Use of tegaserod for the preparation of a medicament for the prevention or treatment of coronavirus infection
阅读说明：本技术 替加色罗在制备用于预防或治疗冠状病毒感染的药物中的应用 (Use of tegaserod for the preparation of a medicament for the prevention or treatment of coronavirus infection ) 是由 柳晓春 徐锡明 袁文敏 赵广健 王娟 赵晨阳 杨金波 于 2020-12-03 设计创作，主要内容包括：本发明公开了替加色罗在制备用于预防或治疗冠状病毒感染的药物中的应用。所述替加色罗的分子式为C-(16)H-(23)N-5O,能够抑制新型冠状病毒PLpro蛋白的活性,且在50μmol/L浓度下的抑制率高达100％,半数抑制浓度IC-(50)＜15μmol/L。本发明经过实验验证替加色罗对多种细胞无毒性作用,能够用于制备预防或治疗新型冠状病毒感染中的药物组合物,且安全可靠。本发明新型冠状病毒的预防或治疗提供了新的化合物以及新的思路。(The invention discloses application of tegaserod in preparing a medicament for preventing or treating coronavirus infection. The molecular formula of the tegaserod is C 16 H 23 N 5 O, can inhibit the activity of a novel coronavirus PLpro protein, and has an inhibition rate of up to 100% at a concentration of 50 mu mol/L and a half inhibition concentration IC 50 < 15. mu. mol/L. Experiments prove that the tegaserod has no toxic effect on various cells, can be used for preparing a pharmaceutical composition for preventing or treating novel coronavirus infection, and is safe and reliable. The prevention or treatment of the novel coronavirus of the invention provides novel compounds and novel ideas.)
1. Use of tegaserod in the manufacture of a medicament for the prevention or treatment of coronavirus infection.
2. Use according to claim 1, wherein tegaserod has the formula C16H23N5O has a structural formula
3. The use according to claim 1, wherein the coronavirus comprises a novel coronavirus.
4. Use according to claim 3, wherein tegaserod is capable of inhibiting the activity of a novel coronavirus PLpro protein.
5. The use according to claim 4, characterized in that the novel coronavirus PLpro protein is a PLpro protein expressed after codon optimization based on the G/C content of its original coding sequence and E.coli.
6. The use according to claim 5, wherein the coding sequence of the optimized PLpro protein is as shown in SEQ ID No. 1.
7. Use according to claim 4, characterized in thatThe inhibitory concentration of the tegaserod for inhibiting the PLpro protein is IC50＜15μmol/L。
8. The use according to claim 1, wherein the tegaserod can be used alone or in combination with other drugs, compounds or carriers to form a pharmaceutical composition.
9. A medicament for the prevention or treatment of coronavirus infection, characterized by: the medicament contains tegaserod.
10. The agent for preventing or treating coronavirus infection according to claim 9, wherein the agent is an oral preparation, an injection preparation, a paint, a lotion, an aerosol, an oil preparation, or a transdermal patch.
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of tegaserod in preparing a medicine for preventing or treating coronavirus infection.
Background
The new type of coronavirus pneumonia (Corona Virus Disease 2019, COVID-19), named as "2019 coronavirus Disease" by the world health organization, is a Disease caused by infection with the medicine named "Severe acute respiratory syndrome coronavirus 2" (SARS-Coronavirus 2, SARS-CoV-2). Currently, confirmed cases of new coronary pneumonia have emerged in numerous countries. COVID-19 is transmitted by inhalation or contact with infected droplets with latency periods of 2 to 14 days or more, with common clinical features including fever, cough, sore throat, headache, fatigue, myalgia, dyspnea, etc. COVID-19 is mild in most people but severe in some elderly and comorbid patients, and can rapidly progress to pneumonia, acute respiratory distress syndrome or multiple organ dysfunction, etc., even leading to death; while some of the population is asymptomatic infected without any significant symptoms. At present, the research on COVID-19 relates to many aspects such as epidemic research, clinical research, virus detection method and the like, but the research on SARS-CoV-2 virus invasion, replication, immune regulation mechanism and the like is still in an exploration stage, and no vaccine can provide sufficient protection for patients without diseases temporarily, so the research on antiviral drugs against SARS-CoV-2 has very important significance.
SARS-CoV-2 belongs to the genus beta coronavirus of the family Coronaviridae, is an enveloped, non-segmented, forward RNA virus whose entry into the host cell is mediated by the S protein, which is divided into a receptor-binding S1 region and a membrane-fusion S2 region. S1 binds to the cell surface angiotensin converting enzyme 2(ACE2) receptor through its Receptor Binding Domain (RBD); s2 fuses host cells and viral membranes to enable viral genomes to enter host cells for replication and disease initiation. The SARS-CoV-2 genome is about 29.8kb in size, contains 14 ORFs, and can encode 27 proteins, including 4 more conserved structural proteins: spike protein S, envelope protein E, membrane glycoprotein M, and nucleocapsid protein N; 8 accessory proteins 3a, 3b, p6, 7a, 7b, 8b, 9b and orf14, etc. The SARS-CoV-2 virus genome has 2 open reading frames orf1a and orf1b at the 5' end, and can code 2 polyprotein pp1a and pp1 b; the virus also contains two cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), and 15 nonstructural proteins NSP (NSP1-NSP10 and NSP12-NSP16) can be obtained by hydrolyzing polyprotein by the two proteases and are involved in the transcription and replication process of the virus.
The PLpro protein is a member of the family of cysteine proteases, and in host cells, viral PLpro recognizes the boundaries of NSP1/2, NSP2/3 and NSP3/4, and hydrolytic cleavage of the peptide bond of polyproteins occurs at the glycine site after P1, which in turn causes the virus to release NSP1, NSP2 and NSP3 to initiate virus-mediated RNA replication. In addition to viral peptide cleavage, PLpro is involved in the escape of the virus from innate immune responses through deubiquitination, ISG15 removal modification, and the like. Therefore, PLpro is crucial to the virus life cycle and is considered as an important target for developing anti-novel coronavirus infection drugs, and research and discovery of drugs or compounds capable of effectively acting on the targets participating in the life cycle of the coronavirus have positive significance for the deep research, drug development, prevention and treatment of SARS-CoV-2.
Disclosure of Invention
The invention provides an application of tegaserod in preparing a medicament for preventing or treating coronavirus infection. The tegaserod has strong effect of inhibiting the activity of novel coronavirus PLpro, can be used for preventing or treating SARS-CoV-2 infection, and has no harm to various cells.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides an application of tegaserod in preparing a medicament for preventing or treating coronavirus infection.
Further, the molecular formula of the tegaserod is C16H23N5O has a structural formula
Further, the coronavirus includes a novel coronavirus.
Further, the tegaserod can inhibit the activity of a novel coronavirus PLpro protein.
Further, the inhibition rate of the tegaserod at the concentration of 50 mu mol/L is 100%.
Furthermore, the novel coronavirus PLpro protein is expressed after being optimized according to the G/C content in the original coding sequence and the Escherichia coli codon.
Furthermore, the coding sequence of the optimized PLpro protein is shown as SEQ ID NO. 1.
Furthermore, the translated amino acid sequence of the coding sequence of the optimized PLpro protein is shown as SEQ ID NO. 2.
Further, the inhibitory concentration of the tegaserod for inhibiting the PLpro protein is IC50＜15μmol/L。
Further, the detection method of the inhibitory concentration of tegaserod comprises the following steps: adding PLpro with the total concentration of 40-200nmol/L in a phosphate buffer solution system with the concentration of 20mmol/L, pH of 6.8, adding tegaserod with different concentrations at the incubation temperature of 25 ℃, oscillating and incubating for 15min at room temperature, rapidly adding a fluorescent substrate, measuring and recording the fluorescence reading for 30min, and calculating the inhibition rate; then combining the tegaserod concentrateLogarithmic value and inhibition rate of the degree, and calculating the inhibition concentration IC of the tegaserod by a four-parameter method50The value is obtained.
Further, fluorogenic substrates include MCA-Dnp-Lys, Cbz-RLRGG-AMC, and Ub-AMC.
Further, the suppression rate is calculated as ir (inhibition rate) ((1-v))i/v0) X 100% where v0Initial velocity of enzymatic reaction without addition of inhibitor, viThe initial rate of the enzymatic reaction to which the inhibitor is added.
Further, when the fluorogenic substrate is MCA-Dnp-Lys and the concentration is 20 mu mol/L, the inhibiting concentration IC of the tegaserod50The concentration was 9.26. mu. mol/L.
Further, when the fluorogenic substrate is Cbz-RLRGG-AMC and the concentration is 2.4. mu. mol/L, the inhibitory concentration IC of the tegaserod50It was 1.42. mu. mol/L.
Further, when the fluorogenic substrate is Ub-AMC and the concentration is 2.4 mu mol/L, the inhibitory concentration IC of the tegaserod50The concentration was 14.01. mu. mol/L.
Further, the tegaserod has no toxic effect on cells.
Further, the cells are A549 cells, caco-2 cells, HepG2 cells, HEK293 cells and vero cells.
Furthermore, the tegaserod can be used alone or combined with other medicines, compounds and carriers to form a pharmaceutical composition.
Further, the pharmaceutical composition is an oral preparation, an injection, a liniment, a lotion, an aerosol, an oil preparation or a transdermal patch.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention obtains the tegaserod by virtual screening, which has good function of inhibiting the activity of the PLpro protein of the novel coronavirus, the inhibition rate is up to 100 percent under the concentration of 50 mu mol/L, and the inhibition concentration of the tegaserod for inhibiting the PLpro protein is IC50Less than 15 mu mol/L, which indicates that the tegaserod can be a potential medicament for preventing or treating the novel coronavirus infection.
2. Tegaserod has no toxic effect on various cells, can be independently used as a raw material medicine, or can be matched with other known medicines, compounds or carriers to prepare medicaments or medicinal compositions in various forms such as oral agents, injections, paints, lotions, aerosols, oil preparations or transdermal patches and the like so as to prevent or treat the novel coronavirus infection, and is safe and reliable in use without generating toxic and side effects on cells and organisms. The invention provides new compounds and new ideas for treating novel coronavirus infections.
Drawings
Fig. 1 shows a virtual screening process of tegaserod.
FIG. 2 is a PLpro binding pattern analysis of tegaserod with SARS-CoV-2.
FIG. 3 is a diagram showing the purification results of the PLpro protein of the novel coronavirus.
FIG. 4 is a graph of the Mie constant of the novel coronavirus PLpro when the fluorogenic substrate is MCA-Dnp-Lys.
FIG. 5 is a Mie's constant curve for the novel coronavirus PLpro when the fluorogenic substrate is Cbz-RLRGG-AMC.
FIG. 6 is a graph of the Mie constant of the novel coronavirus PLpro when the fluorogenic substrate is Ub-AMC.
FIG. 7 is a graph showing the inhibition of the novel coronavirus PLpro by tegaserod when the fluorogenic substrate is 20. mu. mol/L MCA-Dnp-Lys.
FIG. 8 is a graph showing the inhibition of the novel coronavirus PLpro by tegaserod when the fluorogenic substrate is 2.4. mu. mol/L Cbz-RLRGG-AMC.
FIG. 9 is a graph showing the inhibition of the novel coronavirus PLpro by tegaserod when the fluorogenic substrate is 2.4. mu. mol/L Ub-AMC.
Figure 10 is a graph of the effect of tegaserod at various concentrations on the growth activity of a549 cells.
FIG. 11 is a graph of the effect of varying concentrations of tegaserod on caco-2 cell growth activity.
FIG. 12 is a graph of the effect of varying concentrations of tegaserod on the growth activity of HepG2 cells.
FIG. 13 is a graph of the effect of varying concentrations of tegaserod on the growth activity of HEK293 cells.
FIG. 14 is a graph of the effect of tegaserod at various concentrations on vero cell growth activity.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1: virtual screening
The Papain-like protease (PLpro) of SARS-CoV-2 has an extremely high similarity to the amino acid sequence of the PLpro of SARS-CoV, and therefore the PLpro structural MODEL of SARS-CoV-2 (MODEL01) can be obtained by SWISS-MODEL (https:// swissnodel. expasy. org /) homology modeling method using the crystal structure of the PLpro of SARS-CoV (PDBID: 3E 9S). Model01 was subjected to hydrogenation, charging, and protonation state prediction by using lepro, centered around the ligand (TTT) in template structure 3E9SThe range of (1) is a lattice point interval of molecular docking, the lattice point interval contains the whole active pocket, and the length of each lattice point is set asLigand hydrogenation, three-dimensional structure optimization and protonation state prediction are carried out on the ligand through ligaprep, and finally 9175 compound conformations (sdf format) are generated. Performing molecular docking by using ledock _ go, separating a first binding conformation of a docking result, calculating Ligand Efficiency (LE) according to a Score (Score), and selecting 656 compound conformations with LE being less than-0.3; the conformation of these compounds and the binding free energy (. DELTA.G, Kcal/mol) of the receptor protein were then calculated using prime-mmgbsa. Finally, using lewater to calculate Hydrogen Bond Penalty (HBP), and recalculating the calculation result and free energy of binding of receptor-ligand complex, using final AG < -50Kcal/mol as selection standard to obtain 61 compounds as candidate active molecules, and the screening process is shown in (FIG. 1). Finally, tegaserod is found to have high inhibitory activity to PLpro of SARS-CoV-2. The molecular docking model (fig. 2) shows that the indole ring forms a stable hydrogen bond network with the nitrogen atoms on the chain structure and the residues within the PLpro active pocket; as the carbon atom of the enamine structure is simultaneously connected with three nitrogen atoms, the enamine structure carries partial positive charges and can be used as a positive charge center to respectively form a salt bridge and a pi-cation interaction with ASP164 and TYR264, and the enamine structure also forms a water bridge with the main chain amino group of VAL 165; aromatic rings and aliphatic chains form hydrophobic interactions with ASP164, TYR264, GLN269, and these favorable intermolecular interactions play a key role in increasing the binding affinity of compounds and proteins.
The chemical name of Tegaserod (Tegaserod) is: n' - [ (E) - [ (5-methoxy-1H-indol-3-yl) methylidene]amino]-N-pentylguanidine of formula C16H23N5O, molecular weight 301, and the formula:
the tegaserod is an indole selective 5-HT4 receptor partial agonist, stimulates gastrointestinal peristalsis reflex and intestinal secretion by activating gastrointestinal tract 5-HT4 receptors, inhibits high sensitivity of viscera, and has the functions of regulating gastrointestinal tract autonomous function, regulating gastrointestinal dysfunction caused by various factors and restoring the gastrointestinal function to normal. No research report about antiviral activity of tegaserod is found at present.
Example 2: determination of the Mie constant of the novel coronavirus PLpro
1. Expression purification of novel coronavirus PLpro protein
The gene of the novel coronavirus PLpro protein is optimized according to the G/C content in the original coding sequence and the application of a codon suitable for an escherichia coli prokaryotic expression system, the optimized coding nucleotide sequence is shown as SEQ ID NO.1, and the translated amino acid sequence is shown as SEQ ID NO. 2.
PET-28 was selected as an expression vector, and the correctly sequenced pET was obtainedTransformation of-28 recombinant plasmid into E.coli BL21(DE3), selection of monoclonal bacteria for amplification culture, shake culture at 37 ℃ to OD6001.0-1.2, adding IPTG (final concentration of 0.1mmol/L), and inducing overnight expression at 20 ℃; the bacterial liquid is centrifuged at 4500rpm and 4 ℃ for 30min, and the supernatant is discarded to collect the thallus. The cells were resuspended in lysis buffer (20mM HEPES, 0.5M NaCl, pH 7.4, plus 200. mu.g/ml lysozyme and 0.05% Triton X-100) into 50ml centrifuge tubes, incubated on a shaker at 4 ℃ for 30min and then sonicated on ice, and the supernatant was centrifuged at 12000rpm at 4 ℃ and collected for use. Cleaning a nickel column by using double distilled water with 5 times of column volume, balancing by using a balancing solution with 10 times of column volume, then adding a thallus supernatant, incubating for 1h by using a shaking table at 4 ℃, discarding an effluent, adding a washing solution to fully wash impurity proteins, adding an eluent to elute and collecting target proteins; and finally, pouring the target protein into a Millipore Amicon Ultra-15 ultrafiltration tube, centrifuging, concentrating and desalting to obtain the purified recombinant PLpro protein, detecting the protein purity by adopting a Coomassie brilliant blue method, and displaying that the purity of the PLpro protein obtained by the invention is higher by using a purification result (figure 3).
2. Determination of the PLpro Mie constant
The enzyme activity of the novel coronavirus PLpro was measured under the conditions of a total enzyme concentration of 500nmol/L, a substrate concentration of 0.195. mu. mol/L, 0.39. mu. mol/L, 0.78. mu. mol/L, 1.57. mu. mol/L, 3.13. mu. mol/L, 6.25. mu. mol/L, 12.5. mu. mol/L, 25. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L, 150. mu. mol/L, 200. mu. mol/L. The buffer system for the determination of the activity of the novel coronavirus PLpro is: 20mmol/L phosphate buffer (pH 6.8). The experimental group was incubated at 25 ℃ with 500nmol/L of PLpro, rapidly added with fluorogenic substrate 1, and the fluorescence reading was recorded every 1.5min for a total of 30 min. The control group was set to add the same volume of buffer and the remaining experimental conditions were kept the same as the experimental group. Wherein the fluorogenic substrate 1 is MCA-AKIALKGGKIVN-Lys (MCA-Dnp-Lys). The instrument for measuring the fluorescence intensity is a multifunctional microplate reader SpectraMax iD5, and the wavelengths of excitation light and emission light are 320nm and 425nm respectively; the initial rate of enzymatic reaction was calculated using a four parameter analysis, and the results are shown in FIG. 4, for the fluorogenic substrate 1, the Michaelis constant K of PLpromThe value was 105.7. mu. mol/L, the maximum reaction rate VmaxThe ratio was 0.000701. mu. mol/(L · s).
The enzyme activity of the novel coronavirus PLpro was measured under the conditions of a total enzyme concentration of 100nmol/L, a substrate concentration of 0.125. mu. mol/L, 0.25. mu. mol/L, 0.5. mu. mol/L, 1. mu. mol/L, 2. mu. mol/L, 4. mu. mol/L, 8. mu. mol/L, 16. mu. mol/L, 20. mu. mol/L, 24. mu. mol/L, 28. mu. mol/L, 32. mu. mol/L, 40. mu. mol/L. The buffer system for the determination of the activity of the novel coronavirus PLpro is: 20mmol/L phosphate buffer (pH 6.8). The experimental group was incubated at 25 ℃ with 100nmol/L of PLpro, and either fluorogenic substrate 2 or fluorogenic substrate 3 was added rapidly, and the fluorescence readings were recorded every 1.5min for a total of 30 min. The control group was set to add the same volume of buffer and the remaining experimental conditions were kept the same as the experimental group. Wherein the fluorogenic substrate 2 is Cbz-RLRGG-AMC and the fluorogenic substrate 3 is Ub-AMC. The instrument for measuring the fluorescence intensity is a multifunctional microplate reader SpectraMax iD5, and the wavelengths of the excitation light and the emission light are 360nm and 460nm respectively; the initial rate of enzymatic reaction was calculated using a four parameter analysis, and the results are shown in FIGS. 5 and 6 for the fluorescent substrate 2, the Michael constant K of PLpromThe value was 32.39. mu. mol/L, maximum reaction rate Vmax0.00126. mu. mol/(L.s); mie's constant K for the fluorogenic substrate 3, PLpromThe value was 7.32. mu. mol/L, maximum reaction rate VmaxThe purity and the enzyme activity of the PLpro protein obtained by the extraction of the invention are both better as shown by the result of 0.519 nmol/(L.s).
Example 3: inhibitor tegaserod activity screen
The initial rates of the enzymatic reactions of the different compounds on the novel coronavirus PLpro were determined at a concentration of 50. mu. mol/L. The buffer system for the determination of the activity of the novel coronavirus PLpro is: 20mmol/L phosphate buffer (pH 6.8). The total PLpro concentration of the experimental group was 200nmol/L, lysates of different compounds were added at an incubation temperature of 25 deg.C, incubated with shaking at room temperature for 15min, 20. mu. mol/L fluorogenic substrate 1 was added rapidly, fluorescence readings were recorded every 1.5min for a total of 30 min. The control group was added with the same volume of buffer and the rest of the experimental conditions were kept the same as the experimental group. The instrument for measuring the fluorescence intensity is a multifunctional microplate reader SpectraMax iD5, and the wavelengths of excitation light and emission light are 320nm and 425nm respectively.
According to enzymatic reactionsRate the remaining activity ra (residual activity) of each compound was calculatedi/v0) And an inhibition rate ir (inhibition rate) ((1-v))i/v0) X 100% where v0The initial rate of the enzymatic reaction without inhibitor addition; v. ofiThe initial rate of the enzymatic reaction to which the inhibitor is added. The experimental results are shown in table 1, and in various compounds, tegaserod has high inhibitory activity, and the inhibitory rate can reach 100% at the concentration of 50 mu mol/L, which indicates that tegaserod can well inhibit the activity of PLpro protein.
Table 1: inhibition of PLpro of novel coronaviruses by different compounds at a concentration of 50. mu.M
Example 4: half inhibitory concentration IC of inhibitor tegaserod50Measurement of (2)
Tegaserod was formulated with DMSO at stepped concentrations.
For fluorogenic substrate 1, the initial rate of enzymatic reaction of the novel coronavirus PLpro was determined at different concentrations of tegaserod treatment. The buffer system for the determination of the activity of the novel coronavirus PLpro is: 20mmol/L phosphate buffer (pH 6.8). The total concentration of PLpro in the experimental group was 200nmol/L, 1.56. mu. mol/L, 3.13. mu. mol/L, 6.25. mu. mol/L, 12.5. mu. mol/L, 25. mu. mol/L, 37.5. mu. mol/L, 50. mu. mol/L, 75. mu. mol/L concentration of tegaserod was added at an incubation temperature of 25 ℃, shaking incubation at room temperature for 15min, rapid addition of 20. mu. mol/L fluorogenic substrate 1, IC of the compound was performed50Detecting; fluorescence readings were recorded every 1.5min for a total of 30 min. The control group was added with the same volume of buffer and the rest of the experimental conditions were kept the same as the experimental group. The instrument for measuring the fluorescence intensity is a multifunctional microplate reader SpectraMax iD5, and the wavelengths of excitation light and emission light are 320nm and 425nm respectively. In terms of tegaserod concentrationThe logarithmic value of (A) is the abscissa and the corresponding value of the inhibition ratio is the ordinate, a curve is made, and the IC of tegaserod is calculated by a four-parameter method50The value is obtained. The results are shown in FIG. 7, the tegaserod has good inhibitory activity, and the inhibitory concentration IC of the tegaserod50The concentration was 9.26. mu. mol/L.
For fluorogenic substrate 2 and fluorogenic substrate 3, the initial rates of enzymatic reactions of the novel coronavirus PLpro were determined at different concentrations of tegaserod treatment. The buffer system for the determination of the activity of the novel coronavirus PLpro is: 20mmol/L phosphate buffer (pH 6.8). The total concentration of PLpro in the experimental group was 40nmol/L, 0.78. mu. mol/L, 1.57. mu. mol/L, 3.13. mu. mol/L, 6.25. mu. mol/L, 12.5. mu. mol/L, 25. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L tegaserod was added at an incubation temperature of 25 ℃, incubation was performed with shaking at room temperature for 15min, 2.4. mu. mol/L fluorogenic substrate 2 or fluorogenic substrate 3 was added rapidly, and IC of the compound was performed50Detecting; fluorescence readings were recorded every 1.5min for a total of 30 min. The control group was added with the same volume of buffer and the rest of the experimental conditions were kept the same as the experimental group. The instrument for measuring the fluorescence intensity is a multifunctional microplate reader SpectraMax iD5, and the wavelengths of the excitation light and the emission light are 360nm and 460nm respectively. Taking the logarithmic value of the concentration of the tegaserod as an abscissa and the corresponding value of the inhibition rate as an ordinate, making a curve, and calculating the IC of the tegaserod by a four-parameter method50The value is obtained. The results are shown in FIGS. 8-9, and the inhibitory concentration IC of tegaserod is very good when the substrate is 2.4 mu mol/L fluorogenic substrate 2501.42 mu mol/L; inhibitory concentration IC of tegaserod when the substrate is 2.4. mu. mol/L fluorogenic substrate 350The concentration was 14.01. mu. mol/L.
Example 5: inhibitor tegaserod cytotoxicity assay
The cytotoxic effect of tegaserod on various cells was determined. The Vero cell, HepG2 cell, A549 cell, caco-2 cell and HEK-293 cell are selected. Taking cells in logarithmic growth phase, adjusting the cell concentration, inoculating the cells into a 96-well plate at 4000-5000/well, placing the 96-well plate at 37 ℃ and 5% CO2Culturing in an incubator for 24 h. The experimental groups were added with different amounts per wellTegaserod at a concentration (20. mu. mol/L, 10. mu. mol/L, 5. mu. mol/L, 2.5. mu. mol/L, 1.25. mu. mol/L, 0.625. mu. mol/L, 0.313. mu. mol/L, 0.157. mu. mol/L, 0.078. mu. mol/L, 0.04. mu. mol/L, 0.02. mu. mol/L, 0.01. mu. mol/L), a control group to which an equifold diluted solvent was added, 4 to 5 duplicate wells per group, 5% CO at 37 ℃2Culturing in an incubator with saturated humidity for 48h, adding 10 μ l of resazurin solution (1mg/ml) into each well, incubating at 37 deg.C for 4h, and measuring fluorescence intensity with a multifunctional microplate reader SpectraMax iD5, wherein the wavelengths of excitation light and emission light are 549nm and 595nm, respectively. The inhibition rate of tegaserod on the growth of various cells is calculated according to the following formula:
the results are shown in FIGS. 10-14, where tegaserod at a concentration of 20. mu. mol/L had no cytotoxic effect on a variety of cells.
In conclusion, PLpro is used as a protein which is related to RNA replication, translation and proteolytic processing in the novel coronavirus and is important in the life cycle of the novel coronavirus, the invention obtains tegaserod by virtual screening and screening, verifies that tegaserod has strong inhibitory activity on the PLpro of the novel coronavirus, and has IC (integrated circuit) activity on three different fluorescent substrates509.26 mu mol/L, 1.42 mu mol/L and 14.01 mu mol/L respectively show that tegaserod can block the life cycle of the novel coronavirus by inhibiting the activity of PLpro, and further becomes a potential medicament or compound for preventing or treating the novel coronavirus infection. The tegaserod can be used as a raw material medicine alone, or can be matched with other known medicines, compounds or carriers to prepare various forms of medicines such as oral preparations, injections, paints, lotions, aerosols, oil preparations or transdermal patches and the like so as to prevent or treat the novel coronavirus infection.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> China oceanic university
National laboratory development center for Qingdao ocean science and technology pilot
Qingdao Marine biological medicine institute, Inc
Application of <120> tegaserod in preparing medicine for preventing or treating coronavirus infection
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gttgatatga gcatgaccta tggtcagcag tttggcccga cctatctgga tggtgcagat 120
gtgaccaaaa ttaagccgca taatagccat gaaggtaaaa ccttttatgt tctgccgaat 180
gatgataccc tgcgtgttga agcatttgaa tattatcata ccaccgatcc gagttttctg 240
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ctgaccagta ttaagtgggc agataataat tgttacctgg caaccgccct gctgaccctg 360
caacagattg aactgaaatt caatccgccg gcactgcaag atgcctatta tcgtgcacgc 420
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ggcgtggaag ccgtgatgta tatgggcacc ctgagttatg aacagtttaa aaaaggtgtg 660
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Glu Val Arg Thr Ile Lys Val Phe Thr Thr Val Asp Asn Ile Asn Leu
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His Thr Gln Val Val Asp Met Ser Met Thr Tyr Gly Gln Gln Phe Gly
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Pro Thr Tyr Leu Asp Gly Ala Asp Val Thr Lys Ile Lys Pro His Asn
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Ser His Glu Gly Lys Thr Phe Tyr Val Leu Pro Asn Asp Asp Thr Leu
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Arg Val Glu Ala Phe Glu Tyr Tyr His Thr Thr Asp Pro Ser Phe Leu
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Gly Arg Tyr Met Ser Ala Leu Asn His Thr Lys Lys Trp Lys Tyr Pro
85 90 95
Gln Val Asn Gly Leu Thr Ser Ile Lys Trp Ala Asp Asn Asn Cys Tyr
100 105 110
Leu Ala Thr Ala Leu Leu Thr Leu Gln Gln Ile Glu Leu Lys Phe Asn
115 120 125
Pro Pro Ala Leu Gln Asp Ala Tyr Tyr Arg Ala Arg Ala Gly Glu Ala
130 135 140
Ala Asn Phe Cys Ala Leu Ile Leu Ala Tyr Cys Asn Lys Thr Val Gly
145 150 155 160
Glu Leu Gly Asp Val Arg Glu Thr Met Ser Tyr Leu Phe Gln His Ala
165 170 175
Asn Leu Asp Ser Cys Lys Arg Val Leu Asn Val Val Cys Lys Thr Cys
180 185 190
Gly Gln Gln Gln Thr Thr Leu Lys Gly Val Glu Ala Val Met Tyr Met
195 200 205
Gly Thr Leu Ser Tyr Glu Gln Phe Lys Lys Gly Val Gln Ile Pro Cys
210 215 220
Thr Cys Gly Lys Gln Ala Thr Lys Tyr Leu Val Gln Gln Glu Ser Pro
225 230 235 240
Phe Val Met Met Ser Ala Pro Pro Ala Gln Tyr Glu Leu Lys His Gly
245 250 255
Thr Phe Thr Cys Ala Ser Glu Tyr Thr Gly Asn Tyr Gln Cys Gly His
260 265 270
Tyr Lys His Ile Thr Ser Lys Glu Thr Leu Tyr Cys Ile Asp Gly Ala
275 280 285
Leu Leu Thr Lys Ser Ser Glu Tyr Lys Gly Pro Ile Thr Asp Val Phe
290 295 300
Tyr Lys Glu Asn Ser Tyr Thr Thr Thr Ile
305 310