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WO2024237764A9 - Sonde pour mesurer l'activité de la cathepsine k et composition pour diagnostiquer une maladie osseuse la comprenant - Google Patents

Sonde pour mesurer l'activité de la cathepsine k et composition pour diagnostiquer une maladie osseuse la comprenant Download PDF

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Publication number
WO2024237764A9
WO2024237764A9 PCT/KR2024/095801 KR2024095801W WO2024237764A9 WO 2024237764 A9 WO2024237764 A9 WO 2024237764A9 KR 2024095801 W KR2024095801 W KR 2024095801W WO 2024237764 A9 WO2024237764 A9 WO 2024237764A9
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cathepsin
probe
activity
measuring
bone disease
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WO2024237764A2 (fr
WO2024237764A3 (fr
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박석인
김종승
구세영
이은정
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • 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.
  • Bone maintains a dynamic equilibrium state by the balance between osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption.
  • osteoblasts which are responsible for bone formation
  • osteoclasts which are responsible for bone resorption.
  • various bone diseases such as osteoporosis, rheumatoid bone disease, osteoarthritis, periodontal disease, inflammatory bone disease, and cancer bone metastasis
  • osteoporosis rheumatoid bone disease
  • osteoarthritis osteoarthritis
  • periodontal disease inflammatory bone disease
  • cancer bone metastasis bone resorption is overwhelmingly superior to bone formation, resulting in extensive bone destruction (osteolysis), which is caused by the hyperplasia and hyperactivity of osteoclasts. Therefore, drugs that inhibit osteoclasts are utilized as a treatment strategy for the above bone diseases, and many new drugs are currently being developed.
  • Osteoclasts are TRAP (tartrate-resistant acid phosphatase)-positive multinucleated cells formed by the fusion of multiple monocyte precursor cells and are responsible for bone resorption, removing old or weakened bone and maintaining the homeostasis of blood minerals. Osteoclast differentiation is regulated by two cytokines, M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of nuclear factor- ⁇ (NF- ⁇ ) ligand).
  • M-CSF macrophage colony stimulating factor
  • RANKL receptor activator of nuclear factor- ⁇ (NF- ⁇ ) ligand
  • 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 the osteoclast receptor RANK and induces the activation of mitogen-activated protein kinase (MAPK) and NFATc1.
  • MAPK mitogen-activated protein kinase
  • 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).
  • the probes developed to measure osteoclast hyperplasia and hyperactivation to date have had low sensitivity and high signal-to-background ratios, and thus have mostly been used in in vitro experiments.
  • In vivo experiments have been 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.
  • 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 animal models.
  • the inventors of the present invention have made 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 selecting a degradation target amino acid of cathepsin K and developing a cathepsin K-specific probe by linking a fluorophore and a quencher to both ends.
  • One 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.
  • Another exemplary object of the present invention is to provide a composition for measuring osteoclast activity comprising the probe.
  • Another exemplary object of the present invention is to provide a composition for diagnosing bone diseases comprising the probe.
  • Another exemplary object of the present invention is to provide a kit for measuring osteoclast activity comprising the composition.
  • Another exemplary object of the present invention is to provide a kit for diagnosing bone disease comprising the composition.
  • Another exemplary object of the present invention is to provide a method for measuring cathepsin K activity, comprising the steps of treating a subject or sample with the probe for measuring cathepsin K activity; and obtaining an image from the subject or sample by the probe.
  • Another exemplary object of the present invention is a step of administering the probe for measuring cathepsin K activity to a subject or a sample;
  • a method for measuring osteoclast activity comprising a step of obtaining an image by the probe from the object or sample.
  • Another exemplary object of the present invention is a step of administering the probe for measuring cathepsin K activity to a subject or a sample;
  • a method for diagnosing a bone disease comprising a step of obtaining an image from the object or sample using the probe.
  • 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.
  • 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.
  • a method for screening a drug that inhibits cathepsin K activity comprising the step of measuring cathepsin K activity using a probe for measuring cathepsin K activity for a sample treated with the candidate substance.
  • Another exemplary object of the present invention is to provide a use of a probe comprising a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, wherein a fluorophore and a quencher are respectively conjugated to both ends of the polypeptide, for measuring the activity of cathepsin K.
  • 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.
  • 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 diagnosing a bone disease.
  • the present invention provides a probe for measuring cathepsin K activity, which comprises a polypeptide having an amino acid sequence represented by sequence number 1, and in which a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide.
  • polypeptide of the present invention means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.
  • amino acids and their 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).
  • the polypeptide is designed to be cleaved by cathepsin K, which is specifically expressed in osteoclasts, and includes an amino acid sequence of sequence number 1.
  • the polypeptide may include an RG sequence, which is a cleavage site that binds to cathepsin K and is cleaved.
  • 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 decomposed 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.
  • a fluorescent substance and a quencher are respectively conjugated to both terminals of the polypeptide.
  • a fluorescent substance may be conjugated to the N terminal of the polypeptide and a quencher may be conjugated to the C terminal.
  • a quencher may be conjugated to the N terminal of the polypeptide and a fluorescent substance may be conjugated to the C terminal.
  • an amino acid may be additionally included between the polypeptide and the quencher to facilitate synthesis.
  • the amino acid may be, but is not limited to, propargylglycine (pra), a synthetic amino acid.
  • Cathepsin K (CatK) refers to an enzyme specifically expressed in osteoclasts during the bone destruction process.
  • the activity of cathepsin K in cultured osteoclasts can be confirmed by molecular imaging, or the activity of osteoclasts can be measured by measuring and observing cathepsin K secreted from osteoclasts in an animal model.
  • the probe may be decomposed by cathepsin K enzyme that is specifically expressed and activated in osteoclasts, thereby restoring fluorescence.
  • the above cathepsin K activity measuring probe detects cathepsin K by utilizing the FRET (fluorescence resonance energy transfer) principle, in which fluorescence is lost due to a quencher, but when an 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.
  • FRET fluorescence resonance energy transfer
  • the probe for measuring cathepsin K activity of the present invention can specifically detect only cathepsin K enzyme that has penetrated into cells and activated, so that accurate and sensitive measurement is possible. Accordingly, compared to invasive analysis methods that have been used in the past to measure the activity of cathepsin K, it has high sensitivity and can detect cathepsin K with high efficiency in a short period of time, so that various bone diseases associated with osteoclast hyperactivation can be diagnosed.
  • 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.
  • TAMRA TAMRA
  • the TAMRA absorbs a wavelength of 750 to 1000 nm when excited, and thus exhibits fluorescence in a wavelength range of 564 to 585 nm.
  • the TAMRA may include various 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.
  • 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.
  • the fluorescent substances or quenchers listed above are known to exhibit or absorb fluorescence in a specific wavelength range, so that the type of the appropriate fluorescent substance and quencher can be selected depending on the molecule or environment to be detected.
  • BHQ1 very efficiently absorbs fluorescence in the wavelength range of 480 to 580 nm, BHQ2 in the wavelength range of 550 to 650 nm, and BHQ3 in the wavelength range of 620 to 730 nm. Therefore, when using TAMRA as a fluorescent substance, it is preferable to select a BHQ2 quencher so as to absorb the fluorescence.
  • the probe for measuring cathepsin K activity may be represented by the structural formula of FIG. 1.
  • the probe for measuring cathepsin K activity was introduced into a mouse model of bone metastasis, a mouse model of osteoclast hyperplasia, and a mouse model of osteoporosis, and the activity of cathepsin K was measured to confirm hyperplastic osteoclasts.
  • the present invention provides a composition for measuring osteoclast activity, comprising the probe for measuring cathepsin K activity.
  • the present invention provides a composition for diagnosing bone disease, comprising the probe for measuring cathepsin K activity.
  • 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.
  • the “diagnosis” includes not only simply determining whether a disease has developed, but also monitoring the degree of progression of the disease or detecting mild symptoms prior to full-blown onset.
  • the composition may be prepared in a unit dosage form or may be prepared 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 invention pertains, and the composition may be prepared in a unit dosage form or by placing it in a multi-dose container.
  • 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, tablets or capsules, and may additionally include a dispersant or stabilizer.
  • the present invention provides a kit for measuring osteoclast activity, comprising the composition for measuring osteoclast activity.
  • the present invention provides a bone disease diagnostic kit comprising the bone disease diagnostic composition.
  • the present invention comprises a step of treating a probe for measuring cathepsin K activity to a subject or a sample;
  • a method for measuring cathepsin K activity comprising a step of obtaining an image by the probe from the object or sample.
  • the subject 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.
  • the sample includes a sample derived from a living organism, for example, a cell or a collection of cells separated 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.
  • the present invention comprises a step of administering the probe for measuring cathepsin K activity to a subject or a sample;
  • a method for measuring osteoclast activity comprising a step of obtaining an image by the probe from the object or sample.
  • the present invention comprises a step of administering the probe for measuring cathepsin K activity to a subject or a sample;
  • a method for diagnosing a bone disease comprising a step of obtaining an image from the object or sample using the probe.
  • the dosage of the probe varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art.
  • 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.
  • the administration may be administered once a day or in several divided doses, may be administered at intervals of time after fluorescence emission, and may be appropriately increased or decreased depending on the age, sex, weight, metabolic action, and other drugs currently being taken by the subject.
  • 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 a specialized dosing method and formulation method according to the judgment of a specialist who supervises or manages the administration of the drug and the individual's needs, as needed.
  • the administration may be administered to the subject by various routes, for example, orally or by intraperitoneal, intranasal, intrarectal, intravenous, intramuscular, subcutaneous, or intracerebrovascular injection.
  • 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.
  • the probe for measuring cathepsin K activity can determine the presence or absence, activity, activity inhibition, etc. of activated cathepsin K enzyme in hyperplastic osteoclasts of an individual suffering from a bone disease, and thus can be used for the purpose of screening for a drug that inhibits the activity of cathepsin K.
  • 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.
  • the present invention provides a method for screening a drug that inhibits cathepsin K activity, comprising the following steps.
  • a method for screening a drug that inhibits cathepsin K activity comprising the step of measuring cathepsin K activity using the cathepsin K activity measuring probe for a sample treated with the candidate substance.
  • the sample includes a sample derived from a living organism, for example, a cell or a collection of cells separated 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.
  • the candidate substance can be determined as a drug for inhibiting cathepsin K activity.
  • the present invention provides the use of a probe, which comprises a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1, for measuring the activity of cathepsin K, and in which a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide.
  • 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, wherein a fluorescent substance and a quencher are respectively conjugated to both ends of the polypeptide.
  • polypeptide and “probe” are as described above.
  • the present invention provides a composition for use including 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.
  • 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.
  • Figure 1 shows the structural formula of a probe for measuring cathepsin K activity of the present invention.
  • Figure 2 schematically illustrates the manufacturing process (a) and its structural formula (b) of a probe for measuring cathepsin K activity.
  • Figure 3 shows an HPLC chromatogram of a probe for measuring cathepsin K activity.
  • Figure 4 shows the MALDI-TOF mass spectra of probes for measuring cathepsin K activity (a: CatKP1, b: CatKP2, c: CatKP3).
  • 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).
  • Figure 6 shows the UV-vis absorption (a, b)/emission (c) spectra of CatKP1 incubated with cathepsin K.
  • Figure 7 shows the UV-vis absorption (a and b)/emission (c and d) spectra of CatKP2 or CatKP3 incubated with cathepsin K.
  • 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.
  • Figure 9 shows the binding positions (a to c) and the values of each physical parameter according to the molecular docking simulation of CatKP1, CatKP2, and CatKP3.
  • Figure 10 is a linear graph showing the fluorescence intensity of CatKP1 according to the concentration of cathepsin K.
  • Figure 11 shows the Michaelis-Menten graph of CatKP1.
  • Figure 12 shows the fluorescence intensity of CatKP1 according to the concentration of cathepsin K.
  • Figure 13 shows the HPLC (a) and ESI-MS (b and c) analysis results before and after reaction of CatKP1 with cathepsin K.
  • Figure 14 is a graph showing the fluorescence intensity of CatKP1 according to ODN concentration.
  • Figure 15 shows the cytotoxicity of CatKP1 against osteoclasts.
  • Figure 16 shows the results of confirming cathepsin K activity through a fluorescence microscope in cultured osteoclasts (a: appearance where probe fluorescence is not observed when cathepsin K is not expressed, b: appearance where probe fluorescence is observed when cathepsin K is expressed).
  • Figure 17 shows the results of confirming cathepsin K activity using CatKP1 in cultured osteoclasts.
  • Figure 18 shows fluorescence microscopy images of CatKP1 according to cell type.
  • Figure 19 shows confocal microscopy images of osteoclasts cultured on dentin slices treated with cathepsin K antibody (a) or CatKP1 (b).
  • Figure 20 shows the intracellular spatial location of fluorescence in response to CatKP1 treatment within osteoclasts differentiated from dentin slices in confocal images (a and c) and z-projection images (b and d).
  • Figure 21 is a schematic representation of the process of treating the tibia (a) and muscle (b) of a mouse model of bone metastasis with CatKP1, and the results of confirming osteoclasts in the tibia (c) and muscle (d) using a two-photon microscope image.
  • Figure 22a shows a two-photon microscope image of a tumor-free tibia in a mouse model of bone metastasis
  • Figure 22b shows a microscope image of a Csf1r-EGFP transgenic mouse after CatKP1 treatment.
  • FIG 23 briefly illustrates the manufacturing process of an osteoclast hyperplasia mouse model.
  • Figure 24a shows photographs of mouse organs after CatKP1 treatment in an osteoclast hyperplasia mouse model
  • Figure 24b shows cross-sectional z-stack images of each organ.
  • Figure 25 shows the results of monitoring fluorescence in the presence or absence of cathepsin K using a multiphoton microscope.
  • Figure 26a shows two-photon microscope images of the trabecular meshwork (1) and the trabecular meshwork (2) over time (40, 60, 80, 100, and 120 minutes) after CatKP1 treatment
  • Figure 26b shows a graph of fluorescence intensity in the trabecular meshwork
  • Figure 26c shows in vivo two-photon microscope images 24 hours after CatKP1 treatment and after retreatment.
  • 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.
  • Figure 28a shows an image of the uterus and the measured weight of the uterus in an osteoporosis mouse model
  • Figure 28b shows a microscopic image of the tibia in an osteoporosis mouse model and a graph of BV (bone volume)/TV (tissue volume).
  • 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)
  • Figure 29c shows the results of confirming the decrease in osteoclasts according to ZA treatment.
  • cathepsin K probes (CatKP1, CatKP2, and CatKP3) is schematically shown in Fig. 2a.
  • TAMRA (TMR) fluorophore and BHQ-2 were selected as a FRET pair.
  • TMR has excellent brightness when stimulated by two-photon in the range of 750–1000 nm, and BHQ-2 efficiently quenches TMR fluorescence.
  • the cathepsin K-sensitive peptide sequences were selected as KPRGSKQ and HPGGPQ.
  • TAMRA TAMRA
  • Pra propargylglycine
  • the quencher, BHQ2-azide was synthesized through a two-step reaction.
  • the manufacturing process of CatKP1 is as follows. TMR-pep1 (0.01 mmol) and N3-BHQ2 (0.03 mmol) manufactured in 1.1 and 1.2 above were dissolved in 1 mL of DMSO. Sodium ascorbate (0.02 mmol) and CuSO 4 .5H 2 O (0.01 mmol) were separately dissolved in 0.1 mL of deionized water, respectively. Each solution was purged with Ar gas for 10 minutes, all solutions were mixed together, and then stirred at ambient temperature for 5 hours.
  • 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 ⁇ ) and the crude product was purified by HPLC using a C18 column (5 ⁇ m, 250 ⁇ 20 mm) as a stationary phase.
  • Solvent A acetonitrile containing 0.1% TFA
  • solvent B deionized water containing 0.1% TFA
  • CatKP2 was eluted in 25.2 min (elution conditions: solvent A treatment at 30-38% for 27 min, 38-100% for 3 min, 100% for 5 min, and 100-30% for 2 min).
  • 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 ⁇ ) and the crude product was purified by HPLC using a C18 column (5 ⁇ m, 250 ⁇ 20 mm) as a stationary phase.
  • Solvent A acetonitrile containing 0.1% TFA
  • solvent B deionized water containing 0.1% TFA
  • CatKP3 was eluted at 29.1 min (elution conditions: solvent A at 30 to 45% for 30 min, 45-100% for 3 min, 100% for 9 min, and 100-30% for 3 min).
  • the structural formulas of the manufactured cathepsin K probes are briefly shown in Fig. 2(b).
  • the probes containing sequence numbers 1, 2, and 3 are denoted as CatKP1, CatKP, and CatKP3, respectively.
  • 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, using a Dr. Maish GmbH ReproSil 100 C 18, 5 ⁇ m (250 X 20 mm) column) and MALDI-TOF/MS (MALDI-TOF/TOFTM 5800 system (AB SCIEX), Korea Basic Science Institute, Seoul, Korea). Data are expressed as mean ⁇ SEM, and statistical analysis was performed using GraphPad PrismTM 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.
  • each cathepsin K probe was measured at 550 nm, and each sample was prepared with a 1% DMSO (dimethyl sulfoxide) aqueous solution and eluted at a flow rate of 1 mL/min.
  • solvent A acetonitrile containing 0.1% TFA
  • solvent B deionized water containing 0.1% TFA
  • the solvent A was increased from 30% to 45% over 15 min, increased from 45% to 100% over 5 min, and maintained at 100% for 5 min.
  • Fig. 3 As a result, it was confirmed that CatKP1 and CatKP2 were eluted at 13.3 min, and CatKP3 was eluted at 17.0 min.
  • 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 from the difference in HPLC elution time.
  • MALDI-TOF/MS spectra were analyzed using a MALDI-TOF/TOFTM 5800 system (AB SCIEX) at the Korea Basic Science Institute (Seoul, Korea). Spectra were acquired in positive ion mode.
  • Fig. 4 The results are shown in Fig. 4 (a: CatKP1, b: CatKP2, and c: CatKP3).
  • the calculated m/z value obtained through MALDI-TOF MS of CatKP1 was 1967.95 for C 94 H 124 N 27 O 21 +, and the actual found value was 1967.9623.
  • the calculated m/z value obtained through MALDI-TOF MS of CatKP2 was 1909.9250 for C 92 H 121 N 26 O 20 +, and the actual found value was 1909.9366.
  • the calculated m/z value obtained through MALDI-TOF MS of CatKP3 was 1758.7566 for C 86 H 100 N 23 O 19 +, and the actual found value was 1758.6851. That is, each of the cathepsin K probes (CatKP1, CatKP, and CatKP3) was confirmed to match the predicted m/z (mass/charge).
  • the efficiency of FRET depends on the spatial proximity between the fluorophore and the quencher. Therefore, it was expected that the fluorophore would recover its fluorescence 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, thereby enabling the measurement of cathepsin K enzyme activity.
  • TAMRA TMR
  • FIG. 5a schematically shows the process of observing mouse tibia (shin bone) osteoclasts in vivo using intravital two-photon microscopy
  • Fig. 5b schematically shows the chemical structure of the cathepsin K probe and the process of the probe being cleaved by cathepsin K. The following experiment was conducted to confirm this.
  • 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.
  • a 2.5 mM DMSO stock solution of CatKP was prepared and diluted to an appropriate concentration for each solution-phase analysis.
  • CatKP1 (2.5 ⁇ M) 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 ⁇ M) and CatKP3 (2 ⁇ M) 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 prior to the experiment.
  • the fluorescence intensity of the cathepsin K probe at 590 nm upon reaction with active-cathepsin K was recorded as a function of time ( ⁇ ex : 544 nm, excitation and emission apertures 1 mm) using a microplate reader (Hidex Sense). Kinetic parameters were extracted from the initial velocity data using Prism software. In addition, for fluorescence measurements using a two-photon excitation system, the fluorescence intensity of CatKP1 (2.5 ⁇ M) at 400 to 800 nm was observed with microplate readers (Hidex Sense) when reacted with 100 nM active-cathepsin K (SRP6561, Sigma) for 2 h.
  • 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 about 520 nm (Fig. 6a). This was very similar to the simple superposition of the TMR-pep1 and N3-BHQ2 spectra, confirming that TMR-pep and N3-BHQ2 absorption groups were included in the CatKP1 structure (Fig. 6b).
  • the best ligand pose was selected for binding model analysis by PyMOL 2.5.4.
  • LogD values of CatKP were calculated using Marvin 23.16.0 from Chemaxon.
  • the enzyme kinetic parameters Km were determined to be 2.5 ⁇ M, kcat to be 0.19 s -1 , and kcat/Km to be 76000 M -1 s -1 (Fig. 11).
  • the fluorescence intensity of CatKP1 (2.5 ⁇ M) treated with CatK (20 nM) was measured at 590 nm in the presence of various concentrations of odanacatib (ODN), a selective cathepsin K inhibitor.
  • ODN odanacatib
  • the fluorescence intensity measurement was performed in the same manner as in Example 2.
  • Example 4 Detection of cathepsin K in osteoclasts using a cathepsin K-specific probe
  • BMMs bone marrow-derived mononuclear cells
  • RANKL 50 ng/mL
  • CatKP1 was administered to BMMs at various concentrations (0 to 10 ⁇ M) and cultured for an additional 2 days.
  • the cells were fixed with 4% PFA in PBS and stained using a TRAP kit (Sigma-Aldrich).
  • TRAP-positive multinucleated cells (nuclei ⁇ 3) were considered as osteoclasts. Visual images of TRAP staining were obtained using a phase-contrast microscope (EVOS XL Cell Imaging System, InvitrogenTM).
  • 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 5 x 10 7 cells were suspended in 5 mL of medium (alpha MEM medium containing 10% FBS and 1X penicillin/streptomycin antibiotics) per 60 mm culture dish and cultured in an incubator overnight (37°C, 5% CO 2 ). The next day, only the supernatant of the cell suspension was slowly removed to measure the cell number.
  • medium alpha MEM medium containing 10% FBS and 1X penicillin/streptomycin antibiotics
  • the medium was replaced with newly prepared medium containing 1 30 g/mL mouse recombinant M-CSF only and medium containing 2 30 g/mL mouse recombinant M-CSF and 50 ng/mL mouse recombinant RANKL according to the volume of the culture dish.
  • 2uM of the probe (CatKP1) manufactured in Example 1 was added, and cultured in an incubator for 1 hour.
  • a control group without the probe was also prepared.
  • 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, InvitrogenTM or LSM900, Zeiss). Confocal 3D projection images were adjusted in Huygens Professional 22.10 software (Scientific Volume Imaging BV) using a deconvolution wizard to correct for probe signals in osteoclasts.
  • 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.
  • PBS was added to the culture dish and dentin slices that were differentiated into osteoclasts using the medium containing both M-CSF and RANKL, and the cells were washed, and 4% paraformaldehyde was added, blocked from light, and maintained at 4°C overnight.
  • a solution containing 0.1% Triton X-100 in PBS was added for 15 minutes at room temperature in a light-blocking state to increase cell membrane permeability.
  • 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 state.
  • a solution containing 2% Hoechst33342 in PBS was added for 5 minutes at room temperature in a light-blocking state.
  • a mounting solution was added to the stained cells.
  • Phalloidin stains the cellular skeleton and Alexa Fluor 647 fluorescent dye is attached to show the morphology of the cell (green) in the corresponding channel (emission wavelength 660-680 nm).
  • Hoechst33342 binds to DNA and emits fluorescence, showing the nucleus of the cell in the corresponding channel (emission wavelength 450-500 nm) (blue).
  • ODN odanacatib
  • cathepsin K inhibitor was added at a concentration of 30 nM on the third day of differentiation.
  • osteoclasts showed a strong fluorescence 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.
  • Fig. 19 Bone marrow-derived monocytes (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.
  • Fig. 19a cells were fixed and stained with cathepsin K Ab (cathepsin K antibody, red), LAMP2A (lysosomal membrane, green), and Hoechst 33342 (nucleus, blue), while in Fig. 19b, CatKP1 (2 ⁇ M, red) was administered 1 h before observation, and cells were fixed and stained with LAMP2A (lysosomal membrane, green) and Hoechst 33342 (nucleus, blue).
  • osteoclasts were sliced at four locations (slices 1 to 4), and CatKP1 (2 ⁇ M, red) was administered 1 hour before observation.
  • 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
  • LAMP2A lysosomal membrane, purple
  • phalloidin F-actin, green
  • Hoechst 33342 Hoechst 33342
  • Example 5 Detection of cathepsin K in animal models using cathepsin K-specific probes
  • mice Five thousand cells (20 ⁇ L, Hank's balanced salt solution) of the E0771 murine breast cancer cell line (provided by Vanderbilt University) were injected under general anesthesia with 2% isoflurane into the right proximal tibia bone (Fig. 21a) and muscle (Fig. 21b) of mice (C57BL/6 mice, Orient). The contralateral left bone and tumor-injected muscle served as negative controls. Two-photon microscopy was performed according to the steps below. Mice were injected with CatKP1 (50 ⁇ M) via the tail vein and then general anesthesia was performed 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 images of the right tibia were obtained using a two-photon microscope (IVIM-MS3, IVIM Technology) providing an imaging field of view of 1099 ⁇ 1099 ⁇ m 2 with a 10X objective and a laser tuned to 920 nm (Seoul, Korea).
  • the SHG channel (emission wavelength 380-420 nm) of the two-photon microscope measures the nonlinear optical signal to image bone tissue rich in collagen, which is shown in blue, and 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 ⁇ m.
  • ImageJ software was used to analyze the images compiled into two-dimensional projection images from the z-stacks obtained from the RFP channel to quantify the intensity of the CatKP1 signal. Mice were euthanized at 2 weeks after two-photon microscopy.
  • CatKP1 showed particularly bright fluorescence in the right tibia but remained inactive in the contralateral bone (Fig. 21a and Fig. 21c).
  • no fluorescence was detected in the tumor transplanted into the leg muscle (Fig. 21b and Fig. 21d).
  • Histological staining of the tibia bone tissue showed that the cancer cells severely infiltrated the trabecular bone, and osteoclast invasion was confirmed within the tumor mass (Fig. 21c).
  • 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 were 512 X 512 pixels, 465 X 465 x 64 ⁇ m, and a step size of 1 ⁇ m. The acquired images were analyzed using Leica Microsystems' LAS X software and Bitplane's Imaris 9.
  • CatKP1 (50 ⁇ M) 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 Coldy Stimulating Factor 1 Receptor
  • osteoclasts a receptor found on the surface of bone marrow-derived cells such as osteoclasts, and is known to play an important role in regulating the formation and function of osteoclasts.
  • 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, intraperitoneally daily for 3 days at a volume of 100 ⁇ L, and randomly divided into three groups. On the first day of RANKL injection, one group was administered vehicle (Veh, 10% DMSO in corn oil) and the other group was administered odanaka nodule (ODN, 3.6 mg/kg, Selleckchem) via oral gavage. The mice were euthanized on the 4th day after imaging using a two-photon microscope. The remaining groups were set as controls.
  • RANKL 0.5 mg/kg, PeproTech
  • mice were dissected 2 h after intravenous injection of CatKP1.
  • mice treated with RANKL 0.5 mg/kg, PeproTech
  • CatKP1 50 ⁇ M, IV
  • a negative control group that did not receive 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 tibial radiography.
  • Figure 24a shows photographs of the organs of each mouse
  • Figure 24b shows z-stack images of sagittal sections of each organ observed using a two-photon microscope.
  • the upper side is the negative control group that was not administered CatKP1, and the lower side is the administered group. Bone (SHG, blue) and CatKP1 (RFP, red) could be observed.
  • Fig. 26a 1) is the epiphysis
  • Fig. 26a 2) is the observation of the trabecular bone
  • Fig. 26b is a graph showing the fluorescence intensity in the trabecular bone
  • Fig. 26c is a two-photon microscope image 24 hours after CatKP1 injection and after CatKP1 re-injection.
  • 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 osteoclast hyperplasia model was administered the cathepsin K inhibitor odanacatib (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 ⁇ L) and then the probe was introduced (RANKL+ODN).
  • CatKP1 50 ⁇ M was injected through the tail vein of each mouse.
  • 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.
  • the ovariectomy (OVX)-induced osteoporosis mouse model was surgically removed by surgically removing a pair of ovaries from 8-week-old female C57BL/6 mice through a midline dorsal incision under general anesthesia using 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 at the junction of the ovaries with the oviduct using a Bovie electrosurgical device. Bleeding was controlled with electrocautery, and the wounds were closed using surgical clips. Control mice that received sham surgery (Sham) underwent the same surgical procedure without ovisection.
  • the probe (CatKP1) manufactured in Example 1 was introduced at 4, 6, and 8 weeks, and zoledronate (ZA), an osteoporosis treatment drug 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 by fluorescence.
  • ZA zoledronate
  • the fluorescence of CatKP1 increased over time after OVX surgery (Fig. 29b).
  • the fluorescence signal was significantly enhanced about 8.2-fold compared to the normal mouse model (Sham) at 8 weeks after OVX surgery (Fig. 29c).
  • 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.

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Abstract

La présente invention concerne une sonde permettant de mesurer l'activité de la cathepsine K, une composition pour mesurer l'activité des ostéoclastes comprenant la sonde, une composition pour diagnostiquer des maladies osseuses comprenant la sonde, un kit permettant de mesurer l'activité des ostéoclastes comprenant la composition, un kit permettant de diagnostiquer des maladies osseuses, une méthode permettant de mesurer l'activité de la cathepsine K, une méthode permettant de mesurer l'activité des ostéoclastes, une méthode permettant de diagnostiquer des maladies osseuses, une composition pour cribler des médicaments inhibant l'activité de la cathepsine K, et une méthode permettant de cribler des médicaments inhibant l'activité de la cathepsine K. Selon la présente invention, la rapidité et la précision du diagnostic peuvent être augmentées par la conception d'une sonde permettant de mesurer l'activité de la cathepsine K spécifiquement exprimée dans les ostéoclastes, et ainsi la sonde peut être avantageusement utilisée pour diagnostiquer des maladies osseuses et cribler des médicaments inhibant l'activité de la cathepsine K.
PCT/KR2024/095801 2023-05-18 2024-05-17 Sonde pour mesurer l'activité de la cathepsine k et composition pour diagnostiquer une maladie osseuse la comprenant Pending WO2024237764A2 (fr)

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