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Dual-acting stapled peptides target both HIV-1 entry and assembly.

Zhang H, Curreli F, Waheed AA, Mercredi PY, Mehta M, Bhargava P, Scacalossi D, Tong X, Lee S, Cooper A, Summers MF, Freed EO, Debnath AK - Retrovirology (2013)

Bottom Line: Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (K(d) ~ 1 μM) compared to CAI (K(d) ~ 15 μM).This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge.This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Modeling, Drug Design, Lindsley F, Kimball Research Institute of the New York Blood Center, 310 E 67th Street, New York, NY 10065, USA. adebnath@nybloodcenter.org.

ABSTRACT

Background: Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (K(d) ~ 1 μM) compared to CAI (K(d) ~ 15 μM). NYAD-1 disrupts the formation of both immature- and mature-like virus particles in in vitro and cell-based assembly assays. In addition, it displays potent anti-HIV-1 activity in cell culture against a range of laboratory-adapted and primary HIV-1 isolates.

Results: In this report, we expanded the study to i,i + 7 hydrocarbon-stapled peptides to delineate their mechanism of action and antiviral activity. We identified three potent inhibitors, NYAD-36, -66 and -67, which showed strong binding to CA in NMR and isothermal titration calorimetry (ITC) studies and disrupted the formation of mature-like particles. They showed typical α-helical structures and penetrated cells; however, the cell penetration was not as efficient as observed with the i,i + 4 peptides. Unlike NYAD-1, the i,i + 7 peptides did not have any effect on virus release; however, they impaired Gag precursor processing. HIV-1 particles produced in the presence of these peptides displayed impaired infectivity. Consistent with an effect on virus entry, selection for viral resistance led to the emergence of two mutations in the gp120 subunit of the viral envelope (Env) glycoprotein, V120Q and A327P, located in the conserved region 1 (C1) and the base of the V3 loop, respectively.

Conclusion: The i,i + 7 stapled peptides derived from CAI unexpectedly target both CA and the V3 loop of gp120. This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge. This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents.

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i + 7 stapled peptides have no effect on HIV-1 release, but impair Gag processing. (a) 293T cells were transfected with pNL4-3 and 6 h after transfection treated with indicated concentrations of NYAD-36, -66, and -67 for 16–20 h. Cells were metabolically labeled with [35S]Met/Cys for 2 h. Cells were lysed and virions were collected by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig and subjected to SDS-PAGE. Protein band intensities were quantified by phosphorimager analysis, and HIV-1 release was calculated as the amount of virion-associated p24 relative to total (cell- plus virion-associated) Gag. (b) Accumulation of unprocessed Gag in cells was measured by calculating the ratio of Pr55Gag to p24 in cells. P values were calculated by Student’s t-test, with P < 0.05 considered significant. N = 3, ± SD.
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Figure 6: i + 7 stapled peptides have no effect on HIV-1 release, but impair Gag processing. (a) 293T cells were transfected with pNL4-3 and 6 h after transfection treated with indicated concentrations of NYAD-36, -66, and -67 for 16–20 h. Cells were metabolically labeled with [35S]Met/Cys for 2 h. Cells were lysed and virions were collected by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig and subjected to SDS-PAGE. Protein band intensities were quantified by phosphorimager analysis, and HIV-1 release was calculated as the amount of virion-associated p24 relative to total (cell- plus virion-associated) Gag. (b) Accumulation of unprocessed Gag in cells was measured by calculating the ratio of Pr55Gag to p24 in cells. P values were calculated by Student’s t-test, with P < 0.05 considered significant. N = 3, ± SD.

Mentions: We have shown previously that NYAD-1, an i,i + 4 stapled peptide derived from CAI, inhibited HIV-1 release in a dose-dependent manner. This inhibition was specific to HIV-1 as the release of another lentivirus, equine infectious anemia virus (EIAV), was not inhibited [24]. In this study we investigated whether i,i + 7 stapled CAI-derived peptides have any effect on virus release. 293T cells were transfected with the full-length HIV-1 molecular clone pNL4-3, treated with stapled peptides for 16–20 h, and metabolically labeled with [35S]Met/Cys. The cell- and virus-associated proteins were immunoprecipitated with HIV-Ig. Unlike NYAD-1, these analogs showed little to no inhibition of HIV-1 release (Figure 6a; Additional file 1: Figure S3). However, as we had observed previously with NYAD-1 [24], treatment of cells with each of the three stapled peptides (NYAD-36, -66, and -67) led to a modest accumulation of cell-associated Pr55Gag (Figure 6b; Additional file 1: Figure S3).


Dual-acting stapled peptides target both HIV-1 entry and assembly.

Zhang H, Curreli F, Waheed AA, Mercredi PY, Mehta M, Bhargava P, Scacalossi D, Tong X, Lee S, Cooper A, Summers MF, Freed EO, Debnath AK - Retrovirology (2013)

i + 7 stapled peptides have no effect on HIV-1 release, but impair Gag processing. (a) 293T cells were transfected with pNL4-3 and 6 h after transfection treated with indicated concentrations of NYAD-36, -66, and -67 for 16–20 h. Cells were metabolically labeled with [35S]Met/Cys for 2 h. Cells were lysed and virions were collected by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig and subjected to SDS-PAGE. Protein band intensities were quantified by phosphorimager analysis, and HIV-1 release was calculated as the amount of virion-associated p24 relative to total (cell- plus virion-associated) Gag. (b) Accumulation of unprocessed Gag in cells was measured by calculating the ratio of Pr55Gag to p24 in cells. P values were calculated by Student’s t-test, with P < 0.05 considered significant. N = 3, ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842668&req=5

Figure 6: i + 7 stapled peptides have no effect on HIV-1 release, but impair Gag processing. (a) 293T cells were transfected with pNL4-3 and 6 h after transfection treated with indicated concentrations of NYAD-36, -66, and -67 for 16–20 h. Cells were metabolically labeled with [35S]Met/Cys for 2 h. Cells were lysed and virions were collected by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig and subjected to SDS-PAGE. Protein band intensities were quantified by phosphorimager analysis, and HIV-1 release was calculated as the amount of virion-associated p24 relative to total (cell- plus virion-associated) Gag. (b) Accumulation of unprocessed Gag in cells was measured by calculating the ratio of Pr55Gag to p24 in cells. P values were calculated by Student’s t-test, with P < 0.05 considered significant. N = 3, ± SD.
Mentions: We have shown previously that NYAD-1, an i,i + 4 stapled peptide derived from CAI, inhibited HIV-1 release in a dose-dependent manner. This inhibition was specific to HIV-1 as the release of another lentivirus, equine infectious anemia virus (EIAV), was not inhibited [24]. In this study we investigated whether i,i + 7 stapled CAI-derived peptides have any effect on virus release. 293T cells were transfected with the full-length HIV-1 molecular clone pNL4-3, treated with stapled peptides for 16–20 h, and metabolically labeled with [35S]Met/Cys. The cell- and virus-associated proteins were immunoprecipitated with HIV-Ig. Unlike NYAD-1, these analogs showed little to no inhibition of HIV-1 release (Figure 6a; Additional file 1: Figure S3). However, as we had observed previously with NYAD-1 [24], treatment of cells with each of the three stapled peptides (NYAD-36, -66, and -67) led to a modest accumulation of cell-associated Pr55Gag (Figure 6b; Additional file 1: Figure S3).

Bottom Line: Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (K(d) ~ 1 μM) compared to CAI (K(d) ~ 15 μM).This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge.This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Modeling, Drug Design, Lindsley F, Kimball Research Institute of the New York Blood Center, 310 E 67th Street, New York, NY 10065, USA. adebnath@nybloodcenter.org.

ABSTRACT

Background: Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (K(d) ~ 1 μM) compared to CAI (K(d) ~ 15 μM). NYAD-1 disrupts the formation of both immature- and mature-like virus particles in in vitro and cell-based assembly assays. In addition, it displays potent anti-HIV-1 activity in cell culture against a range of laboratory-adapted and primary HIV-1 isolates.

Results: In this report, we expanded the study to i,i + 7 hydrocarbon-stapled peptides to delineate their mechanism of action and antiviral activity. We identified three potent inhibitors, NYAD-36, -66 and -67, which showed strong binding to CA in NMR and isothermal titration calorimetry (ITC) studies and disrupted the formation of mature-like particles. They showed typical α-helical structures and penetrated cells; however, the cell penetration was not as efficient as observed with the i,i + 4 peptides. Unlike NYAD-1, the i,i + 7 peptides did not have any effect on virus release; however, they impaired Gag precursor processing. HIV-1 particles produced in the presence of these peptides displayed impaired infectivity. Consistent with an effect on virus entry, selection for viral resistance led to the emergence of two mutations in the gp120 subunit of the viral envelope (Env) glycoprotein, V120Q and A327P, located in the conserved region 1 (C1) and the base of the V3 loop, respectively.

Conclusion: The i,i + 7 stapled peptides derived from CAI unexpectedly target both CA and the V3 loop of gp120. This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge. This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents.

Show MeSH
Related in: MedlinePlus