)(21). Our previous studies demonstrated that SFT possessed efficient potency to inhibit HIV-1 variants that carry single or double T20-resistant mutations (e.g.…”