KR20110039093A - In vitro kit for detecting protease and method for preparing the same - Google Patents

Abstract

PURPOSE: An in vitro kit for quantitative measurement of protease activity and a method for manufacturing the same are provided to specifically emit strong fluorescence. CONSTITUTION: An in vitro kit for quantitative measurement of protease activity comprises a complex in which fluorescence and quencher are combined and a peptide substrate is conjugated to a polymer. The complex has structural formula 1. The quencher is a blackhole quencher, blackberry quencher, or derivative thereof. A method for manufacturing the kit comprises: a first step of synthesizing a specific peptide substrate to a specific protease and reacting with near infrared ray fluorescence; a second step of binding the peptide substrate with quencher; a third step of binding polymers to fluorescence-peptide substrate-quencher; and a fourth step of fixing the fluorescence-peptide substrate-quencher-polymers onto a 96-well.

Description

IN VITRO KIT FOR DETECTING PROTEASE AND METHOD FOR PREPARING THE SAME}

The present invention relates to an in vitro kit for measuring protease activity for quantitatively measuring the activity of protease in vitro and a method of manufacturing the same.

Recently, proteases have been found to play a pivotal role in various human diseases such as cancer and dementia and to provide a cause. For example, matrix metalloprotease (MMP) has been recognized in the past as a factor that degrades extracellular matrix in cells and the body, but through various studies they have been involved in integrin signaling and cell periphery. It has been shown to be involved in cellular movement following degradation of the pericellular matrix. In addition, MMP has been found to play an important role in cancer growth, such as new angiogenesis, cancer cell infiltration, metastasis.

As a method for quantitatively analyzing the activity of proteolytic enzymes such as the substrate metalloproteinase MMP, the electrophoretic technique using a polyacrylamide gel under nondenaturing conditions zymography, enzyme-linked immunosorbent assay (ELISA), which detects antigens using the enzyme-specific antibody, western blots that detect only the desired protein in a protein mixture, using antibodies that react with the epitope of the antigen Immunohistochemistry, which is a kind of assay that can be directly observed under a microscope, is used to detect a substance for a specific protein by using the sensitivity and specificity of an antibody reaction. However, the zymography method has a disadvantage in that the processing is cumbersome and inferior in specificity, and the ELISA method takes a long time. In addition, Western blot has the disadvantage of being expensive and also takes a long time, and immunohistochemistry requires a large amount of samples and reagents and also has a disadvantage of being time consuming. In addition, these methods are not efficient because they increase the cost and processing time for processing multiple samples at once.

In order to solve the above disadvantages, a method of measuring proteolytic enzymes using molecular imaging techniques has recently been developed.

The most representative technology is a polymer sensor for protease imaging developed at Harvard Medical School in 2001. The sensor consists of a chemical bond of a phosphor-peptide substrate-biocompatible polymer capable of degradation by proteolytic enzymes. When near-infrared phosphors are at close distances (tens of nm), the emission and excitation spectra of the phosphors are shared, and the fluorescence of the phosphors is quenched by the fluorescence resonance energy transfer (FRET) principle. Thus, when the phosphor-peptide is bound to the polymer, the fluorescence is quenched by the physical binding of the phosphor-phosphor, but the peptide is decomposed by protease and the fluorescence is restored when the distance between the phosphor-phosphor is increased. The degree of protease expression can be imaged. However, in this study, the sensor was quenched by using self-quenching, in which the fluorescence was quenched when the near-infrared phosphors were in close proximity. However, self-quenching due to FRET is made when the phosphor is very close and there is a disadvantage that the fluorescence extinction rate is not excellent.

Thus, the present inventors introduced a quencher to increase the signal-to-noise (S / N) ratio which is considered to be the most important in the optical imaging technique. Among the various quencher, the black hole quencher, which is a complete light absorber, has various absorption wavelengths. For example, BHQ-1 absorbs fluorescence very efficiently at wavelengths of 480 to 580 nm, BHQ-2 is 550 to 650 nm, and BHQ-3 is 620 to 730 nm. A single molecule of the phosphor-peptide substrate-quencher is capable of measuring the amount of protease since fluorescence is restored when the peptide substrate is degraded by the protease, and exhibits a higher fluorescence extinction rate than the auto-quenching of the phosphor-phosphor bonds. .

An object of the present invention is a phosphor for quantitatively imaging the activity of proteolytic enzymes expressed in cells and tissues in the body, a peptide substrate specifically degraded by proteolytic enzymes, and a quencher that absorbs the luminescence of the phosphors in the polymer. It is to provide an in vitro kit for measuring the bound protease activity and a method of preparing the same.

In order to achieve the above object, the present invention is in vitro for quantitative measurement of protease activity comprising a complex in which a phosphor substrate and a quencher is coupled, the peptide substrate specifically cleaved by protease is bound to a polymer. Provide the kit.

The complex included in an in vitro kit for quantitative determination of protease activity has the following structural formula:

[Formula 1]

Here, A is a phosphor, B is a peptide substrate specifically cleaved by the proteolytic enzyme, C is a quencher that can absorb the luminescence of the phosphor and exhibit a quenching effect, D is well It is a polymer for attaching a large amount of probes made of ABC to a plate.

According to one embodiment of the invention, the proteolytic enzyme that specifically degrades the peptide substrate of the complex is matrix metalloproteinases (MMP), thrombin, FXIIIa, caspase, urokinase plasmin It may be one selected from the group consisting of urokinase plasminogen activator (uPA), HIV protease, DPP-IV and proteasome.

According to one embodiment of the present invention, the phosphor may be a phosphor emitting red or near infrared fluorescence. More specifically, the phosphor may be selected from the group consisting of cyanine, fluorescein, tetramethylrodamine, Alexa, bodiphy, and derivatives thereof.

According to one embodiment of the present invention, the peptide may be a peptide substrate that is specifically degraded by protease.

According to one embodiment of the invention, the quencher may be selected from the group consisting of blackhole quencher, blackberry quencher quencher and derivatives thereof capable of quenching fluorescence.

According to one embodiment of the present invention, as the polymer to be bonded to the phosphor-peptide substrate-quencher, the molecular weight may be used without limitation if the polymer is between 1,000 and 1,000,000 Da, preferably between 1,000 and 1,000,000 Da Glycol chitosan or poly-l-lysine may be used.

According to one embodiment of the invention, the protease is MMP, the A is Cy5.5, the B is a peptide substrate that is specifically cleaved MMP, the C is BHQ-3 (black hole quencher-3 ) And D may be glycol chitosan.

In another aspect, the present invention provides a method of preparing an in vitro kit for measuring the protease activity, comprising the following steps:

(a) synthesizing a peptide substrate specific for a specific protease and binding to and reacting a near infrared phosphor to the peptide substrate;

(b) binding a quencher to the peptide substrate;

(c) binding a polymer to the phosphor-peptide substrate-quencher; And

(d) immobilizing the phosphor-peptide substrate-quencher-polymer in a 96-well

In another aspect, the present invention uses the in vitro kit for measuring the protease activity, squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, An in vitro kit for diagnosing cancer selected from the group consisting of testicular cancer, kidney cancer, colon cancer, rectal cancer, gastric cancer, kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma and gallbladder cancer may be provided.

In another aspect, the present invention can also diagnose autoimmune diseases using the in vitro kit for measuring protease activity.

According to one embodiment of the invention, the autoimmune disease is an inflammatory disease, may be osteoarthritis or rheumatoid arthritis.

As described above, the phosphorase according to the present invention and a quencher that absorbs the light emission of the phosphor are coupled, and a protease activity measurement comprising a complex in which a peptide substrate specifically cleaved by a protease is bound to a polymer. For in vitro kits, the fluorescence does not luminesce due to the high quenching ability of the quencher's fluorescent material, and then, when the peptide substrate is decomposed by a specific protease, the fluorescence specifically emits strong fluorescence. Therefore, while quantitatively analyzing various protease expressed in cells and tissues, there is an advantage that can be imaged in a short time. These properties can be useful for screening new drugs such as inhibitors that inhibit protease overexpression, and for the diagnosis of various diseases such as cancer, osteoarthritis, rheumatoid arthritis, dementia and other autoimmune diseases.

The present invention maximizes the extinction rate of fluorescence by binding a phosphor and a quencher to any peptide substrate specifically degraded by proteolytic enzymes expressed in cells and the body, and a protein comprising a complex prepared by binding it to a polymer. The present invention relates to a kit for measuring enzyme activity, wherein the peptide substrate of the complex specifically reacts only with a specific proteinase to be targeted to generate fluorescence, thereby making it possible to measure protease activity in a quick and simple manner. to be.

More specifically, but not limited thereto, the complex included in the in vitro kit for measuring protease activity has the following structural formula:

[Formula 1]

Here, A is a phosphor, B is a peptide substrate specifically degraded by the proteolytic enzyme, C is a quencher that can absorb the luminescence of the phosphor and exhibit a quenching effect, and D is a polymer Was done.

Here, the phosphor and the quencher in the peptide substrate may be bonded to the amino terminal, lysine, cysteine, or carboxylic acid of the peptide substrate, the -SH group of the carboxylic acid or cysteine of the C terminal of the peptide substrate The polymer may be bound.

The protease is matrix metalloproteinases (MMP), thrombin, FXIIIa, caspase, urokinase plasminogen activator (uPA), HIV protease, DPP-IV or proteases. Proteasome and the like.

The peptide substrate (B) is a peptide substrate that is specifically degraded by proteolytic enzymes, for example, targets such as matrix metalloprotenase (MMP), thrombin, caspase activated by apoptosis, proteasome, etc. It may be a peptide. The peptide substrate used depends on the particular protease.

The phosphor (A) may be a phosphor that emits fluorescence in the visible region or near infrared rays, for example, fluorescein, fluoresce, BODYPY, tetramethylrhodamine, Alexa Cyanine, allopicocyanine, phosphors that generate other fluorescence, or derivatives thereof may be used. In addition, it is preferable to use a phosphor having a high quantum yield. Among the above-mentioned phosphors, in particular, cyanine-based and Alexa-based emit and absorb near-infrared light, and thus, interference or absorption with cells, blood, and biological tissues is minimized.

The quencher (C) is a quencher chemically bonded to the peptide substrate to which the phosphor is bonded to absorb the light of the wavelength emitted by the phosphor to achieve a strong quenching effect (peptide) by a specific proteolytic enzyme If not resolved, the fluorescence does not emit, and this quenching effect occurs when the distance between the phosphor and the quencher is within several tens of nm. In other words, when the peptide substrate is decomposed by reacting with a specific protease, the phosphor and quencher bound to the peptide are separated from each other, and the quenching effect is lost. Analysis is possible.

In the present invention, the quencher is to maximize the quenching effect by absorbing the wavelength of light emitted from the phosphor, because the quenching effect should be used only when the quencher of the same or almost the same wavelength as the luminescence wavelength of the phosphor can be maximized, The kind of quencher used depends on the emission wavelength range of the phosphor.

In the present invention, the quencher is a dark quencher, which is a material capable of quenching the fluorescence of the phosphor because it absorbs the energy of the excited phosphor but does not emit the fluorescence energy outside. Black hole quenchers (BHQ, WO01 / 86001, Biosearch Technologies, Inc. Novato, CA, USA) and blackberry quencher (Berry & Associates Inc. Dexter, MI, USA) can be used. .

The polymer (D) is preferably capable of immobilizing a large amount of the probe (probe) in the 96-well complex consisting of the phosphor-peptide substrate-quencher-polymer. Preferably, the polymer (D) of the complex may be glyco chitosan or poly-lysine having a molecular weight of 1,000 to 1,000,000 Da.

As a specific example of the present invention, in the probe for measuring protease activity of the present invention, the peptide (B) is a peptide substrate that is specifically degraded to various enzymes overexpressed in cancer and inflammatory disease sites. Phosphor (A) is Cy5.5, which is a cyanine phosphor that binds to the amino terminus of the peptide and exhibits near infrared fluorescence, and the quencher (C) is combined with lysine and cysteine of the peptide to absorb fluorescence of the phosphor It may be a black hole quencher-3 (BHQ-3) to maximize the effect. In addition, the polymer (D) can chemically bind to the -SH portion of the carboxylic acid or cysteine of the C terminal end portion of the peptide (B) to fix a large amount of phosphor ABC in the form of a steel in 96-well It can be lycol chitosan.

In addition, the in vitro kit for measuring the protease activity for imaging of the protease according to the present invention, a complex in which a phosphor and a quencher are coupled to a peptide substrate that is specifically degraded to a specific protease, and the polymer is bound to a polymer. Can be prepared by binding to 96-well.

More specifically, (a) reacting and binding a near infrared phosphor to a peptide substrate specific for a specific protease; (b) binding a quencher to the peptide substrate; And (c) binding a polymer to the phosphor-peptide substrate-quencher; (d) a method for preparing a kit for in vitro imaging of proteolytic enzymes comprising immobilizing the phosphor-peptide substrate-quencher-polymer in a 96-well.

In the above production method, if necessary, further comprising the step of purifying or identifying the phosphor-peptide-quencher-polymer prepared by the method.

In one embodiment, the peptide substrate may be appropriately synthesized, and such synthesis may use various peptide synthesis methods, such as Fmoc strategy according to solid phase synthesis.

In addition, by removing the protecting group at the N-terminal or the middle of the peptide of the synthesized peptide substrate, by reacting a phosphor or a derivative thereof capable of binding to the terminal functional group of the peptide, the peptide is bonded to one end of the peptide The protecting group at the other end of the derivative can be removed and the quencher or derivative thereof capable of binding to this terminal functional group can be reacted to bind.

 The distance between the phosphor (A) and the quencher (C) maximizes the quenching effect occurring between the phosphor and the quencher, so that the fluorescence of the phosphor is minimized. It is preferable to synthesize the phosphor to minimize the fluorescence intensity by the quenching effect caused by keeping the distance between the phosphor and the quencher within several tens of nanometers. In addition, the prepared phosphor-peptide-quencher-nanoparticle derivative, which is a nanoparticle sensor for detecting protease activity, can easily change and control the phosphor, quencher, and peptide substrate. Sensors for various proteases can be designed, such as sensors or various sensors in the desired wavelength range.

In addition, the phosphor-peptide substrate-quencher-polymer is fixed to 96-well, and the polymer can be attached to a 96-well plate to prepare an in vitro kit for quantitative measurement of protease activity according to the present invention.

On the other hand, the in vitro kit for measuring the protease activity is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer , Gastric cancer, kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, gallbladder cancer, and the like, and can be used in the method of imaging and quantifying proteolytic enzymes in inflammatory diseases such as rheumatoid arthritis, osteoarthritis, The in vitro kit for measuring protease activity may be used in a method for imaging and quantifying protease in refractory diseases including dementia, stroke, and the like.

The present invention, for example, the in vitro kit for measuring the protease activity is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney Cancer, colorectal cancer, rectal cancer, gastric cancer, kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, and gallbladder cancer, a composition for diagnosing cancer, or a composition for diagnosing dementia or stroke, or an autoimmune disease, specifically osteoarthritis or rheumatoid arthritis It can be used to diagnose inflammatory diseases.

In addition, the in vitro kit for measuring protease activity according to the present invention can be used to screen the efficacy of drugs or drugs that inhibit the overexpression of the protease, or to perform high-throughput screening required for new drug development or Early disease diagnosis can be made.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only examples of the present invention, and the scope of the present invention is not limited thereto.

Example  1. Phosphor Peptide - Quencher -Polymer Derivatives

1-1. Phosphor Peptide - Quencher  Preparation of Derivatives

First, in order to prepare a peptide combined with a phosphor and a quencher, the peptide is prepared by Fmoc method according to solid phase synthesis, and chemically binds the phosphor and quencher to the prepared peptide sequence. It was.

Phosphors were Cy5.5 (ex / em, 670/690), TRITC (ex / em, 547/572), FITC (ex / em, 490/520), and Cy5.5, TRITC, FITC, respectively. The quenchers capable of quenching the fluorescence of phosphors are BHQ-3 (abs. 620 nm-730 nm), BHQ-2 (abs. 550 nm-650 nm) and BHQ-1 (abs. 48- nm-580 nm) (Biosearch Technologies Inc.) were used respectively.

Fmoc peptide synthesis method using NH ₂ -Gly-Pro-Leu-Gly-Val-Arg (Pbf) -Gly-Lys (Boc) -Gly-Gly-COOH, a peptide substrate that is selectively degraded by MMPs protein enzyme 5 mg of peptide substrate, 8.5 mg of a near-infrared phosphor Cy5.5-HNS ester, 8 μl of N-methylmorpholine, and 0.3 mg of 4-dimethylaminopyridine were dissolved in 200 μl of dimethylformamide and reacted at room temperature for 12 hours. . The reaction solution was precipitated with 4 ml of cold ethyl ether and centrifuged to remove the supernatant and washed again with 2 ml of cold ethyl ether. Remove the ethyl ether from the surface and dry it with a speed vacuum or vacuum oven, Cy5.5-Gly-Pro-Leu-Gly-Val-Arg (Pbf) -Gly-Lys (Boc) -Gly -Gly-COOH precursor was prepared.

In order to deprotect the protecting group of the peptide precursor, the material was reacted at room temperature for 1 hour by adding 1 ml of trifluoroacetic acid, 25 μl of distilled water, and 25 μl of anisole. Remove all the solution using a rotary pump, dissolve it in 1 ml of HPLC eluent (saline with 0.1% TFA: acetonitrile = 1: 1) with 0.1% TFA, and filter (0.45μm). And an organic solvent usable filter). HPLC eluent (saline with 0.1% TFA: acetonitrile = 1: 1 with 0.1% TFA), using Agilent ZORBAX SB-C18 column (9.4 x 150 mm), with 0.1% TFA HPLC was stabilized with 95% saline with 5% acetonitrile, 0.1% TFA. Substance was passed through gradient elution for 20 minutes (5% at 0 minutes, 22% at 5 minutes, 40% acetonitrile (with 0.1% TFA) vs DW (with 0.1% TFA) at 20 minutes). Separated. At this time, after measuring at UV 220nm, FLD ex: 675nm em: 690nm Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Gly-COOH material was separated. The separated material was identified by taking a molecular weight using a mass, and then lyophilized. BHQ3-NHS ester (Biosearch Technologies Inc., 0.71 mg), 1.5 μl of NMM, and 0.2 mg of DMAP were dissolved in 2 mg of the material in 30 μl DMSO and reacted at room temperature for 12 hours. HPLC eluent (saline with 0.1% TFA: acetonitrile = 1: 1) with 0.1% TFA, 5% aceto with 0.1% TFA, using an Agilent ZORBAX SB-C18 column (9.4 x 150 mm) The HPLC was stabilized with 95% saline with nitrile, 0.1% TFA. The material was separated via gradient elution (5% at 0 min, 30% at 5 min, 70% acetonitrile (with 0.1% TFA) vs. saline with 0.1% TFA at 25 minutes) for 25 minutes. At this time, after measuring at UV 220nm, FLD ex: 675nm em: 690nm Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys (BHQ3) -Gly-Gly-COOH material was separated. The separated material was confirmed by taking a molecular weight using a mass (mass) and then lyophilized.

1-2. Phosphor Peptide - Quencher Preparation of Polymer Derivatives

The polymer was used to immobilize the phosphor-peptide-quencher material in a large amount on a 96 well plate. Rather than attaching a phosphor-peptide-quencher directly to a 96-well plate, several phosphor-peptide-quenchers can be attached to a polymer first, and then the polymer can be attached to a 96-well plate to increase efficiency. Biocompatible glycol chitosan (molecular weight 250,000 Da) was used as the polymer.

The phosphor-peptide-quencher made in 1-1 was dissolved in 100 μl DMSO, and then 100 μl PBS (pH 6.0) was added, followed by reaction at room temperature for 15 minutes by addition of EDC (1 mg) and NHS (0.8 mg). After completion of the reaction, the solution was added to a solution in which 10 mg of glycol chitosan was dissolved in 15 ml PBS (pH 7.4) and reacted at room temperature for 12 hours. After completion of the reaction, the phosphor-peptide-quencher which was not reacted for 3 days was removed using a dialysis method.

Example  2. MMP Fluorescence is restored by reacting specifically to in in vitro kit  Produce

The phosphor-peptide-quencher-polymer complex prepared in Example 1 was placed in a 96-well coated with maleic anhydride according to a desired concentration, and stirred at room temperature for 12 hours, whereby the phosphor-peptide-quencher-polymer complex was maleic. Allow chemical bonding with anhydride. After removing the complex, 2 ml of PBS containing 0.5% BSA was added to quench unreacted maleic anhydride and stirred for 1 hour. After removing PBS containing 0.5% BSA, wash twice with TCNB buffer.

Experimental Example  1. Phosphor according to the present invention Peptide - Quencher Phosphor of Polymer Complex Peptide - Quencher  Content

The content of the phosphor-peptide-quencher of the phosphor-peptide-quencher-polymer complex prepared in Example 1 was measured. 22 μM, 11 μM, 5.5 μM, 2.8 μM, 1.4 μM of the phosphor-peptide-quencher and 10 μM of the phosphor-peptide-quencher-polymer complex were UV-scanned to 450 nm to 800 nm (FIG. 2A). UV values corresponding to various concentrations of phosphor-peptide-quencher appeared linear (FIG. 2B), and when the UV values of the phosphor-peptide-quencher-polymer complex were substituted thereto, the phosphor-peptide-quencher -It was confirmed that the phosphor-peptide-quencher 10.6 uM bound to 1.10 uM of polymer.

Experimental Example  2. Phosphor according to the present invention Peptide - Quencher Of polymer complexes MMPs  Fluorescence Changes by Degrading Enzymes

The selection specificity for the MMPs proteolytic enzyme of the phosphor-peptide-quencher-polymer complex prepared in Example 1 was observed. The prepared complex was added to the reaction solution to which various proteolytic enzymes were added, and then fluorescence intensity (FI) which was restored over time was measured.

Specifically, 9 μg / ml of the prepared complex was added to activated MMP-2, MMP-3, MMP-7, MMP-9, or MMP-13 degrading enzyme (150 nM each), followed by fluorescence expression by enzymatic degradation. Observed. MMPs were added to TCNB reaction solution (0.1 M Tris, 5 mM calcium chloride, 200 mM NaCl, 0.1% Brij) to which p-aminophenyl mercuric acid was added to activate each MMPs. The reaction was carried out at 37 ° C. for 1 hour. Fluorescence expression was measured at ex: 675 nm, em: 676 ~ 800 nm by fluorescence spectrometer.

The complex shows a result of 24.6, 1.7, 1.0, 9.8, and 22.3-fold fluorescence recovery after 80 minutes of reaction with MMP-2, MMP-3, MMP-7, MMP-9 and MMP-13, as shown in Table 1 below. It was. In addition, when the inhibitor was treated with MMP-13, fluorescence was hardly restored. As a result of fluorescence restoration, the complex reacted with MMP-2 and MMP-13, indicating that relatively high fluorescence was restored (FIG. 3).

TABLE 1

Size of fluorescence to be restored for MMPs of the complex

time (min) MMP-2 MMP-3 MMP-7 MMP-9 MMP-13 M13 + inhibitor control 0 142.1 283.2 174 151.3 147.6 170.4 133.9 10 1233 261.7 195.9 281.5 815.1 125.3 119.1 20 1964 292.1 187.4 445.2 1399 132.9 117.3 30 2450 312.9 194.1 641.3 1798 133 123.1 40 2818 352.4 188.7 819.4 2191 129.5 118.1 50 3092 389.1 195 988.6 2653 130.4 120.2 60 3174 412.8 197.3 1124 2897 128 119.9 70 3319 435.4 184.6 1233 3144 142.1 115.1 80 3497 468.9 184.2 1483 3293 137 128.9

Experimental Example 3. Fluorescence restoration and imaging for the concentration of protease of the complex according to the present invention

The dependence of the MMP-13 degrading enzyme concentration of the phosphor-peptide-quencher-polymer complex prepared in Example 1 was observed. The prepared complex was added to a reaction solution to which various concentrations of proteolytic enzymes were added, and then fluorescence intensity (FI), which was restored over time, was measured. After the complex was added to the activated MMP-13 of 75, 38, 19, 5, 0 nM in the same experimental manner as in Experimental Example 2 and reacted at 37 ° C. for 60 minutes, the degree of fluorescence was observed with a fluorescence spectrometer.

As shown in FIG. 4, since the fluorescence expression of the complex increases proportionally with the concentration of the added MMP-13 degrading enzyme, the specific enzyme concentration in the sample can be determined by measuring the luminescence of the experimental group. Shows.

Experimental Example 4. Fluorescence restoration and imaging according to the concentration of the immobilized complex of the in vitro kit according to the present invention

In vitro kits as in Example 2, in which the complexes prepared in Example 1, 2.5, 1.25, 0.63, 0.32, and 0.16 mg / ml were immobilized, did not show fluorescence in the quenched state when the enzyme was not treated. When the 15 nM MMP-13 degrading enzyme was treated in each well, the fluorescence was restored in proportion to the concentration of the immobilized complex (Fig. 5a).

Experimental Example  5. According to the present invention in in vitro kit Fluorescence Restoration and Imaging According to the Degrading Enzyme Concentration

In vitro kits as in Example 2, in which 2.5 mg / ml of the complex prepared in Example 1 were immobilized, did not show fluorescence in the quenched state when the enzyme was not treated. When MMP-13 degrading enzymes of 30, 15, 7.5, 3.8, and 1.9 nM were treated in each well, images of fluorescence were restored in proportion to the concentration of the degrading enzymes treated (Fig. 5b).

Experimental Example  6. According to the present invention in in vitro kit Fluorescence Restoration and Imaging According to Various Degrading Enzymes

When MMP-13, Caspase-3, and Proteasome Degrading Enzyme were treated to each well in an in vitro kit as in Example 2 immobilized with 2.5 mg / ml of the complex prepared in Example 1, it was specific only by MMP degrading enzyme. As a result, an image in which fluorescence was strongly restored was obtained (FIG. 6).

1 is in vitro fluorescence quenching in vitro specific assay for protease, strong fluorescence is restored to the proteolytic enzyme activity of the present invention consisting of peptides, near infrared phosphors, quencher and polymer to enable imaging It is a schematic diagram of the kit.

Figure 2 shows that it is possible to quantify the amount of phosphor-peptide substrate-quencher bound to the glycol chitosan polymer.

Figure 3 shows that the phosphor-peptide substrate-quencher-polymer differs in luminescence intensity by various kinds of MMP degrading enzymes. The complex used in this experiment can be seen that the fluorescence is the largest emission by MMP-2 and MMP-13.

Figure 4 shows that as the concentration of the protease MMP-13 increases the fluorescence of the complex used in this experiment constantly increases, it is possible to quantitative analysis of the protease.

FIG. 5A shows that 96 wells are quenched when MMP-13 degrading enzyme is not treated, and the complexes of various concentrations are immobilized in 96-well and the concentrations of the immobilized complexes are treated when MMP-13 degrading enzyme is treated. It shows that fluorescence is emitted in proportion.

FIG. 5B shows that 96 wells are quenched when MMP-13 degrading enzyme is not treated, and the complex of fixed concentration is immobilized in 96-well and treated with various concentrations of MMP-13 degrading enzyme. 13 shows fluorescence emission in proportion to the concentration of the enzyme. This shows that quantitative analysis of MMP degrading enzyme activity is possible.

FIG. 6 shows that when a complex of a certain concentration is immobilized in a 96-well and treated with MMP, caspase, and proteasome degrading enzymes, fluorescence specifically emits strongly against MMP degrading enzymes. To show. This shows that the activity of the MMP degrading enzyme can be specifically measured.

Claims (15)

An in vitro kit for quantitative determination of protease activity comprising a complex in which a phosphor substrate and a quencher are coupled to and a peptide substrate specifically cleaved by a protease is bound to a polymer. The in vitro kit for quantitative determination of protease activity according to claim 1, wherein the complex has the following structure: [Formula 1]

Here, A is a phosphor, B is a peptide substrate specifically degraded by the proteolytic enzyme, C is a quencher that can absorb the luminescence of the phosphor and exhibit a quenching effect, and D is a polymer Was done. The in vitro kit for quantitative measurement of proteolytic enzyme activity according to claim 2, wherein the polymer is bound to the -SH group of the carboxylic acid or cysteine at the C terminus of the peptide substrate. The in vitro kit of claim 2, wherein the phosphor and the quencher bind a peptide substrate bound to an amino terminus, lysine, cysteine, or carboxylic acid of the peptide substrate. The in vitro kit for quantitative determination of protease activity of claim 2, wherein the polymer is glyco chitosan or poly-L-lysine with a molecular weight of 1,000 to 1,000,000 Da. According to claim 1, The protease is matrix metalloproteinases (MMP), thrombin, FXIIIa, caspase (caspase), urokinase plasminogen activator (uPA), HIV protease, An in vitro kit for quantitative determination of protease activity, which is selected from the group consisting of DPP-IV and proteasome. The kit of claim 1, wherein the phosphor is a phosphor emitting red or near infrared fluorescence. The in vitro kit for quantitative determination of protease activity according to claim 7, wherein the phosphor is selected from the group consisting of cyanine, fluorescein, tetramethylrodamine, alexa, bodiphy, and derivatives thereof. The method of claim 1, wherein the quencher is selected from the group consisting of blackhole quencher, blackberry quencher and derivatives thereof capable of quenching fluorescence for quantitative measurement of protease activity In vitro kits. The peptide substrate according to any one of claims 1 to 5, wherein the protease is MMP, A is Cy5.5, and B is specifically degraded to substrate metalloproteinase (MMP). And C is BHQ-3 (black hole quencher-3) and D is glycol chitosan. In vitro kit for quantitative determination of protease activity. (a) a first step of synthesizing a peptide substrate specific for a specific protease and reacting the same with near-infrared phosphors; (b) a second step of binding a quencher to the peptide substrate; (c) a third step of binding a polymer to the phosphor-peptide substrate-quencher; And (d) a method for producing an in vitro kit for quantitative determination of protease activity, comprising a fourth step of immobilizing the phosphor-peptide substrate-quencher-polymer in a 96-well. According to claim 1, wherein the in vitro kit for quantitative measurement of protease activity is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney Kit for quantitative measurement of in vitro protease activity, characterized in that it is used for cancer diagnosis selected from the group consisting of cancer, colorectal cancer, rectal cancer, stomach cancer, kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma and gallbladder cancer. The kit for quantitative measurement of in vitro protease activity according to claim 1, wherein the in vitro kit for quantitative measurement of protease activity is used for diagnosing autoimmune disease. The in vitro kit of claim 13, wherein the autoimmune disease is osteoarthritis or rheumatoid arthritis. The quantitative measurement of protease activity according to claim 1, wherein the kit for quantitative determination of protease activity in vitro is used for screening of the drug or the efficacy of the drug that inhibits overexpression of the protease. In vitro kits for use.

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