Structure-Stability-Function Mechanistic Links in the Anti-Measles Virus Action of Tocopherol-Derivatized Peptide Nanoparticles

Abstract

Measles remains one of the leading causes of child mortality worldwide and is re-emerging in some countries due to poor vaccine coverage, concomitant with importation of measles virus (MV) from endemic areas. The lack of specific chemotherapy contributes to negative outcomes, especially in infants or immunodeficient individuals. Fusion inhibitor peptides derived from the MV Fusion protein C-terminal Heptad Repeat (HRC) targeting MV envelope fusion glycoproteins block infection at the stage of entry into host cells, thus preventing viral multiplication. To improve efficacy of such entry inhibitors, we have modified a HRC peptide inhibitor by introducing properties of self-assembly into nanoparticles (NP) and higher affinity for both viral and cell membranes. Modification of the peptide consisted of covalent grafting with tocopherol to increase amphipathicity and lipophilicity (HRC5). One additional peptide inhibitor consisting of a peptide dimer grafted to tocopherol was also used (HRC6). Spectroscopic, imaging, and simulation techniques were used to characterize the NP and explore the molecular basis for their antiviral efficacy. HRC5 forms micellar stable NP while HRC6 aggregates into amorphous, loose, unstable NP. Interpeptide cluster bridging governs NP assembly into dynamic metastable states. The results are consistent with the conclusion that the improved efficacy of HRC6 relative to HRC5 can be attributed to NP instability, which leads to more extensive partition to target membranes and binding to viral target proteins.

Keywords: antiviral; fusion inhibitor; measles virus; metastable; nanoparticle; peptide; self-assembling.

Figure 1.

HRC5 and HRC6 NP morphology imaged using TEM and AFM. (A) TEM images of deposited HRC5 and HRC6 peptides (30 μM) self-assembled NP. NP selected for measurement are highlighted in 1 × 1 (HRC5) and 2 × 2 μm² (HRC6) zoomed images by a white arrow. The measured NP longest axis are highlighted by a white line in individual zoomed insets (a−f). (B) AFM topographic imaging of deposited HRC6 (30 μM) self-assembled NP. The analysis of HRC5 (30 μM) and control images are included in Figure S3. (C) Height profiles of selected linear sections a−c, highlighted in B (left); NP Feret’s radius frequency distribution, grouped in 12.5 nm intervals (right). Error bars correspond the standard deviation of the mean from three independent experiments. (D) Abnormal HRC6 NP morphologies detected by AFM. Representative height images emphasizing uncharacteristic HRC6 NP clusters, respective 3D projections and height profiles of selected linear sections, highlighted in black, are presented for three independent cases. h, height.

Figure 2.

HRC5 and HRC6 NP time-dependent size distributions. Time-resolved DLS measurements of HRC5 (A−C) and HRC6 (D−F) NP. Number-averaged size distribution profiles (A and D) of each sample (30 μM), collected for a 9 h period (0, 3, 6, and 9 h profiles are presented), were used to determine the respective average R_H (B and E) and sample PDI (C and F) values, plotted for each time point. The dashed line is a guide to the eye. R_H and PDI data sets are the average of three independent replicates. Error bars correspond to the standard deviation of the mean.

Figure 3.

Biophysical characterization of HRC5 and HRC6 NP structure. (A) HRC5 and HRC6 CMC determination through the pyrene 3:1 peak ratio method: pyrene (5 μM) fluorescence emission intensity ratio I₃/I₁ (I₃, 383 nm; I₁, 372 nm) was plotted as a function of the peptide concentration (0.03–30 μM). Lines correspond to linear regressions fitted to the experimental data regimes in the presence (high slope) and absence (low slope) of pyrene excimers. CMC is the x-axis value at the intersecion. Presented data sets are one of three independent replicates. (B) HRC5 and HRC6 NP internal accessibility probed by ANS fluorescence quenching. Stern−Volmer plots of ANS (12.8 μM) fluorescence emission intensity while inserted in HRC5 or HRC6 NP (30 μM), quenched by increasing acrylamide concentrations (0–600 mM). Lines correspond to the best fit of the Stern−Volmer equation (eq 1) to one of three independent replicates for each peptide. (C) HRC1, HRC5, and HRC6 peptide (30 μM) secondary structure in aqueous solution evaluated through CD spectroscopy. HRC1 was studied in aqueous solution and in the presence of 20% (v/v) TFE. Peptide mean residue ellipticity values ([θ]) were calculated according to eq 4. Presented spectra are one of three independent replicates. (D) HRC5 and HRC6 NP ζ-potential. Independent replicates correspond to the average of 15 measurements performed for each peptide sample (30 μM). Error bars correspond to the standard deviation of the mean.

Figure 4.

CG MD simulations of HRC5 and HRC6 assembly into NP. Coarse-grained molecular dynamics simulations of 8 HRC5 or HRC6 peptides, followed for 10 μs. (A) Representative image of the peptide models and most characteristic peptide assembling behavior. The peptide chain backbone is represented in red, the PEG₄ linker in gray, and the Toc moiety in yellow (all other system components were omitted for clarity). Black arrows indicate different association clusters. (B) Stills of each peptide simulation, captured at 0, 2, 4, 6, 8, and 10 μs, collected to emphasize the progression of the peptides assembly during the experiment. (C) Preferential contact maps reporting favored interaction regions between peptides, built from full simulation data. For HRC5, the CG bead numbers correspond to peptide chain −0 to 80; PEG₄ linker −81 to 89; and Toc −90 to 95. For HRC6, CG bead numbers are the same as those for HRC5, with the second peptide chain and PEG₄ linker corresponding to beads 96 through 180. Darker tones in the contact map gradient scale indicate a higher number of detected contacts. For both HRC5 and HRC6, Toc−Toc interactions can be seen to predominate; additional Toc−peptide interactions are visible for HRC6 but not for HRC5.

Figure 5.

Schematic simplified representation of the effect of peptide dimerization in HRC5 and HRC6 NP structural dynamics. (A) Peptide dimerization impacts HRC5 and HRC6 NP internal and surface-related properties. NP formed by HRC5 monomer peptides are small, tightly packed micellar structures. Because internal accessibility to polar solvents is low, HRC5 NP core is expected to be considerably hydrophobic. HRC6 forms larger and looser NP deprived of a well-organized internal architecture. The internal space is permeable to polar molecules, which translates into a more hydrophilic interior. Dimerization introduces repulsion between the two peptide chains and partial loss of extended (helical) conformation of the peptide causing high internal steric hindrance to ordered packing. (B) Stable HRC5 NP collide elastically with very low impact in the overall particle structure (size and dispersity are constant); in distinction, HRC6 NP collisions are more inelastic, leading to the formation of larger aggregated clusters, over time (increase in size and polydispersion). The metastable behavior correlates with the peptides mode of action, as a determining factor for efficacy.