CN112237584A

CN112237584A - Novel use of compound for preventing and/or treating diseases caused by coronavirus infection

Info

Publication number: CN112237584A
Application number: CN202010851779.3A
Authority: CN
Inventors: 山广志; 朱志玲; 仇小丹; 李玉环; 吴硕; 左利民; 赵婷; 刘伊彤
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2021-01-19
Anticipated expiration: 2040-08-21
Also published as: CN112237584B

Abstract

The invention discovers that the noreulaldehyde and cediranib can effectively block the combination of RBD and ACE2 protein, so that the noreulaldehyde and cediranib can be used for preventing and/or treating diseases caused by coronavirus infection.

Description

Novel use of compound for preventing and/or treating diseases caused by coronavirus infection

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a small molecule inhibitor targeting SARS-CoV-2RBD and/or human ACE2 protein.

Background

2019 New coronavirus disease (COVID-19) caused by SARS-CoV-2 infection^[1,2]. Infection with SARS-CoV-2 can cause symptoms such as fever, dry cough, and dyspnea. Unlike SARS, COVID-19 may be converted into a chronic disease similar to influenza and coexist with humans for a long period of time. To date, the number of diagnosed COVID-19 cases worldwide has exceeded 1630 ten thousand, and is on a continuing rising trend^[3]。

Scientists have now obtained detailed biological information about SARS-CoV-2^[4-7]. The S protein is mainly combined with membrane receptor angiotensin converting enzyme 2(ACE2) on host cells to promote viruses to enter the host cells^[8,9]. Among them, the Receptor Binding Domain (RBD) of the S protein is directly involved in recognition of host receptors. Amino acid variations in this region will lead to changes in infection characteristics and the like^[10,11]。

Vaccines are one of the most promising therapeutic approaches. The development of antiviral drugs is another effective strategy against COVID-19. However, no drug with definite clinical efficacy has been found at present. Screening for SARS-CoV-2 inhibitors has now been fully developed and has obtained a variety of promising inhibitors^[12-16]。

Entry into the host cell is the first step in viral infection, and thus prevention of viral entry into the host cell is an effective strategy for antiviral drug development^[17-18]. Recently published virtual screening studies targeting RBD and ACE2 proteins showed that natural products such as glycyrrhizic acid, emodin and hesperidin inhibit the binding of SARS-CoV-2 to ACE2^[19]. RBD and ACE2 proteins achieve protein-protein interactions (PPI) through the interface between them, lacking a clear cavity structure and active centers. This presents a challenge to traditional screening methods.

The Surface Plasmon Resonance (SPR) technique was of the last centuryUniversal biophysical detection techniques developed in the 90 s for the analysis of biomolecular interactions^[20,21]. It does not need marker protein and can monitor molecular interaction in real time. SPR, not only characterizes protein-protein interactions with strong affinity, but also characterizes weak interactions between fragments or low molecular weight compounds and proteins. The SPR technology is utilized for screening, and the information of the kinetics and the affinity of the action of the compound and the protein can be obtained simultaneously. It is furthermore a powerful tool for the discovery of PPI inhibitors^[22]. In contrast to traditional enzyme activity-based inhibitor screening approaches, SPR techniques do not rely on any enzymatic reaction processes.

Disclosure of Invention

The invention takes the RBD protein and ACE2 protein of SARS-CoV-2 as targets, and screens a database containing 960 compounds by using SPR technology. Through establishing a screening model, clearing a library, combining horizontal screening and an affinity determination experiment, 7 Ks are obtained_DThe value is less than 100 μ M for compounds acting on ACE2 protein, 6 for compounds acting on RBD protein and having KD value less than 100 μ M. Wherein, two compounds 02B05 (demethyleularwood aldehyde, CAS number 107316-88-1) and 03D12 (Xidinebu, CAS number 288383-20-0) which can effectively block the interaction of RBD and ACE2 are obtained through SPR competition experiments, and the chemical structures are respectively as follows:

according to one aspect of the invention, the invention relates to the use of noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the same, or a hydrate of the same for the preparation of a medicament for the prevention and/or treatment of a disease caused by a coronavirus infection. Preferably, the coronavirus is SARS-CoV-2. More preferably, the disease is a novel coronavirus disease and/or a complication thereof.

According to another aspect, the present invention relates to a method for preventing and/or treating a disease caused by coronavirus infection, which is characterized in that a therapeutically effective amount of noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the same or a hydrate of the same or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof. Preferably, the coronavirus is SARS-CoV-2. More preferably, the disease is a novel coronavirus disease and/or a complication thereof.

According to another aspect of the invention, the invention relates to a method of destroying coronaviruses in an environment, characterized in that noreulaldehyde or cediranib, or a salt thereof, or a solvate of the same, or a hydrate of the same, is administered. Preferably, the coronavirus is SARS-CoV-2.

According to another aspect of the invention, the invention relates to the use of noreulaldehyde or cediranib, or a salt thereof, or a solvate of the same, or a hydrate of the same, as a SARS-CoV-2RBD inhibitor.

According to another aspect, the present invention relates to the use of noreulaldehyde or cediranib, or a salt thereof, or a solvate of the same, or a hydrate of the same, as an ACE2 inhibitor.

As will be appreciated by those skilled in the art, salts or pharmaceutically acceptable salts of eulaldehyde are formed, for example, with inorganic or organic bases. Inorganic bases include, but are not limited to, alkali metals such as sodium, potassium, and the like, alkaline earth metals such as calcium, magnesium, and the like, organic bases include, but are not limited to, organic amines such as ethanolamine, trimethylamine, tert-butylamine, pyridine, picoline, diethanolamine, triethanolamine, tromethamine, and the like, basic amino acids such as arginine, lysine, ornithine, and the like. Salts or pharmaceutically acceptable salts of cediranib for example with inorganic or organic acids. Examples of inorganic acids include, but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, nitric acid, and the like, and organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, valeric acid, diethylacetic acid, trifluoroacetic acid, maleic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, and the like.

According to another aspect, the invention relates to a pharmaceutical composition comprising noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the same, or a hydrate of the same. Preferably, further comprising a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is used for preventing and/or treating diseases caused by coronavirus infection. More preferably, the coronavirus is SARS-CoV-2. More preferably, the disease is a novel coronavirus disease and/or a complication thereof.

It will be appreciated by those skilled in the art that the pharmaceutical composition of the present invention can be prepared into various conventional forms in the art, such as intravenous, oral, inhalation, ophthalmic, rectal administration, and the like, including but not limited to tablets, capsules, pills, granules, syrups, emulsions, suspensions, aerosols, injections, suppositories, eye drops, and the like.

It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention may contain a variety of pharmaceutically acceptable carriers commonly used in the art.

It will be appreciated by those skilled in the art that the therapeutically effective amount may be determined based on body weight, mode of administration, individual response to the drug, type of formulation, time or interval of administration.

According to another aspect of the invention, the invention relates to a disinfectant for killing coronavirus in the environment, which comprises noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the noreulaldehyde or cediranib, or a hydrate of the noreulaldehyde or cediranib.

The invention discovers that the noreulaldehyde or cediranib can be combined with RBD and ACE2, and can effectively block the interaction of the RBD and ACE2, so that diseases caused by coronavirus infection can be prevented and/or treated.

Drawings

FIG. 1 is a surface plasmon resonance sensorgram of the interaction of RBD-mFc with captured ACE2-His in example 1.

Figure 2 is a sensorgram of immobilized ACE2 in example 3 in combination with horizontal screening of the resulting miaquillage compound. Compound data are shown as solid lines and blank data are shown as dashed lines.

FIG. 3 is a sensorgram for immobilized RBD of the resulting shoot head compound in combination with horizontal screening in example 3. Compound data are shown as solid lines and blank data are shown as dashed lines.

FIG. 4 is a sensorgram of binding of the resulting leptin compounds to ACE2 protein in example 3 with binding level screening.

FIG. 5 is a sensorgram for binding of the resulting leptin compounds to RBD proteins in binding level screening of example 3.

FIG. 6 is an ITC assay of 02B05 binding to RBD-His in example 4.

FIG. 7 is a graph of competitive inhibition of ACE2-RBD by 02B05 in example 5. In the figure, the concentrations of the compounds from top to bottom were 0. mu.M, 31.25. mu.M, 62.5. mu.M, and 125. mu.M, respectively.

FIG. 8 is a graph of competitive inhibition of ACE2-RBD by 03D12 in example 5. In the figure, the concentrations of the compounds from top to bottom were 0. mu.M, 31.25. mu.M, 62.5. mu.M, and 125. mu.M, respectively.

FIG. 9 is a superposition of sensorgrams for binding of the RBD protein (6.25, 12.5 and 25nM, first line from top to bottom), 125 μ M02B 05 and RBD protein (6.25, 12.5 and 25nM, second line from top to bottom), 125 μ M02B 05 (third line from top to bottom) and running buffer (fourth line from top to bottom) to ACE2 protein in example 5.

FIG. 10 is a graph of the cytotoxicity of compound 02B05 in example 6 and its effect on transduction by pseudotyped VSV virus or SARS-CoV-2 virus in 293T-hACE2 cells. In the figure, P < 0.05; p <0.01, left and right Y-axis represent the average percentage of Luc activity and cell viability, respectively.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.

A compound library consisting of 960 preclinical compounds is provided by Targetmol (usa). The RBD protein (mFc tag, cat no:40592-V05H) of SARS-CoV-2S protein and human ACE2 protein (His tag, cat no:10108-H08H) were purchased from Sino Biological (China), and SARS-CoV-2RBD protein with His tag was provided by the institute of medicine of the Chinese academy of medicine. HBS-N buffer, amino coupling kit, NTA kit, acetate buffer, CM5 chip, and NTA chip were purchased from Cytiva (formerly Swedish GE healthcare Co.). Dimethyl sulfoxide (DMSO), Tween 20 and sodium hydroxide were purchased from Sigma-Aldrich (Germany).

A293T derived cell line (293T-hACE2) expressing human ACE2 was purchased from Midagukang Gene technology, Inc. at 5% CO₂The cells were maintained in an incubator at 37 ℃ in DMEM (Invitrogen, USA) supplemented with 10% FBS (Gibbs, USA) and antibiotics (100U/ml penicillin and 100mg/ml streptomycin). PrestoBlue cell viability reagent was purchased from Invitrogen, usa. Vesicular Stomatitis Virus (VSV) and SARS-CoV-2 pseudotype virus particles were purchased from Zhongji Dangkang Gen technology, Inc. (China). Cell lysis buffer and luciferase substrate were purchased from Promega (usa).

Example 1 protein-protein interaction assay

The protein-protein interaction of ACE2 and RBD proteins was determined using a Biacore S200 instrument using NTA chips. The running buffer was HBS-T + buffer (10mM HEPES,150mM NaCl, 0.05% tween 20, pH 8.0,0.22 μm microfiltration). The ligand was ACE2-His, concentration 10mg/mL, and capture time 100 seconds. A series of S-RBD-mFc solutions were prepared at concentrations of 3.13, 6.25, 12.5, 25, 50 and 100nM, flowed over the captured ACE2-His surface, and the Response Units (RUs) obtained were recorded. The flow rate was set at 30. mu.L/min, the binding time 120s, and the dissociation time 500 s. 350mM EDTA as regeneration solution, regeneration time 60 seconds.

Experimental results showed that the amount of ACE2 captured was relatively stable at different cycles (response was approximately 143RU, with no more than 2RU deviation). In addition, sensorgrams obtained by repeatedly feeding RBD protein with the same concentration almost coincide, which shows that the capture method has better reproducibility and robustness. Using this robust interaction method, ACE2-RBD interaction was measured (fig. 1). The measured maximum RBD response signal was 94.2 RU. The above results show that the activity of both proteins in the running buffer remained good. Data analysis was done using Biacore S200 evaluation software version 1.0, with an affinity constant of 0.37nM obtained by software analysis after 1:1 data fitting.

Example 2 establishment of amino coupling SPR screening model

RBD and ACE2 proteins were immobilized on CM5 chips by amino-coupling. The chip was first activated by contacting it with a mixture of EDC and NHS. Then RBD and ACE2-His proteins at concentrations of 20. mu.g/mL and 16. mu.g/mL were dissolved in sodium acetate solution (pH 5.0 and pH 4.5, respectively) and flowed over the chip surface and finally immobilized on Fc2 and Fc4, respectively, of a CM5 chip. Finally, the chip is sealed by ethanolamine.

The coupling amount of RBD protein is about 8500RUs, and the coupling amount of ACE2 protein is about 9100 RUs. When about 20nM of ACE2 or RBD was flowed through a chip with RBD or ACE2 immobilized, the response signals were 242RUs and 319RUs, respectively. The above results show that both target proteins retain activity after immobilization.

EXAMPLE 3 active Compound screening

The whole process of compound screening is divided into three steps: clearing, combining horizontal screening and determining affinity. The experiments were performed using a Biacore T200 or Biacore S200 instrument at a flow rate of 30 μ L/min, and the data were analyzed using Biacore T200/S200 evaluation software. HBS-T + buffer (10mM HEPES,150mM NaCl, 0.05

% tween

20, 5% DMSO, pH 8.0,0.22 μm microfiltration) was used as the running buffer. Compounds were first diluted to 100 μ M and finally to 25 μ M or 10 μ M with running buffer of 5% DMSO.

Based on the difference in solubility, compounds at a concentration of 25. mu.M or 10. mu.M were flowed over the chip surface for inventory. By purging, a total of 13 high viscosity compounds were excluded. The compounds that pass the library are then screened for binding levels in order to identify compounds that bind to the target protein.

For binding level screening, compound screening concentration was 10 μ M and binding and dissociation time was 60 seconds. The running buffer was a negative control during the screening process. Solvent corrected samples were prepared according to the GE Healthcare Laboratory guidelines (8 spots). The final screen yielded 15 compounds that bound to RBD and 15 compounds that bound to ACE 2. The sensing diagrams are shown in fig. 2 and 3. After automated analysis by software, 8 ACE 2-binding compounds (01N 12, 02B05, 02C19, 02D16, 03D12, 03E22, 03I19, and 03P04, respectively) and 5 RBD-binding compounds (02B05, 02C15, 02J06, 03D12, and 03I14, respectively) were labeled as slow-dissociating compounds among the obtained miaquillak compounds screened for binding levels.

Then, the binding level-screened shoot head compound was subjected to affinity assay at a concentration ranging from 0.3125 to 20. mu.M. The binding time was 60 seconds and the dissociation time was 120 seconds. Kinetic and steady-state affinity data were obtained by a 1:1 binding model. The results showed that 5 compounds (02B05, 02C15, 02J06, 02M09, 03D12) showed high binding affinity to RBD, the K of which_DThe value was less than 100 mM. Also, these 5 compounds bind to ACE 2. It was further found that their affinity for RBD or ACE2 was essentially the same, within an order of magnitude of difference. Furthermore, compounds 02C19 and 02F14 only showed strong binding to ACE2, indicating that these compounds are highly specific for both proteins. The affinity values are summarized in table 1 below.

TABLE 1 results of affinity determination of different compounds with ACE2 and RBD

a kinetic analysis data auto-fitting to a 1:1 binding model

b Steady State affinity analysis data auto-fitting to a 1:1 binding model

Finally, kinetic constants of the above compound and ACE2 and RBD were measured using a kinetic fitting model. Compounds were injected onto the chip surface at concentrations of 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 μ M. The binding time was 60 seconds and the dissociation time was 120 seconds. The sensing diagrams are shown in fig. 4 and 5. Overall, the dissociation rate constant for compound 02B05 was the smallest, indicating that it had the longest residence time. In general, a longer residence time is closely related to the magnitude and duration of the drug effect. The research result shows that the compound 02B05 is expected to be a potential RBD-ACE2 binding inhibitor.

Example 4 use iTC for correlation assays

S-RBD-His was placed in iTC200 (British Marvin) sample cells at a concentration of 15. mu.M. Compound 02B05 was diluted from a 2mM stock solution (dissolved in DMSO) into 100 μ M (solvent as running buffer). The experiment was carried out at 25 ℃ with a reference power of 8. 0.2. mu.L was injected initially, followed by 15X 2. mu.L. The heat of dilution of the compound was measured by injecting the same concentration of compound into the buffer. The data fit a 1:1 binding isotherm.

As shown in FIG. 6, the affinity measured by iTC was 2.09. mu.M, which is close to the affinity measured by SPR in example 3. This result indicates that compound 02B05 can bind to S-RBD-His in buffer solution, further demonstrating the reliability and accuracy of the SPR screening method.

Example 5 SPR-based Competition assays

The inhibitory effect of the miaow compounds on RBD protein and ACE2 protein was determined by NTA chip, and the running buffer was identical to the affinity screening assay buffer. The ACE2-His capture concentration was 5. mu.g/mL, and the capture time was kept constant. The assay solution was a 20nM RBD solution dissolved in running buffer and containing 0-125. mu.M (0,31.25,62.5, or 125. mu.M) of compound. The assay solution was injected through ACE2-His with a binding time of 120s and a dissociation time of 500 s. Prior to analysis, RBD were incubated with compound for one hour at room temperature. In a single injection analysis, the analyte was changed to a 125 μ M solution of the compound containing 0 to 25nM RBD.

Compound 02B05 showed the strongest blocking effect, followed by 03D 12. The relationship between response signal and compound concentration is shown in FIGS. 7 and 8. 02B05 and 03D12 significantly reduced the response in a dose-dependent manner, whereas competitive inhibition by the other compounds (125. mu.M) was not evident. To determine the results, we compared the response of buffer solution, compound solution, RBD solution and compound mixture solution flowing across the ACE2 protein surface alone. As can be seen from fig. 9, significant signal changes were observed when RBD solutions (6.25, 12.5 and 25 μ M) were injected alone. However, when 125 μ M compound 02B05 and RBD (6.25, 12.5, and 25nM) were mixed and injected, the signal response decreased, and the sensorgram was similar to that obtained by analyzing the compounds alone. This indicates that once compound 02B05 binds to the RBD, binding of the RBD to ACE2 is blocked. The data indicate that 02B05 is effective in blocking ACE2-RBD interaction.

Example 6 entry of pseudotyped Lentiviral particles into human cells and luciferase assay

To investigate whether Compound 02B05 inhibited viral entry into host cells, 293T-hACE2 cells were infected with a luciferase reporter virus, a pseudovirus expressing SARS-CoV-2 spike envelope protein or VSV G protein, in the presence of Compound 02B 05.

First, 02B05 was tested for cytotoxicity in 293T-hACE2 cells using PrestoBlue cell viability reagent. Cells were seeded into 96-well plates and treated with varying concentrations of 02B 05. After 24 hours incubation at 37 ℃, 10 μ L of PrestoBlue (10 ×) was added to each well and mixed well with the medium. After a further incubation at 37 ℃ for 1 hour, the fluorescence signal was read using an excitation wavelength of 560nm and an emission wavelength of 590nm (Perkin Elmer, Waltham, MA, USA). The results showed that the maximum non-toxic concentration of compound 02B05 in 293T-hACE2 cells was less than 1. mu.M. As shown in FIG. 10, at a concentration of 1.1. mu.M of 02B05, approximately 12% of the cells died.

293T-hACE2 in white wall and clear bottom 96-well plates were inoculated with infection with either pseudotyped VSV virus or SARS-CoV-2 virus in the absence or presence of Compound 02B 05. Ammonium chloride was selected as a positive control. At 24 hours post-infection, cells were lysed with 20. mu.L/well of cell lysis buffer for 15 minutes, and then 50. mu.L/well of luciferase substrate was added. Firefly luciferase activity was measured by luminescence assay in a PerkinElmer ensspire instrument. Luciferase activity was used to characterize the entry efficiency of the virus. The results are shown in FIG. 10, and compound 02B05 moderately reduced infection by VSV pseudovirus, but significantly inhibited infection by SARS-CoV-2 pseudovirus. In addition, 0.37. mu.M of Compound 02B05 inhibited approximately 7% of the transduction by the SARS-CoV-2 pseudovirus, but had no effect on the transduction by the VSV pseudovirus. These results reflect that Compound 02B05 shows inhibitory activity against the entry of SARS-CoV-2 pseudovirus at very low concentrations.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. application of noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the noreulaldehyde or cediranib or a hydrate of the noreulaldehyde or cediranib in preparation of a medicament for preventing and/or treating diseases caused by coronavirus infection. Preferably, the coronavirus is SARS-CoV-2. More preferably, the disease is a novel coronavirus disease and/or a complication thereof.

2. A method for preventing and/or treating a disease caused by a coronavirus infection, comprising administering to a subject in need thereof a therapeutically effective amount of noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the same or a hydrate of the same or a pharmaceutically acceptable salt thereof. Preferably, the coronavirus is SARS-CoV-2. More preferably, the disease is a novel coronavirus disease and/or a complication thereof.

3. A method of destroying coronaviruses in an environment comprising administering noreulaldehyde or cediranib, or a salt thereof, or a solvate of the same, or a hydrate of the same, or a salt thereof. Preferably, the coronavirus is SARS-CoV-2.

4. Use of noreulaldehyde or cediranib, or a salt thereof, or a solvate of the same, or a hydrate of the same, as a SARS-CoV-2RBD inhibitor.

5. Use of noreulaldehyde or cediranib, or a salt thereof, or a solvate of the same, or a hydrate of the same, as an ACE2 inhibitor.

6. A pharmaceutical composition comprising noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the noreulaldehyde or cediranib, or a hydrate of the noreulaldehyde or cediranib. Preferably, further comprising a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is used for preventing and/or treating diseases caused by coronavirus infection. More preferably, the coronavirus is SARS-CoV-2. More preferably, the disease is a novel coronavirus disease and/or a complication thereof.

7. A disinfectant for killing coronavirus in the environment, which is characterized by comprising noreulaldehyde or cediranib, or a pharmaceutically acceptable salt thereof, or a solvate of the noreulaldehyde or cediranib, or a hydrate of the noreulaldehyde or cediranib.