Shedding light on the D1-like receptors : a fluorescence-based toolbox for visualization of the D1 and D5 receptors

Funding: Universitat de Barcelona; Fonds der Chemischen Industrie. Grant Number: 661688; University of Regensburg graduate school “Receptor Dynamics: Emerging Paradigms for Novel Drugs. Grant Number: K-BM-2013-247; Elite Network of Bavaria Deutsche Forschungsgemeinschaft. Grant Numbers: 421152132, SFB1423, SFB1470; Spanish MCIN/AEI/10.13039/501100011033. Grant Number: PID2021-126600OB−I00; European Union Next Generation EU/PRTR.Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.Publisher PDFPeer reviewe

A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D2-Like Receptors

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of beta-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify beta-arrestin2 recruitment to D-2long and D(3)receptors and measure time-resolved beta-arrestin2 recruitment to the D-2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no beta-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the beta-arrestin recruitment to the D-2long R and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/beta-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([S-35]GTP gamma S, [gamma-P-32]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC(50) values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [S-35]GTP gamma S binding assay

A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D2-Like Receptors

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of beta-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify beta-arrestin2 recruitment to D-2long and D(3)receptors and measure time-resolved beta-arrestin2 recruitment to the D-2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no beta-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the beta-arrestin recruitment to the D-2long R and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/beta-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists

Abolishing Dopamine D2long/D3 Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H2 Receptor Agonists

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H2 receptor (H2R) agonists, but their applicability as pharmacological tools to elucidate the largely unknown H2R functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D2-like receptors (especially to the D3R). To improve the selectivity, a series of novel carbamoylguanidine-type ligands containing various heterocycles, spacers, and side residues were rationally designed, synthesized, and tested in binding and/or functional assays at H1–4 and D2long/3 receptors. This study revealed a couple of selective candidates (among others 31 and 47), and the most promising ones were screened at several off-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42, and 7.35) are responsible for the different affinities at the H2- and D2long/3-receptors. These results provide a solid base for the exploration of the H2R functions in the brain in further studies

Shedding light on the D₁-like receptors:a fluorescence-based toolbox for visualization of the D₁and D₅ receptors

Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups

Fluorescent tools for the imaging of dopamine D₂-like receptors

The family of dopamine D2-like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2-like receptors (pKi (D2longR)=8.24, pKi (D3R)=8.58, pKi (D4R)=7.78) with decent selectivity towards D1-like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R, and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2-like receptors in a broad variety of experimental setups

Fluorescent Tools for the Imaging of Dopamine D2-Like Receptors

Funding: S.P. was supported by the Fonds der Chemischen Industrie (No. 661688) and the University of Regensburg (Academic Research Sabbatical Program). Financial support by the graduate school “Receptor Dynamics: Emerging Paradigms for Novel Drugs (K-BM-2013-247)” of the Elite Network of Bavaria (ENB) for S.P., N.R., M.N. and H.S. is gratefully acknowledged. We would like to thank the Deutsche Forschungsgemeinschaft (DFG) for support through project 421152132 SFB1423 subproject C03 (P.A.) and SFB 1470 subproject A01 (P.A.). R.F., as PI, was funded by Spanish MCIN/AEI/10.13039/501100011033 (grant PID2021-126600OB-I00) and by the European Union Next Generation EU/PRTR (ERDF A way of making Europe). Open Access funding enabled and organized by Projekt DEAL.The family of dopamine D2-like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 ( 20 ) as a high-affinity ligand for D2-like receptors (pKi (D2longR)=8.24, pKi (D3R)=8.58, pKi (D4R)=7.78) with decent selectivity towards D1-like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R, and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20 , equipped with a 5-TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2-like receptors in a broad variety of experimental setups.Publisher PDFPeer reviewe

Structural modifications in the distal, regulatory region of histamine H3 receptor antagonists leading to the identification of a potent anti-obesity agent

A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott’s A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound