Antiviral activity of α-helical stapled peptides designed from the HIV-1 capsid dimerization domain

Abstract

BackgroundThe C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs.ResultsDue to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC), sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease.ConclusionsThese preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind CA-CTD.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

“…A peptide mimicking the helix 9 sequence, CAC-1, was shown to inhibit CTD dimerization (21), and a stabilized version of this peptide, NYAD-201, inhibited mature-like virus particle formation (22). These results suggested that targeting CTD dimerization would be a good strategy to identify HIV inhibitors, and we initiated the development of an HTS-TR-FRET assay using the CTD of CA tagged with either GST (GST-CTD) or Flag (CTD-Flag) (Fig.…”

HIV Type 1 Gag as a Target for Antiviral Therapy

“…137 As mentioned above, a Pro-rich loop in the CA NTD binds the peptidyl-prolyl isomerase cyclophilin A. Early studies demonstrated that disruption of the CA-cyclophilin A interaction with cyclosporine inhibited HIV-1 replication.…”

“…Several molecules that disrupted in vitro CA assembly and virus infectivity resulted from this effort. 136 Debnath and co-workers also designed a dimer interface peptide, NYAD-201, containing the sequence AQEVKXW MTXTLLVA [based on the sequence of CA amino acids [178][179][180][181][182][183][184][185][186][187][188][189][190][191][192], where X represents the (S)-2-(2¢-pentenyl) alanines that are connected by hydrocarbon stapling 137 ]. This stapled peptide penetrates into cells and inhibits HIV-1 production by 3-fold.…”

Larger Helical Populations in Peptides Derived from the Dimerization Helix of the Capsid Protein of HIV-1 Results in Peptide Binding toward Regions Other than the “Hotspot” Interface

“…And second, we have assumed that the peptides dissociate before binding to CTD, but self-associated species could also bind to CTD, as recently shown in stapled peptides. 48 Thus, we can only indicate that the apparent K D values for the peptides designed in this work are smaller than those measured for CAC-1, 15 but we cannot provide a real value of such constants under the approaches used because of the difficulties in measuring the different thermodynamic constants for the concomitant equilibria.…”