Application of FABP5 inhibitor in preparation of medicine for inhibiting growth of plasmodium and antimalarial medicine combining FABP5 inhibitor with artemisinin
By developing the FABP5 inhibitor SBFI-26 and using it in combination with artemisinin, the existing antimalarial drug resistance and the unknown lipid metabolism mechanism of Plasmodium were solved, and significant antimalarial effects were achieved and toxic side effects were reduced.
Patent Information
- Application Number
- CN202510432602.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-06-20
AI Technical Summary
The drug resistance of existing antimalarial drugs and the mechanism of lipid metabolic interaction between the Plasmodium and the host are unknown, making it difficult to develop effective drugs to inhibit the growth of Plasmodium.
A FABP5 inhibitor, SBFI-26, was developed and used in combination with artemisinin to enhance antimalarial effects. SBFI-26 inhibits FABP5, affects the lipid metabolism of Plasmodium in the host, and thus inhibits the growth of Plasmodium.
SBFI-26 showed significant antimalarial effects in vitro, with IC50 of 12 μM to 13 μM and no obvious cytotoxicity to human normal cells and tumor cells. When combined with artemisinin, the antimalarial effect is significantly enhanced and there are no obvious toxic side effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of an FABP5 inhibitor in the preparation of a drug for inhibiting the growth of malaria parasites and an antimalarial drug in combination with artemisinin. Background Art
[0002] Malaria is a parasitic disease caused by the genus Plasmodium. Although China announced the elimination of malaria in 2021, the global burden of malaria remains heavy. In addition, the drug resistance of antimalarial drugs is an increasingly serious problem. Therefore, clarifying the interaction between malaria parasites and the host and finding new therapeutic and drug targets have become the current research focus. When malaria parasites parasitize in the human body, their life cycle can be divided into two stages, the liver stage and the erythrocytic stage, and the erythrocytic stage is the main stage causing clinical symptoms and also the main current research direction.
[0003] Lipid metabolism is very important for the life activities of living organisms. All living cells need lipids to provide energy and maintain the homeostasis of biological membranes. Similarly, lipid metabolism is crucial for the growth, proliferation, and transmission of malaria parasites in the asexual and sexual reproductive stages. There are two ways for malaria parasites to obtain fatty acids (FAs): one is through self-synthesis, and there is a special fatty acid synthesis type II pathway (FASII) in malaria parasites. The other is to directly uptake from the host serum during the erythrocytic stage. Existing studies have shown that when parasitizing in human red blood cells, FASII basically does not play a role. Therefore, the lipid metabolism of malaria parasites in the erythrocytic stage mainly relies on directly uptake from the host body, and the interaction and its mechanism between malaria parasites and the host during this process are still unknown.
[0004] Fatty acid-binding protein 5 (FABP5) is a cytoplasmic protein that is abundantly expressed in most mammalian tissues, with a size of about 15 kDa. It has multiple biological functions such as promoting the transport of lipids to specific intracellular compartments, contributing to signal transduction in the endoplasmic reticulum, lipid droplet storage, transport and membrane synthesis, oxidation in mitochondria and peroxisomes, and regulating the activities of other enzymes.
[0005] The compound SBFI-26 is a competitive inhibitor of FABP5, and its therapeutic effect in malaria has not been reported. Summary of the Invention
[0006] The present invention aims to solve at least one of the technical problems existing in the prior art or related technologies.
[0007] In the first aspect of the present invention, there is provided the use of an FABP5 inhibitor in the preparation of a drug for inhibiting the growth of malaria parasites.
[0008] Furthermore, the overall analysis of protein chip scanning and fluorescence intensity shows that the content of FABP5 in the sera of malaria patients is significantly higher than that of healthy individuals.
[0009] Furthermore, the FABP5 inhibitor includes the compound SBFI-26, and the structure of the compound SBFI-26 is shown as follows:
[0010]
[0011] Furthermore, the malaria parasite is Plasmodium falciparum.
[0012] Furthermore, the Plasmodium falciparum is the 3D7 strain of Plasmodium falciparum.
[0013] Furthermore, the compound SBFI-26 has antimalarial effects in vitro, and its IC 50 is 12 μM to 13 μM.
[0014] Furthermore, the compound SBFI-26 inhibits the development of malaria parasites before the schizont stage, and the compound SBFI-26 has no obvious effect on the function of malaria parasite schizonts.
[0015] Furthermore, the compound SBFI-26 has no obvious cytotoxicity to normal human cells and tumor cells.
[0016] In the second aspect of the present invention, there is provided an antimalarial drug in which an FABP5 inhibitor is combined with artemisinin, and the antimalarial drug includes: component A and component B; wherein, the component A is the FABP5 inhibitor, and the component B is one or more of artemisinin and its derivatives. Optionally, the component B is selected from one, two or more of artemisinin, artesunate, artemether and dihydroartemisinin.
[0017] Furthermore, in the drug against the 3D7 strain of Plasmodium falciparum, the molar ratio of the component A to the component B is 0.005:(6.25 - 12.5); in the drug against the resistant 3D7 strain of Plasmodium falciparum C580Y drug, the molar ratio of the component A to the component B is 0.7:(6.25 - 12.5).
[0018] Compared with the prior art, the present invention has at least the following beneficial effects:
[0019] The present invention provides a new use of an FABP5 inhibitor in the preparation of a drug for inhibiting the growth of malaria parasites, providing a basis for the application of the FABP5 inhibitor in the preparation of antimalarial drugs. At the same time, the FABP5 inhibitor can be used in combination with one or more of artemisinin and its derivatives, and the antimalarial effect is significantly enhanced without obvious toxic and side effects, further proving that the FABP5 inhibitor can be used in the preparation of antimalarial drugs. Brief Description of the Drawings
[0020] Figure 1 This is for the detection of the content of FABP5 in the sera of malaria patients and normal individuals provided by Example 1 of this application using protein chip technology.
[0021] Figure 2 This is for the in vitro anti - malaria activity of different concentrations of SBFI - 26 against Plasmodium falciparum 3D7 provided by Example 2 of this application ( Figure 2 A) and the IC of SBFI - 26 against Plasmodium falciparum 3D7 50 ( Figure 2 B).
[0022] Figure 3 This is for the effect of SBFI - 26 on the growth and development of Plasmodium falciparum 3D7 provided by Example 3 of this application.
[0023] Figure 4 This is for the anti - malaria effect of the combination of SBFI - 26 and dihydroartemisinin (DHA) against the Plasmodium falciparum 3D7 strain provided by Example 4 of this application ( Figure 4 A) and the anti - malaria effect of the combination of SBFI - 26 and dihydroartemisinin (DHA) against the artemisinin - resistant Plasmodium falciparum 3D7 C580Y strain ( Figure 4 B).
[0024] Figure 5 This is for the cytotoxicity of SBFI - 26 against normal human cells HEK 293T provided by Example 5 of this application ( Figure 5 A) and the cytotoxicity of SBFI - 26 against tumor cells A549 ( Figure 5 B). Detailed Embodiments
[0025] To better understand the above - mentioned technical solutions, the technical solutions of the present invention will be described in detail through specific examples below.
[0026] In the embodiments of the present invention, the difference in the content of FABP5 in the sera of malaria patients infected with Plasmodium falciparum and healthy individuals was detected. The compound SBFI - 26 was used for drug treatment, and the effect of the compound SBFI - 26 on the growth and proliferation of Plasmodium falciparum cultured in vitro was detected, so as to provide a basis for the application of SBFI - 26 in the preparation of anti - malaria drugs. The results showed that the compound SBFI - 26 could act in combination with artemisinin, and the anti - malaria effect was significantly enhanced, and there were no obvious toxic and side effects, further proving that the compound SBFI - 26 could be used in the preparation of anti - malaria drugs.
[0027] Example 1 Detection of the Content of FABP5 in the Sera of Malaria Patients and Normal Individuals by Protein Chip Technology
[0028] Detecting the content of FABP5 in the sera of patients with falciparum malaria and normal people by protein chip technology, including the following steps:
[0029] (1) Punching and sticking the chip: Use a handheld puncher to punch holes required for the experiment in the polystyrene tape, making the edges of each hole clear and the sizes uniform. Stick the tape on the aminated glass slide. After forcefully removing the air gap between the slide and the tape, place it in an incubator at 37°C for 30 min to make the tape fit tightly with the slide. Then use a high-pressure air compressor to remove the dust layer and particles on the surface of the slide.
[0030] (2) Serum spotting: Use an equal-volume pipette to add 1 μl of serum sample to each well, with a dilution ratio of 1:2. After spotting, place the slide in a wet box and incubate it in an incubator at 37°C for 2 h.
[0031] (3) Washing: After incubation, rinse the slide with running water using PBS. Then put the slide into a small square box, pour 1×PBS - 0.1% Tween 20 to submerge the slide, and place it on a horizontal shaker and shake vigorously for 10 min. Then rinse the slide with running water using DDW to remove the surface foam. Put the slide into a small square box, pour DDW to submerge the slide, and place it on a horizontal shaker and shake vigorously for 5 min. Use a high-pressure air compressor to remove the water on the surface of the slide.
[0032] (4) Blocking with 5% BSA: Use an equal-volume pipette to add 1 μl of 5% BSA to each well for blocking. After spotting, place the slide in a wet box and incubate it in an incubator at 37°C for 1 h. After incubation, repeat the above washing steps.
[0033] (5) Spotting of FABP5 antibody: Use an equal-volume pipette to add 1 μl of antibody to each well. After spotting, place the slide in a wet box and incubate it in an incubator at 37°C for 1 h. After incubation, repeat the above washing steps.
[0034] (6) Spotting of fluorescent secondary antibody: Use an equal-volume pipette to add 1 μl of fluorescent secondary antibody (diluted with PBS at a ratio of 1:100) to each well. After spotting, place the slide in a wet box and incubate it in an incubator at 37°C for 1 h. After incubation, repeat the above washing steps. After processing, use a microarray chip scanner to scan the chip.
[0035] It was found that the content of FABP5 in the sera of patients with falciparum malaria was significantly higher than that in healthy people.
[0036] The results were as Figure 1 shown. The overall analysis of the protein chip scanning results and fluorescence intensity showed that there were differences in the expression of FABP5 in the sera of patients and normal populations, and the content was higher in the sera of patients.
[0037] Inhibitory Effect of FABP5 Inhibitor SBFI-26 on the Growth of Plasmodium falciparum in the In Vitro Culture Stage
[0038] The inhibitory effect of FABP5 inhibitor SBFI-26 on the growth of Plasmodium falciparum in the in vitro culture stage, where the Plasmodium used is Plasmodium falciparum 3D7, includes the following steps:
[0039] (1) Parasite synchronization: Synchronize the Plasmodium with 5% D-sorbitol to make the Plasmodium in the trophozoite stage.
[0040] (2) Parasite density determination: Take a part of the synchronized blood, prepare a blood smear for staining, and calculate the parasite density. Add an appropriate amount of red blood cells to adjust the density to 0.8%.
[0041] (3) Preparation of blood-parasite suspension: Dilute the blood-parasite with CM to make a blood-parasite suspension with a hematocrit of 4%.
[0042] (4) Inhibitor dilution: Dilute the inhibitor with CM to 200 μM. After adding it to the blood-parasite suspension, the final concentration is 100 μM.
[0043] (5) Sampling in 96-well plate: Use a 96-well plate. Add 50 μl of the inhibitor to the first well with a multichannel pipette, and add 50 μl of CM medium to the remaining wells for two-fold serial dilution. Set three wells for each concentration as parallel controls.
[0044] (6) Setting of positive control: Set the final concentration of chloroquine to 160 μM and set three parallel wells as positive controls.
[0045] (7) Addition of blood-parasite suspension: Add 50 μl of the blood-parasite suspension to each well.
[0046] (8) Incubation: Place the 96-well plate in a sterile cylinder containing a small amount of sterile water and incubate it in a 37 °C incubator for 72 hours.
[0047] (9) Freezing treatment and thawing: Place the 96-well plate in a -20 °C refrigerator and freeze it overnight. Take out the 96-well plate from the refrigerator and let it thaw naturally at room temperature.
[0048] (10) Dye addition and incubation: Add 100 μl of SYBR Green dye (add 0.2 μl of SYBR Green to 1 ml of lysis buffer) to each well, and incubate the 96-well plate in the dark for 1 hour.
[0049] (11) Detection: Use an enzyme-linked immunosorbent assay (ELISA) reader for detection (excitation wavelength 490 nm, emission wavelength 530 nm).
[0050] It was found that the FABP5 inhibitor SBFI-26 has antimalarial effects in vitro, with an IC 50 of approximately 12.5 μM.
[0051] The results are as Figure 2 shown. After gradient dilution of the FABP5 inhibitor SBFI-26 and incubation with Plasmodium falciparum in vitro for 72 hours, Figure 2 A shows the in vitro antimalarial activities of different concentrations of SBFI-26 against Plasmodium falciparum 3D7, Figure 2 B shows the IC 50 of SBFI-26 against Plasmodium falciparum.
[0052] Example 3 Specific action time of the FABP5 inhibitor SBFI-26 on the growth inhibition of Plasmodium falciparum in the in vitro culture stage
[0053] The specific action time of the FABP5 inhibitor SBFI-26 on the growth inhibition of Plasmodium falciparum in the in vitro culture stage, using Plasmodium falciparum 3D7, includes the following steps:
[0054] (1) Parasite synchronization: After two rounds of 5% D-sorbitol synchronization, when the parasites grow to the mature schizont stage, further synchronization is carried out using the 40%-70% Percoll-sorbitol gradient separation method. After 3 hours, 5% D-sorbitol synchronization is used again to ensure that the parasites are in a highly consistent developmental stage. Subsequently, red blood cells are added, and the parasite density is adjusted to 1%, and a packed cell suspension with a hematocrit of 2% is prepared.
[0055] (2) Inhibitor treatment and observation: The packed cell suspension is added to a 24-well plate, and the inhibitor (final concentration 50 nM) is added to the corresponding wells every 8 hours, and at the same time, blood smears are prepared for observation, continuing until 56 hours.
[0056] It was found that the FABP5 inhibitor SBFI-26 has a significant impact on the developmental process of Plasmodium falciparum before the schizont stage, showing phenomena of developmental delay or even arrest. However, the addition at the schizont stage has no obvious effect, and merozoites can be normally released and invade new RBCs.
[0057] The results are as Figure 3 shown. After adding 50 μM SBFI-26, continuous developmental assessments of highly synchronized parasites (starting from 0 hpi) are carried out every 8 hours starting from 0.8 hpi, 16 hpi, 24 hpi, 32 hpi, 40 hpi, and 48 hpi. Once added, SBFI-26 is maintained until the last time point of 56 hpi. It was found that SBFI-26 has a significant impact on the growth and development of Plasmodium falciparum before the schizont stage.
[0058] Example 4: Combined effect of FABP5 inhibitor SBFI-26 and artemisinin
[0059] Combined effect of FABP5 inhibitor SBFI-26 and dihydroartemisinin (DHA), using Plasmodium falciparum 3D7 and 3D7 C580Y , including the following steps:
[0060] (1) On the day before the formal experiment, at the trophozoite stage, perform 5% D-sorbitol synchronization.
[0061] (2) On the next day, when the malaria parasites reach the late stage (about 3 hours to form rings, smear and observe after 20 hours of synchronization), perform 40%-70% Percoll-sorbitol synchronization. Prepare 90% Percoll-sorbitol in advance and dilute it to 70% and 40% with it.
[0062] (3) Wash twice with ICM, centrifuge at 2000 rpm for 5 minutes.
[0063] (4) Discard the supernatant and resuspend the parasite blood in 1:1 ICM / CM.
[0064] (5) Spread 3 ml of 70% Percoll-sorbitol at the bottom of a 15-ml centrifuge tube, and spread 40% Percoll-sorbitol above the liquid level of 70% Percoll-sorbitol. Add it again before centrifugation to prevent the two liquid levels from merging.
[0065] (6) Slowly spread the parasite blood suspension above 40% Percoll-sorbitol. Centrifuge at 3500 rpm for 20 minutes. Record the layering situation and adjust the acceleration and deceleration to the lowest.
[0066] (7) Collect the cells in the middle layer into a new test tube and wash three times with ICM.
[0067] (8) After 40%-70% Percoll-sorbitol, obtain schizonts, add fresh red blood cells, and place them in an incubator for 3 hours.
[0068] (9) After 3 hours, perform 5% D-sorbitol synchronization again and plate at intervals.
[0069] (10) Drug dilution: The storage concentration of DHA is 14 mM. When targeting the 3D7 parasite strain, dilute DHA to 10 nM with CM, and then prepare SBFI-26 at 2 times the working concentration in DHA; when targeting 3D7 C580YFor the parasite strain, dilute DHA with CM to 1400 nM and prepare SBFI-26 at twice the working concentration in DHA. Add 250 μl of CM containing the drug to each well. After plating, add 250 μl of a suspension of packed 4% and density 1% parasite blood. So that finally in the anti- Plasmodium falciparum parasite strain 3D7 drug, the molar ratios of DHA and SBFI-26 are 0.005:6.25 and 0.005:12.5 respectively; in the anti- Plasmodium falciparum resistant parasite strain 3D7 C580Y In the drug, the molar ratios of DHA and SBFI-26 are 0.7:6.25 and 0.7:12.5 respectively. At the same time, set up negative control wells and blank control wells.
[0070] (11) After culturing for 6 h, discard the supernatant. Transfer it to a centrifuge tube with a Pasteur pipette and wash twice with ICM. After washing, add 1 ml of CM containing the inhibitor, make it into 2% packed volume and divide it equally into 4 replicate wells, and continue to culture for 66 h.
[0071] (12) After 66 h, mix well, smear, stain and count under the microscope.
[0072] The results showed that the antimalarial effect was significantly enhanced after the combination of SBFI-26 and DHA.
[0073] The results are as Figure 4 shown in A. After the combination of SBFI-26 and DHA, the survival rate of the Plasmodium falciparum 3D7 strain was significantly reduced under the action of the inhibitor. Figure 4 B shows that the survival rate of the artemisinin-resistant Plasmodium falciparum strain 3D7 C580Y also decreased significantly.
[0074] Example 5 Cytotoxicity experiment of the FABP5 inhibitor SBFI-26
[0075] The cytotoxicity experiment of the FABP5 inhibitor SBFI-26 includes the following steps:
[0076] (1) Cell suspension preparation: Prepare cell suspensions of the two cell types with a medium and count them.
[0077] (2) Cell seeding: Add the cell suspension to a 96-well plate, 100 μL per well, and control the number of cells per well at 5000, and culture in a carbon dioxide incubator.
[0078] (3) Cell culture and treatment: After overnight culture, when the cell attachment rate reaches 80% - 90%, set up 6 parallel control wells. Add the corresponding inhibitor etc. to each well and continue to incubate for 48 hours.
[0079] (4) Addition of CCK-8: After the incubation, discard the liquid in the wells, add 100 μL of fresh medium containing CCK-8 solution (prepared by mixing 10 μL of CCK-8 solution with 90 μL of medium) to each well, and place the 96-well plate back into the incubator for 2 hours of incubation.
[0080] (5) Absorbance measurement: Use a microplate reader to measure the absorbance values of each well at 450 nm.
[0081] (6) Data analysis: Use GraphPad Prism 8.0 software to process and analyze the experimental data.
[0082] The results showed that the FABP5 inhibitor SBFI-26 had no obvious cytotoxicity to normal human cells and tumor cells.
[0083] Figure 5 A shows that after SBFI-26 was applied to normal cells (HEK 293T) for 48 hours detected by the CCK-8 method, cell proliferation was not significantly affected. Figure 5 B shows that after SBFI-26 was applied to tumor cells (A549) for 48 hours detected by the CCK-8 method, cell proliferation was not significantly affected either.
[0084] It is easy for those skilled in the art to understand that on the premise of no conflict, the above advantageous ways can be freely combined and superimposed. The above is only the preferred embodiment of this application and is not used to limit this application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of this application shall be included within the protection scope of this application. The above is only the preferred implementation manner of this application. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of this application, several improvements and variations can be made, and these improvements and variations should also be regarded as within the protection scope of this application.
Claims
1. Application of FABP5 inhibitors in the preparation of drugs for inhibiting the growth of Plasmodium.
2. The use according to claim 1, characterized in that: Protein chip scanning and overall analysis of fluorescence intensity showed that the FABP5 content in the serum of patients with malignant malaria was significantly higher than that in healthy people.
3. The use according to claim 1, characterized in that: The FABP5 inhibitor includes compound SBFI-26, and the structure of the compound SBFI-26 is shown in the following formula:
4. The use according to claim 3, characterized in that: The malarial parasite is Plasmodium falciparum.
5. The use according to claim 4, characterized in that: The Plasmodium falciparum is the Plasmodium falciparum 3D7 strain.
6. The use according to claim 5, characterized in that: The compound SBFI-26 has an antimalarial effect in vitro, and its IC 50 It is 12μM~13μM.
7. The use according to any one of claims 3 to 6, characterized in that: The compound SBFI-26 inhibits the development of Plasmodium before the schizont stage, and the compound SBFI-26 has no significant effect on the schizont function of Plasmodium.
8. The use according to claim 3, characterized in that: The compound SBFI-26 has no obvious cytotoxicity to normal human cells and tumor cells.
9. An antimalarial drug in combination with a FABP5 inhibitor and artemisinin, characterized in that: The antimalarial drug comprises: component A and component B; wherein component A is the FABP5 inhibitor according to any one of claims 1 to 8, and component B is one or more of artemisinin and its derivatives.
10. The antimalarial drug used in combination with a FABP5 inhibitor and artemisinin according to claim 9, characterized in that: In the drug against Plasmodium falciparum strain 3D7, the molar ratio of component A to component B is 0.005:(6.25-12.5); C580Y In the medicine, the molar ratio of the component A to the component B is 0.7:(6.25-12.5).