Application of GYY4137 in preparation of medicine for resisting cryptosporidium parvum

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of GYY4137 in preparation of a medicine for resisting cryptosporidium parvum, and the GYY4137 inhibits the activity of the cryptosporidium parvum by inhibiting CpLDH. The invention discloses that GYY4137 is used as a candidate drug with great potential and is used for targeting CpLDH and inhibiting the survival of cryptosporidium. At the nanomole level, the GYY4137 can completely inhibit the enzyme activity of the recombinant CpLDH. In addition, an in vitro cell culture model verifies that GYY4137 inhibits the growth of parasites at low micromole levels. The discovery not only proves that the GYY4137 has the potential of being further developed into the cryptosporidium-resistant medicine, but also emphasizes that the strategy of taking the CpLDH as the target spot to resist parasitic infection is feasible.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of GYY4137 in the preparation of an anti-Cryptosporidium parvum drug. 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 lead to fatal infections in AIDS patients. It belongs to the same phylum Apicomplexan as Plasmodium and coccidia. Because Cryptosporidium oocysts are resistant to chemical treatments (such as chlorine treatment), it often causes waterborne outbreaks worldwide. However, there are limited treatment options for cryptosporidiosis. 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 has been slow, mainly due to two challenges: one is the technical difficulties in the study of Cryptosporidium pathogenic biology, such as the inability to perform routine 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 many metabolic pathways present in other parasites, such as the tricarboxylic acid cycle (TCA cycle), respiratory chain, apicoplast and class II fatty acid synthesis pathway. These characteristics make Cryptosporidium 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 in the glycolysis pathway, catalyzes the conversion of pyruvate into lactate, thereby 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 multiple 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 anticancer effects of H2S donors such as GYY4137 have been studied, and some H2S donors have been used in the treatment of thyroid cancer. In addition, GYY4137 also has anti-inflammatory activity.

[0005] In order to solve the difficult problem of treating cryptosporidiosis, the present invention proposes the use of GYY4137 in preparing an anti-Cryptosporidium parvum drug. Summary of the invention

[0006] The purpose of the present invention is to provide the use of GYY4137 in the preparation of an anti-Cryptosporidium parvum drug, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: Application of GYY4137 in the preparation of anti-Cryptosporidium parvum drugs.

[0008] Furthermore, the GYY4137 inhibits the activity of Cryptosporidium parvum by inhibiting CpLDH.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses GYY4137 as a highly promising drug candidate 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 low micromolar levels. These findings not only demonstrate that GYY4137 has the potential to be further developed as an anti-Cryptosporidium drug, but also emphasize that the strategy of targeting CpLDH to combat parasitic infections is feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the CpLDH-catalyzed enzyme activity reaction.

[0011] Figure 2 The expression and enzyme activity identification of CpLDH recombinant protein; a is the molecular weight result of rCpLDH, b is the molecular weight of substrate pyruvate and NADH in the forward reaction K m value, c is the substrate lactate and NAD in the reverse reaction + of K m value.

[0012] Figure 3 Screening for inhibitors based on CpLDH enzyme activity.

[0013] Figure 4 GYY4137 inhibits the enzyme kinetics of rCpLDH.

[0014] Figure 5 To determine the toxicity of GYY4137 to HCT-8 host cells.

[0015] Figure 6 To evaluate the antiparasitic effect of GYY4137 in an in vitro HCT-8 cell infection model. DETAILED DESCRIPTION

[0016] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0017] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0018] Example 1: Inhibitory effect of GYY4137 on CpLDH protein levels; The gene number of CpLDH is cgd7_480, which can be found at the NCBI / crypto.DB website.

[0019] First, we obtained recombinant CpLDH (rCpLDH) in vitro through a prokaryotic expression system, with a molecular weight of 75 kDa ( Figure 2 a), and can catalyze the mutual conversion between pyruvate and lactate through CpLDH ( Figure 1 ) to verify that rCpLDH has enzymatic activity.

[0020] The specific experimental steps are as follows: Since the enzyme reaction is reversible, under the action of LDH (lactate dehydrogenase), pyruvate and lactate are converted to each other, accompanied by an increase or decrease in NADH (reduced nicotinamide adenine dinucleotide). We use NADH to have an absorbance at 340 nm, while NAD + (oxidized nicotinamide adenine dinucleotide) does not produce an absorption peak at 340 nm, and the enzyme activity reaction is determined by monitoring the reduction or generation of NADH. The total volume of the reaction system is 100 μL, and 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 substrate includes (0-2mM) NAD + , (0-100 mM) lactate, and MBP-CpLDH protein (500 ng). In all assays, MBP-tag was used as a negative control and background subtraction. We determined the enzymatic activity of MBP-CpLDH by monitoring the rate of NADH oxidation at 340 nm using a microplate reader (Bio Tek) spectrophotometrically. The MBP-CpLDH enzyme activity was determined based on the amount of NADH reduced per unit time, and enzyme kinetic parameters such as the Michaelis constant ( K m ).

[0021] like Figure 2As shown in b and c, we measured the bidirectional enzymatic activity catalyzed by LDH and determined the Michaelis constants for each substrate ( K m ). In the forward reaction, the substrate pyruvate K m =471 μM, NADH K m =51 μM. In the reverse reaction, the substrate lactate K m =5.4 mM, NAD + of K m =39 μM. The above data fully prove that rCpLDH can catalyze the enzymatic reaction and can therefore be used in subsequent experiments.

[0022] In the body of Cryptosporidium parvum, the main catalysis is the reaction of pyruvate to lactate. Therefore, when conducting drug screening, the forward reaction was selected. 8892 drugs in the laboratory drug library were screened ( Figure 3 ). According to the previously determined substrate K m We selected an appropriate substrate concentration for drug screening reaction. The specific method is as follows: the reaction system is 100 μL, the forward reaction contains 50 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 1.2 mM pyruvate and purified MBP-CpLDH protein (100 ng), and the drug screening concentration is selected as 10 μM. Figure 3 As shown in the figure, at a drug concentration of 10 μM, 156 drugs had an inhibitory effect on CpLDH of more than 50%. Among the 156 inhibitors screened, we found that GYY4137 had a good inhibitory effect on the enzymatic activity of rCpLDH protein. Specifically, in order to clarify the insect inhibition parameters of GYY4137, the concentration range of GYY4137 (0-20 μM) was selected, and its anti-rCpLDH enzyme kinetics was measured. IC 50 (i.e. the substrate concentration that inhibits half of the maximum enzyme activity reaction rate). Figure 4 As shown, GYY4137 inhibited the expression of rCpLDH IC 50 =940 nM, indicating that GYY4137 can clearly inhibit the activity of CpLDH of Cryptosporidium parvum.

[0023] The structural formula of GYY4137 is as follows: .

[0024] Example 2: Utilization C . parvum(Cryptosporidium parvum) infection in HCT-8 host cell model to verify the efficacy of GYY4137 against Cryptosporidium parvum; To verify the anti-Cryptosporidium parvum efficacy of GYY4137, we used C . parvum Infection of HCT-8 host cell model. The specific experimental steps are as follows: First, HCT-8 cells (5000 / well) were inoculated into 96-well cell culture plates (BioFil) and cultured overnight in an incubator (Thermo) at 37°C and 5% CO2. After the cell number reached 80%, the original 1640 complete medium was removed and 1640 complete medium containing 20,000 / well of Cryptosporidium parvum oocysts was added. Subsequently, the cells were incubated in an incubator (Thermo) at 37°C and 5% CO2 for 3 h to allow sporozoites to excyst and invade host cells. After the incubation, the cells were washed with PBS to remove the unexcysted oocysts, the excysted oocyst walls, and the unsuccessfully invaded sporozoites. Next, 100 μL of 1640 complete medium containing GYY4137 (0-5 μM) was added and cultured in an incubator at 37°C and 5% CO2 for 41 h. After the incubation period, the culture medium was removed and the cell lysis buffer iScript was added TM RT-qPCR reagents. Use a vortex shaker (Multi-Tube, Thermo) at 2000 rpm for 20 minutes, and then use a plate centrifuge at 2000g for 15 minutes. Take the supernatant as a sample for subsequent qRT-PCR experiments.

[0025] The specific steps of the qRT-PCR experiment are as follows: 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 times for the detection of Cp18S and Hs18S transcripts. The reaction system was then set up in a 96-well plate (BioRad Laboratories, Hercules) with a total reaction volume of 20 µL per well, including: 2 µL diluted cell lysate, 10 µL 2x One-Step SYBR Green Mix, 1 µL One-Step SYBR Green Enzyme Mix, 0.4 µL Passive Reference Dye II, 5.8 µL RNase-free water, and specific primers for Cryptosporidium parvum 18S rRNA (Cp18S) and host cell 18S rRNA (Hs18S) (0.4 μL each for upstream and downstream primers): Cp18S_F: 5'-TTGTTCCTTACTCCTTCAGCAC-3' (as shown in SEQ ID NO. 1); Cp18S_R: 5'-TCCTTCCTATGTCTGGACCTG-3' (as shown in SEQ ID NO. 2); Hs18S_F: 5'-GGCGCCCCCTCGATGCTCTTA-3' (as shown in SEQ ID NO. 3); Hs18S_R: 5′-CCCCCGGCCGTCCCTCTTA-3′ (as shown in SEQ ID NO. 4).

[0026] The above primers were synthesized by Sangon Biotechnology (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 at 95°C for 10 s and 60°C for 30 s for 40 cycles; after amplification, melting curve analysis was performed at 65-95°C.

[0027] After the qRT-PCR reaction was completed, the quality and specificity of the reaction were evaluated by detecting the amplification curve and melting peak. T ) to calculate relative parasite load. The specific method is: first calculate the technical replicates C of a single biological replicate T The average of the values ​​was then calculated to find the △C between Cp18S and Hs18S. TValue (i.e. △C T = C T[Cp18S] -C T[Hs18S] ), and the △△C between each experimental sample and the control T Determine the relative level between the sample and the control (△C T is the average value of all biological replicates); finally, by calculating 2 —△△CT The values ​​are used to reflect the relative changes between parasites and host cells.

[0028] According to the above method, we obtained the GYY4137 pair C. parvum In vitro inhibition curves of Figure 6 ), and its half-maximal effective concentration ( EC 50 =1.80 μM). These results indicate that the CpLDH inhibitor GYY4137 is a low-micromolar lead compound against Cryptosporidium parvum.

[0029] Example 3: Detection of the sensitivity of GYY4137 to host cells (HCT-8); First, the cytotoxicity test was performed using the MTS cell proliferation assay kit (Saint-Bio). The specific steps are as follows: HCT-8 cells (5000 / well) were seeded into a 96-well cell culture plate (BioFil) and then cultured overnight in a 37°C, 5% CO2 incubator (Thermo). When the cell number reached 40%, the original 1640 complete medium was removed and replaced with 1640 complete medium containing GYY4137 (concentration of 0-50 μM), and the culture was continued for 24 h. After that, the medium containing GYY4137 was removed, and the MTS working solution was added to each well (90 μL of incomplete 1640 medium was added to every 10 μL of MTS reagent), and then the culture plate was returned to the 37°C incubator for incubation for 1 h. After the incubation, the OD value at 490 nm was measured using a microplate reader (Bio Tek). The drug concentration for half-maximal cytotoxicity ( TC 50 =7.6 μM), such as Figure 5 This indicator reflects the resistance of cells to GYY4137. TC 50 The larger the value, the weaker the drug toxicity and the stronger the cell resistance to the drug. In addition, we also calculated the safety interval (SI) of GYY4137, that is, TC 50 and EC 50 The ratio is 4.22 ( Figure 6). The larger the SI value, the stronger the antiparasitic ability of the drug and the lower its toxicity to cells.

[0030] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of GYY4137 in the preparation of anti-Cryptosporidium parvum drugs.

2. The use according to claim 1, characterized in that: The GYY4137 inhibits the activity of Cryptosporidium parvum by inhibiting CpLDH.

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