A tor inhibitor and its use in inhibiting proliferation of mosquito cells

Abstract

The application discloses a TOR inhibitor and application thereof in inhibition of cell proliferation of mosquitoes, relates to the field of pesticides, and the TOR inhibitor is at least one of RAP, AZD-8055 and JR-AB2-011. The TOR inhibitor with three mechanisms of RAP, AZD-8055 and JR-AB2-011 is used, and it is confirmed at the level of Aedes aegypti Aag2 embryonic cells that the TOR signal channel is a key target point for regulating cell proliferation. The target point is novel, the mechanism is clear, and the channel is completely different from that of a traditional insecticide. The three inhibitors have a significant and dose-dependent inhibitory effect in vitro, have the potential to reduce the risk of drug resistance, have a small influence on non-target organisms, have high environmental friendliness, provide experimental and theoretical support for research and development of a new type of efficient and low-risk anti-mosquito preparation, and have important potential application values for prevention and control of Aedes aegypti and blocking of insect-borne infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, specifically to a TOR inhibitor and its application in inhibiting mosquito cell proliferation. Background Technology

[0002] Aedes aegypti, belonging to the family Culicidae in the order Diptera, is a primary vector for major insect-borne viruses such as dengue fever, Zika virus disease, chikungunya fever, and yellow fever. This mosquito species is highly adapted to urban environments, primarily inhabiting human settlements, and rapidly disseminating pathogens among populations through blood-sucking. In recent years, its geographical distribution has continued to expand, making its control increasingly challenging. The Aedes aegypti embryonic cell line Aag2 is an important in vitro model for studying mosquito physiology, insect-borne virus interactions, and screening for novel control drugs. The mTOR (mammalian target of rapamycin) signaling pathway is a core pathway regulating cell growth, proliferation, and metabolism; it is highly conserved in insects and can serve as a potential insecticidal target.

[0003] Currently, the control of Aedes aegypti mosquitoes still relies primarily on traditional chemical insecticides, including classic agents such as organophosphates and pyrethroids. However, long-term, high-volume, and single-use of these agents has led to the emergence of resistant populations and a continuous increase in resistance levels, significantly reducing the effectiveness of traditional insecticides. Furthermore, traditional chemical insecticides generally have long environmental residue periods, easily polluting water and soil, disrupting ecological balance, and failing to meet the requirements of modern mosquito control for high efficiency, low residue, and environmental friendliness.

[0004] This invention provides a TOR inhibitor and its application in inhibiting mosquito cell proliferation. By using at least one of rapamycin (RAP), AZD-8055, and JR-AB2-011 to act on mosquito cells, it provides a new technical solution and experimental basis for the development of novel, efficient, and environmentally friendly mosquito repellent agents. Summary of the Invention

[0005] The purpose of this invention is to provide a TOR inhibitor and its application in inhibiting mosquito cell proliferation, so as to solve the problem of poor efficacy of traditional chemical insecticides in controlling Aedes aegypti mosquitoes in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a TOR inhibitor, wherein the TOR inhibitor is at least one of RAP, AZD-8055, and JR-AB2-011, and the TOR inhibitor is used to inhibit mosquito cell proliferation.

[0007] Furthermore, the RAP is a first-generation mTOR allosteric inhibitor that specifically inhibits mTORC1 activity by forming a complex with FKBP12 and has no direct effect on mTORC2.

[0008] Furthermore, AZD-8055 is an ATP-competitive dual-target inhibitor of mTORC1 / mTORC2.

[0009] Furthermore, JR-AB2-011 is a selective mTORC2 inhibitor.

[0010] The application of a TOR inhibitor in inhibiting mosquito cell proliferation, wherein the TOR inhibitor is at least one of RAP, AZD-8055, and JR-AB2-011.

[0011] Furthermore, the mosquito is a mosquito of the genus Aedes.

[0012] Furthermore, the mosquito cells are Aedes mosquito embryonic cells.

[0013] Furthermore, the concentration range of the TOR inhibitor acting on mosquito cells is 0.0001 μM-100 μM.

[0014] Compared with existing technologies, this invention provides a TOR inhibitor and its application in inhibiting mosquito cell proliferation. By employing three complementary TOR inhibitors—RAP, AZD-8055, and JR-AB2-011—it confirms at the embryonic cell level of Aedes aegypti Aag2 that the TOR signaling pathway is a key target regulating its cell proliferation. This target is novel, the mechanism is clear, and it is completely different from the traditional insecticide pathway. The three inhibitors show significant and dose-dependent inhibitory effects in vitro, and have the potential to reduce the risk of drug resistance. At the same time, they have minimal impact on non-target organisms and are highly environmentally friendly. This provides experimental and theoretical support for the development of new, highly effective, and low-risk mosquito repellent agents, and has important potential application value for the control of Aedes aegypti and the blocking of vector-borne infectious diseases. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0016] Figure 1 A graph showing the effect of different concentrations of RAP on the growth status of Aag2 cells, provided in an embodiment of the present invention.

[0017] Figure 2 A graph showing the effect of different concentrations of AZD-8055 on the growth status of Aag2 cells, as provided in an embodiment of the present invention.

[0018] Figure 3 A graph showing the effect of different concentrations of JR-AB2-011 on the growth status of Aag2 cells, as provided in an embodiment of the present invention.

[0019] Figure 4 Morphological images of Aag2 cells with different concentrations of RAP, AZD-8055 and JR-AB2-011 provided for embodiments of the present invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Dimethyl sulfoxide (DMSO), purchased from Solarbio;

[0022] Rapamycin (RAP) (Catalyst No.: HY-10219) is a macrolide immunosuppressant produced by Streptomyces hygroscopicus.

[0023] AZD-8055 (catalog number: HY-10422) was purchased from MedChemexpress (MCE). AZD is an ATP-competitive TOR kinase inhibitor.

[0024] JR-AB2-011 (catalog number: HY-122022) was purchased from MedChemexpress (MCE). JR-AB2-011 is a selective mTORC2 inhibitor that inhibits the assembly and activity of the mTORC2 complex by blocking the binding of Rictor to mTOR.

[0025] Aag2 cell line culture medium: Drosophila culture medium (product number: SL0166-500mL), purchased from Coolaber;

[0026] Premium fetal bovine serum was purchased from Shanghai Sangon Biotech (Order No.: E600001).

[0027] CCK8 was purchased from Yisheng Biotechnology Co., Ltd. (item number: 40203ES60);

[0028] All other reagents and instruments are ordinary commercial items.

[0029] The Aag2 cell line was preserved by the Institute of Insect Science and Technology, College of Life Sciences, South China Normal University. It was cultured in a cell culture incubator at 27℃. Experiments were conducted when the Aag2 cells reached 70% adherence to the culture vessel, and subcultured approximately every 2-3 days. The Aag2 cell culture medium for Drosophila was supplemented with 10% serum.

[0030] Example 1:

[0031] Please see Figure 1-3 This embodiment provides the application of TOR inhibitors in inhibiting mosquito cell proliferation.

[0032] All three inhibitors were prepared using the same serial dilution protocol, as shown in Table 1. First, each inhibitor was dissolved in DMSO to prepare a 10 mM stock solution (1 mL). Using this stock solution as the starting concentration, serial dilutions were performed according to the steps shown in Table 1 to obtain intermediate dilutions with concentrations of 5000 μM, 1000 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM. Each serial dilution was aliquoted and stored at -20°C for later use. During drug treatment, 1 μL of the corresponding concentration of dilution was added to 100 μL of a 96-well cell culture plate to achieve final concentrations of 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0.0001 μM, respectively. DMSO was used as a control group.

[0033] Table 1 Solution Preparation Table

[0034] serial number Final concentration of culture medium (μM) Prepare the stock solution concentration (μM). Drug dilution Added amount DMSO 0 0 DMSO 1μL 0# 0.0001 0.01 Take 100 μL of #1 + 900 μL of DMSO 1μL 1# 0.001 0.1 Take 100 μL of #2 + 900 μL of DMSO 1μL 2# 0.01 1 Take 100 μL of #3 + 900 μL of DMSO 1μL 3# 0.1 10 Take 100 μL of #4 + 900 μL of DMSO 1μL 4# 1 100 Take 100 μL of #5 + 900 μL of DMSO 1μL 5# 10 1000 Take 200 μL of #6 + 800 μL of DMSO 1μL 6# 50 5000 Take 500 μL of #7 + 500 μL of DMSO 1μL 7# 100 10000 Stock solution (10 mM in 1 mL DMSO) 1μL

[0035] Aegypti mosquito larval embryo Aag2 cell culture:

[0036] 1. Cell resuscitation

[0037] Non-primary cultured cells are generally cryopreserved in liquid nitrogen and need to be thawed from liquid nitrogen before culture: First, turn on the water bath and adjust the temperature to 37°C; take the desired cells out of the liquid nitrogen tank and immediately place them in the 37°C water bath to thaw them quickly; wipe the outer surface of the cryovials and culture medium bottles clean with medical alcohol and place them in a clean bench; add the cell suspension to a centrifuge tube and add 1 mL of culture medium, and gently pipette; centrifuge at 600 rpm for 3 min; discard the supernatant, add 1 mL of culture medium, and pipette to mix well to suspend the cells; transfer the cell suspension to a 50 mL culture flask and add about 5 mL of culture medium; place in a cell culture incubator for culture.

[0038] 2. Cell culture

[0039] First, sterilize the laminar flow hood for 30 minutes. Then, remove the cell culture flask from the incubator and observe the cell growth status under a 10× inverted microscope. The cells are predominantly fibroblast-like, clustered together to form tightly connected cell clumps. Cells at the clump edges are often spindle-shaped or irregularly polygonal. Good cytoplasmic refractivity is normal. Remove the adherent cells, transfer them to 15 mL centrifuge tubes, centrifuge, discard the supernatant, add fresh culture medium, and continue culturing.

[0040] 3. Cytotoxicity assay

[0041] Aag2 cells from Aedes aegypti larval embryos in the logarithmic growth phase were collected and adjusted to approximately 1.0 × 10⁻⁶ cells. 5 Aag2 cells / mL were seeded into 96-well plates and cultured for 12 h until the cells were fully adhered. Then, 1 μL of each concentration of inhibitor was added. The experiment was repeated 3 times per well. After 48 h of culture, CCK8 was added to detect cell viability.

[0042] CCK-8 (Cell Counting Kit-8) is a cell viability assay based on the water-soluble tetrazolium salt WST-8. Its principle is that dehydrogenases in the mitochondria of living cells can reduce WST-8 to an orange-yellow formazan product. The amount of this product produced is directly proportional to the number of living cells. By measuring the absorbance at a wavelength of 450 nm, the cell viability status can be quantitatively reflected.

[0043] Cell viability after treatment with different concentrations of inhibitors was detected using the CCK-8 assay. CCK-8 reagent was added to each well and incubated for 3 h. Absorbance was measured using a microplate reader. Cell viability was calculated based on the absorbance ratio of each concentration group to the control group. A dose-response curve was fitted using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was then calculated. 50 To evaluate the inhibitory effects of various inhibitors on Aag2 cell viability.

[0044] 4. Results

[0045] like Figure 1 As shown, Aag2 cells were treated with nine different concentrations of RAP solution, from low to high, for 48 h. It was found that as the concentration increased, the inhibitory effect of RAP on the activity of Aag2 cells gradually increased, and its IC50 value... 50 The concentration was 0.77 μM; nine concentrations of AZD-8055 solution, from low to high, were used to treat Aag2 cells for 48 h. Figure 2 As shown, with increasing concentration, the inhibitory effect of AZD-8055 on the activity of Aag2 cells gradually increased, and its IC50 value... 50 The concentration was 0.57 μM; Aag2 cells were treated with nine different concentrations of JR-AB2-011 solution, from low to high, for 48 h. Figure 3 As shown, with increasing concentration, the inhibitory effect of JR-AB2-011 on the activity of Aag2 cells gradually increased, and its IC50 value... 50 The concentration was 1.69 μM. These results indicate that all three TOR inhibitors exhibited dose-dependent inhibitory effects on the proliferation of Aag2 cells.

[0046] Example 2:

[0047] Please see Figure 4This embodiment provides the effects of different inhibitors on the morphology of Aag2 cells, based on Embodiment 1.

[0048] Under normal culture conditions, the Aag2 cell line, isolated from the embryonic tissue of Aedes aegypti mosquito larvae, exhibits adherent growth, predominantly fibroblast-like morphology, with cells clustering into tightly connected clumps. Cells at the clump edges are often spindle-shaped or irregularly polygonal, and the cytoplasm displays good refractive properties. When the cell monolayer is sparsely distributed, cells are scattered, extending slender projections; as density increases, cell connections become tighter, and boundaries become blurred. Additionally, a small number of highly refractive, round, floating cells are observed in the culture system; these are detached cells during normal proliferation, and their proportion remains relatively stable. Overall, cell growth is uniform, the background is clean, and there is no obvious cell debris or vacuolation.

[0049] via TOR inhibitor IC 50 Cell morphology under concentration treatment, such as Figure 4 As shown in the figure, Aag2 cells exhibit typical drug-induced cell damage characteristics compared to normal morphology. With increasing concentrations of RAP, AZD-8055, and JR-AB2-011, the number of adherent cells significantly decreased, cell clumps gradually disintegrated, and most cells shrank from spindle-shaped or polygonal to round and detached from the culture wall. Cell debris in the culture system increased significantly, some cell membranes ruptured, intracellular contents leaked out, and many granular substances were visible in the background.

[0050] The above morphological changes further validated the inhibitory effect of TOR inhibitors on Aag2 cell viability, which is consistent with the cell viability results measured by the CCK-8 assay (Example 1).

[0051] Experimental results showed that all three inhibitors exhibited good dose-dependent inhibitory effects, with AZD-8055 showing the strongest inhibitory effect (IC50). 50 =0.57 μM), JR-AB2-011 is relatively weak (IC). 50 =1.69 μM), suggesting that mTORC1 and mTORC2 may have a synergistic regulatory role in the cell proliferation of Aedes aegypti.

[0052] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. Use of a TOR inhibitor for inhibiting proliferation of mosquito cells, characterized in that, The TOR inhibitor is at least one of AZD-8055, JR-AB2-011; The mosquito is an Aedes mosquito; The mosquito cell is an Aedes embryonic cell.

2. Use according to claim 1, characterized in that, The TOR inhibitor acts on the mosquito cell in a concentration range of 0.0001 μM to 100 μM.

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