enfuvirtide and sifuvirtide

HIV-1 fusion inhibitors are a new class of anti-HIV compounds, which block the entry of HIV into target cells through preventing the fusion between viral and cell plasma membrane and thus interrupt the initial steps of viral replication. T-20 (enfuvirtide), which has been clinically approved as the first fusion inhibitor of HIV-1 by U.S. FDA in 2003, can suppress replication of HIV variants with multi-drug resistance to reverse transcriptase and protease inhibitors. Peptides and small molecules display potent anti-HIV fusion activities by targeting gp41 thus inhibit its fusogenic function. In recent years, with the development of studies on the molecular mechanism of HIV membrane fusion process and the function of gp41, many new fusion inhibitors are found and some have been in advanced clinical trials. This review discusses recent progress in the development of HIV-1 fusion inhibitors targeting the gp41.

Topics: alpha 1-Antitrypsin; Anti-HIV Agents; Drug Resistance, Multiple; Enfuvirtide; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV Infections; HIV-1; Humans; Peptide Fragments; Peptides; Recombinant Fusion Proteins; Virus Replication

The pharmacokinetics assessment in two clinical studies of sifuvirtide (a novel HIV fusion inhibitor) was first reported in Chinese HIV patients. Nineteen treatment-naive HIV patients were treated with s.c.(subcutaneous injection) sifuvirtide [10 or 20 mg q.d.(quaque die)] for 28 days in study 1, and eight treatment-experienced HIV patients were treated with s.c. sifuvirtide (20 mg q.d.) in combination with HAART drugs (lamivudine, didanosine, and Kaletra) for 168 days in study 2. In study 1, T1/2 was 17.8 ± 3.7 h for 10 mg group and 39.0 ± 3.5 h for 20 mg group; the mean Cmax of last dose was 498 ± 54 ng/mL for 10 mg group and 897 ± 136 ng/mL for 20 mg group. In study 2, T1/2 was 6.71 ± 2.17 h in treatment-experienced patients. Cmax was 765 ± 288 ng/mL after last 168th dosage. Sifuvirtide showed improved clinical pharmacokinetics characteristics compared with Enfuvirtide, and showed very different pharmacokinetic characteristics between treatment-naive and treatment-experienced patients. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:4038-4047, 2014.

Topics: Adult; Anti-HIV Agents; Antiretroviral Therapy, Highly Active; Didanosine; Double-Blind Method; Drug Combinations; Enfuvirtide; Female; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV Infections; Humans; Lamivudine; Lopinavir; Male; Middle Aged; Peptide Fragments; Peptides; Ritonavir

Protein- or peptide-based viral inactivators are being developed as novel antiviral drugs with improved efficacy, pharmacokinetics and toxicity profiles because they actively inactivate cell-free human immunodeficiency virus type 1 (HIV-1) virions before attachment to host cells. By contrast, most clinically used antiviral drugs must penetrate host cells to inhibit viral replication. In this study, we pre-treated HIV-1 particles with a gp120-targeting bispecific multivalent protein, 2Dm2m or 4Dm2m, in the presence or absence of the gp41-targeting HIV-1 fusion inhibitory peptides enfuvirtide (T20), T2635, or sifuvirtide (SFT). HIV-1 virions were separated from the inhibitors using PEG-6000, followed by testing of the residual infectivity of the HIV-1 virions. 2Dm2m and 4Dm2m exhibited significant inactivation activity against all HIV-1 strains tested with EC

Topics: Anti-HIV Agents; Drug Discovery; Drug Resistance, Viral; Enfuvirtide; HIV Envelope Protein gp120; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV-1; Humans; Peptide Fragments; Peptides; Protein Binding; Virion; Virus Inactivation; Virus Replication

Most mechanistic studies on human immunodeficiency virus (HIV) peptide fusion inhibitors have focused on the interactions between fusion inhibitors and viral envelope proteins. However, the interactions of fusion inhibitors with viral membranes are also essential for the efficacy of these drugs. Here, we utilized surface plasmon resonance (SPR) technology to study the interactions between the HIV fusion inhibitor peptides sifuvirtide and enfuvirtide and biomembrane models. Sifuvirtide presented selectivity toward biomembrane models composed of saturated dipalmitoylphosphatidylcholine (DPPC) (32-fold higher compared with unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [POPC]) and sphingomyelin (SM) (31-fold higher compared with POPC), which are rigid compositions enriched in the HIV viral membrane. In contrast, enfuvirtide showed no significant selectively toward these rigid membrane models. Furthermore, the bindings of sifuvirtide and enfuvirtide to SM bilayers were markedly higher than those to monolayers (14-fold and 23-fold, respectively), indicating that the inner leaflet influences the binding of these drugs to SM bilayers. No obvious differences were noted in the bindings of either peptide to the other mono- and bilayer models tested, illustrating that both peptides interact with these membranes through surface-binding. The bindings of the inhibitor peptides to biomembranes were found to be driven predominantly by hydrophobic interactions rather than electrostatic interactions, as determined by comparing their affinities to those of positively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPC) to zwitterionic membrane models. The improved efficiency of sifuvirtide relative to enfuvirtide might be related to its ability to adsorb on rigid lipidic areas, such as the viral envelope and lipid rafts, which results in an increased sifuvirtide concentration at the fusion site.

Topics: Enfuvirtide; HIV Envelope Protein gp41; HIV Fusion Inhibitors; Humans; Lipid Bilayers; Peptide Fragments; Peptides; Surface Plasmon Resonance

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.. 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.

Topics: Amino Acid Substitution; Drug Resistance, Viral; Enfuvirtide; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV-1; Mutation; Mutation, Missense; Peptide Fragments; Peptides; Protein Binding

Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide.

Topics: Amino Acid Substitution; Cross Reactions; Drug Resistance, Viral; Enfuvirtide; Genetic Variation; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV-1; Humans; Mutagenesis, Site-Directed; Mutation; Peptide Fragments; Peptides

Mutations that are selected at low frequency and/or reside outside the enfuvirtide target region, amino acid 36-45 of gp41, might still be important determinants for drug resistance. This study aimed to investigate the phenotypic impact against enfuvirtide and sifuvirtide of uncharacterized gp41 mutations 42G, 43T and 50V, selected in patients failing enfuvirtide-containing regimens. As single mutations, neither 42G, 43T nor 50V conferred resistance to enfuvirtide. However, 50V increased slightly resistance levels for 36D, 38M, 43D or 43T as did 43T for 38M. All mutants displayed a reduced replication capacity, except 42S, 50V and 36D+/-50V. None of the mutants displayed resistance to the next-generation fusion inhibitor sifuvirtide. This study highlights the necessity to confirm the in vitro effect of infrequently selected mutations as 42G was not associated with enfuvirtide resistance whereas 43T and 50V should be considered as secondary enfuvirtide resistance mutations.

Topics: Amino Acid Sequence; Anti-HIV Agents; Cell Line; Enfuvirtide; HIV Envelope Protein gp41; HIV Infections; HIV-1; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Mutation, Missense; Peptide Fragments; Peptides; Virus Replication

Enfuvirtide and sifuvirtide, the first- and next-generation HIV-fusion inhibitors, contain different functional domains and have distinct target sites. Here, we found that a combination of enfuvirtide and sifuvirtide resulted in potent synergism in inhibiting HIV-1-mediated cell-cell fusion and infection by X4 and R5 as well as enfuvirtide-resistant HIV-1 strains. These findings suggest that application of enfuvirtide and sifuvirtide in combination may improve their efficacy and resistant profile, leading to a reduction of the dosage and frequency of drug use.

Topics: Drug Evaluation, Preclinical; Drug Synergism; Enfuvirtide; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV-1; Humans; Membrane Fusion; Peptide Fragments; Peptides

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion inhibitor, presently in clinical trials. Because of the aromatic amino acid residues of the peptide, its behavior in aqueous solution and the interaction with lipid-membrane model systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy techniques (both steady-state and time-resolved). No significant aggregation of the peptide was observed with aqueous solution. Various biological and nonbiological lipid-membrane compositions were analyzed, and atomic force microscopy was used to visualize phase separation in several of those mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquid-ordered phase). However, significant partitioning was observed with the positively charged lipid models (K(p) = (2.2 +/- 0.3) x 10(3)), serving as a positive control. Fluorescence quenching using Förster resonance acrylamide and lipophilic probes was carried out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at the surface of these membranes was observed and confirmed by using Förster resonance energy-transfer experiments. These results indicate a targeting of the peptide to gel-phase domains relatively to liquid-disordered or liquid-ordered phase domains. This larger affinity and selectivity toward the more rigid areas of the membranes, where most of the receptors are found, or to viral membrane, may help explain the improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the peptide at the fusion site.

Topics: Acrylamide; Amino Acid Sequence; Enfuvirtide; Fluorescence Resonance Energy Transfer; HIV Envelope Protein gp41; HIV Fusion Inhibitors; Lipid Bilayers; Membrane Fluidity; Membrane Lipids; Molecular Sequence Data; Peptide Fragments; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Solutions; Water

To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion.. Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-(127)I iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC(50)) of sifuvirtide on cell fusion was determined by co-cultivation assay.. The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 microg/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10 626+/-2886 microg/L and 528+/-191 microg/L, and the terminal elimination half-lives (T(1/2)) were 6.3+/-0.9 h and 5.5+/-1.0 h, respectively. After sc, T(max) was 0.25-2 h, and the absolute bioavailability was 49%+/-13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC(50) of 0.33 microg/L.. An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T(1/2) of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC(50) in vitro.

Topics: Animals; Anti-HIV Agents; Area Under Curve; Cell Line, Tumor; Chromatography, Liquid; Enfuvirtide; Female; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV-1; Inhibitory Concentration 50; Injections, Intravenous; Injections, Subcutaneous; Macaca mulatta; Male; Mass Spectrometry; Peptide Fragments; Peptides