Application of GYY4137 in the preparation of drugs against Cryptosporidium parvum

Abstract

The present invention is applicable to the field of biomedical technologies, and provides the use of GYY4137 in the preparation of drugs against Cryptosporidium parvum. The GYY4137 inhibits the activity of Cryptosporidium parvum by inhibiting CpLDH. The present invention reveals that GYY4137 is a highly potential candidate drug for targeting CpLDH and inhibiting the survival of Cryptosporidium. At the nanomolar level, GYY4137 can completely inhibit the enzymatic activity of recombinant CpLDH. In addition, in vitro cell culture models have verified that GYY4137 inhibits the growth of parasites at the low micromolar level. These findings not only demonstrate the potential of GYY4137 for further development as an anti-Cryptosporidium drug, but also emphasize that the strategy of targeting CpLDH to combat parasitic infections is feasible.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of GYY4137 in the preparation of drugs against Cryptosporidium parvum. Background Art

[0002] Cryptosporidium, especially Cryptosporidium parvum ( Cryptosporidium parvum ), is a gastrointestinal parasite that can cause moderate to severe diarrhea in children and adults and even fatal infections in AIDS patients. It belongs to the Apicomplexan protozoa together with Plasmodium and Coccidia. Due to the resistance of Cryptosporidium oocysts to chemical treatments (such as chlorine treatment), it often causes waterborne outbreaks globally. However, the treatment options for cryptosporidiosis are limited. In the United States, only nitazoxanide (NTZ) is approved for the treatment of immunocompetent individuals, but it is not approved for immunocompromised patients. Therefore, the development of new anti-Cryptosporidium drugs is particularly important.

[0003] The research progress of anti-Cryptosporidium drugs is slow, mainly due to two challenges: one is the technical difficulties in the research of Cryptosporidium pathogenic biology, such as the inability to perform conventional in vitro culture to complete its life cycle and the lack of necessary tools and reagents; the other is the unique metabolic and biochemical characteristics of Cryptosporidium, especially its lack of multiple metabolic pathways existing in other parasites, such as the tricarboxylic acid cycle (TCA cycle), respiratory chain, apicoplast, and type II fatty acid synthesis pathway, etc. These characteristics enable Cryptosporidium to mainly obtain energy through the glycolysis pathway. Therefore, the key enzymes of the glycolysis pathway have become important targets for drug development. Among them, Cryptosporidium parvum lactate dehydrogenase (CpLDH), as one of the key enzymes of the glycolysis pathway, catalyzes the conversion of pyruvate to lactate, thus avoiding the accumulation of pyruvate and ensuring the smooth operation of the glycolysis pathway. It is worth noting that CpLDH belongs to the bacterial type and has significant differences in protein sequence and structure from mammalian lactate dehydrogenase.

[0004] As a slow-release H2S donor, GYY4137 has various cell function regulatory effects. H2S can reduce the disulfide bonds of many proteins and may form protein persulfides. In cancer cells, H2S exhibits dual behaviors of proliferation and cell death, promoting proliferation at low concentrations and being cytotoxic at high concentrations. Therefore, the anti-cancer effects of H2S donors such as GYY4137 have been studied, and some H2S donors have been applied to the treatment of thyroid cancer. In addition, GYY4137 also has anti-inflammatory activity.

[0005] To solve the treatment problem of cryptosporidiosis, the present invention proposes the application of GYY4137 in the preparation of drugs against Cryptosporidium parvum. Summary of the Invention

[0006] The object of the present invention is to provide the application of GYY4137 in the preparation of drugs against Cryptosporidium parvum, aiming to solve the problems raised in the above-mentioned background technology.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] The application of GYY4137 in the preparation of drugs against Cryptosporidium parvum.

[0009] Furthermore, the GYY4137 inhibits the activity of Cryptosporidium parvum by inhibiting CpLDH.

[0010] Compared with the prior art, the beneficial effects of the present invention are:

[0011] The present invention reveals that GYY4137 is a highly potential candidate drug for targeting CpLDH and inhibiting the survival of Cryptosporidium. At the nanomolar level, GYY4137 can completely inhibit the enzyme activity of recombinant CpLDH. In addition, the in vitro cell culture model verifies that GYY4137 inhibits the growth of parasites at the low micromolar level. These findings not only prove the potential of GYY4137 for further development as an anti-Cryptosporidium drug, but also emphasize that the strategy of targeting CpLDH against parasite infection is feasible. Brief Description of the Drawings

[0012] Figure 1 It is a schematic diagram of the catalytic enzyme activity reaction of CpLDH.

[0013] Figure 2 It is the expression and enzyme activity identification of CpLDH recombinant protein; among them, a is the molecular weight result of rCpLDH, b is the K m value of the substrates pyruvate and NADH in the forward reaction, and c is the + of K m value.

[0014] Figure 3 It is the screening of inhibitors based on the enzyme activity of CpLDH.

[0015] Figure 4 It is the enzyme kinetics of GYY4137 inhibiting rCpLDH.

[0016] Figure 5 It is the toxicity determination of GYY4137 on HCT-8 host cells.

[0017] Figure 6 It is to evaluate the anti-parasitic effect of GYY4137 in the in vitro HCT-8 cell infection model. Detailed Implementation Modes

[0018] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following provides a detailed description of the technical solution of the present invention, but it should not be construed as a limitation on the implementable scope of the present invention.

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

[0020] Example 1: Inhibitory effect of GYY4137 on the CpLDH protein level;

[0021] The gene number of CpLDH is cgd7_480, which can be queried on the NCBI / crypto.DB website.

[0022] First, we obtained recombinant CpLDH (rCpLDH) in vitro through a prokaryotic expression system, with a molecular weight of 75 kDa ( Figure 2 as shown in a), and verified that rCpLDH has enzymatic activity by the ability of CpLDH to catalyze the interconversion between pyruvate and lactate ( Figure 1 ).

[0023] The specific experimental steps are as follows: Since the enzymatic reaction is reversible, under the action of LDH (lactate dehydrogenase), pyruvate and lactate are interconverted, accompanied by an increase or decrease in NADH (reduced nicotinamide adenine dinucleotide). We utilized the principle that NADH has absorbance at 340 nm, while NAD + (oxidized nicotinamide adenine dinucleotide) does not produce an absorption peak at 340 nm, and determined the enzymatic reaction by monitoring the decrease or generation of NADH. The total volume of the reaction system is 100 μL. The forward reaction contains 50 mM Tris-HCl (pH 8.0), (0 - 2.5 mM) NADH, (0 - 10 mM) pyruvate, and purified MBP-CpLDH protein (100 ng). The reverse reaction uses 50 mM Tris-HCl (pH 9.2) as the reaction buffer, and the substrates include (0 - 2 mM) NAD + 、(0 - 100 mM) lactate, and MBP-CpLDH protein (500 ng). In all assays, the MBP-tag was used as a negative control and for background subtraction. We used a microplate reader (Bio Tek) to monitor the rate of NADH oxidation at 340 nm by spectrophotometry to determine the enzymatic activity of MBP-CpLDH. The enzymatic activity of MBP-CpLDH was determined based on the decrease in NADH per unit time, and the kinetic parameters related to the enzyme, such as the Michaelis constant ( K m ) were calculated accordingly.

[0024] As Figure 2 shown in b and c, we measured the bidirectional enzymatic reaction catalyzed by LDH and determined the Michaelis constants ( K m ) of each substrate. In the forward reaction, the K m of pyruvate as substrate was 471 μM, and the K m of NADH was 51 μM. In the reverse reaction, the K m of lactate as substrate was 5.4 mM, and the + for K m NAD⁺ was 39 μM. The above data fully demonstrated that rCpLDH could catalyze the enzymatic reaction and thus could be used in subsequent experiments.

[0025] In Cryptosporidium parvum, the main reaction catalyzed is from pyruvate to lactate. Therefore, in drug screening, the forward reaction was selected. 8892 drugs in the laboratory drug library were screened ( Figure 3 ). According to the previously determined K m values of the substrates, we selected an appropriate substrate concentration for the drug screening reaction. The specific method was as follows: The reaction system was 100 μL. The forward reaction contained 50 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 1.2 mM pyruvate, and purified MBP-CpLDH protein (100 ng). The drug screening concentration was selected as 10 μM. As Figure 3 shown, at a drug concentration of 10 μM, 156 drugs had an inhibitory effect on CpLDH greater than 50%. Among the 156 inhibitors obtained from the screening, we found that GYY4137 had a good inhibitory effect on the enzymatic activity of rCpLDH protein. Specifically, to clarify the inhibitory parameters of GYY4137, the concentration range of GYY4137 (0 - 20 μM) was selected, and its IC 50 against rCpLDH enzyme kinetics (i.e., the substrate concentration when inhibiting half of the maximum enzymatic reaction rate) was measured. As Figure 4 shown, the IC 50 of GYY4137 inhibiting rCpLDH was 940 nM, indicating that GYY4137 could significantly inhibit the activity of Cryptosporidium parvum CpLDH.

[0026] The structural formula of GYY4137 is as follows:

[0027] .

[0028] Example 2: Using C . parvum The (Cryptosporidium parvum) - infected HCT - 8 host cell model was used to verify the anti - Cryptosporidium parvum efficacy of GYY4137;

[0029] To verify the anti - Cryptosporidium parvum efficacy of GYY4137, we used C . parvum the infected HCT - 8 host cell model. The specific experimental steps are as follows: First, HCT - 8 cells (5000 / well) were seeded into 96 - well cell culture plates (BioFil) and cultured overnight in an incubator (Thermo) at 37 °C and 5% CO₂. When the cell number reached 80%, the original complete 1640 medium was removed, and complete 1640 medium containing 20000 Cryptosporidium parvum oocysts / well was added. Subsequently, it was incubated in an incubator (Thermo) at 37 °C and 5% CO₂ for 3 h to allow sporozoites to excyst and invade host cells. After incubation, the cells were washed with PBS to remove unexcysted oocysts, excysted oocyst walls, and sporozoites that did not successfully invade. Then, 100 μL of complete 1640 medium containing GYY4137 (0 - 5 μM) was added, and the cells were cultured in an incubator at 37 °C and 5% CO₂ for 41 h. After the culture, the medium was removed, and cell lysis buffer iScript TM RT - qPCR reagent was added. The mixture was shaken on a vortex shaker (Multi - Tube, Thermo) at 2000 rpm for 20 minutes, and then centrifuged at 2000 g for 15 min using a tabletop centrifuge. The supernatant was taken as the sample for subsequent qRT - PCR experiments.

[0030] The specific steps of the qRT-PCR experiment are as follows: The one-step SYBR Green qRT-PCR kit (One Step PrimeScript™ RT-PCR Kit, Takara) was used to detect the 18S rRNA transcript levels of Cryptosporidium parvum and host cells (Cp18S and Hs18S respectively) by qRT-PCR. During the experiment, the previously prepared cell lysate was first diluted 100-fold for detecting Cp18S and Hs18S transcripts. Subsequently, a reaction system was set up on a 96-well plate (BioRad Laboratories, Hercules), with a total reaction volume of 20 µL per well, including: 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 DyeⅡ, 5.8 µL of RNase-free water, and specific primers for Cryptosporidium parvum 18S rRNA (Cp18S) and host cell 18S rRNA (Hs18S) (0.4 μL each of forward and reverse primers):

[0031] Cp18S_F: 5’-TTGTTCCTTACTCCTTCAGCAC-3’ (as shown in SEQ ID NO.1);

[0032] Cp18S_R: 5’-TCCTTCCTATGTCTGGACCTG-3’ (as shown in SEQ ID NO.2);

[0033] Hs18S_F: 5’-GGCGCCCCCTCGATGCTCTTA-3’ (as shown in SEQ ID NO.3);

[0034] Hs18S_R: 5’-CCCCCGGCCGTCCCTCTTA-3’ (as shown in SEQ ID NO.4).

[0035] The above primers were synthesized by Sangon Biotech (Changchun) Co., Ltd. The reaction program was set as follows: 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, melting curve analysis was performed at 65 - 95°C.

[0036] After the qRT-PCR reaction was completed, the quality and specificity of the reaction were evaluated by detecting the amplification curve and melting peak. According to the cycle threshold (C T)Calculate the relative parasite load. The specific method is as follows: First, calculate the mean value of the technical replicates C of a single biological replicate T value, and then calculate the △C T value between Cp18S and Hs18S (i.e., △C T = C T[Cp18S] -C T[Hs18S] ), and the △△C T value between each experimental sample and the control. Determine the relative level between the sample and the control (the △C T of the control group is the average value of all its biological replicates); finally, reflect the relative change between the parasite and the host cell by calculating the 2 —△△CT value.

[0037] According to the above method, we obtained the in vitro inhibition curve of GYY4137 against C. parvum ( Figure 6 ), and determined its half-maximal effective concentration ( EC 50 = 1.80 μM). These results indicate that the CpLDH inhibitor GYY4137 is a low micromolar anti- Cryptosporidium parvum lead compound.

[0038] Example 3: Detect the sensitivity of GYY4137 to host cells (HCT-8);

[0039] First, perform a cytotoxicity assay using the MTS cell proliferation assay kit (Saint-Bio). The specific steps are as follows: Seed HCT-8 cells (5000 / well) into a 96-well cell culture plate (BioFil), and then incubate overnight in a 37 °C, 5% CO2 incubator (Thermo). When the cell number reaches 40%, remove the original complete 1640 medium and replace it with complete 1640 medium containing GYY4137 (at a concentration of 0 - 50 μM), and continue to culture for 24 h. Then, remove the medium containing GYY4137, add MTS working solution to each well (add 10 μL of MTS reagent to 90 μL of incomplete 1640 medium), and then place the culture plate back into the 37 °C incubator for 1 h. After incubation, measure the OD value at 490 nm using an enzyme-linked immunosorbent assay reader (Bio Tek). By non-linear regression analysis, calculate the drug concentration of half-maximal cytotoxicity ( TC 50 = 7.6 μM), as shown in Figure 5 . This index reflects the drug resistance of cells to GYY4137. TC 50The larger the value is, the weaker the drug toxicity is and the stronger the drug resistance of the cells to the drug is. In addition, we also calculated the safety interval (SI) of GYY4137, that is TC 50 The ratio of EC 50 is 4.22 ( Figure 6 ). The larger the SI value is, the stronger the anti-parasitic ability of the drug is and the lower the toxicity to cells is.

[0040] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. The application of GYY4137 in the preparation of drugs against Cryptosporidium parvum, characterized in that, The structural formula of GYY4137 is as follows: 。 2. The application according to claim 1, wherein GYY4137 inhibits the activity of Cryptosporidium parvum by inhibiting CpLDH.

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