Functional studies of PP, UR-AK95c, and UR-AK86c.

A) Sequence of PP and structure of UR-AK95c and UR-AK86c. The C-terminal pentapeptide of PP mimicked by the hexapeptides is underlined. Ki values were previously reported (Konieczny et al., 2021). B) Concentration-response curves, EC50, and pEC50 value of PP, UR-AK95c, and UR-AK86c at Y4R obtained by BRET-based Gi3 protein assay performed in COS-7 cells transiently transfected with untagged Y4R(wt) and Gi3 protein-based tricistronic activity sensor (Gi3-CASE). C) Kinetic measurement of ligand-induced G-protein dissociation over time obtained by BRET-based Gi3 protein assay. D) Concentration-response curves of NPY, PP, UR-AK95c, and UR-AK86c at human neuropeptide Y receptor subtypes obtained by IP-one accumulation assays in stably transfected COS-7_Y1/2/4/5R_ GαΔ6qi4myr cells. E) Structure of Y4R allosteric antagonist (S)-VU0637120 and Y4R positive allosteric modulator VU0506013 and concentration-response curves of PP, UR-AK95c, and UR-AK86c in presence of DMSO, 30 µM of (S)-VU0637120, or 30 µM of VU0506013. Data were obtained by Ca2+-flux assay in COS-7 cells stably transfected with C-terminally eYFP-tagged Y4R and GαΔ6qi4myr. All data are presented as the mean ± SEM from at least three independent experiments, each performed in triplicates.

Docking models of UR-AK95c and UR-AK86c at Y4R compared to PP.

A) and C) show the overall binding modes of UR-AK95c (green, A) or UR-AK86c (red, C) superimposed with PP (yellow, A and C) at Y4R, generated using Rosetta based on the PP-Y4R-Gi1 cryo-EM structure (PDB:7X9C). TM4 is omitted for better visualization. B) and D) highlight receptor residues in proximity to UR-AK95c (green, B) or UR-AK86c (red, D) that contribute < −1 Rosetta Energy Units (REU) to the interaction energy with at least one of the hexapeptides, as determined by a per-residue energy breakdown. The corresponding contact maps of this interface analysis for PP, UR-AK95c, and UR-AK86c are provided in Figure 2-figure supplement 1-3.

Mutagenesis studies of PP, UR-AK95c and UR-AK86c at Y4R.

A) Y4R snake plot. Residues selected for mutagenesis studies are marked in grey. Residues are named according to Ballesteros-Weinstein nomenclature (Ballesteros and Weinstein, 1995). Snake plot was adapted from GPCRdb.org (Pándy-Szekeres et al., 2018). B) pEC50 values of PP, UR-AK95c, and UR-AK86c determined for Y4R single- and multiple residue variants. Mutagenesis data were obtained by IP-one accumulation assay conducted in COS-7 cells transiently transfected with C-terminally eYFP-tagged Y4R variant and Δ6Gαqi4myr. Significance was determined by one-way ANOVA with Dunnett’s post-test. *, P < 0.033, **, P < 0.002, ***, P < 0.001. 4xA_ECL2, Y4(F184A, H188A, L192A, F194A). Data represent the mean ± SEM from at least three experiments, each performed in triplicates.

Key Y4R activation interface and receptor residues mediating peptide-specific interactions.

A) Area of Y4R within a distance of 5 Å of PP (blue), UR-AK95c (green), or UR-AK86c (red). Surface area was estimated by subtracting the solvent accessible surface area of the peptide-Y4R-complex from the sum of the solvent accessible interface area of Y4R and the peptide using PyMol (DeLano, 2026). B) Selected Y4R key residues interacting with the peptides’ conserved C-terminal -RY-NH2 motif. C) ΔpEC50 values of Y4R variants displaying more than 5-fold reduction in potency across all three peptides compared to Y4R(wt). D) Selected Y4R residues that contribute differentially to the interactions with the three peptide agonists. E) ΔpEC50 values of Y4R variants displaying pronounced differences between the peptides. For better visualization, TM4 is omitted in all structural snapshots.

Functional studies of UR-AK86c and truncated derivatives at neuropeptide Y receptor subtypes.

A) Structure of UR-AK86c, [Agp5]-UR-AK86c, the linear hexapeptide RYRLRY-NH2, and UR-AK32 (Konieczny et al., 2020) in descending order of solvent-accessible surface area (SASA). B) Concentration-response curves of PP at Y4R; NPY at Y1R, Y2R, and Y5R, and UR-AK86c, [Agp5]-UR-AK86c, RYRLRY-NH2, and UR-AK32 at all neuropeptide Y receptor subtypes obtained through IP-one accumulation assay conducted in stably transfected COS-7_Y1/2/4/5R_GαΔ6qi4myr cells. All data are presented as the mean ± SEM from at least three independent experiments, each performed in triplicates.

Identification of hits from ULLS.

A) Structures of Z8878907875, Z9239258318, and Z9407529782. B) Activation of neuropeptide Y receptor subtypes by 100 µM Z8878907875, 100 µM Z9239258318, and 100 µM Z9407529782 in comparison with 1 µM PP or 1 µM NPY, respectively. Data were obtained through IP-one accumulation assay conducted in stably transfected COS-7_Y1/2/4/5R_ GαΔ6qi4myr cells. All data are presented as the mean ± SEM from at least three independent experiments, each performed in triplicates. C) Overlay of PP and Z9407529782 in the Y4R binding pocket as predicted by FlexPepDock and RosettaLigand, respectively. D) Representative docking pose of Z9407529782 at Y4R, with receptor residues shown as sticks, which were defined as key Y4R residues in this study. Residues are colored according to their minimal distance to Z9407529782.

Functional data of tested peptides obtained by IP-one accumulation and BRET-based Gi3 protein assays.

IP-one accumulation assay was performed in COS-7 cells stably expressing the Y1/2/4/5R-eYFP construct and the chimeric G protein Δ6Gαqi4myr. BRET-based Gi3 protein assay was performed in COS-7 cells transiently co-transfected with untagged Y4R and Gi3-CASE construct. EC50 and pEC50 values were calculated from a minimum of three independent experiments. n.a., not applicable, n.d., not determined.

Functional characterization of PP, UR-AK95c, and UR-AK86c at Y4R in the presence of DMSO, 30 µM (S)-VU0637120, and 30 µM VU0506013.

EC50 values were determined using Ca2+-flux assay in COS-7 cells stably expressing the Y4R-eYFP construct and the chimeric G protein Δ6Gαqi4myr. The EC50 shift represents the ratio of the EC50 value of peptide in the presence of an allosteric modulator to the EC50 value of the same peptide in the presence of DMSO. Each condition was tested in at least three independent experiments. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s post-test. n.a., not applicable, *, P < 0.033, **, P < 0.002, ***, P < 0.001.

Contact map of per-residue energy breakdown of the best-fitting model of PP at Y4R.

Average pairwise energy scores are depicted in Rosetta Energy Units (REU). The residues are named according to Ballesteros-Weinstein nomenclature (Ballesteros and Weinstein, 1995).

Contact map of per-residue energy breakdown of the best-fitting model of UR-AK95c at Y4R.

Average pairwise energy scores are depicted in Rosetta Energy Units (REU). The residues are named according to Ballesteros-Weinstein nomenclature (Ballesteros and Weinstein, 1995).

Contact map of per-residue energy breakdown of the best-fitting model of UR-AK86c at Y4R.

Average pairwise energy scores are depicted in Rosetta Energy Units (REU). The residues are named according to Ballesteros-Weinstein nomenclature (Ballesteros and Weinstein, 1995).

Mutagenesis studies of wild-type Y4R, Y4R single- and multiple-residue variants, wild-type Y4R, and Y4/Y1R loop chimeras with PP, UR-AK95c, and UR-AK86c.

EC50 values were determined by IP-one accumulation assays in COS-7 cells transiently expressing the receptor variant-eYFP constructs and Δ6Gαqi4myr. The EC50 shift represents the ratio of the EC50 value of the peptide at the receptor variant to the EC50 value of the same peptide at wild-type Y4R. Each Y4R variant was tested in at least three independent experiments. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s post-test. Residues are named according to the Ballesteros-Weinstein nomenclature (Ballesteros and Weinstein, 1995). n.a., not applicable, n.s., not significant, *, P < 0.033, **, P < 0.002, ***, P < 0.001. 4xA_ECL2, Y4(F184A, H188A, L192A, F194A).

Functional studies of PP, UR-AK95c, and UR-AK86c at Y4/Y1R loop chimeras.

A) Snake plots of Y4R and Y1R. Interchanged segments are colored in light grey (Y4R) and dark grey (Y1R). Snake plot was adapted from GPCRdb.org (Pándy-Szekeres et al., 2018). B) pEC50 values of PP, UR-AK95c, and UR-AK86c determined at wild-type Y4R, wild-type Y1R, and Y4/Y1R loop chimeras. Functional data were obtained through IP-one accumulation assay conducted in COS-7 cells transiently transfected with C-terminally eYFP-tagged receptor variant and Δ6Gαqi4myr. Significance was determined by one-way ANOVA with Dunnett’s post-test. *, P < 0.033, **, P < 0.002, ***, P < 0.001. All data represent the mean ± SEM from at least three independent experiments, each performed in triplicates.

Membrane expression of Y4R residue variants and Y4/Y1R loop chimeras.

The membrane insertion was studied using fluorescence microscopy in HEK293 cells, transiently expressing Y4R-eYFP fusion protein. The receptor-eYFP constructs are represented in yellow, while the nuclei stained with Hoechst33342 are shown in blue. Scale bar is 5 μm. Pictures are representatives of at least two independent experiments. 4xA_ECL2, Y4(F184A, H188A, L192A, F194A).

Multiple sequence alignment of human neuropeptide Y receptor subtypes.

Sequences were obtained from the National Center for Biotechnology Information (NCBI) database (NIH) and represent the amino acid sequences of Y1R (NP_000900.1), Y2R (NP_001362399.1), Y4R (NP_005963.4), and Y5R (NP_001304021.1). Alignment was performed using CLUSTAL O(1.2.4) multiple sequence alignment tool (Chenna et al., 2003). Receptor segments are annotated based on the location of the residues in the available PP-Y4R cryo-EM structure. Residue coloring indicates side chain properties: negatively charged (blue), positively charged (pink), polar (green), and hydrophobic (red).

Functional data of tested peptides obtained by IP-one accumulation assays.

IP-one accumulation assay was performed in COS-7 cells stably expressing the Y1/2/4/5R-eYFP construct and the chimeric G protein Δ6Gαqi4myr.. EC50 and pEC50 values were calculated from a minimum of three independent experiments. n.a., not applicable.

Analysis of hPP (A), pNPY (B), UR-AK95c (C), UR-AK86c (D), [Agp5]-UR-AK86c (E), RYRLRY-NH2 (F), RLRY-NH2 (G).

The upper panels show RP-HPLC chromatograms recorded at 220 nm using gradients of 20–70% (A, B), 10–60% (C–E), or 5–55% (F, G) eluent B (0.08% TFA in ACN) in eluent A (0.1% TFA in H₂O) over 40 min at 40 °C. Analyses were performed on either an Aeris Peptide 100 Å XB-C18 column (flow rate: 1.55 mL/min) or a Jupiter Proteo 90 Å C12 column (flow rate: 1.0 mL/min). The lower panels show the corresponding MALDI-ToF mass spectra.

List of compounds ordered from Enamine LTD including their Enamine ID and structure as SMILES (Grygorenko et al., 2020).

The compounds, which showed significant Y4R activity, are highlighted in grey.