Specific substrate of cathepsin S, its use and kit containing the same

Abstract

The present invention relates to the field of biological detection technology, and in particular to a specific substrate of cathepsin S, its application and a kit containing the same. The present invention provides a specific substrate of cathepsin S, ACA-HPVGAPWD-Lys(DNP)-DLys-NH2, which is highly specific to cathepsin S. The kit prepared therefrom can be used for the early diagnosis of dry mouth disease belonging to Sjögren's syndrome. The sample is collected by a special saliva collector, and then mixed with the specific substrate and 2× enzyme reaction solution. The enzyme activity of the sample is determined according to the fluorescence intensity of the reaction solution, thereby diagnosing whether the patient suffers from Sjögren's syndrome. The detection method of the kit is highly sensitive, and non-invasive and non-invasive detection and diagnosis can be performed at an early stage of the onset of the disease. The patient can be effectively controlled as soon as possible due to early diagnosis, thereby reducing the pain and suffering caused by the development of the disease; and the kit is easy to use, has a fast detection speed, does not require high-end instruments and high-end technicians, has a low detection cost, and is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a specific substrate of cathepsin S, an application thereof and a kit containing the same. Background Art

[0002] Sjögren's syndrome is a systemic autoimmune disease characterized by decreased tear and saliva production in relatively healthy individuals, leading to dryness and discomfort in the mouth, cornea, and conjunctiva. Dry eye, a component of Sjögren's syndrome, causes severe pain and a foreign body sensation, which can deprive the cornea of ​​its normal ability to resist harmful substances and microorganisms in the external environment, leading to corneal lesions and even blindness. Dry mouth, a component of Sjögren's syndrome, causes difficulty swallowing, a foreign body sensation in the throat, and a decreased antibacterial capacity of the oral cavity, significantly increasing the risk of tooth decay. Furthermore, the disease can cause extreme fatigue, joint pain, and damage to multiple organs, including the kidneys, gastrointestinal tract, blood vessels, lungs, liver, pancreas, and central nervous system. It can even lead to blindness or the development of malignant lymphoma.

[0003] Currently, the diagnosis of this disease relies primarily on clinical manifestations, confirmed by biopsy of mucosal tissue and pathological examination. While this method provides reliable pathological results, it requires a certain degree of organ damage to be detected and confirmed, making it ineffective for early diagnosis. Furthermore, biopsy and histological examination require specialized training and in-person dental work by dental specialists, which can also cause tissue damage to the patient. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a specific substrate of cathepsin S, its application and a kit containing the same. The kit containing the specific substrate has high sensitivity and can be used for diagnosis in the early stages of the disease so that the disease can be controlled as early as possible. It does not require live sampling, avoids damage to the subject's organs, can improve the diagnosis rate and reduce medical costs, and is therefore expected to replace the tissue biopsy method in the future.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The present invention provides a specific substrate of cathepsin S, the structural formula of which is shown below:

[0007]

[0008] This specific substrate is named ACA-HPVGAPWD-Lys(DNP)-DLys-NH2 cathepsin S substrate. ACA stands for 7-aminocoumarin-4-acetamide, which has a near-perfect fluorescence quantum yield (Φ value of 0.861), meaning that 86.1% of the excitation photons absorbed by this molecule are converted into emitted photons, acting as a donor / luminescent group in the substrate. HPVGAPWD consists of the amino acid sequence: histidine-proline-valine-glycine-alanine-proline-tryptophan-aspartic acid. This amino acid sequence is highly specific for cathepsin S, but has only negligible affinity for other members of the cathepsin family, such as cathepsin B, cathepsin H, and cathepsin L. Cathepsin S recognizes this amino acid sequence and catalyzes the hydrolysis of the peptide bond between G and A. Compared to other cathepsins in the same family, cathepsin S has overwhelming specificity and catalytic activity for this substrate (approximately 100-fold greater than other enzymes). Lys(DNP) stands for 2,4-dinitrobenzene-lysine, formed by a covalent bond between 2,4-dinitrobenzene and the 6-amino group of lysine. It serves as an acceptor / quencher group in the substrate. Because DNP's absorption wavelength partially overlaps with the emission wavelength of ACA, when DNP and ACA are located at opposite ends of the substrate, DNP absorbs the emitted light from ACA, quenching it and rendering it invisible to the instrument within the detection wavelength range. DLys-NH2, D-lysine amide, blocks the C-terminus of the substrate, preventing degradation by exoproteinases.

[0009] As can be seen from the above, the substrate molecules provided by the present invention are in a quenched state and produce essentially no detectable emission light under the action of excitation light. However, upon degradation by cathepsin S, the resulting free product, ACA, can be excited by 355 nm excitation light to produce blue fluorescence at a wavelength of 460 nm. This emission is captured and recorded by a fluorescence detector, making it useful as a quantitative tracking group for enzyme activity analysis.

[0010] In previous scientific research practices, the applicant of this patent discovered that the activity of cathepsin S is significantly increased in the tears of patients with dry eye associated with Sjögren's syndrome. Based on the important role of cathepsin S in autoimmune diseases, this pathological phenomenon and anatomical characteristics, a dry eye detection kit suitable for detecting the activity of this enzyme in tears was successfully designed and developed (CN110283233 A). The applicant recently further discovered that the activity of this enzyme is also significantly increased in the saliva of patients with dry mouth associated with Sjögren's syndrome. Since the cathepsin S substrate in the above patented technology has a high background when saliva is used as the measurement sample and is not perfect, the applicant decided to design a more specific substrate for cathepsin S and develop it into a Sjögren's syndrome diagnostic kit using saliva as the measurement sample.

[0011] Currently, the measurement of blood biomarkers associated with autoimmune diseases, such as C-reactive protein (CRP), antinuclear antibodies (ANA), anti-SSA (Ro), and anti-SSB (La), is used as an auxiliary diagnostic tool for diseases such as lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. However, due to the fact that 1) these biomarkers are common to multiple autoimmune diseases and 2) the interaction of substances and biomolecules between blood and all organs and tissues, the test results can only be used as a reference and cannot provide a definitive diagnosis of the specific autoimmune disease.

[0012] Compared with the above traditional detection methods, the design concept of the patent applicant has the following special advantages: 1) The exocrine characteristics of the salivary glands isolate the tested samples from the blood, and the test results are not interfered with by blood and other body fluids. The test results fully reflect the physiological and pathological conditions of the salivary glands, so a clear diagnosis can be made based on this. 2) Saliva is secreted by multiple salivary glands in the mouth, and the autoimmune inflammation of any gland will be reflected as an increase in salivary tissue protease S. Therefore, this method can effectively increase the positive detection rate of Sjögren's syndrome. 3) The measurement object in the present invention is based on the enzymatic amplification principle in biochemistry, and its sensitivity is higher than all other diagnostic methods. Therefore, it can be used as an auxiliary diagnostic method that can draw clear conclusions in the early, middle and late stages of Sjögren's syndrome.

[0013] A second aspect of the present invention provides the use of a specific substrate in the preparation of a kit for diagnosing Sjögren's syndrome. The kit, using this specific substrate as one of its main components, enables a non-invasive clinical test for dry mouth, a symptom of Sjögren's syndrome. The test requires only collecting saliva from the patient and measuring the activity of cathepsin S in the saliva to obtain a diagnostic result, without causing any pain, trauma, or discomfort to the patient, and without any toxic side effects.

[0014] A third aspect of the present invention provides a kit comprising a specific substrate, a 2× enzyme reaction solution, a cathepsin S specific inhibitor, a product standard, and a saliva collector.

[0015] The present invention uses a saliva collector to collect saliva from a patient and incubates it in a 2× enzyme reaction solution containing the specific substrate. The fluorescence intensity of the resulting reaction solution is substituted into a standard curve formula drawn from the concentrations of a series of product standard solutions and their corresponding fluorescence intensities for calculation, thereby obtaining the activity of cathepsin S in the sample. The cathepsin S-specific inhibitor is used to confirm that the enzyme catalytic activity detected by the kit is generated by cathepsin S because it can selectively bind to cathepsin S, causing it to lose its catalytic activity.

[0016] In combination with the third aspect, the concentration of the specific substrate in the kit is 0.2 mM, which is sufficient for accurate quantification of the fluorescent product ACA-HPVG produced by cathepsin S catalysis.

[0017] In conjunction with the third aspect, the 2× enzyme reaction solution comprises: 50 mM sodium acetate, 100 mM sodium chloride, 4 mM dithiothreitol, and a pH value of 5.5. The components and pH value of this 2× enzyme reaction solution are optimal conditions for cathepsin S in saliva to catalyze specific substrates.

[0018] In combination with the third aspect, the cathepsin S specific inhibitor is Z-FL-COCHO, and the inhibitor is used to confirm that the fluorescence intensity detected in the experiment is produced by cathepsin S catalysis.

[0019] In conjunction with the third aspect, the concentration of the cathepsin S specific inhibitor Z-FL-COCHO is 5 μM. The IC 50 The concentration is 7.7 nM. A concentration of 5 μM can completely inactivate the catalytic activity of cathepsin S in saliva samples.

[0020] In combination with the third aspect, the product standard is ACA-HPVG or ACA amide. The reason why ACAamide can be used as the product standard here is that the luminescent group of this molecule is the same as the luminescent group of the product ACA-HPVG produced by cathepsin S catalysis, except that it does not contain oligopeptides. Therefore, the molecular structure of ACA amide is simpler and more stable.

[0021] In conjunction with the third aspect, the saliva collector is composed of a hollow elastic body (2), an open hemisphere (1), and a hollow tube (3), wherein the hollow tube (3) connects the hollow elastic body (2) and the open hemisphere (1). The purpose of inventing this saliva collector is that the saliva secretion of patients with dry mouth syndrome, which is a type of dry mouth, is very small. This method can collect the amount of saliva required for testing. In addition, this saliva collection method can also reduce the contamination of the sample by oral microorganisms.

[0022] The present invention provides a specific substrate for cathepsin S, ACA-HPVGAPWD-Lys(DNP)-DLys-NH2, which is highly specific for cathepsin S but has no affinity or only negligible low affinity for other members of the cathepsin family. A kit for diagnosing dry mouth associated with Sjögren's syndrome, prepared using the substrate, eliminates the need for live sampling during the detection process, thus avoiding damage to the patient's tissues. Furthermore, salivary gland lesions can be detected in the early stages of the disease by measuring the activity of cathepsin S in saliva, providing patients with the opportunity for early treatment and disease control. Since oral saliva is secreted and collected from a variety of different salivary glands, lesions in any of the glands can be reflected as elevated cathepsin S activity in the saliva. Therefore, this diagnostic method improves the diagnosis rate and reduces the missed diagnosis rate. Furthermore, the kit is easy to use, quick to detect, and does not require high-end instruments and equipment, nor does it require the training and operation of high-end technicians. The detection cost is low and readily available. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of a saliva collector device;

[0024] In the picture:

[0025] 1. Open hemisphere; 2. Hollow elastic body; 3. Hollow tube;

[0026] Figure 2 It is the standard curve diagram in Example 2;

[0027] Figure 3 This is the reaction process of cathepsin S catalyzing the substrate. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] The present invention has discovered that the expression of cathepsin S is significantly elevated in the saliva of patients with Sjögren's syndrome, while the expression of other cathepsins, such as cathepsin H, cathepsin B, cathepsin L, and cathepsin Z, remains essentially unchanged compared to healthy individuals. Furthermore, the sole source of saliva is the salivary glands, which are isolated from other organs in the human body and lack a channel for communication between saliva and other body fluids. Therefore, changes in cathepsin S activity in saliva only reflect physiological or pathological changes in the salivary glands. This makes it possible to diagnose dry mouth, a condition associated with Sjögren's syndrome, by detecting cathepsin S activity.

[0030] Cathepsin S has the biological function of recognizing and catalyzing the hydrolysis of various substrate proteins. Kits for diagnosing Sjögren's syndrome that leverage this biological function of cathepsin S have been reported. However, these kits require the patient's tears, which is much more difficult to collect in sufficient quantities compared to saliva. Importantly, tears are produced exclusively by the lacrimal gland and subsequently flow from the eyes or nasal cavity through a closed system, unaffected by interference from other body fluids. Saliva, on the other hand, is produced by multiple glands and is easily affected by other oral health conditions and contaminated by oral bacteria. Its composition, particularly the types of proteases, is much more complex than that of tears. We attempted to use a kit designed for tear analysis on saliva from the same patient, but the results were unsatisfactory, resulting in a high background. This suggests the need for a more specific substrate molecule for cathepsin S to detect its activity in saliva.

[0031] Therefore, based on the above reasons, we leveraged our understanding of the properties of cathepsin S to redesign a short peptide template and coupled a quencher and a luminescent group at either end, ensuring that cathepsin S can catalyze the hydrolysis of this template substrate. Furthermore, this substrate is highly specific for cathepsin S, has no affinity or only negligibly low affinity for other cathepsin family members in saliva, and exhibits excellent stability and resistance to degradation by exoproteinases. The cathepsin S substrate described in this patent application further improves specificity, successfully resolving the high background observed when using tear test kits for saliva testing.

[0032] The embodiments of the present invention are further described below with reference to a number of embodiments.

[0033] The product standards ACA-HPVG or ACA amide used in the examples of the present invention were purchased from Tianjin WuXi AppTec Pharmaceutical Development Co., Ltd., with a purity of ≥98% and molecular weights of 627.63 g / mol and 220.16 g / mol, respectively.

[0034] Example 1

[0035] The present invention provides a specific substrate for cathepsin S, which is named ACA-HPVGAPWD-Lys(DNP)-DLys-NH2 cathepsin S substrate, and its structural formula is shown below:

[0036]

[0037] Example 2

[0038] The present invention provides a kit for diagnosing Sjögren's syndrome, comprising the specific substrate of Example 1 (stock solution concentration 1 mM; final concentration 0.2 mM), 2× enzyme reaction solution, cathepsin S specific inhibitor Z-FL-COCHO (stock solution concentration 0.25 mM; final concentration 5 μM), product standard ACA-HPVG and a saliva collector (such as Figure 1 The final concentrations of the 2× enzyme reaction solution were 50 mM sodium acetate, 100 mM sodium chloride, 4 mM dithiothreitol, and pH 5.5.

[0039] The kit is used as follows:

[0040] (1) Drawing of standard curve

[0041] ACA-HPVG series standard solutions with concentrations of 0, 200pmol / 100μL, 400pmol / 100μL, 600pmol / 100μL, 800pmol / 100μL and 1000pmol / 100μL were prepared, and the fluorescence blue intensity (excitation light 355nm / emission light 460nm) of the above ACA-HPVG series standard solutions with known concentrations was measured using a fluorescence microplate reader. A standard curve was then drawn using the measured fluorescence intensity as the ordinate and the concentration of the ACA-HPVG series standard solution as the abscissa. The measurement results are shown in Table 1. The standard curve is shown in Table 1. Figure 2 shown.

[0042] Table 1 Standard curve determination data

[0043]

[0044]

[0045] The fluorescence intensity X corresponding to the product produced by catalytic activity of cathepsin S in saliva can be accurately calculated using the formula in the obtained standard curve: X (pmol) = (Y – 98.11) / 62.25. According to the definition of cathepsin activity, 1 U (one unit of enzyme activity) = 1 μmol / min at 37°C (i.e., 1 μmol of product produced per minute at 37°C). If a sample is measured at 37°C and for 60 minutes, then the calculated X (pmol) using the formula X = (Y – 98.11) / 62.25 should be divided by 60 minutes to calculate the enzyme activity. The result is pmol / min (i.e., the average rate of picomoles of product produced per minute), which is expressed in units of enzyme activity (μU). When measuring cathepsin S activity in clinical saliva samples, we use the formula for the ACA-HPVG standard curve to convert the obtained fluorescence units (RFU) to units of enzyme activity (μU).

[0046] (2) Sample collection and testing:

[0047] Place the hemispherical portion 1 of the saliva collector on the cheek opening inside the parotid duct of the subject. Massage the parotid gland from the outside. Manually pinch the elastic portion 2 of the saliva collector and draw 0.5 mL of parotid saliva according to the scale on the hollow tube 3 as a sample. (If performing batch testing, the sample can be frozen at -80°C. Enzyme activity will not change significantly when tested within one week.) Place the collected sample at the bottom of a 1.5 mL Eppendorf tube and centrifuge at 3000 rpm for 7 minutes. Set aside. Transfer 50 μL of the 2× enzyme reaction solution to a 1.5 mL Eppendorf tube, add 2 μL of the specific substrate, and add 38 μL of water. Add 10 μL of the centrifuged saliva supernatant to the tube, bringing the final volume to 100 μL. Mix thoroughly and incubate in a 37°C incubator for 60 minutes. Then, 50 μL of each enzyme reaction sample was taken and placed in a 96-well plate (a plate with a flat and transparent bottom and black well wall) and the fluorescence intensity of the reaction solution was measured using a fluorescence quantitative analyzer at an excitation light wavelength of 355 nm and an emission light wavelength of 460 nm (the reaction process is as follows Figure 3 shown).

[0048] While performing the above enzymatic reaction, a parallel blank control experiment needs to be set up. Except for not containing saliva sample, all other components in the blank reaction tube are the same as those in the above sample tube.

[0049] From the same Eppendorf tube, 10 μL of centrifuged saliva supernatant was aspirated and a similar procedure was used, except that the volume of water was changed to 36 μL and 2 μL of the cathepsin S-specific inhibitor Z-FL-COCHO was added before adding the substrate to bring the volume to 98 μL. The mixture was mixed and incubated at room temperature for 10 minutes. Then, 2 μL of substrate was added to bring the final reaction volume to 100 μL. The remaining steps were the same as above. The reaction solution was then transferred to a 96-well plate and the fluorescence intensity of the reaction solution was measured at the same wavelength as above to determine whether the fluorescence intensity of the inhibitor tube and the blank control tube was similar or close. If the fluorescence intensities were similar, it indicated that the fluorescence intensity measured without the cathepsin S-specific inhibitor was largely due to product molecules produced by the catalysis of the specific substrate by cathepsin S in the sample. This fluorescence intensity was calculated using the formula derived from the standard curve to determine that the cathepsin S activity in the sample was zero or close to zero. When the kit of this example was used and the cathepsin S specific inhibitor was added, the fluorescence intensity measured was similar to that of the blank control test, indicating that the specific substrate of the present invention is highly specific for cathepsin S.

[0050] Experimental Example 1

[0051] Specificity test

[0052] (1) Standard samples containing equal amounts of cathepsins B, H, Z, L, and S were taken and their enzyme activities were measured according to the method of Example 2. Cathepsin B, H, Z, L, and S standards can be purchased from BioVision, a US company. The experiment showed that only the sample containing cathepsin S was detected to have a high-intensity 460 nm blue fluorescence, while the other samples did not have a fluorescence intensity significantly higher than that of the negative control sample, demonstrating that, except for cathepsin S, the other family members basically do not recognize this substrate.

[0053] (2) A standard sample of cathepsin S (denoted as sample 1) and a mixed standard sample containing an equal amount of cathepsin S and cathepsins B, H, Z, and L (denoted as sample 2) were taken and enzyme activity was measured according to the method of Example 2. The results showed that the fluorescence intensity of samples 1 and 2 was basically the same. This further indicates that, except for cathepsin S, the other family members basically do not recognize this substrate and have no promoting effect on cathepsin S.

[0054] (3) Saliva samples were collected using a saliva collector, and the cathepsin S activity inhibition assay was performed according to the method of Example 2. Specific inhibitors of cathepsins B, H, Z, L, and S can be purchased from the American company BioVision or the British company Abcam. Enzyme activity assays involving the five inhibitors were performed simultaneously. The results showed that samples containing only the specific inhibitor of cathepsin S no longer produced blue fluorescence above background, while inhibitors of cathepsins B, H, Z, and L had little effect on the fluorescence intensity produced by the saliva samples. This experiment shows that the substrate used in the kit is highly specific for cathepsin S and is not enhanced by several other family members. It also shows that there is basically no enzyme activity of the above family members against the substrate in saliva, that is, all fluorescent product molecules are produced by cathepsin S catalyzing the substrate.

[0055] Experimental Example 2

[0056] Using the kit provided in Example 2 and the method for use thereof, saliva from 150 patients and 150 normal subjects was collected and measured simultaneously. The test results are as follows: the average fluorescence intensity of the product obtained after the cathepsin S in the saliva samples of the 150 patients catalyzed the specific substrate was 8.05 μU; the average fluorescence intensity of the product obtained after the cathepsin S in the saliva samples of the 150 normal subjects catalyzed the specific substrate was 1.47 μU. It can be seen that the enzyme activity in the patient group was about 5.4 times that of the control group. The results of the statistical method (T Test) on the data of the two groups of patients and normal subjects showed that there was a significant difference between the two groups (P<<0.0001). Based on the above clinical sample measurement results, we preliminarily determined that the positive standard critical value of dry mouth syndrome belonging to Sjögren's syndrome is 2.67 μU (please refer to the drawing of the standard curve in Example 2 of this patent application (1) for the calculation method), that is, when the salivary cathepsin S is above 2.67 μU, the possibility of Sjögren's syndrome is considered.

[0057] For example, if the positive cutoff value for dry mouth, a symptom of Sjögren's syndrome, is set at 2.67 μU, only 9 of the 150 patients had test results below this value, resulting in a 94% positive enzyme activity assay compared to histopathological diagnosis. On the other hand, 18 of the 150 normal controls had test results exceeding 2.67 μU, resulting in a false-positive rate of 12.5%. Regarding the false-positive results of this assay, the inventors believe that certain oral infections or inflammatory diseases, such as caries, gingivitis, and oral ulcers, may increase cathepsin S activity in the oral cavity, leading to elevated test results. This hypothesis was confirmed in our experiments: All 18 patients, examined by dental specialists, all had the aforementioned various oral diseases. Among these, 12 patients with subacute oral diseases had test results that returned to normal after treatment. Therefore, the false-positive rate of this assay should be less than 12.5%.

[0058] The above descriptions are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A specific substrate of cathepsin S, characterized in that The structural formula is shown below: 。 2. Use of the specific substrate according to claim 1 in preparing a kit for diagnosing Sjögren's syndrome.

3. A kit, characterized in that The kit comprises the specific substrate according to claim 1, a 2× enzyme reaction solution, a cathepsin S specific inhibitor, a product standard and a saliva collector; the product standard is ACA-HPVG or ACA amide.

4. The kit according to claim 3, wherein The concentration of the specific substrate in the kit is 0.2 mM.

5. The kit according to claim 3, wherein The 2× enzyme reaction solution includes: 50 mM sodium acetate, 100 mM sodium chloride, 4 mM dithiothreitol, and a pH value of 5.

5.

6. The kit according to claim 3, wherein The cathepsin S specific inhibitor is Z-FL-COCHO.

7. The kit according to claim 6, wherein The concentration of the cathepsin S specific inhibitor Z-FL-COCHO was 5 μM.

8. The kit according to claim 3, wherein The saliva collector is composed of a hollow elastic body (2), an open hemisphere (1) and a hollow tube (3), wherein the hollow tube (3) connects the hollow elastic body (2) and the open hemisphere (1).

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