Position and length of fatty acids strongly affect receptor selectivity pattern of human pancreatic polypeptide analogues

Abstract

Pancreatic polypeptide (PP) is a satiety-inducing gut hormone targeting predominantly the Y4 receptor within the neuropeptide Y multiligand/multireceptor family. Palmitoylated PP-based ligands have already been reported to exert prolonged satiety-inducing effects in animal models. Here, we suggest that other lipidation sites and different fatty acid chain lengths may affect receptor selectivity and metabolic stability. Activity tests revealed significantly enhanced potency of long fatty acid conjugates on all four Y receptors with a preference of position 22 over 30 at Y1 , Y2 and Y5 receptors. Improved Y receptor selectivity was observed for two short fatty acid analogues. Moreover, [K(30)(E-Prop)]hPP2-36 (15) displayed enhanced stability in blood plasma and liver homogenates. Thus, short chain lipidation of hPP at key residue 30 is a promising approach for anti-obesity therapy because of maintained selectivity and a sixfold increased plasma half-life.

Keywords: lipidation; pancreatic polypeptide; receptors; selectivity; therapeutic peptides.

Figure 1

a) Amino acid sequence of hPP; residues to be modified are underlined. b) Side view of the comparative model of human Y₄R (blue) with docked bovine PP (red; PDB code: 1LJV);^[21b] lipidated positions are indicated in green.^[17] c) Enlarged section of the same model obtained by horizontal rotation by approximately 90°; lipidated residues Ala²² and Met³⁰ appear green.

Figure 2

a) First evaluation of the binding capacity by 1 μm CF-labeled hPP analogues 1b, 2–6 in transiently hY₄R-expressing COS-7 cells towards [³H]-hPP after a 90 min stimulation period. Total binding was set to 100% and corresponds to binding of radioligand in presence of H₂O in 1 % (w/v) BSA. Statistical analysis was performed by one-way ANOVA, followed by Dunnett's post-hoc test (GraphPad Prism 5.0). ***P ≤ 0.001, as compared to 1b (horizontal lines). b) For competition binding experiments, [³H]-hPP was displaced by increasing concentrations of most promising analogues 1, 3, 5 and 6. Assays were performed in triplicate; mean values ±SEM of independently examined experiments are shown.

Figure 3

Functional characterization of hPP compounds fatty acid acylated at a) position 22 and b) residue 30 at COS-7 cells stably expressing the anorexigenic hY₄R and hY₂R as well as the orexigenic hY₁R and hY₅R. Co-expression of hYR and a chimeric G_i/q protein allowed concentration-dependent radioactive inositol phosphate accumulation. Mean concentration–response curves of at least two independent experiments, fitted by non-linear regression (GraphPad Prism 5.0) are shown with ±SEM. Dashed black lines correspond to the respective native ligands (hPP for hY₄R and porcine NPY (pNPY) for hY₁R, hY₂R and hY₅R).

Figure 4

In vitro stability tests of TAMRA-labeled hY₄R-selective lipidated hPP conjugates (15b, 16b) along with the Pam-variant (18b) and relevant control peptides (14b, 20) performed in a) human blood plasma and b) 50 mgmL⁻¹ porcine liver extract homogenates. 10 μm peptide solutions were incubated at 37°C and 500 rpm. Degradation was followed using RP-HPLC at indicated time points by peptide-specific fluorescence monitoring and referred to control at 0 h (100%). Values are the mean ±SEM of two independent experiments.

Scheme 1

Synthesis of a) [K(E-Lip)]hPP_2–36 and b) TAMRA-[K(E-Lip)]hPP_2–36. Peptides were assembled by automated SPPS up to modification site (22 or 30), substituted with Lys (grey) that was protected by Fmoc at the γ-amino group and by Dde at the N terminus. 1) Fmoc removal and coupling of Fmoc-l-Glu-OtBu. 2) Fmoc deprotection and acylation with fatty acids (Lip): C₂H₅COOH (Prop) for 9, 15a, 15b; C₇H₁₅COOH (Capr) for 10, 16a, 16b; C₁₁H₂₃COOH (Laur) for 11, 17; C₁₅H₃₁COOH (Pam) for 12, 18a, 18b; C₁₉H₃9COOH (Ara) for 13, 19. 3) Dde removal and automated elongation to desired peptide sequence. 4) TAMRA-labeling of peptide N terminus. 5) Cleavage from resin including deprotection of all acid-labile side chain protecting groups (SPG) and methionine reduction.