Peptide-based probes for diagnosis of disease and uses thereof

Abstract

The present invention relates to a probe in which a fluorescence dye-quencher is additionally bound to a fusion peptide to which a cell-penetrating peptide-biomarker binding functional peptide for diseases is bound and, more particularly, to a cell-penetrating peptide-bioactive peptide molecular probe which directly introduces a non-penetrating disease-sensitive important domain into a cell so as to penetrate a target cell and emit fluorescence when bound to a target material, thereby facilitating diagnosis and simultaneously inducing treatment. According to the present invention, compared to conventional staining-based histological disease diagnosis, diseases, such as inflammation, can be identified in a non-invasive and rapid manner, and thus the probe may be useful in diagnosing various inflammatory diseases or monitoring transplant-related inflammatory responses during in vivo transplantation. Also, the probe is applicable to clinical use without undergoing multiple steps and can be mass-produced, and thus, by using this approach, is useful in the development of drug delivery systems and treatment technologies.

Description

Peptide-based probes for disease diagnosis and their uses

[0001] The present invention relates to a fusion peptide of a cell-penetrating peptide and a physiologically active peptide labeled with a fluorophore and a quencher, and a use thereof, and more specifically, to a probe designed to additionally bind a fluorophore and a quencher to both ends of a chemically bonded cell-penetrating peptide and a physiologically active peptide so that fluorescence is not emitted by the quencher until reaching a target, and fluorescence is detected when the physiologically active peptide binds to a target biomarker, and a diagnostic composition and a diagnostic method comprising the same.

[0002]

[0003] Noninvasive and simultaneous imaging and therapy have 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 these imaging approaches have previously relied on targeting specific enzyme activities, primarily expressed on substrates conjugated to activatable probes (Akhatib B, et al., J Biol Chem 2013;288:19280-7). For example, in the case of rheumatoid arthritis, matrix metalloproteinase (MMP)-3-specific substrate-modified polymers have been studied and attempted as near-infrared fluorescent (NIRF) probes (Ryu JH, et al., Arthritis Rheum 2011;63:3824-32). The problem was that the accuracy was only recognized after the mid-term when this marker was present at high concentrations, and the probe was not specific for a single MMP. Rather, designing probes 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 many diagnostic and therapeutic technologies have been developed based on it. The formation of stable covalent bonds between synthetic probes and specific sites on target proteins has many potential applications in biomedical science (Marquez BV, et al., Bioconjug Chem 2012;16:1080-9). Among the more interesting examples are peptide ligands, the most potent of which typically bind to receptors with nanomolar dissociation, which is advantageous for determining specificity (Ehrlich A, et al., Biochem Pharmacol 2013;86:1263-71).

[0004] We applied the E3 ubiquitin enzyme system as a protein binding target. Ubiquitination is a natural proteasome degradation reaction that balances the removal of aged or abnormal proteins, but dysfunction or overactivity of ubiquitination leads to inflammation and disease states. In the ubiquitin proteasome system (UPS), ubiquitin ligation is mediated by a cascade of enzymes E1-E2-E3 (Tian M, Xie Q. J Integr Plant Biol 2013;55:54-63). Ubiquitin is activated by E1, and then a thioester bond formed between the cysteine ​​of ubiquitin C-terminus and E1 is transferred to active ubiquitin. The final linkage of the ubiquitin C-terminus to the target occurs by E3 ligase (Schulman BA, Harper JW. Nat Rev Mol Cell Biol 2009;10: 319-31). Most studies have targeted E3 ligases as key molecules in many diseases associated with the UPS (Wang Z, et al., Neoplasia 2013;15:1028-35). The two main classes of E3 ligases are RING and HECT type E3s, with C2-WW-HECT E3s being the most studied. This subfamily of E3s typically consists of an N-terminal C2 domain, two to four WW domains, a domain for substrate interaction, and a C-terminal HECT domain to maintain E3 activity. Smurf-1, an example of an E3 ubiquitin ligase, is a HECT class ubiquitin ligase that specifically binds Smad proteins and promotes their degradation by the proteasome, and is a frequent marker in inflammatory diseases (Sangadala S, et al., J Biol Chem 2006;281:17212-9). Smads, particularly Smad 1 / 5 / 8, are also associated with osteogenesis. Smurf overexpression is significant in most inflammatory diseases, including rheumatoid arthritis, periodontitis, osteoporosis, other bone resorption diseases, and cancer.In these inflammatory diseases, it is clear that bone formation is impaired due to degradation of Smad proteins. The role of E3-ubiquitin is to induce proteasomal degradation based on specific binding to target proteins. We designed a peptide motif as a therapeutic tool that blocks proteasomal 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 generate mineralized nodules in rat calvarial osteoblasts in vitro (Boden SD, et al., Endocrinology 1998;139:5125-34). Although the precise mechanism or key domain of LMP-1's osteogenic action is unknown, LMP-1 has been shown to strongly bind to Smurf-1 (Sangadala S, et al., Proteins 2007;68:690-701).

[0005] We hypothesized that a specific domain sequence of LMP-1 could be produced by peptide synthesis and mimic the full activity of LMP-1, while also strongly binding to the inflammatory marker Smurf. The resulting peptide must satisfy three essential requirements: (i) retain the same chemical and physical properties as LMP-1, (ii) have a well-defined Smurf-1 binding strength (i.e., greater than the binding strength between Smurf-1 and Smad), and (iii) the sequence must be delivered intracellularly and be fluorescently labeled to monitor Smurf-1 localization and binding. Target binding can be detected by fluorescence resonance energy transfer (FRET) imaging if it alters the peptide conformation. This involves conjugating a fluorescent dye and its quencher to each end of the peptide. Although fluorescence is absent, the conformational change can extend the distance between the two dyes. According to the present invention, a peptide probe is coupled to a sequence together with BHQ-1 and a fluorescent dye, and exhibits fluorescent inactivity until it reaches Smurf-1, and then, upon binding between Smurf-1 and a Smurf1-binding peptide (SBP) sequence, it switches to activity as the distance between the fluorophore and the quencher increases, thereby compensating for interference. Consequently, the peptide motif, called a peptide probe, has been confirmed to provide a convenient, rapid, specific, and sensitive means of monitoring analytes and reporting the presence of a specific target substrate, thereby completing the present invention.

[0006]

[0007] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0008]

[0009] Summary of the invention

[0010] The purpose of the present invention is to provide a probe in which a fusion peptide in which a cell-penetrating peptide and a physiologically active peptide are combined is labeled with a fluorescent substance and a quencher to rapidly diagnose a disease in a non-invasive manner.

[0011] Another object of the present invention is to provide a composition for diagnosing an inflammatory disease including the probe.

[0012] Another object of the present invention is to provide a method for diagnosing an inflammatory disease including the above probe and a method for providing information for diagnosing an inflammatory disease.

[0013]

[0014] To achieve the above purpose, the present invention provides a probe for detecting a biomarker in which a fluorophore and a quencher are combined with a fusion peptide in which a cell-penetrating peptide and a physiologically active peptide are combined.

[0015] The present invention also provides a composition for diagnosing an inflammatory disease comprising the probe.

[0016] The present invention also provides a method for diagnosing an inflammatory disease and a method for providing information for diagnosis, including a step of treating a sample separated from a subject with the probe.

[0017]

[0018] Figure 1 is a schematic diagram of the concept of real-time molecular imaging using a ubiquitin proteasome system (UPS)-based peptide probe and its application to diagnosis and therapy.

[0019] Figure 2 is a schematic diagram of peptide design and production using (A) F-moc solid-phase peptide synthesis, (B) the results of Smurf-1 binding evaluation of SBP and hBCPP-SBP containing WW domain interaction sites using slot blot analysis, (C) sensorgrams confirming the interactions between 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, at 1000, 4000, 6000, and 8000 nM from the top of each graph, and (G) the SPR analysis results for the specific binding of each peptide to immobilized Smurf-1 at 1 μM.

[0020] Figure 3 is a schematic diagram showing (A) the fluorescence imaging of fluorescently labeled peptides (light emission, left panel), BHQ-1-conjugated fluorescently labeled peptides (middle panel), and re-fluorescent peptides using Smurf-1 protein due to conformational changes upon binding to target markers (light re-emission, right panel), (B) the results of treating Smurf-1-overexpressing hMSCs with various doses of fluorescently quenched peptides (fluorescence images were detected with LAS3000 at 488 nm), and (C) the fluorescence intensity was quantified by a fluorimeter. Data are presented as mean ± standard error of the mean (*p-value < 0.05, ***p-value < 0.01).

[0021]

[0022] Detailed description of the invention and preferred embodiments

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0024]

[0025] The present invention relates to a peptide probe that can be more usefully applied in the diagnosis of diseases including inflammation, based on an intracellular delivery technique that allows a non-permeable bioactive substance such as a peptide or drug bound thereto to penetrate into cells using a cell-penetrating peptide.

[0026] A non-permeable bioactive substance such as a peptide or drug to be introduced is treated in vivo and in vitro to allow it to penetrate cells quickly and safely, that is, to be introduced directly into cells without the endocytosis process, which is a conventional cellular uptake method, by chemically bonding a cell-penetrating peptide, and a peptide probe labeled with a fluorescent substance and a quencher is manufactured by labeling the fusion peptide, and only when the physiologically active peptide binds to the biomarker after the probe penetrates the target cell by the cell-penetrating peptide, does the quencher and the fluorescent substance become distant and only then does light shine, thereby facilitating molecular diagnosis even at low concentrations, and thus it was confirmed to be useful for the diagnosis of diseases expressing target biomarkers.

[0027]

[0028] Accordingly, the present invention relates, from one aspect, to a probe for detecting a biomarker in which a fluorophore and a quencher are combined with a fusion peptide in which a cell-penetrating peptide and a physiologically active peptide are combined.

[0029] In the present invention, the "probe" is a peptide or protein-based probe for confirming the presence or absence of a specific biomarker, has cell-penetrating functionality by a cell-penetrating peptide, includes a physiologically active peptide that specifically binds to a specific biomarker, and is characterized in that a fluorescent substance and a quencher are combined within a distance at which the fluorescent substance can be quenched by binding to the quencher.

[0030] In the present invention, the “biomarker” is molecular information based on a single molecule or a pattern of molecules derived from DNA, RNA, metabolites, proteins, and protein fragments, and is an indicator that can detect changes in the body caused by the influence of genetic or epigenetic changes in a living organism.

[0031] In this specification, the “cell-penetrating peptide” is used interchangeably with “cell-penetrating domain” and “protein transport domain (PTD)”, and refers to a permeable peptide or the like that can permeate a peptide, drug, or drug-containing particle into the cytoplasm or nucleus of a cell. By forming a covalent bond with an oligonucleotide, peptide, protein, oligosaccharide, polysaccharide, or nanoparticle, these substances can be introduced into the cell without requiring a separate receptor, carrier, or energy.

[0032] In the present invention, the cell-penetrating peptide may be characterized by being composed of a sequence of 5 to 15 amino acids and having a content ratio of at least one amino acid selected from the group consisting of arginine (R), lysine (K) and histidine (H) of 40 to 70%, but is not limited thereto.

[0033] In the present invention, the cell-penetrating peptide may be characterized by including at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12:

[0034] 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: 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).

[0035] H4Q is a peptide derived from human bone morphogenetic protein (BMP)-4, HBD-3P is a peptide derived from human beta defensin, H2 is derived from human bone morphogenetic protein (BMP)-2, H3 and H3B are derived from human bone morphogenetic protein (BMP)-3, H4-1 is a peptide derived from human bone morphogenetic protein (BMP)-4 with some modification, H6 is derived from human bone morphogenetic protein (BMP)-6, and H8 is a peptide derived from human bone morphogenetic protein (BMP)-8.

[0036] The amino acids constituting the above cell-penetrating peptide can be in L-form or D-form considering stability in the body.

[0037] In the present invention, the cell-penetrating peptide may be any peptide or peptide analogue other than the aforementioned peptide, as long as it can penetrate the cell membrane. Similarly, a fusion peptide of the cell-penetrating peptide and the physiologically active peptide according to the present invention may be created using an existing non-human cell-penetrating functional domain, such as TAT ​​or an arginine-derived peptide. However, in the present invention, human-derived peptides are listed to impart biocompatibility, and this does not mean that they cannot be applied to existing viral or non-human-derived peptides.

[0038] In the present specification, the “biologically active peptide” is used interchangeably with “biologically active protein”, “biologically active domain”, “biomarker conjugate”, “biomarker binding sequence”, etc., and may be a peptide including an amino acid sequence that specifically binds to a specific biomarker and has anti-inflammatory functionality, antimicrobial functionality, anticancer functionality, tissue regeneration functionality, or bone regeneration induction functionality.

[0039] In the present invention, the physiologically active peptide may be characterized by including at least one amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 16:

[0040] Sequence number 13 (GAPPPADSAP), sequence number 14 (PPGY), sequence number 15 (PPAY), which are osteogenic differentiation and bone regeneration inducing sequences, and sequence number 16 (anti care peptide: TRGRKCCRRKK), which is an anti-inflammatory functional sequence.

[0041] In the present invention, a fusion peptide of a cell-penetrating peptide and a physiologically active peptide can be manufactured by chemical synthesis using a peptide synthesis device, and a bone differentiation-inducing sequence or a physiologically active domain is sequentially chemically synthesized at the C-terminal end of a protein transport domain (PTD) having cell-penetrating functionality, so that a fusion peptide can be manufactured by synthesizing in the order of 'N terminus-protein transport domain-physiologically active domain-C terminus' or 'N terminus-physiologically active domain-protein transport domain-C terminus'. The physiologically active domain is mainly used to have the function of binding to a biomarker of a disease. In addition, it can be characterized by being able to play a role in correcting abnormal pathologies caused by deficiency or excessive secretion of substances involved in functional regulation in the body, as a substance that regulates genetic expression and physiological functions in vitro or in vivo, as well as pharmacologically active functions, such as osteogenic differentiation induction, and can be made into L-type or D-type considering stability in the body.

[0042] In the present invention, the fusion peptide can be applied to cell and in vivo biomarker detection and disease diagnosis by covalently bonding with a fluorophore and a quencher. At this time, the fluorophore or quencher can be covalently bonded to the N-terminal or C-terminal portion of the fusion peptide, and preferably, a cysteine ​​can be additionally attached to the terminal portion of a cell-penetrating peptide or a physiologically active peptide to form a covalent bond.

[0043] Additionally, a complex of the fusion peptide with a fluorophore and a quencher can be formed by inducing chemical bonding using a cross-linking agent. When inducing chemical bonding using a cross-linking agent, the cell-penetrating peptide has a free amino group at the N-terminus, making it easy to form a complex using the cross-linking agent. Crosslinking agents that can be used in the present invention include 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4), 1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride (EDC), succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]] (SMCC) and its sulfonated salt (sulfo-SMCC), succinimidyl Examples thereof include, but are not limited to, succimidyl 6-[3-(2-pyridyldithio)-ropionamido] hexanoate (SPDP) and its sulfonated salt (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonated salt (sulfo-MBS), succimidyl[4-(p-maleimidophenyl)butyrate] (SMPB) and its sulfonated salt (sulfo-SMPB).In particular, since it is difficult to make an accurate diagnosis by labeling only the fluorescent dye itself due to non-specific fluorescence expression, it is essential to apply a quencher, which is essential for the fluorescence resonance energy transfer (FRET) technique, which designs the physiologically active domain to fluoresce only when it binds to a disease marker protein.

[0044] In the present invention, the fluorescent dye is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, cyanine series dyes and thiadicarbocyanine, but is not limited thereto, and the quencher is selected from the group consisting of Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, It may be characterized by being selected from the group consisting of BHQ), Qxl, Iowa black FQ, Iowa black RQ and IRDye QC-1. Preferably, the fluorescent agent may be FITC, and the quencher may be BHQ-1, but is not limited thereto.

[0045]

[0046] From another perspective, the present invention relates to a composition for diagnosing an inflammatory disease comprising the above-described probe for detecting a biomarker.

[0047] In the present invention, the “diagnosis” means accurately identifying the condition of a subject with respect to a specific disease or condition, and the contents thereof include the name of the disease, etiology, pathological type, severity, detailed aspects of the condition, and presence or absence of complications. For example, the condition of a subject with respect to a specific disease or condition is used in a broad sense, including not only susceptibility to a specific disease or condition, determination of the disease the subject is currently suffering from, but also confirmation of the characteristics of the disease, such as prognosis of the subject, identification of the disease condition, determination of the stage of the disease, or prediction of the sensitivity and responsiveness of cancer to treatment, obtaining a basis for appropriate treatment according to the patient’s disease and condition, such as confirming the condition of the subject to confirm the therapeutic effect of a specific drug, and further including prediction and confirmation of recurrence in a subject who has been cured from a specific disease or condition.

[0048] In the present invention, the inflammatory disease may be characterized by being selected from the group consisting of bone disease, fibrosis, periodontitis, rheumatoid arthritis, inflammatory bowel disease, biomaterial transplant inflammatory response, asthma, dermatitis, psoriasis, and cancer, but is not limited thereto.

[0049] In the present invention, the bone disease may be characterized by being selected from the group consisting of osteoporosis, osteoarthritis, osteitis, osteogenesis imperfecta, hypercalcemia, osteomalacia, Paget's disease, bone loss due to cancer, and osteonecrosis, but is not limited thereto.

[0050]

[0051] In another aspect, the present invention relates to a kit for diagnosing an inflammatory disease comprising the above-mentioned probe for detecting a biomarker.

[0052] In another aspect, the present invention relates to a method for diagnosing an inflammatory disease and a method for providing information for diagnosis, which comprises a step of treating a sample separated from a subject with a probe for detecting a biomarker.

[0053] In the context of the present invention, definitions and implementation examples of terms not described may share the same characteristics as those described in the context of the composition for diagnosing inflammatory diseases, unless otherwise stated.

[0054]

[0055] 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.

[0056]

[0057] Example 1: Synthesis of fusion peptides

[0058] A fusion peptide containing GAPPPADSAP (SEQ ID NO: 13) as a bone differentiation-inducing sequence derived from LMP1 and H4S (SSRKKNPNCRRH: SEQ ID NO: 1) as a PTD in order from the N-terminus was synthesized by F-moc solid-phase chemical synthesis using a peptide synthesizer (Fig. 1). The peptide was synthesized using Rink resin (0.075 mmol / g, 100-200 mesh, 1% DVB crosslinking) conjugated with Fmoc-(9-Fluorenylmethoxycarbonyl) as a blocking group. After adding 50 mg of Rink resin to the synthesizer, the resin was swelled with DMF, and a 20% piperidine / DMF solution was used to remove the Fmoc-group. From the C-terminus, 5, 10, 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 in sequence and reacted for 1 to 2 hours under a nitrogen flow. After each deprotection and coupling step, the reaction mixture was washed twice with DMF and NMP. After coupling the last amino acid, deprotection was performed to remove the Fmoc-group.

[0059] The synthesis was confirmed using the ninhydrin test method. After the test and the synthesis was completed, the resin was dried with THF or DCM, and then TFA cleavage cocktail was added at a ratio of 20 ml per 1 g of resin, shaken for 3 hours, and then filtered to separate the resin and the cocktail containing the dissolved peptide. The filtered solution was removed using a rotary evaporator, and then cold ether was added or an excessive amount of cold ether was added directly to the TFA cocktail solution containing the dissolved peptide to crystallize the peptide into a solid phase, which was then centrifuged. At this time, the TFA cocktail was completely removed through several washes with ether and centrifugation processes. The peptide thus obtained was dissolved in distilled water and lyophilized.

[0060] Fusion peptide: NH2-GAPPPADSAP-SSRKKNPNCRRH-C-COONH2 (SEQ ID NO: 17)

[0061] The synthesized peptide sequence was cleaved from the resin, washed, lyophilized, and then separated and purified by liquid chromatography. The molecular weight of the purified peptide was confirmed using MALDI analysis.

[0062]

[0063] Comparative Example 1: H4S (SSRKKNPNCRRH: SEQ ID NO: 1) as PTD

[0064] The above peptide was synthesized using a peptide synthesis device using the F-moc solid-phase chemical synthesis method.

[0065]

[0066] Comparative Example 2: Inflammatory marker binding sequence derived from LMP1 (GAPPPADSAP: SEQ ID NO: 13)

[0067] The above peptide was synthesized using a peptide synthesis device using the F-moc solid-phase chemical synthesis method.

[0068]

[0069] Example 2: Confirmation of binding affinity between fusion peptide and inflammatory marker Smurf1.

[0070] In order to chemically confirm the binding affinity between the fusion peptide synthesized by the method of Example 1 and Smurf1, human-derived Smurf1 protein, which acts as a ligand in this experiment, was purchased from Origene (Rockville, MD, USA). 100 mg / ml of the protein was bound to the gold-coated side of a CM5 chip (BIACORE AB, Sweden) with amino groups bonded to the surface using an EDC / NHS kit (BIACORE AB, Sweden) (the above experimental method may also use 'Immobilization' built into the BIACORE T100 (BIACORE AB, Sweden) software used by the present inventors). At this time, a method called 'pH scouting' was performed to find a suitable pH condition under which the protein can bind to the amino group of the CM5 chip, and one pH condition was found. The experimental method of 'pH scouting' may also use the one built into the BIACORE T100 (BIACORE AB, Sweden) software used by the present inventors. After binding the ligand under the pH conditions derived in this manner, 10 mM of each of the analytes (the fusion peptide synthesized in Example 1, the cell-penetrating peptide of Comparative Example 1 to be used as a negative control, and the inflammatory marker binding sequence of Comparative Example 2 to be used as a positive control) is flowed over the CM5 chip and the binding force is measured (the above experimental method may also be used with the 'Binding analysis' built into the BIACORE T100 (BIACORE AB, Sweden) software used by the present inventors).

[0071] As a result, as shown in Fig. 2A, the portion that binds to the WW domain in the Smurf1 protein sequence, Comparative Example 2 with osteogenic differentiation ability, showed a binding affinity for Smurf1 of 750 RU (Resonance Unit; a unit of measurement for binding affinity), the fusion peptide showed 600 RU, and the cell-penetrating peptide of Comparative Example 1 used as a negative control showed 100 RU (Figs. 2B to 2G). This can be said to be a result showing that the fusion peptide has a sequence that induces osteogenic differentiation and that the physiologically active sequence (a portion that can exhibit functionality by binding to Smurf1 in vivo) was not significantly damaged during the synthesis process.

[0072]

[0073] In cases of osteoarthritis and other inflammatory diseases, Smurf1, an inflammatory factor that interferes with tissue maintenance and regeneration, strongly binds to its counterpart, SMAD, and then initiates ubiquitination, which degrades the protein, irreversibly hindering cell regeneration. The inventors determined that the more overexpressed Smurf1, the more severe the inflammatory disease.

[0074] The apparent association constants (KA) for 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) were 96.6 nM, 80.8 nM, and 2422 nM for SBP, hBCPP-SBP, and hBCPP, respectively. In contrast, the apparent dissociation constant of Smad-1, a molecule known to strongly bind to Smurf-1 upon ubiquitination, is 298 nM (Table 1).

[0075]

[0076] Therefore, we can conclude from the binding constants that there is a stronger interaction between SBP and Smurf-1 than between Smad-1 and Smurf-1. Therefore, LMP-1 may compete with Smad1 / 5 / 8 for binding to Smurf-1, thereby inhibiting its targeting for ubiquitination. This relatively high affinity raises the possibility that the Smurf-1 interaction may have biological significance. These results are consistent with the binding assays above and demonstrate that the hBCPP-SBP identified in this study is a critical domain of LMP for Smurf-1 inhibition, which is further activated by the addition of a human cell-penetrating peptide (hBCPP) by the inventors.

[0077] Furthermore, these features suggest that ubiquitination can be regulated by cell-penetrating functional target-binding peptide probes, confirming their potential as therapeutic agents. As shown in Figure 2, Smurf1 strongly binds to SMAD, inducing its proteasomal degradation by ubiquitinating it. However, binding of Smurf1 to the peptide probe inhibited this degradation process, halting SMAD degradation.

[0078]

[0079] Example 3: Simultaneous imaging using fusion peptide probes in cells overexpressing Smurf-1.

[0080] To investigate the binding interaction between the bioactive peptide (SBP) sequence and Smurf-1 using a quenching strategy called FRET, the fusion peptide was further modified with an activatable fluorescent dye. BHQ-1 can quench the fluorescence of fluorescein isothiocyanate (FITC) with higher efficiency. In this study, the fluorescence of the two peptides (hBCPP and SBP) was inactive due to the close proximity of BHQ to the fluorescent dye. However, fluorescence activation was observed after Smurf-1 treatment, which was attributed to the sequestration of BHQ by the binding of Smurf-1 and SBP (Fig. 3A). The fluorescence intensity was proportional to the Smurf-1 concentration, indicating that the peptide probe of the present invention is useful for measuring inflammatory diseases (Figs. 3B and 3C). This is likely a result of a structural change in the peptide caused by the relatively large molecule Smurf-1. To investigate the following results in vitro, FRET analysis was performed on human osteosarcoma (HOS) cells, which are Smurf-1-positive cells, and mouse fibroblasts (NIH3T3) cells, which are Smurf-1-negative cells (Fig. 3). HOS cells treated with the quenched hBCPP-SBP probe restored the fluorescence signal, but NIH3T3 cells did not. On the other hand, neither HOS nor NIH3T3 cells showed fluorescence when the cell-penetrating functional peptide was linked, i.e., cells treated with the target-binding peptide SBP did not show fluorescence because SBP cannot penetrate the cell membrane itself. Furthermore, because the cell-penetrating functional peptide hBCPP does not bind to Smurf-1, no recovery of the fluorescence signal was observed in cells treated with this peptide.

[0081]

[0082] The diagnostic method according to the present invention can identify diseases, such as inflammation, relatively quickly and non-invasively compared to conventional staining-based histological disease diagnosis, making it useful for diagnosing various inflammatory diseases and monitoring inflammatory responses associated with transplantation during in vivo transplantation. Furthermore, it can be applied clinically without requiring multiple steps and can be mass-produced, making it useful for the development of diagnostic drug delivery systems and therapeutic technologies.

[0083]

[0084] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0085]

[0086] Electronic file attached.

Claims

1. A probe for detecting a biomarker in which a fluorophore and a quencher are combined with a fusion peptide in which a cell-penetrating peptide and a physiologically active peptide are combined.

2. A probe for detecting a biomarker, characterized in that in the first paragraph, the cell-penetrating peptide is composed of a sequence of 5 to 15 amino acids and has a content ratio of at least one amino acid selected from the group consisting of arginine (R), lysine (K), and histidine (H) of 40 to 70%.

3. A probe for detecting a biomarker, characterized in that in the second paragraph, 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. A probe for detecting a biomarker, characterized in that in the first paragraph, the physiologically active 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 an inflammatory disease, comprising a probe for detecting a biomarker according to any one of claims 1 to 4.

6. A diagnostic composition according to claim 5, wherein the inflammatory disease is selected from the group consisting of bone disease, fibrosis, periodontitis, rheumatoid arthritis, inflammatory bowel disease, biomaterial transplant inflammatory response, asthma, dermatitis, psoriasis, and cancer.

7. A diagnostic composition according to claim 6, wherein the bone disease is selected from the group consisting of osteoporosis, osteoarthritis, osteitis, osteogenesis imperfecta, hypercalcemia, osteomalacia, Paget's disease, bone loss due to cancer, and osteonecrosis.

8. A kit for diagnosing an inflammatory disease comprising a probe for detecting a biomarker according to any one of claims 1 to 4.

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