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High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors N-Modified Thioflavin T Derivatives 3
Yuka Kataoka, Hiroto Fujita, Arina Afanasyeva, Chioko Nagao, Kenji Mizuguchi, Yuuya Kasahara, Satoshi Obika, and Masayasu Kuwahara Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01181 • Publication Date (Web): 29 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019
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Biochemistry
Yuka Kataoka,† Hiroto Fujita,‡ Arina Afanaseva,§ Chioko Nagao,§ Kenji Mizuguchi,§ Yuuya Kasahara,§,|| Satoshi Obika,§,|| Masayasu Kuwahara*,‡ †
Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan Graduate School of Integrated Basic Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan § National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki City, Osaka 567-0085, Japan || Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan ‡
Supporting Information Placeholder ABSTRACT: Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, further rapidity and simplicity of these methods are required for relevant techniques in laboratory researches and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N3-(d-desthiobiotinyl-PEGlyated) thioflavin T (ThT-PD) and N3-(cortisolylPEGlyated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. As compared with the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N3-ThT derivatives by rapid specific staining of glucocorticoid receptors (GRs) in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took about 24 h for immunostaining to capture the corresponding fluorescence images.
Thioflavin T (ThT) that was developed as a highly water-soluble mordant for natural fibers in 1889 was originally synthesized from dehydrothio-p-toluidine1. After 70 years, ThT was first applied as a fluorescent dye for amyloid detection by Vassar et al. in 19592. Currently, the formation of amyloid fibrils is known to associate with various diseases such as Alzheimer dementia, Parkinson’s disease, and type 2 diabetes3–5. Accordingly, ThT is widely used for diagnoses of these diseases. Furthermore, ThT gains attention to its potential applications to detect not only amyloids but particular nucleic acid structures as well, such as hairpin, G-quadruplex, and imotif ones6–10. ThT can be categorized into a fluorescent molecular rotor because its fluorescence intensity greatly depends on the dihedral angle (φ) between benzothiazole and dimethylaminobenzene rings11– 13. The ground and the excited states of ThT that is free in solution have φ of 37° and 90°, respectively, when its potential energy is at the lowest. The excited state of free ThT (φ = 90°) does not emit
Figure 1. Illustration of the N3-modified thioflavin T derivatives and their detection mechanisms to target proteins (A). Fluorescence spectra of ThT-PD (B) and ThT-P (C) (8 μM) in buffer PBS153NM (10 mM HPO42−, 146 mM Cl−, 153 mM Na+, 2.7 mM K+, and 2.5 mM Mg2+; pH 7.4) with increasing concentration of SA (0, 1, 2, 4, 6, 8, 12, 16, and 20 μM). Fluorescence spectra of ThT-PC (D) and ThT-P (E) (4 μM) with increasing concentration of anti-cortisol antibody [XM210] (0, 1, 2, 3, 4, 5, and 6 μM). Fluorescence spectra were measured at 25 °C with excitations at 430 nm (B and C) and 440 nm (D and E).
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Figure 2. Fluorescence microscopy images of MCF-7 cells with ThT-PC (A) (100 μM), ThT-P (B) (100 μM), and anti-GR antibody [BuGR2] (1:12.5) and goat anti-mouse IgG H&L (Alexa Fluor® 488) (1:200) (C) and DAPI (1 μM). fluorescence (quantum yield: Φ < 0.001) owing to the radiationless deactivation caused by non-radiative twisted internal chargetransfer. Meanwhile, when ThT binds to targets and thereby φ is restricted to be about 20°–40° at the excited state, fluorescence emission derived from the locally excited state can be observed (Φ = 0.3–0.8)14–16. Therefore, high contrast images having low background fluorescence can generally be obtained using ThT without removing excess probe molecules17. To date, we developed ThT derivatives by introducing substituents instead of methyl groups to the N3-position adjacent to the rotational axis18. We demonstrated that these fluorophores enabled to
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selectively emit fluorescence upon binding to parallel G4s18–21. However, so far, targets of ThT are still limited to β-sheet-rich proteins like amyloid fibrils and some abovementioned nucleic acid structures. Accordingly, in this study, we attempted to develop a fluorescence probe having a different target-directing property from the unmodified ThT by the conjugation of a ligand to N3-position. Namely, N3-(d-desthiobiotinyl-PEGlyated) thioflavin T (ThT-PD) and N3-(cortisolyl-PEGlyated) thioflavin T (ThT-PC) that bore d-desthiobiotin as a modifier of d-biotin and cortisol as a kind of glucocorticoids (GCs), respectively, were synthesized from N3-PEGlyated thioflavin T (ThT-P) to target streptavidin (SA) and glucocorticoid receptors (GRs), separately (Fig. 1A and Scheme S1). Based on the X-ray crystallographic data for these target proteins, the PEG linker with a length of about 68 Å was selected, which is long enough to reach the depth of the ligand binding pockets so that the ThT portion did not interfere with the ligand moiety binding to the target. The fluorescence properties of the ThT derivatives for the respective targets were then examined (Figs. 1–4 and Figs. S1–S8). First, we conducted fluorescence titrations of ThT-PD with the target SA, which can form an extremely stable complex with d-biotin and d-desthiobiotin, respectively (Fig. 1B). The dissociation constant (Kd) values were 10−14 and 10−13 M. SA was chosen for the first test because we assumed that the strong specific ligandreceptor interactions22 should provide unambiguous outcomes. ThT-P was used as a negative control, which has a PEG linker but without a ligand moiety binding to the target (Fig. 1C). Consequently, the fluorescence intensity of ThT-PD showed 27-fold increase in sufficient SA concentration. Meanwhile, as with ThT-P, ThT-PD emitted little fluorescence without SA (Fig. 1B and 1C). The titration of ThT-PD with SA provided a sigmoidal curve that had an inflection point at a molar ratio of approximately 0.7 (Fig. S1A). Considering that SA forms a homo-tetramer by each subunit binding to a single ligand23, the inflection point may be caused by
Figure 3. Visual observation of fluorescence microscopy images of MCF-7 cells with ThT-PC (100 µM) at 0, 1, 2, 4, 6, 8, 10, 15, 20, and 30 min (A). Scale bar = 50 µm. Illustrations of the comparison between using ThT-PC and immunostaining methods (B). Immunostaining methods required a 5-min soaking step before washing; however, ThT-PC did not require this step.
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Biochemistry
Figure 4. Binding mode of docking obtained conformations: a biotin-like moiety of ThT-PD molecule bound in SA binding pocket compared with the crystal conformation of biotin (pink) (A); a ThT moiety of ThT-PD bound on the surface of SA (B); a cortisol-like moiety of ThTPC molecule bound in GR binding pocket compared with the crystal conformation of cortisol (colored in pink) (C); ThT moiety of ThT-PC bound on the surface of GR protein. H-bonds are shown with dotted lines (D). the fact that an allosteric conformational change in the SA-binding site is induced by the first binding with ThT-PD24. Thereby, the affinity of the other three ligand-binding pockets can be enhanced22. The titration curve showed that the target SA can be quantitatively detected by ThT-PD in the range of molar ratio (target versus probe) from 0.5 to 1.0 (R2 = 0.996)25,26. The deficiency of d-biotin in blood is known to cause alopecia, loss weight, psychiatric disorders, and increased blood glucose levels27. Therefore, we assumed that ThT-PD can quantitatively detect d-biotin using a significant difference between d-biotin and d-desthiobiotin in binding affinity to SA. We then attempted an exchange reaction between ThT-PD and d-biotin in association of SA (Fig. S2A and S2B). As expected, the fluorescence intensity of ThT-PD was decreased by increasing the concentration of d-biotin. Namely, ThT-PD as a weak SA binder was replaced with d-biotin as a strong SA binder, which is attributable to the dissociation of ThT-PD from SA. Using such competitive methods, ThT-PD enabled the quantitative detection of a small molecular ligand (d-biotin) as well as its receptor protein (SA) (Fig. S2C). Next, we examined the fluorescence response of ThT-PC to an anti-cortisol antibody (XM210) as a target (Fig. 1D and 1E), which has a Kd value for the target cortisol of approximately 10–10 M. The fluorescent intensity of ThT-PC in excess of XM210 was found to be 8-fold higher than that without XM210. As with ThT-P, only little fluorescence intensity of ThT-PC was observed without XM210. The fluorescence titration curve was fitted by a single-site binding model, showing that ThT-PC can quantitatively detect XM210 in the range of molar ratio (target versus probe) from 0.25 to 0.75 (R2 = 0.961). Furthermore, similar to the ThT-PD/SA system, cortisol can be quantitatively detected by ThT-PC in the presence of XM210 under the competitive condition (Fig. S2D–S2F).
Cortisol is known as one of the biomarkers for psychological stress; therefore, the development of convenient methods for cortisol detection is necessary for diagnosis based on an objective index to improve the effectiveness of mental health care28. To evaluate the feasibility of ThT-PC, we attempted to substantiate rapid and facile fixed-cell imaging of GRs that are a member of the nuclear receptor superfamily. Exerting anti-proliferative and anti-apoptotic activities, GRs are overexpressed particularly in breast cancer cell lines29,30. In the progression of breast cancers, psychological stress is known to promote synthesis of GCs31. Accordingly, the working mechanisms of GCs and GRs are currently being investigated in detail32. Immunostaining has generally been used for research and development of methods for cancer therapy, while the techniques require time-consuming processes and technical skills32–34. As Fig. 2A shows, GR specific staining images of immobilized MCF-7 breast cancer cells were yielded using ThT-PC. No fluorescent image was obtained when ThT-P was used for the negative control instead of ThT-PC (Fig. 2B). Meanwhile, immunostaining was also conducted using anti-GR antibody [BuGR2] and Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) as primary and secondary antibodies for the positive control, respectively (Fig. 2C). Intriguingly, ThT-PC could stain only GRs in cytoplasm, although the immunostaining stained them in both cytoplasm and nuclei (Fig. 2A and 2C). The possible explanation for this is that GRs are predominantly localized in the cytoplasm but translocate to the cell nucleus soon after binding with GCs, which is subsequently followed by phosphorylation, and the GRs then immediately bind with glucocorticoid-responsive genes35,36. Since GCs bind tightly to the DNA-binding form of GRs, the ThT-PC could not stain GRs located in the nucleus, unlike the anti-GR antibody, although it was
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still capable of staining free GRs in the cytoplasm37. Also of note, we visually recognized GRs in cytoplasm in only six min after the addition of ThT-PC, whereas it took about 24 h to handle a severalstep process by immunostaining (Fig. 3 and S3)38–41. No fluorescent image was captured using ThT-P or ThT-PD as negative controls (Fig. S4). Similarly, specific staining of GRs in the cytoplasm with ThTPC was also confirmed using a human colon carcinoma cell line of Colo20530 that expresses GRs at a lower level than MCF-7 (Fig. S5). In the case of Colo205, the total intensity of fluorescence per a single cell was approximately 2-fold lower than that obtained in the experiment using MCF-7 (Fig. S6A), which is in good agreement with the results from the immunostaining (Fig. S6B). The dependency of fluorescent emission on the target amount ensured the feasibility of ThT-PC as a dye specific to GRs for cell staining. It is known that the translocation of GRs from cytoplasm to nucleus can be provisionally promoted by exposure to dexamethasone (Dex)31,34. To observe the fluctuation of GR abundance in cytoplasm, we chronologically monitored the change in the fluorescence of ThT-PC after the addition of Dex to cells. As Fig. S7 shows, the fluorescence intensity in the cytoplasm was decreased to about 30% after 30 min since the addition of Dex, which was recovered to the original level after 6 h. This is consistent with the result presented in a previous report34, which employed a conventional immunostaining method, supporting our hypothesis with respect to the specific staining of GRs in cytoplasm with ThT-PC. Finally, we proposed possible binding modes of ThT-PD and ThT-PC to their respective target receptors, SA and GR, using molecular docking (for details of the methodology, see the section “Docking simulation” in Supplementary materials). The complex structure of ThT-PD with SA tetramer is shown in Fig. 4A and 4B. A biotin-like molecular moiety is bound within one of the four equivalent binding pockets (Fig. 4A). This portion forms several H-bonds with pocket residues, similar to the crystal structure of the biotin in complex with SA (PDB ID: 1STP 42). The ThT moiety in this simulation was found to be delved into the crevice formed at the entrance of the opposite binding pocket (Fig. 4B). Although this site has no appropriate aromatic residues to form stacking interactions, this conformation might be favorable for the ThT moiety and can stabilize its configuration in the emitting state with φ ~38.5°. In case of ThT-PC bound to GR (PDB ID for the receptor: 4UDC43), there is also a cortisol-like molecular moiety bound within the main ligand binding pockets of GR (Fig. 4C). This moiety forms a network of H-bonds with surrounding residues. As a whole, the binding mode and conformation of this portion are similar to the conformation of the cortisol in complex with GR (PDB ID: 4UDC). The ThT moiety of ThT-PC is bound on the protein surface where it forms a pi-stacking interaction with Y648 residue. The configuration of ThT moiety is stabilized with its dihedral angle φ close to −38°, which corresponds to the fluorescence emitting state (Fig. 4D). In conclusion, we demonstrated that ThT derivatives tethering a ligand at the N3-position via the linker chain enabled the specific detection of targets, while the properties of the fluorescent molecular rotor were retained. The great merit of the present probes is that high contrast fluorescence images can be facilely obtained in a short period of time owing to their high target specificity and low background emission. Furthermore, another advantage is that these detection probes are very simply designed and created by merely conjugating with a ligand. Accordingly, we expect that molecular probes for various disease targets can be developed based on the present concept for molecular design and attempted to clinical applications such as bedside diagnosis and operative rapid pathologic diagnosis in the future.
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The Supporting Information is available free of charge on the ACS Publications website. Experimental details for all chemical reactions and measurements (PDF)
The atomic coordinates and crystallographic structure factors of SA and GR have been deposited in the Protein Data Bank as entries 1STP and 4UDC and in the UniProtKB as entries P22629 and P04150, respectively.
*
[email protected] Masayasu Kuwahara: 0000-0002-0810-4627
The authors declare no conflict of interest.
This study was partly supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Japan Agency for Medical Research and Development (AMED), and by a Grant-in-Aid for Scientific Research (C), No. 18K05347, from the Japan Society for the Promotion of Science (JSPS). The first author Y. K. is grateful to JSPS Research Fellowships for Young Scientists.
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Illustration of the N3-modified thioflavin T derivatives and their detection mechanisms to target proteins (A). Fluorescence spectra of ThT-PD (B) and ThT-P (C) (8 μM) in buffer PBS153NM (10 mM HPO42−, 146 mM Cl−, 153 mM Na+, 2.7 mM K+, and 2.5 mM Mg2+; pH 7.4) with increasing concentration of SA (0, 1, 2, 4, 6, 8, 12, 16, and 20 μM). Fluorescence spectra of ThT-PC (D) and ThT-P (E) (4 μM) with increasing concentration of anti-cortisol antibody [XM210] (0, 1, 2, 3, 4, 5, and 6 μM). Fluorescence spectra were measured at 25 °C with excitations at 430 nm (B and C) and 440 nm (D and E).
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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 2. Fluorescence microscopy images of MCF-7 cells with ThT-PC (A) (100 μM), ThT-P (B) (100 μM), and anti-GR antibody [BuGR2] (1:12.5) and goat anti-mouse IgG H&L (Alexa Fluor® 488) (1:200) (C) and DAPI (1 μM). 86x62mm (300 x 300 DPI)
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Biochemistry
Figure 3. Visual observation of fluorescence microscopy images of MCF-7 cells with ThT-PC (100 µM) at 0, 1, 2, 4, 6, 8, 10, 15, 20 and 30 min (A). Scale bar = 50 µm. Illustrations of the comparison between using ThT-PC and immunostaining methods (B). Immunostaining methods required a 5-min soaking step before washing; however, ThT-PC did not require this step. 174x103mm (300 x 300 DPI)
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Binding mode of docking obtained conformations: a biotin-like moiety of ThT-PD molecule bound in SA binding pocket compared with the crystal conformation of biotin (pink) (A); a ThT moiety of ThT-PD bound on the surface of SA (B); a cortisol-like moiety of ThT-PC mole-cule bound in GR binding pocket compared with the crystal conformation of cortisol (colored in pink) (C); ThT moiety of ThT-PC bound on the surface of GR protein. H-bonds are shown with dotted lines (D).
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Biochemistry
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