阅读说明：本技术 跨膜丝氨酸蛋白酶2抑制剂在制备治疗和/或预防冠状病毒感染药物中的用途 (Use of transmembrane serine protease 2 inhibitors for the treatment and/or prophylaxis of coronavirus infections ) 是由 余伯阳 刘秀峰 梅杰 于 2021-10-14 设计创作，主要内容包括：本发明公开了跨膜丝氨酸蛋白酶2抑制剂在制备治疗和/或预防冠状病毒感染药物中的用途。本发明通过亲和垂钓及活性导向分离获得3种化合物,证实该类化合物可以直接地与跨膜丝氨酸蛋白酶2结合,K-(D)<13μM,且能够显著抑制跨膜丝氨酸蛋白酶2的催化活性。在细胞水平上可以有效的抑制新型冠状病毒SARS-CoV-2假病毒入侵,表明该类化合物对于制备治疗和/或预防病毒感染药物具有非常积极的作用。化合物1 化合物2 化合物3。(The invention discloses application of a transmembrane serine protease 2 inhibitor in preparing a medicament for treating and/or preventing coronavirus infection. The invention obtains 3 compounds through affinity fishing and activity guiding separation, proves that the compounds can be directly combined with transmembrane serine proteinase 2, K D <13 mu M, and can obviously inhibit the catalytic activity of transmembrane serine protease 2. The compound can effectively inhibit the invasion of a novel coronavirus SARS-CoV-2 pseudovirus at a cellular level, and shows that the compound has very positive effect on preparing medicaments for treating and/or preventing virus infection. Compound 1 Compound 2 Compound3。)

1. Use of a transmembrane serine protease 2 inhibitor for the manufacture of a medicament for the treatment and/or prophylaxis of coronavirus infection, the compound having the formula:

compound 1 or

Compound 2 or

Compound 3.

2. Use according to claim 1, characterized in that: the compounds can inhibit transmembrane serine protease 2 catalytic hydrolytic activity.

3. Use according to claim 1, characterized in that: the median inhibitory concentrations of the compound 1, the compound 2 and the compound 3 on transmembrane serine protease 2 were 2.13 mM, 1.65 mM and 0.39 mM, respectively.

4. Use according to claim 1, characterized in that: the compounds are capable of inhibiting viral entry into target cells.

5. Use according to claim 1, characterized in that: the compounds are capable of specifically binding to TMPRSS 2.

6. Use according to claim 1, characterized in that: the dosage form of the medicine is oral administration dosage form or non-oral administration dosage form.

7. Use according to claim 6, characterized in that: the oral administration dosage form is tablet, powder, granule, capsule, emulsion or syrup.

8. Use according to claim 6, characterized in that: the non-oral administration dosage form is injection.

Technical Field

The invention relates to a pharmaceutical application, in particular to an application of a transmembrane serine protease 2 inhibitor in preparing a medicine for treating and/or preventing coronavirus infection.

Background

The emergence of drug resistant viruses and new viruses has highlighted the need for new antiviral approaches and drugs. However, since viruses are easy to mutate, the development of drugs aiming at the viral self-target is difficult. Therefore, targeting host factors becomes a relatively new antiviral drug development strategy, which can reduce or avoid the emergence of escape mutants.

Transmembrane serine protease 2 (TMPRSS 2) is one of the members of the type ii serine protease (TTSPs) family. In recent years, numerous studies have shown that TMPRSS2 participates in the proteolysis of the viral envelope, becoming one of the common key steps for the entry of multiple types of viruses into cells. When coronavirus invades cells, TMPRSS2 cleaves coronavirus Spike protein (Spike, S) to activate it, thereby promoting fusion of virus and cell membrane. TMPRSS2 is an important link for mediating the effect of coronavirus invasion cells such as severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prior research shows that TMPRSS2 can also bind to protein complex formed by SARS-CoV or S protein of SARS-CoV-2 and angiotensin converting enzyme 2 (ACE 2), thereby cutting and activating S protein of virus and assisting the virus to enter cells. Therefore, development of related inhibitors against the virus invasion common target TMPRSS2, inhibiting its activity, is currently considered to be a key therapeutic strategy for viral infection.

The traditional Chinese medicine has the traditional Chinese medicine theory and a large amount of clinical practice as supports, has wide sources and rich resources, and is one of the important sources for the research and development of new medicines. Aiming at the common target TMPRSS2 of virus invading cells, the screening of high-efficiency inhibitors in clinically effective Chinese medicine formulas has important value and provides a certain foundation for developing or preparing medicines with strong medicine property and good safety.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide application of a compound capable of inhibiting transmembrane serine protease 2 in preparing a medicament for treating and/or preventing coronavirus infection.

The technical scheme is as follows: the invention provides application of a transmembrane serine protease 2 inhibitor in preparing a medicament for treating and/or preventing coronavirus infection, wherein the structure of the compound is as follows:

compound 1 or

Compound 2 or

Compound 3.

Further, the half inhibitory concentrations of compound 1, compound 2, and compound 3 against TMPRSS2 were 2.13 mM, 1.65 mM, and 0.39 mM, respectively.

Further, the compounds were able to specifically bind to TMPRSS 2. The compounds can inhibit the catalytic hydrolytic activity of transmembrane serine protease 2. The compounds are capable of inhibiting viral entry into target cells.

The inhibitor is used for preparing a medicament for treating and/or preventing virus infection.

Further, the drug dosage form is an oral administration dosage form or a non-oral administration dosage form.

Further, the oral administration dosage form is tablet, powder, granule, capsule, emulsion or syrup.

Further, the non-oral administration dosage form is injection.

The preferred preparation method of the compound capable of inhibiting transmembrane serine protease 2 comprises the following steps:

(1) taking a proper amount of scutellaria baicalensis decoction pieces, crushing, sieving, and ultrasonically extracting by using ethanol. And after the ultrasonic treatment is finished, performing suction filtration, collecting filtrate, volatilizing, and dissolving in PBS buffer solution to prepare suspension. Centrifuging the suspension to remove impurities, and collecting the supernatant for later use;

(2) mixing the scutellaria baicalensis sample solution with a pichia pastoris solution with the surface displaying TMPRSS2, incubating at room temperature, centrifuging, removing a supernatant, washing a precipitate with a PBS buffer solution, adding a methanol solution, performing ultrasonic treatment, centrifuging, and collecting the supernatant as a sample to be detected;

(3) mixing a scutellaria baicalensis sample solution with an equivalent wild pichia pastoris solution, incubating at room temperature, centrifuging, removing a supernatant, washing a precipitate with a PBS buffer solution, adding a methanol solution, carrying out ultrasonic treatment, centrifuging, and collecting the supernatant as a blank sample;

(4) and (3) respectively detecting the sample liquid to be detected and the blank sample liquid obtained in the steps (2) and (3) by using a high performance liquid chromatography-mass spectrometry combined technology, and analyzing a compound, namely the TMPRSS2 affinity component in the scutellaria baicalensis, of which the concentration in the sample liquid is obviously higher than that in the blank filtrate.

Since TMPRSS2 is involved in the proteolysis of the viral envelope, it is one of the common key steps for the entry of multiple types of viruses into cells. Thus, targeting TMPRSS2 is a key therapeutic strategy for viral infections. The inventor finds that 3 compounds in the scutellaria baicalensis can inhibit the catalytic hydrolysis activity of TMPRSS2 on a molecular level. Can effectively inhibit the invasion of a novel coronavirus SARS-CoV-2 pseudovirus at a cellular level, and the compound can be directly combined with TMPRSS2, and KD is less than 13 mu M. The compounds have very positive effects on preparing medicaments for treating and/or preventing virus infection.

The invention comprises a pharmaceutical composition, which contains a compound shown in a structural formula or pharmaceutically acceptable salt thereof, or a corresponding isomer, a non-corresponding isomer or a racemate thereof as an active ingredient, and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient refers to any diluent, adjuvant or carrier that can be used in the pharmaceutical field. The compounds of the invention may be used in combination with other active ingredients, provided that they do not produce other adverse effects.

Has the advantages that: the compound can be combined with TMPRSS2, effectively inhibit the catalytic hydrolysis activity of the TMPRSS2, prevent viruses from entering cells, and can be used for preparing medicaments for treating and/or preventing virus infection.

Drawings

FIG. 1 is an HPLC chromatogram of an extract of Scutellariae radix;

FIG. 2 is HPLC chromatogram of affinity adsorption component (solid line) and nonspecific adsorption component (dotted line) with TMPRSS2 in Scutellariae radix medicinal material;

FIG. 3 shows the results of the dose-dependent inhibition of TMPRSS2 catalytic hydrolysis activity and the half inhibitory concentration assay of Compound 1;

FIG. 4 shows the results of the dose-dependent inhibition of TMPRSS2 catalytic hydrolysis activity and the half inhibitory concentration assay of Compound 2;

FIG. 5 shows the results of the dose-dependent inhibition of TMPRSS2 catalytic hydrolysis activity and the half inhibitory concentration assay of Compound 3;

FIG. 6 is a graph of the affinity assay of Compound 1 binding to TMPRSS 2;

FIG. 7 is a graph of the affinity assay of Compound 2 binding to TMPRSS 2;

FIG. 8 is a graph of the affinity assay of Compound 3 binding to TMPRSS 2;

FIG. 9 shows the results of toxicity assay of Compound 1 on 293T (293T-ACE 2) cells overexpressing the ACE2 receptor;

FIG. 10 shows the toxicity assay of compound 2 against 293T-ACE2 cells;

FIG. 11 shows the results of toxicity assay of compound 3 against 293T-ACE2 cells;

FIG. 12 shows the result of the measurement of compound 1 dose-dependently inhibiting the invasion of SARS-CoV-2 novel coronavirus pseudovirus into 293T-ACE2 cells;

FIG. 13 shows the result of the measurement of compound 2 dose-dependently inhibiting the invasion of SARS-CoV-2 novel coronavirus pseudovirus into 293T-ACE2 cells;

FIG. 14 shows the result of the measurement of compound 3 dose-dependently inhibiting the invasion of SARS-CoV-2 novel coronavirus pseudovirus into 293T-ACE2 cells.

Detailed Description

Example 1

This example method for screening TMPRSS2 inhibitor in Scutellaria baicalensis

The experimental method comprises the following steps: yeast surface display system (YSD) is a eukaryotic display system for immobilizing and expressing exogenous protein, after fusing target protein gene with specific carrier protein gene, it is introduced into yeast host cell, the target protein is expressed and positioned on the yeast cell surface under the guide of carrier protein, this technique not only maintains the relatively independent space conformation of protein, but also maintains the original biological activity of host cell. The inventor successfully constructs and cultures the pichia pastoris with TMPRSS2 displayed on the surface, and lays a foundation for subsequent fishing experiments and enzyme activity determination experiments.

(1) Taking a proper amount of scutellaria baicalensis decoction pieces, crushing, sieving by a fourth sieve, weighing 2 g of powder, adding 200 mL of 70% ethanol, and carrying out ultrasonic extraction for 30 min. After the completion of sonication, filtration was carried out by suction, and the filtrate was collected, evaporated to dryness, and dissolved in 200 mL of PBS buffer solution to prepare a suspension of 10 mg/mL. Centrifuging the suspension at 11000 Xg for 10 min, removing impurities, and collecting supernatant for use. HPLC chromatogram of Scutellariae radix extractive solution is shown in figure 1.

(2) 100 μ L of the Scutellariae-like solution was mixed with 100 μ L of a Pichia solution with TMPRSS2 surface-displayed, incubated at room temperature and centrifuged at 3000 Xg for 5 min. Washing the precipitate with PBS buffer solution for 3 times, adding 200 μ L methanol, ultrasonic treating for 30 min, centrifuging at 11000 × g for 10 min, and collecting supernatant as sample to be tested.

(3) 100 μ L of the Scutellariae-like solution was mixed with 100 μ L of the wild-type Pichia solution, incubated at room temperature and centrifuged at 3000 Xg for 5 min. Washing the precipitate with PBS buffer solution for 3 times, adding 200 μ L methanol, ultrasonic treating for 30 min, centrifuging at 11000 × g for 10 min, and collecting supernatant as sample to be tested.

(4) And (3) respectively detecting the sample liquid to be detected and the blank sample liquid obtained in the steps (2) and (3) by using a high performance liquid chromatography-mass spectrometry combined technology, and analyzing a compound, namely the TMPRSS2 affinity component in the scutellaria baicalensis, of which the concentration in the sample liquid is obviously higher than that in the blank filtrate.

Wherein the high performance liquid phase separation conditions of the above steps (1) (4) comprise: the specification of the chromatographic column is Agilent C18, 2.7 mu m, 3.7 mm multiplied by 250 mm; the mobile phase is 0.1% formic acid water solution (A) and acetonitrile (B); the detection wavelength was 254 nm.

TABLE 1 HPLC elution gradient

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0-20	90-70	10-30
20-45	70-30	30-70
			45-52	30-5	70-95
52-61	5-90	95-10
			61-70	90	10

The experimental results are as follows: HPLC chromatogram of TMPRSS-affinity adsorption component (solid line) and nonspecific adsorption component (dotted line) in Scutellariae radix medicinal material is shown in FIG. 2. The peak 1, the peak 2 and the peak 3 are substances with the concentration in the sample liquid obviously higher than that in the blank filtrate, namely substances with specific interaction with transmembrane serine protease. A total of 3 chemical components were identified by HPLC-Q-TOF-MS/MS detection, comparison of controls and fragment analysis combined with mass spectrometry spectra. The results are shown in Table 2.

TABLE 2 Mass Spectrometry identification of structures

Peak number	Chemical formula (II)	MS	MS/MS	Authentication
					1	C21H18O11	445.0747	269.0444, 175.0266	Baicalin
2	C22H20O11	459.0932	283.0616, 268.0348, 175.0208	Wogonoside
					3	C15H10O5	269.0444	225.0527, 195.0430, 183.0119,151.0527, 117.0359	Baicalein

Example 2

The compounds of the present invention inhibit TMPRSS2 catalytic hydrolytic activity.

The experimental method comprises the following steps: 200. mu.L of a Pichia pastoris solution with TMPRSS2 on the surface was taken, and 2. mu.L of compounds 1 to 3 at different concentrations were added, respectively. The mixture was incubated in a rotary incubator at room temperature for 1 hour. Boc-Leu-Gly-Arg-AMC (1 mM) substrate (2. mu.L) was added in the dark, and the mixture was placed in a rotary incubator and incubated in the dark at room temperature for 1 hour. Centrifuging at 10000 Xg for 5 min, sucking 100. mu.L of supernatant into a new centrifuge tube in the dark, and adding 100. mu.L of acetonitrile to terminate the reaction. Centrifuging at 10000 Xg for 10 min, and taking supernatant as a sample to be detected. The content of AMC, a product after the enzymatic reaction, was analyzed under the high performance liquid separation conditions described in Table 1. Raw data were analyzed using GraphPad Prism 6.0 software and relative inhibition was calculated. The measurement values are expressed as mean values of three independent experiments. + -. standard error of the mean value.

The experimental results are as follows: as shown in FIGS. 3-5, each of compounds 1-3 inhibited TMPRSS2 catalytic hydrolysis activity dose-dependently at half-inhibitory concentrations of 2.13 mM, 1.65 mM, and 0.39 mM, respectively. The compound disclosed by the invention can effectively inhibit the catalytic hydrolysis activity of TMPRSS 2.

Example 3

The compounds of the present invention bind directly to TMPRSS 2.

The experimental method comprises the following steps: surface Plasmon Resonance (SPR) experiments. The SPR sensing technology utilizes the principle of surface plasmon resonance, and when light is coupled to a sensing chip, a portion of the light energy is absorbed due to the occurrence of surface plasmon resonance, so that the light reflected from the sensing chip forms an SPR resonance signal. The SPR resonance signals are very sensitive to the change of the refractive index of the sample substance on the surface of the sensing chip, so that the substance information of the sample can be obtained by analyzing the SPR resonance images. Has extremely important application in the biosensing field of detecting protein-protein interaction and the like.

The experiment was tested using a BIAcore T200 instrument at 25 ℃ with the mobile phase being phosphate buffer (pH 7.4). The TMPRSS2 recombinant protein was diluted with 10 mmol/L sodium acetate buffer (pH 5.5, 5.0, 4.5 and 4.0, respectively) to a final concentration of 20. mu.g/ml, and pH screening was performed by manual injection, and the pH with the highest coupling value was selected for subsequent experiments. TMPRSS2 recombinant protein was coupled to CM5 chip by amino coupling, and TMPRSS2 recombinant protein was diluted to a final concentration of 20. mu.g/ml with 10 mmol/L sodium acetate buffer of optimum pH value obtained by the above screening, using phosphate buffer as working buffer. The chip surface was activated with a mixture of 0.2 mol/L EDC and 50 mmol/L NHS at a ratio of 1:1, injected at a flow rate of 10. mu.l/min, injected with TMPRSS2 recombinant protein solution after 7 min of continuous injection, then injected with 1 mol/L ethanolamine hydrochloric acid (pH 8.5) blocking solution for 7 min, the activated chip surface was blocked, and the blocked blank channel was used as a negative control for the assay. Experiments the compound of the invention was selected as ligand at a maximum concentration of 25 μ M and the ligand was injected as a two-fold serial dilution onto a chip of a biosensor to which the TMPRSS2 recombinant protein was immobilized. 1:1 binding model for the assessment of binding kinetics. KD values were calculated by BIAcore T200 analysis software using a kinetic model.

The experimental results are as follows: as shown in FIGS. 6, 7 and 8, the affinity of compound 1 to TMPRSS2 recombinant protein was 1.70. mu.M, the affinity of compound 2 to TMPRSS2 recombinant protein was 13.00. mu.M, and the affinity of compound 3 to TMPRSS2 recombinant protein was 0.61. mu.M. Kinetic analysis data show that although compound 3 has a slightly stronger affinity to TMPRSS2 recombinant protein than compound 1 or 2, but is an order of magnitude, the result suggests that the compound skeleton of the present invention is the key structure for its function, and the change of the substituent has no obvious influence on the activity of the compound. The compounds of the formulae I/II may all have this activity. The compounds of the present invention interact with TMPRSS 2.

Example 4

The compound inhibits SARS-CoV-2 novel coronavirus S protein pseudovirus from invading 293T cells (293T-ACE 2) over expressing ACE2 receptor.

The experimental method comprises the following steps: SARS-CoV-2 new type coronavirus S protein pseudovirus contains luciferase reporter gene, and after virus invades into cell, the activity of luciferase can be evaluated by luciferase detection reagent. The viability of which is reflected in the number of cells invaded by the virus. The combination of the novel coronavirus S protein and ACE2 receptor is a key step of virus invasion cells, so 293T cells (293T-ACE 2) which over-express ACE2 receptor are selected to simulate virus invasion, and the function of inhibiting TMPRSS2 hydrolysis S protein can obviously reduce the number of virus invasion target cells.

First, the MTT method was used to determine whether each compound had cytotoxicity or not, depending on the effect on the viability of the cells. 293T-ACE2 cells were plated into well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ for 24 hours, and after incubation of each well with 5% CO2, each compound was added and incubated for 24 hours. 293T-ACE2 cells were then placed in 100. mu.l 10% FBS-DMEM containing 500. mu.g/ml MTT. After 4 hours of incubation at 37 ℃, 150 μ l of dimethyl sulfoxide was added to each well to dissolve the crystals. The absorbance values were measured at 570 nm using a microplate reader. Then, virus invasion assay was performed by uniformly spreading 293T-ACE2 cells in a well plate, culturing the cells in DMEM medium containing 10% fetal bovine serum at 37 ℃ under 5% CO2, and starting the assay when the cells grow to 30%. The same concentration of SARS-CoV-2 novel coronavirus S protein pseudovirus was mixed with different concentrations of test compound, incubated at 37 ℃ for 6h, and then added to each well. After 6h incubation, the medium was replaced with fresh medium. After normal culture for 48h, luciferase assay reagent equal in volume to the culture medium was added to each well and shaken for 10 min to completely lyse the cells. Then, a chemiluminescence signal is detected by using a microplate reader, the expression quantity of luciferase is quantitatively detected, and the quantity of 293T-ACE2 cells invaded by the virus is analyzed. Raw data were analyzed using GraphPad Prism 6.0 software. Analysis between groups was performed using one-way analysis of variance,p<0.05 was considered statistically significant. The measurement values are expressed as mean values of three independent experiments. + -. standard error of the mean value.

The experimental results are as follows: the compounds 1-3 can prevent the novel coronavirus S protein pseudovirus from entering 293T-ACE2 cells. As shown in figures 9, 10 and 11, compounds 1 to 3 have no influence on the activity of 293T-ACE2 cells within a certain concentration range, which indicates that the compounds are good in safety. FIGS. 12, 13, 14 show that all of compounds 1-3 can prevent the novel coronavirus S protein pseudovirus from entering 293T-ACE2 cells with dose-dependent inhibition. These results all indicate that the compounds of the present invention inhibit TMPRSS2 and inhibit the invasion of the SARS-CoV-2 novel coronavirus S protein pseudovirus into 293T cells overexpressing ACE2 receptor.