Developing Photoaffinity Probes for Dopamine Receptor D2 to Determine Targets of Parkinson's Disease Drugs

Abstract

Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the most therapeutically important and best-studied receptors in these pathways is dopamine receptor D₂ (DRD2). Unfortunately, DRD2 is challenging to study with traditional molecular biological techniques, and most drugs designed to target DRD2 are ligands for many other receptors. Here, we developed probes able to both covalently bind to DRD2 using photoaffinity labeling and provide a chemical handle for detection or affinity purification. These probes behaved like good DRD2 agonists in traditional biochemical assays and were able to perform in chemical-biological assays of cell and receptor labeling. Rat whole brain labeling and affinity enrichment using the probes permitted proteomic analysis of the probes' interacting proteins. Bioinformatic study of the hits revealed that the probes bound noncanonically targeted proteins in Parkinson's disease network as well as the retrograde endocannabinoid signaling, neuronal nitric oxide synthase, muscarinic acetylcholine receptor M1, GABA receptor, and dopamine receptor D₁ (DRD1) signaling networks. Follow-up analysis may yield insights into how this pathway relates specifically to Parkinson's disease symptoms or provide new targets for treatments. This work reinforces the notion that the combination of chemical biology and omics-based approaches provides a broad picture of a molecule's "interactome" and may also give insight into the pleiotropy of effects observed for a drug or perhaps indicate new applications.

Keywords: DRD2; GABA receptor; bioinformatics; dopamine receptors; endocannabinoid pathway; muscarinic receptor M1; photo-cross-linking; photoaffinity labeling (PAL); pramipexole; proteomics; ropinirole.

Figure 1

(a) Photoaffinity labeling for the simultaneous determination of protein targets and sites of probe labeling. Probes bear two handles: one for visualization and one for enrichment. This allows monitoring of probe–target interactions by imaging as well as enrichment for proteomics. (b) Ropinirole, pramipexole, and clickable, photo-cross-linkable target probes based on their pharmacophores.

Scheme 1. Synthesis of Ropinirole-Based Targets with Multifunctional Cross-Linkers (a) (i) MsCl, TEA, CH₂Cl₂, 74%; (ii) NaN₃, H₂O, 72%; (iii) polymer-bound PPh₃, 22%; (iv) N-propylamine, reflux 29%. (b) (i) CsCO₃, KI, 14, 33% for 5, 10% for 3; (ii) EDC-HCl, HATU, DIPEA, 15, 27% for 6; 32% for 4

Scheme 2. Synthesis of Pramipexole-Based Targets with Multifunctional Cross-Linkers. (i) CsCO₃, KI, 14, 79%; (ii) EDC-HCl, HATU, DIPEA, 15, 45%

Figure 2

Intracellular calcium flux assay. HEK293T cell line stably expressing constructs for human DRD2 and a chimeric G-protein is loaded with calcium-sensing dye, Fura-4. After dosing the probe, confocal microscopy is used to determine the calcium flux in the cell by change in dye fluorescence. EC₅₀ curves determined with GraphPad software using a Hill slope of 1.0.

Figure 3

β-Arrestin recruitment analysis. (a) PRESTO-TANGO assay schematic: a ligand binds a chimeric DRD2 receptor, which then recruits β-arrestin fused with a TEV protease. The protease cuts a site between the receptor and a fused transcription factor, which then transits to the nuclease to initiate transcription of a luciferase gene. The luciferase activity is subsequently quantified. (b) Agonism of β-arrestin recruitment is quantified in EC₅₀ curves via the detection of luciferase activity, using a Hill slope of 1.0.

Figure 4

Photo-cross-linking of dye-clicked probe: confocal microscopy. (a) Schematic of the methodology used in the labeling process. Cells stably expressing DRD2 fused to an N-terminal Strep Tag II are treated with DRD2-targeting probes 5 or 7 at 5 μM, photo-cross-linked, and excess probe is washed out. An Alexa Fluor 555 azide is then clicked to the probe, washed out, and cells are treated with an anti-Strep-Tag II antibody and fluorescent secondary to visualize DRD2. Nuclei were stained with DAPI. (b) Confocal microscopy results of labeled cells. All probes show some degree of labeling. However, probes 5 and 7 show a notable increase in the labeling density. Images taken with 40× magnification, scale bar: 10 μm.

Figure 5

Flow cytometry quantification of probe labeling. (a) Schematic of flow cytometry workflow. (b) DRD2-expressing 293T cells or untransduced 293T cells (negative control) are treated with a 100 nm probe, which is photo-cross-linked, and an Alexa Fluor 555 azide is then “clicked” onto the probe. The cells were then analyzed by flow cytometry. P values determined using two-way ANOVA in GraphPad; **** corresponds to P < 0.0001.

Figure 6

Photo-cross-linking of probes 5 and 7 to DRD2 visualized with Western blot. Lanes 1–5 correspond to samples with probe 5 at 100 nm, lanes 6–10 correspond to samples treated with probe 7 at 100 nm, and lanes 11–14 correspond to samples treated with negative control probe 16. Lanes: 1, 6, and 11 are the anti-DRD2 antibody channel, lanes 2, 3, 7, 8, and 12 are fluorescence of the Alexa Fluor 555 clicked to the probes 5, 7, or 16, lanes 4, 9, and 13 are the anti-Strep Tag antibody channel, and lanes 5, 10, and 14 are the overlaid channels for the respective probes.

Figure 7

Workflow for affinity purification and proteomic analysis. Whole brain tissue is homogenized and lysed, and endogenous biotin is removed. The lysate is treated with probe, photo-cross-linked, and biotin is clicked onto the probe. Streptavidin beads pull down proteins and their interactors via the linked biotin. An example Coomassie-stained gel of resulting proteins is shown in the inset. The proteins are trypsin-digested while on-bead, eluted, desalted, and peptides are run on LC-MS. Proteomic analysis (SEQUEST) is performed to ID the proteins, followed by bioinformatic analysis of functional protein association networks (KEGG, STRING).

Figure 8

Protein networks identified by bioinformatic analysis. (a) Both probes 5 and 7 hit multiple genes in Parkinson’s disease network according to STRING and KEGG analyses. (b) Probe 5 (ropinirole-based) hit many genes in the retrograde endocannabinoid signaling pathway according to STRING and KEGG analyses. (c) Probe 7 (pramipexole-based) hit genes in the dopamine receptor D₁ (DRD1) signaling network according to STRING and gene ontology analyses.