Application of Rosmarinol in the Preparation of Drugs against Cryptosporidium parvum

Abstract

The present invention is applicable to the technical field of anti-Cryptosporidium drugs, and provides the use of epirosmanol in the preparation of anti-Cryptosporidium parvum drugs. The present invention also provides an anti-Cryptosporidium drug, and the drug uses epirosmanol as a lead compound. The present invention discovers through screening that Epirosmanol has a definite inhibitory effect on Cryptosporidium infection, and clarifies that this inhibitory effect targets CpLDH.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-Cryptosporidium drugs, and particularly relates to the application of epirosmanol in the preparation of drugs against Cryptosporidium parvum. Background Art

[0002] Cryptosporidium parvum ( Cryptosporidium parvum ) is a gastrointestinal parasite that can cause moderate to severe diarrhea in children and adults and can cause fatal opportunistic infections in AIDS patients. In addition, due to the resistance of Cryptosporidium oocysts to chemical treatments (such as chlorine treatment), it also frequently causes waterborne outbreaks worldwide. However, the current drug research and development for cryptosporidiosis still progresses slowly. Therefore, there is an urgent need to develop new drugs for the prevention and treatment of animal cryptosporidiosis and human cryptosporidiosis.

[0003] Cryptosporidium lacks the tricarboxylic acid cycle and also lacks a cytochrome-based respiratory chain. Therefore, this type of parasite mainly relies on the glycolytic pathway to produce ATP. To promote this "anaerobic metabolism", Cryptosporidium has an l-lactate dehydrogenase (LDH), two alcohol dehydrogenases (ADH), and an acetyl-CoA synthetase, which may produce lactate, alcohol, or acetic acid as organic end products. Among these enzymes, LDH is considered a drug target for some parasites, including Plasmodium falciparum and Toxoplasma gondii among Apicomplexa parasites, as well as anaerobic protozoa, Giardia lamblia, Trypanosoma cruzi, and Entamoeba histolytica, etc. And because Cryptosporidium parvum lactate dehydrogenase (CpLDH) is of the bacterial type and is quite different from the LDH of the host humans and other mammals, using it as a drug target is more convenient for screening anti-Cryptosporidium drugs that have no toxic side effects on humans and other mammals.

[0004] Currently, several anti-Cryptosporidium lead compounds have been discovered and reported. For example, compound 1294 against calcium-dependent protein kinases (CDPKs), P131 against inosine monophosphate dehydrogenase (IMPDH), KDU731 against phosphatidylinositol-4-OH kinase (PI(4)K), triacsin C against acetyl-CoA synthetase (ACS), and SAHA against histone acetyltransferase (HDAC). In the future, one or several of these lead compounds may ultimately be developed into new drugs for cryptosporidiosis, but at the present stage, there is still a need to explore new drug targets and develop new drugs. One is the need for more treatment options, and the other is to consider the emergence of drug resistance in the future.

[0005] Epirosmanol is a natural phenolic diterpene extracted from Salvia officinalis, a traditional Chinese medicine, and has biological activities such as treating melanoma, antioxidant, antibacterial, anti-inflammatory and antioxidant stress. However, the prior art does not disclose that Epirosmanol has an inhibitory effect on Cryptosporidium infection. Summary of the Invention

[0006] An object of an embodiment of the present invention is to provide the use of epirosmanol in the preparation of a drug against Cryptosporidium parvum, aiming to solve the problems raised in the above background art.

[0007] An embodiment of the present invention is implemented as follows: the use of epirosmanol in the preparation of a drug against Cryptosporidium parvum.

[0008] Preferably, the epirosmanol inhibits the enzyme activity of Cryptosporidium lactate dehydrogenase at the submicromolar level.

[0009] Preferably, the epirosmanol inhibits the growth of Cryptosporidium at the low micromolar level.

[0010] Another object of an embodiment of the present invention is to provide a drug against Cryptosporidium, and the drug uses epirosmanol as a lead compound.

[0011] Through screening, an embodiment of the present invention finds that Epirosmanol has a definite inhibitory effect on Cryptosporidium infection, and clarifies that this inhibitory effect targets CpLDH. Brief Description of the Drawings

[0012] Figure 1 It is the SDS-PAGE result of the recombinant protein MBP-CpLDH obtained by prokaryotic expression provided in Example 1 of the present invention;

[0013] Figure 2 It is the biochemical function result of the recombinant protein MBP-CpLDH provided in Example 1 of the present invention;

[0014] Figure 3 It is the drug screening result provided in Example 2 of the present invention;

[0015] Figure 4 It is the detection result of the inhibitory effect of Epirosmanol on the enzyme activity of the recombinant protein CpLDH provided in Example 3 of the present invention;

[0016] Figure 5 It is provided in Example 4 of the present invention for Epirosmanol against C. parvum in vitro inhibition curve;

[0017] Figure 6Detection results of the cytotoxicity of Epirosmanol provided in Example 5 of the present invention. Detailed implementation mode

[0018] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] The following describes the specific implementation of the present invention in detail with reference to specific embodiments.

[0020] Example 1: Enzymatic activity identification of recombinant protein MBP-CpLDH:

[0021] Cryptosporidium lacks the tricarboxylic acid cycle and cytochrome-based respiratory chain. Therefore, the parasite depends on the glycolytic pathway to produce ATP. At the same time, the parasite depends on lactate dehydrogenase (LDH) to prevent the accumulation of pyruvate, which is harmful to anaerobic respiration, and to ensure the recycling of coenzyme factors NAD(P)H and NAD(P) + The cycle of, LDH can catalyze the interconversion of pyruvate and lactate (Lactate) and ensure the NADH / NAD + Cycle, that is: Pyruvate + NADH + H + =Lactate + NAD + ;

[0022] Express the MBP-CpLDH recombinant protein. MBP (Maltose Binding Protein), the tag protein is 40 kDa in size, which is used for protein expression and purification during prokaryotic expression, and its biochemical function is verified. Specifically: Using the pMAL-c5X-cgd7_480 plasmid, express the MBP-CpLDH protein in Escherichia coli Rosetta competent cells. Inoculate a single transformed colony into 50 mL of LB medium containing ampicillin (50 μg / mL) and glucose (2 mg / mL), culture overnight at 37°C. Inoculate the bacterial solution at a ratio of 1:10 to the LB medium, incubate at 37°C for 2 h until the OD600 reaches ~0.5; then add isopropyl-1-thio-β-D-galactoside (IPTG) with a final concentration of 0.3 mM to induce protein expression, and shake the bacteria at low speed at 16°C for ~16 h to induce Escherichia coli Rosetta to overexpress the MBP-CpLDH recombinant protein; According to the manufacturer's instructions (New England Biolabs, Ipswich, MA), centrifuge to collect the bacterial solution precipitate, lyse the bacteria, and purify the MBP-CpLDH recombinant protein using amylose resin affinity chromatography. The obtained protein is subjected to SDS-PAGE, and the results are as shown in Figure 1 ; Use an enzyme-linked immunosorbent assay (Bio Tek) to determine the enzyme activity of MBP-CpLDH by monitoring the rate of NADH oxidation at 340 nm by spectrophotometry. Calculate the enzyme kinetic related parameters of MBP-CpLDH such as the Michaelis constant ( K m ) according to the decrease in NADH per unit time. The reaction system of this enzyme is 100 μL. The forward enzyme activity reaction contains 50 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 1.2 mM pyruvate, and purified MBP-CpLDH protein (50 ng). The reverse enzyme activity reaction uses 50 mM Tris-HCl (pH 9.2) as the reaction buffer, the substrate is 1 mM NAD + and 100 mM lactate. MBP-tag (500 ng) is used as a negative control and background subtraction in all assays. The results are as shown in Figure 2 ; The purified rCpLDH protein is 75 kDa, and rCpLDH can catalyze the interconversion of pyruvate and lactate.

[0023] Example 2. Screening process of Epirosmanol:

[0024] After determining that the MBP-CpLDH recombinant protein has biochemical activity, a large amount of active recombinant CpLDH protein was obtained through prokaryotic expression. The CpLDH protein was used to screen a library of 8,892 compounds. The screening method is as follows:

[0025] In this screening process, 1.2 mM pyruvate was used as the substrate and 0.25 mM NADH was used as the cofactor to detect the inhibitory ability of these compounds on the forward reaction catalyzed by CpLDH. The decrease in NADH during the rCpLDH catalytic reaction can be detected by spectrophotometry at the 340 nm wavelength band. According to the decrease in NADH per unit time, the enzyme activity and the inhibition efficiency of the inhibitor on MBP-CpLDH were calculated. The initial screening drug concentration was 10 μM, and compounds with an inhibition effect greater than 50% were further detected. As Figure 3 shown, compound Epirosmanol was detected to have a clear inhibitory effect on the enzymatic activity function of CpLDH through preliminary screening.

[0026] Example 3: Inhibitory effect of Epirosmanol on the enzymatic activity of recombinant protein CpLDH:

[0027] According to the enzyme activity reaction measurement method, the concentration range of Epirosmanol (0 - 20 μM) was selected to measure its anti-rCpLDH enzyme kinetics IC 50 (substrate concentration at half of the maximum enzyme activity reaction rate), as Figure 4 shown, to determine IC 50 = 0.28 μM.

[0028] Example 4: Verification of the anti-Cryptosporidium effect of Epirosmanol using C . parvum the HCT-8 host cell model infected with Cryptosporidium:

[0029] HCT-8 cells (5000 / well) were seeded into 96-well cell culture plates (BioFil) and cultured overnight in a 37°C, 5% CO2 incubator (Thermo). When the cell number reached 80%, the 1640 complete medium was removed, and 1640 complete medium containing 20,000 Cryptosporidium oocysts / well was added. Incubate in a 37°C, 5% CO2 incubator (Thermo) for 3 h to allow sporozoites to excyst and invade host cells. Wash appropriately with PBS to remove unexcysted oocysts, oocyst walls, and non-invasive sporozoites after excystation; add 100 μL of 1640 complete medium containing gradient concentrations of Epirosmanol and culture in a 37°C, 5% CO2 incubator for 41 h. Remove the medium and add cell lysate iScript TMRT-qPCR reagent, use a vortex shaker (Multi-Tube, Thermo) at 2000 rpm for 20 min, centrifuge at 2000 g for 15 min using a tabletop centrifuge, take the supernatant, and use it as a sample for qRT-PCR experiment. The specific steps are as follows: One-Step SYBR Green qRT-PCR Kit (One Step PrimeScript™ RT-PCR Kit, Takara), use qRT-PCR method to detect the 18S rRNA transcript levels of Cryptosporidium parvum and host cells (Cp18S and Hs18S respectively), dilute the above-prepared cell lysate 100-fold for detecting Cp18S and Hs18S transcripts. In a 96-well plate (BioFil) (20 µL / well), it contains 2 µL of diluted cell lysate, 10 µL of 2x One-Step SYBR Green Mix, 1 µL of One-Step SYBR Green Enzyme Mix, 0.4 µL of Passive Reference Dye Ⅱ, 5.8 µL of RNase-free water and the following primers:

[0030] Cp18S_F (5′- TTGTTCCTTACTCCTTCAGCAC-3′),

[0031] Cp18S-R (5′- TCCTTCCTATGTCTGGACCTG-3′),

[0032] Hs18S_F (5 ′-GGCGCCCCCTCGATGCTCTTA-3 ′),

[0033] Hs18S_R (5 ′-CCCCCGGCCGTCCCTCTTA-3 ′).

[0034] The above primers were synthesized by Sangon Biotech (Changchun) Co., Ltd.; the reaction program is: reverse transcription at 50°C for 3 min; pre-denaturation at 95°C for 30 s; amplification for 40 cycles at 95°C for 10 s and 60°C for 30 s; after amplification, perform melting curve analysis between 65 - 95°C. After the qRT-PCR reaction is completed, detect the amplification curve and melting peak to evaluate the quality and specificity of the reaction;

[0035] Calculate the relative parasite load according to the cycle threshold (C T ) of Cp18S and Hs18S transcripts. First, obtain the mean value of technical replicates of a single biological replicate for C T value, and calculate the △C T value between Cp18S and Hs18S (i.e., △C T = CT[Cp18S] -C T[Hs18S] ), and the ΔΔC between each experimental sample and the control T value was used to determine the relative level between the sample and the control (here, the ΔC of the control group T is the average of all its biological replicates); finally, by calculating the 2 —△△CT value, the relative change between the parasite and the host cell was reflected. According to the above method, the in vitro inhibitory curve of Epirosmanol against C. parvum was determined, and the results are shown in Figure 5 as follows. The half-maximal effective concentration EC 50 was determined to be 2.47 μM, indicating that Epirosmanol is a low micromolar anti-Cryptosporidium compound.

[0036] Example 5. Sensitivity detection of Epirosmanol to host cells (HCT-8):

[0037] The cytotoxicity was detected using the MTS cell proliferation detection kit (Saint-Bio). The specific steps were as follows: HCT-8 cells (4000 - 5000 / well) were seeded into a 96-well cell culture plate (BioFil) and cultured overnight in a 37°C, 5% CO2 incubator (Thermo). When the cell number reached 40%, the complete 1640 medium was removed, and the cells were continued to be cultured for 24 h using the complete 1640 medium containing Epirosmanol (0 - 50 μM). The drug was removed, and the MTS working solution (10 μL of MTS reagent added to 90 μL of incomplete 1640 medium) was added to each well and incubated in a 37°C incubator for 1 h; the OD value at 490 nm was measured using a microplate reader (Bio Tek), and the drug concentration of the half-maximal cytotoxicity was calculated by non-linear regression. The results are shown in Figure 6 as follows. TC 50 was determined to be 13.61 μM. This index represents the drug resistance of the cells to the compound. TC 50 The larger the TC 50 value, the weaker the drug toxicity and the stronger the drug resistance of the cells to the compound. Moreover, the safety interval (SI) of Epirosmanol was determined to be SI = 5.5, that is, EC 50 the ratio of

[0038] . The larger the SI value, the stronger the anti-parasitic ability of the drug and the smaller the cytotoxicity. TC50 The value is 13.61 μM, but its anti-Cryptosporidium effect in the in vitro HCT-8 host cell infection model EC 50 The value is 2.47 μM, indicating that Epirosmanol can basically completely inhibit the growth of Cryptosporidium at concentrations below the cytotoxic concentration. In vitro, C. parvum The low micromolar inhibitory ability on growth indicates that Epirosmanol is likely to act on a single drug target, CpLDH.

[0039] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of carnosol in the preparation of drugs against Cryptosporidium parvum.

2. Use of rosmarinol according to claim 1 in the preparation of a drug against Cryptosporidium parvum, characterized in that, The carnosol inhibits the enzyme activity of Cryptosporidium parvum lactate dehydrogenase at the submicromolar level.

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