Peptide-based probes for disease diagnosis and uses thereof

Abstract

The present application relates to a probe in which a fluorescent dye-quencher is additionally combined with a fusion peptide to which a cell penetrating peptide-biomarker binding functional peptide for a disease is combined, and more particularly, to a cell penetrating peptide-biomarker molecular probe that can directly introduce a non-penetrating important domain of a disease into a cell, thereby penetrating a target cell and emitting fluorescence when binding to a target substance, thereby facilitating diagnosis and simultaneously inducing treatment. According to the present application, compared to conventional dye-based histological disease diagnosis, diseases such as inflammation can be identified in a non-invasive and rapid manner, and thus the probe can be used to diagnose various inflammatory diseases or monitor inflammation associated with transplantation during transplantation in the body. In addition, the probe can be applied to clinical use without going through a number of steps, and can be mass-produced, and thus, by using such a method, can be used to develop a drug delivery system and a treatment technique.

Description

Technical Field

[0001] This invention relates to a fusion peptide and its uses, and more specifically, to a probe, a diagnostic composition containing the probe, and a diagnostic method comprising the probe; the fusion peptide comprises a cell-penetrating peptide and a bioactive peptide, and is labeled with a fluorescent dye and a quencher, the probe being designed such that the fluorescent dye and the quencher are further bound to both ends of the fusion peptide formed by the chemical binding of the cell-penetrating peptide and the bioactive peptide, such that fluorescence is not emitted due to the quencher before the bioactive peptide reaches the target; and fluorescence is detectable when the bioactive peptide binds to the target biomarker. Technical Background

[0002] Non-invasive and simultaneous imaging with therapy has been attempted, with some success in the treatment of cancer and inflammatory diseases (Kim EM, et al., Nucl Med Biol 2009;36:371-8). Most of this type of imaging relies on specific enzyme activity, which is primarily expressed on substrate targets previously conjugated to activatable probes (Akhatib B, et al., J Biol Chem 2013;288:19280-7). For example, in rheumatoid arthritis, polymers specifically modified with matrix metalloproteinase (MMP)-3 substrates have been used as near-infrared fluorescence (NIRF) probes (Ryu JH, et al., Arthritis Rheum 2011;63:3824-32). The problem is that its accuracy is only detectable in the mid-to-late stages of disease progression (when the biomarker is present at high concentrations), and the probe is not specific to a single MMP. In contrast, probes designed based on protein-protein binding may be more advantageous for early detection. This specific binding of one molecule to another is a well-known biological phenomenon and has led to the development of numerous diagnostic and therapeutic techniques based on it. Molecules that form stable covalent bonds between synthetic probes and specific sites on target proteins have many potential applications in biomedical science (Marquez BV, et al., Bioconjugate Chemistry 2012;16:1080-9). Some interesting examples include peptide ligands, whose strongest bindings typically occur with nanomolar dissociation constants, making them advantageous for confirming specificity (Ehrlich A, et al., Biochem Pharmacol 2013;86:1263-71).

[0003] The inventors applied the E3 ubiquitinase system as a protein-binding target. Ubiquitination is a natural proteasome degradation reaction used to balance the removal of aging or abnormal proteins, while dysfunction or overactivity of ubiquitination can lead to inflammatory or disease states. In the ubiquitin-proteasome system (UPS), ubiquitin linkage is mediated by a cascade of E1-E2-E3 enzymes (Tian M, Xie Q. J Integr Plant Biol 2013;55:54-63). Ubiquitin is activated by E1, and then a thioester bond is formed between the C-terminus of ubiquitin and the cysteine ​​residue of E1, thereby generating activated ubiquitin. The final binding between the C-terminus of ubiquitin and the target is formed by the E3 ligase (Schulman BA, Harper JW. Nat Rev Mol Cell Biol 2009;10:319-31). Most studies have identified E3 ligases as key molecules in many diseases associated with UPS (Wang Z, et al., Neoplasia 2013;15:1028-35). The two main classes of E3 ligases are RING-type E3 and HECT-type E3, with C2-WW-HECT E3 being the most extensively studied. The E3 subfamily typically consists of an N-terminal C2 domain and 2 to 4 WW domains for substrate interaction. Smurf-1, an example of an E3 ubiquitin ligase, possesses a domain for substrate interaction and a C-terminal HECT domain for maintaining E3 activity. It is a HECT-like ubiquitin ligase that specifically binds to Smad proteins to promote proteasome-mediated degradation of Smad and is a frequently observed biomarker in inflammatory diseases (Sangadala S, et al., Biochemistry 2006;281:17212-9). Smads, particularly Smad 1 / 5 / 8, are also associated with bone formation. Overexpression of Smurf is significant in most inflammatory diseases such as rheumatoid arthritis, periodontitis, osteoporosis, other bone resorption diseases, and cancer. In these inflammatory diseases, bone formation is significantly impaired due to the degradation of Smad proteins. E3 ubiquitin works by inducing proteasome degradation through specific binding to target proteins. The inventors designed a peptide motif as a therapeutic tool to block the proteasome degradation of Smads by binding to E3 ligases in cells and tissues overexpressing Smurf-1. LIM mineralization protein (LMP)-1 is a recently identified intracellular protein that has been shown to stimulate osteoblast differentiation and the formation of mineralized nodules in mouse cranial osteoblasts in vitro (Boden SD, et al., Endocrinology 1998;139:5125-34).Although the exact mechanism or key regions of LMP-1 osteogenic activity are not yet clear, it has been shown that LMP-1 binds strongly to Smurf-1 (Sangadala S, et al., Proteins 2007;68:690-701).

[0004] The inventors hypothesize that a specific domain sequence of LMP-1 can be prepared via peptide synthesis, and that this sequence can mimic all the activities of LMP-1; they also hypothesize that the domain sequence of LMP-1 will strongly bind to the inflammatory marker Smurf. The generated peptide should meet three basic requirements: (i) the peptide should retain the same chemical and physical properties as LMP-1; (ii) the peptide should have a defined Smurf-1 binding affinity (i.e., greater than the binding affinity between Smurf-1 and Smad); and (iii) the sequence should be deliverable into cells and should be labeled with a fluorescent dye capable of monitoring intracellular localization and Smurf-1 binding. When target binding alters the peptide conformation, detection can be achieved using fluorescence resonance energy transfer (FRET) imaging. That is, a fluorescent dye and a quenching dye are respectively bound to the respective ends of the peptide. At this point, there is no fluorescence, but the conformational change may increase the distance between the two dyes. According to the present invention, a peptide probe, together with BHQ-1 and a fluorescent dye, is conjugated to a sequence. The peptide probe exhibits fluorescence inactivation before reaching Smurf-1, and upon binding between Smurf-1 and the Smurf1-binding peptide (SBP) sequence, the peptide probe transitions to an active state due to the increased distance between the fluorescent dye and the quencher, thereby compensating for interference. As a result, it has been confirmed that this peptide motif, referred to as a peptide probe, can provide a convenient, rapid, specific, and sensitive means for monitoring analytes and reporting the presence of specific target substrates. Based on this discovery, the present invention has been completed.

[0005] The information disclosed in the background section is provided merely to enhance the understanding of the background of the invention, and therefore may not include information that forms the prior art that is obvious to those skilled in the art. Summary of the Invention

[0006] One object of the present invention is to provide a probe in which a fluorescent dye and a quencher are labeled on a fusion peptide, the fusion peptide comprising a cell-penetrating peptide and a bioactive peptide bound together, for rapid diagnosis of diseases in a non-invasive manner.

[0007] Another object of the present invention is to provide a composition for diagnosing inflammatory diseases, the composition containing the probe.

[0008] Another object of the present invention is to provide a method for diagnosing inflammatory diseases including the probe, and a method for providing information for diagnosing inflammatory diseases including the probe.

[0009] According to one aspect of the invention, the above and other objectives can be achieved by providing a probe for detecting biomarkers, the probe comprising a fusion peptide and a fluorescent dye and a quencher bound to the fusion peptide, the fusion peptide comprising a cell-penetrating peptide and a bioactive peptide bound together.

[0010] According to another aspect, a composition for diagnosing inflammatory diseases is provided, which contains the probe.

[0011] According to another aspect, a method for diagnosing inflammatory diseases and a method for providing diagnostic information are provided, each method comprising processing a sample separated from a subject with the probe. Attached Figure Description

[0012] Figure 1 The following schematic diagram illustrates the concept of real-time molecular imaging using peptide probes based on the ubiquitin-proteasome system (UPS) and its applications in diagnosis and therapy.

[0013] Figure 2 (A) illustrates a schematic diagram of peptide design and production using Fmoc solid-phase peptide synthesis; (B) shows the results of evaluating the binding of SBP and hBCPP-SBP, including the WW domain interaction site, to Smurf-1 using narrow-line blot analysis; and sensor maps confirming the interactions between (C) SBP and Smurf-1, (D) hBCPP-SBP and Smurf-1, (E) human recombinant protein Smad 1 and Smurf-1, and (F) hBCPP and Smurf-1, where the concentrations from top to bottom are 1000, 4000, 6000, and 8000 nM, respectively; and (G) shows the SPR analysis results of the specific binding between 1 μM peptides and immobilized Smurf-1.

[0014] Figure 3 (A) illustrates the following schematic diagrams: fluorescence imaging of the fluorescently labeled peptide following structural changes after binding to the target marker (photoemission, left inset); the BHQ-1 conjugated fluorescently labeled peptide (middle inset); and the peptide that re-fluoresces using Smurf-1 protein (photoreemission, right inset). (B) shows the results of treating hMSCs overexpressing Smurf-1 with different doses of the fluorescent-quenching peptide (fluorescence images detected at 488 nm using a LAS3000). (C) shows the quantification of fluorescence intensity using a fluorometer. Data are presented as mean ± standard error of the mean (*p < 0.05, ***p < 0.01). Detailed Implementation

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. Generally, the nomenclature used herein is well-known and commonly used in the art.

[0016] This invention relates to peptide probes based on intracellular delivery technology, which can be more useful in the diagnosis of diseases including inflammation. This intracellular delivery technology enables non-permeable bioactive substances, such as peptides or drugs bound to them, to penetrate into cells using cell-penetrating peptides.

[0017] By chemically conjugating cell-penetrating peptides, non-permeable bioactive substances (such as peptides or drugs) can be rapidly and safely delivered into cells through in vivo and in vitro processing, i.e., directly introduced into cells without endocytosis (a traditional method of intracellular uptake). Peptide probes labeled with fluorescent dyes and quenchers were prepared. Because the distance between the quencher and the fluorescent dye increases after the probe penetrates the target cell via the cell-penetrating peptide, and fluorescence only occurs when the bioactive peptide binds to the biomarker, molecular diagnosis can be easily performed even at low concentrations. This suggests that the probe can be used to diagnose diseases expressing target biomarkers.

[0018] Therefore, in one aspect, the present invention relates to a probe for detecting biomarkers, the probe comprising a fusion peptide and a fluorescent dye and a quencher bound to the fusion peptide, the fusion peptide comprising a cell-penetrating peptide and a bioactive peptide bound together with each other.

[0019] As used herein, the term "probe" refers to a peptide or protein-based probe used to determine the presence or absence of a specific biomarker, wherein the probe is based on a cell-penetrating peptide that is cell-permeable, including a bioactive peptide that specifically binds to a specific biomarker, and is conjugated to a fluorescent dye and a quencher at a distance from which the fluorescent dye can be quenched by binding to the quencher.

[0020] As used herein, the term "biomarker" refers to molecular information based on a single molecule or a molecular pattern derived from DNA, RNA, metabolites, proteins, and protein fragments, and is an indicator capable of detecting changes in an organism caused by genetic or epigenetic alterations.

[0021] As used herein, the term "cell-penetrating peptide" is used interchangeably with "cell-penetrating domain" and "protein transduction domain (PTD)," and refers to a permeable functional peptide capable of delivering peptides, drugs, or drug-containing particles into the cytoplasm or nucleus of a cell. This cell-penetrating peptide can form covalent bonds with oligonucleotides, peptides, proteins, oligosaccharides, polysaccharides, or nanoparticles, and can deliver these substances into the cell without the need for separate receptors, carriers, or energy.

[0022] In this invention, the cell-penetrating peptide may comprise an amino acid sequence of 5 to 15 amino acids, and contains a total of 40% to 70% of at least one amino acid selected from the group consisting of arginine (R), lysine (K) and histidine (H), but is not limited thereto.

[0023] In this invention, the cell-penetrating peptide may include at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12:

[0024] H4S (SEQ ID NO: 1: SSRKKNPNCRRH), H4Q (SEQ ID NO: 2: QRARKKNKNCRRH), HBD-3P (SEQ ID NO: 3: CSTRGRKCCRRKK), H2 (SEQ ID NO: 4: HKREKRQAKHKQRKR), H3 (SEQ ID NO: 5: KSKNKKKQRKGPHRK), H3B (SEQ ID NO: 3: CSTRGRKCCRRKK) NO: 6: KPRPGRKDRRKK), H4-1 (SEQ ID NO: 7: RRRRAKRSPKHHS), H6 (SEQ ID NO: 8: SRRRQQSRNR), H8 SEQ ID NO: 9:RAVRPLRRRQPKKS), H4C (SEQ ID NO: 10: CSSRKKNPNCRRH), H5C (SEQ ID NO: 11:CSSRKKNKNCPRRH) and H6C (SEQ ID NO: 12: CSSRKKNPNCPRRH).

[0025] H4Q is a peptide derived from human bone morphogenetic protein (BMP)-4, HBD-3P is a peptide derived from human β-defense peptide, H2 is a peptide derived from human bone morphogenetic protein (BMP)-2, H3 and H3B are peptides derived from human bone morphogenetic protein (BMP)-3, H4-1 is a peptide obtained by partially modifying a peptide derived from human bone morphogenetic protein (BMP)-4, H6 is a peptide derived from human bone morphogenetic protein (BMP)-6, and H8 is a peptide derived from human bone morphogenetic protein (BMP)-8.

[0026] Considering in vivo stability, the amino acids that make up cell-penetrating peptides can be either L-type or D-type.

[0027] In this invention, other peptides or peptide analogs besides the aforementioned peptides can be used as long as they can penetrate the cell membrane. Similarly, the fusion peptide of the cell-penetrating peptide and the bioactive peptide according to the present invention can also be produced using conventional non-human cell-penetrating functional domains (e.g., peptides derived from TAT or arginine). To improve biocompatibility, embodiments of the present invention use human peptides. However, the present invention is also applicable to conventional viral or non-human peptides.

[0028] As used herein, the term “bioactive peptide” is used interchangeably with “bioactive protein,” “bioactive domain,” “biomarker conjugate,” and “biomarker binding sequence,” and can be a peptide containing an amino acid sequence that specifically binds to a particular biomarker and has anti-inflammatory, antimicrobial, anticancer, tissue regeneration, or bone regeneration induction activities.

[0029] In this invention, the bioactive peptide may include at least one amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 16: SEQ ID NO: 13 (GAPPPADSAP), SEQ ID NO: 14 (PPGY) and SEQ ID NO: 15 (PPAY), which are bone differentiation or bone regeneration inducing sequences; and SEQ ID NO: 16 (anti-inflammatory peptide: TRGRKCCRRKK), which is an anti-inflammatory sequence.

[0030] In this invention, a fusion peptide of a cell-penetrating peptide and a bioactive peptide can be produced chemically using a peptide synthesizer. Specifically, a bone differentiation-inducing sequence or a bioactive domain is sequentially chemically synthesized at the C-terminus of a protein transduction domain (PTD) with intracellular permeability. This allows the fusion peptide to be synthesized in either the N-terminus – protein transduction domain – bioactive domain – C-terminus sequence or the N-terminus – bioactive domain – protein transduction domain – C-terminus sequence. The bioactive domain is primarily used to bind to disease biomarkers. The bioactive domain can also possess pharmacological activity; for example, it can not only have bone differentiation-inducing activity but also the ability to act as a substance that regulates gene expression and physiological function in vitro or in vivo, correcting abnormal pathological conditions caused by the deficiency or over-secretion of substances involved in functional regulation in vivo. Furthermore, its in vivo stability can be considered, allowing it to be L-form or D-form.

[0031] In this invention, the fusion peptide can be applied to the detection of intracellular and in vivo biomarkers and the diagnosis of diseases by covalently binding with a fluorescent dye and a quencher. In this case, the fluorescent dye or quencher can be covalently bound to the N-terminus or C-terminus of the fusion peptide, and preferably, covalent binding can be achieved by additionally linking a cysteine ​​residue to the end of the cell-penetrating peptide or the bioactive peptide.

[0032] Furthermore, complexes of fusion peptides with fluorescent dyes and quenchers can be produced by inducing chemical bonding using cross-linking agents. When cross-linking agents are used to induce chemical bonding, the presence of free amino groups at the N-terminus of the cell-penetrating peptide facilitates the formation of complexes via the cross-linking agent. The crosslinking agents available in this invention include, but are not limited to, 1,4-bismaleimide butane (BMB), 1,11-bismaleimide tetraethylene glycol (BM[PEO]4), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), succinimide-4-[N-maleimide methylcyclohexane-1-carboxy-[6-aminohexanoate]] (SMCC) and its sulfonated salt (sulfo-SMCC), succinimide-6-[3-(2-pyridyldithio)propamido]hexanoate (SPDP) and its sulfonated salt (sulfo-SPDP), m-maleimide benzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonated salt (sulfo-MBS), and succinimide-4-(p-maleimide phenyl)butyrate ester (SMPB) and its sulfonated salt (sulfo-SMPB). In particular, since labeling fluorescent dyes alone can make accurate diagnosis difficult due to nonspecific fluorescence expression, quenchers are required. Fluorescence resonance energy transfer (FRET) is essential for fluorescent transducers designed to fluoresce only when the bioactive domain binds to the biomarker protein of the disease.

[0033] In this invention, the fluorescent dye may be selected from the group consisting of: fluorescein, fluorescein isothiocyanate (FITC), chlorotriazine fluorescein, rhodamine green, rhodamine red, tetramethylrhodamine, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, anthocyanidates, and thiadicarbon anthocyanins; and the quencher may be selected from the group consisting of: Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry quencher, Black Hole quencher (BHQ), Qxl, Iowa Black FQ, Iowa Black RQ, and IRDye QC-1, but is not limited thereto. Preferably, the fluorescent dye may be FITC, and the quencher may be BHQ-1, but is not limited thereto.

[0034] In another respect, the present invention relates to a composition for diagnosing inflammatory diseases, which contains probes for detecting biomarkers.

[0035] As used herein, the term "diagnosis" means accurately determining a subject's condition relative to a specific disease or ailment, and includes determining the name, cause, type, severity, detailed aspects of symptoms, and presence or absence of complications. For example, a subject's condition relative to a specific disease or ailment can be broadly defined as: susceptibility to a specific disease or ailment; determining a subject's current disease; identifying prognostic characteristics of a subject's disease; identifying disease status; determining disease stage; or predicting cancer susceptibility and responsiveness to treatment; providing a basis for appropriate treatment based on the patient's disease and symptoms, such as confirming a subject's symptoms to determine the effectiveness of a specific drug; and additionally, predicting and confirming relapse in subjects who have recovered from a specific disease or ailment.

[0036] In this invention, inflammatory diseases can be selected from the group consisting of: bone diseases, fibrosis, periodontitis, rheumatoid arthritis, inflammatory bowel disease, inflammatory reactions caused by biomaterial implantation, asthma, dermatitis, psoriasis, and cancer, but are not limited thereto.

[0037] In this invention, bone diseases may be selected from the group consisting of: osteoporosis, osteoarthritis, osteitis, osteogenesis imperfecta, hypercalcemia, osteomalacia, Paget's disease, bone loss caused by cancer, and osteonecrosis, but are not limited thereto.

[0038] In another aspect, the present invention relates to a kit for diagnosing inflammatory diseases, comprising probes for detecting biomarkers.

[0039] In another aspect, the present invention relates to methods for diagnosing inflammatory diseases and methods for providing diagnostic information, each method comprising contacting a sample separated from a subject with a probe for detecting biomarkers.

[0040] In aspects of this invention, unless otherwise stated, the definitions and implementations of terms not described herein may have the same characteristics as those described in the context of compositions for diagnosing inflammatory diseases.

[0041] The present invention will now be described in more detail with reference to embodiments. However, it will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0042] Example 1: Synthesis of Fusion Peptide

[0043] Using a peptide synthesizer, a fusion peptide comprising GAPPPADSAP (SEQ ID NO: 13), a bone differentiation inducing sequence derived from LMP1, and H4S (SSRKKNPNCRRH: SEQ ID NO: 1), a PTD, was synthesized via Fmoc solid-phase chemical synthesis. Figure 1The synthesis was performed using Rink resin (0.075 mmol / g, 100 to 200 mesh, 1% DVB crosslinking) with Fmoc (9-fluorenylmethoxycarbonyl) as the protecting group. 50 mg of Rink resin was loaded into the synthesizer and swollen with DMF, followed by removal of the Fmoc group using a 20% piperidine / DMF solution. Starting from the C-terminus of the sequence, 5 equivalents, 10 equivalents, and 5 equivalents of 0.5 M amino acid solution (solvent: DMF), 1.0 M DIPEA (solvent: DMF & NMP), and 0.5 M HBTU (solvent: DMF) were added, respectively, and the reaction was carried out under a nitrogen atmosphere for 1 to 2 hours. After each deprotection and coupling step, the mixture was washed twice with DMF and twice with NMP. Deprotection was also performed after the final amino acid coupling to remove the Fmoc group.

[0044] The synthesis was confirmed using a ninhydrin test. After testing and confirmation of the synthesis, the resin was dried with THF or DCM, and a TFA lysis mixture was added at a ratio of 20 mL per 1 g of resin, followed by shaking for 3 hours. The resin and the mixture containing the dissolved peptides were then separated by filtration. After removing the filtrate using a rotary evaporator, cold diethyl ether was added; or alternatively, the mixture was added directly to the TFA containing the dissolved peptides. Soluble An excess of cold diethyl ether was added to the liquid to crystallize the peptides into a solid phase, followed by centrifugation. The TFA mixture was then completely removed by repeated washing with ether and centrifugation. The resulting peptides were dissolved in distilled water and freeze-dried.

[0045] Fusion peptide: NH2-GAPPPADSAP-SSRKKNPNCRRH-C-COONH2 (SEQ ID NO: 17)

[0046] The synthesized peptide sequences were cleaved from the resin, washed, freeze-dried, and then separated and purified by liquid chromatography. The molecular weight of the purified peptides was measured by MALDI analysis.

[0047] Comparative Example 1: H4S (SSRKKNPNCRRH: SEQ ID NO: 1) as PTD

[0048] The peptide was synthesized using a peptide synthesizer via the Fmoc solid-phase chemical synthesis method.

[0049] Comparative Example 2: Inflammatory marker binding sequence derived from LMP1 (GAPPPADSAP: SEQ ID NO: 13)

[0050] The peptide was synthesized using a peptide synthesizer via the Fmoc solid-phase chemical synthesis method.

[0051] Example 2: Confirmation of the binding affinity between the fusion peptide and the inflammatory marker Smurf1

[0052] To chemically confirm the binding affinity between the fusion peptide synthesized by the method in Example 1 and Smurf1, the human Smurf1 protein used as the ligand in this experiment was purchased from OriGene Technologies (Rockville, MD, USA). The protein was immobilized at 100 mg / mL on a gold-coated surface of a CM5 chip (Cytiva, Sweden) using an EDC / NHS kit (BIACORE AB, Sweden), where amino groups were bound (the above experimental method can also be performed using the "immobilization" function embedded in the BIACORE T100 (BIACORE AB, Sweden) software used by the inventors). To determine the appropriate pH conditions for binding of the protein to the amino groups of the CM5 chip, a method called "pH probing" was performed. Alternatively, the method embedded in the BIACORE T100 (BIACORE AB, Switzerland) software used by the inventors can also be used for the "pH probing" experiment. After immobilizing the ligands under the pH conditions determined by this method, the analytes (the fusion peptide synthesized in Example 1, the cell-penetrating peptide of Comparative Example 1 used as a negative control, and the inflammatory marker binding sequence of Comparative Example 2 used as a positive control) were each passed through a CM5 chip at 10 mM, and the binding affinity was measured (the above experimental method can also be alternatively used by the "binding analysis" embedded in the BIACORE T100 (BIACORE AB, Sweden) software used by the inventors).

[0053] As a result, Figure 2 As shown in Figure A, Comparative Example 2, which binds to the WW domain within the Smurf1 protein sequence and possesses osteodifferentiation ability, exhibited a binding affinity of 750 RU (resonance units; units of binding affinity measurement) for Smurf1, with the fusion peptide showing 600 RU. In contrast, the cell-penetrating peptide of Comparative Example 1, used as a negative control, showed 100 RU. Figure 2 These results indicate that the fusion peptide possesses a bone differentiation-inducing sequence and that no significant damage was caused to the bioactive sequence (the region capable of functioning by binding to Smurf1 in vivo) during synthesis.

[0054] In osteoarthritis or other inflammatory diseases, Smurf1, as an inflammatory factor that interferes with tissue maintenance or regeneration, binds strongly to its corresponding protein SMAD, thereby promoting ubiquitination that degrades the corresponding protein and thus irreversibly interfering with cell regeneration. The inventors have determined that with increased Smurf1 overexpression, the inflammatory state becomes more severe.

[0055] The apparent binding constants (KA) of the LMP-1-derived Smurf1-binding peptide (SBP), cell-penetrating SBP fusion peptide (hBCPP-SBP), and cell-penetrating peptide (hBCPP) were calculated to be 1.036E+7 (M-1), 1.237E+7 (M-1), and 4.129E+5 (M-1), respectively. Therefore, the apparent dissociation constants (KD = 1 / KA) of SBP, hBCPP-SBP, and hBCPP are 96.6 nM, 80.8 nM, and 2422 nM, respectively. In contrast, the apparent dissociation constant of Smad-1, which is known to strongly bind to Smurf-1 during ubiquitination, is 298 nM (Table 1).

[0056] [Table 1]

[0057] Combination parameters of Smurf-1 with SBP and hBCPP-SBP

[0058]

[0059] Therefore, the binding constants suggest that the interaction between SBP and Smurf-1 is stronger than that between Smad-1 and Smurf-1. Thus, LMP-1 may competitively bind to Smurf-1 with Smad1 / 5 / 8, thereby inhibiting ubiquitination targeting. This relatively high affinity increases the likelihood that the Smurf-1 interaction is biologically significant. These results are consistent with the binding analysis and indicate that the hBCPP-SBP identified in this study is a key domain in LMP that inhibits Smurf-1, and is further activated by the inventors' addition of a human cell-penetrating peptide (hBCPP).

[0060] Furthermore, this function indicates that ubiquitination can be modulated by cell-permeable target-binding peptide probes, supporting its potential as a therapeutic agent. Figure 2 As shown, Smurf1 binds strongly to SMAD and induces proteasome degradation through ubiquitination. Furthermore, the degradation process is inhibited by the binding of Smurf1 to the peptide probe, and the degradation of SMAD is stopped.

[0061] Example 3: Synchronous imaging using fusion peptide probes in cells overexpressing Smurf-1

[0062] To investigate the binding interaction between the bioactive peptide (SBP) sequence and Smurf-1, a quenching strategy called fluorescence resonance energy transfer (FRET) was employed, and the fusion peptide was further modified with an activateable fluorescent dye. BHQ-1 efficiently quenched the fluorescence of fluorescein isothiocyanate (FITC). In this study, due to the close proximity of the fluorescent dye and BHQ, the fluorescence of both peptides (hBCPP and SBP) was not activated. However, after treatment with Smurf-1, the binding between Smurf-1 and SBP led to the separation of BHQ, thus resulting in fluorescent activity. Figure 3 A). The fluorescence intensity is directly proportional to the concentration of Smurf-1, indicating that the peptide probe of the present invention can be used for the diagnosis of inflammatory diseases. Figure 3 B and Figure 3 C). This may be a result of structural changes in the peptide caused by the relatively large Smurf-1 expression. To examine the following results in vitro, FRET analysis was performed on human osteosarcoma (HOS) cells that were positive for Smurf-1 expression and mouse fibroblasts (NIH3T3) that were negative for Smurf-1 expression. Figure 3 HOS cells treated with the quenched hBCPP-SBP probe recovered fluorescence signals, while NIH3T3 cells did not. Simultaneously, neither HOS nor NIH3T3 cells showed fluorescence when the cell-penetrating functional peptide was not attached, i.e., when treated with the targeting-binding peptide SBP, because SBP itself cannot penetrate the cell membrane. Furthermore, since hBCPP is a cell-penetrating functional peptide, it does not bind to Smurf-1, therefore no recovery of fluorescence signals was observed in cells treated with this peptide.

[0063] Industrial applicability

[0064] The diagnostic method according to the present invention is non-invasive and, compared to traditional stain-based histological diagnostics, can identify diseases (e.g., inflammatory conditions) relatively quickly. Therefore, it can be used to diagnose various inflammatory diseases or monitor transplant-related inflammatory responses during implantation. Furthermore, since this diagnostic method can be applied clinically without multiple steps and can be mass-produced, it has practical value in developing diagnostic drug delivery systems and therapeutic technologies.

[0065] Although specific configurations of the invention have been described in detail, those skilled in the art will understand that this description is provided to illustrate preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the invention. Therefore, the essential scope of the invention is defined by the appended claims and their equivalents.

[0066] Sequence List Free Text

[0067] An electronic document is attached.

Claims

1. A probe for detecting biomarkers, comprising: Fusion peptides, wherein the fusion peptides comprise cell-penetrating peptides and bioactive peptides bound together; as well as Fluorescent dyes and quenchers that bind to the fusion peptide.

2. The probe according to claim 1, wherein, The cell-penetrating peptide comprises an amino acid sequence of 5 to 15 amino acids, and contains a total of 40% to 70% of at least one amino acid selected from the group consisting of arginine (R), lysine (K), and histidine (H).

3. The probe according to claim 2, wherein, The cell-penetrating peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

12.

4. The probe according to claim 1, wherein, The bioactive peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO:

16.

5. A composition for diagnosing inflammatory diseases, comprising a probe according to any one of claims 1 to 4.

6. The composition according to claim 5, wherein, The inflammatory diseases are selected from the group consisting of: bone diseases, fibrosis, periodontitis, rheumatoid arthritis, inflammatory bowel disease, inflammatory reactions caused by biomaterial implantation, asthma, dermatitis, psoriasis, and cancer.

7. The composition according to claim 6, wherein, The bone diseases are selected from the group consisting of: osteoporosis, osteoarthritis, osteitis, osteogenesis imperfecta, hypercalcemia, osteomalacia, Paget's disease, bone loss and osteonecrosis caused by cancer.

8. A kit for diagnosing inflammatory diseases, comprising a probe according to any one of claims 1 to 4.

Images