Solution structures of long-acting insulin analogues and their complexes with albumin

Abstract

The lipidation of peptide drugs is one strategy to obtain extended half-lives, enabling once-daily or even less frequent injections for patients. The half-life extension results from a combination of self-association and association with human serum albumin (albumin). The self-association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration-dependent self-association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin-detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin-degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size-exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin-detemir and albumin-degludec mixtures are proposed.

Characterization of binding between model protein GA-Z and human serum albumin using asymmetrical flow field-flow fractionation and small angle X-ray scattering

“…For example, insulin-like peptides have shown considerably increase in half-life when lipidated [9]. Ryberg et al have shown that the tertiary structure of the HSA-lipidated peptide complex corresponds to hexamers, indicating that the structure of HSA can change as a consequence of API-binding [10].…”

Investigations of Albumin–Insulin Detemir Complexes Using Molecular Dynamics Simulations and Free Energy Calculations

“…The experimental details can be found in Table 1 and Table 2 as well as the Materials and methods section in Ryberg et al 2019. 22 SASREFCV 79,80 (SASREF) was used for rigid body modelling with standard settings for X-ray data and based on data up to q = 0.43 Å -1 . The input structures were the same albumin and detemir hexamer structures that were used for setting up the simulations.…”

“…The SAXS sample preparation and data collection were carried out as described in Ryberg et al 2019. 22 The samples were titrated with 0-100 mM NaCl. Data analysis and modelling were carried out using the ATSAS Program Package version 2.8.3 82 .…”

“…Recently, small-angle X-ray scattering (SAXS) solution structures of albumin-detemir/degludec complexes in 1:6, 1:12, and 2:12 (albumin:insulin analogue) stoichiometries were presented 22 . Due to the limited resolution of SAXS and the symmetric shape of albumin, an exact binding site for the two insulin analogues could not be identified.…”

Water Distribution and Clustering on the Lyophilized IgG1 Surface: Insight from Molecular Dynamics Simulations

“…Therapeutic proteins (biologics) provide important therapies for a variety of diseases, such as diabetes 1 , cancer 2 , infectious diseases 3 , hemophilia 4 , and anemia 5 . Although liquid formulations are usually preferred for injectable biologics, this form is not always feasible given the high molecular sensitivity of proteins to mechanical stresses, temperature, and other environmental factors that can lead to protein denaturation, aggregation, and degradation 6 .…”