Polypeptides capable of inhibiting human coronavirus infections in a broad spectrum manner, and applications thereof

A coronavirus and human technology, applied in the field of biomedicine, can solve the problems of lack of neutralization activity, no cross-protection effect of human coronavirus, lack of antiviral effect, etc., and achieve the effect of good inhibitory activity and good inhibitory effect.

Active Publication Date: 2017-08-08
SHANXI JINBO BIO PHARMA CO LTD
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AI Technical Summary

Problems solved by technology

[0005] At present, in the research and development of inhibitors for this type of virus, the S1 and S2 regions of the S protein are two important targets. Among them, the entry inhibitors for the S1 region are mainly antibodies, although these antibodies are specific to specific The virus has good neutralizing activity, but it has no cross-protection effect on other human coronaviruses; studies have shown that although SARS-CoV and its similar virus RsSHC014-CoV have high gene homology and the same receptors, However, SARS-CoV-specific antibodies still lack neutralizing activity against RsSHC014-CoV (references: Menachery, V.D., B.L.Yount, Jr., K.Debbink, S.Agnihothram, L.E.Gralinski, J.A.Plante, R.L.Graham, T. .Scobey, X.Y.Ge, E.F.Donaldson, S.H.Randell, A.Lanzavecchia, W.A.Marasco, Z.L.Shi, and R.S.Baric. 2015. A SARS-like cluster of circulating bat coronaviruses showspotential for human emergence. Nat Med 21:1508- 1513.); The entry inhibitors targeting the S2 region are mainly polypeptide drugs, and most of these entry inhibitors are derived from the amino acid sequence of the heptapeptide repeat region 2 (HR2) of the virus S2 protein; the HR2 polypeptide can bind to the heptapeptide of the virus The repeat region 1 (HR1) trimer binds to form a stable heterologous 6HB, which prevents the binding of the viral HR2 to the viral HR1 trimer, preventing them from forming homologous 6HB, thereby inhibiting the fusion of the viral membrane and the target cell membrane , which also inhibits the viral gene from entering the target cell membrane for replication (such as figure 1 shown); at present, polypeptide entry inhibitors with the same mechanism of action have played an

Method used

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  • Polypeptides capable of inhibiting human coronavirus infections in a broad spectrum manner, and applications thereof
  • Polypeptides capable of inhibiting human coronavirus infections in a broad spectrum manner, and applications thereof
  • Polypeptides capable of inhibiting human coronavirus infections in a broad spectrum manner, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Inhibition of human coronavirus S protein-mediated cell-cell fusion experiment (cell-cellfusion)

[0040] (1) Experimental method:

[0041] 1. 293T cells were transfected with a plasmid encoding the human coronavirus S protein and cultured for 36-48 hours. As effector cells, for example, transfected with a plasmid encoding the MERS-CoV S protein -- pAAV-IRES-GFP-MERS-S 293T cells are called 293T / MERS / EGFP cells, and 293T cells are transfected with empty vector plasmid--pAAV-IRES-GFP, which are 293T / EGFP cells, which are used as negative control cells;

[0042] 2. The above cells were digested with 0.02% EDTA, centrifuged, and the cells were resuspended in fresh 10% FBS DMEM medium, and the cell concentration was adjusted to 2×10 5 Individuals / mL, take 50 μL and add it to the drug (50 μL) in serial dilution, and incubate at 37°C for 30 min;

[0043] 3. Take 100 μL of the cell / drug mixture and add it to the target cells (Huh-7) that have been plated on a 96-we...

Embodiment 2

[0047] Embodiment 2 The inhibitory activity assay of polypeptide to human coronavirus pseudovirus

[0048] (1) Experimental method:

[0049] 1. Use DMSO to dissolve the peptide and measure the peptide concentration;

[0050] 2. Prepare the target cell (HuH7) cell suspension expressing the human coronavirus receptor, add 10 4 cells;

[0051] 3. In a 96-well plate, use DMEM medium containing 10% FBS to dilute the peptide drug 2 times in a gradient, 50 μL per well;

[0052] 4. Add a certain titer of MERS-CoV pseudovirus to the drug dilution plate at 50 μL / well. Effect at room temperature for 30 minutes, so that the drug and the virus can fully act. Add 100 μL of the drug and virus mixture to each well and add it to the target cells from which the supernatant has been removed. After culturing at 37° C. for 12 hours, replace with fresh DMEM medium containing 10% FBS. 5. Measure luciferase after 72 hours, calculate and make the inhibition rate curve, and calculate the half inhi...

Embodiment 3

[0056] Example 3. Research experiment on the antiviral mechanism of HR2 polypeptide

[0057] Taking HCoV-EK1 as a representative, detect the interaction between a series of peptides and peptides (HR1Ps) derived from HR1 regions of different human coronaviruses, so as to further explore the antiviral mechanism of HCoV-EK series peptides; due to SARS-CoV and SARS-like The sequence of the virus (RsSHC014-CoV or RsW1V1-CoV) in the HR1 region is completely consistent, so the mechanism of the HCoV-EK series polypeptides against RsSHC014-CoV or RsW1V1-CoV can be explained here; this part of the research is carried out with reference to the literature. (Liu, S., G. Xiao, Y. Chen, Y. He, J. Niu, C. R. Escalante, H. Xiong, J. Farmar, A. K. Debnath, P. Tien, and S. Jiang. 2004. Interaction between heptad Repeat 1 and 2 regions inspike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 363:938-947.)

[0058] ...

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Abstract

The present invention belongs to the field of biomedicine, and relates to polypeptides capable of inhibiting human coronavirus infections, particularly to polypeptides capable of inhibiting human coronavirus infections in a broad spectrum manner, and applications thereof. According to the present invention, the polypeptides capable of providing broad spectrum inhibition effects for infections caused by more than 2 human coronaviruses are provided based on the conservation property of the S2 region of a coronavirus S protein and the similar fusion mechanism; the test results show that the polypeptides can achieve the commonality of the human coronavirus, ie., the similar HR region and the same fusion mechanism mediated by the coronavirus S protein so as to provide a series of HCoV-EK polypeptides, wherein the polypeptides provide good inhibition effects for currently popular human coronaviruses, and further provide good inhibition activity for SARS virus (RsSHC014-CoV or RsW1V1-CoV) possibly infecting human; and the polypeptides of the present invention can provide the prevention and treatment candidate drug for the currently popular human coronaviruses and the novel human coronavirus possibly emerging in the future.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a polypeptide for inhibiting human coronavirus infection, in particular to a polypeptide capable of broadly inhibiting human coronavirus infection and an application thereof. [0002] technical background [0003] Data show that a new type of pathogen was first discovered in Guangzhou, China in 2002, which caused severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS), also known as infectious atypical pneumonia. Respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus, SARS-CoV). As of July 2003, the disease has spread to 29 countries and regions around the world, with more than 8,000 confirmed cases and an average death rate of more than 10%. In 2012, another new type of coronavirus broke out in the Middle East, the Middle East respiratory coronavirus (Middle East respiratory Syndrome coronavirus, MERS-CoV), according to the World Health Organi...

Claims

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Application Information

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IPC IPC(8): C07K14/165C12N15/50A61K38/16A61P31/14
CPCA61K38/00C07K14/005C12N2770/20022C12N2770/20033
Inventor 姜世勃陆路夏帅王茜于飞
Owner SHANXI JINBO BIO PHARMA CO LTD
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