Solution structure of glucagon-like peptide-1 analogues and their interactions with the endogenous receptor and albumin

Glucagon-like peptide-1 (GLP-1) is a hormone peptide, which is secreted to the bloodstream upon glucose uptake through nutrients. GLP-1 has one target, the GLP-1 receptor (GLP-1R), which is present in many different places in the body, e.g. the pancreas, heart, gut, and brain. Here, GLP-1 binding will activate GLP-1R and stimulate tissue specific signalling.

In the pancreas, GLP-1 binding causes glucose dependent insulin secretion, which is relevant in relation to treatment of type 2 diabetes mellitus. In addition GLP-1 has the property to decrease bodyweight, and indications point to an ability to prevent cardiovascular disease, and protecting against dementia.

However, GLP-1 immediatly suffers from its short half-life, making it inappropriate as a drug without sufficient modifications. Even though such modifications will in fact increase half-life, it is important that the biological effect of GLP-1R–GLP-1 binding is maintained. Several strategies have been developed to overcome the short half-life of GLP-1.

One such approach is acylation with fatty acid (FA) chains, promoting self-association and interactions to human serum albumin (HSA), which in turn will increase the systemic half-life. However, little is known about the atomic interactions of the selfassociating oligomers and the effects of different acylation schemes hereon.

Nor is it known how acylation of GLP-1 influences the interactions with HSA. In addition, only little knowlegde exists on GLP-1R–GLP-1 interactions on an atomic level, and none exists on interactions of GLP-1R with acylated GLP-1 analogues. In this work, the solution structures of 11 different GLP-1 analogues, along with their interactions to HSA, were investigated.

Assymetric flow-field-flow fractioning followed by light scattering was used to give insight into both oligomerisation and HSA interactions. The result showed that oligomer size is directly dependent on the length of the attached FA chain as well as the ionic strength, both with positive correlations.

In addition, it was also shown that the presence of a linker decrease the oligomeric state. With regards to HSA interactions, acylated peptides containing medium to long FA chains and linkers interact partially with HSA, wherease peptides containing the same FA chains, but no linkers, interact fully with HSA.

This thesis also presents studies of binding interactions between eight different acylated GLP-1 analogues and GLP-1R by use of in silio modelling. To compare the results with experimental studies, molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) binding energies were calculated, and a methodology for computing entropy values was developed.

The resulting MM-PBSA binding energies correlated with potency measurements. Additionaly, the computational studies could disclose that the acylation site on GLP-1 highly affects binding interactions between the FA chain and the receptor extracellular domain (ECD). If acylation is placed on GLP-1 Lys26 and Lys38, the FA chain can freely interact with the ECD, whereas, if acylation is placed on GLP-1 Lys34, the FA chain will be pointing away from the hydrophobic patch of the ECD.

If there are two acylation sites, on GLP-1 Lys26 and Lys34, the two FA chains can interact with each other rather than the ECD.