Application of triazoloquinoline derivative in preparation of medicine for preventing or treating malaria

Abstract

The invention discloses a novel application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof. The triazoloquinoline derivative can inhibit expression of an invasive gene PfAP2-O5, prevent plasmodium falciparum from invading red blood cells, influence growth and development of the red blood cells and achieve the effect of killing the plasmodium falciparum, so that the triazoloquinoline derivative can be used as an inhibitor of the PfAP2-O5, a medicine for inhibiting the plasmodium falciparum or a medicine for preventing or treating malaria.

Description

Technical Field

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

[0002] Malaria is a parasitic disease caused by infection with the protozoan Plasmodium. It can seriously threaten human health and life safety. Its morbidity and mortality rates are high among infectious diseases. According to the World Malaria Report released by WHO in 2021, malaria is prevalent in 87 countries and regions around the world. In 2020, there were 241 million new cases and about 627,000 deaths.

[0003] The life cycle of malarial parasites is complex, mainly including sexual reproduction in the final host and asexual reproduction in the intermediate host. In the human body, malarial parasites (sporozoites) first invade the liver, undergo a schizont asexual proliferation, and the released merozoites enter the blood and invade red blood cells, starting the schizont proliferation phase in red blood cells. This stage is the period of clinical onset of malaria, and the invasion of merozoites into red blood cells is the key link in the pathogenesis of malaria.

[0004] At present, drug treatment is the main means of malaria prevention and treatment, among which artemisinin-based combination therapy has effectively curbed the spread of malaria worldwide. However, with the long-term and widespread use of artemisinin-based drugs, strains with reduced sensitivity to them have gradually appeared in Southeast Asia and even Africa. The emergence and global spread of such strains have posed severe challenges to the world's malaria prevention and control and malaria elimination strategies. Therefore, finding new targets for antimalarial drugs, especially developing safe and effective transmission-blocking drugs targeting the key links of malarial parasites, is of vital importance to curbing the spread of malaria. Summary of the invention

[0005] The present invention provides a new medicine for preventing or treating malaria to overcome the above-mentioned shortcomings.

[0006] Another object of the present invention is to provide a new application of triazoloquinoline derivatives.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] 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):

[0009]

[0010] The inventors conducted in-depth research on the mechanism of Plasmodium falciparum invading red blood cells and discovered for the first time the function of PfAP2-O5 in positively regulating red blood cell invasion. The triazoloquinoline derivatives shown in formula (I) 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 the killing effect of Plasmodium falciparum.

[0011] The PfAP2-O5 inhibitor of the present invention refers to a compound that achieves the effect of killing Plasmodium falciparum by inhibiting the function of AP2-O5.

[0012] At the same time, in in vitro experiments, the triazoloquinoline derivatives shown in formula (I) showed good growth inhibition effects on Plasmodium falciparum and were almost non-toxic to mammalian cells, making it possible to develop drugs. They also had good killing effects on resistant strains, which was helpful in delaying the generation and spread of drug resistance, and would be more efficient in 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]

[0015] 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):

[0016]

[0017] In the present invention, the "pharmaceutically acceptable salt" includes the salt formed by the triazoloquinoline derivative and an inorganic acid or an organic acid.

[0018] 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.

[0019] In the present invention, the "hydrate" refers to a salt containing water molecules bound in a certain ratio as a component of the crystal.

[0020] 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 auxiliary agent;

[0021]

[0022] 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 serves as an excipient, carrier or medium for the active ingredient.

[0023] 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.

[0024] Therefore, the pharmaceutical composition can be prepared into various known dosage forms. Preferably, the dosage form prepared by the pharmaceutical composition is an oral dosage form or an injection dosage form.

[0025] Oral dosage forms include 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, ground 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, flavoring agents, drainage agents or melting agents. Oral dosage forms can also be liquid oral dosage forms, such as aqueous solutions, emulsions, suspensions, solutions made of non-effervescent particles and / or suspensions or effervescent particles. As the suitable auxiliary agent type for preparing liquid oral agents, include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, coloring agents or flavoring agents.

[0026] The injection dosage forms include: injection solution (normal), lyophilized powder injection, powder injection (normal), injection tablets, etc.

[0027] 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.

[0028] 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.

[0029] The pharmaceutical composition may have a triazoloquinoline derivative having a structure as shown in formula (I) or a pharmaceutically acceptable salt or hydrate thereof as the sole active ingredient, and may also have a known artemisinin compound as a common active ingredient.

[0030] Preferably, the active ingredient also includes an artemisinin compound. Artemisinin compounds have been shown to have a therapeutic effect on malaria. More preferably, the artemisinin compound is a dihydroartemisinin compound. Dihydroartemisinin compounds have better effects on multiple drug-resistant malarial parasites than general artemisinin compounds.

[0031] 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.

[0032] Preferably, the pharmaceutical composition is in the form of an oral dosage form or an injection dosage form.

[0033] The pharmaceutical composition is used in preparing drugs for preventing and / or treating malaria.

[0034] Preferably, the pharmaceutical composition is used in the preparation of a drug for treating malaria resistant to artemisinin and its analogs.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a new application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof, wherein 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 the killing effect of Plasmodium falciparum, so the triazoloquinoline derivative can be used as an inhibitor of PfAP2-O5, a drug for inhibiting Plasmodium, or a drug for preventing or treating malaria. Based on the above findings, the present invention provides a new pharmaceutical composition, which can use a triazoloquinoline derivative alone as an active ingredient, or compound it with an artemisinin compound as a common active ingredient; the pharmaceutical composition can be used to prepare a drug for treating malaria, especially a drug for treating malaria resistant to artemisinin and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 middle, Figure 1 A shows the WB identification results of the PfAP2-O5 gene knockdown strain. After the PfAP2-O5 gene was knocked down, the expression level of PfAP2-O5 protein was reduced; Figure 1 B shows the growth curve test of the PfAP2-O5 gene knockdown strain.

[0038] Figure 2 ChIP-seq analysis shows the distribution of PfAP2-O5 across the genome; as well as the types of PfAP2-O5 binding target genes, most of which are invasion gene families.

[0039] Figure 3 Schematic diagram of the inhibitory effect of triazoloquinoline derivatives on invasive gene expression. Figure 3 A RNA-Seq technology was used to determine the expression of invasion genes in the PfAP2-O5 gene knockdown strain; Figure 3 B shows the expression of invasion genes after the triazoloquinoline derivatives inhibited the function of PfAP2-O5 gene; Figure 3 C shows the inhibitory effect of PfAP2-O5 gene knockdown and triazoloquinoline derivative treatment on invasion gene expression.

[0040] Figure 4 The figure is a schematic diagram of the quantitative growth inhibition effect of triazoloquinoline derivatives on malarial parasites cultured in vitro, showing that the EC50 of the quantitative growth inhibition effect on malarial parasites cultured in vitro is 2.632 μM, and the malarial parasite growth is determined by a 3-day SYBR GreenI cell growth assay.

[0041] Figure 5 The figure is a schematic diagram of the cytotoxic effect of the triazoloquinoline derivative on the human renal epithelial cell line 293T and the human liver cancer cell line HepG2. The selective index between the inhibitory effect of the small molecule inhibitor on Plasmodium and the toxicity to different cell lines is also shown, and the cell growth is measured by the 3-day CCK8 method.

[0042] Figure 6 In order to determine 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

[0043] The technical solutions in the embodiments of the present invention will be described clearly and completely below, but the implementation manner of the present invention is not limited thereto.

[0044] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] 1. Triazoloquinoline derivatives of the structure shown in formula (I): purchased from ChemDiv ID.V003-8956

[0046] 2. Plasmodium falciparum (3D7_G7): purchased from ATCC

[0047] 3. 293T cell line: purchased from ATCC

[0048] 4. HepG2 cell line: purchased from ATCC

[0049] 5. SYBR Green I: purchased from Invitrogen Cat. No. S-7585

[0050] 6. Pancreatin-EDTA: purchased from Gbico Cat. No. 25200-072

[0051] 7. DMEM culture medium: purchased from Gbico Cat. No. 11965-092

[0052] 8. FBS: purchased from Gbico Cat. No. 10082-147

[0053] 9. Double-stranded DNA quantitative detection kit: purchased from ThermoFisher Cat.No.P7581

[0054] 10. Protein A / G magnetic beads: purchased from ThermoFisher Cat.No.88803

[0055] 11. Mouse monoclonal antibody: purchased from Sigma Cat.No.SAB4800032

[0056] 12. 37% formaldehyde solution: purchased from Sigma Cat. No. 47608-250ML-F

[0057] 13. 96-well plate: purchased from Corning

[0058] 14. Red blood cells: purchased from Shanghai Blood Center

[0059] 15. Dihydroartemisinin (CAS: 71939-50-9)

[0060] 16. The components of various buffers are as follows:

[0061] (1) Lysis buffer

[0062] Reagent (stock solution) Final concentration 1M Hepes PH7.9 10mM 0.5M EDTA pH 8.0 0.1mM 0.5M EGTA pH 8.0 0.1mM 2M KCl 10mM 1M DTT (added before use) 1mM 100× protease inhibitor (add before use) 1×

[0063] (2) Ultrasonic buffer

[0064] Reagent (stock solution) Final concentration 10% SDS 0.1％ 0.5M EDTA pH 8.0 1mM 1M Tris-HCl PH8.0 10mM

[0065] (3) ChIP dilution buffer

[0066] Reagent (stock solution) Final concentration 10% SDS 0.01％ 100% Triton X-100 1.1％ 5M NaCl 150mM 0.5M EDTA pH 8.0 1.2mM 1M Tris-HCl PH8.0 16.7mM

[0067] (4) Low-salt immune complex washing buffer

[0068] Reagent (stock solution) Final concentration 10% SDS 0.1％ 100% Triton X-100 1％ 5M NaCl 150mM 0.5M EDTA pH 8.0 2mM 1M Tris-HCl PH8.0 20mM

[0069] (5) High Salt Immune Complex Washing Buffer

[0070]

[0071]

[0072] (6) Lithium chloride immune complex washing buffer

[0073] Reagent (stock solution) Final concentration 10% NP-40 1％ 10% deoxycholic acid 1％ 5M LiCl 250mM 0.5M EDTA pH 8.0 1mM 1M Tris-HCl PH8.0 10mM

[0074] (7)TE buffer

[0075] Reagent (stock solution) Final concentration 1M Tris-HCl PH8.0 10mM 0.5M EDTA pH 8.0 1mM

[0076] (8) Elution buffer

[0077] Reagent (stock solution) Final concentration 10% SDS 1％ <![CDATA[1M NaHCO3]]> 100mM

[0078] Example 1 Plasmodium culture

[0079] Plasmodium falciparum was cultured using RPMI 1640 complete medium in a 37°C incubator (5% CO2, 5% O2, and 90% N2).

[0080] 0.5L RPMI 1640 complete medium contains: 5.22g RPMI-1640 powder; 1.0g sodium bicarbonate; 5g AlbmaxI; 2.98g 25mM HEPES; 13.6mg hypoxanthine; 10mg gentamicin sulfate.

[0081] Example 2 Construction of transgenic insect strain: PfAP2-05 conditional knockdown insect strain

[0082] 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.

[0083] The specific construction steps are as follows:

[0084] 1. Construction of knockdown vector for transgenic insect strain (named pL6cs-ap2-o5-glmS)

[0085] Using the CRISPR / Cas9 gene editing system, clone the guide RNA into the vector as follows:

[0086] (1) Design of gRNA

[0087] The sequence of the PfAP2-O5 gene of Plasmodium falciparum 3D7-G7 strain was downloaded from the PlasmoDB website (from https: / / plasmodb.org / plasmo / app database, Plasmodium falciparum: PF3D7_1449500 (plasmodium falciparum) 715aa PfAP2-O5. The sequence was also disclosed in ncbi: Sequence ID: XM_001348609.1). Through BLAST comparison, a specific sequence with a length of 20 bp was selected as the guide RNA (gRNA), and the sequence was as follows: 5′-ACCAAGTCATATGTCATTAA-3′.

[0088] (2) Primer design

[0089] Primers were designed using SnapGene software:

[0090] P1: 5′-GCCCTAGTCTAG GGGC CAATTTTAATAACAATTTGGCG-3′;

[0091] P2: 5′-CAAGTAAAATAATTGGTCTTTAAGTGACATATGACTTGGTAGTTTACTG-3′;

[0092] P3: 5′-AGCGGCCGCAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATATTATAC-3′;

[0093] P4: 5′-TTTTTTACAAATG CTTAAG AACTAGCGTATGTGATAACTTAG-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-cutting the plasmid;

[0094] P7: 5′-TATTTTACTTGGACAAATTAGGTACCGGAAGTGGTTCTGG-3′;

[0095] P8: 5′-TTTATTTTTTTTGCGGCCGCGCGGCCGCGTCCCCTC-3′ is used to amplify the exogenous green fluorescent protein (GFP) gene.

[0096] (3) Gene amplification

[0097] The genetic DNA of Plasmodium falciparum 3D7-G7 strain was extracted, and the P1+P2 and P3+P4 primers were used to amplify the left and right homologous sequences of the PfAP2-O5 gene and introduce mutation sites, and the P7+P8 primers were used to amplify the glmS ribozyme sequence (the glmS sequence comes from the literature report: doi:10.1371 / journal.pone.0073783).

[0098] (4) Construction of ligation product

[0099] The left and right homologous sequences were connected to the glmS ribozyme sequence using primers to obtain a connection product. The 3' end of the PfAP2-05 gene was labeled with the glmS ribozyme sequence.

[0100] 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 system.

[0101] 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 / 6b, 65°C for 5 min, for a total of 30 cycles; 72°C for 10 min.

[0102] (5) Construction of knockdown vector

[0103] The plasmid pL6cs carrying the resistance gene of the drug dihydrofolate reductase inhibitor (WR) for screening was digested with XhoⅠ and AvrⅡ (the plasmid was built in the laboratory, and the specific sequence can be found in the literature report: doi:10.3389 / fmicb.2020.625862), and the product was recovered and purified by a gel recovery kit. The gRNA fragments were connected by DNA ligase and transformed into Escherichia coli XL-10 competent cells, inoculated on LB agar plates containing 100μg / ml ampicillin and cultured overnight. A single colony was picked, cultured at 34℃ and 200rpm / s overnight, and the plasmid was extracted for sequencing.

[0104] The plasmid connected to the gRNA was confirmed by sequencing, and double digested with AscⅠ and AflⅡ. The product was recovered and connected to 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℃.

[0105] The knockdown vector of the transgenic insect strain was obtained and named pL6cs-ap2-o5-glmS.

[0106] 2. Construction of PfAP2-O5 conditional knockout strain

[0107] A transgenic insect strain was constructed by combining electrofection with drug screening: the PfAP2-O5 conditional knockdown insect strain.

[0108] (1) Preparation of plasmid for electroporation

[0109] The correct strains were inoculated onto LB agar plates containing 100 μg / ml ampicillin and cultured overnight. The next day, single clones were picked out 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 LB culture medium at a ratio of 1:1000. After shaking culture for 14 h, the bacteria were collected and the plasmid was collected using a plasmid DNA purification kit.

[0110] Take 100 μg of the transgenic insect strain knockdown vector pL6cs-ap2-o5-glmS plasmid constructed above and the plasmid pUF1-Cas9 carrying the screening drug blasticidin (BSD) resistance gene and expressing Cas9 protein (the plasmid was built in the laboratory, and the specific sequence can be found in the literature report: doi:10.3389 / fmicb.2020.625862), add 1 / 10 volume of 3MCH3COONa (pH5.2) solution of the plasmid solution, mix well, add 2.5 times the volume of anhydrous ethanol of the plasmid solution and mix well, 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.

[0111] (2) Electroporation and screening of transgenic insect strains

[0112] The electroporator was set to an electroporation parameter of 310V, 950μF, resistance ∞, and a slit of 2mm. An equal volume (150μl) of 2× cell mixture solution was added to the prepared plasmid and mixed, and then 150μl of red blood cells were added, mixed, and transferred to an electroporation cup, placed in an electroporator for electroporation, and after electroporation, the mixture was transferred to a culture bottle, an appropriate amount of culture medium was added, and then purified schizont-stage malarial parasites were added.

[0113] The culture was carried out in a 37°C three-gas incubator. From the second day on, fresh culture medium was replaced every day. On the fourth day, when the electroporation density was about 10% (parasite rate, the ratio of red blood cells infected with malarial parasites to all red blood cells in a certain field of view (~5000)), 200 μl of fresh red blood cells were added, the culture medium was replaced, and screening drugs WR and BSD (blasticidin) were added. The screening drugs can kill wild-type falciparum malarial parasites, while the successfully transgenic malarial parasites are not affected by the drugs because they carry plasmids expressing drug-resistant genes, so transgenic malarial parasites can be screened out.

[0114] After adding the screening drug until most of the wild malarial parasites are killed, the culture medium is changed and the drug is added every 3 to 4 days to maintain the culture. Smear microscopy begins on the 21st day after transfection, and transgenic strains are obtained in 3 to 6 weeks.

[0115] In summary, a transgenic insect strain was constructed: PfAP2-O5 conditional knockout insect strain.

[0116] 3. Gradient dilution cloning

[0117] The transgenic strain (PfAP2-O5 conditional knockdown strain) obtained by genetic modification is an integrated strain, which needs to be cloned by gradient dilution, that is, a completely integrated monoclonal strain is screened by limiting dilution method. The experiment was carried out in a 96-well cell culture plate. The red blood cells infected by the parasite were diluted to theoretically contain 10, 1, 0.5 and 0.1 parasites per 1 ml by gradient dilution. The specific experimental steps are as follows:

[0118] (1) Synchronizing the transgenic insect strains with sorbitol to obtain annular stage insects with relatively uniform properties;

[0119] (2) Smear microscopy of the trophozoite stage of the parasite and strictly count the original protozoa rate P x;

[0120] (3) Collect the trophozoite stage culture, centrifuge at 2000 rpm, room temperature for 5 min, discard the supernatant, and obtain about 200 μl of infected hematocrit (iRBC);

[0121] (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;

[0122] (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;

[0123] (6) According to the formula 50 μl × P x = 1% × (50 μl + Vx), the parasitemia rate of the above 50 μl infected red blood cells is diluted to 1% to obtain culture A (Vx is the volume of fresh red blood cells added), and the parasitemia rate after dilution is confirmed by smear microscopy;

[0124] (7) Preheat fresh culture medium and, according to the principle of gradient dilution, dilute the infected red blood cells with a protozoan rate of 1% prepared above in proportion so that each centrifuge tube corresponds to a gradient:

[0125] Prepare 9 15 ml centrifuge tubes and number them 1, 2, 3, ... 9. Add 10 ml of fresh culture medium to tube 1, 9 ml of fresh culture medium to tubes 2-7 and 9, and 5 ml of fresh culture medium to tube 8. Preheat all centrifuge tubes in a 37°C incubator for later use. Take 100 μl of culture A with a parasite rate of 1% in (6) and add it to tube 1 to obtain 1×10 6 Culture B with iRBCs / ml;

[0126] 1) Take 1 ml of culture B into tube 2 to obtain 1×10 5 Culture C with iRBCs / ml;

[0127] 2) Take 1 ml of culture C into tube 3 to obtain 1×10 4 Culture D of iRBC / ml;

[0128] 3) Take 1 ml of culture D into tube 4 to obtain 1×10 3 Culture E of iRBC / ml;

[0129] 4) Take 1 ml of culture E into tube 5 to obtain 1×10 2 Culture F with iRBCs / ml;

[0130] 5) Take 1 ml of culture F to tube 6 to obtain culture G with 10 iRBCs / ml;

[0131] 6) Take 1 ml of culture G to tube 7 to obtain culture H with 1 iRBC / ml;

[0132] 7) Take 5 ml of culture H to tube 8 to obtain culture I with 0.5 iRBC / ml;

[0133] 8) Take 1 ml of culture H to tube 9 to obtain culture J with 0.1 iRBC / ml.

[0134] (8) The cultures G, H, I, and J with different gradients obtained above were transferred to a 96-well cell culture plate, 200 μl / well, and finally each row of 12 wells in the 96-well plate corresponded to one gradient.

[0135] (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;

[0136] About 2 weeks later, smears are taken for microscopic examination 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.

[0137] Example 3 Identification of PfAP2-O5 transgenic strain (PfAP2-O5 conditional knockdown strain) and growth and development test

[0138] The ring body (10-15h), trophozoite (28-32h), and schizont (40-44h) stage cultures of the PfAP2-O5 transgenic strain (PfAP2-O5 conditional knockdown strain) were collected. The red blood cells infected with Plasmodium were lysed with 0.15% saponin to collect protein samples. After the proteins were separated by SDS-PAGE electrophoresis, the proteins were transferred to nitrocellulose membranes by semi-dry transfer method. The transfer parameters were: 0.1A, 2h. After the transfer, add an appropriate amount 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 1:1000, seal the membrane and incubate overnight at 4°C; incubate the secondary antibody: dilute the secondary antibody (goat anti-mouse) with 5% skim milk powder at 1:5000, incubate at room temperature for 2 hours; color development: take equal volumes of color development solution A and B and mix them evenly (be careful to avoid light), take a certain amount of color development solution and drop it on the membrane, take a photo and save the exposure picture. The results are as follows Figure 1 As shown in A, Figure 1 A shows the western blot identification of the PfAP2-O5 conditional knockdown strain before and after the addition of 5 mM glucosamine (GlcN), indicating that PfAP2-O5 was effectively knocked down.

[0139] The growth curve test of the PfAP2-O5 conditional knockdown strain was conducted. The strain was synchronized to the ring stage several times, and the initial protozoan rate was determined by strict counting. The strain was divided into two bottles (one bottle was added with the corresponding concentration of GlcN, and the other bottle was used as a control without GlcN). The initial protozoan rate of each bottle was about 0.1%. The four life histories were cultured continuously, and smears were taken from the middle and late stages of each life history. The protozoan rate was counted (the culture medium should be replaced if the protozoan rate is high, and the drug-added group should be supplemented with drugs). The protozoan rates of the strains in the drug-added group and the non-drug-added group were recorded, and the growth curve was drawn. The results are shown in Figure 2. Figure 1 As shown in B, Figure 1 B shows the growth curve test of the wild-type strain and the PfAP2-O5 conditional knockdown strain, indicating that the growth of Plasmodium was significantly inhibited after conditional knockdown of PfAP2-O5.

[0140] Example 4 Chromatin Immunoprecipitation (ChIP) Sequencing

[0141] Chromatin immunoprecipitation (ChIP) sequencing uses the specificity of antigen-antibody reaction to truly reflect the distribution of AP2-O5-bound target genes in the whole genome at the chromatin level, and at the same time reflect the binding of the protein to the target gene. The specific steps are as follows:

[0142] The culture of the schizont stage after synchronization was collected, 1% formaldehyde solution was added, and incubated at 37°C for 10 minutes; pre-cooled 0.125M glycine solution was added and incubated on ice for 5 minutes; 2500rpm, 4°C, centrifuged for 5 minutes, discarded the supernatant, 30mL 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; 2mL of pre-cooled Lysis Buffer was added to resuspend the worms and transferred to a pre-cooled grinding tube and ice bathed for 30 minutes; 200μL of ultrasonic buffer was added, and the precipitate was resuspended and then liquid-phase ultrasonication was performed, and the parameters were set as follows: duty cycle 5%, peak incident power 75W, number of cycles 200, temperature 7°C, time 30 minutes; 10μL of the mixed solution after ultrasonication was taken, 90μl ChIP dilution buffer, 4μL 5M NaCl, 2.5μL proteinase K were added, mixed and incubated at 50°C for 2h, 2μL RNase A was added, incubated at 37°C for 45min, QIAGEN Extract with PCR kit, and detect the effect of ultrasound by 2% agarose gel electrophoresis; the ultrasound product with ideal ultrasound effect is 14000pm, collect the supernatant after centrifugation at 4℃ for 10min, and add 10 times volume of ChIP dilution buffer; add protein A / G magnetic beads to the above solution, shake and incubate at 4℃ for 2h, transfer the supernatant to a 1.5mL centrifuge tube, add the corresponding antibody and protein A / G magnetic beads, and incubate at 4℃ overnight; add different buffers to wash the antibody / magnetic beads / chromatin complex in the following order:

[0143] A: Low-salt immune complex washing buffer: 1 mL per tube, rotate and wash at 4°C for 5 min;

[0144] B: High-salt immune complex washing buffer: 1 mL per tube, rotate and wash at 4°C for 5 min;

[0145] C: Lithium chloride immune complex washing buffer: 1 mL per tube, rotate and wash at 4°C for 5 min;

[0146] D: Wash twice with TE buffer, 1 mL per tube, and rotate at 37°C for 5 min;

[0147] Add 200 μL elution buffer for elution, rotate and incubate at 37°C for 30 minutes, collect the supernatant, and add 180 μL elution buffer to the DNA tube before immunoprecipitation; add 16 μL 5M NaCl to each of the above tubes, mix well and incubate at 45°C overnight; after incubation, add 0.8 μL RNase A (20 mg / mL) to each tube, 37°C, 30 minutes; add 3 μL 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 deionized water, and leave 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 library was amplified using the KAPA HiFiPCR kit under the following conditions: 95°C for 1 min, 98°C for 10 s, 65°C for 1 min, 12 cycles, 65°C for 5 min, 4°C ∞. The library was sequenced on the Illumina HiSeq X Ten platform; two biological replicates were performed for each ChIP-seq dataset.

[0148] The results are as follows Figure 2 As shown, the distribution of PfAP2-O5 protein target genes in the whole genome, such as invasion gene families msps, raps, rhophs, ralp1, ama1, etc., proves that most of the target genes of PfAP2-O5 are related to invasion

[0149] Example 5 RNA-seq analysis of the effect of triazoloquinoline derivatives on PfAP2-O5

[0150] The wild-type insect strain and the PfAP2-O5 conditional knockdown insect strain were treated with the triazoloquinoline derivatives shown in formula (Ⅰ), respectively. After the insect strains were strictly synchronized, 1 μM of the triazoloquinoline derivatives were added one life cycle in advance. After one life cycle, the ring body (10-15h), trophozoite (28-32h), and schizont (40-44h) stage cultures of the wild-type insect strain and the PfAP2-O5 conditional knockdown insect strain treated with the triazoloquinoline derivatives were collected, respectively. After the total RNA was purified by Direct-zol RNA kit, the strand-specific RNA sequencing library was prepared with KAPA mRNA beads, and the RNA was fragmented. The library was sequenced on the Illumina HiSeq X Ten system, and the differential gene expression of the two insect strains after the inhibitor was analyzed.

[0151] The results are as follows Figure 3 Show, Figure 3A RNA-Seq technology was used to determine the expression of invasion genes after conditional knockdown of PfAP2-O5; Figure 3 B RNA-Seq technology was used to determine the inhibitory effect of invasion gene expression after triazoloquinoline derivatives inhibited the function of PfAP2-O5. The results showed that after conditional knockdown of PfAP2-O5, invasion-related target genes were downregulated, and after the wild-type strain was treated with triazoloquinoline derivatives, PfAP2-O5 target genes were downregulated. For example, the expression levels of Plasmodium merozoite surface structural protein family genes such as Rhoptry Nec6, Peripheral surface protein, Rhoptry protein, Rhoptry bulb, Micronemes protein, and GPI-Anchored MSP showed a significant decrease of more than 1.5 times (mRNA level). (Plasmodium merozoite surface structural protein family genes mainly regulate Plasmodium merozoites invading 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 3 C shows the intersection of invasion-related target genes after conditional knockdown of PfAP2-O5 and inhibition of PfAP2-O5 function by triazoloquinoline derivatives.

[0152] Example 6 EC50 determination of the growth inhibition of Plasmodium in vitro by triazoloquinoline derivatives

[0153] 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 gradient dilutions were performed at a ratio of 1 / 2 (11 concentration gradients) according to the multiple 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.

[0154] 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), and set up three replicate wells for all samples; after the addition of samples, place the 96-well plate in a 37°C incubator (5% CO2, 5% O2) and culture for accurately 72 hours. After the incubation, 100 μL Lysis buffer (1x SYBRGreen I, 0.12 mg / mL saponin, 0.12% v / v Triton X-100, 30 mM Tris-HCl, and 7.5 mM EDTA) was added to each well and mixed thoroughly, and then incubated at room temperature in the dark for 2 h. After the incubation, the fluorescence intensity was recorded on a microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm, and the inhibition rate of small molecule inhibitors on the growth of Plasmodium was calculated based on the fluorescence intensity.

[0155] Survival rate = (Fl drug treatment - Fl background) / (Fl control - Fl background)

[0156] Among them, F1 drug treatment, F1 background and F1 control represent the fluorescence intensity values ​​of candidate small molecule drugs, red blood cell control and control group without drug treatment, respectively.

[0157] The drug inhibition rate was calculated according to the survival rate at different concentrations, and the growth inhibition curve was summarized as inhibition rate-drug concentration, and the IC50 was calculated using SPSS 18.0 software.

[0158] The results are as follows Figure 4 As shown, the IC50 of the quantitative growth inhibition effect of triazoloquinoline derivatives on Plasmodium cultured in vitro was 2.63 μM.

[0159] Example 7 Determination of IC50 of Cytotoxicity of Small Molecule Inhibitors

[0160] Mammalian 293T and HepG2 cells were cultured and subcultured, digested with trypsin, resuspended in DMEM medium, counted with a hemocytometer, and diluted to 10 5cell / mL, the cell suspension was evenly added to a 96-well plate, 100 μL per well, and the inhibitor of the corresponding concentration was added after culturing in a 37°C incubator (5% CO2) for 24 hours. The gradient dilution was performed at a ratio of 1 / 2 (11 concentration gradients) according to the multiple gradient dilution method. The complete medium was used as a negative control and the DMEM medium was used as a background control. After culturing in a 37°C incubator (5% CO2) for 72 hours, 10 μL of CCK8 reagent was added to each well, and the cells were 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.

[0161] Survival rate = (Ab drug treatment - Ab background) / (Ab control - Ab background)

[0162] Ab drug treatment, Ab background and Ab control represent the absorbance values ​​of small molecule drugs, DMEM control and control group without drug treatment, respectively. The drug inhibition rate was calculated according to the survival rate at different concentrations, and the growth inhibition curve was summarized by inhibition rate-drug concentration, and IC50 was calculated by SPSS 18.0 software.

[0163] The results are as follows Figure 5 shown. Figure 5 It can be seen that the IC50 of the triazoloquinoline derivative of the formula (I) of the present invention against the novel merozoite protein of Plasmodium falciparum 3D7 strain is 2.63 μM. Figure 5 The triazoloquinoline derivatives of the formula (I) of the present invention have low toxicity to mammalian cells and good biosafety.

[0164] Example 8 Pharmacokinetic Analysis of Small Molecule Inhibitors

[0165] The purpose of this experiment was to study the species differences in the metabolic rates of triazoloquinoline derivatives of formula (I) in mouse, rat and human liver microsomes.

[0166] 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 the 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 test the reliability of the experimental system. The specific method is as follows

[0167] The incubation system consisted of 0.1M Tris-HCl buffer (pH 7.4), 1mM MgCl2, 1mM NADPH and 0.5mg mL-1 microsomes, and the final reaction volume was 200μL. After pre-incubating 1M inhibitor and testosterone with liver microsomes of different species in a 37°C water bath for 5 minutes, 100μL NADPH was added to start the reaction, and 400μL stop solution was added at 0min, 5min, 15min, 30min, 45min, and 60min to stop the reaction. 40μL internal standard was added and vortexed for 5min, centrifuged at 20000g and 4°C for 5min, 100μL supernatant was taken and diluted with 100μL water, and the sample was injected for analysis. The retention time of the compound and the internal standard, the chromatogram acquisition and the integration of the chromatogram were processed by the software Analyst 1.6.3, and the compound was 1 / X 2 The concentration at each time point was calculated by linear regression using the weighted coefficient. The natural logarithm of the percentage of residual amount at each time point was linearly regressed against the incubation time to calculate the in vitro elimination rate constant 6. The half-life (T 1 / 2 ) and hepatic intrinsic clearance CL int(liver) .

[0168] Hepatic microsomal clearance C Lint(mic) =0.693 / half-life / mg microsomal protein per mL; liver intrinsic clearance CL int(liver) = liver microsomal clearance × mg microsomal protein / g liver weight × liver weight to body weight ratio. The liver weight to body weight ratios of mice, rats and humans are 88, 40 and 20 g / 6 g, respectively; the average value of mg microsomal protein / g liver weight in different species is 45. The results of mouse / rat / human microsomal metabolism of triazoloquinoline derivatives are shown in Table 1

[0169] Table 1

[0170]

[0171] The results of liver microsomal metabolism showed that the inhibitor was a medium-clearance compound in rat and human liver microsomes and a high-clearance compound in mice.

[0172] Example 9 Survival experiment of annular bodies of insect strains resistant to triazoloquinoline derivatives

[0173] This experiment aims to evaluate the killing effect of triazoloquinoline derivatives as a combination drug against drug-resistant insect strains using the gold standard method for drug resistance. The specific implementation steps are as follows:

[0174] 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;

[0175] 2. Repeat Sorbitol synchronization after 30-48h (depending on the insect stage);

[0176] 3. After culturing for about 30 hours, 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;

[0177] 4. Collect the culture to enrich the late-stage parasites (Plasmion or Percoll); smear microscopy of the enriched parasites, the S stage must be greater than 10%, and the R stage must be less than 10%;

[0178] 5. Add 10 ml of culture medium and 200 ul (appropriate amount) of fresh red blood cells, and maintain the culture for 3 hours (must be accurate);

[0179] 6. Mix the culture and take out about 200ul of it for centrifugation and smear examination (can be done about 10 minutes in advance), and quickly calculate the protozoan rate (must be greater than 0.5%);

[0180] 7. Collect the culture and synchronize with Sorbitol for 10 minutes, vortex for 5 seconds, centrifuge and discard the supernatant;

[0181] 8. In a 24-well plate: Control group: Add 100ul of DMSO control solution to each well

[0182] Experimental group: 100ul of 7mM DHA solution and 20μM triazoloquinoline derivative solution (dissolved in culture medium) were added to each well;

[0183] 9. Add 900ul of resuspended iRBC culture to each well and mix well; maintain the culture for 6 hours;

[0184] After 10.6 hours, transfer the culture in each well to a 1.5 ml EP tube and centrifuge to remove the supernatant;

[0185] 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;

[0186] 12. Add 1 ml of preheated culture medium, mix well, transfer to a new well plate, and continue to culture for 66 hours;

[0187] 13. After 66 hours of culture, smear microscopy was performed to calculate the parasite rate and the survival rate.

[0188] The results are as follows Figure 6As shown, triazoloquinoline derivatives (inhibitor) are represented, "-" indicates that the corresponding substance is not contained, and "+" indicates that the corresponding substance is contained. The results show that the combination 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.

[0189] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope 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 drug for treating malaria resistant to artemisinin and its analogs.

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