Complex formation between albumin and long-acting insulin analogues : A Small-Angle X-ray Scattering and Molecular Dynamics Study

Abstract

The use of biopharmaceuticals in the treatment of diseases such as diabetes, cancer, and hemophilia has increased dramatically over the past decades. Despite their many advantages such as high potency, specificity, and low toxicity, many biopharmaceuticals suffer from inherent chemical and physical instabilities and short plasma half-lives, which make their formulation development and delivery challenging. Lipidation is a successful strategy for extending the half-lives of peptide drugs through lipidation-induced selfassociation and association to albumin. Though albumin association is exploited by several approved lipidated peptide drugs, structural knowledge about the albumin-peptide complexes formed and their interactions on the atomic level is limited. This thesis aims to shed light on self-association and albumin-association of two lipidated insulin analogues, insulin detemir and insulin degludec, through an interdisciplinary approach using smallangle X-ray scattering (SAXS) and molecular dynamics (MD) simulations. We succeeded in modelling the solution structures of a detemir trihexamer, and albumininsulin analogue complexes in 1:6, 1:12, and 2:12 stoichiometries based on SAXS data, and proposed equilibria for albumin-detemir and albumin-degludec mixtures. The structures are the first detemir trihexamer structure and the first structures of complexes between albumin and lipidated insulin analogues, and contribute to an understanding of detemir and degludec’s prolonged actions. The albumin-detemir hexamer solution structure is ambiguous and shows four possible detemir binding sites. In order to determine the most favorable binding site and obtain knowledge on the specific interactions in the complex, these binding sites were investigated by MD simulations and molecular mechanics Poisson-Boltzmann surface area free energy calculations. The overlapping FA3-FA4 binding site on albumin was found to be the most favorable detemir binding site, and two lipidated detemir residues were found to contribute to the binding with favorable electrostatic and van der Waals interactions. The atomic-level insights on the albumin-detemir binding could be utilized in a more rational design of future lipidated peptide drugs. The study, furthermore, highlights the strength of combining SAXS with MD simulations. The effect of albumin-detemir association on detemir’s stability was explored through different stress tests to investigate whether albumin-association could potentially be utilized in a formulation perspective. The presence of albumin was found to enhance detemir’s stability against freeze-thaw and agitation stresses almost independently on complex formation, suggesting that albumin-detemir complex formation does not lead to further stabilization.