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Rational Development of Stable PYY3–36 Peptide Y2 Receptor Agonists

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Abstract

Purpose

The anorectic effect of PYY3–36 makes it a potential pharmacological weight loss treatment. Modifications of the endogenous peptide to obtain commercially attractive pharmacological and biophysical stability properties are examined.

Methods

Half-life extended PYY3–36 analogues were prepared and examined regarding Y2-receptor potency as well as biophysical and stability properties.

Results

Deamidation of asparagine in position 18 and 29 was observed upon incubation at 37°C. Asparagine in position 18 – but not position 29 – could be substituted to glutamine without detrimental effects on Y2-receptor potency. Covalent dimers were formed via the phenol impurity benzoquinone reacting with two N-terminal residues (Isoleucine-Lysine). Both residues had to be modified to suppress dimerization, which could be done without negatively affecting Y2-receptor potency or other stability/biophysical properties. Introduction of half-life extending modifications in position 30 and 35 eliminated aggregation at 37°C without negatively affecting other stability properties. Placement of a protracting moiety (fatty acid) in the receptor-binding C-terminal region reduced Y2-receptor potency substantially, whereas only minor effects of protractor position were observed on structural, biophysical or stability properties. Lipidated PYY3–36 analogues formed oligomers of various sizes depending on primary structure and solution conditions.

Conclusions

By rational design, a chemically and physically stable Y2-receptor selective, half-life extended PYY3–36 peptide has been developed.

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Abbreviations

2D-LC-MS:

2D liquid chromatography mass spectroscopy

3-Mba:

3-methylbutanoic acid

Ado:

8-amino-3,6-dioxaoctanoic acid

AUC:

Analytical ultracentrifugation

Bis-AgGPSA:

Bis-aminodietoxyacetyl gamma glutamate palmitic sulphonic acid

CD:

Circular dichroism

DLS:

Dynamic light scattering

HMWP:

High molecular weight protein

HPLC:

High performance liquid chromatography

NmeArg:

N-methyl-arginine

Ph Eur:

European Pharmacopoeia

PYY:

Peptide YY

SEC:

Size exclusion chromatography

SLS:

Static light scattering

ThT:

Thioflavin T

UPLC:

Ultra-performance liquid chromatography

USP:

United States Pharmacopoeia

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Authors and Affiliations

  1. Global Research Technology, Novo Nordisk A/S, Måløv, Denmark

    Christian Poulsen, Marie Østergaard Pedersen, Per-Olof Wahlund, Annika Sjölander, Kilian W. Conde-Frieboes & Søren Østergaard

  2. Hellerup, Denmark

    Jens Kaalby Thomsen

  3. Global Drug Discovery, Novo Nordisk A/S, Måløv, Denmark

    Johan F Paulsson & Birgitte S Wulff

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  1. Christian Poulsen
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Poulsen, C., Pedersen, M.Ø., Wahlund, PO. et al. Rational Development of Stable PYY3–36 Peptide Y2 Receptor Agonists. Pharm Res 38, 1369–1385 (2021). https://doi.org/10.1007/s11095-021-03077-x

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  • DOI: https://doi.org/10.1007/s11095-021-03077-x

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