Compound sensing clathrin-mediated endocytosis, fluorescent probe comprising same, and sensor and sensing method

Abstract

The present invention relates to a compound sensing clathrin-mediated endocytosis, a fluorescent probe comprising same, and a sensor and a sensing method. The compound according to the present invention forms a micelle in a quenching state, and when in contact with a cell, the compound is transported into the cell through clathrin-mediated endocytosis and becomes fluorescent. Accordingly, the compound can be used to sense clathrin-mediated endocytosis or stain and label cells in which clathrin-mediated endocytosis takes place. The compound used in the present invention is particularly excellent in that the compound is stably transported via endocytosis into cells, such as live neurons or embryonic stem cells, within a relatively short time and maintains fluorescence for a long time at high fluorescence intensity. In addition, not only somatic cells but also cell-membranes and spikes can be stained.

Description

Compounds for sensing the operation of clathrin-mediated endocytosis, fluorescent probes comprising the same, sensors, and sensing methods

[0001] The present invention relates to a compound for sensing the operation of clathrin-mediated endocytosis, a fluorescent probe for labeling cells in which clathrin-mediated endocytosis is performed, a sensor using the compound, and a sensing method.

[0002] Bioimaging is a technology that visually depicts biochemical changes occurring at the cellular or subcellular molecular level. It is being researched and utilized in a wide range of fields. In particular, because it allows for real-time visual observation of changes within living organisms and cells, as well as objective quantification, it can be a key technology in disease diagnosis and treatment, as well as new drug development.

[0003] Bioimaging typically uses probes to stain specific cells and, through sensors, reveal internal biological structures and various biochemical reactions that are invisible to the naked eye. Organic fluorescent materials can be used as probes. Their small size, diverse wavelengths, ease of chemical modification, and biocompatibility make them highly applicable and promising. Their small size makes them essential in single-molecule and high-resolution imaging.

[0004] However, conventional organic fluorescent substances have the disadvantage of being easily decomposed or rapidly released from cells, making them difficult to use for long-term tracking and observation of cellular development or changes. Furthermore, they stain only a small portion of the cell, such as the soma of a neuron, leaving other parts, such as the cell membrane or protrusions, unstained. Therefore, observing the entire cell may require the use of multiple different fluorescent substances. Furthermore, imaging the movement or changes of specific substances at the subcellular level may require conditions hostile to cellular metabolism, potentially causing oxidative damage to the cell. Therefore, there is a need for the development of small-molecule fluorescent substances that enable selective, long-term imaging of substances at the cellular or subcellular level without altering the cellular environment.

[0005] Meanwhile, the process of endocytosis is crucial for cell physiology. It is the process by which cells absorb metabolites, hormones, proteins (including viruses), etc., through invagination of the plasma membrane. Specifically, the plasma membrane invaginates around a target substance bound to an extracellular receptor, forming a vacuole. Once this vacuole matures, it is cleaved from the plasma membrane to form a vesicle, allowing the substance to enter the cell.

[0006] Because endocytosis is strictly mediated by receptors on the cell surface, only receptor-specific substances can enter the cell through this process. Therefore, targeting specific receptors on the cell surface can be utilized in research to improve drug delivery, such as to immune cells. Furthermore, the trafficking rate of receptors can regulate the duration and intensity of signal transduction, ultimately determining cell growth, motility, and cell fate. Therefore, a thorough understanding and utilization of the molecular mechanisms of endocytosis will contribute to the regulation of cellular biological processes and the development of effective therapeutics.

[0007] The purpose of the present invention is to find a fluorescent organic compound that can selectively stain the entire cell without creating a hostile environment and has excellent fluorescence intensity and fluorescence retention time, and that can solve the problems of the prior art, and to provide a method for sensing the operation of clathrin-mediated endocytosis using the same, a sensor for sensing the operation of clathrin-mediated endocytosis, and a fluorescent probe for labeling cells in which clathrin-mediated endocytosis is performed.

[0008] The present inventors have discovered that a compound of the following chemical formula 1 can achieve the above-mentioned purpose, thereby completing the present invention:

[0009] [Chemical Formula 1]

[0010]

[0011] In the above chemical formula 1, R is a straight-chain alkyl group having 5 to 8 carbon atoms. According to one embodiment, R may be a straight-chain alkyl group having 6 to 8 carbon atoms, for example, a straight-chain alkyl group having 7 carbon atoms, i.e., n-heptyl.

[0012] As can be confirmed in the experimental examples described below, the compound of formula 1 forms micelles. The compound of formula 1 in the form of micelles is in a quenched state, and when this micelle comes into contact with a cell, it is translocated into the cell through clathrin-mediated endocytosis and emits fluorescence. This is surprising in that, unlike the present invention, in the case of a compound in which the substituent R in formula 1 is n-propyl (3 carbon atoms), neither micelle formation nor clathrin-mediated endocytosis occurs. Therefore, the compound of formula 1 of the present invention can be utilized to sense the operation of clathrin-mediated endocytosis or to stain cells in which clathrin-mediated endocytosis is activated. Here, sensing includes not only the presence or absence of fluorescence emission but also the measurement of the activity of clathrin-mediated endocytosis by measuring the fluorescence intensity. For example, healthy neurons have high clathrin-mediated endocytosis activity, whereas degenerating or dying neurons exhibit low activity. As can be confirmed in the experimental examples described below, the compound according to the present invention or the fluorescent probe comprising the same is remarkable in that the fluorescence intensity and fluorescence retention time are 3 to 4 times or more higher than those of a comparative compound in which the substituent R in Chemical Formula 1 is n-propyl (3 carbon atoms).

[0013] In particular, the compound of chemical formula 1 of the present invention is excellent in that it can rapidly and stably undergo endocytosis in cells, specifically, live neurons or embryonic stem cells, and maintain fluorescence for a long period of time with high fluorescence intensity. In particular, it is possible to stain all parts of the cell, including the soma, filopodia, and lamellipodia of neurons, as well as the cell membrane and protrusions.

[0014] The present invention relates to a compound for sensing the operation of clathrin-mediated endocytosis, a fluorescent probe for labeling cells in which clathrin-mediated endocytosis is performed, a sensor using the compound, and a sensing method.

[0015] The compound of the present invention forms micelles in a quenched state, and upon contact with cells, is translocated into the cells through clathrin-mediated endocytosis and emits fluorescence. Thus, the compound can be utilized to sense the operation of clathrin-mediated endocytosis or to stain and label cells undergoing clathrin-mediated endocytosis.

[0016] The compound of the present invention is stably and relatively rapidly endocytosed into cells, specifically, living neural cells or embryonic stem cells. Furthermore, the compound of the present invention or a fluorescent probe comprising the compound is particularly advantageous in that it is selective for the cells without altering the cellular environment and can maintain fluorescence at high fluorescence intensity for a very long time. Furthermore, it is capable of staining the entire cell membrane and protrusions, including soma, filopodia, and lamellipodia.

[0017] Accordingly, the compound of the present invention, or a fluorescent probe comprising the same, can be used for high-resolution imaging, and can also be used to track and observe the development or changes of cells over a long period of time, and in particular, can be sufficient to observe entire cells alone without the addition of other compounds or fluorescent probes.

[0018] Figure 1 shows the results of evaluating the neuronal staining properties of the derivative, including (a) the chemical formula of the derivative, (b) the alkane side chain of the derivative, and (c) a fluorescent image of primary neurons treated with the derivative.

[0019] Figure 2 shows the results of evaluating the neuronal staining properties of the derivatives, where (a) the fluorescence intensity of the derivatives was quantified at different time points, and (b, c) are representative fluorescence images of primary neurons co-stained with C3 or C7 (NeuM) using SR101, where (b) is C3 and (c) is NeuM (Scale bar, 50 μm; fluorescence intensities in the white boxes were analyzed below). (d, e) Representative images of fluorescence and immunofluorescence of C3 or C7 (NeuM) using β-III tubulin antibody in hippocampal neurons, where (d) is C3 and (e) is NeuM (Scale bar, 25 μm).

[0020] Figures 3 and 4 show that NeuM is incorporated into the neuronal cell membrane. (A) Representative images of hippocampal neurons stained with NeuM and βIII-tubulin antibodies (Scale bar, 10 μm), (B) Higher magnification images of βIII-tubulin immunostained neurons and neurites stained with NeuM (50 nM) (Scale bar, 5 μm), (C) Cytoplasmic and membrane fraction diagrams of NeuM-treated neurons, (D) NeuM fluorescence images of whole, cytoplasmic, and membrane fractions under a UV lamp (325 nm), (E) Fluorescence spectra of NeuM in whole cell, cytoplasmic, and membrane fractions, (F) Heat maps of fluorescence intensities of C3 derivatives in each fraction, (G) Time-dependent fluorescence intensity profiles of C3 derivatives in the presence of phosphatidylcholine (PC) liposomes, (H) Screenshots of selected MDs showing C3 and C7 (NeuM) in PC membranes, (I) Graph panels show the normalized partial densities of boron atoms in the core and phosphate groups (dotted lines) of PC for C3 and NeuM in the inner and outer membrane leaflets at 303.15 K (results are averaged and follow MD simulations over a time range of 25–100 ns). (J) Representative screenshots of molecular interactions between membrane lipids and C3 or NeuM taken from MD simulations. H-bonds, hydrophobic interactions, and unfavorable donor–donor interactions are indicated by dotted lines. The bar chart shows the number of H-bonds between small molecules and the lipid membrane over a time range of 25–100 ns.

[0021] Figures 5 and 6 show that NeuM forms micelles. (A) is a fluorescence image of NeuM, (B) is a transmission electron microscope image of NeuM in PBS or PBS containing 10% TritonX-100 (scale bar, 50 nm), (c) is a quantification of the size of NeuM micelles in PBS based on the TEM image, and the average diameter of 684 micelles was calculated (12.8±2.8 nm). (D) is a schematic depicting the incorporation of NeuM into the neuronal membrane, and (E) is a schematic depiction of NeuM (50 nM) and MemGlow. TM Representative fluorescence images of primary neuronal cells (DIV7) stained with 640 (50 nM) (white arrows indicate MemGlow TM (F) NeuM and MemGlow labeled with various liposomal vesicles; Scale bar, 50 μm) TM Time-dependent fluorescence intensity profiles of 640 and (G) representative fluorescence images of neuronal and non-neuronal cells treated with NeuM, where NeuM fluorescence response was quantified; ****p<0.0001,nsnonsignificant (compared to 25 nM NeuM-treated cells).

[0022] Figures 7-8 demonstrate that clathrin-mediated endocytosis is crucial for neuronal trafficking of NeuM. (A) Representative NeuM fluorescence images of primary neurons treated with inhibitors of endocytosis and exocytosis pathways, (B) quantification of NeuM fluorescence intensity at various time points (***p<0.001,nsnonsignificant), (C) time-dependent fluorescence images of NeuM showing that NeuM association at the soma and neurite tips is delayed by dansylcadaverine treatment, and the 3D surface plot represents the NeuM fluorescence intensity profile at 120 min (Scale bar, 10 μm). (D) Higher magnification image of primary neurons treated with dansylcadaverine and endocytidine 2 (Scale bar, 10 μm). (E) Schematic showing NeuM entry into cells via clathrin-mediated endocytosis (CME), and (F-H) Immunoblot analysis of proteins involved in clathrin-mediated endocytosis, clathrin-independent endocytosis, and endosomes in lysates of primary hippocampal neurons, C8-D1A, SIM-A9, SH-SY5Y, and HeLa cells (****p<0.0001,***p<0.001,**p<0.01,nsnonsignificant (compared to primary neurons)).

[0023] Figures 9 and 10 show the results of evaluating the neural selectivity of NeuM at the neural differentiation stage. (A) is a representative fluorescent image of NeuM and β-III tubulin immunostaining (Scale bar, 50 μm), (B) is the quantification of neurite length, root number, and extremity analyzed through NeuM and β-III tubulin fluorescent images (****p<0.0001,***p<0.001,**p<0.01,*p<0.05,nsnonsignificant (compared to β-III tubulin)), (C) is the quantification of NeuM (+) or β-III tubulin neurons at DIV 1, 3, 6, 9, and 13, and (D) is an enlarged image (at DIV3 and DIV9) of the boxed area (i-iv) in (a) above (Scale bar, 10 μm). (E) Representative images of NeuM-labeled neurons at five stages of differentiation (Scale bar, 5 μm). (F) High-resolution images of neurites from NeuM-labeled neurons (Scale bar, 5 μm).

[0024] Figure 11 shows the results of neuronal integrity assessment of primary neurons treated with derivatives. (a) Primary hippocampal neurons (DIV7) treated with 50 nM derivatives and stained with anti-βIII-tubulin antibody. The results are neuron number, root number, limb number, and maximum neurite length (ns nonsignificant (compared to reference)), and (b) cell viability results (ns nonsignificant (compared to reference)).

[0025] Figure 12 shows the fading of C3 and C7 (NeuM) after fixation and permeabilization. (a): Primary hippocampal neurons (DIV7) were treated with 50 nM C3 or C7 (NeuM) for 2 h, the nuclei were counterstained with Hoechst, and fluorescence images were acquired. Then, C3 or C7 (NeuM)-stained primary neurons were fixed with 3.7% paraformaldehyde for 20 min and then permeabilized in 0.3% Triton X-100 in PBS for 10 min. Fluorescence images were acquired after the fixation and permeabilization. (b) Quantification of C3 and C7 (NeuM) fluorescence intensities, which were normalized to the level of fluorescence intensity in live neurons (****p<0.0001 (compared to live neurons)).

[0026] Figure 13 shows the results of cytosolic and membrane fraction evaluation. (a) is the result of immunoblot analysis of tubulin and cadherin in whole cell, cytosolic, and membrane fractions of NeuM-treated neurons, and (b) is the result of quantifying tubulin and cadherin protein levels in whole cell, cytosolic, and membrane fractions (****p<0.0001, **p<0.01 (compared to whole cell fraction)).

[0027] Figure 14 compares the membrane preferences of the derivatives. (a) Fluorescence intensities of the derivatives in whole cell, cytosol, and membrane fractions (***p<0.001,**p<0.01,nsnonsignificant (compared to whole cell fraction)), and (b) quantifies the membrane fluorescence percentage, which represents the ratio of fluorescence intensity of the membrane fraction to that of the total cell fraction.

[0028] Figure 15 shows NeuM uptake in the presence of an exocytosis inhibitor (***p<0.001,nsnonsignificant (compared to reference)).

[0029] Figure 16 shows the expression levels of CME-related proteins at DIV 3, 6, 9, and 12. (a) Immunoblot analysis of clathrin, AP, and transferrin in lysates of primary hippocampal neurons at four maturation stages (DIV3, 6, 9, 12, and C8-D1A cells). b-dorxs was used as a loading control. (b) Quantification of the expression levels of proteins related to clathrin-mediated endocytosis (**p<0.01, *p<0.05, n.s. nonsignificant (compared to PN at DIV3)).

[0030] According to one aspect of the present invention, a compound of the following chemical formula 1 is provided:

[0031] [Chemical Formula 1]

[0032]

[0033] In the above chemical formula 1, R is a straight-chain alkyl group having 5 to 8 carbon atoms. According to one embodiment, R may be a straight-chain alkyl group having 6 to 8 carbon atoms, for example, a straight-chain alkyl group having 7 carbon atoms, i.e., n-heptyl.

[0034] According to one aspect of the present invention, a fluorescent probe for labeling cells in which clathrin-mediated endocytosis operates, comprising a compound of the above chemical formula 1, is provided.

[0035] According to another aspect of the present invention, a sensor for sensing the operation of clathrin-mediated endocytosis, comprising a compound of the above chemical formula 1, is provided.

[0036] According to another aspect of the present invention, a method for sensing the operation of clathrin-mediated endocytosis in a subject is provided, comprising the steps of (a) contacting a compound of the above chemical formula 1 with a subject, and (b) determining whether the subject emits fluorescence.

[0037]

[0038] According to one embodiment of the present invention, the compound used in the sensing method, sensor, and fluorescent probe of the present invention may exist in the form of a micelle prior to endocytosis. In the present specification, the term "micelle" may be used to encompass any aggregate form, with or without internal space. According to one embodiment of the present invention, the micelle may have a diameter of 10 to 16 nm, for example, 11 to 14 nm or 12 to 13 nm.

[0039] According to one embodiment of the present invention, the compound used in the sensing method, sensor, and fluorescent probe of the present invention may have an AlogP value as a measure of lipophilicity of 7 to 7.4, for example, 7.2 to 7.3. Since the AlogP value of cholesterol is 7.376, it can be seen that the compound of the present invention having an AlogP value in the above numerical range is lipophilic. In addition, cholesterol is an essential component of the plasma membrane, maintaining structural integrity and regulating the fluidity of the cell membrane. Therefore, the fact that the AlogP value of the compound of the present invention is similar to that of cholesterol means that the compound of the present invention can be incorporated into the plasma membrane and stably maintained in the membrane.

[0040] According to one embodiment of the present invention, the compound of the present invention or the fluorescent probe comprising the same may have a time to reach maximum fluorescence intensity after contact with a subject, which may be 5 to 7 hours, for example, 6 hours. Furthermore, according to one embodiment of the present invention, the compound or the fluorescent probe may have a fluorescence retention time of more than 25 hours, specifically, 26 hours or more, 35 hours or more, 40 hours or more, or 48 hours or more. For example, according to one embodiment of the present invention, the compound may have a time to reach maximum fluorescence intensity after contact with a subject, which may be 5 to 7 hours, and a fluorescence retention time of more than 25 hours.

[0041] According to one embodiment of the present invention, the compound or the fluorescent probe comprising it may fluorescently label living nerve cells, particularly, living nerve cells in which clathrin-mediated endocytosis operates, or embryonic stem cells. Furthermore, according to one embodiment of the present invention, the compound or the fluorescent probe comprising it may fluorescently label the cell membrane of a nerve cell. Furthermore, according to one embodiment of the present invention, the compound or the fluorescent probe comprising it may fluorescently label a protrusion of a nerve cell, for example, a dendrite, and may also stain both the soma and the protrusions of a nerve cell. Furthermore, according to one embodiment of the present invention, the compound or the fluorescent probe comprising it may fluorescently label an axon terminal region of a nerve cell.

[0042] In addition, according to one embodiment of the present invention, the compound and the fluorescent probe comprising the compound can fluorescently label filopodia and lamellipodia of a cell. For example, the filopodia and lamellipodia may be located at the axon terminals of nerve cells, and the filopodia and lamellipodia at the axon terminals of nerve cells are rarely observed by conventional immunostaining such as βIII-tubulin.

[0043] The compounds of the present invention can selectively stain cells undergoing clathrin-mediated endocytosis in vivo, such as neural cells or embryonic stem cells, and maintain fluorescence for the long-term described above. Therefore, they can be used to sense or label cells undergoing clathrin-mediated endocytosis.

[0044] Furthermore, the compounds of the present invention can be used as liposomes or liposome components for selective substance delivery into the aforementioned cells, such as nerve cells. Furthermore, the compounds of the present invention can be used as drug delivery vehicles into cells via clathrin-mediated endocytosis, through binding or combination with DNA, RNA, drugs, etc.

[0045] Hereinafter, the present invention will be described in more detail with reference to examples to aid understanding. However, the following examples are provided solely to facilitate understanding of the present invention and are not intended to limit the scope of the present invention.

[0046]

[0047] [Synthesis Example] Synthesis of compound derivatives

[0048]

[0049]

[0050] A boron-dipyrromethene (BODIPY)-loaded resin was swelled in DMF for 30 min. NaN3 (9 equiv) in DMF was added, and the reaction mixture was shaken at room temperature for 1 h. The reaction was washed five times with DMF. Then, benzylamine (15 equiv) and N,N-diiopropylethylamine (DIEA) (30 equiv) in DMF were added, and the reaction mixture was shaken at room temperature for 2 h. After washing five times with DMF, alkyne (10 equiv), CuI (4 equiv) and ascorbic acid (4 equiv) were added, and the suspension was shaken for an additional 1 h. The resin was then washed with DMF (X 5) and DCM (X 5), followed by cleavage with 0.5% TFA / DCM (10 min X 5). The residue was concentrated and purified by HPLC.

[0051] The crude product was dissolved in DCM and treated with acetoxylacetyl chloride (5 equiv) and saturated aqueous NaHCO3 (10 equiv). The reaction mixture was stirred at room temperature for 5 h and concentrated. Next, the residue was dissolved in MeOH (3 mL) and water (0.3 mL), to which Na2CO3 (10 equiv) was added, and the mixture was stirred at 50°C for 30 min. The reaction mixture was concentrated and purified by HPLD.

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] [Other general experimental procedures]

[0063] 1. Primary neuronal cell culture and preparation of lysates

[0064] Primary hippocampal and cerebral cortex neurons were isolated from day 18 embryonic Sprague-Dawley rat brains. Neurons were seeded at 1.5 × 10 per well in poly-D-lysine-coated 96-well plates. 4Cells were seeded at a density of 10 μg / mL and maintained in neurobasal medium at 37°C in a humidified atmosphere of 5% CO2. The neurobasal medium was supplemented with 2% B27 (Gibco), 0.5 mM GlutaMAX (Gibco), 100 units / mL penicillin, and 100 μg / mL streptomycin (Welgene). Fifty percent of the medium was replaced with fresh neurobasal medium every 3 days. At DIV3, 6, 9, and 12, hippocampal neurons were washed with PBS and lysed in CelLyticM lysis reagent (Sigma) containing a protease / phosphatase inhibitor cocktail (Sigma).

[0065]

[0066] 2. Molecular modeling

[0067] All small molecules were prepared by sketching 2D structures in ChemDraw 16.0, and then generating 3D conformations in Discovery Studio software (DS) via the Ligand Preparation module. Optimization and energy minimization of the molecular 3D shapes were performed under the standard CHARMM force field. Molecular properties (logP, pka, molecular surface area, molecular volume, etc.) were then estimated using DS tools. In particular, DS calculates the octanol-water partition coefficient (LogP) using the atom-based method published by Ghose and Crippen, and is therefore annotated as AlogP.

[0068] Explicit membrane bilayer systems, topologies, and parameters for molecular dynamics simulations (MD) were prepared using the CHARMM-GUI generator Membrane Builder. Two systems were generated: one for the C7 (NeuM) probe and one for the C3 probe. Simulations were performed using the GROMACS 2016.4 package under the CHARMM36m forcefield. Long-range electrostatics were evaluated using PME (Particle Mesh Ewald) with a cutoff of 1.2 nm. The systems were subjected to a stress of 100 kJ / mol. -1 ㆍnm-1 The maximum force was minimized using a steepest descent algorithm with up to 50000 steps until convergence. Equilibration at 303.15 K and 1 bar was achieved using a Berendsen thermostat, Berendson semi-isotropic pressure coupling (coupling constant 5.0 and compressibility 4.5ㆍ10 -5 bar -1 ) and applying LINCS h-bond constraints. The harmonic constraints during the six-step equilibration were from 2000 to 0 kJㆍmol -1 ㆍnm -2 was gradually released. Production was carried out with a Nose'-Hoover temperature control device and semi-isotropic coupling (coupling constant 5.0 and compressibility 4.5ㆍ10 -5 bar -1 ) was performed for 100 ns using a Parrinello-Rahman pressure control device with 3D periodic boundary conditions.

[0069]

[0070] 3. Quantum yield measurements of NeuM in various solvents

[0071] To measure the fluorescence quantum yield of NeuM in various solvents, NeuM (50 nM) was dissolved in PBS containing 0.1% DMSO, 10% TritonX-100, or PC liposomes (1 mg / mL). The quantum yield values ​​of NeuM were determined by comparison with rhodamine 123. NeuM and rhodamine 123 were excited at the same wavelength (λex = 475 nm), and the integrated emission intensities were measured, and the quantum yields were calculated using the following equation:

[0072]

[0073] QYx represents the quantum yield of the experimental probe, while QYs represents the quantum yield of the standard (rhodamine 123; QYs = 0.89). F is the integrated fluorescence intensity (area), and f is the absorption coefficient (f = 1-10 -A, where A = absorbance) and n is the refractive index of the solvent used.

[0074]

[0075] 4. Transmission electron microscope (TEM)

[0076] For TEM sample preparation, NeuM (50 nM) was dissolved in PBS or PBS containing 10% TritonX-100. Samples were placed on carbon-coated electron microscopy grids and negatively stained with 2% (w / v) aqueous uranyl acetate for 1 min. To image the quenched state of NeuM (micelles) in PBS, the grids were observed at an accelerating voltage of 120 kV using a Tecni G2 F20 transmission electron microscope (FEI). For each experimental condition, 25 random images were captured by an operator acting as a blinded observer. The size of 684 NeuM micelles in PBS was measured using ImageJ software (NIH).

[0077]

[0078] 5. Cell culture and preparation of lysates

[0079] C8-D1A, SIM-A9, SH-SY5Y, and Hela cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. For lysate preparation, SH-SY5Y, C8-D1A, SIM-A9, and Hela cells were washed with phosphate-buffered saline (PBS) and lysed in CelLyticM lysis reagent (Sigma) containing a protease / phosphatase inhibitor cocktail (Sigma).

[0080]

[0081] 6. Immunoblot analysis

[0082] Cell lysates were mixed with 4x Laemmli buffer containing 10% β-mercaptoethanol (Sigma) and boiled at 97°C for 5 min. For immunoblot analysis, 10 μg of each lysate was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The expression levels of proteins involved in clathrin-mediated endocytosis, clathrin-independent endocytosis, and endosomes were detected with the following antibodies. Anti-clathrin light chain (1:1000, Abcam), anti-AP2A1 (1:500, Abcam), anti-transferrin (1:1000, Abcam), anti-DNM1 (1:500, Cell Signaling), anti-caveolin-1 (1:1000, Abcam), anti-caveolin-2 (1:1000, Abcam), anti-EEA1 (1:250, Cell Signaling), and anti-RAP7B (1:1000, Abcam) antibodies. β-Actin (Sigma) was used as a loading control. Band intensities were quantified using Image J software (NIH).

[0083]

[0084] 7. Analysis of NeuM trafficking under inhibition of endocytosis / exocytosis pathways

[0085] At DIV7, cortical neurons grown in 96-well plates were pretreated with inhibitors of endocytosis and exocytosis pathways: dansylcadaverine (60 μM), trifluoperazine (3 μM), genistein (60 μM), endocytidine 2 (60 μM), or exo1 (60 μM). After pretreatment with inhibitors for several minutes, neurons were stained with NeuM (50 nM). Fluorescence images (λex = 460–490 nm and λem = 515–580 nm) were acquired at various time points (1, 7, 15, 30, 60, and 120 min) using an Operetta® CLS system. NeuM fluorescence intensities were analyzed using Harmony 4.9 software. The percentage change in NeuM uptake (slope) and R2 within 30 min were calculated using simple linear regression analysis in GraphPad Prism 8.0 (GraphPad Software).

[0086]

[0087] [Experimental Example 1] Evaluation of fluorescence characteristics - fluorescence intensity, fluorescence retention time, and fluorescence staining pattern -

[0088] Various derivatives containing a series of alkane side chains (C1–C14) in the 4R-1,2,3 triazole group were synthesized (see Figures 1a and 1b). To evaluate the neuronal staining properties of the derivatives, primary hippocampal neurons were prepared from E18 embryonic Sprague-Dawley rat brains and cultured in 96-well plates. After 7 days of in vitro culture (DIV 7), hippocampal neurons were treated with each derivative (50 nM), and fluorescence images were acquired for 48 h using Operetta® (λex= 460–490 nm and λem= 515–580 nm). The fluorescence intensity was quantified using Harmony 4.9 software (Figures 1c and 2d). Depending on the length of the side chain, the derivatives showed unique neuronal staining patterns.

[0089] Among the derivatives, C7 (NeuM) exhibited the strongest and longest-lasting neuronal staining properties for 48 h. The fluorescence intensity of NeuM reached a maximum after 6 h of incubation, and the maximum fluorescence intensity level was 3.5±0.1 times higher than that of C3 (NeuO). Compared with C7 (NeuM), the C3 (NeuO) derivative had a shorter alkyl chain and exhibited faster neuronal uptake and fade-out. C1 stained neurons immediately after treatment, but the fluorescence gradually disappeared within 6 h. For C3 (NeuO) and C4, the fluorescence intensity was stable within 6 h but gradually decreased thereafter. Compared with C7, derivatives with longer alkyl chains showed slower cellular uptake. C10 took 10 h, and C12 took 25 h to reach maximum intensity. C14 barely stained neurons. These results indicate that C7 (NeuM) exhibited the strongest intensity and longest retention time, and thus was the optimal length for neuronal labeling. Forty-eight hours after inducer treatment, hippocampal neurons were fixed and stained with anti-βIII-tubulin antibody to assess neuronal integrity (Fig. 1c and Fig. 11a). Neurite outgrowth assays indicated that treatment with all inducers had no significant effect on neuronal integrity. MTS assays also demonstrated that treatment with the inducers of the present invention did not significantly alter mitochondrial activity (Fig. 11b).

[0090] Interestingly, when neurons were fixed, the fluorescence intensity was significantly reduced (Fig. 12). To compare the staining pattern at high resolution with anti-βIII-tubulin, primary hippocampal neurons at DIV13 were prepared and stained with C3 or C7 (NeuM) for 2 h, then fluorescence images were acquired using Operetta®, and then fixed. Immunostaining with βIII-tubulin was performed (Fig. 2d, e). βIII-tubulin images were acquired using Operetta® and merged with fluorescence images of C3 or C7 (NeuM) for cytofluorescence analysis. At DIV13, hippocampal neurons showed significant neurogenesis and differentiation, exhibiting a single axon with multiple dendrites, and their neurites had begun to mature. While the C3 derivative strongly stained the somatic region, C7 (NeuM) was able to visualize both the soma and neurites. Cytofluorescence images using Pearson correlation coefficients showed stronger co-localization of NeuM-stained neurons and β-III tubulin-immunostained neurons (r=0.7) compared to neurons stained by C3 derivatives (r=0.5).

[0091] Next, primary cortical neuronal cultures were prepared to evaluate the neuronal selectivity of C7 (NeuM). For live-cell imaging, primary cortical neuronal cultures at DIV7 were treated with C3 or C7 (NeuM) and co-stained with SR101, a fluorescent probe for astrocytes (Fig. 2 b and c). 3D surface plot analysis showed that neurons labeled with C3 or C7 (NeuM) were clearly distinct from astrocytes labeled with SR101. These results indicate that C7 (NeuM) has neuronal selectivity similar to that of C3 in mixed neuronal cultures. In summary, compared to C3, which contains a propyl side chain, NeuM, which contains a heptyl side chain, showed improved neuronal staining properties in terms of neuronal retention time, fluorescence intensity, and neurite staining.

[0092]

[0093] [Experimental Example 2] Neuronal cell membrane staining with C7 (NeuM)

[0094] Primary hippocampal neurons at DIV7 were treated with a lower concentration of NeuM (20 nM) for 2 hours. DIV7 represents an early stage of neural development and maturation, allowing for the visualization of developing neurons at the single-cell level. NeuM was able to clearly visualize both soma and neurites, including the terminal regions of dendrites and axons (Fig. 3A). In particular, NeuM was able to visualize neuronal membrane protrusions, such as filopodia and lamellipodia of axonal terminals and dendrites, which were not observed in βIII-tubulin-immunostained neurons. High-resolution images also showed NeuM-stained membrane and vesicular structures at neurite terminals (Fig. 3B).

[0095] DIV7 hippocampal neurons were labeled with NeuM (200 nM) and separated into two compartments. Cytosolic and membrane fractions were extracted using a membrane protein extraction kit (Fig. 3C). The cytosolic and membrane fractions were evaluated by Western blot analysis using anti-tubulin and cadherin antibodies (Fig. 13). NeuM-fluorescence responses in each fraction were visualized using a UV lamp (Fig. 3D), and fluorescence spectra were measured using a SpectraMax® M2 (λex = 475 nm) (Fig. 3E). Compared to the whole cell fraction, nearly all NeuM-fluorescence signals were detected in the membrane fraction, with a fluorescence intensity of 91±9% of that of the whole cell lysate. In contrast, no observable fluorescence signal was observed in the cytosolic fraction. These results indicate that NeuM labels the neuronal membrane and not the cytosol.

[0096] Examination of other derivatives revealed that, regardless of side chain length, all derivatives exhibited a pattern similar to NeuM (Fig. 14). The fluorescence response of all derivatives was detected in the membrane fraction, not the cytosolic fraction (Fig. 3F). However, NeuM containing a heptyl side chain exhibited the strongest fluorescence intensity in neuronal lysates.

[0097] To closely examine the membrane incorporation characteristics of the derivatives of the present invention, artificial liposome membranes (100 nm) composed of phosphatidylcholine (PC) were prepared (Fig. 4G). The liposomes were then mixed with the derivatives, and the fluorescence response was measured for 60 minutes using a SpectraMaxM2. The fluorescence of the derivatives was quenched in phosphate-buffered saline (PBS, pH 7.4), resulting in very low fluorescence intensity. TrionX-100 was used as a detergent to solubilize the derivatives to estimate the maximum fluorescence intensity of the derivatives. After incubation with PC liposomes, C7 (NeuM) exhibited a unique fluorescence pattern, and the fluorescence intensity of NeuM gradually increased over 60 minutes, reaching a plateau. When the alkyl side chain was shorter, the fluorescence increase was lower and the plateau was reached much more quickly. For C5, liposome uptake was saturated within 5 minutes. For NeuM, liposome uptake reached saturation over 60 minutes, with fluorescence slowly increasing. Liposome uptake was inefficient when the side chain was longer than C10.

[0098] The AlogP values ​​of the derivatives were calculated to predict the lipophilicity required for membrane localization. The AlogP increased with increasing alkyl side chain length. Interestingly, the AlogP of NeuM was 7.221, which is similar to that of cholesterol (7.376). Cholesterol is an essential component of the plasma membrane, maintaining its structural integrity and regulating membrane fluidity. The similar AlogP values ​​of NeuM and cholesterol suggest that NeuM can be incorporated into the plasma membrane and remain stable there.

[0099] To elucidate how the longer alkyl tail of NeuM compared to C3 affects dye orientation within a model lipid bilayer, we performed molecular dynamics simulations. The simulations were performed by placing the molecule in a POPC bilayer membrane system and modeling the system for 100 ns at 303.15 K. During the first 25 ns, the compound repositioned itself in the membrane, so positional analysis was performed over a time span of 25–100 ns. The 2-hydroxy-N-phenylacetamide moiety of NeuM was located in a region of high lipid phosphate headgroup density, whereas the core and triazole arm moieties were inserted into the region of lipid tail density (Figure 4H). The alkyl side chain of NeuM extended into a region of low tail density, also known as the hydrophobic core of the bilayer, closer to the membrane center.

[0100] Local partitioning density analysis throughout the simulations (Fig. 4I) revealed that the boron atoms of the NeuM compound core are located on average 0.86–1.08 nm from the membrane center, which is deeper than the boron atoms of C3 (1.23 nm). The striking difference in membrane insertion depth between NeuM and C3 likely results from the increased lipophilicity of NeuM, which clearly increases the number of favorable hydrophobic contacts between the probe and the hydrophobic core of the bilayer. In addition to hydrophobic interactions, H-bonding interactions can also occur between the fluorescent probe and the lipid bilayer (Fig. 4J). Deeper membrane insertion of NeuM did not decrease the average number of H-bonds compared to C3, and both compounds showed H-bond formation between the 2-hydroxy-N-phenylacetamide group and the lipid phosphate. However, for NeuM, inserting the longer alkyl tail closer to the membrane center increases the tilt of the probe, favoring the formation of additional H-bonds between the amine “arm” groups and the PC ester carbonyl oxygen atom.

[0101] Overall, molecular dynamics simulations support that NeuM has an optimal alkyl chain length and can be favorably positioned within the lipid membrane model.

[0102]

[0103] [Experimental Example 3] C7 (NeuM) forms micelles that are integrated into lipid membranes.

[0104] Next, we measured the fluorescence quantum yield (Φ) of NeuM in various solvents, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and PBS containing TritonX-100 (10%) or PC liposomes (1 mg / mL) (Fig. 5A). NeuM showed a moderate quantum yield in DMSO (Φ = 0.43). When dissolved in PBS containing 0.1% DMSO, NeuM fluorescence was quenched (Φ = 0.001), and the addition of TritonX-100 (Φ = 0.63) or PC liposomes (Φ = 0.58) markedly increased the quenched fluorescence. Transmission electron microscopy (TEM) analysis was performed to investigate the quenching state of NeuM in PBS (Fig. 5B). TEM images showed the micellar structure of NeuM in PBS. ImageJ analysis identified 684 NeuM micelles with an average diameter of 12.8 nm (Fig. 5C). NeuM micelles were not observed in PBS containing TritonX-100. This result suggests that NeuM forms micelles in aqueous buffer, and their fluorescence is completely quenched. NeuM micelles dissociate in detergent solution, exhibiting high fluorescence quantum yields. In the presence of PC liposomes, NeuM micelles bind to the liposome membrane and are slowly incorporated into the membrane. Fluorescence is turned on as NeuM molecules diffuse into the membrane (Figure 5D).

[0105] Lipid membrane selectivity does not explain NeuM's neuronal selectivity. Several membrane-selective fluorescent probes are available, one of which is MemGlow, a recently developed membrane probe. TM was compared to NeuM. Similar to NeuM, MemGlow TM It is known that MemGlow forms micelles in PBS and when bound to lipid membranes, the micelles diffuse as single molecules and fluoresce. When treated on primary hippocampal neurons at DIV7, TM 647 stained all types of lipid membranes in culture, showing the strongest fluorescent signal in cell debris indicated by white arrows (Fig. 6E). Similarly, MemGlow TM 647 showed non-selective uptake into liposomal vesicles.

[0106] To investigate membrane affinity, we prepared liposomal vesicles composed of various lipid mixtures, phosphatidylserine (DOPS), phosphatidylcholine (DOPC), and brain-purified phosphatidylincholine (Brain PC) (Fig. 6F). DOPC liposomes represent intact outer cell membranes, while DOPC:DOPS liposomes mimic dying neurons or cell debris, exposing phosphatidylserine with negatively charged head groups. TM 647 did not exhibit selectivity for a specific liposome and was incorporated into all types of liposome membranes within 5 minutes. In contrast, NeuM exhibited differential uptake patterns depending on the type of liposome. Among the three liposomes, NeuM showed the slowest binding to DOPC. The DOPS liposome membrane suggests that NeuM prefers binding to viable cell membranes rather than to dying or dead cell membranes. NeuM fluorescence increased dramatically in Brain PC liposomes compared to DOPC, indicating that NeuM may prefer a specific type of membrane, such as the neuronal membrane.

[0107] To closely examine the cell type selectivity of NeuM, we treated various cell types, including primary neurons, C8-D1A, SIM-A9, SH-SY5Y, and HeLa, with NeuM at five different concentrations (25, 50, 100, 250, and 500 nM). Hoechst was used for nuclear counterstaining. After 2 h of incubation, fluorescence images were acquired using Operetta®, and fluorescence intensities were quantified using Harmony 4.9 software (Fig. 6G). At all concentrations, NeuM showed a higher fluorescence response toward primary neurons. At 25, 50, and 100 nM, NeuM staining was evident only in primary neurons, and NeuM fluorescence was only visible in other cell types, with fluorescence intensities less than 9% of those in primary neurons. At 100 nM, NeuM was still selective for primary neurons, with 5.2±1.2% for C8-D1A, 3.5±0.5% for SIM-A9, 8.6±1.4% for SH-SY5Y, and 7.3±0.1% for HeLa (Fig. 6H). Although NeuM was still selective for primary neurons at 250 nM, NeuM fluorescence was visible in other cell types. Among the cell types tested, SH-SY5Y cells, a human cell line derived from neuroblastoma, showed a significant increase in fluorescence compared to primary neurons, with C8-D1A (13.4±0.1%), SIM-A9 (9.3±0.3%), SH-SY5Y (36.5±4.2%), and HeLa (14.9±0.9%) (Fig. 6H). These results demonstrate the excellent selectivity of NeuM for primary neuronal membranes compared to non-neuronal cell types such as C8D1A, SIM-A9, and HeLa.

[0108]

[0109] [Experimental Example 4] The entry route for NeuM trafficking is clathrin-mediated endocytosis.

[0110] To identify the cellular pathways responsible for NeuM trafficking, we collected small molecules that regulate various endocytic pathways. Primary neurons at DIV6 were pretreated with dansylcadeverine (60 μM), trifluoperazine (3 μM), genistein (60 μM), endosidin2 (60 μM), or exo1 (60 μM) before NeuM treatment. To assess changes in neuinuptake, NeuM fluorescence images were acquired using Operetta®, and fluorescence intensities were measured using Harmony software 4.9 (Fig. 7A, B). Under basal conditions, neuronal fluorescence intensities were expressed as slopes. Basal = 0.43±0.03 (R 2 =0.99) increased rapidly within 30 minutes and reached a plateau over 2 hours. Among those tested, NeuM uptake by dansylcadeverine and trifluoperazine, inhibitors of clathrin-mediated endocytosis (CME), was SlopeDansylcadeverine= 0.13±0.01 (R 2 =0.98) and SlopeTrifluoperazine=0.14±0.02 (R 2 =1.00) was shown (Fig. 15). High-resolution images showed a significant decrease in NeuM uptake in somatic cells and nerve terminals by dansylcadaverine (Fig. 7C and Fig. 8D), and endosidin 2, an inhibitor of exocytosis and endoderm membrane recycling, was also shown (SlopeEndosidin2= 0.19±0.02 (R 2 =1.00) significantly reduced NeuM absorption.

[0111] In contrast, NeuM uptake was affected by genistein, an inhibitor of caveolae-mediated endocytosis, and exo1, an inhibitor of Golgi-ER trafficking, as shown in Slope Genistein= 0.42±0.02 (R 2 =0.99) and Slope Exo1 = 0.39±0.02 (R 2 =0.99) was shown.

[0112] These results imply that clathrin-mediated endocytosis and exocytosis are the major pathways mediating NeuM uptake in primary neurons. Interestingly, these two inhibition patterns were quite different in the high-resolution images (Fig. 8D). Treatment with dansylcadaverine resulted in a homogeneous decrease in NeuM fluorescence across the entire neuronal area, suggesting that clathrin-mediated endocytosis is the primary entry point for NeuM micelles. In contrast, treatment with endosidine-2 resulted in a faint but concentrated NeuM-fluorescent signal in intracellular vesicles. This result suggests that inhibition of exocytosis prevents NeuM from being integrated into the plasma membrane and instead retains it in endosomal vesicles (Fig. 8E). These results suggest that clathrin-mediated endocytosis and exocytosis are important for NeuM trafficking in primary neurons.

[0113]

[0114] [Experimental Example 5] NeuM stains the entire developmental stage of primary neurons.

[0115] We evaluated the ability of NeuM to stain various stages of neuronal development, from premature to mature neurons (Fig. 9A). Primary hippocampal neurons were treated with 50 nM NeuM at five different maturation stages: DIV 1, 3, 6, 9, and 13, and Hoechst was used for nuclear counterstaining. Operate after 2 h. TM Fluorescent images were obtained using . Afterwards, fixed neurons were immunostained with βIII-tubulin antibody for neuronal labeling and Operareta TMFluorescent images were acquired using . Next, total number of neurons, maximum neurite length, number of roots, and number of limbs were calculated using Harmony software 4.9 (Fig. 9B). NeuM was able to label neurons similar to βIII-tubulin immunostaining throughout all stages of neuronal differentiation.

[0116] For maximum neurite length, βIII-tubulin and NeuM staining showed similar increasing patterns from DIV 1 to DIV9. At DIV13, the maximum neurite length was slightly lower in NeuM staining, at 140 ± 54 m for NeuM and 156 ± 43 m for βIII-tubulin. Compared to βIII-tubulin, NeuM staining showed relatively fewer root numbers, with decreases of 67%, 75%, 67%, and 77% at DIV 3, 6, 9, and 13, respectively. On the other hand, NeuM staining showed higher numbers of limbs, with increases of 122%, 121%, and 113% at DIV 6, 9, and 13, respectively.

[0117] These results suggest that NeuM is more advantageous for visualizing neurite terminals because NeuM labels the neuronal membrane as shown in Figures 3A and B.

[0118] For the same reason, NeuM is not suitable for counting the number of roots, where cell membranes are not clearly distinguished. It is noteworthy that the NeuM image is much clearer than the βIII-tubulin image. Massive neuronal cell death occurs during primary neuronal culture (Fig. 5c). βIII-tubulin immunostaining shows dying and dead neurons in primary neuronal cultures, whereas NeuM staining is used only for live neurons. The selectivity of NeuM for live neurons is clearly shown in the enlarged images of neurons stained with NeuM and βIII-tubulin at DIV3 and DIV9 (Fig. 5D). Healthy neurons stained with NeuM and βIII-tubulin are indicated by arrows, while shrunken and dying neurons stained with βIII-tubulin are indicated by arrows in Fig. 8D.

[0119] In summary, NeuM can stain neurons at all stages of primary neuronal development (Fig. 10E). Figure 10E shows representative images of NeuM-stained neurons showing characteristics of five different stages of neurogenesis. NeuM can visualize lamellar formation in stage 1, small neurite outgrowth in stage 2, axonal outgrowth in stage 3, dendritic outgrowth and axonal growth cone development in stage 4, and maximum maturation in stage 5. NeuM can also visualize membranes and vesicles at the neurite terminals.

[0120]

[0121] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. A compound of the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, R is a straight-chain alkyl group having 5 to 8 carbon atoms.

2. A compound in the first paragraph, wherein R is a straight-chain alkyl group having 7 carbon atoms.

3. A fluorescent probe for labeling cells in which clathrin-mediated endocytosis operates, comprising a compound according to claim 1.

4. A fluorescent probe in the third paragraph, wherein R is a straight-chain alkyl group having 7 carbon atoms.

5. In the third paragraph, the compound is a fluorescent probe having an AlogP value as a lipophilicity measure of 7 to 7.

4.

6. A fluorescent probe according to claim 3, wherein the compound exists in the form of micelles before the endocytosis.

7. A fluorescent probe according to claim 6, wherein the micelle has a diameter of 10 to 16 nm.

8. In the third paragraph, the fluorescent probe is a fluorescent probe that fluorescently labels live neural cells in which clathrin-mediated endocytosis is activated.

9. In the third paragraph, the fluorescent probe is a fluorescent probe that fluorescently labels the membrane of a cell in which clathrin-mediated endocytosis operates.

10. In the third paragraph, the fluorescent probe is a fluorescent probe that fluorescently labels filopodia and lamellipodia of cells in which clathrin-mediated endocytosis operates.

11. A sensor for sensing the operation of clathrin-mediated endocytosis, comprising a compound according to claim 1. 12.(a) a step of bringing the compound according to paragraph 1 into contact with the subject, and (b) a step of checking whether fluorescence is emitted from the object, A method for sensing the operation of clathrin-mediated endocytosis in a target organism.

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