Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: GPR61 gene locus is associated with (A) BMI and (B) weight at genome-wide level of significance. The plots show all the GPCR genes found in the top 1000 genes for each of the 2 associations as analyzed with the CMDKP. Please see the section for more details.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Genome Wide

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: Protocol for exon sequencing and SNP variant calling. The same procedure was used for GPR61 and MC 4 R.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Sequencing, Variant Assay

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: UK10K obesity screen reveals presence of GPR61 mutations. (A) Mutational landscape of GPR61 in normal population and then the same mutations in severe obesity patients. The data are presented as frequencies. The allele counts can be found in Supplemental Fig. 6 . (B) GPR61 mutations mapped onto a 2-dimensional model of GPR61.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques:

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: Severe obesity–associated mutations of GPR61 have no statistically significant impact on cell surface trafficking of the overexpressed receptor. NanoBiT-based measurement of total cell and cell surface expression of 34 mutants of GPR61. Data were analyzed for differences between the mutants with the WT by one-way ANOVA with Dunnett’s post hoc analysis. Data are presented as mean of n = 3 independent experiments ± SD. Significance levels are given as: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: Identification and molecular characterization of missense mutations in orphan G protein–coupled receptor GPR61 occurring in severe obesity

doi: 10.1016/j.molpha.2025.100026

Figure Lengend Snippet: Severe obesity–associated mutations of GPR61 have an impact on cAMP production and G s translocation. (A) EPAC-derived FRET-based biosensor reveals that 3 mutations lead to the statistically significant reduction in cAMP production elicited by overexpression of GPR61 in the absence of an agonist (increase in the FRET ratio reflects decrease in cAMP levels). Data are presented as the mean of n = 4 independent experiments ± SD. (B) The presence of overexpressed GPR61 R236C 5.66 leads to a statistically significant difference in the bystander BRET ratio between G α s-67- R luc2 and rGFP-CAAX in comparison with the WT. The reduction in ebBRET signal vs pcDNA3.1 is indicative of G s translocation away (activation) from the cell membrane. The receptor plasmids were overexpressed at 3 different levels to account for any differences in their expression and to confirm that higher receptor expression leads to an increase in its constitutive activity. Data are presented as mean of n = 3 independent experiments ± SD. (C) The presence of overexpressed GPR61 R236C 5.66 led to statistically significant differences in the bystander BRET ratio between G α s-67- R luc2 and rGFP-giantin in comparison with the WT. Data were analyzed for differences between the mutants with the WT by one-way ANOVA with Dunnett’s post hoc analysis. Data are presented as mean of n = 3 independent experiments ± SD. Significance levels are given as: ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: HiBiT-GPR61 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR61 from GPR61-Tango plasmid DNA (#66366 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3 A signal peptide plasmid DNA as a backbone ( ).

Techniques: Translocation Assay, Derivative Assay, Over Expression, Comparison, Activation Assay, Membrane, Expressing, Activity Assay

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) The results from the 10 μM compounds screen using the ebBRET-based Gi activation assay with an overexpressed HiBiT-GPR183. The compounds were added to the cells and the ebBRET measured over 15 minutes. The data are presented as net AUC of ΔebBRET % over the three baseline reads measured prior to the compound addition; vehicle (0.1% DMSO) was not subtracted. The data are shown as mean ± s.e.m. of three independent experiments. The data were analysed for differences with ANOVA with Dunnett’s post-hoc analysis; *P < 0.05; **P < 0.01; ****P < 0.0001. The selected compounds, 3 and 43 , are shown as purple and green bars, respectively. b) The concentration response curve of compounds 3 and 43 in the same assay. The compounds were added, and the ebBRET signal was measured over 15 minutes. The data are presented as net AUC of ΔebBRET % over the three baseline reads measured prior to the compound addition; vehicle (0.1% DMSO) was subtracted. The data are shown as mean ± s.e.m. of three independent experiments.

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Activation Assay, Concentration Assay

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) The results from the screen with the 34 analogues of 43. The compounds were used at 10 μM and their activity to modulate HiBiT-GPR183 activation assesses using the ebBRET-based Gi activation assay. The compounds were added to the cells and the ebBRET measured over 15 minutes. The data are presented as net AUC of ΔebBRET % over the three baseline reads measured prior to the compound addition; vehicle (0.1% DMSO) was not subtracted. The data are shown as mean ± s.e.m. of three-four independent experiments. The data were analysed for differences with ANOVA with Dunnett’s post-hoc analysis; *P < 0.05; **P < 0.01. The selected compound 78 is shown in dark green bars. b) The concentration response curve of compounds 43 , 78 and NIBR189 in the same assay. The compounds were added, and the ebBRET signal was measured over 15 minutes. The data are presented as net AUC of ΔebBRET % over the three baseline reads measured prior to the compound addition; vehicle (0.1% DMSO) was subtracted. The data are shown as mean ± s.e.m. of three independent experiments. The same data for the 43 are also shown in the . c) The concentration response curve of compounds 78 and NIBR189 following cells incubation with 1 μM 7α,25-dihydroxycholesterol for 5 minutes. The data are presented as net AUC of ΔebBRET % over the three baseline reads measured prior to the compounds’ addition; vehicle (0.1% DMSO) was subtracted. The data are shown as mean ± s.e.m. of three-four independent experiments. d) Agonist-stimulated HiBiT-GPR183-Nluc- or HiBiT-GPR183-mediated recruitment of β-arrestin2-mVenus ( left ) and β-arrestin2- R luc2 ( right ). The concentration response curve of 7α,25-dihydroxycholesterol in the presence or absence (vehicle, 1% DMSO) of 10 μM of 78 . 78 or vehicle were added, and the BRET/ebBRET signal was measured over 10 minutes, followed by the addition of different agonist concentrations. The plate was measured for another 15 minutes. The data are presented as net AUC of ΔBRET %/ΔebBRET over the first 10-minute-long read measured prior to the agonist addition; vehicle (1% DMSO) was subtracted. The data are shown as mean ± s.e.m. of three independent experiments. The difference in agonist potency was assessed by comparing log EC 50 values for the two curves with the extra sum-of-squares F test; P = 0.0048.

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Analogues, Activity Assay, Activation Assay, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) The schematic representation on the HiBiT-GPR183-mNG-Nluc conformational sensor. The mNeonGreen (mNG) is inserted into the intracellular loop 3 (ICL3) and the Nanoluciferase (Nluc) is fused directly to the C-terminus. b) Agonist stimulation of the three generated sensors, in which the mNG tag was inserted directly after P231, T233 or K235, leads to a reduction in BRET. The data are presented as 15-min kinetic plots with ΔBRET % (over the three baseline reads); vehicle (1% DMSO) was not subtracted. The data are shown as mean ± s.e.m. of three independent experiments . c) The concentration response curve of the agonist. The data are presented as net AUC of ΔBRET % over the three baselines reads measured prior to the agonist addition; vehicle (1.0% DMSO) was subtracted. The plate was measured for another 15 minutes. The data are shown as mean ± s.e.m. of five independent experiments. d) The concentration response curve of the inverse agonist NIBR189. Different concentrations of NIBR189 were added, and the BRET signal was measured over 10 minutes, followed by the addition of 50 nM 7α,25-dihydroxycholesterol. The plate was measured for another 15 minutes. The data are presented as net AUC of ΔBRET % (over the first 10-minute-long read); vehicle (1% DMSO) was subtracted. The data are shown as mean ± s.e.m. of three independent experiments. e) The concentration response curve of 7α,25-dihydroxycholesterol in the presence or absence (vehicle) of 10 μM, 3.0 μM or 1.0 μM of 78 . 78 or vehicle (0.1% DMSO) were added and the plate incubated for 10 minutes at 37°C. The three baseline measurements were read and they were followed by the addition of different agonist concentrations and the plate was measured for 15 minutes. The data are presented as net AUC of ΔBRET % (over the three baseline measurements); vehicle (1% DMSO) was subtracted. The data are shown as mean ± s.e.m. of four to five independent experiments. The data were analysed using the Gaddum/Schild EC50 shift function in GraphPad Prism. f) The concentration response curve of 78 . Different concentrations of 78 were added, and the BRET signal was measured over 10 minutes, followed by the addition of 50 nM 7α,25-dihydroxycholesterol. The plate was measured for another 15 minutes. The data are presented as net AUC of ΔBRET % (over the first 10-minute-long read); vehicle (1.0% DMSO) was subtracted. The data are shown as mean ± s.e.m. of three independent experiments.

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Generated, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) Z-factor evaluation of the HiBiT-GPR183-(T233)-mNG-Nluc sensor using the HEK293A cells stably overexpressing the construct. The data are presented as the mean ΔBRET% (over the three initial baseline reads) from the 15-minute-long read following the addition of the agonist or vehicle (0.1% DMSO) for three separate plates. b) Left top : pcDNA.3.1-transfected cell nor ( left middle ) HA-GPR183-(T233)-mNG2 (11) -transfected cells do not respond to agonist stimulation to activate Gi in GPR183-specific manner as opposed to ( left bottom ) HA-GPR183-HiBiT, which does not have an ICL3-inserted tag. 0.1% DMSO was used as a vehicle. The data come from a representative experiment of three independent experiments and are shown as mean ± s.d. of three technical replicates. Right top : Ligand-unbound HA-GPR183-(T233)-mNG2 (11) does not constitutively activate Gi. Right bottom : HA-GPR183-(T233)-mNG2 (11) is trafficked to the cell membrane. The data are shown as mean ± s.e.m. of three independent experiments. The data were analysed for differences using one-way ANOVA with multiple comparison Dunnett’s post-hoc analysis; * P<0.05, ****P < 0.0001. c) Left : Upon the overexpression of the HiBiT-GPR183-(T233)-Halo-Nluc conformational sensor and addition of the Halo substrate, there is an energy transfer between the Nluc and Halo (detectable a luminescence peak over 600 nm). Right : The addition of 10 μM of the agonist does not result in a notable change in the BRET signal in comparison with the vehicle (0.1% DMSO).

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Stable Transfection, Construct, Transfection, Membrane, Comparison, Over Expression

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) Overlap between 78 and 7α,25-dihydroxycholesterol. Compound 78 is represented as orange sticks, and 7α,25-dihydroxycholesterol as green sticks, while the GPR183 protein is shown as blue ribbons and sticks. b) Overview of 78 binding mode. The ligand is depicted as orange spheres. c) Predicted binding mode of 78 in complex with GPR183 protein. H-bonds are represented as green dashes

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Binding Assay

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: RMSD (a) , distance (b) , and angles (c) plots over time (in nanoseconds) of compound 78 . d) H-bond interactions vs trajectory time between 78 and C201, H261, Y112, and Y240. e) Representative structure of the single, well-defined cluster of 78 in complex with GPR183 protein. The ligand is depicted as orange sticks while the protein is shown as blue sticks and ribbons. H-bond are represented as green dashes.

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques:

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) Addition of 10 μM of 78 (time = 0, marked as dashed lines) leads to a reduction in the ebBRET signal indicative of a decrease in GPR183-mediated Gi activation. The data are presented as raw ebBRET ratio; vehicle (1% DMSO) was not subtracted. b) Left : Addition of 10 μM of 78 did not modify constitutive GPR183 H261A 6.52 -mediated Gi activation. The data are presented as raw ebBRET ratio; vehicle (1% DMSO) was not subtracted. Right : Preincubation with 10 μM of 78 did not modify agonist-simulated GPR183 H261A 6.52 -mediated Gi activation. The data are presented as net AUC of ΔebBRET % (over the three baseline measurements); vehicle (1% DMSO) was subtracted). c) The concentration response curve of 7α,25-dihydroxycholesterol on the HiBiT-GPR183-(T233)-mNG-Nluc H261A 6.52 in the presence or absence (vehicle, 1% DMSO) of 10 μM of 78 . 78 or vehicle were added, and the BRET signal was measured over 10 minutes, followed by the addition of different concentrations of 7α,25-dihydroxycholesterol. The plate was measured for another 15 minutes, but since the mutations also impact the agonist binding kinetics, only the first three minutes were used in the analysis. The data are presented as net AUC of ΔBRET % (over the 10-minute-long read); vehicle (1% DMSO) was subtracted. The differences in agonist potency and BRET max were assessed by comparing log EC 50 and top values, respectively, for the two curves with the extra sum-of-squares F test. d) Left : Addition of 10 μM of 78 induced GPR183 Y260F 6.51 -mediated Gi activation. The data are presented as raw ebBRET ratio; vehicle (1% DMSO) was not subtracted. Right : Preincubation with 10 μM of 78 did impaired agonist-simulated GPR183 H261A 6.52 -mediated Gi activation. The data are presented as net AUC of ΔebBRET % (over the 10-minute-long read); vehicle (1% DMSO) was subtracted). The differences in ebBRET max were assessed by comparing top values for the two curves with the extra sum-of-squares F test, and the difference is indicated with the line and marked for statistical significance. e) The concentration response curve of 7α,25-dihydroxycholesterol on the HiBiT-GPR183-(T233)-mNG-Nluc Y260F 6.51 in the presence or absence (vehicle, 1% DMSO) of 10 μM of 78 . 78 or vehicle were added, and the BRET signal was measured over 10 minutes, followed by the addition of different concentrations of 7α,25-dihydroxycholesterol. The plate was measured for another 15 minutes, but since the mutations also impact the agonist binding kinetics, only the first three minutes were used in the analysis. The data are presented as net AUC of ΔBRET % (over the first 10-minute-long read); vehicle was subtracted. The differences in agonist potency and BRET max were assessed by comparing log EC 50 and top values, respectively, for the two curves with the extra sum-of-squares F test, and the difference is indicated with the line and marked for statistical significance. The data in this figure are shown as mean ± s.e.m. of three to four independent experiments.

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Activation Assay, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: a) Addition of 10 μM of 78 leads to a subtle but visible change in BRET in HiBiT-GPR183-(T233)-mNG-Nluc Y260F 6.51 conformational biosensor in comparison to vehicle (1% DMSO). This is further supported by the statistical analysis in (b) . The data are presented as the mean ΔBRET% (over the three initial baseline reads) from the 10-minute-long read following the addition of 78 or vehicle. The data are shown as mean ± s.e.m. of three independent experiments. The data in b) were analysed with a paired t -test; ** P<0.01

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Comparison

Journal: bioRxiv

Article Title: AI-Driven Virtual Screening and First-in-Class Conformational Biosensor Enable the Discovery of a GPR183 Inverse Agonist

doi: 10.1101/2025.06.19.659764

Figure Lengend Snippet: ( a, left panel ), median fluorescent intensity (MFI) of GPR183 (middle panel), and frequency of GPR183-positive cells (right panel) of GPR183 in CD4+T, B and CD8+T cells in healthy control (HC, n=4). b) Frequency of migratory lymphocytes treated with 78 , NIBR189, or no-inhibitor (NI) after stimulation in HC. c) Frequency of viable cells treated with 78 , NIBR189, or NI following 7α,25-dihydroxycholesterol stimulation. d) Frequency of transmigrated CD8+T cells after addition of 78 and NIBR189 to 7α,25-dihydroxycholesterol-stimulated (0.1% DMSO final concentration) samples from HC.

Article Snippet: HiBiT-GPR183 plasmid DNA was generated with Gibson cloning using a codon-optimized GPR183 from GPR183-Tango plasmid DNA (#66342 Addgene, deposited by Bryan Roth) as an insert and HiBiT-FZD 6 with a 5-HT 3A signal peptide plasmid DNA as a backbone [ ].

Techniques: Control, Concentration Assay