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


CONCLUSIONS
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.
Hongtao
H
Zhang
Francesca
F
Curreli
Abdul A
AA
Waheed
Peter Y
PY
Mercredi
Mansi
M
Mehta
Pallavi
P
Bhargava
Daniel
D
Scacalossi
Xiaohe
X
Tong
Shawn
S
Lee
Alan
A
Cooper
Michael F
MF
Summers
Eric O
EO
Freed
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.
Asim K
AK
Debnath
D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D016655:HIV Core Protein p24 / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D019065:Virus Assembly / Q000187:drug effects*; D053586:Virus Internalization / Q000187:drug effects*
D019380:Anti-HIV Agents; D016655:HIV Core Protein p24; D015699:HIV Envelope Protein gp120; D010455:Peptides; C514793:gp120 protein, Human immunodeficiency virus 1