Application of epirosmarinol in preparation of medicine for resisting cryptosporidium parvum

Abstract

The invention is suitable for the technical field of cryptosporidium parvum resisting drugs, and provides application of epirosmarinol in preparation of cryptosporidium parvum resisting drugs. The invention also provides an anti-cryptosporidium medicine, and the medicine takes the epirosmarinol as a lead compound. Through screening, it is found that Epirosmanol has a clear inhibition effect on cryptosporidium infection, and it is clear that CpLDH serves as an action target of the inhibition effect.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-cryptosporidiosis drugs, and in particular relates to application of epi-rosmarinol in preparing anti-cryptosporidiosis drugs. Background Art

[0002] Cryptosporidium parvum ( Cryptosporidium parvum ) are gastrointestinal parasites that can cause moderate to severe diarrhea in children and adults and can cause fatal opportunistic infections in AIDS patients. In addition, Cryptosporidium oocysts are resistant to chemical treatments (such as chlorine treatment) and frequently cause waterborne outbreaks worldwide. However, the development of drugs for cryptosporidiosis is still slow, so there is an urgent need to develop new drugs for the prevention and treatment of cryptosporidiosis in animals and humans.

[0003] Cryptosporidium lacks a tricarboxylic acid cycle and a cytochrome-based respiratory chain. Therefore, this type of parasite mainly relies on the glycolysis pathway to produce ATP. In order 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 acetate as organic end products. Among these enzymes, LDH is considered to be a drug target for some parasites, including Plasmodium and Toxoplasma gondii in apicomplexan parasites, as well as anaerobic protozoa, Giardia lamblia, Trypanosoma cruzi and Entamoeba histolytica. And because Cryptosporidium lactate dehydrogenase (CpLDH) is a bacterial type, it is quite different from the LDH of the host human and other mammals. It is more convenient to use it as a drug target to screen anti-Cryptosporidium drugs that are non-toxic and have no side effects on humans and other mammals.

[0004] Several lead compounds against Cryptosporidium have been discovered and reported, such as compound 1294 targeting calcium-dependent protein kinases (CDPKs), P131 targeting inosine nucleotide dehydrogenase (IMPDH), KDU731 targeting phosphatidylinositol-4-OH kinase (PI(4)K), triacsin C targeting acetyl-CoA synthetase (ACS), and SAHA targeting histone acetyltransferase (HDAC). In the future, one or more of these lead compounds may eventually be developed into new drugs against Cryptosporidium, but at this stage, it is still necessary to explore new drug targets and develop new drugs. First, there is a need for more treatment options, and second, the emergence of drug resistance in the future needs to be considered.

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

[0006] The purpose of the embodiments of the present invention is to provide an application of epirosmarinol in the preparation of an anti-Cryptosporidium parvum drug, aiming to solve the problems raised in the above background technology.

[0007] The embodiment of the present invention is achieved by using epi-rosmarinol in the preparation of anti-Cryptosporidium parvum drugs.

[0008] Preferably, the epirosmarinol inhibits the enzymatic activity of Cryptosporidium lactate dehydrogenase at submicromolar levels.

[0009] Preferably, the epirosmarinol inhibits Cryptosporidium growth at low micromolar levels.

[0010] Another object of an embodiment of the present invention is to provide an anti-Cryptosporidium drug, wherein the drug uses epirosmarin as a lead compound.

[0011] The present invention example found through screening that Epirosmanol has a clear inhibitory effect on Cryptosporidium infection, and clearly identified CpLDH as the target of this inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The SDS-PAGE results of the MBP-CpLDH recombinant protein obtained by prokaryotic expression provided in Example 1 of the present invention; Figure 2 The biochemical function results of the MBP-CpLDH recombinant protein provided in Example 1 of the present invention; Figure 3 The drug screening results provided in Example 2 of the present invention; Figure 4 The results of the inhibition test of Epirosmanol on the activity of the recombinant protein CpLDH provided in Example 3 of the present invention; Figure 5 The Epirosmanol provided by Example 4 of the present invention is C. parvum In vitro inhibition curves; Figure 6 This is the test result of the cytotoxicity of Epirosmanol to HCT-8 cells provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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.

[0014] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0015] Example 1, Identification of the enzyme activity of the recombinant protein MBP-CpLDH: Cryptosporidium lacks the tricarboxylic acid cycle and the cytochrome-based respiratory chain, so the worm relies on the glycolysis pathway to produce ATP. At the same time, the worm relies on lactate dehydrogenase (LDH) to prevent the accumulation of pyruvate, which is harmful to anaerobic respiration, and to ensure the coenzyme factor NAD(P)H and NAD(P) + LDH can catalyze the conversion of pyruvate and lactate and ensure the NADH / NAD + Cycle, namely: Pyruvate + NADH + H + =Lactate + NAD + ; The MBP-CpLDH recombinant protein was expressed. MBP (maltose binding protein) was 40 kDa in size and used for protein expression and purification in the prokaryotic expression process. Its biochemical function was verified. Specifically, the MBP-CpLDH protein was expressed in Escherichia coli Rosetta competent cells using the pMAL-c5X-cgd7_480 plasmid. The transformed single colony was inoculated into 50 mL LB medium containing ampicillin (50 μg / mL) and glucose (2 mg / mL). The culture was carried out at 37°C overnight. The ratio of bacterial liquid to LB medium was 1:10. The culture was incubated at 37°C for 2 h until the OD600 reached ~0.5. Then, isopropyl-1-thio-β-D-galactoside (IPTG) was added to a final concentration of 0.3 mM to induce protein expression. The cells were shaken at 16°C for ~16 h, induced Escherichia coli Rosetta to overexpress MBP-CpLDH recombinant protein; according to the manufacturer's instructions (New England Biolabs, Ipswich, MA), the bacterial precipitate was collected by centrifugation, the bacteria were broken, and the MBP-CpLDH recombinant protein was purified using amylose resin affinity chromatography. The obtained protein was subjected to SDS-PAGE, and the results were as follows: Figure 1As shown; the enzyme activity of MBP-CpLDH was determined by spectrophotometrically monitoring the oxidation rate of NADH at 340 nm using a microplate reader (Bio Tek), and the enzyme kinetic parameters of MBP-CpLDH, such as the Michaelis constant ( K m The reaction system of the enzyme was 100 μL. The forward enzyme activity reaction contained 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 used 50 mMTris-HCl (pH 9.2) as the reaction buffer and 1 mM NAD as the substrate. + and 100 mM lactate, MBP-tag (500 ng) was used in all assays as a negative control and for background subtraction, and the results were obtained as shown Figure 2 As shown, the purified rCpLDH protein was 75 kDa, and rCpLDH was able to catalyze the interconversion of pyruvate and lactate.

[0016] Embodiment 2, the screening process of Epirosmanol: After confirming that the MBP-CpLDH recombinant protein has biochemical activity, a large amount of active recombinant CpLDH protein was obtained by prokaryotic expression. The CpLDH protein was used to screen a total of 8,892 compounds in the drug library. The screening method is as follows: The screening process used 1.2 mM pyruvate as a substrate and 0.25 mM NADH as a cofactor to detect the inhibitory ability of these compounds on the forward reaction catalyzed by CpLDH. The reduction of NADH during the rCpLDH catalytic reaction can be detected by spectrophotometry at 340 nm. The enzyme activity and the inhibitory efficiency of the inhibitor on MBP-CpLDH are calculated based on the reduction of NADH per unit time. The drug concentration for the initial screening was 10 μM. Compounds with an inhibitory effect greater than 50% were further tested, such as Figure 3 As shown, the compound Epirosmanol was detected through preliminary screening to have a clear inhibitory effect on the enzymatic function of CpLDH.

[0017] Example 3, Inhibitory effect of Epirosmanol on the enzyme activity of recombinant protein CpLDH: According to the enzyme activity assay method, the concentration range of Epirosmanol (0-20 μM) was selected to determine its anti-rCpLDH enzyme kinetics. IC 50 (the substrate concentration that inhibits the maximum enzyme activity reaction rate by half), such as Figure 4 As shown, confirm IC50 =0.28 μM.

[0018] Example 4: Utilization C . parvum Epirosmanol's anti-Cryptosporidium effect was validated in the HCT-8 host cell model: HCT-8 cells (5000 / well) were inoculated into 96-well cell culture plates (BioFil) and cultured overnight in a 37°C, 5% CO2 incubator (Thermo). When the cell number reached 80%, 1640 complete medium was removed, and 1640 complete medium containing 20,000 Cryptosporidium oocysts / well was added. The cells were incubated at 37°C, 5% CO2 incubator (Thermo) for 3 h to allow sporozoites to excyst and invade host cells. Unexcysted oocysts, excysted oocyst walls and uninvaded sporozoites were removed by appropriate washing with PBS. 100 μL of 1640 complete medium containing gradient concentrations of Epirosmanol was added, and the cells were cultured at 37°C, 5% CO2 incubator for 41 h. The medium was removed, and the cell lysis buffer iScript was added. TM RT-qPCR reagents were vortexed (Multi-Tube, Thermo) at 2000 rpm for 20 min, centrifuged at 2000 g for 15 min on a plate centrifuge, and the supernatant was used as a sample for qRT-PCR experiments. The specific steps are as follows: One-Step SYBR Green qRT-PCR Kit (One Step PrimeScript™ RT-PCRKit, Takara), qRT-PCR method was used to detect the 18S rRNA transcript levels of Cryptosporidium parvum and host cells (Cp18S and Hs18S, respectively), the cell lysate prepared above was diluted 100 times for the detection of Cp18S and Hs18S transcripts, and 2 µL of diluted cell lysate, 10 µL 2x One-StepSYBR Green Mix, 1 µL One-Step SYBR Green Enzyme Mix, 0.4 µL Passive ReferenceDyeⅡ, 5.8 µL RNase-free water and the following primers were plated on a 96-well b plate (BioFil) (20 µL / well): Cp18S_F (5′- TTGTTCCTTACTCCTTCAGCAC-3′), Cp18S-R (5′-TCCTTCCTATGTCTGGACCTG-3′), Hs18S_F (5′-GGCGCCCCCTCGATGCTCTTA-3′), Hs18S_R (5′-CCCCCGGCCGTCCCTCTTA-3′).

[0019] The above primers were synthesized by Sangon Biotechnology (Changchun) Co., Ltd. The reaction procedure was as follows: reverse transcription at 50°C for 3 min; pre-denaturation at 95°C for 30 s; amplification at 95°C for 10 s and 60°C for 30 s for 40 cycles; after amplification, melting curve analysis was performed between 65 and 95°C. After qRT-PCR reaction was completed, amplification curve and melting peak were detected to evaluate the quality and specificity of the reaction. According to the cycle threshold of Cp18S and Hs18S transcripts (C T ) To calculate relative parasite load, first obtain technical replicates C for a single biological replicate. T The average of the values ​​was used to calculate the △C between Cp18S and Hs18S. T Value (i.e. △C T = C T[Cp18S] -C T[Hs18S] ), and the △△C between each experimental sample and the control T value, determine the relative level between the sample and the control (here the △C T is the average value of all biological replicates); finally, by calculating 2 —△△CT The value reflects the relative change of parasite and host cells. According to the above method, the effect of Epirosmanol on C. parvum The in vitro inhibition curve of Figure 5 As shown, the half maximum effective concentration was determined EC 50 =2.47 μM, indicating that Epirosmanol belongs to the low micromolar level anti-Cryptosporidium compound.

[0020] Example 5, Epirosmanol sensitivity test on host cells (HCT-8): Cytotoxicity assay was performed using the MTS cell proliferation assay kit (Saint-Bio). The specific steps were as follows: HCT-8 cells (4000-5000 / well) were inoculated 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 1640 complete medium was removed and the cells were cultured for 24 h using 1640 complete medium containing Epirosmanol (0-50 μM). The drug was removed and MTS working solution was added to each well (90 μL of incomplete 1640 medium was added to every 10 μL of MTS reagent) and the cells were incubated in a 37°C incubator for 1 h. The OD value at 490 nm was measured using an ELISA reader (Bio Tek), and the drug concentration at half the maximum cytotoxicity was calculated by nonlinear regression. The results were as follows: Figure 6 As shown, TC 50 =13.61 μM, which represents the cell resistance to the compound. TC 50 The larger the value, the weaker the drug toxicity and the stronger the cell's resistance to the compound. In addition, the safety interval (SI) of Epirosmanol was determined to be 5.5, i.e. TC 50 and EC 50 The larger the SI value, the stronger the antiparasitic ability of the drug and the smaller the cytotoxicity.

[0021] In summary, although Epirosmanol has certain cytotoxicity to HCT-8 host cells, TC 50 The value was 13.61 μM, but its anti-Cryptosporidium activity in the in vitro HCT-8 host cell infection model was EC 50 The value was 2.47 μM, indicating that Epirosmanol completely inhibited the growth of Cryptosporidium in vitro at concentrations below cytotoxicity. C. parvum The low micromolar inhibition of growth suggests that Epirosmanol is likely to act on a single drug target, CpLDH.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 present invention.

Claims

1. Application of epi-rosmarinol in the preparation of drugs against Cryptosporidium parvum.

2. The use of epirosmantin according to claim 1 in the preparation of an anti-Cryptosporidium parvum drug, characterized in that: The epirosmarinol inhibits the enzymatic activity of Cryptosporidium lactate dehydrogenase at submicromolar levels.

3. The use of epirosmantin according to claim 1 in the preparation of an anti-Cryptosporidium parvum drug, characterized in that: The epirosmarinol inhibits Cryptosporidium growth at low micromolar levels.

4. An anti-cryptosporidiosis drug, characterized in that: The drug uses epirosmarin as a lead compound.

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