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Keywords:

  • peptide modifications;
  • peptide YY;
  • protein ligation;
  • solid-phase peptide synthesis;
  • structure–activity relationships

Abstract

The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N-terminal truncated version PYY(3–36) decreases food intake and has potential as an anti-obesity agent. The anorectic effect of PYY(3–36) is mediated through Y2 receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C-terminal tetrapeptide sequence of PYY(3–36) is crucial for Y2 receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C-terminally modified PYY(3–36) analogues. By using an intein-based expression system, PYY(3–29) was generated as a C-terminal peptide α-thioester. Heptapeptides bearing an N-terminal cysteine and modifications at one of the four C-terminal positions were synthesized in a 96-well plate by parallel solid-phase synthesis. In the plate format, an array of [Ala30]PYY(3–36) analogues were generated by ligation, desulfurization, and subsequent solid-phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non-proteinogenic amino acids, were tested in a functional Y2 receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y2 receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.