Journal: Inflammatory bowel diseases

Article Title: Potential for Developing Purinergic Drugs for Gastrointestinal Diseases

doi: 10.1097/MIB.0000000000000047

Figure Lengend Snippet: Drugs in clinical trials targeting purinergic receptors

Article Snippet: N/A , STW5/II , A 2A AR Antag , IBS , Steigerwald Arzneimittelwerke GmbH , II , Completed 189 , Yes.

Techniques: Functional Assay, Inhibition

Journal: Nature Communications

Article Title: Specific pharmacological and G i/o protein responses of some native GPCRs in neurons

doi: 10.1038/s41467-024-46177-z

Figure Lengend Snippet: a Effect of the indicated α 2 AR agonists on the net BRET of the G i1 and G oA sensors in CGNs co-transfected with Gα i1 Nluc and Venus Gγ 2 or Gα oA Nluc and Venus Gγ 2 (amounts of cDNA as in Fig. ). b Effect of the indicated α 2 AR agonists on the net BRET of the G i1 and G oA sensors in HEK293 cells co-transfected with the mouse α 2A AR (10 ng, 20 ng or 50 ng), and Gβ 1, Venus Gγ 2 and Gα i1 Nluc or Gα oA Nluc as in Fig. . Saturating concentrations of brimonidine (10 μM), oxymetazoline (100 μM), xylazine (50 μM), clonidine (100 μM), tizanidine (100 μM) were used in ( a , b ). c Detection of α 2 AR in cell membranes of CGNs, mock-transfected HEK293 cells and HEK293 cells transfected with indicated amount of α 2 AR, by western blotting. Values are mean ± SEM normalized as fold of α 2 AR expression in CGNs from four independent experiments. d The pEC 50 of brimonidine in CGNs ( n = 3) and transfected HEK293 cells ( n = 4) with indicated amount of α 2 AR measured by G i1 or G oA sensor. e Percentage of change in BRET ratio between Gα Nluc and Venus Gγ 2 induced by brimonidine between Gα Nluc and Venus Gγ 2 in HEK293 cells ( n = 3) (α 2 AR: 15 ng/well per 96-well plate) or CGNs ( n = 4) for the indicated Gα i1 , Gα i2 , Gα i3 , Gα oA , Gα oB or Gα z sensors (amounts of cDNA as in Fig. ). Values are mean ± SEM from at least three biologically independent experiments each performed in triplicate or quadruplicate in ( a , b , d , e ). a , n = 4; b , n = 3; ( d , e ), CGNs, n = 3; HEK293 cells, n = 4. Data are analysed using one-way ANOVA with a Dunnett’s post-hoc multiple comparison test to determine significance (compared with brimonidine in a , b ). Data are analysed using unpaired t -test (two-tailed) in ( e ). **** p < 0.0001, *** p < 0.001, ** p < 0.01 and not significant (ns). The raw data and p -values are available in source data provided as a Source Data file.

Article Snippet: The blots were incubated with the primary monoclonal antibodies anti-GB1 mAb (1:1000, ab55051, Abcam, Shanghai, China), anti-Gβ 2 rabbit (1:1000, A9643, ABclonal Technology, Wuhan, China), anti-β-actin (1:3000, KM9001T, Sungene Biotech., Tianjin, China), the rabbit polyclonal antibodies anti-CB1 (1:1000, A1447, ABclonal, Wuhan, China), anti-α 2A AR (1:1000, A2809, ABclonal, Wuhan, China), anti-Gβ 1 (1:1000, A1867, ABclonal, Wuhan, China), anti-Gβ 3 (1:1000, A1387, ABclonal, Wuhan, China) and anti-Gβ 5 (1:1000, A4447, ABclonal, Wuhan, China).

Techniques: Transfection, Western Blot, Expressing, Comparison, Two Tailed Test

Journal: Journal of medicinal chemistry

Article Title: 2012 Philip S. Portoghese Medicinal Chemistry Lectureship: Structure-Based Approaches to Ligands for G Protein-Coupled Adenosine and P2Y Receptors, From Small Molecules to Nanoconjugates +

doi: 10.1021/jm400422s

Figure Lengend Snippet: A. Structures of selected AR ligands discussed in the text, including those that have been co-crystallized with the A 2A AR ( 1 , 2 , 5 , and 6 ). Dashed lines indicated the major H-bonding and π-bonding contacts between the receptor and ligand present in X-ray structures. Other ligands shown include pharmacological probes ( 4 and 9–12 ) and: advanced clinical candidates 3a and 3b for Parkinson’s disease; diagnostic agents for myocardial perfusion imaging 7 (approved and in trials for sickle cell anemia and ischemic conditions) and 8 (clinical candidate). B. The helical bundle of Family A GPCRs defines a cavity for the recognition of diverse ligands. The phospholipid bilayer is not shown. Figure courtesy of Stefano Costanzi (American Univ., Washington, DC). C. Historical progression of knowledge of the AR binding site(s). 19 , 20 , 37 , 39 , 41 , 45 Arrows in upper panels indicate the following interactions: Yellow, position of terminal amino group intended for covalent linking 37 ; orange, H-bonding interaction predicted between the exocyclic amine of adenine and a conserved Asn6.55 39 ; white, proximity of 3′ and 2′-hydroxyl groups with conserved His7.43 predicted using a neoceptor approach 41 . Lower panels: left, predicted docking of agonist 5 using the antagonist-bound X-ray structure 19 , 45 ; right, actual position of agonist 6 in X-ray structure. 20 , 24

Article Snippet: 19 , 20 A 2A AR complexes were crystallized by Heptares Therapeutics using receptors that are thermostabilized by systematic scanning mutagenesis (StaRs) include several other agonists and antagonists, including one with the antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl)oxy]phenyl]-1,3-dipropylxanthine 2 (xanthine amine congener, XAC).

Techniques: Diagnostic Assay, Imaging, Binding Assay

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Human A 2A AR in different membrane or membrane-mimetic environments and sample preparation workflow. (a) A 2A AR compared in three different environments in this study: (left) detergent micelles, (middle) lipid nanodiscs, and (right) lipid vesicles. (b) Schematic of the sample preparation workflow for preparing lipid vesicles containing 19 F-labeled human A 2A AR for solid-state MAS NMR experiments.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Membrane, Sample Prep, Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Characterization of lipid vesicles containing human A 2A AR and pharmacological validation of A 2A AR in vesicles. ( a and b ) Representative negative stain electron micrographs of unilameller vesicles composed of POPC and POPS (70:30 molar ratio) ( a ) without and ( b ) with reconstituted A 2A AR. ( c ) Dynamic light scattering measurements of the distribution of the sizes of vesicles composed of POPC and POPS (70:30 molar ratio) without (green) and with (purple) A 2A AR. ( d and e ) Pharmacological characterization of A 2A AR in lipid vesicles. ( d ) Saturation binding experiment with 3 H-ZM241385 and A 2A AR in lipid vesicles containing POPC and POPS (70:30 molar ratio). The reported B max value represents the mean and associated error is the s.e.m. from 3 independent trials done in triplicate. ( e ) Radioligand competition experiments. K D and K i values are reported for the antagonist ZM241385 and the agonist NECA, respectively. The reported error represents the s.e.m. from 3 independent trials done in triplicate.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Staining, Binding Assay

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Determination of the orientation of A 2A AR in lipid vesicles. ( a ) Schematic of the fluorescence-quenching assay used to quantify receptor orientation within vesicles. Green circles represent position C289 labeled with Cy3. Receptors with Cy3 covalently attached to position C289 facing toward the vesicle interior are labeled “A 2A AR inside”, while receptors with Cy3 facing away from the vesicle interior are labeled “A 2A AR outside”. Grey circles represent Cy3-labels that have been chemically quenched. ( b ) Quantitative comparison of the orientation of A 2A AR in vesicles made from POPC or POPC and POPS (70:30 molar ratio). The orientations “A 2A AR inside” and “A 2A AR outside” are as defined in ( a ). Error bars indicate the s.e.m. calculated from n≥3 independent experiments. Statistically significant values are illustrated as ***p<0.005 using a 2-tailed unpaired t-test.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Fluorescence, Labeling, Comparison

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Fluorescence thermal melting profiles of the A 2A AR complex with the antagonist in three different membrane mimetics. ( a ) Schematic of the fluorescence thermal shift assay as applied to A 2A AR in lipid vesicles. The inactive fluorescent dye (grey stars) shows increased emission upon covalent attachment with cysteines that become solvent accessible upon protein unfolding (orange stars). ( b ) Representative thermal melting profiles for A 2A AR in DDM/CHS detergent micelles, lipid nanodics containing POPC and POPS (70:30 molar ratio), and lipid vesicles containing POPC and POPS (70:30 molar ratio). ( c ) The melting temperature for A 2A AR in each membrane mimetic is reported as the mean of three independent experiments ± s.e.m.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Fluorescence, Membrane, Thermal Shift Assay, Solvent

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F MAS NMR-observed conformational equilibria of human A 2A AR in complex with an antagonist in lipid vesicles measured with different experimental parameters. ( a ) 1-dimensional 19 F-MAS spectra of A 2A AR[A289C TET ] in complex with the antagonist ZM241385 reconstituted into POPC/POPS lipid vesicles recorded with 105 kHz 1 H TPPM decoupling at three different MAS frequencies. ( b ) NMR spectra from (a) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 and P3. ( c ) 1-dimensional 19 F-MAS spectra of the same sample used to measure the data in (a) recorded with 1 H TPPM decoupling power set to one-quarter of the applied MAS frequency. ( d ) NMR spectra from (c) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 and P3. ( e and f ) ( e ) 1-dimensional 19 F-MAS spectra of the same sample used to measure the data in (a) recorded with no 1 H decoupling and ( f ) NMR spectra from ( e ) are shown superimposed with Lorentzian deconvolutions. ( g-j ) Linewidths measured for populations P1 and P3 for A 2A AR in ( g-i ) lipid vesicles or ( j ) lipid nanodiscs or detergent micelles. Components colored green are from free TET, consistent with earlier NMR studies (see text).

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F MAS NMR-observed conformational equilibria of human A 2A AR in complex with the agonist NECA in lipid vesicles measured with low power TPPM 1 H decoupling at several MAS frequencies. ( a ) 1-dimensional 19 F-MAS spectra of A 2A AR[A289C TET ] in complex with the agonist NECA reconstituted into POPC/POPS lipid vesicles recorded with three different MAS frequencies and 1 H TPPM decoupling at an applied 1 H power of one-quarter of the MAS frequency. ( b ) NMR spectra from (a) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 through P5. Components colored green are from free TET, consistent with earlier NMR studies (see text).

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F-NMR systematic comparison of the conformational equilibria of antagonist-bound and agonist-bound human A 2A AR[A289C TET ] across three membrane or membrane-mimetic systems by solution NMR in (a) detergent micelles and (b) lipid nanodiscs and by MAS solid-state NMR in (c) lipid vesicles. Same color scheme as in and .

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Comparison, Membrane