WO2025001748A1 - 六氢喹啉衍生物在制备预防或治疗疟疾的药物中的应用 - Google Patents

六氢喹啉衍生物在制备预防或治疗疟疾的药物中的应用 Download PDF

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WO2025001748A1
WO2025001748A1 PCT/CN2024/096683 CN2024096683W WO2025001748A1 WO 2025001748 A1 WO2025001748 A1 WO 2025001748A1 CN 2024096683 W CN2024096683 W CN 2024096683W WO 2025001748 A1 WO2025001748 A1 WO 2025001748A1
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pfap2
hexahydroquinoline
drug
culture
pharmaceutical composition
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French (fr)
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曹俊
张青锋
杨勇
俞心愉
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Jiangsu Insititute Of Parasitic Diseases
China Pharmaceutical University
Tongji University
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Jiangsu Insititute Of Parasitic Diseases
China Pharmaceutical University
Tongji University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of medical technology, and more specifically, to the application of hexahydroquinoline derivatives in preparing medicines for preventing or treating malaria.
  • 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.
  • malarial parasites The life cycle of malarial parasites is complex, mainly including sexual reproduction in the final host and asexual reproduction in the intermediate host.
  • malarial parasites sporozoites
  • 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.
  • the present invention provides a new medicine for preventing or treating malaria to overcome the above-mentioned shortcomings.
  • Another object of the present invention is to provide a new application of hexahydroquinoline derivatives.
  • hexahydroquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a transcription factor PfAP2-05 inhibitor of Plasmodium falciparum, wherein the hexahydroquinoline derivative has a structure as shown in formula (I):
  • the hexahydroquinoline derivatives shown in formula (I) can inhibit the expression of the invasion gene PfAP2-O5, block Plasmodium falciparum from invading red blood cells, affect its growth and development, and achieve the killing effect of Plasmodium falciparum.
  • 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.
  • the hexahydroquinoline 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.
  • hexahydroquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug for inhibiting Plasmodium falciparum;
  • the hexahydroquinoline derivative has a structure as shown in formula (I):
  • hexahydroquinoline 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 hexahydroquinoline derivative has a structure as shown in formula (I):
  • the "pharmaceutically acceptable salt” includes the salt formed by the hexahydroquinoline derivative and an inorganic acid or an organic acid.
  • 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.
  • the "hydrate” refers to a salt containing water molecules bound in a certain ratio as a component of the crystal.
  • a pharmaceutical composition comprising a hexahydroquinoline 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;
  • 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.
  • a carrier which may be in the form of a capsule or sachet.
  • 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.
  • excipients examples include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup and methylcellulose.
  • 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.
  • the pharmaceutical composition can be prepared into various known dosage forms.
  • the dosage form prepared by the pharmaceutical composition is an oral dosage form or an injection dosage form.
  • 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.
  • 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.
  • suitable auxiliary agent type for preparing liquid oral agents include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, coloring agents or flavoring agents.
  • the injection dosage forms include: injection solution (normal), lyophilized powder injection, powder injection (normal), injection tablets, etc.
  • 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.
  • the EC50 value of the present invention on the growth inhibition of Plasmodium falciparum reached 2.158 ⁇ M, showing good growth inhibition function.
  • the pharmaceutical composition may contain a hexahydroquinoline derivative having a structure shown in formula (I) or a pharmaceutically acceptable salt or hydrate thereof as the sole active ingredient, and may also contain a known artemisinin compound as a common active ingredient.
  • 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.
  • the molar ratio of the hexahydroquinoline 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.
  • the pharmaceutical composition is in the form of an oral dosage form or an injection dosage form.
  • the pharmaceutical composition is used in preparing drugs for preventing and/or treating malaria.
  • the pharmaceutical composition is used in the preparation of a drug for treating malaria resistant to artemisinin and its analogs.
  • the present invention has the following beneficial effects:
  • the present invention provides a new application of a hexahydroquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof, wherein the hexahydroquinoline 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 hexahydroquinoline derivative can be used as an inhibitor of PfAP2-O5, a drug for inhibiting Plasmodium, or a drug for preventing or treating malaria.
  • the present invention provides a new pharmaceutical composition, which can use a hexahydroquinoline 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 that is resistant to artemisinin and its analogs.
  • Figure 1A 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 1B shows the growth curve test of the PfAP2-O5 gene knockdown strain.
  • FIG 2 shows the distribution of PfAP2-O5 in the whole genome by ChIP-seq analysis; as well as the types of PfAP2-O5 binding target genes, most of which are invasion gene families.
  • Figure 3 is a schematic diagram of the inhibitory effect of hexahydroquinoline derivatives on invasion gene expression.
  • Figure 3A uses RNA-Seq technology to determine the expression of invasion genes in the PfAP2-O5 gene knockout strain;
  • Figure 3B shows the expression of invasion genes after hexahydroquinoline derivatives inhibit the function of the PfAP2-O5 gene;
  • Figure 3C shows the inhibitory effect of PfAP2-O5 gene knockout and hexahydroquinoline derivative treatment on invasion gene expression.
  • Figure 4 is a schematic diagram of the quantitative growth inhibition effect of hexahydroquinoline derivatives on in vitro cultured malarial parasites. It shows that the quantitative growth inhibition effect EC50 of hexahydroquinoline derivatives on in vitro cultured malarial parasites is 2.158 ⁇ M, and the malarial parasite growth is determined by a 3-day SYBR GreenI cell growth assay.
  • Figure 5 is a schematic diagram of the cytotoxic effects of the hexahydroquinoline derivatives on the human renal epithelial cell line 293T and the human liver cancer cell line HepG2. It also shows the selectivity index between the inhibitory effect of the small molecule inhibitor on Plasmodium and the toxicity to different cell lines, and the cell growth was measured using the 3-day CCK8 method.
  • FIG6 shows the killing effect of the hexahydroquinoline derivatives as a combination drug with dihydroartemisinin on resistant insect strains, and a ring body survival experiment is used to clarify the growth inhibitory effect of the combined drug on resistant insect strains.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
  • HepG2 cell line purchased from ATCC
  • SYBR Green I purchased from Invitrogen Cat. No. S-7585
  • Pancreatin-EDTA purchased from Gbico Cat. No. 25200-072
  • DMEM culture medium purchased from Gbico Cat. No. 11965-092
  • FBS purchased from Gbico, Cat. No. 10082-147
  • Double-stranded DNA quantitative detection kit purchased from ThermoFisher Cat.No.P7581
  • Protein A/G magnetic beads purchased from ThermoFisher Cat.No.88803
  • Mouse monoclonal antibody purchased from Sigma, Cat. No. SAB4800032
  • Red blood cells purchased from Shanghai Blood Center
  • Plasmodium falciparum was cultured using RPMI 1640 complete medium in a 37°C incubator (5% CO 2 , 5% O 2 and 90% N 2 ).
  • 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.
  • the CRISPR/Cas9 gene editing system was used to clone the guide RNA into a vector and construct a PfAP2-O5 conditional knockout strain.
  • the guide RNA is cloned into the vector as follows:
  • Plasmodium falciparum 3D7-G7 strain The sequence of PfAP2-O5 gene of Plasmodium falciparum 3D7-G7 strain was downloaded from 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).
  • gRNA guide RNA
  • the sequence is as follows: 5′-ACCAAGTCATATGTCATTAA-3′.
  • P2 5′-CAAGTAAAATAATTGGTCTTTAAGTGACATATGACTTGGTAGTTTACTG-3′;
  • P4 5′-TTTTTTACAAAATG CTTAAG AACTAGCGTATGTGATAACTTAG-3′ was used to amplify the homology arm of the PfAP2-O5 gene and introduce a synonymous mutation site.
  • P7 5′-TATTTTACTTGGACAAATTAGGTACCGGAAGTGGTTCTGG-3′;
  • P8 5′-TTTATTTTTTTTTTGCGGCCGCGCGGCCGCGTCCCCTC-3′ is used to amplify the exogenous green fluorescent protein (GFP) gene.
  • GFP green fluorescent protein
  • 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).
  • 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-O5 gene was labeled with the glmS ribozyme sequence.
  • the reaction system was as follows: 2 ⁇ Phanta Max Master Mix (Dye Plus): 25 ⁇ L, P1 primer (10 ⁇ M): 2 ⁇ L, P4 primer (10 ⁇ M): 2 ⁇ L, 1 ⁇ L each of the left and right homologous sequences and glmS ribozyme sequence, and ddH 2 O was added to make up to 50 ⁇ L system.
  • 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.
  • the plasmid pL6cs carrying the resistance gene of the drug dihydrofolate reductase inhibitor (WR) for screening was double-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 100 ⁇ g/ml ampicillin and cultured overnight. A single colony was picked, cultured at 34°C and 200rpm/s overnight, and the plasmid was extracted for sequencing.
  • 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°C.
  • the knockdown vector of the transgenic insect strain was obtained and named pL6cs-ap2-o5-glmS.
  • a transgenic insect strain was constructed by combining electrofection with drug screening: the PfAP2-O5 conditional knockdown insect strain.
  • the correctly sequenced bacterial strain was inoculated onto an LB agar plate containing 100 ⁇ g/ml ampicillin and cultured overnight. The next day, a single clone was picked out and cultured in LB culture medium. It was shaken at 34°C and 200 rpm/s for 8 h, and 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.
  • the electroporator was set to an electroporation parameter of 310V, 950 ⁇ F, resistance ⁇ , and a slit of 2mm.
  • An equal volume (150 ⁇ l) of 2 ⁇ cell mixture solution was added to the prepared plasmid and mixed, and then 150 ⁇ l 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.
  • 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 ⁇ l 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.
  • the culture medium 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.
  • a transgenic insect strain was constructed: PfAP2-O5 conditional knockout insect strain.
  • 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:
  • PfAP2-O5 transgenic worm strain PfAP2-O5 conditional knockdown worm strain
  • the ring body (10-15h), trophozoite (28-32h), and schizont (40-44h) stage cultures of the PfAP2-O5 transgenic 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.
  • Figure 1A shows the 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.
  • the growth curve test of the PfAP2-O5 conditional knockdown strain was performed.
  • 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 not added as a control).
  • the initial protozoan rate of each bottle was about 0.1%.
  • Four life histories were cultured continuously, and smear microscopy was performed on the worms in 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), and the protozoan rates of the drug-added group and the non-drug-added group were recorded, and the growth curve was drawn.
  • Figure 1B shows the growth curve test of the wild strain and the PfAP2-O5 conditional knockdown strain, indicating that the growth of Plasmodium was significantly inhibited after the PfAP2-O5 conditional knockdown.
  • 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:
  • Low-salt immune complex washing buffer 1 mL per tube, rotate and wash at 4°C for 5 min;
  • High-salt immune complex washing buffer 1 mL per tube, rotate and wash at 4°C for 5 min;
  • Lithium chloride immune complex washing buffer 1 mL per tube, rotate and wash at 4°C for 5 min;
  • the wild-type insect strain and the PfAP2-O5 conditional knockdown insect strain were treated with the hexahydroquinoline derivatives shown in formula (I), respectively.
  • 1 ⁇ M of the hexahydroquinoline derivatives was added one life cycle in advance.
  • 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 hexahydroquinoline derivatives were collected, respectively.
  • the strand-specific RNA sequencing library was prepared using 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 action of the inhibitor was analyzed.
  • 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 invasion gene expression after hexahydroquinoline derivatives inhibit PfAP2-O5 function.
  • 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 hexahydroquinoline derivatives, PfAP2-O5 target genes were downregulated.
  • genes of 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 times (mRNA level).
  • the genes of the Plasmodium merozoite surface structural protein family mainly regulate the invasion of Plasmodium merozoites into red blood cells.
  • Example 6 EC50 determination of hexahydroquinoline derivatives for the inhibition of malarial parasite growth in vitro
  • DHA Dihydroartemisinin
  • 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) to each well, mix thoroughly, and incubate at room temperature in the dark for 2 hours.
  • 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 the small molecule inhibitor on the growth of Plasmodium was calculated based on the fluorescence intensity.
  • F1 drug treatment, F1 background and F1 control represent the fluorescence intensity values of candidate small molecule drugs, red blood cell control and non-drug treatment control group, respectively.
  • 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 SPSS18.0 software.
  • the mammalian cells 293T and HepG2 were cultured and subcultured, they were digested with trypsin and resuspended in DMEM medium. The cells were counted and diluted to 10 5 cell/mL using a hemocytometer. The cell suspension was evenly added to a 96-well plate, 100 ⁇ L per well, and cultured in a 37°C incubator (5% CO 2 ) for 24 hours. The inhibitors of corresponding concentrations were added and gradient dilutions were performed according to the multiple gradient dilution method at a ratio of 1/2 (11 concentration gradients). Complete culture medium was used as a negative control and DMEM culture medium as a background control.
  • 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 SPSS18.0 software.
  • the purpose of this experiment is to study the species differences in the metabolic rates of hexahydroquinoline derivatives of formula (I) in mouse, rat and human liver microsomes.
  • 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
  • the incubation system consisted of 0.1M Tris-HCl buffer (pH 7.4), 1mM MgCl 2 , 1mM NADPH and 0.5mg mL-1 microsomes, and the final reaction volume was 200 ⁇ L.
  • 100 ⁇ L NADPH was added to start the reaction, and 400 ⁇ L stop solution was added to stop the reaction at 0min, 5min, 15min, 30min, 45min, and 60min, 40 ⁇ L internal standard was added, vortexed for 5min, centrifuged at 20000g, 4°C for 5min, 100 ⁇ L supernatant was taken and diluted with 100 ⁇ L water, and injected for analysis.
  • the retention time of the compound and internal standard, chromatogram acquisition and chromatogram integration were processed by Analyst 1.6.3 software, and the compound was linearly regressed with 1/X 2 as the weighting coefficient to calculate the concentration at each time point.
  • 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 k.
  • the half-life (T 1/2 ) and liver intrinsic clearance CL int(liver) of the test article were calculated according to the following formula.
  • the results of mouse/rat/human microsomal metabolism of hexahydroquinoline derivatives are shown in Table 1
  • liver microsomal metabolism showed that the inhibitor was a medium-clearance compound in mouse and human liver microsomes and a high-clearance compound in rats.
  • This experiment aims to evaluate the killing effect of hexahydroquinoline 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:
  • step 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;
  • Control group Add 100ul of DMSO control solution to each well

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Abstract

六氢喹啉衍生物或其药学上可以接受的盐或水合物的新应用。该六氢喹啉衍生物可以抑制入侵基因PfAP2-O5的表达，阻断恶性疟原虫入侵红细胞，影响其生长发育，实现恶性疟原虫的杀灭效果，因此六氢喹啉衍生物可以作为PfAP2-O5的抑制剂、抑制恶性疟原虫的药物或预防或治疗疟疾的药物。

Description

六氢喹啉衍生物在制备预防或治疗疟疾的药物中的应用 技术领域
本发明涉及医药技术领域，更具体地，涉及六氢喹啉衍生物在制备预防或治疗疟疾的药物中的应用。
背景技术
疟疾是由原生动物疟原虫感染引起的，能够严重威胁人类健康和生命安全的寄生虫病，其发病率和死亡率在传染病中居高不下。根据2021年WHO发布的《世界疟疾报告》，全球范围内共有87个国家和地区存在疟疾流行，2020年共有2.41亿新发病例并造成约62.7万患者死亡。
疟原虫生活史复杂，主要包括在终末宿主体内的有性繁殖和中间宿主体内的无性繁殖阶段。在人体中，疟原虫(子孢子)先入侵肝脏，经过一次裂体无性增殖，释放的裂殖子进入血液入侵红细胞，开始位于红细胞内的裂体增殖期，这个阶段是疟疾临床发病的时期，而裂殖子入侵红细胞是疟疾致病的关键环节。
目前以药物治疗作为疟疾防治的主要手段，其中以青蒿素为基础的联合用药方法有效的遏制了疟疾在世界范围内的传播。但是随着青蒿素类药物长期广泛使用，东南亚甚至非洲地区逐渐出现了对其敏感性下降的虫株，此类虫株的产生以及全球扩散蔓延，对世界疟疾防控以及消除疟疾战略提出了严峻的挑战。因此，寻找抗疟药物新靶点，尤其是针对疟原虫致病的关键环节研发安全有效的传播阻断药物对于遏制疟疾传播的意义至关重大。
发明内容
本发明为克服上述不足提供一种新的预防或治疗疟疾的药物。
本发明的另一目的在于提供六氢喹啉衍生物的新应用。
本发明的上述目的通过如下技术方案予以实现：
六氢喹啉衍生物或其药学上可以接受的盐或水合物在制备恶性疟原虫的转录因子PfAP2-O5抑制剂中的应用，所述六氢喹啉衍生物具有如式(Ⅰ)所示结构：
发明人在对恶性疟原虫入侵红细胞的机理进行深入的研究，首次发现PfAP2-O5的正向调控红细胞入侵的功能。而式(Ⅰ)所示六氢喹啉衍生物能够通过抑制入侵基因PfAP2-O5的表达，阻断恶性疟原虫入侵红细胞，影响其生长发育，实现恶性疟原虫的杀灭效果。
本发明所述PfAP2-O5抑制剂，是指化合物是通过抑制AP2-O5发挥功能而达到杀灭恶性疟原虫的效果。
同时，体外实验中，式(Ⅰ)所示六氢喹啉衍生物现出了良好的恶性疟原虫生长抑制效果，同时对哺乳动物细胞几乎无毒性，使得成药成为可能。针对抗性虫株也具有良好的杀灭作用，对于延缓药物抗性的产生以及蔓延具有良好的帮助，同时将会更高效的进行抗疟治疗。
六氢喹啉衍生物或其药学上可以接受的盐或水合物在制备抑制恶性疟原虫的药物中的应用；所述六氢喹啉衍生物具有如式(Ⅰ)所示结构：
六氢喹啉衍生物或其药学上可以接受的盐或水合物在制备预防或治疗由恶性疟原虫引起的疟疾的药物中的应用，所述六氢喹啉衍生物具有如式(Ⅰ)所示结构：
本发明中，所述“药学上可接受的盐”包括所述六氢喹啉衍生物与无机酸或有机酸形成的盐。
所述无机酸例如是盐酸、氢溴酸、硫酸、磷酸、硝酸等。所述有机酸例如是乙酸、羟基乙酸、丙酸、乳酸、丙酮酸、丙二酸、琥珀酸、马来酸、富马酸、苹果酸、酒石酸、柠檬酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、环酸、环状水杨酸、对氨基水杨酸等。
本发明中，所述“水合物”是指含有以一定比例结合的水分子作为晶体的组成部分的盐。
一种药物组合物，以包含如式(Ⅰ)所示结构的六氢喹啉衍生物或其药学上可以接受的盐或水合物作为活性成分，以及药学上可接受的载体、稀释剂或赋形剂作为助剂；
在制备这些组合物时，通常将活性成分与赋形剂混合，或用赋形剂稀释，或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时，它可以是固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。
合适的赋形剂的例子包括乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、阿拉伯胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆和甲基纤维素。除此以外，组合物中还可包括润滑剂(如滑石粉、硬脂酸镁和矿物油)，湿润剂，乳化剂和悬浮剂，防腐剂(如羟基苯甲酸甲酯和羟基苯甲酸丙酯)，甜味剂或矫味剂。
因此，所述药物组合物可以制备成各种已知的剂型，优选地，所述药物组合物制成的剂型为口服剂型或注射剂型。
口服剂型，包括固态剂型如片剂、滴丸、速释滴丸、胶囊(如液体凝胶胶囊和固体凝胶胶囊)、颗粒剂或粉剂。片剂可以是压缩片剂、研磨片剂、肠溶衣片剂、糖衣片剂、薄膜衣片、或多层压制片。作为制备片剂的合适助剂类型，包括但不限于粘合剂、润滑剂、稀释剂、崩解剂、着色剂、调味剂、引流剂或融化剂。口服剂型还可以是液态口服剂型，如水溶液、乳液、悬浮液、由非泡腾颗粒制成的溶液和/或悬浮液、或泡腾颗粒制成的泡腾剂。作为制备液态口服剂的合适助剂类型，包括溶剂、防腐剂、乳化剂、悬浮剂、稀释剂、甜味剂、融化剂、着色剂或调味剂。
所述注射剂型包括：注射液(普通)、冻干粉针、粉针(普通)、注射用片剂等。
具体地，所述药物组合物可以是片剂、丸剂、粉剂、酏剂、混悬剂、乳剂、溶液剂、糖浆剂、软和硬明胶胶囊、栓剂、灭菌可注射溶液和灭菌包装粉剂。
通过恶性疟原虫生长抑制实验，以青蒿素作为对照，本发明对恶性疟原虫的生长抑制EC50值达到2.158μM，表现出良好的生长抑制功能。
所述药物组合物，可以是以式(Ⅰ)所示结构的六氢喹啉衍生物或其药学上可以接受的盐或水合物作为唯一活性成分，还可以将已知的具有青蒿素类化合物作为共同的活性成分。
优选地，所述活性成分中还包括青蒿素类化合物。青蒿素类化合物已经被证明对疟疾有治疗作用。更优选地，所述青蒿素类化合物为双氢青蒿素类化合物。双氢青蒿素类化合物对多种抗药性疟原虫具有比一般青蒿素类化合物更好的效果。
优选地，所述组合物中，式(Ⅰ)所示结构的六氢喹啉衍生物或其药学上可以接受的盐或水合物与青蒿素类化合物的摩尔比为1:500～500:1。
优选地，所述药物组合物制成的剂型为口服剂型或注射剂型。
所述药物组合物在制备预防和/或治疗疟疾药物中的应用。
优选地，所述药物组合物在制备治疗对青蒿素及其类似物有抗性的疟疾的药物中的应用。
与现有技术相比，本发明的有益效果是：
本发明提供了六氢喹啉衍生物或其药学上可以接受的盐或水合物的新应用，该六氢喹啉衍生物可以抑制入侵基因PfAP2-O5的表达，阻断恶性疟原虫入侵红细胞，影响其生长发育，实现恶性疟原虫的杀灭效果，因此所述六氢喹啉衍生物可以作为PfAP2-O5的抑制剂、抑制疟原虫的药物或预防或治疗疟疾的药物。基于上述发现，本发明提供了一种新的药物组合物，该药物组合物可以单独以六氢喹啉衍生物作为活性成分，或将其与青蒿素类化合物复配作为共同活性成分；该药物组合物可以用于制备治疗疟疾药物，尤其是制备治疗对青蒿素及其类似物有抗性的疟疾的药物。
附图说明
图1中，图1A显示了PfAP2-O5基因敲减虫株的WB鉴定结果，PfAP2-O5基因敲减后，PfAP2-O5蛋白表达水平降低；图1B显示了PfAP2-O5基因敲减虫株的生长曲线测试。
图2显示了ChIP-seq分析PfAP2-O5在全基因组上的分布情况；以及PfAP2-O5结合靶基因类型，其中，大部分是入侵基因家族。
图3为六氢喹啉衍生物对于入侵基因表达抑制效果示意图。图3A采用了RNA-Seq技术来确定PfAP2-O5基因敲减虫株入侵基因表达情况；图3B显示了六氢喹啉衍生物抑制PfAP2-O5基因功能后入侵基因表达情况；图3C显示了PfAP2-O5基因敲减与六氢喹啉衍生物处理对于入侵基因表达抑制效果。
图4为六氢喹啉衍生物对体外培养疟原虫的定量生长抑制效果的效果示意图。显示了其对体外培养疟原虫的定量生长抑制效果EC50为2.158μM，采用3-day SYBR GreenI细胞生长测定的方法来确定疟原虫生长情况。
图5为所述六氢喹啉衍生物对人肾上皮细胞系293T和人肝癌细胞系HepG2的细胞毒性作用效果示意图。同时显示了小分子抑制剂对疟原虫的抑制效果与对不同细胞系毒性间的选择指数，采用采用3-day CCK8的方法测定细胞生长情况。
图6为所述六氢喹啉衍生物作为与双氢青蒿素的配伍用药对抗性虫株杀灭作用，采用环状体存活实验来明确联合用药对抗性虫株生长抑制作用。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，但本发明的实施方式不限于此。
本发明所采用的试剂、方法和设备，如无特殊说明，均为本技术领域常规试剂、方法和设备。
1、式(Ⅰ)所示结构的六氢喹啉衍生物：购自ChemDiv ID.4023-0687
2、恶性疟原虫(3D7_G7)：购自ATCC
3、293T细胞株：购自ATCC
4、HepG2细胞株：购自ATCC
5、SYBR Green I：购自Invitrogen Cat.No.S-7585
6、胰酶-EDTA：购自Gbico Cat.No.25200-072
7、DMEM培养基：购自Gbico Cat.No.11965-092
8、FBS：购自Gbico Cat.No.10082-147
9、双链DNA定量检测试剂盒：购自ThermoFisher Cat.No.P7581
10、蛋白A/G磁珠：购自ThermoFisher Cat.No.88803
11、小鼠单克隆抗体：购自Sigma Cat.No.SAB4800032
12、37％甲醛溶液：购自Sigma Cat.No.47608-250ML-F
13、96孔板：购自Corning
14、红细胞：购自上海市血液中心
15、双氢青蒿素(CAS:71939-50-9)
16、各种缓冲液成分如下：
(1)裂解缓冲液
(2)超声缓冲液
(3)ChIP稀释缓冲液
(4)低盐免疫复合物洗涤缓冲液
(5)高盐免疫复合物洗涤缓冲液
(6)氯化锂免疫复合物洗涤缓液
(7)TE缓冲液
(8)洗脱缓冲液
实施例1疟原虫培养
恶性疟原虫使用RPMI 1640完全培养基在37℃培养箱(5％ CO2、5％ O2和90％N2)中培养。
0.5L RPMI 1640完全培养基包含：5.22g RPMI-1640粉末；1.0g碳酸氢钠；5g AlbmaxI；2.98g 25mM HEPES；13.6mg次黄嘌呤；10mg硫酸庆大霉素。
实施例2构建转基因虫株：PfAP2-O5条件性敲减虫株
本实验利用CRISPR/Cas9基因编辑系统，将向导RNA克隆到载体，构建PfAP2-O5条件性敲减虫株。
具体构建步骤如下：
1、转基因虫株敲减载体的构建(命名为pL6cs-ap2-o5-glmS)
利用CRISPR/Cas9基因编辑系统，将向导RNA克隆到载体，具体做法如下：
(1)gRNA的设计
从PlasmoDB网站中下载恶性疟原虫3D7-G7株PfAP2-O5基因的序列(来自https://plasmodb.org/plasmo/app数据库，恶性疟原虫：PF3D7_1449500(plasmodium falciparum)715aa PfAP2-O5。同时该序列也在ncbi有披露：Sequence ID:XM_001348609.1)，通过BLAST比对，选取长度为20bp的特异性序列作为引导RNA(guide RNA，gRNA)，序列如下：5′-ACCAAGTCATATGTCATTAA-3′。
(2)引物的设计
应用SnapGene软件设计引物：
P1：5′-GCCCTAGTCTAGGGcGCGCCCAATTTTAATAACAATTTGGCG-3′；
P2：5′-CAAGTAAAATAATTGGTCTTTAAGTGACATATGACTTGGTAGTTTACTG-3′；
P3：5′-AGCGGCCGCAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATATTATAC-3′；
P4：5′-TTTTTTACAAAATGCTTAAGAACTAGCGTATGTGATAACTTAG-3′用于扩增PfAP2-O5基因的同源臂并引入同义突变位点，引入同义突变位点是为了阻止gRNA对质粒自我切割；
P7：5′-TATTTTACTTGGACAAATTAGGTACCGGAAGTGGTTCTGG-3′；
P8：5′-TTTATTTTTTTTGCGGCCGCGCGGCCGCGTCCCCTC-3′用于扩增外源的绿色荧光蛋白(GFP)基因。
(3)基因扩增
提取恶性疟原虫3D7-G7株基因DNA，采用P1+P2，P3+P4引物分别扩增PfAP2-O5基因的左侧和右侧同源序列并引入突变位点、P7+P8引物扩增glmS核酶序列(glmS序列来自于该篇文献报道：doi:10.1371/journal.pone.0073783)。
(4)连接产物的构建
用引物分别将左侧和右侧同源序列与glmS核酶序列连接，得到连接产物。PfAP2-O5基因的3’末端标记有glmS核酶序列。
反应体系如下：2×Phanta Max Master Mix(Dye Plus)：25μL，P1引物(10μM)：2μL，P4引物(10μM)：2μL，左侧和右侧同源序列与glmS核酶序列各1μL，ddH2O补足至50μL体系。
PCR条件如下：95℃3min，95℃15s，65℃15s，65℃30s/kb，65℃5min，共30个循环；72℃10min。
(5)敲减载体的构建
采用XhoⅠ和AvrⅡ双酶切携带筛选药物二氢叶酸还原酶抑制剂(WR)抗性基因的质粒pL6cs(质粒为实验室自建，具体序列参见文献报道：doi:10.3389/fmicb.2020.625862)，产物经胶回收试剂盒回收纯化，经DNA连接酶将gRNA片段连接后转化至大肠埃希菌(Escherichia coli)XL-10感受态细胞，接种至含100μg/ml氨苄青霉素的LB琼脂平板上培养过夜。挑取单个菌落，34℃、200rpm/s振荡培养过夜，提取质粒测序。
经测序确定连上gRNA的质粒，采用AscⅠ和AflⅡ双酶切，产物经回收后与含有glmS核酶序列的同源臂连接转化，挑取单克隆进行培养，经PCR鉴定正确质粒进行测序，菌种-80℃保存。
得到转基因虫株敲减载体，命名为pL6cs-ap2-o5-glmS。
2、构建PfAP2-O5条件性敲减虫株
利用电转染结合药物筛选的方式，构建转基因虫株：PfAP2-O5条件性敲减虫株。
(1)电击转染质粒的制备
取经测序正确的菌种接种至含有100μg/ml氨苄青霉素LB琼脂平板上培养过夜，次日挑取单克隆于LB培养液中，34℃、200rpm/s振荡培养8h，按1：1000稀释至400ml LB培养液中，振荡培养14h后收集菌体，经质粒DNA纯化试剂盒收集质粒。
取上述构建的转基因虫株敲减载体pL6cs-ap2-o5-glmS质粒，与携带筛选药物杀稻瘟菌素(BSD)抗性基因且表达Cas9蛋白的质粒pUF1-Cas9(质粒为实验室自建，具体序列参见文献报道：doi:10.3389/fmicb.2020.625862)各100μg，加入质粒溶液1/10体积的3M CH3COONa(pH5.2)溶液，混匀后加入质粒溶液2.5倍体积的无水乙醇充分混匀，13000×g离心30min，弃上清，将沉淀用75％的乙醇洗涤2次后，在超净台中晾干并用150μl无菌水溶解沉淀。
(2)电击转染及转基因虫株的筛选
将电转仪设定条件为电击参数为310V，950μF，电阻∞，狭缝2mm。向制备好的质粒中加入等体积(150μl)的2×细胞混合溶液并混匀，再加入150μl红细胞，混匀后转入电击杯中，置于电转仪中电击，电击后将混合液转入培养瓶中，加入适量培养液，随后加入纯化后的裂殖体期疟原虫。
在37℃三气培养箱中进行培养。第二天起，每天更换新鲜培养基，第四天电击转染密度10％左右时(原虫率，计数一定视野下(～5000个)感染疟原虫的红细胞占所有红细胞的比例)补加200μl的新鲜红细胞，更换培养液并加入筛选药物WR和BSD(杀稻瘟菌素)，筛选药物可将野生型的恶性疟原虫杀死，而转基因成功的疟原虫因携带表达抗药基因的质粒而不受药物影响，因此可筛选出转基因的疟原虫。
加筛选药物直至绝大部分野生疟原虫被杀死后，改为每3～4天更换培养液并加药，维持培养。在转染后的第21天开始涂片镜检，3～6周获得转基因虫株。
综上，便构建得到转基因虫株：PfAP2-O5条件性敲减虫株。
3、梯度稀释克隆化
转基因得到的转基因虫株(PfAP2-O5条件性敲减虫株)为整合型虫株，需要进行梯度稀释克隆化，即通过有限稀释法，筛选得到完全为整合型的单克隆株，该实验在96孔细胞培养板中进行，通过梯度稀释，分别将虫体感染的红细胞稀释到理论上每1ml中含有10个、1个、0.5个和0.1个虫子，具体实验步骤如下：
(1)对转基因虫株进行sorbitol同步化，得到性质较为单一的环状体期虫体；
(2)于虫体滋养体期涂片镜检，严格计数原始原虫率P x；
(3)收集滋养体期培养物，于2000rpm，室温，离心5min，弃上清，得到约200μl感染红细胞压积(iRBC)；
(4)向iRBC中加入800μl新鲜培养基，轻柔混匀后，转入1.5ml离心管中，至终体积1ml；
(5)1500rpm，室温离心2-3min，弃上清，取50μl iRBC至新的离心管中备用；
(6)按照公式50μl×P x＝1％×(50μl+Vx)，将上述50μl感染红细胞的原虫率稀释至1％，获得培养物A(Vx为加入的新鲜红细胞体积)，并涂片镜检确认稀释后的原虫率；
(7)预热新鲜培养基，按照梯度稀释的原理，将上述准备的原虫率为1％的感染红细胞按比例依次稀释，使得每一个离心管对应一个梯度：
准备9支15ml的离心管并依次编号为1、2、3……9，管1中加入10ml新鲜培养基，管2-7和9中加入9ml新鲜培养基，管8中加入5ml新鲜培养基，所有离心管于37℃培养箱中预热备用。将(6)中原虫率为1％的培养物A取100μl加入到上述管1中，获得1×106个/ml iRBC的培养物B；
1)取1ml培养物B至管2中，获得1×105个/ml iRBC的培养物C；
2)取1ml培养物C至管3中，获得1×104个/ml iRBC的培养物D；
3)取1ml培养物D至管4中，获得1×103个/ml iRBC的培养物E；
4)取1ml培养物E至管5中，获得1×102个/ml iRBC的培养物F；
5)取1ml培养物F至管6中，获得10个/ml iRBC的培养物G；
6)取1ml培养物G至管7中，获得1个/ml iRBC的培养物H；
7)取5ml培养物H至管8中，获得0.5个/ml iRBC的培养物I；
8)取1ml培养物H至管9中，获得0.1个/ml iRBC的培养物J。
(8)将上述获得的不同梯度的培养物G、H、I、J分别转移到96孔细胞培养板中，200μl/孔，最终96孔板每一行12个孔中对应一个梯度。
(9)后期培养按照每2天换一次新鲜培养基，每6天添加一次新鲜红细胞(2μl/孔)维持培养；
约2周后涂片镜检，直到镜检到活虫，便可以用24孔、12孔、6孔板慢慢扩大培养，基因组检测正确后，得到完全整合型的单克隆PfAP2-O5转基因虫株(PfAP2-O5条件性敲减虫株)，保种并进行后续实验。
实施例3 PfAP2-O5转基因虫株(PfAP2-O5条件性敲减虫株)鉴定及生长发育测试
收集PfAP2-O5转基因虫株(PfAP2-O5条件性敲减虫株)的环状体(10-15h)、滋养体(28-32h)、裂殖体(40-44h)期培养物。0.15％皂素裂解感染疟原虫的红细胞收集蛋白样本，SDS-PAGE电泳分离蛋白后，以半干转法将蛋白转移至硝酸纤维素膜，转膜参数为：0.1A，2h。转膜结束后，加入适量体积的封闭液(5％脱脂奶粉：脱脂奶粉溶于PBST溶液)，室温封闭2h；孵育一抗(鼠来源的anti-Ty1抗体)：一抗(anti-Ty1)用5％脱脂奶粉按照1:1000进行稀释，将膜密封4℃过夜孵育；孵育二抗：二抗(羊抗鼠)用5％脱脂奶粉按照1:5000进行稀释，室温孵育2h；显色：取等体积的显色液A、B混匀(注意避光)，取一定量显色液滴加于膜上，拍照保存曝光图片。结果如图1A所示，图1A显示了PfAP2-O5条件性敲减虫株加入5mM氨基葡萄糖(GlcN)前后western blot鉴定，表明PfAP2-O5被有效敲降。
PfAP2-O5条件性敲减虫株生长曲线测试，将虫株经过几次同步化至环状体期，严格计数确定起始原虫率，分成2瓶(一瓶加入相应浓度的GlcN，另一瓶作为对照不加)，每一瓶的起始原虫率约为0.1％。连续培养4个生活史，在每个生活史的虫子中晚期涂片镜检，原虫率计数(原虫率高要更换培养基，加药组补加药物)，记录加药组与不加药组虫株的原虫率，绘制生长曲线。结果如图1B所示，图1B显示了野生虫株和PfAP2-O5条件性敲减虫株的生长曲线测试，表明PfAP2-O5条件性敲减后疟原虫的生长受到明显的抑制。
实施例4染色质免疫共沉淀(ChIP)测序
染色质免疫共沉淀(ChIP)测序为利用抗原抗体反应的特异性，在染色质水平上真实反映AP2-O5结合的靶基因在全基因组上的分布，同时反应蛋白与靶基因的结合情况，具体做法如下：
收集经同步化处理后的裂殖体期的培养物，加入1％的甲醛溶液，37℃孵育10min；加入预冷的0.125M甘氨酸溶液冰上孵育5min；2500rpm，4℃，离心5min，弃上清，加入30mL的1×PBS重悬红细胞，加入0.15％皂素裂解红细胞收集虫体；加入2mL预冷的Lysis Buffer重悬虫体转移到预冷的研磨管中冰浴30min；加入200μL的超声缓冲液，重悬沉淀后液相超声，参数设置为：占空比5％,峰值入射功率75W，循环次数200，温度7℃，时间30分钟；取10μL超声后的混合液加入90Μl ChIP稀释缓冲液，4μL 5M NaCl，2.5μL蛋白酶K，混匀后50℃孵育2h，加入2μLRNase A，37℃孵育45min，QIAGEN PCR试剂盒抽提，2％琼脂糖凝胶电泳检测超声效果；超声效果理想的超声产物14000pm，4℃离心10min后收集上清，加入10倍体积的ChIP稀释缓冲液；在上述溶液中加入蛋白A/G磁珠，4℃摇晃孵育2h，上清转移到1.5mL离心管中，加入相应抗体和蛋白A/G磁珠，4℃过夜孵育；抗体/磁珠/染色质复合物，按照如下顺序加入不同的缓冲液进行洗涤：
A：低盐免疫复合物洗涤缓冲液：每管1mL，4℃旋转洗涤5min；
B：高盐免疫复合物洗涤缓冲液：每管1mL，4℃旋转洗涤5min；
C：氯化锂免疫复合物洗涤缓冲液：每管1mL，4℃旋转洗涤5min；
D：TE缓冲液洗涤2次，每管1mL，37℃旋转洗涤5min；
加入200μL洗脱缓冲液进行洗脱，37℃旋转孵育30min，收集上清，同时在免疫沉淀前的DNA管加入180μL洗脱缓冲液；上述各管中加入16μL的5M NaCl，混匀后于45℃过夜孵育；孵育结束后每管加入0.8μL RNase A(20mg/mL)，37℃，30min；每管加入3μL的蛋白酶K，45℃孵育2h；采用QIAGEN PCR纯化试剂盒提取样本DNA，洗脱时用10μL去离子水洗脱两次，每次洗脱前室温放置10min；测定浓度，文库构建。为了制备测序文库，将1.5ng ChIP-DNA进行末端修复、3’腺苷酸化和接头连接；Agencourt AMPure XP磁珠纯化后，使用KAPA HiFi PCR试剂盒在以下条件下扩增文库：95℃1min，98℃10s，65℃1min，12个循环，65℃5min，4℃∞。在Illumina HiSeq X Ten平台上对文库进行测序；对每个ChIP-seq数据集进行两次生物重复。
结果如图2所示，表明PfAP2-O5蛋白靶基因在全基因组上的分布情况，例如入侵基因家族msps、raps、rhophs、ralp1、ama1等，证明PfAP2-O5的靶基因大部分与入侵相关
实施例5转录组测序(RNA-seq)分析六氢喹啉衍生物对PfAP2-O5的作用
利用式(Ⅰ)所示六氢喹啉衍生物分别处理野生型虫株和PfAP2-O5条件性敲减虫株，虫株严格同步后，提前一个生活史加入1μM六氢喹啉衍生物，作用一个生活史周期后，分别收集六氢喹啉衍生物处理后野生型虫株和PfAP2-O5条件性敲减虫株的环状体(10-15h)、滋养体(28-32h)、裂殖体(40-44h)期培养物，Direct-zol RNA试剂盒纯化总RNA后，以KAPA mRNA beads制备链特异性RNA测序文库，并将RNA片段化；在Illumina HiSeq X Ten系统上对文库进行测序，并分析两种虫株经抑制剂作用后差异基因表达。
结果如图3示，图3A采用了RNA-Seq技术来确定PfAP2-O5条件性敲减后入侵基因表达情况；图3B采用了RNA-Seq技术来确定六氢喹啉衍生物抑制PfAP2-O5功能后，入侵基因表达抑制效果。结果表明，PfAP2-O5条件性敲减后，入侵相关靶基因下调，野生型虫株经六氢喹啉衍生物作用后，PfAP2-O5靶基因下调。如Rhoptry Neck，Peripheral surface protein，Rhoptry protein，Rhoptry bulb，Micronemes protein，GPI-Anchored MSP等疟原虫裂殖子表面结构蛋白家族基因表达水平出现1.5倍以上的显著下降(mRNA水平)。(疟原虫裂殖子表面结构蛋白家族基因主要调控疟原虫裂殖子入侵红细胞，主要参考文献如下：DOI:10.1016/j.chom.2017.07.003；DOI:10.1016/j.chom.2017.05.006)；图3C显示了PfAP2-O5条件性敲减以及六氢喹啉衍生物抑制PfAP2-O5功能后，入侵相关靶基因交集。
实施例6六氢喹啉衍生物疟原虫体外生长抑制EC50测定
100μL完全培养基加入到96孔板中，在第一孔中加入200μL浓度为200μM的化合物储备液，按照倍比梯度稀释法按照1/2的比例进行梯度稀释(11个浓度梯度)，以双氢青蒿素(DHA)作为阳性对照，完全培养基作为阴性对照，不加疟原虫和化合物的红细胞作为背景对照。
准确向每孔中加入100μL野生型疟原虫培养物(原虫率为1％，红细胞压积为4％)，使得最终每孔原虫率为0.5％，红细胞压积为2％，化合物终浓度梯度为(100μM,50μM,25μM,12.5μM,6.25μM,3.125μM,1.5625μM,0.78125μM,0.390625μM,0.195313μM,0.097656μM)，所有样本设置三个复孔；完成加样后，96孔板放置到37℃培养箱(5％ CO2,5％ O2)准确培养72h。培养完成后，每孔加入100μL Lysis buffer(1x SYBRGreen I,0.12mg/mL皂素,0.12％v/vTriton X-100,30mM Tris-HCl,and 7.5mM EDTA)充分混匀后室温避光孵育2h。孵育完成后， 在酶标仪上以485nm为激发光波长，535nm为发射波长记录荧光强度，根据荧光强度计算小分子抑制剂对疟原虫生长的抑制率。
存活率＝(Fl药物处理-Fl本底背景)/(Fl对照-Fl本底背景)
其中Fl药物处理、Fl本底背景和Fl对照分别表示候选小分子药物，红细胞对照和无药物处理对照组的荧光强度值。
根据不同浓度的存活率计算药物抑制率，并以抑制率-药物浓度汇总生长抑制曲线，通过SPSS18.0软件计算IC50。
结果如图4所示，六氢喹啉衍生物对体外培养疟原虫的定量生长抑制效果IC50为2.158μM。
实施例7小分子抑制剂细胞毒性IC50测定
哺乳动物细胞293T,HepG2培养传代后，胰酶消化，DMEM培养基重悬后，以血球计数板计数并稀释至105cell/mL，将细胞悬液均匀加入96孔板，每孔100μL，37℃培养箱(5％CO2)贴壁培养24h后加入相应浓度抑制剂，按照倍比梯度稀释法按照1/2的比例进行梯度稀释(11个浓度梯度)，以完全培养基作为阴性对照，DMEM培养基作为背景对照。37℃培养箱(5％CO2)培养72h后，每孔加入10μL CCK8试剂，37℃培养箱(5％CO2)孵育1h，酶标仪读取450nm波长吸光度，根据吸光度计算存活率
存活率＝(Ab药物处理-Ab本底背景)/(Ab对照-Ab本底背景)
其中Ab药物处理、Ab本底背景和Ab对照分别表示小分子药物，DMEM对照和无药物处理对照组的吸光度值。根据不同浓度的存活率计算药物抑制率，并以抑制率-药物浓度汇总生长抑制曲线，通过SPSS18.0软件计算IC50。
结果如图5所示。图5可以看出，本发明所述式(Ⅰ)结构的六氢喹啉衍生物对恶性疟原虫3D7株新型裂殖子蛋白质的IC50为2.16μm。同时，图5反映本发明所述式(Ⅰ)结构的六氢喹啉衍生物对于哺乳动物细胞毒性较低，具有良好的生物安全性。
实施例8小分子抑制剂药物代谢动力学分析
本实验旨在研究式(Ⅰ)结构的六氢喹啉衍生物在小鼠、大鼠和人肝微粒体中代谢速率的种属差异。
微粒体代谢试验通过将抑制剂与不同种属的肝微粒体孵育，在不同时间点终止反应，利用LC-MS/MS方法测定各时间点样品中抑制剂的剩余量，计算原药剩余百分比，最终得出肝固有清除率CLint(liver)。睾酮在本研究中用作对照物来检验实验体系的可靠性。具体做法如下
温孵体系由0.1M的Tris-HCl缓冲液(pH 7.4)、1mM MgCl2、1mM NADPH和0.5mg mL-1微粒体组成，反应的终体积为200μL。将1M抑制剂及睾酮与不同种属动物的肝微粒体在37℃水浴中预温孵5min后，加入100μL NADPH启动反应，分别于0min、5min、15min、30min、45min、60min的时间点加入400μL终止液终止反应，加入40μL内标涡旋5min，20000g，4℃离心5min，取100μL的上清液并加入100μL水稀释，进样分析。化合物和内标的保留时间、色谱图采集和色谱图的积分由软件Analyst 1.6.3进行处理，化合物以1/X2为加权系数进行线性回归，计算各时间点浓度。将各时间点的百分剩余量的自然对数对孵育时间作线性回归，算得体外消除速率常数k。按下面公式计算供试品的半衰期(T1/2)和肝固有清除率CLint(liver)。
半衰期肝微粒体清除率CLint(mic)＝0.693/半衰期/mg微粒体蛋白每mL；肝固有清除率CLint(liver)＝肝微粒体清除率×mg微粒体蛋白/g肝重×肝重体重比，小鼠、大鼠和人的肝重体重比分别为88、40和20g/kg；mg微粒体蛋白/g肝重在不同种属的均数值为45。六氢喹啉衍生物小鼠/大鼠/人微粒体代谢结果如表1所示
表1
肝微粒体代谢结果表明，抑制剂在小鼠和人肝微粒体中属于中清除率化合物，在大鼠中属于高清除率化合物。
实施例9六氢喹啉衍生物抗性虫株环状体存活实验
本实验旨在通过药物抗性金标准方法，评价六氢喹啉衍生物作为配伍用药对药物抗性虫株的杀灭作用。具体实施步骤如下：
1.疟原虫培养，原虫率大于4％且大部分为R期时，Sorbitol同步10min，涡旋震荡5s；
2.30-48h(视虫体阶段决定)后重复Sorbitol同步；
3.培养约30h后涂片镜检：若成熟S期(10-12个裂殖子)的比例大于0.5％，进行第4步；若成熟S期(10-12个裂殖子)的比例小于0.5％，重复第2步；
4.收集培养物进行晚期虫体富集(Plasmion或Percoll)；涂片镜检富集的虫体，S期必须大于10％，R期必须小于10％；
5.加入10ml培养基，200ul(适量)新鲜的红细胞，维持培养3h(必须精确)；
6.将培养物混匀后吸取约200ul离心后涂片镜检(可提前约10min)，迅速计算原虫率(必须大于0.5％)；
7.收集培养物进行Sorbitol同步10min，涡旋震荡5s，离心弃上清；
8.在24孔板中：对照组：每孔加入100ul的DMSO对照溶液
实验组：每孔加入100ul的7mM DHA溶液以及20μM六氢喹啉衍生物溶液(溶于培养基)；
9.每孔加入900ul重悬的iRBC培养物，混匀；精确维持培养6h；
10. 6h后，转移每孔中的培养物至1.5mlEP管中，离心去上清；
11.加入1ml预热培养基洗涤，离心去上清；重复洗涤一次，共洗涤两次；
12.加入1ml预热的培养基混匀，转移至新的孔板中，继续维持培养66h；
13.培养66h后涂片镜检计算原虫率，并计算生存率。
结果如图6所示，六氢喹啉衍生物(inhibitor)表示，“-”表示不含对应物质、“+”表示含有对应物质，结果表明与DHA联合用药后能够更有效杀灭青蒿素抗性虫株，提示小分子能够作为配伍药物改善青蒿素抗性的蔓延。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 六氢喹啉衍生物或其药学上可以接受的盐或水合物在制备恶性疟原虫的转录因子PfAP2-O5抑制剂中的应用，所述六氢喹啉衍生物具有如式(Ⅰ)所示结构：
  2. 六氢喹啉衍生物或其药学上可以接受的盐或水合物在制备抑制恶性疟原虫的药物中的应用，所述六氢喹啉衍生物具有如式(Ⅰ)所示结构：
  3. 六氢喹啉衍生物或其药学上可以接受的盐或水合物在制备预防或治疗由恶性疟原虫引起的疟疾的药物中的应用，所述六氢喹啉衍生物具有如式(Ⅰ)所示结构：
  4. 一种药物组合物，其特征在于，以包含如式(Ⅰ)所示结构的六氢喹啉衍生物或其药学上可以接受的盐或水合物作为活性成分，以及药学上可接受的载体、稀释剂或赋形剂作为助剂；
  5. 根据权利要求4所述药物组合物，其特征在于，所述活性成分中还包括青蒿素类化合物。
  6. 根据权利要求5所述药物组合物，其特征在于，所述青蒿素类化合物为双氢青蒿素类化合物。
  7. 根据权利要求5所述药物组合物，其特征在于，所述组合物中，式(Ⅰ)所示结构的六氢喹啉衍生物或其药学上可以接受的盐或水合物与青蒿素类化合物的摩尔比为1:500～500:1。
  8. 根据权利要求4所述药物组合物，其特征在于，所述药物组合物制成的剂型为口服剂型或注射剂型。
  9. 权利要求4所述药物组合物在制备预防和/或治疗疟疾药物中的应用。
  10. 权利要求4所述药物组合物在制备治疗对青蒿素及其类似物有抗性的疟疾的药物中的应用。
PCT/CN2024/096683 2023-06-28 2024-05-31 六氢喹啉衍生物在制备预防或治疗疟疾的药物中的应用 Ceased WO2025001748A1 (zh)

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