Determination method of UXS1 enzyme activity

Abstract

The invention relates to the technical field of biological detection, in particular to a UXS1 enzyme activity determination method. The method comprises the following steps: a) hydrolyzing a substrate UDP-GlcA by using UXS1 to generate UDP-xyl; b) adding XYLT1 to catalyze the UDP-xyl to generate UDP (User Datagram Protocol); c) converting the UDP into ATP (adenosine triphosphate); and d) quantitatively detecting ATP, and deducing the enzyme activity of UXS1 through the obtained signal intensity. The method can effectively reduce the cost of UXS1 enzyme activity detection, can help a pharmaceutical enterprise to quickly screen drugs of related targets at high throughput, and is beneficial to promoting the research and development process of related drugs.

Description

Technical Field

[0001] The present disclosure generally relates to the field of biological detection technology, and in particular, to a method for determining UXS1 enzyme activity. Background Art

[0002] UXS1 (UDP Glucuronate Decarboxylase 1) is an enzyme found in the Golgi apparatus and is encoded by the UXS1 gene. Its main function is to decompose the intermediate product UDP-glucuronic acid (UDP-GlcA) produced by UGDH, form UDP-xylose (UDP-Xyl) from UDP-GlcA, and then use UDP-xylose to initiate the biosynthesis of glycosaminoglycans on the core protein of proteoglycans. UXS1 not only reduces the level of UDP-GlcA, but also forms negative feedback to reduce the expression of upstream UGDH. Multiple transcript variants encoding different subtypes have been found for this gene. Diseases associated with it include Walker-Warburg syndrome. In addition, many tumor cells highly express UGDH relative to healthy cells, which means that only cancer cells need the detoxification effect of UXS1. Therefore, by inhibiting the activity of UXS1 and seizing this weakness of tumor cells, it is expected to achieve precise anti-tumor effects. Other studies have found that high UGDH expression is associated with tumor cell resistance to chemotherapy drugs such as paclitaxel, gemcitabine, and doxorubicin. In mouse experiments, knocking out UXS1 has a synergistic effect with chemotherapy (cisplatin) to inhibit tumor growth.

[0003] The existing technical solutions generally use high performance liquid chromatography (HPLC) to detect the UXS1 enzyme activity (by measuring the amount of UDP-xyl generated). This method has the following three main shortcomings:

[0004] 1. High testing cost. The purchase cost of liquid chromatography system is high, which may limit its popularity and application in some laboratories.

[0005] 2. Complex sample processing. Liquid chromatography analysis usually requires sample pretreatment, such as solid phase extraction (SPE) or liquid-liquid extraction (LLE), to remove interfering substances in the sample and enrich the target analyte. These steps may increase the complexity and time cost of the analysis.

[0006] 3. Challenges of solvents and mobile phases: The selection and optimization of mobile phases are crucial to improving analytical efficiency and reducing background interference.

[0007] In addition, UXS1 enzyme activity can also be measured by immunoassay methods such as nuclear magnetic resonance (NMR), MALDI-TOF, Western Blot or ELISA. However, considering the experimental cycle, sensitivity and throughput, it is difficult to meet the high-throughput screening of multiple batches of compounds that affect UXS1 enzyme activity. Summary of the invention

[0008] This disclosure covers the following technical solutions:

[0009] One of the purposes of the present disclosure is to provide a method for determining UXS1 enzyme activity, comprising:

[0010] a) UXS1 hydrolyzes the substrate UDP-GlcA to generate UDP-xyl;

[0011] b) Adding XYLT1 catalyzes UDP-xyl to generate UDP;

[0012] c) Convert UDP into ATP;

[0013] d) ATP was quantitatively detected and the UXS1 enzyme activity was inferred from the obtained signal intensity.

[0014] According to another aspect of the present disclosure, a UXS1 enzyme activity assay kit is also provided, comprising:

[0015] XYLT1, xylosyltransferase substrate peptides, and UDP-Glo TM The components in .

[0016] According to yet another aspect of the present disclosure, there is provided a method as described above, or use of the kit as described above in screening drugs related to UXS1 enzyme activity.

[0017] The beneficial effects of the present disclosure are:

[0018] (1) The detection method is sensitive, and the proteins and substrates used can be detected at low concentrations, which can greatly save reagent costs;

[0019] (2) The experimental method system is stable, which reduces repeated detection of XYLT1 targets and reduces labor costs;

[0020] (3) The operation steps are simple and the reagents are easily available, which can increase the speed of drug screening and save time and cost;

[0021] (4) It can help pharmaceutical companies to quickly and high-throughput screen the drug effects of related targets, which is conducive to advancing the research and development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of the assay process of UXS1 enzyme activity disclosed in the present invention;

[0024] Figure 2 The UDP-GlcA Km titration result provided by one embodiment of the present disclosure;

[0025] Figure 3 The β-NAD+ titration result provided by one embodiment of the present disclosure;

[0026] Figure 4 The UXS1 enzyme activity multiple titration result provided in one embodiment of the present disclosure;

[0027] Figure 5 and Figure 6 This is the result of the data difference analysis between the plates disclosed in this paper. DETAILED DESCRIPTION

[0028] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0029] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0030] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0031] The terms "and / or", "or / and", and "and / or" used herein have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present disclosure, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0032] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0033]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0034] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0035] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0036] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0037] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0038] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In the present disclosure, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0040] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0041] The present disclosure relates to a method for determining UXS1 enzyme activity, comprising:

[0042] a) UXS1 hydrolyzes the substrate UDP-GlcA to generate UDP-xyl;

[0043] b) Adding XYLT1 catalyzes UDP-xyl to generate UDP;

[0044] c) Convert UDP into ATP;

[0045] d) ATP was quantitatively detected and the UXS1 enzyme activity was inferred from the obtained signal intensity.

[0046] The XYLT1 referred to in the present disclosure includes the complete protein, or protein fragments and mutants having the catalytic activity of XYLT1.

[0047] The method of converting UDP into ATP can be carried out by any known method, such as an enzyme-catalyzed reaction, or other chemical synthesis methods.

[0048] Any method capable of accurately quantifying ATP is applicable to the present disclosure. As an example of the most commonly used method, in some embodiments, in step d), ATP is used to oxidize the substrate luciferin under the catalysis of luciferase to generate a light signal.

[0049] In some embodiments, in steps c) and d), UDP-Glo TM Conduct testing.

[0050] UDP-Glo TM UDP-Glo TM Glycosyltransferase Assay is a bioluminescent detection system for detecting glycosyltransferase activity, provided by Promega.

[0051] In some embodiments, the concentration of UDP-GlcA in step a) is 50 μM to 150 μM, for example 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM.

[0052] In some embodiments, the concentration of UXS1 in step a) is 70 nM to 130 nM, such as 80 nM, 90 nM, 100 nM, 110 nM, 120 nM.

[0053] According to yet another aspect of the present disclosure, it also relates to a UXS1 enzyme activity assay kit, which comprises:

[0054] XYLT1, xylosyltransferase substrate peptides, and UDP-Glo TM The components in .

[0055] The term "kit" in the present disclosure may refer to any article (e.g., package or container) comprising at least one device, which includes a detection agent as described in the present disclosure. In some embodiments, the kit may further include instructions for use used in the method described in the present disclosure or its steps, preferably also include supplementary reagents and / or components or components.

[0056] The components in the kit can be packaged separately or in combination as needed. The components in the kit can be packaged in the form of a solution, a solid or a test strip. In some embodiments, at least one component in the kit is a solid, and the solid includes at least one of a lyophilized microsphere, a lyophilized cake, a lyophilized powder, and a spot that depends on the presence of a solid medium. Therefore, the components required for the detection of UXS1 enzyme activity, in particular various enzymes, peptide components, and reaction buffer components, etc., can be provided in a lyophilized form.

[0057] According to yet another aspect of the present disclosure, it also relates to the use of the above-mentioned method or the above-mentioned kit in screening drugs related to UXS1 enzyme activity.

[0058] In some embodiments, the drug is an agonist or inhibitor of the UXS1 enzyme.

[0059] In some embodiments, the medicament is used to treat a disease selected from Walker-Warburg syndrome and cancer.

[0060] In some embodiments, the cancer is caused by any one or more lesions in bones, bone joints, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, cardia, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicle, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid, parathyroid, adrenal glands, pituitary, pineal gland, pancreatic islets, thymus, gonads, sublingual glands and parotid glands; optionally, the cancer is any one or more of lung cancer, gastric cancer, esophageal cancer, liver cancer, colon cancer, gallbladder cancer and cardia cancer.

[0061] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0062] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0063] The following provides specific examples of UXS1 high-throughput screening assays:

[0064] 1. UXS1 enzyme activity titration

[0065] Based on previous experimental experience and data, 2mM UDP-GlcA and 2.5mM β-NAD+ were initially selected for UXS1 enzyme activity titration. 200nM was selected as the starting titration concentration of UXS1 for 2x gradient dilution, and the concentration points were 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78nM, 0.39nM, and 0nM. Experimental data showed that 200nM UXS1 presented a 6-7-fold window, so 200nM UXS1 was selected for UDP-GlcA titration.

[0066] 2. UDP-GlcA titration

[0067] UDP-GlcA Km titration was performed under 200nM UXS1 and 2.5mM β-NAD+. 4mM was selected as the starting concentration of UDP-GlcA for 2x gradient dilution, and the concentration points were 4000μM, 2000μM, 1000μM, 500μM, 250μM, 125μM, 62.5μM, 31.25μM, 15.625μM, 7.8125μM, and 0μM. The UDP-GlcAKm value was analyzed to be 247.4μM ( Figure 2 ), and select 100 μM UDP-GlcA for β-NAD+ titration in the data window.

[0068] 3. β-NAD+ titration

[0069] β-NAD+ titration was performed under the conditions of 200nM UXS1 and 100μM UDP-GlcA. 5mM was selected as the starting titration concentration of β-NAD+ for 2x gradient dilution, and the concentration points were 5000μM, 2500μM, 1250μM, 625μM, 312.5μM, 156.25μM, 78.125μM, 39.06μM, 19.53μM, 9.765μM, and 0μM. The titration results showed that the gradient dilution of β-NAD+ had no significant effect on the titration signal ( Figure 3 ) and will not be added in subsequent verification.

[0070] 4. UXS1 enzyme titration

[0071] According to the above substrate titration data, 100μM UDP-GlcA was finally selected for UXS1 enzyme activity re-titration. 800nM was selected as the starting titration concentration of UXS1 for 2x gradient dilution, and the concentration points were 800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0nM. The experimental data showed that UXS1 had a 7-8-fold window at 100nM and the data linearity was good ( Figure 4 ), thus 100 nM UXS1 and 100 μM UDP-GlcA can be selected for compound screening experiments.

[0072] According to the above experiments, the specific detection steps are determined as follows:

[0073] Step 1: Obtain UXS1, XYLT1, substrate and other related reagents. The specific reagents, consumables and equipment involved are shown in Table 1;

[0074] Step 2: Transfer 2 μL of 200 nM UXS1 working solution to Greiner 784075 and centrifuge at 1000 rpm for 1 min;

[0075] Step 3: Transfer 2 μL of 200 μM UDP-GlcA working solution to Greiner 784075, centrifuge at 1000 rpm for 1 min; incubate at 25°C for 60 min;

[0076] Step 4: Transfer 4 μL of 40 nM XYLT1 working solution and 200 μM xylT-substratepeptide to Greiner 784075, centrifuge at 1000 rpm for 1 min; incubate at 25°C for 60 min;

[0077] Step 5: Transfer 8 μL UDP-Glo ​​detection (prepare an appropriate amount of detection reagent with Enzyme working reagent: ATP detection substrate = 1:100) to Greiner 784075, centrifuge at 1000 rpm for 1 min; incubate at 25°C for 60 min;

[0078] Step 6: Use a BMG microplate reader to read the luminescent fluorescence signal value (Gain: 3600).

[0079] Table 1 Experimental reagents

[0080]

[0081]

[0082] Table 2 English abbreviations and annotations

[0083] Abbreviations Chinese Notes UXS1 Uridine glucuronidase 1 XYLT1 Xylosyltransferase 1 UDP-GlcA UDP-glucuronide xylT-substrate peptide Xylosyltransferase substrate peptide β-NAD+ β-Nicotinamide adenine dinucleotide UDP-Glo Uridine diphosphate detection kit UDP-xyl UDP-Xylose UDP Uridine diphosphate

[0084] The data stability of the above method is analyzed. The experimental data show that ( Figure 5-Figure 6 ), the negative or positive control values ​​Z' (standard normal distribution value) between wells or plates were all around 0.8; the signal-to-noise ratio and standard normal distribution value deviation between plates were controlled within 10%, and the target development data was relatively stable.

[0085] The traditional method LC-MS needs to set the UDP-xyl concentration range as the experimental standard curve when testing, which involves the risk that the upper and lower limit signal values ​​of the standard curve are not easy to obtain during the optimization process, so it is not easy to effectively calculate the enzymatic reaction window value. The method provided by the present disclosure does not involve this drawback and the sensitivity meets the enzymology development standards, and the experimental window and data repeatability are easy to obtain. In addition, the traditional method LC-MS detection requires pre-treatment such as extraction of the sample, which requires a long optimization time and cumbersome operation. Considering the actual operation and verification cost, the development steps provided by the present disclosure are simple and the experimental cost is low. Although this development method involves a cascade reaction step, the conversion efficiency of the enzyme activity is not affected, and almost no systematic error is introduced, and the detection stability is good.

[0086] The above-described embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. The method for determining the activity of UXS1 enzyme comprises: a) UXS1 hydrolyzes the substrate UDP-GlcA to generate UDP-xyl; b) Adding XYLT1 catalyzes UDP-xyl to generate UDP; c) Convert UDP into ATP; d) ATP was quantitatively detected and the UXS1 enzyme activity was inferred from the obtained signal intensity.

2. The method according to claim 1, wherein in step d), the substrate luciferin is oxidized by ATP under the catalysis of luciferase to generate a light signal.

3. The method according to claim 2, wherein in steps c) and d), UDP-GloTM is used for detection.

4. The method according to any one of claims 1 to 3, wherein the concentration of UDP-GlcA in step a) is 50 μM to 150 μM.

5. The method according to claim 4, wherein the concentration of UXS1 in step a) is 70 nM to 130 nM.

6. UXS1 enzyme activity assay kit, comprising: XYLT1, xylosyltransferase substrate peptides, and components of UDP-GloTM.

7. Use of the method according to any one of claims 1 to 5, or the kit according to claim 6, in screening drugs related to UXS1 enzyme activity.

8. The use according to claim 7, wherein the drug is an agonist or inhibitor of UXS1 enzyme.

9. The use according to claim 7 or 8, wherein the drug is used to treat a disease selected from Walker-Warburg syndrome and cancer.

10. The use according to claim 9, wherein the cancer is caused by lesions in any one or more of bones, bone joints, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, cardia, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicle, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus gland, gonads, sublingual gland and parotid gland; optionally, the cancer is caused by any one or more of lung cancer, gastric cancer, esophageal cancer, liver cancer, colon cancer, gallbladder cancer and cardia cancer.

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