Identification of key Y4R residues enables the discovery of selective non-peptide small-molecule agonists
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eLife Assessment
This important study combines peptide engineering, molecular docking, and functional assays to define the molecular basis of ligand recognition and activation of the human Y4 receptor and to identify three novel small-molecule agonists. The evidence supporting the conclusions is convincing, with complementary experimental and computational approaches providing strong support for the proposed receptor-ligand interactions. While concentration-response analyses of the small-molecule agonists and additional structural or mutagenesis studies would further strengthen the work, these are not essential to support the main conclusions. The work will be of interest to researchers studying GPCR pharmacology, structural biology, and ligand discovery.
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Abstract
G protein-coupled receptors (GPCRs) are central regulators of human physiology and disease, classifying them as relevant targets for therapeutic interventions. As transmembrane proteins, they convert extracellular signals into intracellular responses through agonist-induced conformational changes. Understanding how agonists stabilize active receptor conformations is decisive for rational drug design. In this study, we used the endogenous ligand pancreatic polypeptide (PP) and the cyclic hexapeptides UR-AK95c and UR-AK86c as molecular tools to determine key interactions critical for Y4R activation, which plays a crucial role in metabolic diseases. Guided by molecular docking, we systematically replaced Y4R residues and assessed activation. The in vitro and in silico studies delineated a key Y4R activation interface centered around the conserved C-terminal RXRY-NH2 motif of the peptides and, separately, identified receptor residues with distinct peptide-specific functional effects. Next, we performed an ultra-large library screening (ULLS) and experimentally validated three predicted hits as selective Y4R agonists that engage in a substantial subset of the identified critical receptor contacts. This study demonstrates how GPCR activation interface knowledge can be translated into the discovery of novel small-molecule agonists and outlines a general strategy for advanced GPCR drug discovery.
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eLife Assessment
This important study combines peptide engineering, molecular docking, and functional assays to define the molecular basis of ligand recognition and activation of the human Y4 receptor and to identify three novel small-molecule agonists. The evidence supporting the conclusions is convincing, with complementary experimental and computational approaches providing strong support for the proposed receptor-ligand interactions. While concentration-response analyses of the small-molecule agonists and additional structural or mutagenesis studies would further strengthen the work, these are not essential to support the main conclusions. The work will be of interest to researchers studying GPCR pharmacology, structural biology, and ligand discovery.
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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 …
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.
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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, …
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.
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