A pearl polypeptide for improving ACE2 enzyme activity and inhibiting specific binding of novel coronavirus S protein to ACE2 and application thereof

Abstract

The present application provides a kind of bioactive peptide derived from seawater pearl, its amino acid sequence is Ile-Pro-Ser-Thr-Thr-Pro-Phe-Pro-Ser-Thr-Thr-Val-Ala-Thr-Thr-Thr-Met, named SCOL polypeptide.The active peptide can improve ACE2 enzyme activity to 3.76 times, can be used to prepare the drug for improving ACE2 activity, for preparing the drug for treating hypertension, anti-heart failure, anti-tissue fibrosis, anti-inflammation, treating type 2 diabetes and its complications and relieving neuropathic pain.The active peptide can be specifically combined with ACE2, and effectively inhibit 65% of the binding of new coronavirus S protein and ACE2, as a blocking agent, can be applied to prepare a kind of anti-coronavirus infection drug, especially to prepare anti-novel coronavirus drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a pearl polypeptide derived from seawater pearls that can enhance ACE2 enzyme activity and inhibit the specific binding of the SARS-CoV-2 S protein to ACE2, and its application. Background Technology

[0002] Angiotensin-converting enzyme 2 (ACE2) is a type I transmembrane glycoprotein with a single extracellular catalytic domain. ACE2 is widely distributed on the surface of various cells, including those in the heart, kidneys, lungs, cardiovascular system, intestines, testes, liver, pancreas, and central nervous system. ACE2 is a potent negative regulator of the renin-angiotensin system (RAS) and an important therapeutic target for diseases related to RAS imbalance. ACE2 can convert angiotensin I to angiotensin 1–9, balancing the effects of ACE by reducing substrate levels; and it can convert angiotensin II to angiotensin 1–7. Angiotensin 1–7 has the opposite effect to angiotensin II, inducing vasodilation and lowering blood pressure. As a beneficial vasodilator and antiproliferator, ACE2 has shown protective effects in the cardiovascular system and many other organs.

[0003] Imbalances in the RAS system lead to vascular dysfunction, a major cause of atherosclerosis and cardiovascular disease (CVD). One approach to repairing vascular dysfunction and other pathological changes is to balance the effects of angiotensin II by increasing ACE2 activity. Craceower et al. found that ACE2 knockout mice exhibited severe cardiac dysfunction, suggesting that ACE2 may have the potential to remodel and regulate cardiac function. Uri et al. verified that decreased serum ACE2 activity is a selective biomarker for abnormal cardiac systolic function. ACE2 deficiency enhances susceptibility to heart failure, and further studies confirmed that patients with left ventricular systolic dysfunction have increased plasma ACE2 activity, which is a significant risk factor for coronary heart disease, sudden death, heart failure, and stroke. However, increasing ACE2 levels can prevent and reverse the heart failure phenotype. ACE2 and angiotensin I-7 have become major protective pathways against heart failure.

[0004] Imbalances in the renal artery system (RAS) are also closely related to tissue fibrosis. The RAS is currently considered one of the local endocrine systems present in tissues such as the heart, kidneys, lungs, and liver. Angiotensin II, as the main effector molecule of this system, mediates the promotion of tissue fibrosis by fibroblasts through the angiotensin receptor (ATR). For example, Konigshoff M et al. found that angiotensin II promotes pulmonary fibrosis by mediating the migration of primary lung fibroblasts through the AT1R receptor. In the kidneys, kidney damage has been reported to be largely mediated by angiotensin II. Hypertensive nephropathy is a common complication of hypertension, and one of its main mechanisms is inflammation, oxidative stress, and renal tissue fibrosis associated with angiotensin II. Studies have shown that ACE2 gene knockout leads to increased blood pressure, glomerular damage, and renal fibrosis in diabetic mice. In the heart, RAS imbalance in diabetic patients participates in the formation of myocardial fibrosis, and the PI3K / Akt / mTOR signaling pathway regulates the role of myocardial fibrosis. Myocardial fibrosis caused by excessive proliferation, increased collagen synthesis, and imbalanced ratio of cardiac fibroblasts (CFs) is one of the cytopathological bases of left ventricular hypertrophy in hypertension. ACE2 has thus become a target for the treatment of multi-tissue fibrosis. Angiotensin I-7 not only participate in blood pressure regulation but also play a role in the anti-fibrotic process. Studies have shown that serum ACE2 expression levels are negatively correlated with the degree of liver fibrosis.

[0005] Imbalances in the Renin-angiotensin System (RAS) play a driving role in the pathogenesis of some inflammatory diseases. Some studies have found that abnormal activation of the RAS promotes the progression of atherosclerotic diseases. Specific mechanisms may include the accumulation of angiotensin II in blood vessels, promoting inflammatory responses. Experimental evidence shows that major inflammatory signaling pathways, such as the NF-κB pathway, can be activated by reduced ACE2 expression, leading to excessive release of inflammatory factors, endothelial cell dysfunction, oxidative stress (a series of reactions that can cause tissue damage), thrombosis, and other mechanisms that promote atherosclerosis. Conversely, activation of signaling pathways related to ACE2 and its metabolites angiotensin I-7 is considered to have anti-inflammatory and antioxidant effects. Studies have found a possible mechanism: a positive correlation between ACE2 expression and the activation of the antioxidant pathway Nrf2. Clinical evidence shows that ACE2 protein expression is reduced within existing atherosclerotic plaques, inevitably leading to the accumulation of angiotensin II and a reduction in the production of angiotensin I-7. Increasing ACE2 activity can play an important anti-inflammatory or protective role in the inflammatory process.

[0006] ACE2 is an important target for the treatment of type 2 diabetes and its complications. Hypertension often coexists with insulin resistance, which is a common pathophysiological basis of diabetes and metabolic syndrome. Therefore, improving the body's insulin resistance status is one of the important measures for the prevention and treatment of diabetes and metabolic syndrome. Studies have shown that endothelial dysfunction induced by angiotensin II and inflammation play an important role in the development of insulin resistance. Therefore, the effects of overactive angiotensin II in diabetes and its complications can be eliminated by increasing ACE2 expression or activity. ACE2 can effectively delay the progression of insulin function decline in diabetic patients by increasing pancreatic islet blood perfusion, inhibiting cell apoptosis, and promoting insulin secretion. In the pathophysiology of diabetic microvascular and macrovascular complications, ACE2 exerts an anti-ACE effect, regulating the balance of vasoconstriction and vasodilation in the heart, retina, and kidneys. ACE2 and its activators have extremely broad clinical application prospects in the prevention and treatment of diabetes and its complications.

[0007] The central nervous system contains an independent autocrine RAS system. Angiotensin II may act as a neurotransmitter or neuromodulator to participate in the transmission of nociceptive information in the central nervous system and play an important role in the generation of pain.

[0008] In recent years, there have been reports of angiotensin receptor antagonists or other related peptides being used in the clinical treatment of pain. Rice et al. conducted a double-blind, randomized, placebo-controlled phase 2 clinical trial using the small molecule AT2R antagonist EMA401 in 183 patients with postherpetic neuralgia. The results showed that EMA401 produced good analgesia compared to placebo and was well-tolerated. Angiotensin I-7 is an important neuromodulator in areas related to arterial baroreflex control, such as the hypothalamus and ventrolateral medulla oblongata. In these sites, the cardiovascular effects of angiotensin I-7 are blocked by A-779 (a MasR antagonist), indicating that angiotensin I-7 may act on brain Mas receptors. Studies have shown that angiotensin I-7 can act on both MasR and AT1R, and the anti-nociceptive effect produced by acting on MasR is far greater than the pain response caused by acting on AT1R. Current research suggests that the angiotensin I-7 / Mas receptor axis may be an effective approach for the future treatment of neuropathic and inflammatory pain. Neuropathic pain observed in type 2 diabetic mice involves downregulation of the ACE2 / angiotensin 1-7 / Mas receptor pathway, which is caused by the loss of ACE2-expressing dorsal horn neurons in the spinal cord. Since activation of the ACE2 / angiotensin 1-7 / Mas receptor pathway can alleviate hyperalgesia, exogenous supplementation of angiotensin 1-7 is an effective therapeutic strategy for relieving neuropathic pain in type 2 diabetes. Therefore, increasing ACE2 activity is an important target for developing drugs to alleviate neuropathic pain.

[0009] In conclusion, research on how to amplify the enzymatic activity of ACE2 to convert more angiotensin II into angiotensin I-7 and thus exert beneficial biological effects has important therapeutic value.

[0010] The S protein of coronaviruses can specifically bind to ACE2 and mediate the process of viral entry into host cells, which is the initiation process of coronavirus recognition and invasion of host cells. ACE2 and the S protein bind in a lock-and-key-like manner, allowing the virus to enter human cells. XU et al., through biological analysis, found that the S protein of the novel coronavirus is structurally similar to that of the SARS coronavirus (SARS-CoV), and can also infect host epithelial cells by interacting with the ACE2 protein molecule on the surface of host cells. Therefore, the ACE2 molecule is a key molecule for novel coronavirus (2019-CoV-2) infection and is an essential protein for SARS-CoV-2 infection of cells. Since ACE2 plays a crucial role in the invasion of host cells by SARS-CoV-2, developing drugs that can block or delay its function as a viral receptor is an ideal prevention and treatment strategy and is of great significance. Summary of the Invention

[0011] This invention provides a bioactive peptide that can be used as a direct ACE2 activator. This bioactive peptide can be used to prepare drugs that enhance ACE2 activity, and can be used to prepare drugs for treating hypertension, heart failure, tissue fibrosis, inflammation, type 2 diabetes and its complications, and relieving neuropathic pain. This bioactive peptide can specifically bind to ACE2, thereby inhibiting the specific binding of the coronavirus S protein to ACE2. As a blocker, it can be used to prepare a drug against coronavirus infection, especially a drug against the novel coronavirus.

[0012] The technical solution provided by the present invention to achieve the above objectives is as follows:

[0013] A bioactive peptide derived from seawater pearls that can bind to and activate ACE2 has been named SCOL polypeptide, with the amino acid sequence Ile-Pro-Ser-Thr-Thr-Pro-Phe-Pro-Ser-Thr-Thr-Val-Ala-Thr-Thr-Thr-Met.

[0014] Taking Hepu pearl oyster pearl as an example, a bioactive peptide isolated and purified from the matrix protein of Hepu pearl oyster pearl is prepared by the following steps:

[0015] (1) Extraction of pearl matrix protein: Decalcification of Hepu pearl oyster nucleus powder was carried out by dialysis and freeze drying to obtain pearl matrix protein.

[0016] (2) Enzymatic hydrolysis of pearl matrix protein: The lyophilized powder of pearl matrix protein was enzymatically hydrolyzed by trypsin for 2 hours. After inactivation by boiling water bath, the supernatant was collected by centrifugation, filtered, and then subjected to ultrafiltration by protein concentration centrifuge tube with a molecular weight cutoff of 3KD. The filtrate with a molecular weight less than 3KD was collected, concentrated, and then freeze-dried to obtain a mixture of enzymatically hydrolyzed pearl matrix protein peptides.

[0017] (3) Isolation and purification of ACE-inhibiting peptides of pearl matrix protein: The enzymatically hydrolyzed pearl matrix protein peptide mixture was purified by high performance liquid chromatography. The chromatographic column was C18, the mobile phase A was deionized water containing 0.1% trifluoroacetic acid, the mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid, the flow rate was 1 ml / min, the UV detection wavelength was 280 nm, and the sample with a retention time of 9.5 min was collected. After concentration, the sample was freeze-dried to obtain angiotensin-converting enzyme 2 binding peptide with the sequence Ile-Pro-Ser-Thr-Thr-Pro-Phe-Pro-Ser-Thr-Thr-Val-Ala-Thr-Thr-Thr-Met, which was named the SCOL peptide.

[0018] This bioactive peptide can also be prepared artificially based on the sequence disclosed in this invention.

[0019] The beneficial effects of this invention are:

[0020] (1) The bioactive peptide provided by this invention is derived from pearl. Pearl has been proven to be safe and non-toxic and can be consumed in ancient times. Therefore, the bioactive peptide has good biosafety, and has a small molecular weight and high activity. It is not only easy to separate and purify, but also easy to synthesize artificially.

[0021] (2) The bioactive peptides provided by the present invention can increase ACE2 enzyme activity by 3.76 times and can be used to treat related diseases caused by RAS system imbalance, such as preparing drugs for treating hypertension, anti-heart failure, anti-tissue fibrosis, anti-inflammation, treating type 2 diabetes and its complications, and relieving neuropathic pain.

[0022] (3) The bioactive peptide provided by the present invention can specifically bind to ACE2 and effectively inhibit 65% of the binding of the novel coronavirus S protein to ACE2. As a blocker, it can be used to prepare a drug against coronavirus infection, especially a drug against the novel coronavirus.

[0023] (4) This invention provides a reliable basis for the further development of pearl craftsmanship and pharmaceuticals. Attached Figure Description

[0024] Figure 1This is a graph showing the effect of the SCOL peptide of the present invention on ACE2 activity (significant differences exist between different lowercase letters, p≤0.05).

[0025] Figure 2 This diagram illustrates the binding and dissociation process of the SCOL peptide with ACE2 in this invention.

[0026] Figure 3 This is a diagram showing the competitive binding of the SCOL peptide and S protein to ACE2 in this invention (significant differences exist between different lowercase letters, p≤0.05).

[0027] Figure 4 This is the molecular formula of the SCOL polypeptide of the present invention. Detailed Implementation

[0028] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0029] Example 1: Preparation of SCOL peptide

[0030] (1) Extraction of pearl matrix protein

[0031] Nucleus-free pearls from the Hepu pearl oyster in Zhanjiang, Guangdong Province, were washed, dried, and thoroughly pulverized using a traditional Chinese medicine pulverizer. During pulverization, the process was paused periodically to allow the pulverizing container to cool, preventing overheating and damage to the pearl's active ingredients. The pearl powder was collected and placed in a beaker, with five times its volume of 1.5M EDTA added. The mixture was then placed in a 4°C chromatography freezer and stirred to remove calcium. After 24 hours, the mixture was transferred to a 50ml high-speed centrifuge tube and centrifuged (10,000 rpm). The supernatant was collected and filtered through a 0.2μm microporous membrane. The filtrate was then subjected to low-temperature dialyzing at 4°C for 48 hours, with multiple water changes during this period. Finally, the mixture was freeze-dried to obtain the pearl matrix protein.

[0032] (2) Enzymatic hydrolysis of pearl matrix proteins

[0033] Take the lyophilized pearl matrix protein powder and add trypsin for enzymatic hydrolysis. The trypsin activity is 10000 U / g, and the amount of trypsin added is 5% of the weight of the pearl matrix protein. The pH of the enzymatic hydrolysis system is adjusted to 8.0. Enzymatic hydrolysis is carried out in a 50℃ constant temperature water bath for 2 hours, followed by inactivation in a boiling water bath. The solution is then transferred to a 50ml high-speed centrifuge tube and centrifuged (10000rpm). The supernatant is collected and filtered through a 0.2μm microporous membrane. The filtrate is then subjected to ultrafiltration (4000rpm) using a Millipore protein concentration centrifuge tube with an internal low-adsorption Ultracel-PL ultrafiltration membrane with a molecular weight cutoff of 3KD. The filtrate with a molecular weight less than 3KD is collected, concentrated, and then freeze-dried to obtain a mixture of enzymatically hydrolyzed pearl matrix protein peptides.

[0034] (3) Isolation and purification of ACE-inhibiting peptides from pearl matrix proteins

[0035] The enzymatically hydrolyzed pearl matrix protein peptide mixture was purified using high-performance liquid chromatography (HPLC) with a C18 column. Mobile phase A consisted of deionized water containing 0.1% trifluoroacetic acid, and mobile phase B consisted of acetonitrile containing 0.1% trifluoroacetic acid. UV detection was performed at 280 nm, the flow rate was 1 mL / min, and samples with a retention time of 9.5 min were collected. After concentration, the sample was freeze-dried to obtain the target peptide, named the SCOL peptide, with the sequence Ile-Pro-Ser-Thr-Thr-Pro-Phe-Pro-Ser-Thr-Thr-Val-Ala-Thr-Thr-Thr-Met.

[0036] Example 2: Determination of the effect of SCOL peptide on ACE2 activity

[0037] The Beyotime ACE2 activity fluorescence detection kit was used for detection.

[0038] Principle: When ACE2 does not cleave the substrate, the two fluorescent groups are close enough to undergo fluorescence resonance energy transfer, resulting in undetectable fluorescence. When the substrate is cleaved by ACE2, the two ends of the peptide separate, the two fluorescent groups separate, and fluorescence can be detected. Thus, fluorescence detection can be used to detect the enzyme activity of ACE2 protease very sensitively.

[0039] (1) Sample preparation: Take 50mg of mouse liver tissue, add 500μl of lysis buffer for homogenization, centrifuge at 12000×g at 4℃ for 5 minutes, and take the supernatant for the next step.

[0040] (2) Set up a standard curve: Dilute the standard solution with buffer solution to concentrations of 0, 0.25, 0.5, 1, 2, 3, 4, and 5 μM. Add 100 μl to each well and repeat three times to obtain the standard curve.

[0041] (3) First, add 2 μl of substrate to a 96-well plate. Then, add 2.2 μl of SCOL peptide at concentrations of 0.004, 0.008, 0.012, 0.016, and 0.02 μg / μl in the total system to the PCR tubes, respectively. Next, add 8.8 μl of the supernatant sample from step (1), and then add 96.8 μl of buffer. Add 11 μl of lysis buffer and 96.8 μl of buffer to the blank control, mix well, and use a pipette to add 98 μl of the above solution to the 96-well plate containing the substrate. After mixing, perform fluorescence detection using a fluorescence microplate reader, setting the temperature to 37℃, the excitation wavelength to 325 nm, and the emission wavelength to 393 nm.

[0042] (4) Experimental Results

[0043] Calculation: Let T be the time interval during which the fluorescence intensity of the test sample group shows a linear relationship, and let ΔRFU be the change in fluorescence intensity within T. Substituting ΔRFU into the standard curve gives the amount produced in the sample, A. Let V be the sample volume, C be the sample protein concentration, and the formula for calculating ACE2 protease activity is:

[0044] ACE2 Activity=A×V×T×C(U / mg)

[0045] Experimental results are as follows Figure 1 As shown, the ACE2 activity in the sample increased with the increase of SCOL peptide concentration, reaching a maximum of 3.76 times the initial activity when the SCOL peptide concentration was 0.02 μg / μl.

[0046] Example 3: Determination of the binding ability of SCOL peptide to ACE2

[0047] The binding affinity of SCOL peptide to ACE2 was determined using a Biacore X100 microarray (GE, lot: 20139043) with a 10×HBS-EP buffer. ACE2 protein was immobilized on the microarray surface using a biotin-streptavidin method. Different concentration gradients of SCOL peptide were prepared (3.125, 6.25, 12.5, 25, 50, and 100 μg / ml), with a flow rate of 50 μl / min, a binding time of 300 s, a reaction temperature of 25 °C, a dissociation flow rate of 30 μl / min, and a dissociation time of 600 s. The results are shown below. Figure 2 As shown, the test curves for SCL peptide at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μg / ml, from top to bottom, indicate that SCL peptide has a high affinity for ACE2, and the dissociation constant KL is [value missing]. D The value of (M) is 7.687 × 10 -5 .

[0048] Example 4: Competitive binding analysis of SCOL peptide and S protein to ACE2

[0049] use The COVID-19 Spike-ACE2 Binding Assay Kit is used for testing.

[0050] Principle: The 96-well plate in the kit contains recombinantly expressed S protein. Then, in the presence of ACE2 protein, the peptide is added to the well. The small peptide can bind to the key site of ACE2, thereby reducing the binding rate between S protein and ACE2 protein.

[0051] (1) Preparation of a mixture of SCOL peptide and ACE2 protein

[0052] Dilute the 100×ACE2 protein concentrate in the kit to a 10×ACE2 protein solution using the diluent. Dilute the SCOL peptide to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml using the diluent. In each PCR tube, add 110 μl of each concentration of SCOL peptide, followed by 11 μl of the 10×ACE2 protein diluent, and vortex to mix. Set up a control group without SCOL peptide, i.e., add 110 μl of the diluent and 11 μl of the 10×ACE2 protein diluent to the control group and mix well. Each group has three replicates.

[0053] (2) Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding rate of ACE2 protein to S protein.

[0054] The bottom of the 96-well plate is coated with the S protein binding domain. 100 μl of solution from each PCR tube in step (1) is pipetted into the 96-well plate, sealed with a plate sealing film, and incubated overnight at 4°C with gentle shaking. The solution is discarded and the plate is washed 4 times with washing buffer. 300 μl of washing buffer is injected into each well for washing. 100 μl of the prepared 1x detection antibody, i.e., goat-derived anti-ACE2 antibody that can bind to the S protein-ACE2 protein complex, is added to each well. The plate is incubated at room temperature with gentle shaking for 1 hour and then washed with washing buffer. 100 μl of the prepared 1×HRP binding antibody, i.e., HRP-labeled anti-"goat-lgG", is added to each well. The plate is incubated at room temperature with gentle shaking for 1 hour and then washed with washing agent. 100 μl of the substrate chromogenic reagent TMB is added to each well. The plate is incubated at room temperature in the dark with gentle shaking for 30 minutes. The reaction is terminated by adding stop solution. The absorbance is measured at 450 nm. The absorbance is proportional to the amount of the S protein-ACE2 protein complex.

[0055] (3) Experimental Results

[0056] like Figure 3 As shown, compared with the control group, all concentrations of SCOL peptides showed inhibitory effects. The highest inhibition rate was found in the 0.8 mg / ml peptide group, reaching 65.8%, indicating that SCOL peptides have a strong binding ability to ACE2 and can competitively inhibit the binding of the SARS-CoV-2 S protein to ACE2. This suggests that SCOL peptides have a promising application prospect as a competitive inhibitor of the SARS-CoV-2 S protein.

[0057] Example 5: Sequence analysis of SCOL peptide

[0058] The SCOL polypeptide derived from pearl matrix protein prepared in Example 1 was analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / TOF-MS). Its molecular weight was 1794.8 Da, and its amino acid sequence was: Ile-Pro-Ser-Thr-Thr-Pro-Phe-Pro-Ser-Thr-Thr-Val-Ala-Thr-Thr-Thr-Met (as shown in SEQ ID NO: 1).

[0059] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A bioactive peptide derived from seawater pearl, named SCOL polypeptide, characterized in that, Its amino acid sequence is Ile-Pro-Ser-Thr-Thr-Pro-Phe-Pro-Ser-Thr-Thr-Val-Ala-Thr-Thr-Thr-Met.

2. The use of the SCOL polypeptide according to claim 1 in the preparation of drugs against SARS-CoV-2.

3. The use of the SCOL polypeptide according to claim 1 in the preparation of drugs for treating heart failure.

Images