Cryptosporidium-resistant small-molecule lead compound targeting cryptosporidium helicase

Abstract

The invention relates to an anti-cryptosporidium micromolecular lead compound of a target cryptosporidium helicase. Comprising the following steps: S1, detecting enzyme activity parameters of a drug target helicase (CpeIF4A) and an inhibition effect of Roc-A on the drug target helicase: expressing and purifying a CpeIF4A recombinant protein containing an MBP tag; detecting the enzyme activity; detecting the inhibition effect of the Roc-A on the enzyme activity; determining the affinity between the Roc-A and the CpeIF4A through a heat transfer experiment; s2, in-vitro drug effect determination: evaluating the direct killing capability of Roc-A on sporozoites; the inhibition effect of Roc-A on cryptosporidium is determined through an in-vitro cell infection model, an EC50 value is obtained, and the intracellular period of the Roc-A mainly playing the inhibition effect is determined; detecting the toxicity of Roc-A to host cells to obtain a TC5 value), and obtaining an in-vitro insect-resistant selectivity index (SI); and S3, in-vivo drug effect test: establishing a C57BL / 6IFN-gamma gene knockout mouse infection model to evaluate the effectiveness and safety of Roc-A. The invention determines that the Roc-A has the potential of being further developed into the cryptosporidium-resistant drug, and verifies that the targeted eIF4A can resist cryptosporidium infection.

Description

Technical Field

[0001] The present invention relates to a small molecule lead compound, in particular to an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase. Background Art

[0002] Cryptosporidiosis is a parasitic disease caused by parasitic protozoa of the genus Cryptosporidium that infect humans or animals. At present, the development of drugs for cryptosporidiosis is still progressing slowly. Globally, only nitazoxanide is approved by the US FDA for the treatment of human cryptosporidiosis. Nitazoxanide has a certain therapeutic effect in infected people with normal immune capacity, but it is basically ineffective for the treatment of immunodeficient people (such as AIDS patients). At the same time, there are currently no FDA-approved veterinary drugs for the treatment of Cryptosporidium infections in animals. Therefore, there is an urgent need to develop new therapeutic drugs and explore new drug targets. Studies have shown that helicases such as eukaryotic initiation factor 4A (eIF4A) play a key role in the growth and reproduction of eukaryotic organisms and are potential drug targets. Existing studies have shown that rocaglamide (Roc-A) is an effective eIF4A inhibitor, but there is still a lack of research reports on its inhibitory effect on Cryptosporidium helicase eIF4A and its anti-Cryptosporidium effects in vitro and in vivo.

[0003] In view of this, the existing small molecule structures were studied and applied, and an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase was proposed to provide new ideas and solutions for solving the above-mentioned problems. Summary of the invention

[0004] The present invention studies an anti-Cryptosporidium small molecule lead compound Roc-A that targets Cryptosporidium helicase, finds that the lead compound has the potential to be further developed into an anti-Cryptosporidium drug, and emphasizes the feasibility of resisting Cryptosporidium infection by targeting eIF4A.

[0005] An anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase comprises the following steps:

[0006] S1. Functional parameters of drug target helicase (CpeIF4A) and detection of the inhibitory effect of Roc-A on it:

[0007] The recombinant protein of Cryptosporidium parvum (C. parvum) eIF4A (CpeIF4A) with MBP (maltose-binding protein) tag was expressed by using prokaryotic Escherichia coli expression system;

[0008] The MBP-CpeIF4A recombinant protein is obtained by purification, and its enzyme activity (such as the unwinding activity of double-stranded RNA) is detected, and its Km value and other enzyme activity parameters are recorded;

[0009] The inhibitory effect of Roc-A on CpeIF4A activity was evaluated and its 50% inhibitory concentration (IC 50 value) and other parameters;

[0010] Thermal shift assay (TSA) was used to determine the affinity of Roc-A binding to CpeIF4A, including the dissociation constant (K d value), etc.

[0011] S2. In vitro drug efficacy verification:

[0012] C. parvum sporozoites were obtained by in vitro excystation, treated with drugs (such as Roc-A), and the survival of sporozoites under different treatment conditions was quantified by qRT-PCR. The 50% effective inhibitory concentration (EC 50 value);

[0013] The in vitro efficacy of drugs (such as Roc-A) against Cryptosporidium parvum was determined using an in vitro culture model of Cryptosporidium parvum infection and quantitative qRT-PCR to obtain a 50% effective inhibitory concentration (EC 50 Value). In addition, the above model was used to further evaluate the effect of Roc-A on the developmental stages of Cryptosporidium by changing the different drug action times;

[0014] The cytotoxicity of Roc-A to host cells was detected using an in vitro cell culture method, and its 50% cytotoxic concentration (TC 50 ; also known as CC 50 Value), to obtain the in vitro insecticide selectivity index (SI);

[0015] S3. In vivo drug testing:

[0016] In vivo drug testing C57BL / 6IFN-γ knockout mice were inoculated with C. parvum to establish an in vivo infection model;

[0017] The number of Cryptosporidium oocysts in mouse feces was quantitatively monitored by qPCR, the changes in the worm load in fecal samples of the Roc-A treatment group and the control group during the infection process were recorded, and the effect of treatment on the health status of mice was monitored.

[0018] Due to the adoption of the above technical solution, the beneficial effects of the anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention are:

[0019] The present invention demonstrates Roc-A as a promising lead compound and drug candidate against Cryptosporidium infection for treating Cryptosporidium infection in humans and animals by targeting eIF4A.

[0020] The interaction between Roc-A and CpeIF4A has been established, demonstrating its inhibitory effect on the helicase activity of CpeIF4A. In vitro experiments have also confirmed the effectiveness of Roc-A in inhibiting parasite growth and killing sporozoites, as well as its irreversible killing of Cryptosporidium.

[0021] In addition, in vivo studies in mice demonstrated the effectiveness of Roc-A in inhibiting the growth of Cryptosporidium and did not cause toxicity to experimental animals within safe dosages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 The present invention is a flowchart for evaluating the efficacy of Roc-A in an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase.

[0024] Figure 2 It is a multiple sequence alignment diagram of apicomplexan parasite elF4A in an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention.

[0025] Figure 3 This is a graph of the relative mRNA levels of CpelF4A and CpLDH in the anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase in different developmental stages of C. parvum of the present invention.

[0026] Figure 4 This is a diagram showing that Roc-A in an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention binds to CpeIF4A and inhibits its RNA double-stranded unwinding activity.

[0027] Figure 5 This is a graph showing the direct killing activity of Roc-A in an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention against C. parvum sporozoites.

[0028] Figure 6This is a diagram of evaluating the anti-Cryptosporidium effect of Roc-A in an in vitro HCT-8 cell infection model in an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention.

[0029] Figure 7 Roc-A in the anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention is a safe and parasite-targeted drug.

[0030] Figure 8 This is a diagram showing the identification of the anti-insect activity of Roc-A in a mouse infection model in an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present application fully explains the problems of the prior art in the technical background, and there are no superordinate or general problems. The problems to be solved by the present application are described in detail in the following invention content and specific implementation methods, the technical problems to be solved by the technical scheme are clarified, and it is determined that the technical scheme of the present application has beneficial effects relative to the objective prior art.

[0033] See also Figure 1-8 As shown: 1. An anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase, comprising the following steps:

[0034] S1. Functional parameters of drug target helicase (CpeIF4A) and detection of the inhibitory effect of Roc-A on it:

[0035] The recombinant protein of Cryptosporidium parvum (C. parvum) eIF4A (CpeIF4A) with MBP (maltose-binding protein) tag was expressed by using prokaryotic Escherichia coli expression system;

[0036] The MBP-CpeIF4A recombinant protein is obtained by purification, and its enzyme activity (such as the unwinding activity of double-stranded RNA) is detected, and its Km value and other enzyme activity parameters are recorded;

[0037] The inhibitory effect of Roc-A on CpeIF4A activity was evaluated and its 50% inhibitory concentration (IC 50value) and other parameters;

[0038] Thermal shift assay (TSA) was used to determine the affinity of Roc-A binding to CpeIF4A, including the dissociation constant (K d value), etc.

[0039] S2. In vitro drug efficacy verification:

[0040] C. parvum sporozoites were obtained by in vitro excystation, treated with drugs (such as Roc-A), and the survival of sporozoites under different treatment conditions was quantified by qRT-PCR. The 50% effective inhibitory concentration (EC 50 value);

[0041] The in vitro efficacy of drugs (such as Roc-A) against Cryptosporidium parvum was determined using an in vitro culture model of Cryptosporidium parvum infection and quantitative qRT-PCR to obtain a 50% effective inhibitory concentration (EC 50 Value). In addition, the above model was used to further evaluate the effect of Roc-A on the developmental stages of Cryptosporidium by changing the different drug action times;

[0042] The cytotoxicity of Roc-A to host cells was detected using an in vitro cell culture method, and its 50% cytotoxic concentration (TC 50 ; also known as CC 50 Value), to obtain the in vitro insecticide selectivity index (SI);

[0043] S3. In vivo drug testing:

[0044] In vivo drug testing used C57BL / 6IFN-γ knockout mice inoculated with C. parvum to establish a chronic infection model;

[0045] The number of Cryptosporidium oocysts in mouse feces was quantitatively monitored by qPCR, the changes in the worm load in fecal samples of the Roc-A treatment group and the control group during the infection process were recorded, and the effect of treatment on the health status of mice was monitored.

[0046] In step S1, the dissociation constant K between Roc-A and CpeIF4A is determined by a thermal shift experiment. d The binding strength of Roc-A and CpeIF4A was determined by quantifying the binding strength of Roc-A and CpeIF4A.

[0047] In step S2, the in vitro drug efficacy of Roc-A on excysted C. parvum sporozoites was detected by qRT-PCR method, and the 50% effective inhibitory concentration (EC50 The in vitro inhibitory effect of Roc-A on Cryptosporidium parvum was determined by using an in vitro culture Cryptosporidium parvum infection model and quantitative qRT-PCR method, and the IC value of the drug was determined. 50 The experimental control group used time point recording, statistical analysis, and chart display to display the results and record the specific effects of drug treatment on the development of Cryptosporidium, analyze the effects of different concentrations and treatment times on the inhibitory effect, and put forward experimental conclusions.

[0048] In step S3, the mouse model infected with C. parvum is constructed using 6-8 week old C57BL / 6 transgenic mice (IFN-γ gene knockout) to study the in vivo efficacy of Roc-A against Cryptosporidium.

[0049] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. An anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase, comprising the following steps: S1. Functional parameters of drug target helicase (CpeIF4A) and detection of the inhibitory effect of Roc-A on it: The recombinant protein of Cryptosporidium parvum (C. parvum) eIF4A (CpeIF4A) with MBP (maltose-binding protein) tag was expressed by using prokaryotic Escherichia coli expression system; The MBP-CpeIF4A recombinant protein is obtained by purification, and its enzyme activity (such as the unwinding activity of double-stranded RNA) is detected, and its Km value and other enzyme activity parameters are recorded; The inhibitory effect of Roc-A on CpeIF4A activity was evaluated and its 50% inhibitory concentration (IC 50 value) and other parameters; Thermal shift assay (TSA) was used to determine the affinity of Roc-A binding to CpeIF4A, including the dissociation constant (K d value), etc. S2. In vitro drug efficacy verification: C. parvum sporozoites were obtained by in vitro excystation, treated with drugs (such as Roc-A), and the survival of sporozoites under different treatment conditions was quantified by qRT-PCR. The 50% effective inhibitory concentration (EC 50 value); The in vitro efficacy of drugs (such as Roc-A) against Cryptosporidium parvum was determined using an in vitro culture model of Cryptosporidium parvum infection and quantitative qRT-PCR to obtain a 50% effective inhibitory concentration (EC 50 Value). In addition, the above model was used to further evaluate the effect of Roc-A on the developmental stages of Cryptosporidium by changing the different drug action times; The cytotoxicity of Roc-A to host cells was detected using an in vitro cell culture method, and its 50% cytotoxic concentration (TC 50 ; also known as CC 50 Value), to obtain the in vitro insecticide selectivity index (SI); S3. In vivo drug testing: In vivo drug testing used C57BL / 6IFN-γ knockout mice inoculated with C. parvum to establish a chronic infection model; The number of Cryptosporidium oocysts in mouse feces was quantitatively monitored by qPCR, the changes in the worm load in fecal samples of the Roc-A treatment group and the control group during the infection process were recorded, and the effect of treatment on the health status of mice was monitored.

2. The anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase according to claim 1, characterized in that: In step S1, the dissociation constant K between Roc-A and CpeIF4A is determined by a thermal shift experiment. d The binding strength of Roc-A and CpeIF4A was determined by quantifying the binding strength of Roc-A and CpeIF4A.

3. The anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase according to claim 1, characterized in that: In step S2, the in vitro drug efficacy of Roc-A on excysted C. parvum sporozoites was detected by qRT-PCR method, and the 50% effective inhibitory concentration (EC 50 The in vitro inhibitory effect of Roc-A on Cryptosporidium parvum was determined by using an in vitro culture Cryptosporidium parvum infection model and quantitative qRT-PCR method, and the IC value of the drug was determined. 50 The experimental control group used time point recording, statistical analysis, and chart display to display the results and record the specific effects of drug treatment on the development of Cryptosporidium, analyze the effects of different concentrations and treatment times on the inhibitory effect, and put forward experimental conclusions.

4. The anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase according to claim 1, characterized in that: In step S3, the mouse model infected with C. parvum is constructed using 6-8 week old C57BL / 6 transgenic mice (IFN-γ gene knockout) to study the in vivo efficacy of Roc-A against Cryptosporidium.

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