Synthesis and Characterization of Click Chemical Probes for Single-Cell Resolution Detection of Epichaperomes in Neurodegenerative Disorders

Abstract

Neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.

Keywords: click probes; drug discovery; epichaperomes; fluorescence imaging; imaging probe; neurodegenerative disease; therapeutic strategy.

Figure 1

The epichaperome click probe design and principles of epichaperome detection in biological samples. (a) Chemical structures of the epichaperome drug candidates (left) and the designed probe, PU-TCO (right). PU-TCO incorporates the pharmacophore of the drug candidates and features a linker attached at N9, along with a trans–cyclooctene (TCO) moiety for in situ probe labeling. A negative control probe, PU-NTCO, containing a chemically similar but epichaperome-inactive moiety, is also depicted. (b) The schematic illustrates the concept of epichaperome illumination in biological samples. The probe should effectively permeate into cells and, once inside, interact specifically with epichaperomes by binding to a core component, HSP90, without interfering with the abundant HSP90 pools involved in physiologic chaperone folding functions. Upon in situ reaction with a fluorescent dye carrying a tetrazine functionality—the click step—the probe becomes fluorescent and can be detected by various methods. The schematic demonstrates the specific use of cy5-tetrazine as the fluorescent reporter.

Figure 2

Synthetic scheme for the synthesis of the PU-TCO epichaperome probe and the PU-NTCO control probe. Compound numbering, see Methods, Section 2.3.1, Section 2.3.2, Section 2.3.3, Section 2.3.4, Section 2.3.5, Section 2.3.6 and Section 2.3.7. Abbreviations: DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; rt, room temperature; NEt3, triethylamine; TFA, trifluoroacetic acid.

Figure 3

Evaluation of effective epichaperome binding by the designed probes in vitro, in cell homogenates, and in cellulo, in live cells. (a) A fluorescence polarization (FP) assay was used to measure the equilibrium competitive binding of the click probes to epichaperomes in cell homogenates obtained from an epichaperome-positive cell line (MDA-MB-468 breast cancer cells). Data are presented as the mean ± s.e.m., n = 3. PU-H71, positive control. (b) Detection of epichaperome components (chaperones and co-chaperones HSP90, HOP, and CDC37) through native-PAGE (left), followed by immunoblotting in cells treated in duplicate with DMSO (vehicle control) or PU-TCO (1 and 2 µM) for 1 h. Blue brackets indicate the approximate position of epichaperome-incorporated chaperones. Western blotting analysis (right, SDS-PAGE) was used to evaluate the total levels of these proteins. β-Actin serves as the protein loading control. Gel images are representative of two independent experiments, with each condition performed at two concentrations of the agent.

Figure 4

Evaluation of effective and selective epichaperome binding by PU-TCO in cellulo, in live cells. (a) Overview of the experimental design showing several control experiments designed to test probe specificity. Epichaperome-high (MDA-MB-468) and epichaperome-low (ASPC1) cancer cell lines were stained with the cy5 fluorescence reporter following the addition of PU-TCO (1 µM), PU-NTCO (1 µM, control epichaperome-inert probe), or PU-TCO after the pretreatment of the cells for 1 h with PU-H71 (1 µM) (competition). (b) Graph depicting the median, dotted line, and quartiles, dashed lines; n = 50 cells from 3 replicate experiments; one-way ANOVA with Sidak’s post-hoc. Each data point represents the mean fluorescence intensity recorded per cell. (c) Representative micrographs from three individual experiments, per panel (b). Images were captured using an LSM880 confocal microscope (Zeiss) with the 20×/0.8NA objective. Scale bars represent 50 µm.

Figure 5

Side-by-side evaluation of selective epichaperome binding by PU-TCO in cellulo compared to immunoblotting with an HSP90 antibody. (a) Schematic illustrating the biochemical and functional distinction between chaperones and epichaperomes. The HSP90 antibody detects all cellular pools of HSP90, whether involved in folding functions or incorporated into epichaperome platforms. Conversely, the PU-TCO probe should detect HSP90 only when part of epichaperomes, independent of the cellular concentration of HSP90. Epichaperome-high (MDA-MB-468) cancer cells, epichaperome-low (ASPC1) cancer cell lines and CCD-18Co, non-transformed colon fibroblasts, in culture, were stained with the cy5 fluorescence reporter following the addition of PU-TCO (1 µM), and with an Alexa488-labeled HSP90 antibody. (b) Representative micrographs from three individual experiments, per panel (a), are shown. Images were captured using an LSM880 confocal microscope (Zeiss) with the 20×/0.8NA objective. Scale bars represent 50 µm. (c) The data per panel (a) are presented as the mean ± s.e.m., n = 3, one-way ANOVA with Dunnetts’s post-hoc. Each data point represents the average of the mean fluorescence intensity recorded per cell from each experiment.

Figure 6

Evaluation of selective epichaperome detection by PU-TCO in post-mortem murine brains. (a,b) Frozen brains harvested from male M83 homozygous mice (a) and wild-type mice (b) at 13 months of age were sectioned (20 μm) for staining. Sagittal slices were incubated with PU-TCO (0.1, 0.5, and 1 µM), and then the cy5 fluorescent reporter was attached via click chemistry. Negative controls included PU-NTCO and blocking by pre-treating slices with PU-H71 (1 µM, 1 h) before incubation with the PU-TCO clickable probe. Epichaperomes, orange; Hoechst (blue), for visualization, and staining of cell nuclei. The slides were scanned on a Pannoramic Scanner (3DHistech) using a 20×/0.8NA objective. Scale bars represent 2 mm.

Figure 7

Evaluation of single-cell-level epichaperome illumination by PU-TCO in post-mortem murine brains. (a) Left side: Representative brain slice of male M83 homozygous mice (13 months of age) stained with Hoechst (blue) to detect individual brain cells and with an antibody against NeuN (Neuronal Nuclei, green) to discriminate neurons from glia. The approximate location of the ventral striatum is shown. Right side: Images for the anatomical location of brain areas closest to those used for epichaperome detection were obtained from the Allen Institute for Brain Science, Mouse, P56. Ventral striatum is highlighted in purple. Arrows point to the location of the nucleus accumbens and the olfactory tubercule. (b) Slices stained with PU-TCO (1 µM) and clicked to cy5, as shown in Figure 6, were re-imaged using a high-resolution microscope (Airyscan, Zeiss) to detect epichaperomes in individual cells. The micrograph shows the brain region encompassing the ventral striatum. The approximate location of its two subregions, the olfactory tubercle (OT), and the nucleus accumbens, as well as small clusters of neurons located within the ventral striatum of the brain, the Islands of Calleja, specifically found within the OT, are also shown. (c) Cell clusters, chosen from regions illustrated in panel (b), show the individual cells susceptible to epichaperome formation. The PU-TCO probe indicates that both neurons and glia are affected by epichaperomes (red). Blue represents Hoechst staining, while green represents NeuN.