Use of wye-125132 in the manufacture of a medicament for combating monkeypox virus

Abstract

The application discloses application of WYE-125132 in preparation of an anti-monkeypox virus drug and belongs to the technical field of biological medicines. The application first finds that WYE-125132 can significantly inhibit replication of monkeypox virus, and the inhibition is dose-dependent. Meanwhile, the WYE-125132 has good anti-monkeypox virus activity, provides important scientific basis and scheme for development of a new, safe and effective monkeypox treatment drug and clinical treatment, and therefore, the WYE-125132 has good application prospect in development of the anti-monkeypox virus drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of WYE-125132 in the preparation of anti-monkeypox virus drugs. Background Technology

[0002] Monkeypox is a zoonotic infectious disease caused by the monkeypox virus (MPXV). MPXV is a double-stranded DNA virus belonging to the genus Orthopoxvirus of the family Poxviridae, and is related to smallpox virus (Variola virus) and vaccinia virus (Vaccinia virus). The virus was first discovered in laboratory monkeys in 1958, and the first human case was reported in the Democratic Republic of Congo in 1970. For a long time, monkeypox was mainly endemic in the tropical rainforests of west-central Africa, occasionally spreading to other regions, but it did not attract widespread global attention. However, since 2022, monkeypox outbreaks have occurred and continued to spread in several non-endemic countries around the world, posing a serious threat to public health. It has been declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization twice. Monkeypox virus is mainly divided into two genetic branches: the Congo Basin branch (branch I, with a higher mortality rate) and the West African branch (branch II, with a lower mortality rate). Currently, imported cases in my country are mainly from the West African branch. Monkeypox virus is primarily transmitted through contact with the rash, bodily fluids, respiratory droplets, and contaminated items (such as clothing and bed sheets) of infected individuals. Clinical symptoms typically include fever, severe headache, swollen lymph nodes, back pain, myalgia, and fatigue, followed by a characteristic rash that spreads from the face to other parts of the body. In recent years, monkeypox virus has continued to evolve, with accelerated mutations and increasingly complex strains. For example, the new MPXV strain reported by the UK Department of Health in 2025 was a recombinant "hybrid" of subtypes Ib and IIb, increasing viral transmissibility and the potential risk of immune escape, posing new challenges to prevention and control efforts.

[0003] Currently, there are no approved specific treatments for monkeypox virus. Clinical treatment primarily focuses on symptomatic and supportive care and management of complications, aiming to alleviate symptoms and prevent and manage secondary infections. Although antiviral drugs for smallpox, such as Tecovirimat, are available for treating monkeypox under emergency use authorization, accessibility is limited, and there is a potential risk of viral resistance. Therefore, developing safe, effective, and readily available novel anti-monkeypox drugs is a critical and urgent issue that needs to be addressed.

[0004] Drug repurposing (drug repurposing) strategies have become a rapid and effective way to deal with the threat of emerging infectious diseases because they can make full use of known drug pharmacological, efficacy, safety, and pharmacokinetic data, significantly reducing R&D risks, shortening the R&D cycle and costs.

[0005] WYE-125132 (CAS No.: 1144068-46-1; Molecular Formula: C) 27 H 33 N7O4 is a highly specific ATP-competitive mTOR kinase inhibitor that inhibits mTOR kinase activity by competitively binding to the ATP-binding site of the mTOR kinase domain, thereby blocking the mTOR signaling pathway. It inhibits both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), simultaneously suppressing cell growth, metabolism, and proliferation signaling mediated by these two complexes. WYE-125132 exhibits extremely strong inhibitory activity against recombinant mTOR kinase, with an IC50 concentration of [missing information]. 50 The value is approximately 0.19 nM. WYE-125132 exhibits significant anti-proliferative effects against various tumor cell lines (such as breast cancer, prostate cancer, and lung cancer), and its IC50 value is approximately 0.19 nM. 50 Values ​​are typically in the nanomolar range. WYE-125132 can induce cell cycle arrest (mainly in the G1 phase), inhibit protein synthesis, reduce cell size, and induce apoptosis. In various tumor xenograft models (such as MDA-MB-361, U87MG, etc.), oral or intravenous administration of WYE-125132 can significantly inhibit tumor growth, induce tumor regression, and has low toxicity to normal tissues.

[0006] However, there are currently no research reports on WYE-125132 as an anti-monkeypox virus drug. Summary of the Invention

[0007] The purpose of this invention is to provide the application of WYE-125132 in the preparation of anti-monkeypox virus drugs, thereby addressing the problem of the lack of highly effective anti-monkeypox virus drugs in the prior art.

[0008] In a first aspect, the present invention provides the use of WYE-125132 in the preparation of anti-monkeypox virus drugs.

[0009] In this invention, the inventors discovered for the first time that WYE-125132 can significantly inhibit the replication of monkeypox virus, and that this inhibition is dose-dependent. At the same time, WYE-125132 has good anti-monkeypox virus activity, providing important scientific basis and solutions for the development of novel, safe and effective monkeypox treatment drugs and clinical treatment.

[0010] In some implementations, the drug inhibits the proliferation of monkeypox virus by inhibiting at least one of the viral titer of monkeypox virus or the replication of monkeypox virus.

[0011] In some implementations, the concentration of WYE-125132 is 0.5-5 μM.

[0012] In a second aspect, the present invention provides the use of WYE-125132 in the preparation of products that inhibit monkeypox virus titers.

[0013] In a third aspect, the present invention provides the use of WYE-125132 in the preparation of products that inhibit monkeypox virus replication.

[0014] In a fourth aspect, the present invention provides a pharmaceutical composition for treating monkeypox virus, the pharmaceutical composition comprising WYE-125132.

[0015] In some embodiments, the concentration of WYE-125132 in the pharmaceutical composition is 0.5-5 μM.

[0016] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0017] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0018] In some implementations, solid dosage forms include at least one of tablets, capsules, powders, pills, granules, and suppositories; semi-solid dosage forms include at least one of ointments, creams, and patches; and liquid dosage forms include at least one of injections, oral liquids, syrups, and inhalants.

[0019] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention is the first to discover that WYE-125132 can significantly inhibit the replication of monkeypox virus, and this inhibition is dose-dependent; at the same time, WYE-125132 has good anti-monkeypox virus activity, providing important scientific basis and solutions for the development of novel, safe, and effective monkeypox treatment drugs and clinical treatment; therefore, WYE-125132 has good application prospects in the development of anti-monkeypox virus drugs. Attached Figure Description

[0020] Figure 1 The graph shows the cytotoxicity assay results of WYE-125132 on A549 cells in Example 1 of this invention. The horizontal axis represents the concentration of WYE-125132 (μM), and the vertical axis represents the percentage of cytotoxicity (%). The yellow curve in the graph represents the WYE-125132 treatment group, and the gray curve represents the positive drug Tecovirimat treatment group. Figure 2 This is a pharmacodynamic curve of WYE-125132 inhibiting monkeypox virus replication in Example 2 of the present invention. The horizontal axis represents the concentration of WYE-125132 (μM), and the vertical axis represents the virus replication inhibition rate (%). The yellow curve in the figure represents the WYE-125132 treatment group, and the gray curve represents the positive drug Tecovirimat treatment group. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0023] Currently, there is a lack of highly effective drugs against monkeypox virus in existing technologies.

[0024] To address the lack of highly effective anti-monkeypox virus drugs in the existing technology, this invention provides the application of WYE-125132 in the preparation of anti-monkeypox virus drugs.

[0025] In a first aspect, the present invention provides the use of WYE-125132 in the preparation of anti-monkeypox virus drugs.

[0026] In the application provided by this invention, it was discovered for the first time that WYE-125132 can significantly inhibit the replication of monkeypox virus, and this inhibition is dose-dependent; at the same time, WYE-125132 has good anti-monkeypox virus activity, providing important scientific basis and solutions for the development of novel, safe and effective monkeypox treatment drugs and clinical treatment.

[0027] In some implementations, the drug inhibits the proliferation of monkeypox virus by inhibiting at least one of the viral titer of monkeypox virus or the replication of monkeypox virus.

[0028] In this invention, the drug exerts its anti-monkeypox effect by inhibiting the viral titer or replication of monkeypox virus.

[0029] In some implementations, the concentration of WYE-125132 is 0.5-5 μM, for example, it can be 0.5 μM, 1 μM, 1.5 μM, 2 μM, 25 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM or other values ​​within this range.

[0030] In this invention, WYE-125132 can effectively inhibit the replication of monkeypox virus at low toxic concentrations, and this inhibition is dose-dependent.

[0031] In a second aspect, the present invention provides the use of WYE-125132 in the preparation of products that inhibit monkeypox virus titers.

[0032] Understandably, the form and type of products that inhibit monkeypox virus titers can be selected according to actual usage needs, as long as they can effectively inhibit monkeypox virus titers.

[0033] In a third aspect, the present invention provides the use of WYE-125132 in the preparation of products that inhibit monkeypox virus replication.

[0034] Understandably, the form and type of products that inhibit monkeypox virus replication can be selected conventionally according to actual usage needs, as long as they can effectively inhibit monkeypox virus titers.

[0035] In a fourth aspect, the present invention provides a pharmaceutical composition for treating monkeypox virus, the pharmaceutical composition comprising WYE-125132.

[0036] The pharmaceutical composition provided by this invention has good anti-monkeypox virus activity.

[0037] In some embodiments, the concentration of WYE-125132 in the pharmaceutical composition is 0.5-5 μM, for example, it can be 0.5 μM, 1 μM, 1.5 μM, 2 μM, 25 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM or other values ​​within this range.

[0038] In this invention, WYE-125132 in the pharmaceutical composition can effectively inhibit the replication of monkeypox virus at low toxic concentrations, and this inhibition is dose-dependent.

[0039] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0040] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0041] The pharmaceutical compositions provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0042] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0043] In some implementations, solid dosage forms include at least one of tablets, capsules, powders, pills, granules, and suppositories; semi-solid dosage forms include at least one of ointments, creams, and patches; and liquid dosage forms include at least one of injections, oral liquids, syrups, and inhalants.

[0044] The pharmaceutical compositions provided by this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0045] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0046] Example 1: Detection of cytotoxicity of WYE-125132 on A549 cells In this embodiment, the aim is to evaluate the in vitro toxicity of WYE-125132 to host cells A549 in order to determine the safe concentration range for subsequent antiviral experiments.

[0047] Specifically, it includes the following steps: 1) Cell Culture: Human non-small cell lung cancer cells A549 (purchased from the American Type Culture Collection, ATCC) were resuscitated and cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments.

[0048] 2) Experimental grouping and treatment: After digesting A549 cells in the logarithmic growth phase, they were subjected to 2×10⁻⁶... 4 Cells were seeded at a density of 100 cells / well in 48-well plates, with 200 μL of culture medium per well. After 16 hours of culture until the cells reached approximately 80% confluence, the supernatant was discarded and replaced with DMEM maintenance medium containing 2% FBS. The experiment included a WYE-125132 treatment group, a Tecovirimat positive control group, a blank control group, and a cell control group. WYE-125132 and Tecovirimat were prepared at four concentration gradients: 0, 0.5 μM, 1 μM, and 5 μM, with three replicates for each concentration. The blank control group had three replicates. The control group (DMSO) had three replicates.

[0049] 3) Toxicity assay: After 72 hours of drug treatment, add 25 μL of CCK-8 solution to each well, mix gently, and incubate for 1-4 hours at 37°C in a 5% CO2 incubator. After sufficient color development, measure the absorbance (OD value) at 450 nm using a microplate reader.

[0050] Test results as follows Figure 1 As shown.

[0051] from Figure 1 As can be seen, WYE-125132 inhibited A549 cells by approximately 25.56% at a concentration of 5 μM. The results indicate that WYE-125132 exhibits low cytotoxicity and good safety within the tested concentration range.

[0052] Example 2: Evaluation of the efficacy of WYE-125132 in inhibiting monkeypox virus replication in vitro. In this embodiment, the aim is to evaluate the inhibitory effect of WYE-125132 on monkeypox virus replication in an A549 cell model.

[0053] Specifically, it includes the following steps: 1) Virus and Cells: Monkeypox virus (MPXV, West African branch strain, deposited at Wuhan Institute of Virology, Chinese Academy of Sciences) was amplified in A549 cells, and the viral titer was determined using a plaque formation assay. A549 cell culture was performed as in Example 1.

[0054] 2) Drug treatment and viral infection: A549 cells were treated at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 48-well plates and incubated overnight. The culture medium was discarded, and 2% FBS DMEM maintenance medium containing different concentrations of WYE-125132 (0, 0.5, 1, 5 μM) was added to each well, with three replicates for each concentration. A positive control group (Tecovirimat at the same concentration) and a virus control group (no drug) were also included. After 1 hour of drug pretreatment, the drug-containing culture medium was discarded and replaced with fresh maintenance medium containing the corresponding drug concentration. Monkeypox virus was inoculated at a multiplicity of infection (MOI) of 0.1, and the plates were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0055] 3) Viral load detection: a) RNA extraction: 48 hours after infection, cell culture supernatant and cell lysate were collected, and total viral RNA was extracted according to the instructions of the Qiagen ViralRNA Mini Kit.

[0056] b) qRT-PCR detection: The viral gene copy number was quantitatively detected using a one-step qRT-PCR method with TaqMan probes. The primers and probe sequences used targeted conserved genes of MPXV. Forward primer: 5'-AGTGGATTAACACCGGAACAA-3' (SEQ ID NO.1); Reverse primer: 5'-CGACCGCGCTAGAATTACAA-3' (SEQ ID NO.2); Probe: 5'-CGTACCAGCTATGTTTACTGCTGCGT-3' (SEQ ID NO.3); FAM is connected to the 5' end of the probe, and TAMRA is connected to the 3' end.

[0057] Reaction system (25 μL): 2×One Step RT-PCR Buffer III 12.5 μL, Takara Ex Taq HS 0.5 μL, PrimeScript RT Enzyme Mix II 0.5 μL, forward primer (10 μM) 1.5 μL, reverse primer (10 μM) 1.5 μL, probe (10 μM) 0.5 μL, RNA template 2 μL, and RNase-free water to 25 μL.

[0058] The reaction procedure was as follows: 42℃, 5 min (reverse transcription); 95℃, 10 s (pre-denaturation); then 95℃, 5 s, 60℃, 30 s (collect fluorescence signal), for 40 cycles.

[0059] Test results as follows Figure 2 As shown.

[0060] from Figure 2 As can be seen, the inhibitory effect of WYE-125132 on monkeypox virus is dose-dependent, and its half-maximal effective concentration (EC50) for monkeypox virus with MOI=0.1 is 0.49 μM.

[0061] In summary, this invention is the first to discover that WYE-125132 can significantly inhibit the replication of monkeypox virus in a dose-dependent manner. Furthermore, WYE-125132 exhibits good anti-monkeypox virus activity, providing important scientific evidence and guidelines for the development of novel, safe, and effective monkeypox treatment drugs and clinical treatment. Therefore, WYE-125132 has promising application prospects in the development of anti-monkeypox virus drugs.

[0062] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0063] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Application of WYE-125132 in the preparation of anti-monkeypox virus drugs.

2. The application according to claim 1, characterized in that, The drug inhibits the proliferation of monkeypox virus by inhibiting at least one of the viral titer of monkeypox virus and the replication of monkeypox virus.

3. The application according to claim 1, characterized in that, The concentration of WYE-125132 is 0.5-5 μM.

4. Application of WYE-125132 in the preparation of products that inhibit monkeypox virus titers.

5. Application of WYE-125132 in the preparation of products that inhibit monkeypox virus replication.

6. A pharmaceutical composition for treating monkeypox virus, characterized in that, The pharmaceutical composition includes WYE-125132.

7. The pharmaceutical composition according to claim 6, characterized in that, In the pharmaceutical composition, the concentration of WYE-125132 is 0.5-5 μM.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition includes at least one of solid dosage form, semi-solid dosage form, and liquid dosage form.

10. The pharmaceutical composition according to claim 9, characterized in that, The solid dosage form includes at least one of tablets, capsules, powders, pills, granules, and suppositories; the semi-solid dosage form includes at least one of ointments, creams, and patches; and the liquid dosage form includes at least one of injections, oral liquids, syrups, and inhalers.

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