Use of triazoloquinoline derivative in preparation of drug for preventing or treating malaria

Abstract

A use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in inhibiting the expression of an invasive gene PfAP2-O5, blocking the invasion of plasmodium falciparum into red blood cells, affecting the growth and development of plasmodium falciparum, and killing plasmodium falciparum. A pharmaceutical composition comprising the triazoloquinoline derivative can also comprise an artemisinin compound.

Description

Application of triazoloquinoline derivatives in the preparation of drugs for preventing or treating malaria Technical Field

[0001] The present invention relates to the field of medical technology, and more particularly to the use of triazoloquinoline derivatives in preparing medicines for preventing or treating malaria. Background Art

[0002] Malaria, caused by infection with the protozoan Plasmodium, is a parasitic disease that poses a serious threat to human health and life. Its morbidity and mortality rates are among the highest among infectious diseases. According to the 2021 World Malaria Report released by the WHO, malaria is endemic in 87 countries and regions worldwide, with 241 million new cases and approximately 627,000 deaths in 2020.

[0003] The life cycle of the malaria parasite is complex, primarily consisting of sexual reproduction within the definitive host and asexual reproduction within the intermediate host. In humans, the parasite (sporozoites) first invades the liver, undergoes a period of asexual schizont proliferation, and then releases merozoites that enter the bloodstream and invade red blood cells, beginning the intracellular schizont phase. This phase is when clinical malaria develops, and merozoite invasion of red blood cells is a key step in the pathogenesis of malaria.

[0004] Currently, drug treatment is the primary means of preventing and treating malaria, with artemisinin-based combination therapy effectively curbing the spread of malaria worldwide. However, with the long-term and widespread use of artemisinin-based drugs, strains of parasites with reduced sensitivity to them have gradually emerged in Southeast Asia and even Africa. The emergence and global spread of these strains pose a severe challenge to global malaria prevention and control, as well as malaria elimination strategies. Therefore, identifying new targets for antimalarial drugs, especially developing safe and effective transmission-blocking drugs that target key links in the pathogenesis of Plasmodium, is crucial to curbing the spread of malaria.

[0005] Summary of the Invention

[0006] The present invention provides a new medicine for preventing or treating malaria in order to overcome the above-mentioned shortcomings.

[0007] Another object of the present invention is to provide new applications of triazoloquinoline derivatives.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a transcription factor PfAP2-05 inhibitor of Plasmodium falciparum, wherein the triazoloquinoline derivative has a structure as shown in formula (I):

[0010] The inventors conducted in-depth research on the mechanism of Plasmodium falciparum invasion of erythrocytes and discovered for the first time that PfAP2-O5 positively regulates erythrocyte invasion. The triazoloquinoline derivatives represented by formula (I) can inhibit the expression of the invasion gene PfAP2-O5, blocking the invasion of erythrocytes by Plasmodium falciparum, affecting its growth and development, and achieving a parasite-killing effect.

[0011] The PfAP2-O5 inhibitors of the present invention refer to compounds that achieve the effect of killing Plasmodium falciparum by inhibiting the function of AP2-O5.

[0012] In vitro experiments also showed that the triazoloquinoline derivatives of formula (I) exhibited significant growth inhibition of Plasmodium falciparum and were virtually non-toxic to mammalian cells, making them viable as drugs. They also demonstrated potent killing effects against resistant strains, significantly helping to slow the development and spread of drug resistance and leading to more efficient antimalarial treatment.

[0013] Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug for inhibiting Plasmodium falciparum; the triazoloquinoline derivative has a structure as shown in formula (I):

[0014] Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug for preventing or treating malaria caused by Plasmodium falciparum, wherein the triazoloquinoline derivative has a structure as shown in formula (I):

[0015] In the present invention, the "pharmaceutically acceptable salt" includes the salt formed by the triazoloquinoline derivative and an inorganic acid or an organic acid.

[0016] The inorganic acid is, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc. The organic acid is, for example, acetic acid, glycolic acid, propionic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclic acid, cyclic salicylic acid, p-aminosalicylic acid, etc.

[0017] In the present invention, the "hydrate" refers to a salt containing water molecules bound in a certain ratio as a component of the crystal.

[0018] A pharmaceutical composition comprising a triazoloquinoline derivative having a structure as shown in formula (I) or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient as an adjuvant;

[0019] In preparing these compositions, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed in a carrier which may be in the form of a capsule or sachet. When the excipient acts as a diluent, it may be a solid, semisolid or liquid material which acts as an excipient, carrier or medium for the active ingredient.

[0020] Examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup and methylcellulose. In addition, the composition may also include lubricants (such as talc, magnesium stearate and mineral oil), wetting agents, emulsifiers and suspending agents, preservatives (such as methyl hydroxybenzoate and propyl hydroxybenzoate), sweeteners or flavoring agents.

[0021] Therefore, the pharmaceutical composition can be prepared into various known dosage forms. Preferably, the dosage form prepared from the pharmaceutical composition is an oral dosage form or an injection dosage form.

[0022] Oral dosage form includes solid dosage forms such as tablets, dripping pills, quick-release dripping pills, capsules (such as liquid gel capsules and solid gel capsules), granules or powders.Tablets can be compressed tablets, abrasive tablets, enteric-coated tablets, sugar-coated tablets, film-coated tablets or multilayer compressed tablets.As the suitable auxiliary agent type for preparing tablets, include but are not limited to adhesives, lubricants, diluents, disintegrants, coloring agents, flavorings, drainage agents or melting agents.Oral dosage form can also be liquid oral dosage form, such as aqueous solution, emulsion, suspension, solution and / or suspension made by non-effervescent granules or the effervescent agent made by effervescent granules.As the suitable auxiliary agent type for preparing liquid oral agents, include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, coloring agents or flavorings.

[0023] The injection dosage forms include: injection solution (ordinary), freeze-dried powder injection, powder injection (ordinary), tablets for injection, etc.

[0024] Specifically, the pharmaceutical composition can be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

[0025] Through the Plasmodium falciparum growth inhibition experiment, with artemisinin as a control, the EC50 value of the present invention on the growth inhibition of Plasmodium falciparum reached 2.632 μM, showing good growth inhibition function.

[0026] The pharmaceutical composition may contain a triazoloquinoline derivative having a structure represented by formula (I) or a pharmaceutically acceptable salt or hydrate thereof as the sole active ingredient, and may also contain a known artemisinin-like compound as a common active ingredient.

[0027] Preferably, the active ingredient also includes an artemisinin compound. Artemisinin compounds have been shown to have therapeutic effects on malaria. More preferably, the artemisinin compound is a dihydroartemisinin compound. Dihydroartemisinin compounds have better effects against multi-drug-resistant malarial parasites than standard artemisinin compounds.

[0028] Preferably, in the composition, the molar ratio of the triazoloquinoline derivative having the structure represented by formula (I) or its pharmaceutically acceptable salt or hydrate to the artemisinin compound is 1:500 to 500:1.

[0029] Preferably, the pharmaceutical composition is in the form of an oral dosage form or an injection dosage form.

[0030] The pharmaceutical composition is used in preparing drugs for preventing and / or treating malaria.

[0031] Preferably, the pharmaceutical composition is used in the preparation of a drug for treating malaria resistant to artemisinin and its analogs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a novel application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof. The triazoloquinoline derivative can inhibit the expression of the invasion gene PfAP2-O5, block the invasion of Plasmodium falciparum into red blood cells, affect its growth and development, and achieve a killing effect on Plasmodium falciparum. Therefore, the triazoloquinoline derivative can be used as a PfAP2-O5 inhibitor, a drug for inhibiting Plasmodium, or a drug for preventing or treating malaria. Based on the above discovery, the present invention provides a novel pharmaceutical composition, which can use a triazoloquinoline derivative as a single active ingredient or be combined with an artemisinin compound as a co-active ingredient; the pharmaceutical composition can be used to prepare a drug for treating malaria, especially a drug for treating malaria that is resistant to artemisinin and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In Figure 1, Figure 1A shows the WB identification results of the PfAP2-O5 gene knockout strain. After the PfAP2-O5 gene was knocked down, the expression level of PfAP2-O5 protein was reduced; Figure 1B shows the growth curve test of the PfAP2-O5 gene knockout strain.

[0035] Figure 2 shows the distribution of PfAP2-O5 across the genome by ChIP-seq analysis; as well as the types of PfAP2-O5-binding target genes, most of which are invasion gene families.

[0036] Figure 3 illustrates the inhibitory effects of triazoloquinoline derivatives on invasion gene expression. Figure 3A uses RNA-Seq technology to determine invasion gene expression in a PfAP2-O5 knockout strain. Figure 3B shows invasion gene expression after PfAP2-O5 gene function is inhibited by triazoloquinoline derivatives. Figure 3C shows the inhibitory effects of PfAP2-O5 knockout and triazoloquinoline derivative treatment on invasion gene expression.

[0037] Figure 4 is a schematic diagram showing the quantitative growth inhibition effect of triazoloquinoline derivatives on in vitro cultured Plasmodium. The EC50 of the quantitative growth inhibition effect on in vitro cultured Plasmodium is 2.632 μM, and the growth of Plasmodium is determined using a 3-day SYBR Green I cell growth assay.

[0038] Figure 5 shows the cytotoxic effects of the triazoloquinoline derivatives on the human renal epithelial cell line 293T and the human liver cancer cell line HepG2. Also shown are the selectivity indices between the inhibitory effects of the small molecule inhibitors on Plasmodium and their toxicity to different cell lines. Cell growth was measured using a 3-day CCK8 assay.

[0039] FIG6 shows the killing effect of the triazoloquinoline derivatives as a combination drug with dihydroartemisinin on resistant insect strains. A ring body survival experiment was used to clarify the growth inhibitory effect of the combined drug on resistant insect strains. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention will be described clearly and completely below, but the implementation of the present invention is not limited thereto.

[0041] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] 1. Triazoloquinoline derivatives of the structure shown in formula (I): purchased from ChemDiv ID.V003-8956

[0043] 2. Plasmodium falciparum (3D7_G7): purchased from ATCC

[0044] 3. 293T cell line: purchased from ATCC

[0045] 4. HepG2 cell line: purchased from ATCC

[0046] 5. SYBR Green I: purchased from Invitrogen Cat. No. S-7585

[0047] 6. Pancreatin-EDTA: purchased from Gbico Cat.No.25200-072

[0048] 7. DMEM culture medium: purchased from Gbico Cat. No. 11965-092

[0049] 8. FBS: purchased from Gbico Cat. No. 10082-147

[0050] 9. Double-stranded DNA quantitative detection kit: purchased from ThermoFisher Cat.No.P7581

[0051] 10. Protein A / G magnetic beads: purchased from ThermoFisher Cat.No.88803

[0052] 11. Mouse monoclonal antibody: purchased from Sigma Cat.No.SAB4800032

[0053] 12. 37% formaldehyde solution: purchased from Sigma Cat. No. 47608-250ML-F

[0054] 13. 96-well plate: purchased from Corning

[0055] 14. Red blood cells: purchased from Shanghai Blood Center

[0056] 15. Dihydroartemisinin (CAS: 71939-50-9)

[0057] 16. The components of various buffers are as follows:

[0058] (1) Lysis buffer

[0059] (2) Ultrasonic buffer

[0060] (3) ChIP dilution buffer

[0061] (4) Low salt immune complex washing buffer

[0062] (5) High salt immune complex washing buffer

[0063] (6) Lithium chloride immune complex washing buffer

[0064] (7)TE buffer

[0065] (8) Elution buffer

[0066] Example 1 Plasmodium culture

[0067] Plasmodium falciparum was cultured using RPMI 1640 complete medium in a 37°C incubator (5% CO2, 5% O2, and 90% N2).

[0068] 0.5 L RPMI 1640 complete medium contains: 5.22 g RPMI-1640 powder; 1.0 g sodium bicarbonate; 5 g AlbmaxI; 2.98 g 25 mM HEPES; 13.6 mg hypoxanthine; and 10 mg gentamicin sulfate.

[0069] Example 2 Construction of transgenic insect strain: PfAP2-05 conditional knockdown insect strain

[0070] In this experiment, the CRISPR / Cas9 gene editing system was used to clone the guide RNA into a vector and construct a PfAP2-O5 conditional knockout strain.

[0071] The specific construction steps are as follows:

[0072] 1. Construction of a knockdown vector for transgenic insect strains (named pL6cs-ap2-o5-glmS)

[0073] Use the CRISPR / Cas9 gene editing system to clone the guide RNA into the vector as follows:

[0074] (1) gRNA design

[0075] The sequence of the PfAP2-O5 gene of Plasmodium falciparum strain 3D7-G7 was downloaded from the PlasmoDB website (from the database https: / / plasmodb.org / plasmo / app, Plasmodium falciparum: PF3D7_1449500 (plasmodium falciparum) 715aa PfAP2-O5. This sequence is also disclosed in NCBI: Sequence ID: XM_001348609.1). Through BLAST comparison, a specific sequence of 20 bp in length was selected as the guide RNA (gRNA). The sequence is as follows: 5′-ACCAAGTCATATGTCATTAA-3′.

[0076] (2) Primer design

[0077] Primers were designed using SnapGene software:

[0078] P4: 5′-TTTTTTACAAAATGCTTAAGAACTAGCGTATGTGATAACTTAG-3′ is used to amplify the homology arm of the PfAP2-O5 gene and introduce synonymous mutation sites. The introduction of synonymous mutation sites is to prevent gRNA from self-cleaving the plasmid;

[0079] P8: 5′-TTTATTTTTTTTGCGGCCGCGCGGCCGCGTCCCCTC-3′ is used to amplify the exogenous green fluorescent protein (GFP) gene.

[0080] (3) Gene amplification

[0081] The genetic DNA of Plasmodium falciparum strain 3D7-G7 was extracted, and the left and right homologous sequences of the PfAP2-O5 gene were amplified using primers P1+P2 and P3+P4, respectively, to introduce mutation sites. The glmS ribozyme sequence was amplified using primers P7+P8 (the glmS sequence was obtained from the literature report: doi:10.1371 / journal.pone.0073783).

[0082] (4) Construction of ligation product

[0083] The left and right homologous sequences were ligated to the glmS ribozyme sequence using primers to obtain a ligation product. The 3' end of the PfAP2-O5 gene was labeled with the glmS ribozyme sequence.

[0084] The reaction system was as follows: 2×Phanta Max Master Mix (Dye Plus): 25 μL, P1 primer (10 μM): 2 μL, P4 primer (10 μM): 2 μL, 1 μL each of the left and right homologous sequences and glmS ribozyme sequence, and ddH2O was added to make up to 50 μL of the system.

[0085] PCR conditions were as follows: 95°C for 3 min, 95°C for 15 s, 65°C for 15 s, 65°C for 30 s / kb, 65°C for 5 min, for a total of 30 cycles; 72°C for 10 min.

[0086] (5) Construction of knockdown vector

[0087] The plasmid pL6cs (a self-developed plasmid, for its specific sequence, see the literature: doi:10.3389 / fmicb.2020.625862) carrying the resistance gene for the drug dihydrofolate reductase inhibitor (WR) was digested with XhoⅠ and AvrⅡ. The product was recovered and purified using a gel extraction kit. The gRNA fragments were ligated with DNA ligase and transformed into Escherichia coli XL-10 competent cells. The cells were inoculated onto LB agar plates containing 100 μg / ml ampicillin and cultured overnight. A single colony was picked and cultured overnight at 34°C with shaking at 200 rpm / s. The plasmid was then extracted and sequenced.

[0088] The plasmid connected to the gRNA was confirmed by sequencing and double-digested with AscⅠ and AflⅡ. The product was recovered and ligated with the homology arm containing the glmS ribozyme sequence for transformation. A single clone was picked for culture, and the correct plasmid was identified by PCR for sequencing. The strain was stored at -80℃.

[0089] The knockdown vector of the transgenic insect strain was obtained and named pL6cs-ap2-o5-glmS.

[0090] 2. Construction of PfAP2-O5 conditional knockout strain

[0091] A transgenic insect strain, PfAP2-O5 conditional knockdown strain, was constructed by electrofection combined with drug screening.

[0092] (1) Preparation of electroporation plasmid

[0093] The correct strain after sequencing was inoculated onto an LB agar plate containing 100 μg / ml ampicillin and cultured overnight. The next day, a single clone was picked and cultured in LB culture medium. The culture was shaken at 34°C and 200 rpm / s for 8 h, and then diluted to 400 ml of LB culture medium at a ratio of 1:1000. After shaking culture for 14 h, the bacteria were collected and the plasmid was purified using a plasmid DNA purification kit.

[0094] Take the above-constructed transgenic insect strain knockdown vector pL6cs-ap2-o5-glmS plasmid and the plasmid pUF1-Cas9 carrying the screening drug blasticidin (BSD) resistance gene and expressing Cas9 protein (the plasmid was built in the laboratory, the specific sequence can be found in the literature report: doi:10.3389 / fmicb.2020.625862) 100 μg each, add 1 / 10 volume of 3M CH3COONa (pH 5.2) solution of the plasmid solution, mix well, add 2.5 times the volume of anhydrous ethanol of the plasmid solution and mix thoroughly, centrifuge at 13000 × g for 30 minutes, discard the supernatant, wash the precipitate twice with 75% ethanol, dry it in the clean bench and dissolve the precipitate with 150 μl sterile water.

[0095] (2) Electroporation and screening of transgenic insect strains

[0096] Set the electroporator to 310V, 950μF, resistance ∞, and a 2mm slit. Add an equal volume (150μl) of 2× cell mixture to the prepared plasmid and mix thoroughly. Then, add 150μl of red blood cells. After mixing, transfer the mixture to an electroporation cuvette and place it in the electroporator for electroporation. After electroporation, transfer the mixture to a culture flask, add an appropriate amount of culture medium, and then add purified schizont-stage Plasmodium.

[0097] Culture was performed in a 37°C, three-gas incubator. Fresh culture medium was replaced daily starting on the second day. On the fourth day, when the electroporation density reached approximately 10% (parasite yield, defined as the ratio of infected red blood cells to all red blood cells within a given field of view (~5,000)), 200 μl of fresh red blood cells were added, the culture medium was replaced, and the screening drugs WR and BSD (blasticidin) were added. These screening drugs kill wild-type Plasmodium falciparum, while successfully transgenic parasites, carrying plasmids expressing drug-resistance genes, are unaffected by the drugs, allowing the selection of transgenic parasites.

[0098] After the screening drug is added until most wild parasites are killed, the culture medium is changed and the drug is added every 3-4 days to maintain the culture. Smear microscopy begins 21 days after transfection, and transgenic parasites are obtained in 3-6 weeks.

[0099] In summary, a transgenic insect strain was constructed: PfAP2-O5 conditional knockdown insect strain.

[0100] 3. Gradient dilution cloning

[0101] The transgenic strain (PfAP2-O5 conditional knockdown strain) obtained by genetic engineering is an integrated strain and requires gradient dilution cloning. That is, by screening through limiting dilution, a completely integrated monoclonal strain is obtained. This experiment is performed in a 96-well cell culture plate. The infected red blood cells are diluted to theoretical concentrations of 10, 1, 0.5, and 0.1 worms per ml, respectively. The specific experimental steps are as follows:

[0102] (1) Synchronize the transgenic insect strain with sorbitol to obtain annular stage insects with relatively uniform properties;

[0103] (2) Smear microscopy of the trophozoite stage of the parasite and strictly count the original protozoa rate P x;

[0104] (3) The trophozoite stage culture was collected and centrifuged at 2000 rpm at room temperature for 5 min. The supernatant was discarded to obtain approximately 200 μl of infected hematocrit (iRBC);

[0105] (4) Add 800 μl of fresh culture medium to the iRBCs, mix gently, and transfer to a 1.5 ml centrifuge tube to a final volume of 1 ml;

[0106] (5) Centrifuge at 1500 rpm for 2-3 min at room temperature, discard the supernatant, and transfer 50 μl of iRBC to a new centrifuge tube for later use;

[0107] (6) Dilute the parasite content of the 50 μl infected erythrocytes to 1% according to the formula 50 μl × P x = 1% × (50 μl + V x ) to obtain culture A (V x is the volume of fresh erythrocytes added), and confirm the parasite content after dilution by smear microscopy.

[0108] (7) Preheat fresh culture medium and, according to the principle of gradient dilution, dilute the infected red blood cells with a parasite rate of 1% prepared above in proportion so that each centrifuge tube corresponds to a gradient:

[0109] Prepare nine 15ml centrifuge tubes and number them 1, 2, 3, ... 9. Add 10ml of fresh culture medium to tube 1, 9ml of fresh culture medium to tubes 2-7 and 9, and 5ml of fresh culture medium to tube 8. Preheat all centrifuge tubes in a 37℃ incubator. Take 100μl of culture A with a 1% parasite rate in (6) and add it to the above tube 1 to obtain 1×10 6 Culture B with iRBCs / ml;

[0110] 1) Take 1 ml of culture B and transfer it to tube 2 to obtain 1×10 5 Culture C with iRBCs / ml;

[0111] 2) Take 1 ml of culture C and transfer it to tube 3 to obtain 1×10 4 Culture D of iRBCs / ml;

[0112] 3) Take 1 ml of culture D and transfer it to tube 4 to obtain 1×10 3 / ml iRBC culture E;

[0113] 4) Take 1 ml of culture E and transfer it to tube 5 to obtain 1×10 2 Culture F with iRBCs / ml;

[0114] 5) Transfer 1 ml of culture F to tube 6 to obtain culture G with 10 iRBCs / ml;

[0115] 6) Transfer 1 ml of culture G to tube 7 to obtain culture H containing 1 iRBC / ml;

[0116] 7) Transfer 5 ml of culture H to tube 8 to obtain culture I with 0.5 iRBCs / ml;

[0117] 8) Transfer 1 ml of culture H to tube 9 to obtain culture J with 0.1 iRBC / ml.

[0118] (8) The cultures G, H, I, and J with different gradients obtained above were transferred to a 96-well cell culture plate at 200 μl / well. Finally, each row of the 96-well plate contained 12 wells corresponding to one gradient.

[0119] (9) In the later stage of culture, fresh culture medium was replaced every 2 days, and fresh red blood cells (2 μl / well) were added every 6 days to maintain the culture;

[0120] After about 2 weeks, smears are examined under a microscope until live worms are detected. Then, the culture can be slowly expanded using 24-well, 12-well, and 6-well plates. After the genome is detected correctly, a fully integrated monoclonal PfAP2-O5 transgenic worm strain (PfAP2-O5 conditional knockdown worm strain) is obtained, which can be preserved and used for subsequent experiments.

[0121] Example 3 Identification and Growth and Development Test of PfAP2-O5 Transgenic Insect Strain (PfAP2-O5 Conditional Knockout Insect Strain)

[0122] Cultures of PfAP2-O5 transgenic parasites (conditionally knocked-down PfAP2-O5) were collected at the ring (10-15 hours), trophozoite (28-32 hours), and schizont (40-44 hours) stages. Protein samples were collected by lysing infected red blood cells with 0.15% saponin. Proteins were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes using a semi-dry transfer method at 0.1A for 2 hours. After transfer, add an appropriate volume of blocking solution (5% skim milk powder: skim milk powder dissolved in PBST solution) and block at room temperature for 2 hours. Incubate the primary antibody (mouse anti-Ty1 antibody): dilute the primary antibody (anti-Ty1) with 5% skim milk powder at a 1:1000 dilution, seal the membrane and incubate at 4°C overnight. Incubate the secondary antibody: dilute the secondary antibody (goat anti-mouse) with 5% skim milk powder at a 1:5000 dilution and incubate at room temperature for 2 hours. Develop the color: mix equal volumes of color development solutions A and B (be careful to avoid light), add a certain amount of color development solution to the membrane, and photograph and save the exposed image. The results are shown in Figure 1A. Figure 1A shows western blot identification of the PfAP2-O5 conditional knockdown strain before and after the addition of 5mM glucosamine (GlcN), indicating that PfAP2-O5 was effectively knocked down.

[0123] To test the growth curve of the PfAP2-O5 conditional knockout strain, the strains were synchronized several times to the ring stage, and the initial parasite prevalence was rigorously counted. The strains were then divided into two bottles (one bottle was treated with the corresponding concentration of GlcN, and the other bottle was left untreated). The initial parasite prevalence in each bottle was approximately 0.1%. Four life cycles were cultured continuously. Smears were taken from the mid- and late-stage stages of each life cycle, and the parasite prevalence was counted (high parasite prevalence necessitated medium replacement, and drug was added to the treated group). The parasite prevalence in the treated and untreated groups was recorded, and growth curves were plotted. The results are shown in Figure 1B, which shows the growth curves of the wild-type strain and the PfAP2-O5 conditional knockout strain, demonstrating that the growth of the Plasmodium was significantly inhibited after the PfAP2-O5 conditional knockout.

[0124] Example 4 Chromatin Immunoprecipitation (ChIP) Sequencing

[0125] Chromatin immunoprecipitation (ChIP) sequencing utilizes the specificity of the antigen-antibody reaction to truly reflect the distribution of AP2-O5-bound target genes across the genome at the chromatin level, while also analyzing the binding of the reaction protein to the target gene. The specific steps are as follows:

[0126] The synchronized schizont stage cultures were collected, 1% formaldehyde solution was added, and the cells were incubated at 37°C for 10 min. Pre-cooled 0.125 M glycine solution was added and incubated on ice for 5 min. The cells were centrifuged at 2500 rpm and 4°C for 5 min, the supernatant was discarded, 30 mL of 1× PBS was added to resuspend the red blood cells, and 0.15% saponin was added to lyse the red blood cells and collect the worms. 2 mL of pre-cooled Lysis Buffer was added to resuspend the worms and transfer them to a pre-cooled grinding tube and incubate on ice for 30 min. 200 μL of ultrasonic buffer was added to resuspend the precipitate and liquid phase ultrasonication was performed with the following parameters: duty cycle 5%, peak incident power 75 W, number of cycles 200, temperature 7°C, time 30 min. 10 μL of the sonicated mixture was added to 90 μL ChIP dilution buffer, 4 μL 5 M NaCl, 2.5 μL proteinase K, mixed well, and incubated at 50°C for 2 h. 2 μL RNase A was added and incubated at 37°C for 45 min. Extract with a PCR kit and examine the sonication effect with 2% agarose gel electrophoresis. Ideal sonication product is 14,000 pm. Centrifuge at 4°C for 10 minutes, collect the supernatant, and add 10 volumes of ChIP dilution buffer. Add protein A / G magnetic beads to the above solution and incubate at 4°C with shaking for 2 hours. Transfer the supernatant to a 1.5 mL centrifuge tube, add the corresponding antibody and protein A / G magnetic beads, and incubate at 4°C overnight. Wash the antibody / magnetic bead / chromatin complex with different buffers in the following order:

[0127] A: Low-salt immune complex wash buffer: 1 mL per tube, rotate and wash at 4°C for 5 min;

[0128] B: High-salt immune complex wash buffer: 1 mL per tube, rotate and wash at 4°C for 5 min;

[0129] C: Lithium chloride immune complex wash buffer: 1 mL per tube, rotate and wash at 4°C for 5 minutes;

[0130] D: Wash twice with TE buffer (1 mL per tube) at 37°C for 5 min with rotation.

[0131] Add 200 μL of elution buffer for elution, incubate with rotation at 37°C for 30 minutes, collect the supernatant, and add 180 μL of elution buffer to the DNA tube before immunoprecipitation; add 16 μL of 5 M NaCl to each of the above tubes, mix well, and incubate at 45°C overnight; after the incubation, add 0.8 μL of RNase A (20 mg / mL) to each tube and incubate at 37°C for 30 minutes; add 3 μL of proteinase K to each tube and incubate at 45°C for 2 hours; use QIAGEN PCR purification kit to extract sample DNA, elute twice with 10 μL of deionized water, and let it stand at room temperature for 10 minutes before each elution; determine the concentration and construct the library. To prepare sequencing libraries, 1.5 ng of ChIP-DNA was end-repaired, 3' adenylated, and adapter-ligated. After purification using Agencourt AMPure XP magnetic beads, the libraries were amplified using the KAPA HiFi PCR kit under the following conditions: 95°C for 1 minute, 98°C for 10 seconds, 65°C for 1 minute, 12 cycles, 65°C for 5 minutes, and 4°C for ∞. The libraries were sequenced on the Illumina HiSeq X Ten platform. Two biological replicates were performed for each ChIP-seq dataset.

[0132] The results are shown in Figure 2 , which shows the distribution of PfAP2-O5 protein target genes in the whole genome, such as the invasion gene family msps, raps, rhophs, ralp1, ama1, etc., proving that most of the target genes of PfAP2-O5 are related to invasion

[0133] Example 5 Transcriptome sequencing (RNA-seq) analysis of the effect of triazoloquinoline derivatives on PfAP2-O5

[0134] Wild-type and PfAP2-O5 conditional knockdown strains were treated with triazoloquinoline derivatives represented by formula (I). After strict strain synchronization, 1 μM triazoloquinoline derivatives were added one life cycle in advance. After one life cycle, cultures of the wild-type and PfAP2-O5 conditional knockdown strains were collected at the ring (10-15 hours), trophozoite (28-32 hours), and schizont (40-44 hours) stages. Total RNA was purified using the Direct-zol RNA kit, and strand-specific RNA sequencing libraries were prepared using KAPA mRNA beads. The RNA was then fragmented and sequenced on an Illumina HiSeq X Ten system. The differential gene expression between the two strains after inhibitor treatment was analyzed.

[0135] The results are shown in Figure 3. Figure 3A uses RNA-Seq technology to determine the expression of invasion genes after conditional knockdown of PfAP2-O5; Figure 3B uses RNA-Seq technology to determine the inhibitory effect of triazoloquinoline derivatives on invasion gene expression after inhibiting PfAP2-O5 function. The results show that after conditional knockdown of PfAP2-O5, invasion-related target genes are downregulated. In wild-type strains, treatment with triazoloquinoline derivatives also downregulated PfAP2-O5 target genes. For example, the expression levels of genes in the Plasmodium merozoite surface structural protein family, such as Rhoptry Neck, Peripheral surface protein, Rhoptry protein, Rhoptry bulb, Micronemes protein, and GPI-Anchored MSP, showed a significant decrease of more than 1.5-fold (at the mRNA level). (Genes of the Plasmodium merozoite surface structural protein family mainly regulate the invasion of Plasmodium merozoites into red blood cells. The main references are as follows: DOI: 10.1016 / j.chom.2017.07.003; DOI: 10.1016 / j.chom.2017.05.006); Figure 3C shows the intersection of invasion-related target genes after conditional knockdown of PfAP2-O5 and inhibition of PfAP2-O5 function by triazoloquinoline derivatives.

[0136] Example 6 EC50 determination of the growth inhibition of Plasmodium in vitro by triazoloquinoline derivatives

[0137] 100 μL of complete culture medium was added to a 96-well plate, 200 μL of a 200 μM compound stock solution was added to the first well, and a gradient dilution was performed at a ratio of 1 / 2 (11 concentration gradients) according to the doubling gradient dilution method. Dihydroartemisinin (DHA) was used as a positive control, complete culture medium as a negative control, and red blood cells without malarial parasites and compounds were used as background controls.

[0138] Accurately add 100 μL of wild-type Plasmodium culture (parasitemia rate is 1%, hematocrit is 4%) to each well, so that the final parasitemia rate in each well is 0.5%, the hematocrit is 2%, and the final concentration gradient of the compound is (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, 0.195313 μM, 0.097656 μM). Three replicates are set for all samples. After the addition is completed, the 96-well plate is placed in a 37°C incubator (5% CO2, 5% O2) and cultured for 72 hours. After incubation, add 100 μL of Lysis buffer (1x SYBR Green I, 0.12 mg / mL saponin, 0.12% v / v Triton X-100, 30 mM Tris-HCl, and 7.5 mM EDTA) to each well, mix thoroughly, and incubate at room temperature in the dark for 2 hours. After incubation, record fluorescence intensity on a microplate reader using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The inhibition rate of the small molecule inhibitor on Plasmodium growth is calculated based on the fluorescence intensity.

[0139] Survival rate = (F1 drug treatment - F1 background) / (F1 control - F1 background)

[0140] F1 drug treatment, F1 background, and F1 control represent the fluorescence intensity values ​​of the candidate small molecule drug, red blood cell control, and non-drug treatment control group, respectively.

[0141] The drug inhibition rate was calculated based on the survival rate at different concentrations, and the growth inhibition curve was summarized as inhibition rate-drug concentration, and the IC50 was calculated using SPSS18.0 software.

[0142] The results are shown in FIG4 . The IC50 of the triazoloquinoline derivative on the quantitative growth inhibition of Plasmodium cultured in vitro was 2.63 μM.

[0143] Example 7 Determination of Cytotoxicity IC50 of Small Molecule Inhibitors

[0144] Mammalian 293T and HepG2 cells were cultured and passaged, digested with trypsin, resuspended in DMEM medium, counted using a hemocytometer, and diluted to 10 5Cell / mL, the cell suspension was evenly added to a 96-well plate, 100 μL per well, and cultured in a 37°C incubator (5% CO2) for 24 hours. After that, the corresponding concentration of inhibitor was added and the gradient dilution was performed at a ratio of 1 / 2 (11 concentration gradients) according to the doubling gradient method. Complete medium was used as a negative control, and DMEM medium was used as a background control. After 72 hours of culture in a 37°C incubator (5% CO2), 10 μL of CCK8 reagent was added to each well and incubated in a 37°C incubator (5% CO2) for 1 hour. The absorbance at a wavelength of 450 nm was read by a microplate reader, and the survival rate was calculated based on the absorbance.

[0145] Survival rate = (Ab drug treatment - Ab background) / (Ab control - Ab background)

[0146] Ab treatment, Ab background, and Ab control represent the absorbance values ​​for the small molecule drug, DMEM control, and non-drug treatment control groups, respectively. Drug inhibition rates were calculated based on survival rates at different concentrations. Growth inhibition curves were summarized as inhibition rate versus drug concentration, and IC50 values ​​were calculated using SPSS 18.0 software.

[0147] The results are shown in Figure 5. As shown in Figure 5, the triazoloquinoline derivatives of formula (I) described herein have an IC50 of 2.63 μM against the novel merozoite protein of Plasmodium falciparum strain 3D7. Furthermore, Figure 5 demonstrates that the triazoloquinoline derivatives of formula (I) described herein have low cytotoxicity toward mammalian cells and exhibit good biosafety.

[0148] Example 8 Pharmacokinetic Analysis of Small Molecule Inhibitors

[0149] This study aimed to investigate the species differences in the metabolic rates of triazoloquinoline derivatives of formula (I) in mouse, rat and human liver microsomes.

[0150] The microsomal metabolism test is carried out by incubating the inhibitor with liver microsomes of different species, terminating the reaction at different time points, and using the LC-MS / MS method to determine the remaining amount of inhibitor in the sample at each time point, and calculating the remaining percentage of the original drug, and finally obtaining the liver intrinsic clearance (CL). int(liver) Testosterone was used as a control in this study to verify the reliability of the experimental system. The specific method is as follows

[0151] The incubation system consisted of 0.1M Tris-HCl buffer (pH 7.4), 1mM MgCl2, 1mM NADPH, and 0.5mg mL-1 microsomes, with a final reaction volume of 200μL. 1M inhibitors and testosterone were pre-incubated with liver microsomes from different species in a 37°C water bath for 5 minutes, and then 100μL NADPH was added to initiate the reaction. 400μL stop solution was added to terminate the reaction at 0min, 5min, 15min, 30min, 45min, and 60min, respectively. 40μL internal standard was added and vortexed for 5min, centrifuged at 20000g and 4°C for 5min, and 100μL of the supernatant was taken and diluted with 100μL water for injection and analysis. The retention time of the compound and internal standard, chromatogram acquisition, and chromatogram integration were processed by Analyst 1.6.3 software. The compound was quantified at 1 / X 2 Perform linear regression with % as the weighting coefficient and calculate the concentration at each time point. Perform linear regression of the natural logarithm of the percentage remaining at each time point against the incubation time to calculate the in vitro elimination rate constant k. Calculate the half-life (T) of the test article according to the following formula 1 / 2 ) and hepatic intrinsic clearance CL int(liver) .

[0152] half life Hepatic microsomal clearance C Lint(mic) = 0.693 / half-life / mg microsomal protein per mL; intrinsic hepatic clearance CL int(liver) = Hepatic microsomal clearance × mg microsomal protein / g liver weight × liver weight to body weight ratio. The liver weight to body weight ratios for mice, rats, and humans are 88, 40, and 20 g / kg, respectively. The mean value of mg microsomal protein / g liver weight across species is 45. The microsomal metabolism results of triazoloquinoline derivatives in mice, rats, and humans are shown in Table 1.

[0153] Table 1

[0154] Liver microsomal metabolism results showed that the inhibitor was a moderately cleared compound in rat and human liver microsomes and a highly cleared compound in mice.

[0155] Example 9 Survival experiment of ring-shaped insects resistant to triazoloquinoline derivatives

[0156] This study aimed to evaluate the efficacy of triazoloquinoline derivatives as a combination agent against drug-resistant insect strains using the gold standard method for drug resistance assessment. The specific implementation steps are as follows:

[0157] 1. When the parasite rate is greater than 4% and most of them are in the R stage, culture with Sorbitol for 10 minutes and vortex for 5 seconds;

[0158] 2. Repeat Sorbitol synchronization after 30-48 hours (depending on the insect stage);

[0159] 3. After approximately 30 hours of incubation, perform smear microscopy: If the proportion of mature S-phase (10-12 merozoites) is greater than 0.5%, proceed to step 4; if the proportion of mature S-phase (10-12 merozoites) is less than 0.5%, repeat step 2.

[0160] 4. Collect the culture and enrich for late-stage parasites (Plasmion or Percoll); smear microscopy of the enriched parasites should show that the S-stage fraction must be greater than 10% and the R-stage fraction must be less than 10%;

[0161] 5. Add 10 ml of culture medium and 200 μl (appropriate amount) of fresh red blood cells and culture for 3 hours (must be accurate);

[0162] 6. After mixing the culture, aspirate about 200 μl of the sample and centrifuge it for smear examination (this can be done about 10 minutes in advance). Quickly calculate the parasite rate (must be greater than 0.5%).

[0163] 7. Collect the culture and synchronize it with Sorbitol for 10 minutes, vortex for 5 seconds, centrifuge and discard the supernatant;

[0164] 8. In a 24-well plate: Control group: Add 100ul of DMSO control solution to each well

[0165] Experimental group: 100 μl of 7 mM DHA solution and 20 μM triazoloquinoline derivative solution (dissolved in culture medium) were added to each well;

[0166] 9. Add 900 μl of resuspended iRBC culture to each well and mix thoroughly; maintain the culture for 6 hours;

[0167] 10. After 6 hours, transfer the culture in each well to a 1.5 ml EP tube and centrifuge to remove the supernatant;

[0168] 11. Add 1 ml of preheated culture medium to wash, centrifuge and remove the supernatant; repeat the washing once, for a total of two washes;

[0169] 12. Add 1 ml of preheated culture medium, mix well, transfer to a new well plate, and continue to culture for 66 hours;

[0170] 13. After 66 hours of culture, smear microscopy was performed to calculate the parasite rate and the survival rate.

[0171] The results are shown in Figure 6, where triazoloquinoline derivatives (inhibitor) are represented, "-" indicates that they do not contain the corresponding substance, and "+" indicates that they contain the corresponding substance. The results show that combined use with DHA can more effectively kill artemisinin-resistant insect strains, suggesting that small molecules can be used as compatible drugs to improve the spread of artemisinin resistance.

[0172] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of an inhibitor of the transcription factor PfAP2-05 of Plasmodium falciparum, wherein the triazoloquinoline derivative has a structure as shown in formula (I):

2. Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug for inhibiting Plasmodium falciparum, wherein the triazoloquinoline derivative has a structure as shown in formula (I):

3. Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug for preventing or treating malaria caused by Plasmodium falciparum, wherein the triazoloquinoline derivative has a structure as shown in formula (I):

4. A pharmaceutical composition, characterized in that A triazoloquinoline derivative having a structure as shown in formula (I) or a pharmaceutically acceptable salt or hydrate thereof is used as an active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient is used as an auxiliary agent; 5. The pharmaceutical composition according to claim 4, characterized in that The active ingredients also include artemisinin compounds.

6. The pharmaceutical composition according to claim 5, characterized in that The artemisinin compound is a dihydroartemisinin compound.

7. The pharmaceutical composition according to claim 5, characterized in that In the composition, the molar ratio of the triazoloquinoline derivative of the structure represented by formula (I) or its pharmaceutically acceptable salt or hydrate to the artemisinin compound is 1:500 to 500:

1.

8. The pharmaceutical composition according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form.

9. Use of the pharmaceutical composition according to claim 4 in the preparation of a drug for preventing and / or treating malaria.

10. Use of the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating malaria resistant to artemisinin and its analogs.

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