Probe for measuring cathepsin k activity and composition for diagnosing bone disease comprising same

Abstract

The present invention relates to a probe for measuring cathepsin K activity, a composition for measuring osteoclast activity comprising the probe, a composition for diagnosing bone diseases comprising the probe, a kit for measuring osteoclast activity comprising the composition, a kit for diagnosing bone diseases, a method for measuring cathepsin K activity, a method for measuring osteoclast activity, a method for diagnosing bone diseases, a composition for screening drugs inhibiting cathepsin K activity, and a method for screening drugs inhibiting cathepsin K activity. According to the present invention, rapidity and accuracy of diagnosis can be increased by designing a probe for measuring the activity of cathepsin K specifically expressed in osteoclasts, and thus the probe can be advantageously used for diagnosing bone diseases and screening cathepsin K activity-inhibiting drugs.

Description

Probe for measuring cathepsin K activity and composition for diagnosing bone disease comprising the same

[0001] The present invention relates to a probe for measuring cathepsin K activity, a composition for measuring osteoclast activity comprising the probe, a composition for diagnosing bone disease comprising the probe, a kit for measuring osteoclast activity comprising the composition, a kit for diagnosing bone disease, a method for measuring cathepsin K activity, a method for measuring osteoclast activity, a method for diagnosing bone disease, a composition for screening a drug that inhibits cathepsin K activity, and a method for screening a drug that inhibits cathepsin K activity.

[0002]

[0003] Bone maintains a dynamic equilibrium through the balance between osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption. In various bone diseases such as osteoporosis, rheumatoid bone disease, osteoarthritis, periodontal disease, inflammatory bone disease, and cancer bone metastasis, bone resorption overwhelmingly outpaces bone formation, resulting in extensive bone destruction (osteolysis). This is caused by the hyperplasia and hyperactivity of osteoclasts. Therefore, drugs that inhibit osteoclasts are utilized as treatment strategies for these bone diseases, and many new drugs are currently being developed.

[0004] Osteoclasts are TRAP (tartrate-resistant acid phosphatase)-positive multinucleated cells formed by the fusion of multiple monocyte precursor cells. They are responsible for bone resorption, removing old or weakened bone and maintaining blood mineral homeostasis. Osteoclast differentiation is regulated by two cytokines, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κ (NF-κ) ligand (RANKL). M-CSF, produced by immune cells / osteoblasts, induces RANK expression in osteoclast precursor cells and osteoclast survival signals, while RANKL, secreted by osteoblasts / activated T cells, binds to its receptor RANK on osteoclasts and induces the activation of mitogen-activated protein kinase (MAPK) and NFATc1. The above NFATc1 is a key transcription factor for osteoclast formation and regulates the expression of osteoclast differentiation and activation factors such as TRAP, cathepsin K, and DC-STAMP (dendritic cell-specific transmembrane protein).

[0005] Until now, probes developed to measure osteoclast hyperplasia and hyperactivation have had low sensitivity and high signal-to-background ratios, so most have been used in in vitro experiments. In vivo experiments are performed by staining autopsy bone tissue to measure osteoclast hyperplasia and hyperactivation in animal models such as mice and rats. Therefore, there is an urgent need to develop probes that can be used in vivo and to study more accurate measurement methods.

[0006] Meanwhile, cathepsin K is an enzyme specifically expressed in osteoclasts during the bone destruction process. The activity of cathepsin K can be confirmed in cultured osteoclasts using molecular imaging, or the activity of osteoclasts can be measured by measuring and observing cathepsin K secreted from osteoclasts in an animal model.

[0007] Against this backdrop, the inventors of the present invention have conducted extensive research efforts to develop a method capable of rapidly and accurately measuring the activity of cathepsin K in vitro and in vivo, and have completed the present invention by developing a cathepsin K-specific probe by selecting a cathepsin K degradation target amino acid and linking a fluorophore and a quencher to both ends.

[0008]

[0009] An exemplary object of the present invention is to provide a probe for measuring cathepsin K activity, wherein a fluorophore and a quencher are respectively conjugated to both ends of a polypeptide comprising an amino acid sequence represented by sequence number 1.

[0010] Another exemplary object of the present invention is to provide a composition for measuring osteoclast activity comprising the probe.

[0011] Another exemplary object of the present invention is to provide a composition for diagnosing bone diseases comprising the probe.

[0012] Another exemplary object of the present invention is to provide a kit for measuring osteoclast activity comprising the composition.

[0013] Another exemplary object of the present invention is to provide a kit for diagnosing bone disease comprising the composition.

[0014] Another exemplary object of the present invention is to provide a method for measuring cathepsin K activity, comprising the steps of treating a probe for measuring cathepsin K activity to an object or sample; and obtaining an image using the probe from the object or sample.

[0015] Another exemplary object of the present invention is a step of administering the above cathepsin K activity measuring probe to a subject or sample; and

[0016] A method for measuring osteoclast activity is provided, comprising a step of obtaining an image from the object or sample using the probe.

[0017] Another exemplary object of the present invention is a step of administering the above cathepsin K activity measuring probe to a subject or sample; and

[0018] A method for diagnosing a bone disease is provided, comprising a step of obtaining an image from the object or sample using the probe.

[0019] Another exemplary object of the present invention is to provide a composition for screening drugs that inhibit cathepsin K activity, comprising the above cathepsin K activity measuring probe as an active ingredient.

[0020] Another exemplary object of the present invention is to provide a method for screening a drug that inhibits cathepsin K activity, comprising the following steps.

[0021] (a) a step of treating a sample having cathepsin K activity with a candidate substance; and

[0022] (b) A method for screening a drug that inhibits cathepsin K activity, comprising a step of measuring cathepsin K activity using a probe for measuring cathepsin K activity for a sample treated with the candidate substance.

[0023] Another exemplary object of the present invention is to provide a probe for measuring the activity of cathepsin K, comprising a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, and having a fluorophore and a quencher conjugated to both ends of the polypeptide.

[0024] Another exemplary object of the present invention is to provide a use of a composition comprising a probe, wherein the probe comprises a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, and a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide, for measuring the activity of osteoclasts.

[0025] Another exemplary object of the present invention is to provide a use of a composition comprising a probe, wherein the probe comprises a polypeptide having an amino acid sequence represented by SEQ ID NO: 1, and a fluorescent substance and a quencher are conjugated to both ends of the polypeptide, for diagnosing a bone disease.

[0026] The technical problem to be achieved according to the technical idea of ​​the invention disclosed in this specification is not limited to the problem to solve the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0027]

[0028] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0029] As one aspect for achieving the above object, the present invention provides a probe for measuring cathepsin K activity, which comprises a polypeptide comprising an amino acid sequence represented by sequence number 1, and a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide.

[0030] [Sequence number 1]

[0031] GKPRGSKQ

[0032] The term "polypeptide" of the present invention means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0033] Representative amino acids and their respective abbreviations are: alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), tryptophan (Trp, W), valine (Val, V), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gln, Q), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), tyrosine (Try, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), and lysine (Lys, K).

[0034] In the present invention, the polypeptide is designed to be cleaved by cathepsin K, which is specifically expressed in osteoclasts, and includes the amino acid sequence of SEQ ID NO: 1.

[0035] In the present invention, the polypeptide may include an RG sequence, which is a cleavage site that binds to and is cleaved by cathepsin K.

[0036] In the present invention, the polypeptide may further include additional amino acid residues at both ends of the amino acid sequence of SEQ ID NO: 1 so as to be effectively degraded by cathepsin K, facilitate synthesis, or better express fluorescence. The number of amino acid residues that can be added is not particularly limited within a range that can be detected using the principle of fluorescence-quenching. As an example, the sequence of the entire polypeptide may be about 20. In the present invention, when a fluorophore and a quencher exist together within 1 to 10 nm, detection is possible using the principle of fluorescence-quenching, but is not limited thereto.

[0037] In the present invention, a fluorescent substance and a quencher are respectively conjugated to both terminals of the polypeptide. For example, a fluorescent substance may be conjugated to the N terminal of the polypeptide and a quencher may be conjugated to the C terminal. For example, a quencher may be conjugated to the N terminal of the polypeptide and a fluorescent substance may be conjugated to the C terminal.

[0038] In the present invention, an amino acid may be additionally included to facilitate synthesis between the polypeptide and the quencher. The amino acid may be, but is not limited to, propargylglycine (pra), a synthetic amino acid.

[0039] The term "cathepsin K (CatK)" of the present invention refers to an enzyme specifically expressed in osteoclasts during the bone destruction process, and the activity of osteoclasts can be measured by confirming the activity of cathepsin K in cultured osteoclasts through molecular imaging or by measuring and observing cathepsin K secreted from osteoclasts in an animal model.

[0040] In the present invention, the probe may be decomposed by cathepsin K enzyme that is specifically expressed and activated in osteoclasts, thereby restoring fluorescence.

[0041] Specifically, the above cathepsin K activity measurement probe detects cathepsin K by utilizing the FRET (fluorescence resonance energy transfer) principle, in which fluorescence is lost due to a quencher, but when the amino acid peptide is cleaved by the cathepsin K enzyme, the fluorophore and quencher are separated and the distance between them increases, the quenching effect disappears and fluorescence is expressed by the fluorophore.

[0042] The cathepsin K activity measuring probe of the present invention can specifically detect only activated cathepsin K enzymes by penetrating into cells, enabling accurate and sensitive measurements. Therefore, compared to conventional invasive analytical methods used to measure cathepsin K activity, it offers higher sensitivity and enables high-efficiency cathepsin K detection within a short period of time, enabling the diagnosis of various bone diseases associated with osteoclast hyperactivation.

[0043] In the present invention, the fluorescent substance may be any one selected from the group consisting of TAMRA (TMR, Carboxy tetramethylrhodamine), fluorescein, FITC (fluorescein isothiocyanate), Oregon green, Texas red, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocarbocyanine, rhodamine, oxacarbocyanine, thiacarbocyanine, merocyanine, and phycoerythrin, and may be, for example, TAMRA (TMR, Carboxy tetramethylrhodamine), but is not limited thereto.

[0044] In the present invention, for example, the TAMRA absorbs a wavelength of 750 to 1000 nm when excited, and thus exhibits fluorescence in the wavelength range of 564 to 585 nm.

[0045] In the present invention, the TAMRA may include several derivatives having different functional groups, and examples thereof include, but are not limited to, 5(6)-TAMRA, 5-TAMRA, 6-TAMRA, 5-TAMRA-PEO3-amine, 5(6)-TAMRA-SE, 5-TAMRA SE, 6-TAMRA SE, 5-TAMRA-X SE, 5-TAMRA-PEO8 SE, 5-TAMRA-PEO12 SE, 5(6)-TAMRA-C5-maleimide, or TAMRA-MTS.

[0046] In the present invention, the quencher may be any one selected from the group consisting of black hole quencher 2 (BHQ2), black hole quencher 1 (BHQ1), black hole quencher 3 (BHQ3), nonfluorescent quencher (NFQ), dabcyl, Eclipse, Deep Dark Quencher (DDQ), blackberry quencher, and Iowa black, and may be specifically BHQ2, but is not limited thereto.

[0047] In the present invention, the fluorescent substance or quencher listed above is known to exhibit or absorb fluorescence in a specific wavelength range, so that the type of fluorescent substance and the type of quencher can be selected appropriately depending on the molecule or environment to be detected.

[0048] In the present invention, for example, BHQ1, BHQ2, and BHQ3 absorb fluorescence in the wavelength range of 480 to 580 nm, 550 to 650 nm, and 620 to 730 nm very efficiently. Therefore, when using TAMRA as a fluorescent substance, it is preferable to select a BHQ2 quencher so as to absorb the fluorescence.

[0049] In one embodiment of the present invention, the probe for measuring cathepsin K activity may be represented by the structural formula of FIG. 1.

[0050] Specifically, in an embodiment of the present invention, the probe for measuring cathepsin K activity was introduced into a bone metastasis mouse model, an osteoclast hyperplasia mouse model, and an osteoporosis mouse model, and the activity of cathepsin K was measured to confirm hyperplastic osteoclasts.

[0051] As another aspect for achieving the above object, the present invention provides a composition for measuring osteoclast activity, comprising the above cathepsin K activity measuring probe.

[0052] The above “cathepsin K” and “probe” are as described above.

[0053] As another aspect for achieving the above purpose, the present invention provides a composition for diagnosing bone disease, comprising the above cathepsin K activity measuring probe.

[0054] The above “cathepsin K” and “probe” are as described above.

[0055] The term "bone disease" of the present invention may be at least one selected from the group consisting of osteoporosis, rheumatoid bone disease, osteoarthritis, periodontal disease, inflammatory bone disease, metastatic cancer, and Paget disease, but is not limited thereto.

[0056] In the present invention, the “diagnosis” includes not only simply determining whether a disease has developed, but also monitoring the progression of the disease or detecting mild symptoms prior to full-blown onset.

[0057] In the present invention, the composition may be manufactured in a unit dosage form or may be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersant or stabilizer.

[0058] As another aspect for achieving the above object, the present invention provides a kit for measuring osteoclast activity, comprising the composition for measuring osteoclast activity.

[0059] As another aspect for achieving the above purpose, the present invention provides a bone disease diagnosis kit comprising the bone disease diagnosis composition.

[0060] The above “bone disease” is as described above.

[0061] As another aspect for achieving the above object, the present invention comprises the steps of treating a probe for measuring cathepsin K activity to an object or a sample; and

[0062] A method for measuring cathepsin K activity is provided, comprising a step of obtaining an image by the probe from the object or sample.

[0063] The above “cathepsin K” and “probe” are as described above.

[0064] In the present invention, the object is not particularly limited, but includes, for example, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit or a guinea pig, and preferably means a mammal, more preferably a human.

[0065] In the present invention, the sample includes a sample derived from a living organism, for example, a cell or a collection of cells isolated from a living organism or obtained through genetic engineering or tissue engineering, a tissue culture medium, or various artificial living organism models obtained through bioprinting technology.

[0066] As another aspect for achieving the above object, the present invention provides a method for measuring cathepsin K activity, comprising the steps of: administering the probe for measuring cathepsin K activity to a subject or a sample; and

[0067] A method for measuring osteoclast activity is provided, comprising a step of obtaining an image from the object or sample using the probe.

[0068] The above “cathepsin K”, “subject” and “sample” are as described above.

[0069] As another aspect for achieving the above object, the present invention provides a method for measuring cathepsin K activity, comprising the steps of: administering the probe for measuring cathepsin K activity to a subject or a sample; and

[0070] A method for diagnosing a bone disease is provided, comprising a step of obtaining an image from the object or sample using the probe.

[0071] The above “cathepsin K”, “probe”, “bone disease”, “diagnosis”, “subject” and “sample” are as described above.

[0072] In the present invention, the dosage of the probe varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the period of administration, but can be appropriately selected by a person skilled in the art.

[0073] In one embodiment of the present invention, the dosage may be 0.0001 mg / kg to 100 mg / kg per day, specifically 0.001 mg / kg to 60 mg / kg, for adults.

[0074] In one embodiment of the present invention, the administration may be administered once a day or in several divided doses, and may be administered after fluorescence emission at intervals of time, and may be administered by appropriately increasing or decreasing the dose depending on the age, sex, weight, metabolic function, and other drugs currently being taken by the subject.

[0075] The probe for measuring cathepsin K activity of the present invention is manufactured by taking into account the range of effective doses, and the formulated preparation may be administered several times at regular intervals using specialized dosing and preparation methods according to the judgment of a specialist who supervises or manages the administration of the drug and the individual's needs, as needed.

[0076] In one embodiment of the present invention, the administration may be administered to the subject by various routes, for example, orally or by intraperitoneally, intranasally, intrarectally, intravenously, intramuscularly, subcutaneously, or by intracerebrovascular injection.

[0077] As another aspect for achieving the above object, the present invention provides a composition for screening drugs that inhibit cathepsin K activity, comprising the above cathepsin K activity measuring probe as an active ingredient.

[0078] In one embodiment of the present invention, the probe for measuring cathepsin K activity can determine the presence or absence, activity, inhibition of activity, etc. of activated cathepsin K enzyme in hyperplastic osteoclasts of an individual with a bone disease, and thus can be used for the purpose of screening for a drug that inhibits the activity of cathepsin K.

[0079] In one embodiment of the present invention, the drug screening composition can be applied both in vivo and in vitro, and can be used for various purposes such as a high-throughput screening method required for new drug development and early disease diagnosis.

[0080] As another aspect for achieving the above object, the present invention provides a method for screening a drug that inhibits cathepsin K activity, comprising the following steps.

[0081] (a) a step of treating a sample having cathepsin K activity with a candidate substance; and

[0082] (b) A method for screening a drug that inhibits cathepsin K activity, comprising a step of measuring cathepsin K activity using a cathepsin K activity measuring probe for a sample treated with the candidate substance.

[0083] In the present invention, the sample includes a sample derived from a living organism, for example, a cell or a collection of cells isolated from a living organism or obtained through genetic engineering or tissue engineering, a tissue culture medium, or various artificial living organism models obtained through bioprinting technology.

[0084] In the present invention, if the cathepsin K activity measured after treatment with the candidate substance decreases, the candidate substance can be determined as a drug for inhibiting cathepsin K activity.

[0085] As another aspect for achieving the above object, the present invention provides the use of a probe, which comprises a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, and a fluorophore and a quencher are respectively conjugated to both ends of the polypeptide, for measuring the activity of cathepsin K.

[0086] The above “cathepsin K”, “polypeptide” and “probe” are as described above.

[0087] In another aspect for achieving the above purpose, the present invention provides a composition for use in measuring the activity of osteoclasts, comprising a probe centered on a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, and having a fluorescent substance and a quencher conjugated to both ends of the polypeptide.

[0088] The above “polypeptide” and “probe” are as described above.

[0089] In another aspect for achieving the above purpose, the present invention provides a composition for use in diagnosing bone disease, comprising a probe centered on a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, and having a fluorescent substance and a quencher conjugated to both ends of the polypeptide.

[0090] The above “bone disease”, “diagnosis”, “cathepsin K”, “polypeptide” and “probe” are as described above.

[0091] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0092]

[0093] According to the present invention, a probe for measuring the activity of cathepsin K specifically expressed in osteoclasts can be designed to increase the speed and accuracy of diagnosis, and thus can be usefully utilized in the diagnosis of bone diseases and screening for drugs that inhibit cathepsin K activity.

[0094]

[0095] Figure 1 shows the structural formula of a probe for measuring cathepsin K activity of the present invention.

[0096] Figure 2 schematically shows the manufacturing process (a) and structural formula (b) of a probe for measuring cathepsin K activity.

[0097] Figure 3 shows an HPLC chromatogram of a probe for measuring cathepsin K activity.

[0098] Figure 4 shows the MALDI-TOF mass spectra of probes for measuring cathepsin K activity (a: CatKP1, b: CatKP2, c: CatKP3).

[0099] Figure 5 schematically illustrates the process of observing mouse tibial osteoclasts using a two-photon microscope (a) and the process of cleaved probes with cathepsin K (b).

[0100] Figure 6 shows the UV-vis absorption (a, b) / emission (c) spectra of CatKP1 incubated with cathepsin K.

[0101] Figure 7 shows the UV-vis absorption (a and b) / emission (c and d) spectra of CatKP2 or CatKP3 incubated with cathepsin K.

[0102] Figure 8 is a graph showing the fluorescence intensity (a) and initial velocity (b) of CatKP1, CatKP2, or CatKP3 depending on the presence or absence of cathepsin K.

[0103] Figure 9 shows the binding positions (a to c) and the respective physical parameter values ​​according to the molecular docking simulation of CatKP1, CatKP2, and CatKP3.

[0104] Figure 10 is a linear graph showing the fluorescence intensity of CatKP1 according to the concentration of cathepsin K.

[0105] Figure 11 shows the Michaelis-Menten graph of CatKP1.

[0106] Figure 12 shows the fluorescence intensity of CatKP1 according to the concentration of cathepsin K.

[0107] Figure 13 shows the results of HPLC (a) and ESI-MS (b and c) analyses before and after the reaction of CatKP1 with cathepsin K.

[0108] Figure 14 is a graph showing the fluorescence intensity of CatKP1 according to ODN concentration.

[0109] Figure 15 shows the cytotoxicity of CatKP1 against osteoclasts.

[0110] Figure 16 shows the results of confirming cathepsin K activity in cultured osteoclasts through fluorescence microscopy (a: when cathepsin K is not expressed, probe fluorescence is not observed; b: when cathepsin K is expressed, probe fluorescence is observed).

[0111] Figure 17 shows the results of confirming cathepsin K activity using CatKP1 in cultured osteoclasts.

[0112] Figure 18 shows fluorescence microscopy images of CatKP1 according to cell type.

[0113] Figure 19 shows confocal microscopy images of osteoclasts cultured on dentin slices treated with cathepsin K antibody (a) or CatKP1 (b).

[0114] Figure 20 shows the intracellular spatial location of fluorescence in osteoclasts differentiated from dentin slices according to CatKP1 treatment in confocal images (a and c) and z-projection images (b and d).

[0115] Figure 21 is a schematic representation of the process of treating the tibia (a) and muscle (b) of a bone metastasis mouse model with CatKP1, and shows the results of confirming osteoclasts in the tibia (c) and muscle (d) using a two-photon microscope image.

[0116] Figure 22a shows a two-photon microscope image of a tumor-free tibia in a bone metastasis mouse model, and Figure 22b shows a microscope image of a Csf1r-EGFP transgenic mouse after CatKP1 treatment.

[0117] Figure 23 briefly illustrates the manufacturing process of an osteoclast hyperplasia mouse model.

[0118] Figure 24a shows photographs of mouse organs after CatKP1 treatment in an osteoclast hyperplasia mouse model, and Figure 24b shows cross-sectional z-stack images of each organ.

[0119] Figure 25 shows the results of monitoring fluorescence in the presence or absence of cathepsin K using a multiphoton microscope.

[0120] Figure 26a shows two-photon microscope images of the epiphysis (1) and trabecular bone (2) over time (40, 60, 80, 100, and 120 minutes) after CatKP1 treatment, Figure 26b shows a graph of fluorescence intensity in the trabecular bone, and Figure 26c shows in vivo two-photon microscope images 24 hours after CatKP1 treatment and after re-treatment.

[0121] Figure 27 is a graph showing the microscopic images and BV (bone volume) / TV (tissue volume) according to H&E staining in an osteoclast hyperplasia mouse model.

[0122] Figure 28a shows an image of the uterus and the measured weight of the uterus in an osteoporosis mouse model, and Figure 28b shows a microscopic image of the tibia in an osteoporosis mouse model and a graph of BV (bone volume) / TV (tissue volume).

[0123] Figure 29a is a schematic representation of the manufacturing process of an osteoporosis mouse model, Figure 29b shows the results of confirming hyperplastic osteoclasts after OVX treatment (4, 6, and 8 weeks), and Figure 29c shows the results of confirming the reduction of osteoclasts according to ZA treatment.

[0124]

[0125] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0126]

[0127] Example 1: Preparation of cathepsin K-specific probe

[0128] 1.1. Preparation of TMR-pep

[0129] The fabrication process of cathepsin K probes (CatKP1, CatKP2, and CatKP3) is schematically illustrated in Figure 2a. To develop a two-photon FRET-based probe for detecting cathepsin K (CatK), the TAMRA (TMR) fluorophore and BHQ-2 were selected as a FRET pair. TMR exhibits excellent brightness when excited by two-photon light in the 750–1000 nm range, and BHQ-2 efficiently quenches TMR fluorescence. After screening candidate sequences based on their selectivity and reactivity toward cathepsin K, KPRGSKQ and HPGGPQ were selected as cathepsin K-sensitive peptide sequences.

[0130] Afterwards, TMR-pep1, TMR-pep2, and TMR-pep3, in which a TAMRA (TMR) fluorophore was linked to the N-terminal portion of the polypeptides (pep1, pep2, and pep3) represented by sequence numbers 1 to 3 by a carbon chain and an unnatural amino acid, propargylglycine (Pra), was introduced to the C-terminal portion, were designed, and then purchased from Bionics Co., Ltd. The propargylglycine and the N-terminal glycine of sequence numbers 1 and 3 were introduced to enhance the structural flexibility and diversity of the peptide sequence.

[0131] [Sequence number 1]

[0132] GKPRGSKQ

[0133] [Sequence number 2]

[0134] KPRGSKQ

[0135] [Sequence number 3]

[0136] GHPGGPQ

[0137]

[0138] 1.2. BHQ-2 Preparation

[0139] The quencher BHQ2-azide was synthesized through a two-step reaction.

[0140] 1) 2-(N-Methylanilino)ethanol was reacted with Mesyl chloride (MsCl) at room temperature for 1 hour under the addition of dry TEA. Impurities were removed with Brine by washing with Brine and DCM solvent, and the DCM solution was collected and the solvent was removed through an evaporator. The obtained reaction intermediate was reacted with NaN3 at high temperature for 5 hours, and after removal of the solvent, the product was obtained by purification using silica column chromatography. Silica gel 60 (70-230 mesh) was used as the stationary phase. 60 silica gel (0.25 mm thick pre-coated sheet) was used for analytical thin layer chromatography.

[0141] 2) The aniline structure synthesized in step 1) above and Fast Black K salt were reacted under the conditions of NaOAc buffer:ACN=1:1, and the resulting precipitate was purified using a filter to obtain the product (N3-BHQ2).

[0142]

[0143] 1.3. Preparation of cathepsin K-specific probes (CatKP1, CatKP2, and CatKP3)

[0144] The manufacturing process of CatKP1 is as follows. TMR-pep1 (0.01 mmol) and N3-BHQ2 (0.03 mmol) prepared in 1.1 and 1.2 above were dissolved in 1 mL of DMSO. Sodium ascorbate (0.02 mmol) and CuSO4.5H2O (0.01 mmol) were each separately dissolved in 0.1 mL of deionized water. Each solution was purged with Ar gas for 10 min, and all solutions were mixed together and stirred at ambient temperature for 5 h. The resulting mixture was filtered using Strata C18-E (55 mm, 70 Å), and the crude product was purified by HPLC using a C18 column (5 μm, 250 × 20 mm) as a stationary phase. Solvent A (acetonitrile containing 0.1% TFA) and solvent B (deionized water containing 0.1% TFA) were used as mobile phases. CatKP1 eluted in 21.4 min (elution conditions: solvent A at 20-45% for 20 min, 45-100% for 5 min, and 100-20% for 5 min).

[0145] CatKP2 was synthesized according to the same procedure as CatKP1 except that TMR-pep2 was used instead of TMR-pep1. The resulting mixture was filtered using Strata C18-E (55 mm, 70 Å), and the crude product was purified by HPLC using a C18 column (5 μm, 250 × 20 mm) as the stationary phase. Solvent A (acetonitrile containing 0.1% TFA) and solvent B (deionized water containing 0.1% TFA) were used as the mobile phase. CatKP2 eluted at 25.2 min (elution conditions: solvent A treated at 30-38% for 27 min, 38-100% for 3 min, 100% for 5 min, and 100-30% for 2 min).

[0146] CatKP3 was synthesized according to the same procedure as CatKP1, except that TMR-pep3 was used instead of TMR-pep1. The resulting mixture was filtered using Strata C18-E (55 mm, 70 Å), and the crude product was purified by HPLC using a C18 column (5 μm, 250 × 20 mm) as the stationary phase. Solvent A (acetonitrile containing 0.1% TFA) and solvent B (deionized water containing 0.1% TFA) were used as the mobile phase. CatKP3 eluted at 29.1 min (elution conditions: solvent A at 30-45% for 30 min, 45-100% for 3 min, 100% for 9 min, and 100-30% for 3 min).

[0147] The structural formulas of the manufactured cathepsin K probes are briefly illustrated in Fig. 2(b). The probes containing sequence numbers 1, 2, and 3 are denoted as CatKP1, CatKP, and CatKP3, respectively.

[0148]

[0149] Example 2. Characterization of the manufactured cathepsin K probe

[0150] 2.1. Analysis of the components of the manufactured cathepsin K probe

[0151] The cathepsin K probes (CatKP1, CatKP, and CatKP3) prepared in Example 1 were analyzed for their components using reversed-phase high-performance liquid chromatography (HPLC, YL9100, Dr. Maish GmbH ReproSil 100 C 18, 5 μm (250 X 20 mm) column) and MALDI-TOF / MS (MALDI-TOF / TOF™ 5800 system (AB SCIEX), Korea Basic Science Institute, Seoul, Korea). Data are expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism™ version 10.0, and statistical significance was determined by one-way ANOVA with Tukey's post hoc analysis. This is common to all the examples below.

[0152] Specifically, HPLC chromatography of each cathepsin K probe was measured at 550 nm, and each sample was prepared with a 1% dimethyl sulfoxide (DMSO) aqueous solution and eluted at a flow rate of 1 mL / min. Using solvent A (acetonitrile containing 0.1% TFA) and solvent B (deionized water containing 0.1% TFA), the mobile phase gradient was applied, increasing solvent A from 30% to 45% over 15 min, increasing from 45% to 100% over 5 min, and holding at 100% for 5 min. The results are shown in Fig. 3. As a result, it was confirmed that CatKP1 and CatKP2 eluted in 13.3 min, and CatKP3 eluted in 17.0 min. In addition, it was confirmed by HPLC chromatography that all three probes were well purified. In this regard, CatKP1 and CatKP2 have similar peptide sequences and contain several hydrophilic amino acids (especially, three positively charged amino acids), whereas CatKP3 has a different peptide sequence and contains fewer hydrophilic amino acids (especially, one positively charged amino acid). Therefore, it was expected that the degree of hydrophilicity of the entire molecule may vary depending on the peptide sequence difference, as predicted by the difference in HPLC elution time.

[0153] MALDI-TOF / MS spectra were analyzed using a MALDI-TOF / TOF™ 5800 system (AB SCIEX) at the Korea Basic Science Institute (Seoul, Korea). Spectra were acquired in positive ion mode.

[0154] The results are shown in Fig. 4 (a: CatKP1, b: CatKP2, and c: CatKP3). The calculated m / z values ​​of CatKP1 obtained through MALDI-TOF MS are C 94 H 124 N 27 O 21 For +, it is 1967.95, and the actual found value was 1967.9623. The calculated m / z value obtained through MALDI-TOF MS of CatKP2 is C 92 H 121 N 26 O 20 + For 1909.9250, the actual found value was 1909.9366. The calculated m / z value obtained through MALDI-TOF MS of CatKP3 is C 86 H 100 N 23 O 19 For +, it was 1758.7566, and the actual found value was 1758.6851. That is, it was confirmed that each cathepsin K probe (CatKP1, CatKP, and CatKP3) all matched the predicted m / z (mass / charge).

[0155]

[0156] 2.2. Enzymatic Characterization of Cathepsin K Probe

[0157] The efficiency of FRET depends on the spatial proximity between the fluorophore and the quencher. Therefore, it was expected that the fluorescence of the fluorophore would recover as the spatial proximity between the fluorophore and the quencher increased due to the hydrolysis of the cathepsin K probe by cathepsin K. That is, the inventors predicted that although the fluorescence of the manufactured probe was lost due to the quencher (BHQ2), the fluorescence of the TMR (TAMRA) fluorophore would appear when the substrate amino acid peptide was cleaved by cathepsin K, allowing the measurement of cathepsin K enzyme activity. Figure 5a schematically shows the process of observing mouse tibia (shin bone) osteoclasts in vivo using intravital two-photon microscopy, and Figure 5b schematically shows the chemical structure of the cathepsin K probe and the process of the probe being cleaved by cathepsin K. To confirm this, the following experiment was conducted.

[0158]

[0159] 2.2.1. Fluorescence confirmation of cathepsin K probe according to the presence or absence of cathepsin K

[0160] First, UV / Vis absorption and emission spectra and fluorescence spectra were observed in the presence of cathepsin K. UV / Vis absorption and emission spectra were measured using a Jasco V-750 spectrophotometer, and fluorescence spectra were measured using a Jasco FP-8500 spectrofluorometer. For UV / Vis and fluorescence measurements, a 2.5 mM DMSO stock solution of CatKP was prepared and diluted to an appropriate concentration for each solution-phase analysis. The spectrum of CatKP1 (2.5 μM) in 50 mM MES buffer (pH 5.5, containing 5 mM DTT and 2.5 mM EDTA) was treated with cathepsin K (50 nM) and incubated for 2 hours, and the spectra of CatKP2 (2 μM) and CatKP3 (2 μM) were treated with cathepsin K (20 nM) and incubated for 4 hours. Pro-cathepsin K (Enzo Life Sciences #ALX-201-239-C010) was activated according to the recommended protocol immediately before the experiment. Upon reaction with active-cathepsin K, the cathepsin K probe was measured for fluorescence intensity at 590 nm as a function of time (λ) using a microplate reader (Hidex Sense). ex : 544 nm, excitation and emission aperture 1 mm) were recorded. Kinetic parameters were extracted from the initial velocity data using Prism software. In addition, for fluorescence measurement using a two-photon excitation system, the fluorescence intensity of CatKP1 (2.5 μM) at 400 to 800 nm was observed using microplate readers (Hidex Sense) when reacted with 100 nM activated-cathepsin K (SRP6561, Sigma) for 2 h.

[0161] As a result, CatKP1 exhibited a broad absorption band ranging from 450 to 600 nm, and after incubation with cathepsin K at 37°C, a new pattern appeared with an absorption spectrum maximum at 554 nm and a shoulder at approximately 520 nm (Fig. 6a). This was very similar to the simple superposition of the TMR-pep1 and N3-BHQ2 spectra, confirming that the TMR-pep and N3-BHQ2 absorption groups were included within the CatKP1 structure (Fig. 6b). In addition, although CatKP1 initially exhibited little fluorescence, exposure to cathepsin K resulted in a significant fluorescence enhancement with an off / on ratio of approximately 73 at 585 nm (Fig. 6c). On the other hand, CatKP2 showed a similar pattern to CatKP1 (Fig. 7a and Fig. 7c), but CatKP3 showed a relatively limited response to cathepsin K (Fig. 7b and Fig. 7d).

[0162] Additionally, the fluorescence intensities of CatKP1, CatKP2, or CatKP3 were observed in the presence or absence of cathepsin K (20 nM). As a result, in the absence of cathepsin K, CatKP1, CatKP2, and CatKP3 showed little increase in fluorescence (in the absence of cathepsin K in Fig. 8a, the graphs of CatKP1, CatKP2, and CatKP3 were overlapped, and only the graph of CatKP1 was displayed). It was observed that CatKP3 had the lowest measured fluorescence intensity, indicating a significantly lower activation potential than CatKP1 or CatKP2 (Fig. 8a). In addition, the initial rate of fluorescence activation following cathepsin K treatment was observed to be significantly lower for CatKP2 and CatKP3 than for CatKP1 (Fig. 8b).

[0163]

[0164] 2.2.2. Molecular docking simulation of the cathepsin K probe

[0165] Molecular docking simulations of CatKP1, CatKP2, and CatKP3 were performed. The binding poses of CatKP1 (Fig. 9a), CatKP2 (Fig. 9b), and CatKP3 (Fig. 9c) in the active site of cathepsin K (5TDI) were predicted using Autodock Vina. Furthermore, the physical parameters and calculated binding energies are presented (Fig. 9d). Specifically, the chemical 3D structure of CatKP was obtained by Chem 3D, and the crystal structure of human CTSK was obtained from the PDB bank (PDB entry: 5TDI). The optimized structures (ligands) were docked to the target enzyme (receptor) using Autodock Vina. For the Autodock Vina method, the grid size was set to 70.7716 х 85.3822 х 60.8986 (center = 7.016-2.683-18.896). The best ligand pose was selected for binding model analysis using PyMOL 2.5.4. LogD values ​​of CatKP were calculated using Chemaxon's Marvin 23.16.0.

[0166] From the molecular docking simulation results, it was thought that the highest enzymatic reaction occurred in CatKP1 due to a slight disturbance in the optimal orientation of the substrate at the catalytic site for peptide bond cleavage, given that CatKP1 and CatKP2 differ by only one glycine residue (Fig. 9a and Fig. 9b). In addition, CatKP3 had a distinct peptide sequence different from CatKP1, and had physical differences in its properties, such as fewer positively charged AAs and a higher LogD value, which significantly influenced its affinity for the cathepsin K catalytic site. Furthermore, CatKP3 exhibited the lowest binding energy for cathepsin K, providing evidence supporting the lowest enzymatic reaction of CatKP3 (Fig. 9c).

[0167] That is, from the above examples, it was confirmed that not only the sequence of the peptide but also the structure of the dye conjugated to the end of the peptide sequence affects the activation kinetics of the cathepsin K probe. Therefore, below, among the manufactured cathepsin K probes, only CatKP1, which has the best enzymatic activity toward cathepsin K, was selected as the cathepsin K-specific probe of the present invention, and experiments for measuring cathepsin K activity were performed.

[0168]

[0169] 2.2.3. Measurement of fluorescence intensity of cathepsin K probe according to cathepsin K

[0170] Additionally, the linear relationship between the fluorescence intensity of CatKP1 (2.5 μM) and cathepsin K levels according to cathepsin K concentration is graphically shown in Figure 10. The fluorescence intensity of CatKP1 at 590 nm at the initial time point showed an excellent linear correlation (r) with the concentration of cathepsin K in the range of 0-10 nM. 2 = 0.99) and the limit of detection (LOD) of CatK was determined to be 0.26 nM.

[0171]

[0172] 2.2.4. Enzyme kinetic properties of cathepsin K probe

[0173] To demonstrate the enzymatic kinetics of CatKP1, the rate of CatKP1 at different concentrations in CatK (2.5 nM) was plotted as a Michaelis-Menten graph in Figure 11. The reaction was performed using 50 mM MES buffer (pH 5.5, containing 5 mM DTT and 2.5 mM EDTA). Data were collected as a function of time using microplate readers (Hidex Sense), and λ ex The excitation and emission apertures were 544 nm and 1 mm in size. Data are expressed as mean ± SD (standard deviation) and are based on the results of three repeated measurements.

[0174] As a result, the enzyme kinetic parameters Km was 2.5 μM and kcat was 0.19 s. -1 , kcat / Km is 76000 M -1 s -1 It was decided as (Fig. 11).

[0175]

[0176] Example 3. Confirmation of the reaction of cathepsin K-specific probe according to cathepsin K.

[0177] 3.1. Confirmation of cathepsin K-specific probe activity following cathepsin K treatment

[0178] The fluorescence intensity of CatKP1 (2 μM) was measured at 590 nm in the presence of various concentrations of cathepsin K (CatK).

[0179] As a result, as shown in Fig. 12, it was confirmed that the reaction intensity of CatKP1 was positively correlated with the concentration of cathepsin K.

[0180] In addition, to observe whether CatKP1 was completely consumed when a sufficient amount of cathepsin K was present, HPLC and electrospray ionization (ESI)-MS analyses were performed. Specifically, samples were prepared by incubating CatKP1 with cathepsin K (CatKP1+CatK) or without cathepsin K (CatKP1) for 3 h in 50 mM MES buffer (pH 5.5, containing 5 mM DTT, 2.5 mM EDTA), and analyzed by HPLC using a mobile phase consisting of a binary gradient (solvent A: acetonitrile containing 0.1% TFA, solvent B: deionized water containing 0.1% TFA). Solvent A was treated with 20-45% for 20 min, 45-100% for 5 min, and 100% for 10 min, and the eluted solution was analyzed by ESI-MS. HPLC (Fig. 13a) was performed in the same manner as in Example 1, and the HPLC spectrum of CatKP1 was measured at 550 nm before (blue) and after (red) reaction with cathepsin K. ESI-MS spectra were measured using a Shimadzu LC / MS-2020.

[0181] As a result, a peak eluted at 27.4 min for the solution containing only CatKP1 and at 17.8 to 18.3 min for the CatKP1+CatK solution was observed (Fig. 13a). The major peak at 984.50 m / z, which corresponds to the predicted m / z for CatKP1, was detected in the solution containing only CatKP1 (Fig. 13b), but was not detected in the CatKP1+CatK solution, confirming complete consumption of CatKP1 (Fig. 13c). In other words, complete consumption of CatKP1 was observed when a sufficient amount of cathepsin K was present.

[0182]

[0183] 3.2. Confirmation of hydrolysis of cathepsin K-specific probe following administration of cathepsin K inhibitor

[0184] The fluorescence intensity of CatKP1 (2.5 μM) treated with CatK (20 nM) was measured at 590 nm in the presence of various concentrations of odanacatib (ODN), a selective cathepsin K inhibitor. The fluorescence intensity measurement was performed in the same manner as in Example 2.

[0185] As a result, it was confirmed that the enzymatic hydrolysis of CatKP1 was completely inhibited by administration of odanakatib (Fig. 14). Therefore, it was confirmed that the cathepsin K-specific probe was hydrolyzed depending on the presence or absence of cathepsin K.

[0186]

[0187] Example 4: Detection of cathepsin K in osteoclasts using a cathepsin K-specific probe.

[0188] 4.1. Cytotoxicity evaluation of cathepsin K-specific probes

[0189] The cytotoxicity of CatKP1 against osteoclasts was evaluated. First, bone marrow-derived mononuclear cells (BMMs) were treated with M-CSF (30 ng / mL) and RANKL (50 ng / mL) and cultured for a total of 5 days to generate mature osteoclasts. On the third day of osteoclastogenesis, CatKP1 was administered to BMMs at various concentrations (0 to 10 μM) and cultured for an additional 2 days. On the fifth day, cells were fixed with 4% PFA in phosphate-buffered saline (PBS) and stained using the TRAP kit (Sigma-Aldrich). TRAP-positive multinucleated cells (≥ 3 nuclei) were considered osteoclasts. Visual images of TRAP staining were obtained using a phase-contrast microscope (EVOS XL Cell Imaging System, Invitrogen™).

[0190] As a result, the number of differentiated osteoclasts in the CatKP1 treatment group was consistently maintained compared to the control group at all concentrations, confirming that there was no toxicity to osteoclasts (Fig. 15).

[0191]

[0192] 4.2. Detection of cathepsin K in osteoclasts using a cathepsin K-specific probe

[0193] Cathepsin K fluorescence was confirmed in cultured osteoclasts using fluorescence microscopy. Specifically, bone marrow cells were harvested from the hind limbs of 6- to 8-week-old normal mice (C57BL / 6 mice, purchased from Orient), and approximately 5x10 per 60 mm culture dish. 7 The cells were suspended in 5 mL of medium (alpha MEM medium containing 10% FBS and 1X penicillin / streptomycin antibiotics) and cultured overnight (37°C, 5% CO2) in an incubator. The next day, only the supernatant of the cell suspension was slowly removed to count the cell number. ① Medium containing only 30 g / mL mouse recombinant M-CSF, or ② Medium containing both 30 g / mL mouse recombinant M-CSF and 50 ng / mL mouse recombinant RANKL were each suspended in a concentration of 1 x 10 per mL. 6 The number of cells was adjusted to 10. The cell suspension was added to the culture dish or dentin slice according to the volume (e.g., 2x10 because the medium volume of a 96-well culture plate is 200 uL). 5(The cells were added). After culturing in an incubator for 2 days, the medium was replaced with newly prepared medium containing ① 30 g / mL mouse recombinant M-CSF only and ② medium containing both 30 g / mL mouse recombinant M-CSF and 50 ng / mL mouse recombinant RANKL according to the volume of the culture dish. The culture was continued in an incubator for 2 days, and 2 uM of the probe (CatKP1) prepared in Example 1 was added, followed by culturing in an incubator for 1 hour. A control group without the probe was also prepared. Afterwards, osteoclast differentiation in the culture dish was confirmed by taking images using the Brightfield channel and RFP channel (emission wavelength 574-626 nm) of a fluorescence microscope. Fluorescence images were obtained using a fluorescence or confocal microscope (EVOS FL Auto2 Cell Imaging System, Invitrogen™ or LSM900, Zeiss). To correct the probe signal in osteoclasts, confocal 3D projection images were adjusted in Huygens Professional 22.10 software (Scientific Volume Imaging BV) using a deconvolution wizard.

[0194] As a result, when the probe was injected and observed after differentiation into macrophages using a medium containing only M-CSF, cathepsin K was not expressed and therefore no fluorescence of the probe was observed (Fig. 16a). On the other hand, when the probe was injected and observed after differentiation into osteoclasts using a medium containing both M-CSF and RANKL, it was confirmed through brightfield images that the osteoclasts had differentiated into multinucleated cells, and cathepsin K was expressed in the osteoclasts and therefore fluorescence of the probe was observed (Fig. 16b).

[0195] In addition, culture plates and dentin slices differentiated into osteoclasts using a medium containing both M-CSF and RANKL were stained with phalloidin and observed using a fluorescence microscope and a confocal microscope, respectively. Dentin slices are known to have the same composition of type I collagen, calcium, and phosphate as human bone tissue, and thus serve as a biological in vitro substitute for bone.

[0196] Specifically, the culture dishes and dentin slices differentiated into osteoclasts using medium containing both M-CSF and RANKL were washed with PBS, and then 4% paraformaldehyde was added, blocked from light, and maintained overnight at 4℃. After washing the cells with PBS, a solution containing 0.1% Triton X-100 in PBS was added for 15 minutes at room temperature in a light-blocking condition to increase cell membrane permeability. After washing the cells again with PBS, a solution containing 1% BSA and 0.25% phalloidin stock solution in PBS was added for 1 hour at room temperature in a light-blocking condition. After washing the cells again with PBS, a solution containing 2% Hoechst33342 in PBS was added for 5 minutes at room temperature in a light-blocking condition. After washing the cells again with PBS, a mounting solution was added to the stained cells. Phalloidin stains the cellular skeleton, and Alexa Fluor 647 fluorescent dye is attached to it, showing the morphology of the cell in the corresponding channel (emission wavelength 660-680 nm) (green). Hoechst33342 binds to DNA and emits fluorescence, showing the nucleus of the cell in the corresponding channel (emission wavelength 450-500 nm) (blue). As a control group, a group was prepared in which odanacatib (ODN), a cathepsin K inhibitor, was added at a concentration of 30 nM on the third day of differentiation.

[0197] As a result, in the group to which the probe was added (CatKP1), the fluorescence of the probe was observed in multinucleated osteoclasts (red), but in the group to which the probe was not added (None) and the group to which the probe and Odanaka tip were added (CatKP1+ODN), no red fluorescence was observed, confirming that the probe of the present invention (CatKP1) manufactured in Example 1 has specific sensitivity to cathepsin K (Fig. 17). In addition, it was confirmed that the fluorescence patterns in the culture dish and dentin slices were inconsistent due to the formation of a tight actin ring by osteoclasts cultured in dentin slices during bone resorption, but when treated with ODN, the two fluorescence patterns were completely suppressed, confirming that CatKP1 enabled the visualization of functional osteoclasts in a cathepsin K-dependent manner.

[0198]

[0199] 4.3. Confirmation of the specificity of the cathepsin K-specific probe for osteoclasts

[0200] To confirm the specificity of the cathepsin K-specific probe for osteoclasts, fluorescence microscopy images of CatKP1 were observed in various cells, including osteoclasts. 2x10 5 BMM-derived osteoclasts, E0771 breast tumor cells, RAW264.7 macrophages, and NIH3T3 fibroblasts were placed in 12-well plates, injected with CatKP1 (2 μM, red), and cultured for more than 1 hour, after which fluorescence microscopy images were observed.

[0201] As a result, as shown in Figure 18, osteoclasts exhibited a strong fluorescent signal following CatKP1 treatment, but no significant cellular response was observed in other cell types. In other words, the specificity of the cathepsin K-specific probe for osteoclasts was confirmed.

[0202]

[0203] 4.4. Confirmation of the localization of cathepsin K-specific probes in osteoclasts

[0204] The subcellular localization of CatKP1 in osteoclasts was further confirmed by z-stacked 3D confocal microscopy images. Bone marrow-derived mononuclear cells (BMMs) were cultured in medium with M-CSF (30 ng / mL) and RANKL (50 ng / mL) for 3 days (Fig. 19) or 9 days (Fig. 20). On day 3, cells were fixed with 4% PFA in PBS. In Fig. 19a, cells were fixed and stained with cathepsin K Ab (cathepsin K antibody, red), LAMP2A (lysosomal membrane, green), and Hoechst 33342 (nucleus, blue). In Fig. 19b, CatKP1 (2 μM, red) was administered 1 h before observation, and cells were fixed and stained with LAMP2A (lysosomal membrane, green) and Hoechst 33342 (nucleus, blue).

[0205] Similarly to Fig. 19, osteoclasts were sliced ​​at four locations (slices 1 to 4), and CatKP1 (2 μM, red) was administered 1 hour before observation. After cell fixation, the cells were stained with LAMP2A (lysosomal membrane, purple), phalloidin (F-actin, green), and Hoechst 33342 (nucleus, blue). Cathepsin K Ab (cathepsin K antibody, red) was co-stained with LAMP2A (lysosomal membrane, purple), phalloidin (F-actin, green), and Hoechst 33342 (nucleus, blue) after cell fixation.

[0206] As a result, we confirmed that CatKP1 fluorescence in osteoclasts was mainly localized within intracellular lysosomes (Fig. 19). Furthermore, as osteoclasts matured to a fully differentiated stage characterized by secretion of active CatK into dentin slices for bone resorption, we confirmed that the CatKP1 signal was mainly concentrated under the actin ring, particularly in resorption lacunae (Figs. 20c and 20d). In contrast, the cathepsin K antibody, which can detect both the pro- and active-forms of cathepsin K, stained the entire cytoplasm of osteoclasts (Figs. 20a and 20b). From this, we confirmed the selectivity and intracellular localization of CatKP1 toward active cathepsin K in bone-resorbing osteoclasts.

[0207]

[0208] Example 5: Detection of cathepsin K in an animal model using a cathepsin K-specific probe

[0209] 5.1. Detection of Cathepsin K in a mouse model of bone metastasis

[0210] All animal experiments described below were approved by the Institutional Animal Care and Use Committee of Korea University Medical Center (KOREA-2023-0110) and the Garvan and St Vincent's Animal Ethics committee (ARA21_17).

[0211] Five thousand cells (20 μL, Hank's balanced salt solution) of the E0771 murine breast cancer cell line (provided by Vanderbilt University) were injected intraperitoneally into the right proximal tibia bone (Fig. 21a) and muscle (Fig. 21b) of mice (C57BL / 6 mice, Orient) under general anesthesia using 2% isoflurane. The contralateral left bone and tumor-injected muscle served as negative controls. Two-photon microscopy was performed according to the following steps. Mice were injected with CatKP1 (50 μM) via the tail vein and then under general anesthesia using Avertin (200 mg / kg, ip). A small skin incision (approximately 1.5–2 cm below the level of the knee joint) was made to expose the proximal medial surface of the tibia. The mice were placed in a lateral recumbent position, and a cover glass was placed over the region of interest. Sagittal section image of the right tibia, 1099×1099 μm with a 10X objective lens 2The imaging was performed using a two-photon microscope (IVIM-MS3, IVIM Technology) with an imaging field of view of 100 nm and a laser tuned to 920 nm (Seoul, Korea). In parallel, complementary experiments were performed using similar equipment, specifically an IVIM-CMS3 (a two-photon microscope integrated with a confocal microscope, IVIM Technology) equipped with a 20x objective for image acquisition (Daejeon, Korea). The average laser power applied to the proximal tibia was adjusted to 50 mW. To visualize the inner surface of the trabecular bone, the second harmonic generation (SHG) signal of 2,8-DHA crystals was detected using a bandpass filter set to 380–420 nm. In addition, the signal of the RFP channel was detected using a bandpass filter set to 574–626 nm. The SHG channel (emission wavelength 380–420 nm) of the two-photon microscope measures nonlinear optical signals to image bone tissue rich in collagen, which is shown in blue, while the RFP channel (emission wavelength 574–626 nm) measures the fluorescence signal of the probe, which is shown in red. The inner surface of the trabecular bone could be observed to a depth of approximately 100 μm. ImageJ software was used to quantify the intensity of the CatKP1 signal by analyzing the images compiled into two-dimensional projection images from the z-stacks obtained from the RFP channel. Mice were euthanized two weeks after two-photon microscopy. After euthanasia, the hind limbs were resected and fixed in 4% PFA dissolved in PBS at 4°C for 3 days, and then decalcified with 14% EDTA at 4°C for 3 days. Paraffin-embedded samples were cut into 4-μm-thick sections and stained using a modified hematoxylin-eosin (H&E) staining method containing phloxine B and Orange G dyes. BV / TV parameters were determined using ImageJ software and H&E images were taken from three square frames (0, 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, 4The average of the measurements (measured as 5 x 0.5 mm) was used for evaluation.

[0212] As a result, after intravenous injection, CatKP1 showed particularly bright fluorescence in the right tibia but remained inactive in the contralateral bone (Figs. 21a and 21c). Furthermore, no fluorescence was detected in the tumors implanted in the leg muscles (Figs. 21b and 21d). Histological staining of tibial bone tissue revealed that cancer cells severely infiltrated the trabecular bone, and osteoclast invasion was confirmed within the tumor mass (Fig. 21c). These results indicate that the marked increase in CatKP1 fluorescence in the tumor-bearing bone was not due to the cancer cells themselves, but rather to abnormal osteoclast activation within the tumor microenvironment.

[0213] Additionally, we validated the two-photon imaging results in ex vivo bone tissue in a microscope setup and evaluated the stability of CatKP1 in cryopreserved specimens. Mice with intratibial bone metastases were administered CatKP1 (50 μM) before euthanasia. Tumor-free tibias (contralateral) were resected and rapidly frozen at -80°C. Frozen bone samples were then observed for bone (SHG, brown), CatKP1 (RFP, red), and osteoclasts (EGFP, green) using a Stellaris 8 Dive multiphoton microscope. The multiphoton microscope was equipped with a Leica NDD 4Tune HyD detector, a Leica HC FLUPTAR L 25x / 0.95 W VISIR 0.17 objective, and Spectra Physics Insight X3 single and Mai Tai eHP Deep See lasers. Images had a resolution of 512 x 512 pixels, 465 x 465 x 64 μm, and a step size of 1 μm. The acquired images were analyzed using Leica Microsystems' LAS X software and Bitplane's Imaris 9.

[0214] As a result, two-photon microscopy images of the non-tumor-bearing hindlimb (contralateral tibia) of a bone metastasis mouse model. CatKP1 signal (red) was significantly amplified and clearly visible in osteoclasts located in resorption pits (Fig. 22a).

[0215] In addition, to confirm the specificity of CatKP1 for osteoclasts, CatKP1 (50 μM) was intravenously injected into Csf1r-EGFP transgenic mice that specifically express EGFP in osteoclasts, followed by ex vivo sampling and storage at -80°C until microscopic observation. Csf1r (Colony Stimulating Factor 1 Receptor) is a receptor found on the surface of cells of bone marrow origin, such as osteoclasts, and is known to play an important role in regulating the formation and function of osteoclasts.

[0216] As a result, the CatKP1 signal (red) was confirmed to be colocalized with Csf1r+ osteoclasts (green), and the two-photon microscopy image of the tibia of Csf1r-EGFP transgenic mice showed colocalization of EGFP and CatKP1 (red) signals (Fig. 22b).

[0217]

[0218] 5.2. Detection of Cathepsin K in a mouse model of osteoclast hyperplasia

[0219] The process of constructing an osteoclast hyperplasia mouse model is briefly shown in Figure 23. Specifically, an osteoclast hyperplasia mouse model was constructed by injecting 8-week-old female C57BL / 6 mice with an excessive dose of RANKL (0.5 mg / kg, PeproTech) dissolved in PBS, administered intraperitoneally daily for 3 days at a volume of 100 μL) and randomly divided into three groups. On the first day of RANKL injection, one group was administered vehicle (Veh, 10% DMSO in corn oil) via oral gavage, and the other group was administered odanakinra (ODN, 3.6 mg / kg, Selleckchem). The mice were euthanized on the fourth day after imaging using a two-photon microscope. The remaining groups were set as controls.

[0220] First, we examined the ex vivo distribution of CatKP1 in various organs of RANKL-induced bone loss mice. Organs (liver, heart, lung, and kidney) were dissected 2 hours after intravenous injection of CatKP1. For two-photon imaging of ex vivo tissue samples, mice treated with RANKL (0.5 mg / kg, PeproTech) daily for 3 days were administered CatKP1 (50 μM, IV), while a negative control group was not administered CatKP1.

[0221] Two hours after intravenous injection of CatKP1, each organ (liver, heart, lung, and kidney) was removed from each mouse, and sagittal cross-sectional images of the organs were taken using a two-photon microscope under the same conditions as for the proximal tibia imaging. Figure 24a shows photographs of the organs from each mouse, and Figure 24b shows z-stack images of sagittal sections of each organ observed using a two-photon microscope. The upper group is the negative control group that did not receive CatKP1, and the lower group is the group that received it. Bone (SHG, blue) and CatKP1 (RFP, red) were observed.

[0222] As a result, selective activation of CatKP1 was confirmed in tissues where active osteoclasts are present, as no fluorescence was detected in the epiphysis or other organs (Fig. 24).

[0223] Additionally, multiphoton microscope (λ ex : 920 nm, STELLARIS 8 DIVE, Leica Microsystems), a clear fluorescence activation reaction of CatKP1 by CatK was confirmed (Fig. 25).

[0224] Afterwards, real-time in vivo two-photon microscopy images were observed in the proximal tibia of the mouse for 40 to 120 minutes after injection of CatKP1. Figure 26a 1) shows the epiphysis, Figure 26a 2) shows the trabecular bone, Figure 26b shows a graph of the fluorescence intensity in the trabecular bone, and Figure 26c shows two-photon microscopy images 24 hours after CatKP1 injection and after CatKP1 re-injection.

[0225] As a result, bright fluorescent spots were observed in the proximal tibia within 40 minutes, persisting for at least 2 hours. Furthermore, CatKP1 fluorescence in the tibia disappeared 24 hours after injection (Fig. 26c), confirming that this in vivo clearance allows for repeated assessment of osteoclast activity in the tibia.

[0226] In addition, a total of three groups were prepared: a group in which the probe was introduced into mice that were not injected with RANKL as an experimental group (No RANKL Control), a group in which the probe was introduced into an osteoclast hyperplasia model (RANKL+Veh), and a group in which the probe was introduced after administering the cathepsin K inhibitor odanacatib to an osteoclast hyperplasia model (3.6 mg of odanacatib per kg of mouse weight dissolved in corn oil containing 10% DMSO, administered orally once in a volume of 100 μL) (RANKL+ODN). On the 4th day, CatKP1 (50 μM) was injected through the tail vein of each mouse.

[0227] As a result, as shown in Fig. 26d, osteoclast activity was observed in the group in which the probe was introduced into the osteoclast hyperplasia mouse model administered with RANKL, and it was confirmed that the intensity of fluorescence was reduced in the group in which Odanaka tip was administered compared to the group in which the probe was introduced into the osteoclast hyperplasia model. This indicates a decrease in bone resorption activity.

[0228] These results are consistent with those in Figure 27, which shows microscopic images of modified H&E staining and histomorphometric analysis results quantifying the trabecular bone volume fraction (bone volume (BV) to tissue volume (TV) ratio).

[0229]

[0230] 5.3. Detection of Cathepsin K in a Mouse Model of Osteoporosis

[0231] In an osteoporosis mouse model induced by ovariectomy, hyperplastic osteoclasts were observed using a two-photon microscope using the probe (CatKP1) prepared in Example 1.

[0232] Specifically, the ovariectomy (OVX)-induced osteoporosis mouse model was constructed by surgically removing a pair of ovaries from 8-week-old female C57BL / 6 mice through a midline dorsal incision under general anesthesia with 2% isoflurane. The skin was incised with surgical instruments to open the abdominal cavity, and the ovaries on both sides were exposed. Each oviduct was incised using a Bovey electrosurgical device at the junction of the ovaries. Bleeding was controlled with electrocautery, and the wounds were closed using surgical clips. Control mice (Sham) that underwent sham surgery underwent the same surgical procedure without ovariectomy. After 4 weeks, OVX mice were randomly divided into two groups (n=9 / group) and administered vehicle (PBS) or zoledronate (ZA, 120 μg / kg, Sigma-Aldrich) via the tail vein once a week. Mice were euthanized 8 weeks after two-photon imaging.

[0233] First, to confirm the successful creation of an osteoporosis mouse model, uterine images and uterine weight observed 8 weeks after surgery were graphically represented (Fig. 28a). H&E staining microscopic images, reduced uterine weight, and bone volume (BV) / tissue volume (TV) measurements were also graphically represented (Fig. 28b). This confirmed the successful creation of an osteoporosis mouse model.

[0234] After preparing the two models, the probe (CatKP1) manufactured in Example 1 was introduced at 4, 6, and 8 weeks, and zoledronate (ZA), an osteoporosis treatment agent that inhibits the activity of cathepsin K, was administered weekly from 4 to 8 weeks (120 ug of zoledronate per 1 kg of mouse weight dissolved in PBS once a week, intravenously administered in a volume of 100 uL), and osteoclasts were observed using fluorescence.

[0235] As a result, the fluorescence of CatKP1 increased over time after OVX surgery (Fig. 29b). Furthermore, at 8 weeks after OVX surgery, the fluorescence signal was significantly enhanced approximately 8.2-fold compared to the normal mouse model (Sham) (Fig. 29c). Notably, the CatKP1 signal in ZA-treated mice was significantly reduced to the level observed in the normal mouse model (Sham), demonstrating that CatKP1 can be used to measure osteoclast activity during the progression of bone loss in an osteoporosis model.

[0236] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A probe for measuring cathepsin K activity, comprising a polypeptide comprising an amino acid sequence represented by sequence number 1, with a fluorescent substance and a quencher conjugated to each end of the polypeptide.

2. In paragraph 1, A probe for measuring cathepsin K activity, wherein the fluorescent substance is any one selected from the group consisting of TAMRA (Carboxy tetramethylrhodamine), fluorescein, FITC (fluorescein isothiocyanate), Oregon green, Texas red, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocarbocyanine, rhodamine, oxacarbocyanine, thiacarbocyanine, merocyanine, and phycoerythrin.

3. In paragraph 1, A probe for measuring cathepsin K activity, wherein the quencher is any one selected from the group consisting of black hole quencher 2 (BHQ2), black hole quencher 1 (BHQ1), black hole quencher 3 (BHQ3), nonfluorescent quencher (NFQ), dabcyl, Eclipse, Deep Dark Quencher (DDQ), Blackberry Quencher, and Iowa black.

4. In paragraph 1, The above probe is a probe for measuring cathepsin K activity, which is decomposed by cathepsin K enzyme that is specifically expressed and activated in osteoclasts, and fluorescence is restored.

5. In paragraph 1, A probe for measuring cathepsin K activity, wherein the polypeptide comprises an RG sequence, which is a cleavage site that binds to and is cleaved by cathepsin K.

6. A composition for measuring osteoclast activity, comprising the probe of paragraph 1.

7. A composition for diagnosing bone disease, comprising the probe of paragraph 1.

8. In paragraph 7, A composition for diagnosing a bone disease, wherein the bone disease is at least one selected from the group consisting of osteoporosis, rheumatoid bone disease, osteoarthritis, periodontal disease, inflammatory bone disease, metastatic cancer, and Paget disease.

9. A kit for measuring osteoclast activity, comprising the composition of Article 6.

10. A kit for diagnosing bone disease, comprising the composition of Article 7.

11. In paragraph 10, A kit for diagnosing bone disease, wherein the above bone disease is at least one selected from the group consisting of osteoporosis, rheumatoid bone disease, osteoarthritis, periodontal disease, inflammatory bone disease, metastatic cancer, and Paget disease.

12. A step of treating a probe for measuring cathepsin K activity according to Article 1 to an object or sample; and A method for measuring cathepsin K activity, comprising the step of obtaining an image by the probe from the object or sample.

13. A step of administering a probe for measuring cathepsin K activity according to paragraph 1 to an individual or sample; and A method for measuring osteoclast activity, comprising the step of obtaining an image by the probe from the object or sample.

14. A step of administering a probe for measuring cathepsin K activity according to paragraph 1 to an individual or sample; and A method for diagnosing a bone disease, comprising the step of obtaining an image from the object or sample using the probe.

15. In paragraph 14, A method for diagnosing a bone disease, wherein the probe is decomposed by a cathepsin K enzyme that is specifically expressed and activated in osteoclasts, thereby restoring fluorescence.

16. In paragraph 14, A method for diagnosing a bone disease, wherein the above bone disease is at least one selected from the group consisting of osteoporosis, rheumatoid bone disease, osteoarthritis, periodontal disease, inflammatory bone disease, metastatic cancer, and Paget disease.

17. A composition for screening drugs that inhibit cathepsin K activity, comprising the probe for measuring cathepsin K activity of paragraph 1 as an active ingredient.

18. A method for screening a drug that inhibits cathepsin K activity, comprising the following steps: (a) a step of treating a sample having cathepsin K activity with a candidate substance; and (b) A method for screening a drug that inhibits cathepsin K activity, comprising a step of measuring cathepsin K activity using the cathepsin K activity measuring probe of claim 1, for a sample treated with the candidate substance.

19. In paragraph 18, A drug screening method further comprising a step of determining the candidate substance as a drug for inhibiting cathepsin K activity when the measured cathepsin K activity decreases after treatment with the candidate substance.

20. Use of a probe comprising a polypeptide comprising an amino acid sequence represented by sequence number 1, for measuring the activity of cathepsin K, wherein a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide.

21. Use of a composition comprising a probe, wherein the probe comprises a polypeptide having an amino acid sequence represented by sequence number 1, and a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide, for measuring the activity of osteoclasts.

22. Use of a composition comprising a probe, wherein the probe comprises a polypeptide having an amino acid sequence represented by sequence number 1 for diagnosing a bone disease, and a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide.

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