Application of SARS-CoV-2S protein in cardiovascular diseases of novel coronavirus infected patients

Abstract

The invention provides an application of a binding site of a beta receptor (beta-AR) and an SARS-CoV-2Spike protein as a therapeutic target in preparation of a medicine for treating cardiovascular diseases of a COVID-19 patient. The invention finds that Spike protein is in targeted binding with beta1-AR-ECL2 through an RBD region, but is different from a classical beta-AR agonist binding mode, and plays a role of a beta-AR pathological ligand to cause cardiac inflammation, fibrosis and cardiac dysfunction. Therefore, a beta receptor blocker or targeted therapy for specific binding of beta-AR and Spike protein can relieve cardiac inflammatory response and fibrosis of COVID-19 patients, improve diastolic function and cardiac remodeling, and become a potential therapeutic target.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to the use of SARS-CoV-2S protein (SARS-CoV-2 Spike protein) to activate beta receptors and their specific binding sites in cardiovascular diseases of patients infected with the new coronavirus. Background Art

[0002] Coronavirus (COVID-19) can damage the cardiovascular system and cause chronic cardiovascular symptoms, inducing and / or aggravating a variety of cardiovascular diseases. About 30-70% of COVID survivors continue to have symptoms such as palpitations, tachycardia, chest pain, and decreased activity tolerance after 4 weeks, which can affect normal life and work in severe cases; the incidence of cardiovascular disease increases by 40-50% after COVID-19 infection, and the risk of death increases by 3-4 times. Infected people have cardiac inflammation and fibrosis, accompanied by diastolic dysfunction, which is also very common in non-severe COVID-19 recovered patients.

[0003] COVID-19 can cause acute and chronic systemic multi-organ damage, among which myocardial damage and cardiovascular symptoms are common in both acute and chronic stages. Current studies have shown that cardiac involvement is still quite common (30-78%) in COVID-19 survivors several months later. Cardiac magnetic resonance imaging shows that about 60% of patients have myocardial congestion and edema, indicating a persistent cardiac inflammatory state. One-third of patients have obvious cardiac fibrosis, which is the main reason for the development of chronic cardiac symptoms and the long-term development of heart failure and the increased risk of other cardiovascular diseases.

[0004] Although there is no clear evidence of cardiovascular disease in patients who have recovered from COVID-19, they still have cardiac symptoms and / or abnormal signs such as palpitations, atypical chest pain, and decreased activity tolerance 4-12 weeks after infection or longer. Palpitations are the most common symptom (25-50%), and about 9-14% still have tachycardia (at least 5 beats / min higher than the pre-COVID baseline resting heart rate) 4-6 months after infection. A survey and follow-up of a cohort of patients with mild COVID-19 showed that 73% of the enrolled patients still had cardiac symptoms 4 weeks after infection. Compared with asymptomatic infected persons, patients who recovered from COVID-19 had a faster heart rate and more significant cardiac inflammation and fibrosis. One year later, 57% of patients who recovered from COVID-19 still had cardiac symptoms, and their diffuse cardiac inflammation was more obvious than that of patients with improved symptoms. The persistent inflammatory state explains the chronic cardiac symptoms to a certain extent.

[0005] Therefore, it is necessary to explore the mechanism of myocardial injury after COVID-19 infection and find early intervention targets. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention has found that SARS-CoV-2Spike protein exists in the blood of some COVID-19 patients for a long time after recovery, and it can continuously activate cardiac β-adrenergic receptors (β-ARs) and downstream pathways. The present invention also found that SARS-CoV-2Spike protein specifically binds to β receptors, and cardiovascular diseases caused by new coronavirus infection can be solved through the specific sites where the two bind. Based on this, the purpose of the present invention is to provide a specific binding site of β receptors and SARS-CoV-2Spike protein as a therapeutic target in the preparation of drugs for treating cardiovascular diseases in some COVID patients after recovery.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides the use of a specific binding site of SARS-CoV-2 Spike protein and β receptor as a therapeutic target in the preparation of a drug for treating cardiovascular diseases in patients infected with the new coronavirus.

[0009] Furthermore, the specific site is the binding site between the RBD region of the SARS-CoV-2 Spike protein and the short helical structure on the β-AR-ECL2 of the β receptor.

[0010] Furthermore, the SARS-CoV-2 Spike protein overactivates cardiomyocyte beta receptors.

[0011] Furthermore, the cardiovascular diseases include myocardial fibrosis, arrhythmia, heart failure, coronary heart disease, myopericarditis and Takotsubo cardiomyopathy.

[0012] In a second aspect, the present invention provides the use of a beta-blocker in the preparation of a medicament for treating cardiovascular diseases in patients infected with the novel coronavirus.

[0013] Furthermore, the beta-receptor blocker is used in the preparation of a drug for inhibiting the binding of SARS-CoV-2 Spike protein to beta-receptors of myocardial cells of patients infected with the new coronavirus.

[0014] Preferably, the beta-receptor blocker is used in the preparation of a drug for inhibiting the increase in cAMP content caused by S protein stimulating myocardial cells.

[0015] Preferably, the beta-blockers include metoprolol, ICI-118551, and Carazolol.

[0016] In a third aspect, the present invention provides a drug for treating cardiovascular diseases in patients infected with the new coronavirus, the drug comprising a beta-receptor blocker and / or a targeted drug that inhibits the specific binding of β-AR to SARS-CoV-2S protein, and pharmaceutically acceptable pharmaceutical excipients.

[0017] In a fourth aspect, the present invention provides a method for screening drugs for treating cardiovascular diseases in patients infected with the new coronavirus by using the specific binding site of SARS-CoV-2 Spike protein and β receptor as drugs or reagents.

[0018] Furthermore, the specific site is the binding site between the RBD region of the SARS-CoV-2 Spike protein and the short helix structure on the β-AR-ECL2 of the β receptor; optionally, the cardiovascular diseases include myocardial fibrosis, arrhythmia, heart failure, coronary heart disease, myopericarditis and Takotsubo cardiomyopathy; optionally, the method is an in vitro drug screening method.

[0019] The beneficial effects of the present invention are as follows: Through the research of the present invention, it is found that SARS-CoV-2 Spike protein (SARS-CoV-2S protein) persists in the body of patients infected with the new coronavirus; it may overactivate β-AR (including β1-AR, β2-AR) by specifically binding to β-AR. The S protein targets and binds to β-AR-ECL2 through the RBD region, plays the role of β-AR pathological ligand, and continuously over-excites cardiac β-AR, leading to cardiac inflammation, fibrosis and cardiac dysfunction. Therefore, beta-blockers or targeted therapeutic drugs based on the specific binding sites of β-AR and Spike protein can be used to reduce S protein-mediated cardiac inflammatory response and fibrosis after new coronavirus infection, improve diastolic function and cardiac remodeling, and become a potential therapeutic target. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 Schematic diagram of plasma S protein levels in the recovered group, asymptomatic infection group, and healthy control group;

[0022] Figure 2 Schematic diagram of the comparison results of heart rate and cardiac sympathetic nerve activity indexes in the recovered group, asymptomatic infection group and healthy control group (where NLF: normalized low-frequency power in heart rate variability);

[0023] Figure 3Schematic diagram of the correlation between plasma S protein level and nLF (where NLF: normalized low-frequency power in heart rate variability);

[0024] Figure 4 This is a schematic diagram of the comparison of early cardiac function impairment indicators between the recovered group and the asymptomatic infection group (GLS: left ventricular long axis strain);

[0025] Figure 5 The MST curves of S protein binding to β1-AR and β2-AR respectively (ICI151: β2 receptor blocker, metoprolol: β1 receptor blocker);

[0026] Figure 6 Schematic diagram of cAMP, a molecule in the classic signaling pathway of β-AR in cardiomyocytes, activated by S protein;

[0027] Figure 7 The diagrams show the binding patterns of S protein RBD with β1-AR and β2-AR respectively (RBD: receptor binding domain, ECL2: second extracellular loop, Carazolol: a β-receptor blocker). DETAILED DESCRIPTION

[0028] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0029] 1. Experimental part

[0030] 1: To verify the correlation between S protein levels in the circulation of recovered patients and cardiac sympathetic activation, cardiac function and prognosis, the experimental process is as follows:

[0031] (1) A total of 45 recovered patients who visited the outpatient department of the Department of Cardiology of Peking University Third Hospital due to cardiovascular symptoms were enrolled, and 45 asymptomatic infected persons and 10 healthy controls from the physical examination center were matched according to gender and age. All patients met the inclusion and exclusion criteria and signed the informed consent.

[0032] Note: Inclusion criteria: 1) Age 18-60 years old; 2) No obvious abnormalities in myocardial injury markers, echocardiography, chest X-ray / chest CT. 3) Able to cooperate with follow-up.

[0033] Exclusion criteria: 1) Patients who have been diagnosed with severe COVID-19 infection, or have clear cardiac complications during COVID-19 infection; 2) Patients with known cardiovascular diseases in the past; 3) Patients with serious diseases of other systems.

[0034] (2) Peripheral blood samples were collected from each group, centrifuged, packaged, and frozen, and the plasma S protein level was detected using the ELISA method.

[0035] (3) All patients completed a dynamic electrocardiogram immediately and calculated the heart rate variability index: frequency domain index: normalized low-frequency power (nLF) 0.04-0.15 Hz, nLF mainly represents sympathetic nerve activity, and the correlation between plasma S protein level and the above indexes was analyzed.

[0036] (4) All patients were immediately given an echocardiogram to collect indicators reflecting contractile function, including early indicators of impaired cardiac function, such as left ventricular global longitudinal strain (GLS), etc. The correlation between plasma S protein levels and the above indicators was analyzed.

[0037] 2. Revealing the potential molecular mechanism of S protein as a pathological ligand of β-AR

[0038] (1) Molecular docking technology was used to clarify the structural basis of the S protein's targeted binding to β-AR.

[0039] The PBD codes of S protein, β1-AR and β2-AR were queried on the protein databank (PDB), which were 7N9T, 7BTS and 6KR8 respectively. The docking software ZDock was used to perform amino acid sequence docking calculations, filtering out the docking results of the transmembrane region and the intramembrane region of β1-AR and β2-AR, and retaining the binding mode of the extramembrane region and S protein.

[0040] (2) Microscale thermophoresis (MST) technology was used to prove that S protein interacts with β receptor.

[0041] HEK293T cells were transfected with β1-AR-GFP or β2-AR-GFP plasmids, membrane proteins were extracted and analyzed, and then the MST method was used to quantify the binding of β receptors to Spike proteins; β1 receptor blocker metoprolol (meto) and β2 receptor blocker ICI-118551 were used for verification.

[0042] 2. Experimental Results

[0043] 1. S protein persists in the circulation of recovered patients ( Figure 1 ).

[0044] By analyzing 45 recovered patients, 45 asymptomatic infected persons and 10 healthy persons, it was found that the detection rate of plasma S protein in recovered patients was higher than that in the asymptomatic infected group and the control group.

[0045] 2. Plasma S protein level can reflect the activation state of cardiac sympathetic nerves in recovered patients ( Figure 2 , 3 ).

[0046] The dynamic electrocardiograms of 45 recovered patients and 45 asymptomatic infected patients were analyzed. The results showed that compared with the healthy group and the asymptomatic infected group, the average heart rate of the recovered group was faster, and the heart rate variability index nLF was significantly increased ( Figure 2 ), indicating that the cardiac sympathetic nerves are overactivated. The plasma S protein level is positively correlated with nLF, indicating that the level of S protein can reflect the degree of cardiac sympathetic activation ( Figure 3 ), p<0.05.

[0047] 3. The left ventricular GLS decreased in the high plasma S protein group ( Figure 4 ).

[0048] The results showed that analysis of echocardiographic parameters revealed that the left ventricular GLS in the recovered group was lower than that in the asymptomatic infection group, and echocardiography in patients with high plasma S protein levels indicated early cardiac function impairment.

[0049] 4. S protein can bind to myocardial cell β1 receptor and β2 receptor ( Figure 5 )

[0050] The results showed that S protein could bind to β1 receptor and β2 receptor of cardiomyocytes, and the binding could be antagonized by β1 receptor blockers and β2 receptor blockers, respectively.

[0051] 5. S protein can activate the classic signaling pathway molecule cAMP ( Figure 6 ).

[0052] Neonatal rat cardiomyocytes were isolated and cultured, and incubated with S protein (S-trimer 10nmol / L) and β-AR agonist isoproterenol (ISO 10μmol / L) for 1 hour, and the intracellular cAMP content was detected. Similar to the effect produced by ISO, S protein can significantly increase the cAMP content in cardiomyocytes, and β1 receptor blocker metoprolol (meto 10uM) and β2 receptor blocker ICI-118551 can inhibit the increase in cAMP content in cardiomyocytes caused by S protein stimulation.

[0053] 6. Structural basis for the targeted binding of S protein to β-AR ( Figure 7 )

[0054] The RBD structure of S protein can bind to β1-AR-ECL2 and β2-AR-ECL2 respectively.

[0055] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Application of the specific binding site of SARS-CoV-2 Spike protein to β receptor as a therapeutic target in the preparation of drugs for the treatment of cardiovascular diseases in patients infected with the new coronavirus.

2. The use according to claim 1, characterized in that The specific site is the binding site between the RBD region of the SARS-CoV-2 Spike protein and the short helical structure on the β-AR-ECL2 of the β receptor.

3. The use according to claim 1, characterized in that The SARS-CoV-2 Spike protein overactivates cardiomyocyte β receptors.

4. The use according to claim 1, characterized in that The cardiovascular diseases include myocardial fibrosis, arrhythmia, heart failure, coronary heart disease, myopericarditis and Takotsubo cardiomyopathy.

5. The use of beta-blockers in the preparation of drugs for the treatment of cardiovascular diseases in patients infected with the new coronavirus.

6. The use according to claim 5, characterized in that The beta-receptor blocker is used in the preparation of a drug for inhibiting the binding of SARS-CoV-2 Spike protein to beta-receptors of myocardial cells of patients infected with the new coronavirus. Preferably, the beta-receptor blocker is used in the preparation of a drug for inhibiting the increase in cAMP content caused by S protein stimulating myocardial cells.

7. The use according to claim 5, characterized in that The cardiovascular diseases include myocardial fibrosis, arrhythmia, heart failure, coronary heart disease, myopericarditis and Takotsubo cardiomyopathy.

8. A drug for treating cardiovascular diseases in patients infected with the new coronavirus, the drug comprising a beta-receptor blocker and / or a targeted drug that inhibits the specific binding of β-AR to SARS-CoV-2S protein, and pharmaceutically acceptable excipients.

9. A method for using the specific binding site of SARS-CoV-2 Spike protein to β receptors as a drug or reagent to screen drugs for the treatment of cardiovascular diseases in patients infected with the new coronavirus.

10. The method according to claim 9, characterized in that The specific site is the binding site between the RBD region of the SARS-CoV-2 Spike protein and the short helix structure on the β-AR-ECL2 of the β receptor; optionally, the cardiovascular diseases include myocardial fibrosis, arrhythmia, heart failure, coronary heart disease, myopericarditis and Takotsubo cardiomyopathy; optionally, the method is an in vitro drug screening method.

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