Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors.

In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein-coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y1, Y2, and Y4 receptors in complex with NPY or PP, and the Gi1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y1 receptor, but not with the Y2 and Y4 receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.

The effect of fatty diacid acylation of human PYY3-36 on Y2 receptor potency and half-life in minipigs.

Peptides are notoriously known to display very short in vivo half-lives often measured in minutes which in many cases greatly reduces or eliminates sufficient in vivo efficacy. To obtain long half-lives allowing for up to once-weekly dosing regimen, fatty acid acylation (lipidation) have been used to non-covalently associate the peptide to serum albumin thus serving as a circulating depot. This approach is generally considered in the scientific and patent community as a standard approach to protract almost any given peptide. However, it is not trivial to prolong the half-life of peptides by lipidation and still maintain high potency and good formulation properties. Here we show that attaching a fatty acid to the obesity-drug relevant peptide PYY3-36 is not sufficient for long pharmacokinetics (PK), since the position in the backbone, but also type of fatty acid and linker strongly influences PK and potency. Furthermore, understanding the proteolytic stability of the backbone is key to obtain long half-lives by lipidation, since backbone cleavage still occurs while associated to albumin. Having identified a PYY analogue with a sufficient half-life, we show that in combination with a GLP-1 analogue, liraglutide, additional weight loss can be achieved in the obese minipig model.

Design of Y2 Receptor Selective and Proteolytically Stable PYY3-36 Analogues.

In recent years peptide YY (PYY) has attracted attention within the area of diabetes and obesity due to its involvement in food intake regulation and glucose homeostasis. It is well-known that PYY1-36 is rapidly cleaved by dipeptidyl peptidase-4 to the more Y2 receptor selective analogue PYY3-36, which is further cleaved to the inactive analogue PYY3-34. In order to improve the selectivity and proteolytic stability of the C-terminus, we synthesized several analogues incorporating N-methyl amino acids or ß-homo amino acids and other non-natural amino acids. These were tested against all four NPY receptors, and highly potent and Y2 receptor selective analogues were identified by combining a tryptophan residue in position 30 with either N-methyl or ß-homo arginine in position 35. We also identified an analogue with a MeGln34 substitution that surprisingly displayed high affinity toward all four receptors. In addition, these analogues displayed improved stability toward C-terminal proteolysis compared to native PYY3-36.

α-Helix or ß-Turn? An Investigation into N-Terminally Constrained Analogues of Glucagon-like Peptide 1 (GLP-1) and Exendin-4.

Peptide agonists acting on the glucagon-like peptide 1 receptor (GLP-1R) promote glucose-dependent insulin release and therefore represent important therapeutic agents for type 2 diabetes (T2D). Previous data indicated that an N-terminal type II ß-turn motif might be an important feature for agonists acting on the GLP-1R. In contrast, recent publications reporting the structure of the full-length GLP-1R have shown the N-terminus of receptor-bound agonists in an α-helical conformation. To reconcile these conflicting results, we prepared N-terminally constrained analogues of glucagon-like peptide 1 (GLP-1) and exendin-4 and evaluated their receptor affinity and functionality in vitro; we then examined their crystal structures in complex with the extracellular domain of the GLP-1R and used molecular modeling and molecular dynamics simulations for further investigations. We report that the peptides' N-termini in all determined crystal structures adopted a type II ß-turn conformation, but in vitro potency varied several thousand-fold across the series. Potency correlated better with α-helicity in our computational model, although we have found that the energy barrier between the two mentioned conformations is low in our most potent analogues and the flexibility of the N-terminus is highlighted by the dynamics simulations.

Metabolism of peptide YY 3-36 in Göttingen mini-pig and rhesus monkey.

Peptide YY 3-36-amide (PYY3-36) is a peptide hormone, which is known to decrease appetite and food-intake by activation of the Y2 receptor. The current studies were designed to identify the metabolites of PYY3-36 in mini-pig and rhesus monkey. Plasma samples were analyzed by high resolution LC-MS (and MS/MS) in order to unambiguously identify the metabolites of PYY3-36. In summary, the metabolism of PYY3-36 was similar in mini-pig and rhesus monkey. Several metabolites were identified and PYY3-34 was identified at the highest levels in plasma. In addition, mini-pigs were also dosed with PYY1-36-amide, PYY3-35, PYY3-34 and [N-methyl 34Q]-PYY3-36-amide in order to investigate the mechanisms by which PYY was metabolized. PYY3-35 was rapidly converted to PYY3-34 whereas dosing of PYY3-34 to mini-pigs only showed circulating degradation products at low levels, i.e., PYY3-34 was metabolically more stable than PYY3-36 and PYY3-35. [N-methyl 34Q]-PYY3-36-amide was hypothesized to be stable toward cleavage between 34Q and 35R and after i.v. administration to mini-pigs, one major cleavage product was identified as [N-methyl 34Q]-PYY3-35. Overall, this showed that cleavage between 35R and 36Y was possible as well as between 34Q and 35R (as shown for PYY3-35), which indicated that metabolism of PYY3-36 to PYY3-34 may be a two-step process. PYY1-36 was also dosed to mini-pigs, which showed that PYY1-36 was metabolized in the C-terminal as PYY3-36. The overall degradation pattern of PYY1-36 was more complex due to the simultaneous enzymatic degradation in the N-terminal to form PYY2-34/36 and PYY3-34/36. In vitro incubations with heparin stabilized plasma showed that PYY3-36 was degraded with a half-life of 175 min, whereas incubations with PYY3-35 (half-life of 6 min) showed a rapid formation of PYY3-34. In conclusion, the present studies showed that PYY3-36 underwent enzymatic degradation in the C-terminal part and that the major circulating metabolite was PYY3-34. Furthermore, it may be a sequential two-step process leading to the formation of PYY3-35 and subsequently the metabolically more stable PYY3-34.

Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide.

Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

Comparing dendritic with linear esterase peptides by screening SPOT arrays for catalysis.

Fluorescence screening of a 96-membered SPOT library of histidine containing dendritic and linear peptides revealed the remarkable esterolytic activity of short histidine oligomers that show catalytic proficiencies within one order of magnitude of histidine-containing esterase peptide dendrimers.

Identification of protease substrates by combinatorial profiling on TentaGel beads.

Reacting a 65,536 member combinatorial library of octapeptides on TentaGel beads with various proteases followed by selective staining of the free amino termini at the reacted bead surface and sequence determination by amino acid analysis allowed the rapid identification of protease substrates.

A general method for designing combinatorial peptide libraries decodable by amino acid analysis.

Herein we describe an algorithm for designing combinatorial peptide libraries for split-and-mix synthesis on solid support that are decodable by amino acid analysis (AAA) of the beads. AAA is a standard service analysis available in most biochemical laboratories, and it allows one to control the quality of the peptide on each bead, an important feature that is missing from most library decoding protocols. In the algorithm, each AA is assigned to two variable positions in the sequence grouped in a "unique pair". This arrangement limits sequence design because both the number of unique pairs U (setting the maximum number of variable AA) and the maximum number S of different AA per variable position depend on the peptide length N (U=N(N-1)/2), S=N-1). The method is therefore only suitable for focused libraries. An application example is shown for the selection of peptides with N-terminal proline or hydroxyproline catalyzing an aldol reaction from a combinatorial library of 65536 octapeptides. A simple enumeration program is available to help design combinatorial libraries decodable by amino acid analysis. The method applies to linear and cyclic peptides, can be used for nonnatural building blocks, including beta-amino acids, and should help to explore the vast chemistry of linear and cyclic peptide for catalysis and bioactivity.

Artificial aldolases from peptide dendrimer combinatorial libraries.

Peptide dendrimers were investigated as synthetic models for aldolase enzymes. Combinatorial libraries were prepared with aldolase active residues such as lysine and proline placed at the dendrimer core or near the surface. On-bead selection for aldolase activity was carried out using the dye-labelled 1,3-diketone 1a, suitable for covalent trapping of enamine-reactive side-chains, and the fluorogenic enolization probe 6. Aldolase dendrimers catalyzed the aldol reaction of acetone, dihydroxyacetone and cyclohexanone with nitrobenzaldehyde. Much like enzymes, the dendrimers exhibited strong aldolase activity in aqueous medium, but were also active in organic solvent. Dendrimer-catalyzed aldol reactions reached complete conversion in 3 h at 25 degrees C with 1 mol% catalyst and gave aldol products with up to 65% ee. A positive dendritic effect in catalysis was observed with both lysine and proline based aldolase dendrimer catalysts.

Dual mechanism of zinc-proline catalyzed aldol reactions in water.

The aldol reaction of acetone with aldehydes in aqueous medium under catalysis by zinc-proline (Zn(L-Pro)2) and secondary amines such as proline, (2S,4R)-4-hydroxyproline (Hyp) and (S)-(+)-1-(2-pyrrolidinomethyl)pyrrolidine (PMP) is shown to proceed by an enamine mechanism, as evidenced by reductive trapping of the iminium intermediate, while the aldol reaction of dihydroxyacetone (DHA) under catalysis by zinc-proline and by general bases such as N-methylmorpholine (NMM) is shown to occur under rate-limiting deprotonation of the alpha-carbon and formation of an enolate intermediate.

Dendrimers as artificial enzymes.

Dendrimers are regular tree-like macromolecules accessible by chemical synthesis from a variety of building blocks. Their topology enforces a globular shape that offers a unique opportunity to design artificial enzymes. Catalytic groups such as metal complexes and cofactors can be placed at the dendrimer core to exploit microenvironment and selectivity effects of the dendritic shell. In a second approach, attaching catalytic groups in multiple copies at the end of the dendritic branches may lead to cooperativity effects. Finally, exploration of dendritic structural space by screening combinatorial libraries of peptide dendrimers for catalytic activity can lead to discovery of functional dendrimers with enzyme-like properties, in a process mimicking natural selection.

Prebiotic carbohydrate synthesis: zinc-proline catalyzes direct aqueous aldol reactions of alpha-hydroxy aldehydes and ketones.

Zn-proline catalyzed aldolisation of glycoladehyde gave mainly tetroses whereas in the cross-aldolisation of glycoladehyde and rac-glyceraldehyde, pentoses accounted for 60% of the sugars formed with 20% of ribose.

Zinc-proline catalyzed pathway for the formation of sugars.

Zn-proline catalyzes the aldolisation of unprotected glycolaldehyde in water to give tetroses and hexoses; threose (33% of the product mixture) was formed with 10% enantiomeric excess of the D-isomer.

Discovery of new peptide-based catalysts for the direct asymmetric aldol reaction.

A series of oligo-peptide based catalysts were prepared using Fmoc solid-phase peptide synthesis. It was found that peptides with N-terminal proline residues catalyzed an aldol reaction yielding enantiomeric enriched product. Peptide H-Pro-Glu-Leu-Phe-OH catalyzed the reaction with good activity and moderate enantioselectivity (66% ee). Furthermore, it was shown that an acidic side chain and/or C-termini are essential to catalysis.