A heparin sulfate derivative with inhibitory activity against SARS-CoV-2 spike protein and its preparation method

Abstract

This invention discloses a heparin sulfate derivative derived from donkey-hide gelatin with inhibitory activity against the SARS-CoV-2 spike protein and its preparation method. The heparin sulfate derivative is prepared by first enzymatically hydrolyzing a donkey-hide gelatin solution with streptomycin, followed by separation and purification using DEAE anion exchange chromatography to obtain donkey-hide gelatin glycosaminoglycans. These glycosaminoglycans are then enzymatically hydrolyzed again using chondroitin sulfate ABC enzyme, followed by separation and purification using DEAE anion exchange chromatography. The derivative has an average molecular weight of 13.2 kDa and is composed of disaccharide units linked by hexuronic acid and glucosamine via 1-4 glycosidic bonds. The total sulfation degrees at the C-2-O position of hexuronic acid, the C-6-O position of glucosamine, and the N-position are 28.2%, 44.3%, and 38.9%, respectively. This heparin sulfate derivative exhibits competitive inhibition of the binding activity between the SARS-CoV-2 spike protein and heparin, enabling effective prevention of early-stage SARS-CoV-2 infection and laying an important foundation for developing new clinical applications of donkey-hide gelatin.

Description

Technical Field

[0001] This invention belongs to the field of natural product preparation and purification, specifically relating to a heparin sulfate-based compound containing donkey-hide gelatin with inhibitory activity against the SARS-CoV-2 spike protein and its preparation method. Background Technology

[0002] Donkey-hide gelatin (Ejiao) is a traditional health food and Chinese medicine ingredient. Its main pharmacological effects include replenishing blood, improving calcium metabolism balance, anti-shock, and enhancing immunity. It is often used to treat gynecological diseases and has been proven to have effects in treating blood diseases, anti-inflammation, anticoagulation, and anti-aging. The main components of donkey-hide gelatin include amino acids, proteins, polysaccharides, trace elements, and polypeptides. Current research on the active ingredients in donkey-hide gelatin mainly focuses on amino acids, proteins, and trace elements, with relatively little research on heparin sulfate in donkey-hide gelatin.

[0003] Heparin sulfate is a highly complex sulfated polysaccharide composed of repeating disaccharide units. These disaccharide units are formed by hexuronic acid (GlcA / IdoA) and glucosamine (GlcN) linked by 1-4 glycosidic bonds, with sulfation sites at the C-2-O position of hexuronic acid, the C-6-O and C-3-O positions of glucosamine, and the N-position of glucosamine. Heparin sulfate interacts with over 700 proteins in the body, playing crucial regulatory roles in immune responses, inflammation regulation, viral infection, thrombosis, and angiogenesis. Many viruses, such as herpes simplex virus, dengue virus, HIV, and various coronaviruses, including the recently prevalent SARS-CoV-2, use heparin sulfate as a receptor or co-receptor, binding to various viral proteins and controlling viral infection through its specific sequence structure. Therefore, heparin sulfate / heparin derivatives with specific structures can act as competitive inhibitors, blocking or interfering with the binding of the SARS-CoV-2 spike protein to its co-receptor, thereby inhibiting SARS-CoV-2 infection.

[0004] This invention, through the preparation and purification of heparin sulfate derived from donkey-hide gelatin, reveals a novel function: inhibiting the binding activity of the SARS-CoV-2 spike protein. Since there are no reports domestically or internationally regarding the inhibitory activity of heparin sulfate derived from donkey-hide gelatin on the SARS-CoV-2 spike protein, this invention lays an important foundation for developing new clinical applications of donkey-hide gelatin. Summary of the Invention

[0005] The purpose of this invention is to provide a heparin sulfate derivative of donkey-hide gelatin with inhibitory activity against the SARS-CoV-2 spike protein and its preparation method. This invention realizes a novel function of donkey-hide gelatin polysaccharides in the field of SARS-CoV-2 and lays the foundation for developing new clinical applications of donkey-hide gelatin.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A heparin sulfate derivative with inhibitory activity against the SARS-CoV-2 spike protein is a heterogeneous mixture with an average molecular weight of 13.2 Kda. It is composed of disaccharide units of hexuronic acid (GlcA / IdoA) and glucosamine (GlcN) linked by 1-4 glycosidic bonds. The total sulfation degree of hexuronic acid at the C-2-O position, glucosamine at the C-6-O position, and N-position is 28.2%, 44.3%, and 38.9%, respectively.

[0008] The preparation method of the heparin sulfate derived from donkey-hide gelatin includes the following steps:

[0009] 1) Preparation and extraction of donkey-hide gelatin glycosaminoglycans: After heating donkey-hide gelatin in a water bath, it is enzymatically hydrolyzed with streptomycin protease. After centrifugation, the supernatant is collected and separated and purified by DEAE anion exchange chromatography. Then, it is dialyzed and freeze-dried to obtain donkey-hide gelatin glycosaminoglycans.

[0010] 2) Preparation and extraction of heparin sulfate from donkey-hide gelatin: The donkey-hide gelatin glycosaminoglycan obtained by enzymatic hydrolysis of chondroitin sulfate ABC enzyme in step (1) was centrifuged and the supernatant was separated and purified by DEAE anion exchange chromatography. Then, the donkey-hide gelatin heparin sulfate was obtained by dialysis and freeze drying.

[0011] Specifically, step 1) includes the following operations:

[0012] a) Dissolve 1-2 g of donkey-hide gelatin in 10-20 ml of ultrapure water under a water bath heating at 50-70℃ and keep warm for 30-60 minutes;

[0013] b) Dissolve 1-5 mg of streptomycin in 1-5 ml of streptomycin hydrolysate, then add it to the donkey-hide gelatin solution obtained in step a), heat in a water bath at 36-40℃ for 24-48 h, and then inactivate with boiling water.

[0014] c) Centrifuge the enzymatic hydrolysate obtained in step b) at 10,000 rpm for 15-30 min, and collect the supernatant for later use;

[0015] d) Add the supernatant obtained in step c) to a DEAE anion exchange column and elute using a 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. Detect the collected fractions using ultraviolet light at a wavelength of 212 nm. Then, dialyze the collected target fractions at 2-5℃ using a 1000-5000 Da dialysis membrane for 2-4 days (with deionized water replaced every five hours) to remove NaCl from the eluent. Finally, freeze-dry to obtain glycosaminoglycans.

[0016] Step 2) involves: enzymatically hydrolyzing the obtained donkey-hide gelatin glycosaminoglycans with 300-500 mU chondroitin sulfate ABC enzyme at 36-40℃ for 24-48 h. After enzymatic hydrolysis, the gelatin is inactivated by boiling water and centrifuged at 10000 r for 10-20 min to collect the supernatant. The supernatant is added to a DEAE anion exchange column and eluted with 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. The collected fraction is detected under ultraviolet light at a wavelength of 212 nm. The collected target fraction is then dialyzed with a 5000-8000 Da dialysis membrane at 2-5℃ for 2-4 days to remove NaCl from the eluent. Finally, the fraction is freeze-dried to obtain donkey-hide gelatin sulfate heparin.

[0017] Furthermore, the streptomycin protease hydrolysate contains 0.24 M sodium acetate and 1.9 M sodium chloride, and its pH is 6.5.

[0018] The obtained heparin sulfate derivative can inhibit the binding activity of the SARS-CoV-2 spike protein to its co-receptor, and therefore can be used as an inhibitor of the SARS-CoV-2 spike protein binding activity.

[0019] The significant advantages of this invention are:

[0020] This invention prepares a novel heparin sulfate-like substance from donkey-hide gelatin through conventional operations such as enzymatic hydrolysis, ion exchange chromatography separation and purification, and dialysis. This substance competitively inhibits the binding activity of the SARS-CoV-2 spike protein to heparin, which can effectively prevent early-stage SARS-CoV-2 infection and lay an important foundation for developing new clinical applications of donkey-hide gelatin. Attached Figure Description

[0021] Figure 1 Elution curves for the preparation of donkey-hide gelatin glycosaminoglycan (A) and donkey-hide gelatin sulfate heparin (B) in Example 1.

[0022] Figure 2 This is the mass spectrometry analysis diagram of the disaccharide structure obtained in Example 2.

[0023] Figure 3 Electrophoresis diagram (A) and standard curve (B) of the heparin sulfate obtained in Example 3.

[0024] Figure 4 This is a comparison curve of the response curves of the heparin sulfate obtained in Example 4 to the inhibitory activity of spike protein and heparin chip. Detailed Implementation

[0025] A heparin sulfate derivative based on donkey-hide gelatin, the preparation method of which includes the following steps:

[0026] 1) Preparation and extraction of glycosaminoglycans from donkey-hide gelatin:

[0027] a) Dissolve 1-2 g of donkey-hide gelatin in 10-20 ml of ultrapure water under a water bath heating at 50-70℃ and keep warm for 30-60 minutes;

[0028] b) Dissolve 1-5 mg of streptomycin in 1-5 ml of streptomycin hydrolysate (0.24 M sodium acetate and 1.9 M sodium chloride, pH 6.5), then add it to the donkey-hide gelatin solution obtained in step a), heat in a water bath at 36-40℃ for 24-48 h, and then inactivate with boiling water.

[0029] c) Centrifuge the enzymatic hydrolysate obtained in step b) at 10,000 rpm for 15-30 min, and collect the supernatant for later use;

[0030] d) Add the supernatant obtained in step c) to a DEAE anion exchange column (column volume 5-20 mL) that has been equilibrated with 10-50 mM sodium phosphate buffer containing 0.1 M NaCl. After elution with equilibration buffer, elute with 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. Collect the eluted fraction at 3 mL / tube and detect the collected fraction with ultraviolet light at a wavelength of 212 nm. Then, dialyze the collected target fraction at 2-5℃ using a 1000-5000 Da dialysis membrane for 2-4 days (replacing deionized water every five hours during this period) to remove NaCl from the eluent. Finally, freeze-dry to obtain donkey-hide gelatin glycosaminoglycan.

[0031] 2) Preparation and extraction of heparin sulfate from donkey-hide gelatin:

[0032] The glycosaminoglycans obtained from donkey-hide gelatin were enzymatically hydrolyzed with 300-500 mU chondroitin sulfate ABC enzyme for 24-48 h at 36-40℃. After enzymatic hydrolysis, the glycosaminoglycans were inactivated by boiling water and centrifuged at 10000 r for 10-20 min to collect the supernatant. The supernatant was added to a DEAE anion exchange column (column volume 5-10 mL) that had been equilibrated with 10-50 mM sodium phosphate buffer containing 0.2 M NaCl. After thorough elution with equilibration buffer, the column was eluted with 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. The collected fraction was detected under ultraviolet light at a wavelength of 212 nm. The collected target fraction was then dialyzed with a 5000-8000 Da dialysis membrane at 2-5℃ for 2-4 days to remove NaCl from the eluent. Finally, the fraction was freeze-dried to obtain donkey-hide gelatin sulfate heparin.

[0033] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0034] Example 1: Preparation and extraction of heparin sulfate from donkey-hide gelatin as raw material

[0035] (1) Preparation and extraction of glycosaminoglycans from donkey-hide gelatin: 1 g of donkey-hide gelatin was soaked in 10 ml of ultrapure water and heated in a water bath at 70℃ for 30 min. 1 mg of streptomycin was dissolved in 1 ml of streptomycin hydrolysate (0.24 M sodium acetate and 1.9 M sodium chloride, pH 6.5) and added to the prepared donkey-hide gelatin solution. The mixture was heated in a water bath at 37℃ for 48 h. After hydrolysis, the enzyme was inactivated by boiling water. The supernatant was then collected by centrifugation at 10,000 rpm for 15 min. The supernatant was added to DEAE-Sephacel, which had been equilibrated with 50 mM phosphate buffer containing 0.1 M NaCl. TM In an anion exchange column (20 mL column volume), after thorough elution with equilibration buffer, further elution was performed with 50 mM sodium phosphate buffer containing 2.0 M NaCl. The eluted fraction was collected in 3 mL tubes, and the collected fraction was detected under UV light at 212 nm to plot the elution curve. The target fraction was collected based on the elution curve (see...). Figure 1 (A) The collected target fraction was dialyzed at 4°C for 4 days using a 3000 Da dialysis membrane to remove NaCl from the eluent. During this period, deionized water was replaced every five hours. Finally, the fraction was freeze-dried to obtain donkey-hide gelatin glycosaminoglycan.

[0036] (2) Preparation and extraction of heparin sulfate from donkey-hide gelatin: Under 37℃, the donkey-hide gelatin glycosaminoglycan obtained in step (1) was specifically enzymatically hydrolyzed with 300 mU chondroitin sulfate ABC enzyme for 24 h, so that the chondroitin sulfate, dermatin sulfate and hyaluronic acid contained therein were degraded to disaccharides. After enzymatic hydrolysis, the enzyme was inactivated by boiling water, and the supernatant was collected by centrifugation at 10000 r for 10 min. The supernatant was added to DEAE-Sephacel, which had been equilibrated with 50 mM sodium phosphate buffer containing 0.2 M NaCl. TM In an anion exchange column (column volume 10 mL), after thorough elution with equilibration buffer, separation and elution were performed with 50 mM sodium phosphate buffer containing 2.0 M NaCl. The eluted fraction was detected under UV light at 212 nm, and the elution curve was plotted and the target fraction was collected (see [link to article]). Figure 1 (B) The collected target fraction was dialyzed against a 5000 Da dialysis membrane at 4℃ for 4 days to remove NaCl, and finally freeze-dried to obtain heparin sulfate.

[0037] Example 2: Analysis of the structure and composition of heparin disaccharide sulfate from donkey-hide gelatin

[0038] The heparin sulfate prepared in Example 1 was enzymatically hydrolyzed with heparinase to obtain heparin sulfate disaccharide, which was then collected and purified by size exclusion chromatography. The composition and structure of the heparin sulfate disaccharide were analyzed by LC / MS-ITTOF. Specifically, the heparin sulfate was enzymatically hydrolyzed with 10 mU heparinase at 37°C for 48 h to obtain the heparin sulfate disaccharide. After enzymatic hydrolysis, the disaccharide was inactivated by boiling water, and the supernatant was collected by centrifugation at 10000 r for 10 min. The disaccharide was then collected and purified by size exclusion chromatography using a Superdex™ peptide column with a mobile phase of 0.1 M NH4HCO3 and a flow rate of 0.4 mL / min. The disaccharide peak was collected, and the mobile phase was removed by evaporation at 65°C to obtain the heparin sulfate disaccharide. The composition and structure of heparin disaccharide derived from donkey-hide gelatin sulfate were analyzed using LC / MS-ITTOF. The chromatographic conditions were as follows: ACQUITY UPLC BEH C18 column (2.1 × 150 mm 1.7 μm); mobile phase A was an aqueous solution containing 30 mM N-hydroxyethyl acrylamide (HXA) (pH 8.86), and mobile phase B was an 80% acetonitrile aqueous solution containing 30 mM HXA (pH 8.86); flow rate was 0.1 mL / min; column temperature was 45℃; mass spectrometry conditions: negative ion mode; CDL and Heat Block temperatures were 100–200℃; spray gas flow rate was 1.5 mL / min; cone voltage was -3.5 kV. The structure and component content of the disaccharide were determined based on the standard curve. The mass spectrometry chromatogram and disaccharide composition table are shown below. Figure 2 See Table 1.

[0039] Table 1. Disaccharide composition table

[0040]

[0041] The results showed that eight heparin sulfate disaccharides were detected, with ΔUA-GlcNAc having the highest content (30.4%), followed by three disaccharides: ΔUA-GlcNS, ΔUA-GlcNS(6S), and ΔUA(2S)-GlcNS(6S), at 16.7%, 16.7%, and 15.2%, respectively. The total sulfation degrees at the C-2-O site of hexuronic acid, the C-6-O site of glucosamine, and the N-site were 28.2%, 44.3%, and 38.9%, respectively. These results indicate that heparin sulfate possesses a low-sulfation structure, similar to the co-receptor of the SARS-CoV-2 spike protein on the cell surface, and therefore holds promise as a competitive inhibitor of the SARS-CoV-2 spike protein.

[0042] Example 3: Determination of the molecular weight of heparin sulfate derived from donkey-hide gelatin

[0043] Electrophoretic analysis was performed using a discontinuous gel system with a gel thickness of 1 mm, consisting of a 4% T-type concentrator gel and a 15% separating gel. The electrophoresis buffer was 0.25 M tris-glycine, pH 8.3. The sample of heparin sulfate was 40 μg (EJ), and standards were prepared using dp18 (3 μg), low molecular weight heparin (15 μg), heparin (10 μg), CS (10 μg), and DS (10 μg) of known molecular weight (denoted as S1, S2, S3, S4, and S5, respectively). Staining was performed with 0.5% alnicotinic blue (w / v) for 30 min, followed by destaining with copious amounts of distilled water. A standard curve was plotted using the known molecular weight glycosaminoglycans and their electrophoretic mobility, and the molecular weight of heparin sulfate was calculated based on this curve.

[0044] like Figure 3 As shown, the heparin sulfate derivative is a heterogeneous mixture (molecular weight distribution ranges from 20.4 Kda to 6.0 Kda), with an average molecular weight of 13.2 Kda. This indicates that a portion of the heparin sulfate derivative has a lower molecular weight than heparin, but is closer to low molecular weight heparin. Previous reports have indicated that low molecular weight heparin exhibits 200 times higher competitive inhibitory activity against the spike protein than heparin; therefore, this heparin sulfate derivative shows promise as a highly active competitive inhibitor.

[0045] Example 4: Application of heparin sulfate in inhibiting the binding activity of SARS-CoV-2 spike protein to co-receptor.

[0046] Using the heparin sulfate prepared in Example 1 as a competitive inhibitor, the interaction between the SARS-CoV-2 spike protein and its co-receptor heparin / heparin sulfate in vivo was simulated using the SPR heparin-SA-chip system. The inhibitory response curves of different concentrations of heparin sulfate to the spike protein and heparin chip were characterized, and the inhibitory activity was calculated.

[0047] The heparin-SA chip used was prepared by the following method: ① Preparation of biotinylated heparin: 2 mg of heparin and biotin (amine-PEG3-Biotin) were dissolved in 200 μL of ultrapure water, 10 mg of sodium cyanoborohydride was added, and the mixture was reacted in a water bath at 70 ℃ for 24 h. Then, another 10 mg of sodium cyanoborohydride was added. After cooling to room temperature, the biotinylated heparin was purified using a 3 kDa dialysis bag and freeze-dried for later use. ② Direct crosslinking of biotinylated heparin with SA chip: At 25 ℃, inject 10 μg / mL of biotinylated heparin in HBS-EP solution manually at a flow rate of 10 μL / min for 120 s. The mobile phase is HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4). After injection, replace with 2 mol / L NaCl solution to regenerate the chip surface for 3 min. Finally, equilibrate the chip with HBS-EP buffer. If the baseline response value increases by more than 200 RU and remains unchanged, it indicates that the heparin-SA chip ligand immobilization is successful.

[0048] Different concentrations of heparin sulfate solutions were prepared and mixed with spike protein (final reaction concentration 85 nM). The mixture was then injected into a heparin-SA chip at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 300 s. After analysis, the chip surface was regenerated with 2 mol / L NaCl solution for 3 min. The inhibition response curves of heparin sulfate to spike protein and the heparin chip are shown below. Figure 4 The inhibition rate of the spike protein was calculated based on the response value of the blank group.

[0049] Experimental results showed that, compared with the 0 nM control sample, the 29 nM and 145 nM solutions of heparin sulfate-based solutions inhibited the binding of the SARS-CoV-2 spike protein to the heparin-SA chip by 67.6% and 82.4%, respectively. Simulation calculations yielded the IC50 (inhibition rate) of heparin sulfate-based solutions against the binding of the SARS-CoV-2 spike protein to heparin. 50 Reaching 12 nM, its inhibition rate is 3-5 times higher than that of heparin and other heparin sulfates. This indicates that heparin sulfate has strong inhibitory activity against spike protein binding, and can achieve the effect of preventing and inhibiting early-stage infection of the novel coronavirus.

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A heparin sulfate derivative containing donkey-hide gelatin with inhibitory activity against the SARS-CoV-2 spike protein, characterized in that, The heparin sulfate is a heterogeneous mixture with an average molecular weight of 13.2 kDa. It is composed of disaccharide units of hexuronic acid and glucosamine linked by 1-4 glycosidic bonds. The total sulfation degree of hexuronic acid at the C-2-O site, glucosamine at the C-6-O site and N-site is 28.2%, 44.3% and 38.9%, respectively. The contents of ΔUA-GlcNAc, ΔUA-GlcNS, ΔUA-GlcNS(6S) and ΔUA(2S)-GlcNS(6S) are 30.4%, 16.7%, 16.7% and 15.2%, respectively.

2. A method for preparing heparin sulfate as described in claim 1, characterized in that, Includes the following steps: 1) Preparation and extraction of donkey-hide gelatin glycosaminoglycans: After heating donkey-hide gelatin in a water bath, it is enzymatically hydrolyzed with streptomycin protease. After centrifugation, the supernatant is collected and separated and purified by DEAE anion exchange chromatography. Then, it is dialyzed and freeze-dried to obtain donkey-hide gelatin glycosaminoglycans. 2) Preparation and extraction of heparin sulfate from donkey-hide gelatin: The donkey-hide gelatin glycosaminoglycan obtained by enzymatic hydrolysis of chondroitin sulfate ABC enzyme in step (1) was centrifuged and the supernatant was separated and purified by DEAE anion exchange chromatography. Then, the donkey-hide gelatin heparin sulfate was obtained by dialysis and freeze drying.

3. The method for preparing heparin sulfate based on donkey-hide gelatin according to claim 2, characterized in that, Step 1) Specific operations include: a) Dissolve 1-2 g of donkey-hide gelatin in 10-20 mL of ultrapure water under a water bath heating at 50-70℃ and keep warm for 30-60 min; b) Dissolve 1-5 mg of streptomycin in 1-5 mL of streptomycin hydrolysate, then add it to the gelatin solution obtained in step a), heat in a water bath at 36-40℃ for 24-48 h, and then inactivate with boiling water. c) Centrifuge the enzymatic hydrolysate obtained in step b) at 10,000 rpm for 15-30 min, and collect the supernatant for later use; d) Add the supernatant obtained in step c) into a DEAE anion exchange column and elute using a 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. Detect the collected fractions using ultraviolet light at a wavelength of 212 nm. Then, dialyze the collected target fraction through a 1000-5000 Da dialysis membrane to remove NaCl from the eluent. Finally, freeze-dry the fraction to obtain the final product.

4. The method for preparing heparin sulfate based on donkey-hide gelatin according to claim 3, characterized in that, The streptomyces protease hydrolysate contains 0.24 M sodium acetate and 1.9 M sodium chloride, and its pH is 6.

5.

5. The method for preparing heparin sulfate based on donkey-hide gelatin according to claim 2, characterized in that, Step 2) involves the following steps: At 36-40℃, the obtained glycosaminoglycans of donkey-hide gelatin are enzymatically hydrolyzed using 300-500 mU of chondroitin sulfate ABC enzyme for 24-48 hours. After enzymatic hydrolysis, the gelatin is inactivated by boiling water, and the supernatant is collected by centrifugation at 10000 rpm for 10-20 minutes. The supernatant is then added to a DEAE anion exchange column and eluted with 10-50 mM sodium phosphate buffer containing 2.0 M NaCl. The collected fraction is detected under ultraviolet light at a wavelength of 212 nm. The target fraction is then dialyzed using a 5000-8000 Da dialysis membrane to remove NaCl from the eluent. Finally, the fraction is freeze-dried to obtain the final product.

6. The use of the heparin sulfate derivative as described in claim 1 in the preparation of an inhibitor of SARS-CoV-2 spike protein binding activity.

Images