A Spectroscopic and Molecular Dynamics Study on the Aggregation Properties of a Lipopeptide Analogue of Liraglutide, a Therapeutic Peptide against Diabetes Type 2

Abstract

The pharmacokinetics of peptide drugs are strongly affected by their aggregation properties and the morphology of the nanostructures they form in their native state as well as in their therapeutic formulation. In this contribution, we analyze the aggregation properties of a Liraglutide analogue (LG18), a leading drug against diabetes type 2. LG18 is a lipopeptide characterized by the functionalization of a lysine residue (K26) with an 18C lipid chain. To this end, spectroscopic experiments, dynamic light scattering measurements, and molecular dynamics simulations were carried out, following the evolution of the aggregation process from the small LG18 clusters formed at sub-micromolar concentrations to the mesoscopic aggregates formed by aged micromolar solutions. The critical aggregation concentration of LG18 in water (pH = 8) was found to amount to 4.3 μM, as assessed by the pyrene fluorescence assay. MD simulations showed that the LG18 nanostructures are formed by tetramer building blocks that, at longer times, self-assemble to form micrometric supramolecular architectures.

Keywords: liraglutide analogue; molecular dynamics simulations; peptide aggregation; peptide nanostructures; therapeutic peptides.

Figure 1

Molecular formula of the Liraglutide analogue investigated (LG18). The lipid chain, functionalizing the K26 residue, comprises 18 C atoms. The Y and W fluorescent residues are shown in red.

Figure 2

Circular dichroism spectra of LG18. (A) 2 μM; red: just prepared; black: after two weeks; (B) 10 μM; blue: just prepared; black: after two weeks.

Figure 3

(A) Emission spectra of LG18 at λ_ex = 280 (red line) and 295 (blue line) nm; (B) two-dimensional excitation/emission spectrum of LG18 (PBS, pH 8.1).

Figure 4

UV-vis absorption (A) and emission (B) spectra of LG18 (21 μM, pH = 8.1) at different times. Red: freshly prepared solution. Blue: after two weeks. The emission spectra were normalized by the absorbance at the excitation wavelength (295 nm).

Figure 5

Fluorescence spectra of one month aged solutions of 10 μM LG18. (A) Emission spectrum (λ_ex = 350 nm). The sharp peak at 395 nm is the Raman band of the solvent when excited at 350 nm. (B) Fluorescence excitation spectrum (λ_em = 450 nm).

Figure 6

Diameter size distribution (nm) of LG18 aggregates as obtained by DLS experiments on LG18 aged (one month) solutions. (A) 2 μM; (B) 10 μM.

Figure 7

Ratio of the first and third vibronic components, i.e., I₁/I₃, of the 360–420 pyrene emission band as a function of micromolar LG18 concentrations (semi-log plot). The red and blue dots refer to the transition from a polar to an apolar environment, respectively.

Figure 8

Structure of the LG18 monomer (A) and dimer (B) after 100 ns. Hydrophobic, negatively charged, and positively charged residues are shown in green, red, and blue colors, respectively. The K26m side chain is yellow.

Figure 9

(A) Side and (B) top views of the aggregate formed by four LG18 units after 100 ns. Hydrophobic, negatively charged, and positively charged residues are shown in green, red, and blue colors, respectively. The K26m side chain is yellow. For the sake of clarity, on B only the backbones and the K26m side chains are shown.