Use of clofarabine in the preparation of a medicament for the prevention or treatment of poxvirus infection

Abstract

The application relates to application of clofarabine in preparation of a medicine for preventing or treating poxvirus infection. 50 The IC50 of inhibiting vaccinia virus is 0.44 muM, and the IC50 of inhibiting monkeypox virus is 0.06 muM. 50 Since the drug target points of deoxyribonucleotide large subunits of monkeypox virus, vaccinia virus, smallpox virus and other poxviruses are extremely conservative, clofarabine has the potential of broad-spectrum anti-poxvirus, and the toxic effect of clofarabine on Vero cells is extremely low. The application has important significance for prevention and treatment of poxviruses.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of clofarabine in the preparation of drugs for the prevention or treatment of poxvirus infection. Background Technology

[0002] Poxviruses are a class of structurally complex DNA viruses, classified into six subgenera based on serotype. The genus *Orthopoxvirus* is the first subgenus of poxviruses. Four types of orthopoxviruses—smallpox virus, vaccinia virus, vaccinia virus, and monkeypox virus—can cause human infection. Monkeypox, caused by monkeypox virus, is a zoonotic disease primarily occurring in Africa. The main route of transmission is through bites or contact with the bodily fluids or blood of infected animals. Additionally, monkeypox can be transmitted from person to person through close contact, such as through saliva, bodily fluids, and rashes.

[0003] Poxviruses are relatively large, with a diameter of 200-250 nm, and appear brick-shaped or oval under an electron microscope. During replication, they exist in two distinct infectious forms: mature intracellular virion (MV) and encapsulated virion (EV). Like other poxviruses, the monkeypoxvirus genome is a closed, double-stranded linear DNA, 194-199 kbp in length, with a low G+C content of only 31.1%, encoding approximately 200 proteins, including various enzymes required for viral replication. Therefore, the virus can replicate independently outside the cell nucleus.

[0004] Currently, there are no approved drugs specifically for treating monkeypox. Studies have found that the anti-smallpox drugs tecovirimat, cidofovir, and brincidofovir can inhibit monkeypox virus activity both in vitro and in vivo. Tecovirimat targets the viral membrane protein VP37, inhibiting viral assembly. Brincidofovir targets DNA polymerase, inhibiting viral DNA synthesis. With the emergence of poxvirus mutants, drug resistance will also be a problem; cidofovir-resistant strains have already appeared. Therefore, there is a need to develop new drugs targeting different viral targets to treat monkeypox and other poxviruses.

[0005] Clofarabine is a second-generation purine nucleoside analog, chemically named 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranyl)-9H-purine-6-amine, with the structural formula shown in formula (I) (molecular formula C10H11ClFN5O3, molecular weight 303.68, CAS number 123318-82-1). In 2004, the US FDA approved this drug for the treatment of relapsed or refractory acute lymphoblastic leukemia in children, administered orally or intravenously. Clofarabine targets human ribonucleotide reductase. Current research and clinical applications of this drug are focused on anti-cancer applications; no reports or findings have yet demonstrated its ability to combat poxviruses.

[0006] Summary of the Invention

[0007] Purpose of the invention

[0008] The purpose of this invention is to provide the use of clofarabine in the preparation of drugs for the prevention or treatment of poxvirus infection.

[0009] This invention has found that clofarabine can significantly inhibit the replication of poxvirus in Vero cells, indicating that clofarabine is of great significance in the prevention and treatment of poxvirus.

[0010] Solution

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] In a first aspect, the present invention provides the use of clofarabine in the preparation of medicaments for the prevention, treatment or adjunctive treatment of poxvirus infection and / or diseases caused by poxvirus.

[0013] Furthermore, the poxvirus is an orthopoxvirus.

[0014] Furthermore, the poxvirus includes one or more of monkeypox virus, vaccinia virus, smallpox virus, and cowpox virus.

[0015] The drugs include injections, oral liquids, powders, tablets, granules, capsules, syrups, decoctions, sustained-release preparations, enteric solvents, aerosols, or suspensions.

[0016] Furthermore, the drug comprises a preventive and / or therapeutically effective amount of clofarabine or a pharmaceutically acceptable derivative thereof, as well as a pharmaceutically acceptable carrier and / or excipient.

[0017] Furthermore, the concentration of clofarabine in the drug is ≥0.06 μM.

[0018] Furthermore, the method of administration of the drug is selected from one or more of the following: oral, injection, implantation, spray and / or inhalation.

[0019] Furthermore, the mechanism by which clofarabine prevents, treats, or assists in the treatment of poxvirus infection and / or poxvirus-induced diseases includes: targeting the large subunit of deoxyribonucleotide reductase and inhibiting its synthesis.

[0020] Furthermore, the mechanism by which clofarabine prevents, treats, or assists in the treatment of poxvirus infection and / or poxvirus-induced diseases includes: inhibiting poxvirus replication.

[0021] Furthermore, the application includes the use of clofarabine in combination with other drugs in the preparation of medicaments for the prevention, treatment, or adjunctive treatment of poxvirus infection and / or diseases caused by poxviruses.

[0022] Secondly, a method for preventing or treating or adjunctive treating poxvirus infection or disease caused by poxvirus infection is provided by administering a preventive or therapeutically effective amount of clofarabine or a pharmaceutically acceptable derivative thereof to a subject in need.

[0023] Beneficial effects

[0024] This invention utilizes structural biology to analyze antiviral targets, and based on this analysis, it was determined that clofarabine can target the catalytic center of the large subunit of vaccinia virus deoxyribonucleotides, thereby inhibiting vaccinia virus replication.

[0025] This invention has discovered that clofarabine can significantly inhibit the replication of vaccinia virus or monkeypox virus in Vero cells and inhibit the IC50 of vaccinia virus. 50 The IC50 value for inhibiting monkeypox virus was 0.44 μM. 50 The concentration was 0.06 μM. Because the large subunits of deoxyribonucleotides targeting poxviruses such as monkeypoxvirus, vaccinia virus, and smallpox virus are highly conserved, clofarabine has the potential for broad-spectrum anti-poxvirus activity, and it exhibits extremely low toxicity to Vero cells. This invention is of great significance for the prevention and control of poxviruses. Attached Figure Description

[0026] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0027] Figure 1This is the alignment result of the large subunit sequences of ribonucleotide reductases of monkeypox virus (MPXV), vaccinia virus (VACV), smallpox virus (VARV), and vaccinia virus (CPXV) in Example 1 of this invention.

[0028] Figure 2 The results are SDS-PAGE identification and structural analysis of the large subunit protein of ribonucleotide reductase isolated in Example 2 of this invention; where A is the SDS-PAGE identification result and B is the structural analysis result.

[0029] Figure 3 The results of the structural comparison analysis between the large subunit of monkeypox virus deoxyribonucleotide reductase and the deoxyribonucleotide reductase of vaccinia virus in Example 3 of this invention are shown. In this example, A represents the protein structure comparison result, and B represents the enzyme active center comparison analysis result.

[0030] Figure 4 This is a nonlinear curve of the logarithm of the drug concentration of clofarabine versus the inhibition rate of vaccinia virus in Example 4 of this invention.

[0031] Figure 5 This is a nonlinear curve of the logarithm of the drug concentration of clofarabine and the inhibition rate of monkeypox virus in Example 5 of the present invention.

[0032] Figure 6 This is a nonlinear curve of the logarithm of the drug concentration of clofarabine versus cell viability in Example 6 of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0034] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0035] The present invention will now be described in detail.

[0036] The reagents, enzymes, culture media, and other chemical materials used in the following embodiments of the present invention are all commercially available products.

[0037] Some commonly used biological materials, such as Vero cells and cell culture media, are also commercially available products.

[0038] The vaccinia virus strain WR was obtained from the Institute of Microbiology, Chinese Academy of Sciences (it is a common vaccinia virus strain, but other vaccinia virus strains can also be used).

[0039] Some synthetic biological materials, such as the large subunit gene sequence of deoxyribonucleic acid reductase, which require artificial synthesis, are all outsourced to synthetic companies.

[0040] Although ribonucleotide reductases (RRs) are widely found in various organisms, the amino acid sequence similarity between different types of RRs is very low, and their active sites also differ to some extent. To investigate whether clofalabene has an inhibitory effect on vaccinia virus ribonucleotide reductases, the inventors conducted further research, specifically:

[0041] Example 1: Protein sequence analysis of the large subunit of poxvirus deoxyribonucleotide reductase

[0042] The large subunit sequences of ribonucleotide reductase from monkeypox virus, vaccinia virus, smallpox virus, and vaccinia virus were downloaded from NCBI and sequenced using the online software https: / / espript.ibcp.fr / ESPript / ESPript / index.php. The results are as follows. Figure 1 ,from Figure 1 The comparison results show that the homology of the four is over 97%.

[0043] Example 2: Expression and purification of the large subunit protein of monkeypox virus deoxyribonucleotide reductase, and structural analysis of protein expression and purification:

[0044] The amino acid sequence of the large subunit of deoxyribonucleotide reductase from monkeypox virus (GISAID: EPI_ISL_406798) was purified by adding an 8×His tag to the N-terminus (the amino acid sequence of the complete protein is shown in SEQ ID No:1, with positions 10-782 being the amino acid sequence of the large subunit of deoxyribonucleotide reductase protein). The sequence was then optimized using insect cell codons (optimized species: insect) and synthesized by Nanjing Genscript Biotech Co., Ltd., and ligated into the pFastBac 1 vector (Invitrogen™ 10360014). The optimized gene sequence is shown in SEQ ID No:2.

[0045] SEQ ID NO:1 is as follows:

[0046] MHHHHHHHHGSMFVIKRNGYKENVMFDKITSRIRKLCYGLNTDHIDPIKIAMKVIQGIYNGVTTVELDTLAAEIAATCTTQHPDYAILAARIAISNLHKETKKLFSEVMEDLFNYVNPKNGKHSPIISSITMDIVNKYKDKLNSVIIYERDFSYNYFGFKTLEKSYLLKINNKIVERPQHMLMRVAVGIHQWDIDSAIETYNLLSEKWFTHASPTLFNAGTTRHQMSSCFLLNMIDDSIEGIYDTLKRCALISKMAGGIGLSISNIRASGSYISGTNGISNGIIPMLRVYNNTARYIDQGGNKRPGVMAIYLEPWHSDIMAFLDLKKNTGNDEHRTRDLFIALWIPDLFMKRVKDDGEWSLMCPDECPGLDNVWGDEFERLYTLYERERRYKCIIKARVVWKAIIESQIETGTPFILYKDACNKKSNQQNLGTIKCSNLCTEIIQYADANEVAVCNLASVALNMFVIDGRFDFLKLKDVVKVIVRNLNKIIDINYYPIPEAEISNKRHRPIGIGVQGLADAFILLNYPFDSLEAQDLNKKIFETIYYGALEASCKLAEKEGPYDTYVGSYASNGILQYDLWNVVPSDLWNWEPLKDKIRTYGLRNSLLVAPMPTASTAQILGNNESVEPYTSNIYTRRVLSGEFQVVNPHLLRVLTERKLWNDEIKNRIMVDGGSIQNTNLPEDIKRVYKTIWEIPQKTIIKMAADRGAFIDQSQSMNIHIADPSYSKLTSMHFYGWSLGLKTGMYYLRTKPASAPIQFTLDKDKIKPQVVCDSEICTSCSG

[0047] The optimized gene sequence is as shown in SEQ ID NO:2:

[0048]

[0049] 1. The large subunit protein of deoxyribonucleotidyl reductase was expressed in insect cells HighFive. First, baculovirus containing the target gene (gene sequence as shown in SEQ ID No. 2) was prepared (preparation method referred to the literature Yuan B, Peng Q, Cheng J, Wang M, Zhong J, Qi J, Gao GF, Shi Y. Structure of the Ebola virus polymerase complex. Nature. 2022 Oct; 610(7931):394-401. doi:10.1038 / s41586-022-05271-2). Then, HighFive cells were infected at a ratio of 1:30 and cultured at 28℃ and 125 rpm for 48 h. After centrifugation at 3000 rpm for 10 min, the cells were collected and treated with Buffer A (20 mM Hepes, 500 mM NaCl, 5% glycerol, pH 10). 7.0) Resuspension; Place the resuspended cells in an ice-water mixture, add PMSF to a final concentration of 1 mM, and sonicate the cells (5 s sonication, 8 s intermittent sonication, 30 min); After complete cell disruption, centrifuge at 12000 rpm, 4 °C for 120 min. Collect the supernatant and filter it through a 0.22 μm pore size filter to remove particles or insoluble matter;

[0050] 2. Combine the protein supernatant from step 1 with HisTrap TM After HP binding, impurities were washed with Buffer B (20 mM Hepes, 500 mM NaCl, 5% glycerol, 20 mM Imidazole, 1 mM TCEP, pH 7.0), and then the target protein was eluted with Buffer C (20 mM Hepes, 500 mM NaCl, 5% glycerol, 200 mM Imidazole, pH 7.0).

[0051] 3. The target protein obtained from step 2 was further separated and purified using molecular sieves, and identified by SDS-PAGE. The results showed that the molecular weight was consistent with expectations, and the purified protein had high purity. Figure 2 A).

[0052] Structural analysis of the large subunit protein of deoxyribonucleotide reductase, as follows: Figure 2 B.

[0053] 4. Concentrate the purified deoxyribonuclease large subunit to about 3 mg / mL, and dilute it 5 times with buffer (20 mM Hepes, 300 mM NaCl, pH 7.0) before sample preparation to instantly reduce the glycerol concentration;

[0054] 5. Select Quantifoil 1.2 / 1.3, 300 mesh to prepare frozen samples. The hydrophilization conditions are 14mA, 90s. Load the selected frozen samples onto Titan Krios, select an area with a thin ice layer and uniform particle distribution, take a map, select the shooting coordinate point, set appropriate parameters, and use SerialEM software to automatically collect data.

[0055] After data collection, the structure was analyzed using software such as MotionCor2, CTFFIND4.1, and RELION-2.0.

[0056] Example 3: Structural Comparison and Analysis of the Large Subunit of Monkeypoxvirus Deoxyribonucleic Acid Reductase and the Large Subunit of Vaccinia Virus Deoxyribonucleic Acid Reductase

[0057] AlphaFold2 was used to predict the structure of the large subunit of vaccinia virus deoxyribonuclease, and PyMOL was used to compare the structures of the two proteins (results are shown below). Figure 3 A), and enzyme activity center analysis (results as follows) Figure 3 B) This indicates that the three-dimensional structure and enzyme active site of the large subunit of monkeypoxvirus deoxyribonucleic acid reductase are very similar to those of the large subunit of vaccinia virus deoxyribonucleic acid reductase, suggesting that this enzyme is highly conserved within the poxvirus genus.

[0058] Example 4: Inhibitory effect of different concentrations of clofarabine on vaccinia virus replication

[0059] 1. One day in advance, seed the Vero cells in good condition into 12-well plates, with a loading volume of 1 mL per well;

[0060] 2. On the second day, remove the culture medium and add 100 PFU of vaccinia virus strain WR into the cells. Incubate at 37°C for 2 hours, shaking the culture plate every 25-30 minutes during this period.

[0061] 3. Use a pipette to remove the liquid from the wells and wash away any residual virus with PBS. Then, preheat 2% carboxymethyl cellulose to 50°C and mix it 1:1 with 2×DMEM high-glucose medium (CR12902 Zhejiang Senrui Biotechnology) at room temperature. Set up three replicates of the drug-free wells as negative control wells. Add different concentrations of the small molecule drug clofarabine to the other wells, with three replicates of each concentration. The final concentrations are 4μM, 2μM, 1μM, 0.5μM, 0.25μM, and 0μM, respectively.

[0062] After mixing, store at 37°C until ready to use;

[0063] 4. After adding the drug, continue culturing for 48 hours, then discard the methylcellulose. Directly add 4% paraformaldehyde, stain for 2 hours, then add 0.5% crystal violet solution for 1-2 hours, and count the cells. Use GraphPad Prism to fit a nonlinear curve of the logarithm of drug concentration versus inhibition rate. The results are as follows: Figure 4 Based on the curve, the concentration at which clofabradine achieves a 50% inhibition rate against vaccinia virus is defined as the IC50 concentration. 50 . Figure 4 The results showed that clofabradine inhibited vaccinia virus at an IC50 level. 50 The concentration was 0.44 μM, which is far lower than the dosage for anticancer treatment.

[0064] Example 5: Inhibitory effect of different concentrations of clofarabine on monkeypox virus replication

[0065] This invention also investigated the inhibitory effects of different concentrations of clofarabine on monkeypox virus replication. The relevant experiments were commissioned to the Biosafety Level 3 Laboratory of Shenzhen Third People's Hospital. Specifically:

[0066] 1. One day in advance, seed the Vero cells in good growth condition into 24-well plates to prevent incubation in an incubator;

[0067] 2. On the second day, remove the culture medium and dilute the monkeypox virus with DMEM medium containing 2% FBS to a final concentration of 2 x 10⁻⁶. 5 Add cells / mL to the cells and incubate for 1 hour.

[0068] 3. Dilute the small molecule drug clofarabine with DMEM medium containing 2% FBS. Set up three replicates for different concentrations, with final concentrations of 5 μM, 1 μM, 0.2 μM, 0.04 μM, 0.008 μM, 0.0016 μM, 0.00032 μM, and 0 μM. Incubate at 37℃ for 48 h.

[0069] 4. Nucleic acid was extracted and qPCR was performed. A non-linear curve of the logarithm of drug concentration versus inhibition rate was fitted using GraphPad Prism. Based on the curve, the IC50 concentration of clofarabine at which the inhibition rate against monkeypox virus was 50% was determined. 50 The IC50 of clofarabine inhibiting monkeypox virus. 50 0.06 μM ( Figure 5 ).

[0070] Example 6: Clofarabine cytotoxicity CC 50 test

[0071] Cytotoxicity was detected using the MCE CCK-8 kit (catalog number: HY-K0301), a cytotoxicity assay based on WST-8. In the presence of an electron coupling reagent, WST-8 can be reduced to orange-yellow formazan by mitochondrial dehydrogenases. The stronger the cytotoxicity, the lighter the color.

[0072] 1. Seed cells: Seed Vero cell suspension (100 μL / well) into 96-well plates and place the culture plate in an incubator for pre-culture at 37°C and 5% CO2 for 24 h.

[0073] 2. Add different concentrations of clofarabine (final concentrations of 200 μM, 40 μM, 8 μM, 1.6 μM, and 0.32 μM, respectively) to the culture plate;

[0074] 3. Continue to incubate the culture plate in the incubator for 48 hours, replace with fresh culture medium, and add 10 μL of CCK-8 solution to each well;

[0075] 4. Incubate the culture plate in an incubator for 2 hours, and measure the absorbance at 450 nm using a microplate reader to detect cell viability. Calculate the cytotoxicity of the small molecule drug on Vero cells. Cell viability = [(As-Ab) / (Ac-Ab)] × 100%.

[0076] As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and drug solution);

[0077] Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drugs);

[0078] Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0079] The nonlinear curve of the logarithm of drug concentration versus cell viability was fitted using GraphPad Prism. Based on the curve, the drug concentration at which 50% viability was achieved was determined to be the CC (cell survival rate). 50 The result is as follows Figure 6 The results showed that the CC of chlorofarabine 50 A concentration greater than 200 μM indicates that clopidogrel has extremely low toxicity to Vero cells and good safety profile.

[0080] In summary, clofalabane inhibits vaccinia virus with an IC50 value. 50 The IC50 value for monkeypox virus was 0.44 μM. 50 The concentration was 0.06 μM, which is far lower than the cytotoxic CC of clofarabine. 50 This indicates that chlorofralbine can effectively inhibit vaccinia virus and monkeypox virus within the safe range of use.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of clofarabine in the preparation of medicaments for the prevention, treatment or adjunctive treatment of monkeypox virus infection and / or diseases caused by monkeypox virus infection.

2. The application according to claim 1, characterized in that, The drug is selected from any one of the following: injection, oral liquid, powder, tablet, granule, capsule, syrup, decoction, sustained-release preparation, enteric solvent, aerosol, and suspension.

3. The application according to claim 1, characterized in that, The drug comprises a preventive and / or therapeutically effective amount of clofarabine, and a pharmaceutically acceptable carrier and / or excipient.

4. The application according to any one of claims 1 to 3, characterized in that, The concentration of clofarabine in the drug is ≥0.06 μM.

5. The application according to claim 1, characterized in that, The drug can be administered via one of the following methods: oral, injection, implantation, or spray.

6. The application according to claim 1, characterized in that, The drug is administered via inhalation.

7. The application according to claim 1, characterized in that, The mechanism by which clofarabine prevents, treats, or assists in the treatment of monkeypox virus infection and / or diseases caused by monkeypox virus includes: targeting the large subunit of deoxyribonucleotide reductase and inhibiting its synthesis.

8. The application according to claim 1, characterized in that, The mechanism by which clofarabine prevents, treats, or assists in the treatment of monkeypox virus infection and / or diseases caused by monkeypox virus includes: inhibiting the replication of the poxvirus.

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