Exploiting natural, phylogenetically ancient, PYY peptides in the development of long-acting, NPYR-specific analogues for obesity-diabetes

Abstract

PYY has long been accepted to play a role in energy regulation, with administration of the N-terminally-cleaved PYY(3-36) being demonstrated to reduce food intake in several species, via NPY2R interactions. Ablation of PYY expressing cells was shown to lead to loss of pancreatic insulin, resulting in hyperglycaemia, which was rescued by administration of intact PYY(1-36); highlighting the importance of NPY1R interactions. Additionally, raised PYY levels following Roux-en-Y gastric bypass indicate T2DM remission post-surgery may be mediated by PYY, suggesting PYY-based therapies may restore pancreatic islet function.
The present study aimed to develop NPY1R-specific analogues, resistant to DPP-4-mediated, N-terminal cleavage, retaining NPY1R agonism. Initially, (P3L31P34)PYY(1-36) and PYY(1-36)(Lys12PAL) were developed. While intelligent design produced an N-terminally stable peptide in (P3L31P34)PYY(1-36), both analogues underperformed against human PYY(1-36) when tested in in vitro systems of insulin release and beta-cell survival. Thus, a different approach was taken, and PYY peptides previously sequenced in phylogenetically-ancient fish were synthesised and tested in a series of in vitro and acute in vivo scenarios. While all piscine PYY peptides were resistant to DPP-4, they also maintained positive pancreatic functions on beta-cell rest and survival which were similar to, or superior than that of, human PYY(1-36), with these effects confirmed to be mediated primarily by the NPY1R via the use of NPYR-KO cell-lines. Following promising in vitro and acute in vivo data, sturgeon and sea lamprey PYY(1-36) peptides were taken into a chronic in vivo setting. Intraperitoneal administration of sea lamprey PYY(1-36) was able to significantly improve the condition of STZ-induced diabetic mice decreasing fluid intake, non-fasting glucose and glucagon. Glucose tolerance, insulin sensitivity, pancreatic insulin and glucagon content were also significantly improved. Metabolic benefits were linked to positive changes in pancreatic islet morphology, as a result of augmented proliferation and decreased apoptosis of beta-cells. Thus, sea lamprey PYY(1-36) was highlighted as a promising therapeutic lead.
Recently, another important truncation of PYY peptides was discovered, involving inactivation of PYY(3-36) via the removal of a C-terminal dipeptide. The present data confirmed that this truncation not only ameliorates effects on appetite, but also ablates the positive effects of PYY(1-36) at pancreatic NPY1Rs. Importantly, sea lamprey and human PYY(1-36) share remarkable sequence homology at these C-terminal residues and are thus, equally susceptible to this inactivation. Therefore, C-terminally resistant analogues, NPY1R agonist (D-Arg35)-sea lamprey, and the NPY1/2R dual-agonist [Iso3](D-Arg35)-Sea lamprey were developed. Both were resistant to C-terminal degradation, whilst also retaining functionality at target receptors. Additionally, while (D-Arg35)-sea lamprey was able to delay the progression of STZ-induced diabetes compared to the unaltered peptide and [Iso3](D-Arg35)-sea lamprey was able to elicit clinically significant reductions in body weight in HFF mice, the end-points for both studies proved that there was not much difference between the C-terminally stabilised and unaltered forms.
When taken together, these studies reveal new lessons in the development of PYY analogues, which may ultimately benefit the development of a PYY-based therapy for the treatment of obesity-diabetes.

Date of Award	Mar 2020
Original language	English
Sponsors	Department for the Economy
Supervisor	Peter Flatt (Supervisor) & Nigel Irwin (Supervisor)