Anti-toxoplasma gondii small molecule compound and application thereof

Abstract

The invention belongs to the field of toxoplasma gondii prevention and control, and particularly relates to an anti-toxoplasma gondii small molecule compound and application thereof. The small molecule compound is shown as a formula 1. The small molecule compound provided by the invention can inhibit growth, invasion and intracellular replication of toxoplasma gondii.

Description

Technical Field

[0001] This invention belongs to the field of Toxoplasma gondii control, specifically relating to an anti-Toxoplasma gondii small molecule compound and its application. Background Technology

[0002] Toxoplasma gondii is a parasitic protozoan belonging to the phylum Apicocomplexa, capable of infecting almost all warm-blooded animals, including humans, causing the zoonotic disease toxoplasmosis, which is widespread globally. Currently, the global human infection rate ranges from 10% to 60%, with an average infection rate of approximately 23% in livestock. Toxoplasmosis infection poses serious risks to immunocompromised or weakened populations such as the elderly, children, pregnant women, AIDS patients, and organ transplant recipients, causing toxoplasmic encephalopathy, chorioretinitis, hydrocephalus, miscarriage, stillbirth, and is a leading cause of death in immunocompromised individuals. In healthy individuals, toxoplasmosis infection is often asymptomatic, but the parasite persists infecting the host's central nervous system in cyst form, causing alterations in mental and behavioral patterns. In animals, toxoplasmosis infection can cause subcutaneous hemorrhage, diarrhea, fever, and even death, severely impacting livestock production and food safety. Toxoplasmosis poses a significant threat to public health and is a global health hazard that cannot be ignored.

[0003] The existing classic clinical treatment regimen, the combination therapy of pyrimethamine and sulfadiazine, can only eliminate the tachyzoites of Toxoplasma gondii in the acute infection stage. It is characterized by a long treatment course, significant side effects, poor efficacy, and a high relapse rate. Furthermore, some chemically synthesized drugs and compounds, such as ciprofloxacin, dimethyl ketone, 1,3,4-thiadiazole-12b, and some traditional Chinese medicine extracts, such as artemisinin and glycyrrhizin chalcone A, have been shown to have some killing effect on Toxoplasma gondii. However, due to the non-specific targets and unclear mechanisms of action of these drugs, they still suffer from strong side effects, incomplete treatment, and poor selectivity, and therefore cannot be used clinically. In fact, since 1958, the FDA has not approved any new drugs for the treatment of toxoplasmosis.

[0004] Recently, the synthesis of lead compounds by screening effective drug targets has become a hot topic in Toxoplasma gondii drug research. Deng et al. synthesized a 2-piperazine-quinazoline-4(3H)-1one derivative targeting the small molecule binding pocket of calcium-dependent protein kinase 1 (TgCDPK1), which showed partial inhibitory activity against Toxoplasma gondii. McLeod's team synthesized triclosan targeting Toxoplasma gondii acyl reductase (TgENR), and in vitro experiments showed that it could inhibit the growth of Toxoplasma gondii at low concentrations. However, triclosan has low solubility and oral bioavailability, and it cannot effectively kill Toxoplasma gondii tachyzoites and cysts. Recent studies have reported that the compound MC2590 can target and inhibit histone deacetylase (TgHDAC), and in vivo administration can improve the survival rate of mice in the acute phase of Toxoplasma gondii infection, but it is ineffective in mice in the chronic phase of infection. Summary of the Invention

[0005] To address the above problems, the present invention provides a small molecule compound, the structural formula of which is shown in Formula 1:

[0006]

[0007] This invention also provides the application of small molecule compounds in the preparation of drugs for the prevention and treatment of Toxoplasma gondii.

[0008] Furthermore, the drug can be administered orally, by injection, or by topical application.

[0009] The present invention has the following beneficial effects:

[0010] By analyzing the bioactivity of 40 small molecules selected through pocket screening targeting ROP18, we obtained a small molecule with high safety and good antiparasitic activity. This small molecule has a strong affinity for ROP18 protein and can inhibit ROP18 protein kinase activity. In in vitro experiments, we found that this small molecule can inhibit the growth, invasion, and intracellular replication of Toxoplasma gondii. In in vivo experiments, this small molecule can significantly improve the survival rate of Toxoplasma gondii-infected mice and reduce the Toxoplasma gondii load in the brain, liver, and spleen tissues of mice. The antiparasitic mechanism of this small molecule is relatively clear. It achieves good in vivo antiparasitic effects by specifically inhibiting the biological effects of ROP18. Therefore, this small molecule shows promise as a candidate drug for the treatment of toxoplasmosis. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 The inhibition of ROP18 kinase activity by small molecules was dose-dependent. A: Effect of small molecule #2 on ROP18 kinase activity. B: Effect of small molecule #38 on ROP18 kinase activity. C: Determination of the affinity of small molecule #2 for ROP18. D: Determination of the affinity of small molecule #38 for ROP18. **P<0.01, ***P<0.001

[0013] Figure 2 The inhibitory effects of small molecules 2 and 38 on Toxoplasma gondii growth were dose-dependent. A, B) Molecular weights and structures of small molecules 2 and 38. C: EC50 of small molecules 2 and 38 on Toxoplasma gondii growth. 50 D: IC50 of small molecules 2 and 38 against HFF cytotoxicity. 50 .

[0014] Figure 3 Effects of different concentrations of small molecules No. 2 and No. 38 on Toxoplasma gondii growth. A: Fluorescence graphs showing the inhibitory effects of different concentrations of small molecules No. 2 and No. 38 on Toxoplasma gondii growth. B, C: Statistical analysis of the effects of different concentrations of small molecules No. 2 and No. 38 on Toxoplasma gondii growth. **P<0.01, ****P<0.001

[0015] Figure 4 Effects of different concentrations of small molecules No. 2 and No. 38 on Toxoplasma gondii growth. A: Effects of different concentrations of small molecules No. 2 and No. 38 on plaque formation in Toxoplasma gondii. B, C: Statistical analysis of the area of ​​plaques formed by different concentrations of small molecules No. 2 and No. 38 in Toxoplasma gondii. *P<0.05,**P<0.01,***P<0.001

[0016] Figure 5 The effects of different concentrations of small molecules No. 2 and No. 38 on Toxoplasma gondii invasion. A: No. 2 B: No. 38

[0017] Figure 6 Effects of different concentrations of small molecules No. 2 and No. 38 on intracellular proliferation of *Toxoplasma gondii*. A, B: Effects of different concentrations of small molecule No. 2 on intracellular replication of *Toxoplasma gondii* at 24 h and 48 h, respectively. C, D: Effects of different concentrations of small molecule No. 38 on intracellular replication of *Toxoplasma gondii* at 24 h and 48 h, respectively. 1, 2, 4, 8, 16, 32, 64 represent the number of intracellular tachyzoites. *P<0.05, **P<0.01

[0018] Figure 7 The effects of small molecules 2 and 38 on ROP18 recruitment of Irga6. A: The effects of small molecules 2 and 38 on ROP18 recruitment of Irga6 in the RH strain and ΔROP18 RH strain. B: Statistical analysis of the effects of small molecules 2 and 38 on ROP18 recruitment of Irga6 in the RH strain and ΔROP18 RH strain.

[0019] Figure 8 Detection of anti-Toxoplasma gondii activity of small molecules 2 and 38 in mice. A: Effect of small molecules 2 and 38 on the survival rate of infected mice. B, C, D: qPCR detection of parasite load in the brain, spleen, and liver tissues of mice after treatment with small molecules 2 and 38. Detailed Implementation

[0020] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Discovery Process

[0026] 1. Experimental Background

[0027] Previously, our research group discovered TgROP18 as a potential drug target for Toxoplasma gondii. TgROP18 is a secretory effector encoded by Toxoplasma gondii, secreted into the host cell via the apical complex, mediating the formation of parasitic vesicles (PVMs). It has been demonstrated that TgROP18 is a key virulence determinant for highly virulent type I and moderately virulent type II strains distributed in Europe and North America, and also a virulence determinant for the Chinese type I Toxoplasma gondii isolate (T.gHB1). Studies have shown that infection of mice with type I TgROP18 knockout strains significantly increases mouse survival. Knocking out TgROP18 in type II strains almost completely eliminates chronic Toxoplasma gondii infection in mice and significantly reduces virulence mortality (>100-fold). Therefore, TgROP18 is an important virulence factor at different stages of Toxoplasma gondii infection. TgROP18 belongs to the ROP2 protein family and possesses serine-threonine protein kinase activity. It phosphorylates the host's intracellular immune-related GTPases (IRGs) Irga6 and Irgb6, inhibiting their recruitment to the Toxoplasma gondii PVM, preventing PVM lysis, and thus blocking the IFN-γ-induced innate immune response. This allows the parasite to evade clearance by host immune cells, leading to persistent Toxoplasma gondii infection. TgROP18 can also disrupt the host's acquired immune response by phosphorylating important protein factors such as ATF6β and TRIM21. Furthermore, TgROP18 can inhibit the NF-κB signaling pathway and suppress inflammatory responses by degrading the transcription factor p65, thereby promoting its own survival and growth. Moreover, ROP18 possesses a protein kinase folding pattern and true kinase activity, making it a more ideal drug design target with greater application potential.

[0028] In the early stages, the project team successfully designed a pharmacophore model targeting the ATP-binding pocket of TgROP18 using structure-based drug design methods, based on precise analysis of the ATP-binding pocket of the natural substrate TgROP18. This resulted in the screening of 32 small molecule inhibitors of ROP18. Building on this foundation, this project aims to establish a Toxoplasma gondii infection model to conduct preliminary screening for the anti-Toxoplasma gondii activity of these 32 selected small molecules and 8 homologous compounds. The study will assess the cytotoxicity, kinase activity inhibition, protein affinity, parasite invasion inhibition rate, intracellular proliferation inhibition rate, and growth inhibition rate of these selected small molecules. The anti-parasitic activity of the small molecules will be evaluated in the in vivo infection model, and their anti-parasitic mechanism will be elucidated, laying a theoretical foundation for the development of novel, highly effective drugs against Toxoplasmosis.

[0029] 2. Experimental Materials and Methods

[0030] 2.1 Cells and Toxoplasma gondii

[0031] The RH strain, GFP-RH strain, and ΔROP18 RH strain were all passaged in HFF cells. HFF cells were passaged in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin and cultured at 37°C in a 5% CO2 incubator. Toxoplasma gondii were cultured in a monolayer of HFF cells. After the parasites ruptured, they were filtered through a 5μm filter, centrifuged at 2500 rpm for 10 min to collect the parasites, and stored at -80°C.

[0032] 2.2 Chemical Reagents

[0033] Thirty-two small molecules and eight small molecule homologs were purchased from Shanghai Taoshu Biotechnology Co., Ltd., and sulfadiazine (SDZ) was purchased from Shanghai Yuanye Co., Ltd. All small molecules and SDZ were dissolved in DMSO at a storage concentration of 50 mM and diluted using culture medium or DMEM. The concentration of DMSO in the small molecules added intracellularly was less than 0.1%.

[0034] 2.3 ROP18 protein purification

[0035] The ROP18-expressing strain was inoculated into 1 L of autoclaved LB medium and incubated overnight at 37°C and 2000 rpm in a shaker. The OD of the bacterial culture was then measured. 600 Induction was performed overnight at a glutathione level of 0.6-0.8, followed by overnight incubation with isopropyl thio-β-D-galactopyranoside (IPTG). The bacterial pellet was collected by centrifugation at 4000 rpm for 20 min, resuspended in resuspending buffer, and lysed using an ultrasonic homogenizer with 1 mM PMSF. The supernatant was then separated by centrifugation at 14000 rpm for 50 min at 4 °C. The supernatant was purified twice using a nickel-affinity column, and the sample was collected and eluted to remove contaminating proteins. Finally, elution buffer of 250 mM imidazole was added for protein elution, and the protein eluent was purified again using an anion exchange chromatography column. The purified protein was concentrated to 2 mL using a concentrate tube, and the concentrate was slowly transferred to an EP tube and centrifuged at 14000 rpm for 8 min at 4 °C. Finally, the obtained protein was further purified by gel filtration molecular sieve chromatography. The protein sample was collected from the chromatography column and detected by SDS-PAGE to confirm the purity and molecular weight of the protein. The protein concentration was determined using a BCA protein quantification kit.

[0036] 2.4 Kinase Activity Assay

[0037] ROP18 protein was diluted to 1 μM with enzyme activity reaction buffer and incubated for 20 min in 96-well plates with substrate peptide (0.2 μM) and different concentrations of the small molecule (0 μM, 1 μM, 10 μM, 25 μM, 50 μM, 100 μM), with DMSO as a negative control. ATP (40 μM) was added to initiate the kinase reaction, which was carried out for 30 min. Simultaneously, different concentrations of ATP standards were added to other wells to plot a standard curve. After the reaction, Kinase-Lumi was added to each well. TM An enhanced chemiluminescence immunoassay reagent was used to detect the chemiluminescence signal by reacting at room temperature for 10 minutes using a microplate reader. A standard reaction curve was plotted based on the concentration of ATP standard and the measured fluorescence value. The amount of ATP remaining in the sample wells was calculated based on the standard curve, and then the ROP18 kinase activity was calculated.

[0038] 2.5 Biomembrane Interference Technology (BLI)

[0039] The purified ROP18 protein was pretreated with desalting, followed by elution with PBST buffer and protein collection. The treated protein was biotinylated at 37°C for 30 min, and excess biotin was removed using a desalting column. The biotinylated protein was then immobilized onto an SA probe using streptavidin (SA) coupling. The target small molecules were diluted at different concentrations, and the biotinylated and immobilized protein probes were exposed to solutions of different concentrations of the target small molecules according to the instruction manual. The experiment was run using Data Acquisition software, and signal subtraction and data fitting were performed using analysis software after the experiment.

[0040] 2.6 Small molecule cytotoxicity IC 50 Measurement

[0041] HFF cells were used for the assay of small molecule toxicity, 2×10 4 HFF cells were seeded in 96-well plates and cultured overnight at 37°C with 5% CO2. Diluted small molecules (1000 μM, 800 μM, 500 μM, 300 μM, 200 μM, 150 μM, 100 μM, 50 μM, 25 μM, 5 μM) were added sequentially to the cells, and the cells were incubated for 48 h. After 48 h, aCellTiter was added to the 96-well plates according to the manufacturer's instructions. AQueous One Solution Cell Proliferation Assay. After 2 hours of incubation, cell absorbance was measured at 490 nm using a microplate reader. The IC50 of each small molecule was calculated using Graphpad Prism 8.0. 50 value.

[0042] 2.7 Small molecule insecticidal activity EC 50 Measurement Experiment

[0043] 5×10 4 One HFF cell was seeded in each well of a 24-well plate and incubated overnight at 37°C in 5% CO2. 1×10⁻⁶ HFF cells were then added. 5 The purified GFP-RH was co-cultured for 4 h. After 4 h, the culture medium was replaced with medium containing different drug concentrations (100 μM, 50 μM, 25 μM, 5 μM), and cultured for another 48 h. DMSO was used as a negative control. Five random fields of view were photographed using an Olympus fluorescence microscope at 40×. Fluorescence intensity was analyzed using ImageJ, and the EC50 of each small molecule was calculated using Graphpad Prism 8.0. 50 value.

[0044] 2.8 Plaque Experiment

[0045] 1.5×10 5 HFF cells were seeded in 96-well plates and cultured overnight at 37°C in 5% CO2. Purified Toxoplasma gondii GFP-RH cells were added to 6-well plates at 200 cells per well and cultured for 7 days. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After staining with 2% crystal violet at room temperature for 30 min, followed by PBS washing, images were taken using an inverted microscope, and the area of ​​empty spots was statistically analyzed using ImageJ.

[0046] 2.9 Toxoplasma gondii invasion experiment

[0047] 5×10 4 One HFF cell was seeded in each well of a 24-well plate and incubated overnight at 37°C with 5% CO2. 1 x 10⁻⁶ HFF cells were added to each well. 5 Purified GFP-RH and small molecules at different concentrations (100 μM, 50 μM, 25 μM, 5 μM) were co-incubated for 1 h. Uninvaded Toxoplasma gondii were washed away, and the cells were cultured for another 24 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and HFF cell nuclei were stained with DAPI. Five different fields of view were randomly photographed using a fluorescence microscope. The number of host cells and Toxoplasma gondii vacuoles in each field of view was statistically analyzed using ImageJ. The invasion rate was calculated as the number of Toxoplasma gondii vacuoles per image divided by the number of host cells.

[0048] 2.10 Toxoplasma gondii Replication Experiment

[0049] 5×10 4 One HFF cell was seeded in each well of a 24-well plate and incubated overnight at 37°C in 5% CO2. 1×10⁻⁶ HFF cells were then added. 5The purified GFP-RH was co-cultured for 4 h. After 4 h, the culture medium was replaced with medium containing different drug concentrations (100 μM, 50 μM, 25 μM, 5 μM), and cultured for another 24 h and 48 h. DMSO was used as a negative control, and sulfadiazine was used as a positive control. The number of tachyzoites in 100 vesicles was observed at 40× using an Olympus fluorescence microscope at 24 h and 48 h, and statistical analysis was performed using Graphpad Prism 8.0.

[0050] 2.11 Irga6 Recruitment Experiment

[0051] RAW264.7 cells (3 × 10⁻⁶) 4 Cells were seeded (6 × 10⁶ cells / well) into 24-well plates with climbing sheets and cultured overnight at 37°C in 5% CO₂. IFNγ (20 ng / mL) and LPS (10 ng / mL) were added to the plates and co-cultured for 24 h to induce cell differentiation. The purified RH cell line (6 × 10⁶ cells / well) was then seeded into the plates. 4 Cells were added to wells with 100 μM of a small molecule and cultured for 2 h. DMSO and ΔROP18RH strains were used as controls. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.5% TRITON-X100 PBS at room temperature for 20 min. After PBS washing, cells were blocked with 5% goat serum and incubated at 37°C for 1 h. Rabbit anti-Irga6 polyclonal antibody (1:500) and mouse anti-SGA1 monoclonal antibody (1:500) were added, and the cells were incubated overnight at 4°C in a humidified chamber. After PBS washing, goat anti-mouse IgG labeled with Fluor594 and goat anti-rabbit IgG fluorescent secondary antibodies (1:200) were added, and the cells were incubated at 37°C for 1 h. After PBS washing, mounting medium containing DAPI was added to the center of the slide for mounting. The intensity of green and red fluorescence was observed using an Olympus fluorescence microscope at 40×, and five fields of view were randomly photographed. The recruitment of Irga6 in the experimental and control groups was statistically analyzed using ImageJ and Graphpad Prism 8.0.

[0052] 3. Results

[0053] 3.1 Preliminary screening of small molecules that can inhibit ROP18 kinase activity

[0054] To determine the inhibitory activity of 40 small molecules against ROP18 kinase activity, we selected Kinase-Lumi. TM Kinase activity was measured using an enhanced chemiluminescence immunoassay kit. Results are as follows: Figure 1As shown in A and B, small molecules 2 and 38 exhibited inhibition of ROP18 protein kinase at 25 μM (P < 0.01). At a concentration of 100 μM, both small molecules significantly inhibited ROP18 protein kinase activity (P < 0.001), with both inhibitory effects showing a dose-dependent manner. The affinity of small molecules 2 and 38 for ROP18 protein was further determined using BIL. It was found that the binding of small molecule 2 to ROP18 protein was slow-binding and slow-dissociation, with a KD value of 16.5 μM, while the binding of small molecule 38 to ROP18 protein was fast-binding and fast-dissociation, with a KD value of 96.9 μM. Figure 1 (C, D) This indicates that both small molecules 2 and 38 have a strong binding ability to ROP18, and both can inhibit the kinase activity of ROP18.

[0055] 3.2 Determination of cytotoxicity and anti-Toxoplasma gondii activity of small molecules No. 2 and No. 38

[0056] We utilize a CellTiter The AQueous One Solution Cell Proliferation Assay kit determined the levels of small molecules 2 and 38 (structural formula shown) at different concentration gradients. Figure 2 A and B) showed cytotoxicity to HFF, respectively. The results indicated that the IC50 of small molecule 2 was [missing information]. 50 The IC50 value for the 38th small molecule was 302.9 μM. 50 At concentrations greater than 1000 μM, both showed low toxicity to HFF cells. Next, we determined the effects of small molecules 2 and 38 on Toxoplasma gondii activity at safe concentrations, obtaining dose-Toxoplasma gondii growth inhibition curves for small molecules 2 and 38, respectively. Figure 2 C, D). The results indicate that the EC of small molecule number 2 50 The EC50 of small molecule number 38 was 31.68 μM. 50 The concentration was 34.24 μM, and the antiparasitic effects of both drugs showed a clear dose-dependent manner. Further analysis calculated the therapeutic index of molecule 2 to be 8.9, while that of molecule 38 was greater than 29.2. Therefore, both molecule 2 and molecule 38 can be considered potential candidate drugs for treating Toxoplasma gondii.

[0057] 3.3 Determination of the effects of small molecules No. 2 and No. 3 on Toxoplasma gondii growth

[0058] We used fluorescence-based growth assays and plaque assays to determine the effects of small molecules No. 2 and No. 38 on the growth of *Toxoplasma gondii*. The results are as follows: Figure 3As shown in Figure A, compared with the control group, the fluorescence intensity gradually decreased with increasing concentrations of small molecules 2 and 38. When the concentrations of small molecules 2 and 38 exceeded 25 μM, the fluorescence intensity significantly decreased; the average fluorescence intensity was only 9% at a concentration of 100 μM for small molecule 2 and only 12.4% at a concentration of 100 μM for small molecule 38. Figure 3 (B, C) This indicates that both small molecules 2 and 38 can significantly inhibit Toxoplasma gondii growth. Plaque assay results showed that, compared to the control group, when the concentrations of small molecules 2 and 38 were 25 μM, the number and size of plaques formed by Toxoplasma gondii in the wells were reduced. With increasing concentrations of both small molecules, the number and size of plaques in the wells decreased significantly. When the concentrations of the two small molecules reached 100 μM, no plaques formed by Toxoplasma gondii were observed in the wells, which is consistent with the effect of administering 200 μM sulfadiazine. Figure 4 (A, B, C). The above results indicate that both small molecules No. 2 and No. 38 can inhibit the growth of Toxoplasma gondii, and their inhibitory effect is dose-dependent.

[0059] 3.4 Determination of the effects of small molecules No. 2 and No. 38 on Toxoplasma gondii invasion

[0060] To further determine whether small molecules No. 2 and No. 38 affect Toxoplasma gondii invasion, we conducted Toxoplasma gondii invasion experiments with both small molecules. The results showed that when small molecules No. 2 and No. 38 were administered at 5 μM, Toxoplasma gondii invasion began to be inhibited (P > 0.05). Compared with the control group, when the concentration of small molecules No. 2 and No. 38 reached 25 μM, the invasion rate of Toxoplasma gondii decreased by 60.39% (P < 0.05) and 16.08% (P > 0.05), respectively. When the concentration of both small molecules reached 50 μM, the invasion rate of Toxoplasma gondii decreased by 68.47% (P < 0.05) and 39.75% (P < 0.05), respectively. When the dosage of small molecule No. 2 was increased to 100 μM, the invasion rate of Toxoplasma gondii was only 8.7%, a decrease of 82.85% compared with the control group (P < 0.001). When the dosage of small molecule drug 38 reached 100 μM, the Toxoplasma gondii invasion rate was 24.94%, which was 46.28% lower than the control group. Figure 5 (A, B). These results indicate that both small molecules 2 and 38 can inhibit the invasion process of Toxoplasma gondii, and the higher the dose concentration, the more significant the inhibitory effect on Toxoplasma gondii invasion.

[0061] 3.5 Effects of small molecules No. 2 and No. 38 on intracellular replication of Toxoplasma gondii

[0062] Next, we determined the effects of small molecules No. 2 and No. 38 on intracellular replication of the GFP-RH strain. We treated *Toxoplasma gondii* with two small molecules at safe concentrations (5 μM, 25 μM, 50 μM, 100 μM), a negative control (DMSO), and a positive control (sulfadiazine). One hundred vesicles were randomly selected from each sample group for statistical analysis. The results are as follows: Figure 6 As shown in Figures A and B, 24 hours after administration of small molecules No. 2 and No. 38, respectively, the proportions of vesicles containing 4 and 8 Toxoplasma gondii tachyzoites in the 5 μM group were as high as 79% and 81%, respectively, which were not statistically significant compared with the control group. In the 25 μM group, vesicles containing 4 and 8 Toxoplasma gondii tachyzoites also had the highest proportions, at 72.5% and 69.5%, respectively, which were not statistically significant. In the 50 μM group, the proportions of vesicles containing 1 and 2 Toxoplasma gondii were approximately 76% and 61%, respectively (P < 0.01 and 61%, P < 0.05). In the 100 μM group, all vesicles contained only 1 or 2 Toxoplasma gondii tachyzoites (P < 0.01). 48 hours after the addition of small molecules No. 2 and No. 38, at concentrations ≥ 50 μM, Toxoplasma gondii replication was significantly inhibited. In the 100 μM group, all vesicles also contained only 1 or 2 Toxoplasma gondii tachyzoites (P < 0.01). Figure 6 (C, D). The above results indicate that both small molecules No. 2 and No. 38 can inhibit the proliferation of Toxoplasma gondii in HFF cells, and the inhibitory effect is dose-dependent.

[0063] 3.6 Effects of small molecules No. 2 and No. 38 on the recruitment of Irga6 by Toxoplasma gondii

[0064] ROP18 can recognize and eliminate Irga6 in the host, protecting the parasite's PVM membrane from clearance. This is one of the main mechanisms by which Toxoplasma gondii evade the host's immune system. To investigate whether small molecules 2 and 38 exert their antiparasitic effects intracellularly by blocking the mechanism of action of ROP18, we conducted an Irga6 recruitment experiment. We randomly counted the recruitment of Irga6 in 100 Toxoplasma gondii vesicles from different groups after administration of 100 μM of small molecules 2 and 38. The results showed that, compared with the control group, Irga6 recruitment was detected in approximately 30.47% of the vesicles in the small molecule 2 group (P < 0.01), and in approximately 14.23% of the vesicles in the small molecule 38 group (P < 0.05). These results indicate that both small molecules 2 and 38 can inhibit the biological effects of ROP18 in vivo. To further investigate the specificity of small molecules 2 and 38 in inhibiting ROP18, we administered small molecules 2 and 38 to the ΔROP18 RH strain, respectively. The results showed no significant difference in the recruitment of Irga6 into the vesicles of the larvae in groups 2 and 38 compared to the control group (P>0.05). Figure 7 (A, B) This suggests that small molecules 2 and 38 do not disrupt the recruitment of Irga6 in the host, and that the inhibition of ROP18 by both small molecules is specific. These results indicate that small molecules 2 and 38 have high safety and can inhibit Toxoplasma gondii through the mechanism of inhibiting ROP18.

[0065] in conclusion

[0066] Bioactivity analysis of 40 small molecules selected through pocket screening targeting ROP18 revealed two small molecules with high safety and good antiparasitic activity: molecule 2 and molecule 38. Both small molecules exhibit strong affinity for ROP18 protein and inhibit ROP18 protein kinase activity. In in vitro experiments, we found that both molecule 2 and molecule 38 inhibited the growth, invasion, and intracellular replication of Toxoplasma gondii. The antiparasitic mechanism of these two small molecules is relatively clear; they achieve good in vivo antiparasitic effects by specifically inhibiting the biological effects of ROP18. Therefore, molecule 2 and molecule 38 show promise as candidate drugs for the treatment of toxoplasmosis.

[0067] Example 2: In vivo anti-Toxoplasma gondii activity assay of small molecules No. 2 and No. 38

[0068] Experimental Materials and Methods

[0069] In vivo anti-Toxoplasma gondii activity assay of small molecules No. 2 and No. 38

[0070] Female SPF-grade BALB / c mice aged 6 to 8 weeks were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All mice were housed under specific pathogen-free (SPF) conditions at an appropriate temperature (25±2℃), with adequate light, food, and water. Mice were housed for one week prior to the experiment. Mice No. 2 and No. 38 were first intraperitoneally injected with a dose of 100 mg / kg for 7 consecutive days, and observed for 30 days. No obvious abnormalities were observed during this period, indicating that the dosage of the two small molecules was safe. BALB / c mice were randomly divided into 4 groups of 15 mice each, and each mouse was intraperitoneally injected with 100 RH strain tachyzoites. Treatment with the small molecule compound began 4 hours after Toxoplasma gondii infection. The in vivo small molecule administration formulation consisted of 10% DMSO, 40% PEG-300, 5% Tween 80, and 45% ddH2O. The treatment doses for each group of mice were as follows: small molecule treatment group No. 2 (100 mg / kg), small molecule treatment group No. 38 (100 mg / kg), positive control group (100 mg / kg sulfadiazine), and negative control group (same volume of DMSO stock solution). Each group of mice was injected once daily for 7 consecutive days, and observed for 30 days. Before the onset of disease, five mice from each group were euthanized, and liver, spleen, and brain tissue were collected from each mouse. DNA was extracted from each tissue using a DNA extraction kit. The expression of the Toxoplasma gondii B1 gene in each tissue of each group of mice was detected using qPCR, and a standard curve was constructed using the Toxoplasma gondii RH strain to calculate the Toxoplasma gondii load in different tissues of each mouse.

[0071] result

[0072] Determination of the in vivo anti-Toxoplasma gondii activity of small molecules No. 2 and No. 38

[0073] To detect the in vivo antiparasitic activity of small molecules No. 2 and No. 38, we conducted in vivo anti-Toxoplasma gondii experiments using mice infected with Toxoplasma gondii RH strain as a model. Mice were randomly divided into a No. 2 small molecule treatment group, a No. 38 small molecule treatment group, a negative control group, and a positive control group. Each mouse in each group was intraperitoneally injected with 100 Toxoplasma gondii RH strain tachyzoites. Four hours after infection, mice were treated with 100 mg / kg of No. 2 or No. 38 small molecules for 7 days (the negative control group received the same volume of saline, and the positive control group received 100 mg / kg of sulfadiazine). Mice survival was observed for 30 days. The survival results showed that compared with the negative control group, mice treated with No. 2 small molecule had a 55% increased survival rate, and mice treated with No. 38 small molecule had a 42% increased survival rate. Figure 8A). Next, we measured the Toxoplasma gondii load in the brain, liver, and spleen tissues of mice in each group. We found that compared to the control group, the spleen swelling was significantly less in the No. 2 small molecule treatment group, and the number of Toxoplasma gondii in the brain, spleen, and liver tissues of the mice in the treatment group was significantly reduced (P<0.05, P<0.01, P<0.001). In the No. 38 small molecule treatment group, the number of Toxoplasma gondii in the brain, spleen, and liver tissues of mice was also significantly reduced (P<0.05, P<0.001, P<0.001). Figure 8 (B, C, D). The above results indicate that small molecules No. 2 and No. 38 also have good anti-insect effects in vivo.

Claims

1. A small molecule compound, characterized in that, The structural formula of the compound is shown in Formula 1:

2. The use of the small molecule compound as described in claim 1 in the preparation of drugs for the prevention and treatment of Toxoplasma gondii.

3. The application according to claim 2, characterized in that, The drug can be administered orally, by injection, or by topical application.

Images