A small molecule lead compound against Cryptosporidium that targets Cryptosporidium helicase

Abstract

The present invention relates to a small molecule lead compound against Cryptosporidium that targets the helicase of Cryptosporidium; it includes the following steps: S1. Detection of the enzymatic activity parameters of the drug target helicase (CpeIF4A) and the inhibitory effect of Roc-A thereon: expressing and purifying the CpeIF4A recombinant protein containing an MBP tag; detecting its enzymatic activity; detecting the inhibitory effect of Roc-A on the enzymatic activity; determining the affinity between Roc-A and CpeIF4A through a thermal shift experiment; S2. In vitro efficacy determination: evaluating the direct killing ability of Roc-A against sporozoites; determining the inhibitory effect of Roc-A on Cryptosporidium through an in vitro cell infection model, obtaining the EC50 value, and determining the intracellular stage at which it mainly exerts an inhibitory effect; detecting the toxicity of Roc-A to host cells, obtaining the TC50 value, and obtaining the in vitro anti-parasite selectivity index (SI); S3. In vivo efficacy test: establishing a C57BL / 6 IFN-γ gene knockout mouse infection model to evaluate the effectiveness and safety of Roc-A. The present invention determines that Roc-A has the potential for further development as an anti-Cryptosporidium drug, and confirms that targeting eIF4A can resist Cryptosporidium infection.

Description

Technical Field

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

[0002] Cryptosporidiosis is a parasitic disease caused by the parasitic protozoa of the genus Cryptosporidium infecting humans or animals. At present, the research and 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 immunocompetent infected individuals, but is basically ineffective in the treatment of immunocompromised populations (such as AIDS patients, etc.). At the same time, there is currently no FDA-approved veterinary drug for the treatment of Cryptosporidium infection in animals. Therefore, there is an urgent need to develop new therapeutic drugs and explore new drug targets. Research has shown that helicases such as eukaryotic initiation factor 4A (eIF4A) play a key role in the growth and reproduction of eukaryotes and are potential drug targets. Existing research has shown that rocaglamide (Rocaglamide; abbreviated as 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, by researching and applying the existing small molecule structures, an anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase is proposed, providing new ideas and solutions for solving the above existing problems. Summary of the Invention

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

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

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

[0007] Using a prokaryotic Escherichia coli expression system, express the recombinant protein of Cryptosporidium parvum (C.parvum) eIF4A (CpeIF4A) with an MBP (maltose-binding protein) tag.

[0008] The MBP-CpeIF4A recombinant protein was obtained by purification, and its enzyme activity (such as the helicase activity against double-stranded RNA) was detected, and enzyme activity parameters such as its Km value were recorded;

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

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

[0011] S2. In vitro drug efficacy verification:

[0012] Sporozoites of C. parvum 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, and the 50% effective inhibitory concentration (EC 50 value) was recorded;

[0013] Using an in vitro culture model of Cryptosporidium parvum infection and the quantitative qRT-PCR method to determine the in vitro inhibitory effect (in vitro efficacy) of drugs (such as Roc-A) on Cryptosporidium, and obtain the 50% effective inhibitory concentration (EC 50 value). Moreover, using the above model, by changing different drug action times, the effects of Roc-A on different developmental stages of Cryptosporidium were further evaluated;

[0014] Using an in vitro cell culture method, the cytotoxicity of Roc-A to host cells was detected, and its 50% cytotoxic concentration (TC 50 ; also known as CC 50 value) was determined, and the in vitro anti-parasite selectivity index (selectivity index; SI) was obtained;

[0015] S3. In vivo drug testing:

[0016] For in vivo drug testing, C57BL / 6 IFN-γ knockout mice were used, inoculated with C. parvum, and an in vivo infection model was established;

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

[0018] Due to the above technical solution, the beneficial effects of a small molecule lead compound against Cryptosporidium targeting Cryptosporidium helicase of the present invention are as follows:

[0019] The present invention demonstrates that Roc-A is a promising lead compound and drug candidate for anti-Cryptosporidium infection, and is used to treat 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. At the same time, in vitro experiments have confirmed the effectiveness of Roc-A in inhibiting parasite growth and killing sporozoites, etc., and its irreversibility in killing Cryptosporidium.

[0021] In addition, in vivo studies on mice have demonstrated the effectiveness of Roc-A in inhibiting the growth of Cryptosporidium, and it does not cause toxicity to experimental animals within the safe dosage range. Description of the Drawings

[0022] The 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 to the present invention. In the drawings:

[0023] Figure 1 It is a flow chart of the pharmacodynamic evaluation of Roc-A in a small molecule lead compound against Cryptosporidium targeting Cryptosporidium helicase of the present invention.

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

[0025] Figure 3 It is a relative mRNA level diagram of CpelF4A and CpLDH at different developmental stages of C. parvum in a small molecule lead compound against Cryptosporidium targeting Cryptosporidium helicase of the present invention.

[0026] Figure 4 It is a display diagram of Roc-A binding to CpeIF4A and inhibiting its RNA double-strand unwinding activity in a small molecule lead compound against Cryptosporidium targeting Cryptosporidium helicase of the present invention.

[0027] Figure 5 It is a display diagram of the direct killing activity of Roc-A against C. parvum sporozoites in a small molecule lead compound against Cryptosporidium targeting Cryptosporidium helicase of the present invention.

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

[0029] Figure 7 This is a diagram showing that Roc-A is a safe and parasite-targeted drug among the anti-Cryptosporidium small molecule lead compounds targeting Cryptosporidium helicase of the present invention.

[0030] Figure 8 This is a diagram for identifying the anti-parasitic activity of Roc-A in a mouse infection model among the anti-Cryptosporidium small molecule lead compounds targeting Cryptosporidium helicase of the present invention. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] The problems of the prior art are fully interpreted in the technical background of this application. There are no general and vague problems. The problems to be solved by this application are specifically described in the following invention content and detailed implementation manners, clarifying the technical problems to be solved by the technical solutions, and determining that the technical solutions of this application have beneficial effects compared with the objective prior art.

[0033] Please refer to Figure 1-8 as shown in the following: 1. An anti-Cryptosporidium small molecule lead compound targeting Cryptosporidium helicase includes the following steps:

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

[0035] Using a prokaryotic Escherichia coli expression system to express a recombinant protein of Cryptosporidium parvum (C. parvum) eIF4A (CpeIF4A) with an MBP (maltose-binding protein) tag;

[0036] Obtaining the MBP-CpeIF4A recombinant protein by purification and detecting its enzyme activity (such as the helicase activity on double-stranded RNA), and recording its enzyme activity parameters such as the Km value;

[0037] Evaluating the inhibitory effect of Roc-A on the activity of CpeIF4A and recording its 50% inhibitory concentration (IC 50parameters such as the value);

[0038] Determine the binding affinity of Roc-A to CpeIF4A, including the dissociation constant (K d value), etc., through a thermal shift assay (TSA);

[0039] S2. In vitro drug efficacy verification:

[0040] Obtain sporozoites of C. parvum by in vitro excystation, treat the sporozoites with a drug (such as Roc-A), and quantitatively determine the survival of sporozoites under different treatment conditions by qRT-PCR, and record the 50% effective inhibitory concentration (EC 50 value);

[0041] Use an in vitro culture model of Cryptosporidium parvum infection and the quantitative qRT-PCR method to determine the in vitro inhibitory effect (in vitro efficacy) of a drug (such as Roc-A) on Cryptosporidium, and obtain the 50% effective inhibitory concentration (EC 50 value). Moreover, use the above model to further evaluate the effect of Roc-A on different developmental stages of Cryptosporidium by changing the different drug action times;

[0042] Use an in vitro cell culture method to detect the cytotoxicity of Roc-A to host cells and determine its 50% cytotoxic concentration (TC 50 ; also known as CC 50 value), and obtain the in vitro anti-parasite selectivity index (selectivity index; SI);

[0043] S3. In vivo drug testing:

[0044] For in vivo drug testing, use C57BL / 6 IFN-γ knockout mice, inoculate them with C. parvum, and establish a chronic infection model;

[0045] Quantitatively monitor the number of Cryptosporidium oocysts in the feces of mice by qPCR, record the changes in the parasite load in the fecal samples of the Roc-A treatment group and the control group during the infection process, and monitor the effect of the treatment on the health status of the mice.

[0046] In step S1, determine the dissociation constant K d value between Roc-A and CpeIF4A through a thermal shift assay to determine the binding strength between Roc-A and CpeIF4A.

[0047] In step S2, detect the in vitro drug efficacy of Roc-A on C. parvum sporozoites after excystation by qRT-PCR, and record the 50% effective inhibitory concentration (EC50 value). By using an in vitro culture model of Cryptosporidium parvum infection and the quantitative qRT-PCR method, the in vitro inhibitory effect of Roc-A on Cryptosporidium was determined, and the IC 50 value and in vitro safety were determined. The experimental control group used time point recording, statistical analysis, and chart display to present 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 draw experimental conclusions.

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

[0049] The above describes the present invention and its implementation manners. Such a description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural manners and embodiments without creative efforts without departing from the purpose of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. Use of Roc-A in the preparation of a drug for anti-Cryptosporidium infection, characterized in that, The Roc-A specifically binds to the Cryptosporidium helicase CpeIF4A and inhibits its activity.

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