Use of an inhibitor of nucleoprotein of the tartar virus 1

Abstract

The application belongs to the technical field of biological medicine, and provides application of an anti-Tachyirus 1 nucleoprotein inhibitor, and proposes application of a compound candesartan as an anti-Tachyirus 1 nucleoprotein inhibitor. The compound candesartan is obtained in the following manner: through expression and purification of Tachyirus 1 nucleoprotein by using an E. coli system, and through screening of natural medicines and FDA medicine libraries by using a molecular thermal shift assay (TSA) technology, an effective screening system is established, and the compound candesartan capable of interacting with the nucleoprotein is discovered; further, a biological membrane interference technology (BLI) and other experiments are used to determine the affinity, analyze the affinity of the compound candesartan and the nucleoprotein, and simultaneously verify and analyze the function characteristics of the nucleoprotein, so as to verify that the compound candesartan can effectively inhibit the combination of the nucleoprotein and nucleic acid, and can inhibit the nucleic acid enzymolysis ability of the nucleoprotein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of an anti-Tacheng virus 1 nucleoprotein inhibitor. Background Technology

[0002] The Nairoviridae family contains a variety of viruses, some of which are distributed globally. For example, the Crimean-Congo hemorrhagic fever virus (CCHFV) is a medically important virus in this family, with widespread prevalence in Asia, Africa, the Middle East, and Southeast Europe. Additionally, Tamdy virus (TAMV), a tick-borne virus belonging to the genus *Ortho-Nerovirus* within the Nairoviridae family, has been detected in the Xinjiang Uygur Autonomous Region of my country. It is primarily transmitted by the Asian water tick (*Hyalomma asiaticum*) and the steppe tick (*Dermacentornuttalli*). Tacheng tick virus 1, discovered in Xinjiang Uygur Autonomous Region of my country in 2019, is one of the Tamdy virus genus viruses capable of causing human disease. It belongs to the genus *Ortho-Nerovirus* within the Nairoviridae family. Clinical symptoms primarily include fever and muscle aches; severe cases can lead to meningitis and other neurological disorders.

[0003] However, there is currently no effective specific treatment for this type of virus, making the development of effective antiviral drugs of great significance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an application of an anti-Tacheng virus 1 nucleoprotein inhibitor to resolve the issues raised in the background art.

[0005] This invention proposes the use of the compound candesartan ester as an inhibitor of Tacheng virus 1 nucleoprotein.

[0006] Preferably, the compound candesartan medoxomil is used as an anti-Tacheng virus 1 nucleoprotein inhibitor in the preparation of pharmaceutical compositions for the treatment or prevention of diseases caused by Tacheng virus 1 infection.

[0007] Preferably, the pharmaceutical composition comprises candesartan cilexetil as the active ingredient.

[0008] Preferably, the compound candesartan medoxomil is obtained through the following steps:

[0009] S1. Expression and purification of Tacheng virus 1 nucleoprotein using E. coli system;

[0010] S2. A thermal drift experiment based on protein stability was performed using a real-time PCR instrument, and candesartan ester, a compound that significantly changed the Tm value of Tacheng virus 1 nucleoprotein, was selected as a candidate inhibitor through screening of natural drugs and FDA drug databases.

[0011] S3. The affinity of the compound candesartan cilexetil to nucleoprotein was determined by biolayer interferometry.

[0012] S4. The inhibitory effect of the compound candesartan cilexetil on nucleoprotein was verified by gel retardation assay.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. This invention uses TSA technology to discover that the candidate compound CC can change the Tm value of nucleoprotein, and the change value is greater than 3 degrees Celsius, indicating that the compound candesartan cilexetil interacts with nucleoprotein. Furthermore, the compound candesartan cilexetil has a high affinity for nucleoprotein, with an affinity Kd of 7.8 μM.

[0015] 2. This invention utilizes gel migration imaging to discover that the compound candesartan medoxomil can effectively inhibit the binding ability of nucleoproteins to nucleic acids, and at the same time, it can also effectively inhibit the nuclease digestion ability. Attached Figure Description

[0016] Figure 1 This is a gel filtration chromatography diagram of the purification of Tacheng virus 1 nucleoprotein according to the present invention;

[0017] Figure 2 This invention presents the results of compound screening using TSA technology based on protein stability.

[0018] Figure 3 The TSA results of candesartan cilexetil and nucleoprotein in this invention;

[0019] Figure 4 This invention is based on the determination of the affinity constant between nucleoprotein and candesartan cilexetil using BLI.

[0020] Figure 5 This invention utilizes EMSA to analyze the inhibitory effects of candesartan cilexetil on the binding ability of nucleoprotein nucleic acids and its ability to digest nucleases. Detailed Implementation

[0021] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0022] This invention proposes the use of the compound candesartan ester as an inhibitor of Tacheng virus 1 nucleoprotein.

[0023] The compound candesartan medoxomil, as an anti-Tacheng virus 1 nucleoprotein inhibitor, is used in the preparation of pharmaceutical compositions for the treatment or prevention of diseases caused by Tacheng virus 1 infection.

[0024] The pharmaceutical composition contains candesartan cilexetil as the active ingredient.

[0025] The compound candesartan medoxomil is obtained through the following steps:

[0026] S1. Expression and purification of Tacheng virus 1 nucleoprotein using E. coli system;

[0027] S2. A thermal drift experiment based on protein stability was performed using a real-time PCR instrument, and candesartan ester, a compound that significantly changed the Tm value of Tacheng virus 1 nucleoprotein, was selected as a candidate inhibitor through screening of natural drugs and FDA drug databases.

[0028] S3. The affinity of the compound candesartan cilexetil to nucleoprotein was determined by biolayer interferometry.

[0029] S4. The inhibitory effect of the compound candesartan cilexetil on nucleoprotein was verified by gel retardation assay.

[0030] Example 1: Expression and purification of Tacheng virus 1 nucleoprotein using an E. coli system

[0031] The expression, extraction, and crude purification of nuclear proteins are used for protein preparation. The specific operational steps are as follows:

[0032] The target gene sequence of TcTV-1NP protein was obtained from the NCBI website. This sequence was optimized using codons preferred by *E. coli*. The optimized sequence was then synthesized by a company. The optimized nucleotide sequence of the nucleoprotein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The target gene was then constructed into the pET-28a vector, which was constructed in this experiment and contains a polyhistidine and SUMO fusion tag, using a seamless cloning method. The plasmid extraction process was used for the construction of the expression vector; specific operating procedures were performed according to the plasmid extraction kit operating instructions.

[0033] SEQ ID NO.1 is as follows:

[0034]

[0035] SEQ ID NO.2 is as follows:

[0036] MAPLPKSLLTFSDASGLDSWFKDFEAKNIMSEEYTNSKSFFDLRMATQWKKLPTRAENDAMIAQLVHERLKTCAPIKEFAWTACDGMVERGLNWFDRNKDSETMTWAANYEALKGRLPTTTAEVNQYQKAALQWRTDTNYAINKYTAAISDSVVKIYQVNNKIVTDIRDLLSDM VARRNKALGIKPGEERVPAEHVDSFSNWLKQGDWSAPCPWGDWEKKNKKGNSLIVTACAGVINRALFKEEELKERLKSLAGDASLASKTEGFDPKKCEDTAKILLDLYGKAKAFISGGDGSSQSGGFVQQGSALDTVFSSYFWAWKCGVKKDVFPALSSMLYALGKNPTGKTKI IKVLKASPYTWAHKMTEMFSTLSTDPIHMHPGVLTAGRLTEMVASFGAFPVSDPSKAADGASSPRFLLNLKSSDMNPAATTVSRMFYEYRQGYPDWRDEEIVPVEHLLHQTFLSKLGPYVNVSQVQGNALAVKITEYIVTK.

[0037] The fusion plasmid pET-21a-10×His-SUMO-TcTV-1NP was transformed into competent cells of Escherichia coli expression strain for transformation. After incubation on ice for 30 minutes, heat shock at 42°C for 90 seconds, and incubation on ice for 5 minutes, 600 μl LB liquid was added for recovery for 1 hour.

[0038] The revived bacterial culture was spread onto agarose plates containing 50 μg / ml kanamycin and incubated overnight at 37°C. Transformed positive colonies were picked and added to 50 ml of fresh LB broth (with kanamycin added to inhibit the growth of other bacteria) and incubated overnight at 37°C.

[0039] Single clones of each bacterial strain were picked and placed in LB medium, and kanamycin antibiotic (final concentration 50 μg / ml) was added. The culture was carried out in a shaker at 220 rpm and 37°C for 3.5 hours. When the concentration OD value of the bacterial culture was about 0.8, IPTG was added as an inducer for induction culture.

[0040] Protein extraction: After the culture was completed, the bacterial cells were collected by centrifugation and resuspended in lysis buffer (5mM imidazole, 20mM Tris-HCl pH=7.5, 300mM NaCl). PMSF was added to a final concentration of 2mM and the cells were disrupted by a combination of low temperature high pressure and ultrasonic disruption. The cells were centrifuged at 25000g for 35 minutes at 4℃. The supernatant was collected and the precipitate was discarded.

[0041] The supernatant was initially purified by Ni-NTA affinity chromatography: the centrifuged supernatant was incubated with equilibrated Ni-NTA packing material at 4°C for 1 hour. The resulting mixture was then passed through a gravity chromatography column and eluted with buffers of different imidazole concentrations (5 mM, 20 mM, 40 mM, 250 mM, 500 mM), and the corresponding eluates were collected. Each eluate was then mixed with protein loading buffer and denatured at 100°C for 10 minutes. The purification efficiency of the target protein was then assessed by SDS-PAGE.

[0042] To obtain high-purity target protein, a secondary purification process—ion exchange chromatography—was performed using the AKTA purification system. The specific steps of ion exchange chromatography are as follows: The ion exchange column was installed according to the AKTA purification system operating standards and cleaned with ultrapure water; the pre-packed ion exchange column was cleaned and equilibrated using high-salt and low-salt buffers to remove contaminating proteins; a suitable concentration device was selected based on the sample volume and the molecular weight of the target protein to replace and equilibrate the protein buffer; once the baselines were stable, the protein sample was loaded onto the pre-packed ion exchange column, and gradient elution was performed using high-salt buffer. The protein elution peaks were collected and identified by SDS-PAGE gel electrophoresis.

[0043] like Figure 1 As shown, to further ensure the purity of the protein, gel filtration chromatography was performed. The specific steps of gel filtration chromatography are as follows: the target protein peak purified by ion exchange chromatography was collected, concentrated, and the protein buffer was replaced with gel filtration chromatography buffer. The final concentration volume depends on the chromatography column. The gel filtration column was equilibrated with gel filtration chromatography buffer. After baseline equilibration, the concentrated protein was loaded into the chromatography column, and the target protein was eluted at a flow rate of 0.5 ml / min. The protein elution peak was collected.

[0044] Example 2: Figure 2-3 As shown, drug screening is performed based on nuclear proteins using thermal drift assays.

[0045] A system was established using a real-time PCR instrument to test the interaction between different compounds and nucleoproteins, with SYRO-Orange as the fluorescent dye. Each test could be performed in 96 or 384 wells to increase the number of test samples.

[0046] The interaction between compounds and nucleoproteins was characterized by the degree of change in the Tm value of the nucleoprotein, with a Tm change value greater than 3°C used as the criterion for selection. Compound 2, candesartan cilexetil, induced a Tm change value of 3.8°C in the nucleoprotein.

[0047] Example 3: Determination of the affinity between nucleoproteins and compounds using BLI technology

[0048] Sample preparation: Prepare nucleoprotein samples, ensuring a purity greater than 95% and a concentration greater than 5 mg / mL; prepare the compound to be tested at a concentration of 20 mM / L, and perform serial dilutions according to experimental requirements.

[0049] Baseline equilibration: The Ni-NTA biosensor was placed in a blank buffer solution for 60 seconds to eliminate nonspecific adsorption and background signal on the sensor surface.

[0050] Nucleoprotein-probe coupling: A sample solution containing nucleoproteins is added to the biosensor, allowing the biomolecules to bind to the probe on the sensor surface. Binding time, temperature, and other conditions need to be optimized based on the experimental objectives and sample characteristics; the final determined binding time is 300 seconds.

[0051] Washing unbound nucleoproteins: Rinse the biosensor with buffer to remove unbound nucleoproteins to reduce interference from nonspecific binding. Washing time is 120 seconds.

[0052] Compound-nucleoprotein binding: A sample solution containing the analyte compound was added to a black 96-well deep plate, and a biosensor was placed inside to observe the binding process between the two biomolecules. The binding time was 500 seconds, and the temperature was 25°C.

[0053] Dissociation of the compound and nucleoprotein: The NTA sensor was rinsed with blank buffer to remove bound biomolecules, and the dissociation process between the two biomolecules was observed. The dissociation time was 500 seconds, and the temperature was 25°C.

[0054] like Figure 4 As shown, data on biomolecular interactions, including binding curves, binding rates, dissociation rates, and affinity, were acquired in real time using a BLI instrument. The acquired data were analyzed using specialized data analysis software. During the analysis, attention must be paid to data quality and reliability, eliminating interference from non-specific binding and noise. The results showed that the affinity (Kd) between the compound candesartan cilexetil and the nucleoprotein was 7.8 μM.

[0055] Example 4: Analysis of the inhibitory effect of the compound candesartan cilexetil on the function of nucleoproteins

[0056] EMSA (gel retardation assay) was performed at 4°C using 0.5x TBE buffer and a 2.5% agarose gel. When a specific protein binds to a labeled nucleic acid probe, the resulting protein-nucleic acid complex has a larger molecular weight and migrates more slowly in the gel, thus distinguishing it from unbound nucleic acid probes. Figure 5 As shown, EMSA was used to analyze the inhibitory effects of candesartan cilexetil on nucleoprotein nucleic acid binding and nuclease digestion.

[0057] It is important to note that the constructions and arrangements of this application shown in several different exemplary embodiments are merely illustrative. Although only a few embodiments are described in detail in this disclosure, those who consult this disclosure will readily understand that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in this application. Other substitutions, modifications, changes, and omissions may be made in the design, operation, and arrangement of the exemplary embodiments without departing from the scope of the invention. Therefore, the invention is not limited to the particular embodiments but extends to various modifications that still fall within the scope of the appended claims.

[0058] Furthermore, in order to provide a concise description of exemplary embodiments, not all features of actual embodiments (i.e., those features that are not relevant to the best mode of carrying out the invention as currently considered, or those features that are not relevant to implementing the invention) may be omitted.

[0059] It should be understood that numerous specific implementation decisions can be made during the development of any actual implementation method, and in any engineering or design project. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0060] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of an inhibitor of nucleoprotein of the Tachyirus 1 virus for the preparation of a pharmaceutical composition for the treatment or prevention of a disease caused by an infection with the Tachyirus 1 virus, characterized in that: Anti-tachy virus 1 nucleoprotein inhibitors are candesartan cilexetil.

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