The glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1, liraglutide has an added fatty acid (FA) moiety that causes oligomerization of liraglutide as suggested by small-angle x-ray scattering (SAXS) and multiangle static light scattering (MALS) results. SAXS data suggested a global shape of a hollow elliptical cylinder of size hexa-, hepta-, or octamer, whereas MALS data indicate a hexamer. To elaborate further on the stability of these oligomers and the role of the FA chains, a series of molecular-dynamics simulations were carried out on 11 different hexa-, hepta-, and octameric systems. Our results indicate that interactions of the fatty acid chains contribute noticeably to the stabilization. The simulation results indicate that the heptamer with paired FA chains is the most stable oligomer when compared to the 10 other investigated structures. Theoretical SAXS curves extracted from the simulations qualitatively agree with the experimentally determined SAXS curves supporting the view that liraglutide forms heptamers in solution. In agreement with the SAXS data, the heptamer forms a water-filled oligomer of elliptical cylindrical shape.

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In the present study, small-angle X-ray scattering (SAXS) and static light scattering (SLS) have been used to study the solution properties and self-interaction of recombinant human serum albumin (rHSA) molecules in three pharmaceutically relevant buffer systems. Measurements are carried out up to high protein concentrations and as a function of ionic strength by adding sodium chloride to probe the role of electrostatic interactions. The effective structure factors (Seff) as a function of the scattering vector magnitude q have been extracted from the scattering profiles and fit to the solution of the Ornstein–Zernike equation using a screened Yukawa potential to describe the double-layer force. Although only a limited q range is used, accurate fits required including an electrostatic repulsion element in the model at low ionic strength, while only a hard sphere model with a tunable diameter is necessary for fitting to high-ionic-strength data. The fit values of net charge agree with available data from potentiometric titrations. Osmotic compressibility data obtained by extrapolating the SAXS profiles or directly from SLS measurements has been fit to a 10-term virial expansion for hard spheres and an equation of state for hard biaxial ellipsoids. We show that modeling rHSA as an ellipsoid, rather than a sphere, provides a much more accurate fit for the thermodynamic data over the entire concentration range. Osmotic virial coefficient data, derived at low protein concentration, can be used to parameterize the model for predicting the behavior up to concentrations as high as 450 g/L. The findings are especially important for the biopharmaceutical sector, which require approaches for predicting concentrated protein solution behavior using minimal sample consumption.

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though GLP-1 is interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of <3 min limits its clinical use. A strategy for extending the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days because of its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants, one with increased and one with decreased binding affinity for human FcRn. We have investigated the impact of conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1-albumin conjugates. The solution structures, determined by small-angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures with the available structural information about the FcRn and GLP-1 receptor obtained from X-ray crystallography, we can explain the observed in vitro and in vivo behavior. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease in the potency of GLP-1 can be explained by a steric hindrance of binding of GLP-1 to its receptor.

Protein amyloid fibrillation is obtaining much focus because it is connected with amyloid-related human diseases such as Alzheimer's disease, diabetes mellitus type 2, or Parkinson's disease. The influence of metal ions on the fibrillation process and whether it is implemented in the amyloid fibrils has been debated for some years. We have therefore investigated the influence and binding geometry of zinc in fibrillated insulin using extended X-ray absorption fine-structure and X-ray absorption near-edge structure spectroscopy. The results were validated with fibre diffraction, Transmission Electron Microscopy and Thioflavin T fluorescence measurements. It is well-known that Zn ions coordinate and stabilize the hexameric forms of insulin. However, this study is the first to show that zinc indeed binds to the insulin fibrils. Furthermore, zinc influences the kinetics and the morphology of the fibrils. It also shows that zinc coordinates to histidine residues in an environment, which is similar to the coordination seen in the insulin R hexamers, where three histidine residues and a chloride ion is coordinating the zinc.