Aib(2)oxyntomodulin Peptide Molecular Self-Assembly: Smart Nanostructures Toward Long Acting Formulation for Obesity and Diabetes Type 2

Abstract

Type 2 diabetes mellitus (T2DM) and obesity are the most prevalent metabolic complications in the world. Previous studies have revealed that dual agonist gut hormone, oxyntomodulin (Oxm), has a promising beneficial role to treat obesity and T2DM through suppressing the food intake, raising the energy expenditure, and increasing insulin secretion to maintain the normal blood glucose level in the body. Nevertheless, due to the fast renal clearance of Oxm via proteolytic degradation, Oxm fibrils were explored and discovered to enhance Oxm bioactivity in vivo up to few days. Despite to the prolong bioactivity of Oxm fibrils in subcutaneous (s.c.) space, the plasma half-life of Oxm peptide, upon release from the fibril, was short in serum due to the proteolytic degradation by DPP4, which limits the Oxm biological role. Furthermore, previous study reported that an analogue of Oxm, aminoisobutyric acid (2) oxyntomodulin known as Aib2-Oxm, is resistant to DPP4 proteolytic degradation and enhances the plasma half-life bioactivity in serum. Despite to the promising potential of long-bioactive role of Aib2-Oxm fibrils in the body, the thoroughly identified kinetic, thermodynamic and mechanical parameters affecting the self-assembly process of Aib2-Oxm free peptides as well as the fundamental physical interactions underpinning self-assembly and controlling stability of their fibrillar structures remained to be resolved. A key element to completing our understanding of the physical interactions is the elucidation of the near-atomic level structure of the fibrils and the determination of their mechanical properties. In this PhD study I studied the detailed mechanical and structural characterization of the individual fibrils using atomic force microscopy (AFM) and cryogenic electron microscopy (cryo-EM). The reversibly self-assembled multi-filament fibrils, formed by free Oxm and Aib2-Oxm, were shown to have a mechanical stiffness, in terms of Young's modulus of elasticity, of 0.6 - 1.7 GPa, comparable to those of natural assemblies found within cells, such as actin and tubulin (0.3 - 1.2 GPa), which were previously found to be the reversible self-assembled fibrils, in vivo. This finding supports the possibility of the controlled dissociation mechanism of Oxm and Aib2-Oxm fibrils, in vivo. Moreover, the comparisons of elastic moduli of Oxm and Aib2-Oxm fibrils with those of previously studied materials signify the fact that both dense hydrogen-bonding network and amphiphilic (i.e., hydrophobic and hydrophilic) interactions are comparably responsible for stability of Oxm and Aib2-Oxm fibrils. By combining data from both AFM and cryo-EM images, alongside modelling based on semiflexible polymer theory, we show that Oxm and Aib2-Oxm fibrils exhibit ribbon-like multifilament structures as opposed to the closely packed multifilament structures as previously speculated. In order to understand the fibrillation and dissociation processes of Oxm and Aib2-Oxm, a through quantitative knowledge of the kinetics and thermodynamics of fibril formation and dis-assembly were conducted. Here, I reported for the first time an extensive thermodynamic and kinetic study of fibril formation and dissociation using the quartz crystal microbalance with dissipation (QCM-D) and bulk experiments for Oxm and Aib2-Oxm. I investigated the speed of peptide binding and detachment as well as the related thermodynamic processes via the Gibbs free energy change for the fibril formation and dissociation. My study showed that fibril formation of Oxm and Aib2-Oxm are both exothermic (i.e., spontaneous process) as the Gibbs free energy change associated with them is negative due to the peptide binding whilst Oxm fibrils are more thermodynamically stable than Aib2-Oxm fibrils due to their higher Gibbs free energy loss which is attributed to the larger α-helix contents in the secondary structure of Aib2-Oxm fibrils as compared to Oxm fibrils. Moreover, the speed at which Oxm seeds elongation is higher than Aib2-Oxm seed elongation rate which is related to the larger amount of required Gibbs free energy of activation for Aib2-Oxm peptide unfolding for nucleation as compared to Oxm peptide. The findings acquired from kinetics and thermodynamics of fibril formation indicated the higher propensity of Oxm peptide for fibril elongation than Aib2-Oxm. Moreover, I have found that Aib2-Oxm fibrils are thermodynamically less stable than Oxm fibrils which proves that Aib2-Oxm fibrils are more feasible and faster to dissociate in vivo relative to Oxm fibrils and signifies that Aib2-Oxm exhibits much higher bioactivity in serum relative to Oxm due to the fact that Aib2-Oxm fibrils might send more free peptides inside the serum for a given time as compared to Oxm fibrils. Furthermore, in order to find a systematic approach for Aib2-Oxm fibril formation in vitro as well as Aib2-Oxm fibril dissociation in vivo I examined Aib2-Oxm peptide-seed binding and the peptide release from fibrils via considering three parameters: temperature, concentration and size of pre-formed seeds. In this Ph.D. study, I reported a comprehensive knowledge on kinetics and thermodynamics of Aib2-Oxm fibril elongation and dissociation along with understanding the mechanics and structures of mature Aib2-Oxm fibrils under influence of the three parameters mentioned earlier. Regardless of physical conditions (varying temperature, concentration and size of pre-formed fibril seeds) over which Aib2-Oxm fibrils were prepared, they all exhibit helical and twisted structures with similar materials strength under influence of same forces: hydrogen bonds and hydrophobic-hydrophilic interactions; and provide a promising potential of in vivo reversibility in human body via comparing their materials compatibility with naturally self-assembled fibrils in human cells. Moreover, Aib2-Oxm fibrillation follows Arrhenius behavior, as opposed by its analogue, Oxm. Detailed analysis of thermally driven Aib2-Oxm fibril elongation by QCM-D revealed that the temperature rise from 23 to 37 oC induced twice more probable Aib2-Oxm peptide-seed binding. Similarly, Aib2-Oxm peptide release from the Aib2-Oxm fibril enhances with temperature rise with lower release rate in subcutaneous tissues (32 oC) than other physiological temperatures (i.e., 37 and 42 oC) which indicates strong potential of long-lasting therapeutic effects of Aib2-Oxm peptides via subcutaneous injection in patients.