Identification of key Y₄R residues enables the discovery of selective non-peptide small-molecule agonists

Reviewer #1 (Public review):

Summary:

This manuscript describes an investigation of peptide analogue agonists selective for the human Y4 receptor for pancreatic polypeptide over Y1, Y2 and Y5 receptors. After studies of mutated Y4R in transiently transfected COS-7 cells, binding models were calculated. Then, screening of a virtual library identified three non-peptidergic (albeit somewhat peptide-like) compounds with potential agonist activity that were subsequently confirmed and furthermore were found to have receptor interactions similar to the peptide analogues. This study provides fundamental new information that improves understanding of the Y4R structure and mechanism of activation by the native agonist and the selective peptide analogues. The non-peptide agonists have potential for future pharmacotherapy.

Strengths:

All of the experiments seem to be well performed, using state-of-the-art methods. The manuscript is quite comprehensive and has used a broad range of methods. The conclusions are convincingly supported by the experimental results.

Weaknesses:

The mutagenesis was almost exclusively based on the replacement of potentially interesting amino acid residues with alanine. Replacement with other residues, based on modelling and docking, could have refined the model further. Neither molecular dynamics nor cryo-EM was used to study the agonists' interactions with the Y4 receptor and these are therefore likely next steps in the characterization of the Y4R mechanism of activation.

Reviewer #2 (Public review):

Summary:

Pelczyk et al. investigated the binding site of the neuropeptide Y Y4 receptor with the aim of identifying novel small-molecule agonists. The authors first assessed small cyclic peptides as tool compounds and then identified interactions between peptides and receptor residues, which were confirmed by single-point mutagenesis combined with functional assays for intracellular signalling. It is interesting that a peptide receptor can be activated by the relatively small cyclic peptides used in the study. The authors identified both common and peptide-specific interactions. The identified interactions guided ultra-large library screening, which yielded 53 compounds, 3 of which were confirmed as Y4R-specific agonists in an IP-one accumulation assay.

Strengths:

The combination of techniques (docking, mutagenesis and functional assays) strongly supports the identification and evaluation of small molecules as agonists at the neuropeptide Y Y4 receptor. Functional assays highlight residues that are important for the binding of all tested peptides, as well as residues with peptide-specific importance.

The structure-activity relationship component of the study nicely highlights which components of the peptide are important for binding to the different members of the neuropeptide Y receptor family.

Weaknesses:

It would have been great to see concentration-response curves for the three identified small-molecule agonists, as this would have stengthened the case for these agonists.