Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Structure Determination of Active and Inactive GPR17. a) Active state structure of GPR17 in complex with Gαi1/β1/γ2 and MDL. Also shown: MDL (yellow stick) in cryoEM density, representative 2D class, and proposed binding mode. b) Inactive state structure of bRIL-modified GPR17 in complex I-185. Also shown: I-185 (cyan stick) in cryoEM density, representative 2D class, and proposed binding mode. c) Binding modes of MDL (yellow sticks in green ribbon, left) and I-185 (cyan sticks in fuchsia ribbon, center) overlaid, with residues impacting microswitch cascade (cylinders, right). d) Molecular structures of MDL and I-185 with atoms numbered. e) ECL2 from apo (grey, PDB: 7Y89), MDL-bound (green), and I-185-bound (fuchsia) GPR17 overlaid.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Binding Assay, Modification

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Molecular Mechanism of GPR17 (In)activation. a) Ribbon diagrams of MDL-bound (green) and I-185-bound (fuchsia) GPR17 overlaid. b) Comparison of known microswitch residues between MDL-bound (green) and I-185-bound (fuchsia) GPR17.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Activation Assay, Comparison

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Pharmacology of I-185 and MDL-29951 interactions. a) Diagram of GPR17 signaling and assay principle for measuring cAMP as readout for GPR17 activity. b) Potency of MDL was determined in a 1321N1 astrocytoma line stably expressing GPR17. Normalized percentage is the change in signal from agonist relative to forskolin alone. c) Dose response curves in Schild analysis of I-185 versus MDL showing a right shift in parallel pattern of I-185 with increasing concentrations of MDL. d) Schild regression showing a linear agonist versus antagonist relationship indicative of competitive binding. e) Schematic of a BacMam-based cAMP sensor assay to measure I-185 and MDL activity in human OPCs. f) EC50 determination of MDL in hOPCs. Normalized data are expressed as the percentage of fluorescent signal relative to the baseline forskolin signal pre-agonist addition. g) IC50 determination of I-185 against MDL in hOPCs. Normalized data are expressed as the percentage ratio of fluorescent signal after antagonist addition divided by the difference in FSK baseline signal to agonist signal. Plots and error bars represent the mean ± SEM. N = 4 independent experiments with 2-4 technical replicates. Schematics were created in BioRender.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Activity Assay, Stable Transfection, Expressing, Binding Assay

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Effects of I-185 and MDL Interactions on GPR17 Signaling in OPCs. a) Assay principle and flow scheme for measurement of nuclear pCREB as a readout of GPR17 signaling. b) Representative immunohistochemical images demonstrating induction of nuclear pCREB downstream of GPR17 by Forskolin (FSK) activation of adenylyl cyclase (b.1); reduction of FSK-induced pCREB signal by MDL-29951 (b.2); and antagonism of MDL-29951 effect by I-185 (b.3). c) Determination of MDL potency against increasing levels of FSK. d) MDL activity in GPR17 wild type and knockout derived mouse OPCs. e) Dose response curve of I-185 against MDL. f) IC50 of MDL in human OPCs. g) I-185 dose response in human OPCs. Plots and error bars represent the mean ± SEM. N = 4 independent experiments with 2 to 4 technical replicates. Scale bar in b.3 = 50 µm.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Immunohistochemical staining, Activation Assay, Activity Assay, Knock-Out, Derivative Assay

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Agonism of GPR17 by select oxysterols. a) Structures of 24S-Hydroxycholesterol (24S-OHC), a previously discovered GPR17 agonist, and 20,22-Dihydroxycholesterol (20,22-DHC), a novel oxysterol agonist revealed by lipid screening. b) Schild analysis for 20,22-DHC against MDL. Normalized percentage is the change in signal from agonist relative to forskolin alone. c) Schild regression for 20,22-DHC activity. d) Dose response curves demonstrating GPR17 agonism by 24S-OHC and 20,22-DHC against MDL. e) GC-MS measurements of GPR17 over the course of myelin development in mouse (P7, P14, and P21). f) GC-MS measurements of 24S-OHC in human multiple sclerosis and control brain (cerebral white matter). g) Measurements of mouse GPR17 protein levels over the course of myelin development. h) Measurements of GPR17 protein level in control and MS brain (forebrain white matter). Plots and error bars in a to d are mean ± SEM. N = 4 independent experiments with 2 to 4 technical replicates. Dots in e-h represent individual brain samples. Plots and error bars are mean ± SEM.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Effects and location of lipid binding to GPR17. a) Representative in-gel analysis of PhotoClick cholesterol labeling of recombinant GPR17 shown in grayscale. GPR17 was incubated with indicated lipid followed by 3µM PhotoClick cholesterol analog, crosslinked at 365 nm UV light, conjugated to TAMRA fluorophore and separated by SDS-PAGE. Photolabeled protein was imaged by Typhoon and total protein loaded in each sample was measured by silver stain. b) Competition ratio of GPR17 photolabeling by sterols at each concentration in gel-based assay. Probe labeling was quantified by normalizing fluorescent intensity (TAMARA) to protein loading (silver stain) in each lane via ImageJ. Data are mean ± SEM of n =4 independent replicates. c) MaxQuant database search identifying several N-terminal residues in indicated peptide are modified with a mass consistent with photolabeling (+454.344695) colored in gray. Red brackets label product ions that contain the probe adduct. Ballesteros-Weinstein numbering of residues in blue above sequence. d) Fragmentation (HCD) spectrum of the indicated photolabeled peptide mapped to TM6 (mass error: 2.5ppm). Red and blue represent b and y ion fragments that do and do not contain probe modification, respectively. e) Sterol like densities from the GPR17-MDL structure surrounding the GPR17-MDL model without (right) and overlaid with (left) lipid molecules from similar structures (orange, cholesterol from GPR161 (PDB 8KH4); yellow, oleic acid from CysLT1 (PDB 6RZ5); pink, oleic acid from CysLT2 (PDB 6RZ6).

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Binding Assay, Labeling, Recombinant, Incubation, SDS Page, Silver Staining, Concentration Assay, Modification, Sequencing