Fluorescent Peptide Tracers for Simultaneous Oxytocin Receptor Activation and Visualization

Abstract

The oxytocin receptor (OTR) regulates critical physiological functions and has been implicated in a range of diseases, including psychiatric and neurodevelopmental disorders such as autism spectrum disorder. However, a lack of reliable molecular tools hampers the progress in understanding OTR's mechanistic roles in (patho)physiological processes. In this work, we addressed this gap and developed potent, selective, and bright fluorescent peptide tracers that enable precise spatial and functional investigations of OTR actions. Our tracers showed efficient OTR labeling, activation, and internalization in cellular bioassays in both live and fixed overexpression and primary cell systems, including those subjected to immunocytochemical protocols, highlighting their versatility as reliable new imaging tools. Additionally, they facilitated single-molecule tracking of OTR with live-cell super-resolution microscopy and were able to separate OTR-positive cells from mixed oxytocin and vasopressin receptor-containing cell populations via fluorescence-activated cell sorting, underscoring their wider scope for live-cell applications. In summary, we developed versatile fluorescent tracers based on the endogenous ligand oxytocin for both live-cell and post-hoc imaging that have additional functional capabilities beyond traditional antibody labeling, offering new avenues to explore OTR's role in health and disease.

Keywords: Bioactive peptide tracers; Imaging; Immunocytochemistry; Oxytocin receptor visualization; Structure–activity relationship (SAR) study.

Figure 1

Overview of the most selective OTR tracers. Compound A,^{[ 19 ]} compound 19,^{[ 25 ]} and compound 7.^{[ 27 ]}

Figure 2

Docking of OT and d(Orn)⁸OT to the human OTR binding pocket. a) OT‐hOTR (7RYC; grey) overlaid with the lowest energy docked OT structure (cyan). The amino acids of both macrocycles align well, with only Leu⁸ pointing in a different direction. Comparable geometries and highly similar LigandScout binding affinity score values of −30.86 and −30.76 for the cryo‐EM and docked OT, respectively, led to the conclusion of a valid docking approach. b) OT‐hOTR (grey), overlaid with a low‐energy docked d(Orn)⁸OT structure (yellow). The Orn⁸ side chain was pointing from the binding pocket outwards, supporting the hypothesis that this position (red circle) could be used for linker attachment of other moieties without interfering with the active site. LigandScout binding affinity score of −30.60 indicated only a slightly weaker binding affinity to hOTR.

Figure 3

Molecular structure of d(Orn)⁸OT parent peptide and fluorophores and fluorescent tracer synthesis. a) Overview of synthesized tracer structures. The d in d(Orn)⁸OT stands for desamino (N‐terminal α‐amino group absent). Note: The chemical structure is shown with cis amide bonds for illustration purposes only and does not reflect the actual amide bond configuration, which is trans. b) Chemical structures of the Cy3_s, Cy5_s, AF488, and TAMRA fluorophores. c) Synthetic strategy to produce the different fluorescent OTR tracers. Backbone and linker assemblies were carried out via Fmoc‐SPPS, followed by TFA global side chain deprotection and resin cleavage, oxidative folding, and in‐solution fluorophore attachment using NHS‐activated dyes.

Figure 4

Evaluation of tracer design regarding OTR specificity and compatibility with immunocytochemistry (ICC) protocols. a) and b) OT (100 nM) potently induced internalization of hOTR‐GFP (green) away from the membranes into the cytoplasm (arrowheads versus double arrowheads). Note that the GFP⁺ vesicles are appearing in the cytoplasm. Z‐stacks show complete 3D images of cells reduced to a single plane. Phalloidin‐555 (F‐actin, red) was used to indicate membranes. c) and d) Comparison of d(Orn)⁸OT‐[PEG₅‐Cy5_s]⁸ (3) and d(Orn)⁸OT‐[PEG₅‐k^ε‐Cy5_s]⁸ (7) revealed GFP internalization (arrows) after 1 h of stimulation to accrue strong signals for confocal microscopy at 100 nM and ICC protocols for both tracers. The presence of d‐Lys in d(Orn)⁸OT‐[PEG₅‐k^ε‐Cy5_s]⁸ (7) for ICC cross‐linking with formaldehyde resulted in more retention (arrowheads, c versus d) than d(Orn)⁸OT‐[PEG₅‐Cy5_s]⁸ (3) lacking d‐Lys. (c₁,d₁) A 30‐min pretreatment with 10 µM of OTR antagonist (OTA) before 1‐h stimulation prevented GFP internalization and tracer accumulation for both tracers. Note the diffuse presence of membrane‐bound GFP signals (open arrows) compared to strong punctate cytoplasmic signals (arrows). (e,e₁) Signal intensity was increased for compound (7) but not for (3) after prolonged stimulation (24 h), indicating that d‐Lys was required for post‐fixation ICC protocols and subsequent imaging.

Figure 5

Validation of OTR selectivity and biological activity for tracers d(Orn)⁸OT‐[PEG₅‐k^ε‐Cy3_s]⁸ (8) and d(Orn)⁸OT‐[d‐PEG₅‐k^ε‐Cy3_s]⁸ (11). a‐b₁) Cy3_s intensity measurements of both tracers in hOTR‐GFP and hV_1aR‐GFP HEK293 cells post ICC (normalized to control). Tracers (8) and (11) displayed a significant concentration‐dependent accumulation of fluorescent Cy3_s (red) signals in hOTR‐expressing cells (a,a₁; arrowheads). (b,b₁) In hV_1aR‐containing cells, only tracer (8), but not (11), displayed fluorescent signals and internalization at 100 nM. (b₁) Selectivity was lost at 1 µM for both compounds, in line with the determined affinities (Table 1). n = 6–9 cell clusters per condition. c,c₁) Treatment with tracer (11) confirmed a concentration‐dependent GFP and compound internalization (arrowheads pointing to co‐localizing red and green puncta). CREB phosphorylation events were observed from 1 nM onward. Data are presented as mean ± SEM (n = ∼150 cells over two coverslips), and statistical analysis was done applying Student's t‐test (*P < 0.05; ***P < 0.001; ns, not significant).

Figure 6

Effects of paraformaldehyde (PFA) fixation and immunocytochemistry on d(Orn)⁸OT‐[d‐PEG₅‐k^ε‐Cy3_s]⁸ (11) detectability. a) After 20‐min stimulation and PFA fixation, a strong accumulation of tracer (11) was observed (arrowheads). b) After applying immunocytochemistry protocols, including nonspecific protein blocking, primary/secondary antibody labeling, and extensive washing steps, fluorescent intensity was reduced but still readily detectable with confocal microscopy.

Figure 7

Live single‐particle tracking (SPT) of hOTR using d(Orn)⁸OT‐[d‐PEG₅‐Cy5_s]⁸ (10). HEK293 parent cells or HEK293 cells stably expressing hOTR‐GFP were imaged on glass‐bottom dishes in an isotonic imaging buffer. a) d(Orn)⁸OT‐[d‐PEG₅‐Cy5_s]⁸ (10) was applied to the dish (1 nM), and images were acquired immediately (50 Hz for 16 000 frames). Single‐molecule detections were localized and tracked using PALMtracer software. Super‐resolved trajectory images of representative cells are shown. b) A representative image of each track, pseudo‐colored by its diffusion coefficient (D) (Log₁₀D, confined molecules represented by warmer colors). (b₁) The number of tracks (per µm²) was calculated for both groups and normalized to the HEK293 control line. (b₂) The apparent diffusion coefficient was calculated for all tracks in each HEK293‐hOTR‐GFP cell and plotted as a frequency distribution. Data are presented as mean ± SEM. An unpaired t‐test with Welch's correction was used to compare the track density (p < 0.05, * was considered significant).

Figure 8

Fluorescence‐activated cell sorting (FACS) of OTR‐ and V_1aR‐expressing HEK293 cells with tracer (11). a‐a₂) FACS plots of collected cells sorted from a mixed pool of free‐floating OTR⁺ and V_1aR⁺ cells after treatment with d(Orn)⁸OT‐[d‐PEG₅‐k^ε‐Cy3_s]⁸ (11) at 1, 10, or 100 nM. Positive (+, black) and negative (‐, green) fractions collected were marked with a rectangle. b,b₁) RT‐qPCR quantifications for human OTR (gene: OXTR) and V_1aR (gene: AVPR1A) revealed substantial enrichment of OTR in the positive pool and V_1aR in the negative pool from 1 nM onwards. Optimal separation was achieved with 10 nM of tracer (11) (a₁), reflected by the highest fold enrichment of OTR (b). Values were normalized to the negative (b) and positive (b₁) 10 nM fraction. qPCR products after 40 cycles are indicated below. Data are presented as mean ± SEM from technical triplicates.

Figure 9

Stimulation of human uterine smooth muscle cells (UtSMC) with tracer (11). a,a₁) Live‐cell imaging with an IncuCyte SX‐5 revealed rapid contraction of UtSMC cells (arrowheads versus arrows) when exposed to 100 nM tracer (11), which was prevented by OTA pretreatment (t in min). b) and c) Tracer (11) accumulated in vesicle‐like structures in UtSMC somata (arrows) but was not taken up in cells pretreated with OTA (open arrows). d) The sequence‐scrambled control tracer (SCR) was not internalized by the cells. Note that treatment with tracer (11) induced membrane blebbing (arrowheads in b₁), which is associated with strong actin‐myosin‐based contractions.^{[ 52 ]} Whole cells were visualized with differential interference contrast (DIC, in left figure panels) to reveal fine details and structures.

Figure 10

Uptake of tracer (11) in mouse hippocampal neurons. a,a₁) Tracer (11) was internalized in a subpopulation of embryonic (E16.5) hippocampal neurons cultured for 14 days (arrows versus open arrows). b) and c) Pretreatment with mOTR‐specific OTA prevented compound (11) internalization, while the sequence‐scrambled control tracer (SCR) was not taken up by neurons. Whole cells were visualized with differential interference contrast (DIC, in left figure panels) to reveal fine details and structures.