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Application of a kind of hoip inhibitor for preparing medicine for treating type II human telangiectasia

A telangiectasia drug technology, applied in the preparation of drugs for the treatment of type II human telangiectasia, in the field of HOIP inhibitors, can solve problems that have not been reported in the research, and achieve the effect of enhancing the level of linear ubiquitination

Active Publication Date: 2022-02-08
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the family screening found that the loss of linear ubiquitination is associated with multi-organ autoimmune deficiency and chronic autoinflammation and other diseases, there is no report on the regulation of linear ubiquitination of HHT2 and the application of HOIP inhibitors to treat HHT2

Method used

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  • Application of a kind of hoip inhibitor for preparing medicine for treating type II human telangiectasia
  • Application of a kind of hoip inhibitor for preparing medicine for treating type II human telangiectasia
  • Application of a kind of hoip inhibitor for preparing medicine for treating type II human telangiectasia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 demonstrates that ALK1 270-320aa (amino acids) domain specifically binds to HOIP

[0045] In order to confirm the specific domain of ALK1 binding to HOIP, HEK293T purchased from ATCC was preferred to overexpress the Flag-ALK1 series truncated body ( figure 2 ) and Myc-HOIP plasmid, and the interaction between the two was detected by immunoprecipitation technique.

[0046] The protein co-immunoprecipitation steps are:

[0047] 1. Collect the cells that have been transfected with the plasmid for 48 hours (you can choose culture flask or 6-well plate for transfection);

[0048] 2. Wash 3 times with pre-cooled PBS, centrifuge at 2000rpm for 3min;

[0049] 3. Add 500 μl of lysate (choose HEPES lysate or RIPA lysate according to specific experimental requirements), then choose to lyse on ice for 30 minutes or directly sonicate the cells, and then centrifuge at 12,000 rpm for 10 minutes to obtain the supernatant for later use;

[0050] 4. Take 50 μl of the supern...

Embodiment 2

[0068] Example 2 demonstrates the relationship between HHT2-related mutations and linear ubiquitination

[0069] To further explore the physiological significance of HOIP binding to ALK1, we combined the existing UniProt (https: / / www.uniprot.org / uniprot / P37023) database to screen 8 HHT2-related mutations (T277K, H280R, L285F , L289P, L294R, A306P, L313V, H314Y), using the same method as in Example 1 to overexpress the Flag-ALK1 series mutants and Myc-HOIP plasmids in HEK293T to detect the linear ubiquitination level ( image 3 ), six of the eight mutations (T277K, H280R, L285F, L289P, A306P, H314Y) were found to have significantly enhanced linear ubiquitination levels relative to wild-type ALK1 ( image 3 and Figure 4 ).

[0070] At the same time, it was found that the binding ability of these six mutants to HOIP was significantly enhanced by immunoprecipitation technique ( Figure 5 ). The ubiquitination experiment operation is as follows:

[0071] 1. Collect the cells ...

Embodiment 3

[0077] Example 3 Linear ubiquitination modification inhibits the kinase activity of ALK1

[0078] In order to verify the effect of these mutations on enhancing the ability to bind HOIP and increasing the ubiquitination level, we need to first figure out how the ubiquitination modification affects ALK1 itself. ALK1 is a classic serine-threonine kinase, and its kinase activity is the key to its function. The common detection of ALK1 kinase activity is to detect by constructing and purifying the constitutive activator of ALK1. Therefore, we found that the constitutive activator of ALK1 can phosphorylate the substrate Smad1 through in vitro phosphorylation experiments, but the constitutive activator modified by linear ubiquitination lost its activity. Therefore, the activity of ALK1 kinase after enhanced ubiquitination of mutants was proved by in vitro phosphorylation experiments. The in vitro phosphorylation experiment steps are as follows:

[0079] 1. HEK293T cells were transf...

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Abstract

The invention discloses an application of a HOIP inhibitor for treating type II human telangiectasia (HHT2). First disclosed the binding domain and binding mechanism of ALK1 kinase and HOIP, and found that the enhanced binding of ALK1 mutants to HOIP led to a significant increase in the ubiquitination level. By inhibiting the HOIP ubiquitin ligase activity, the ubiquitination level of ALK1 mutants was reduced, thereby restoring the ALK1‑Smad1 / 5 signaling pathway and alleviating the symptoms of HHT2. The present invention proves that the pathogenesis of ALK1-mutated HHT2 is caused by the reduction of ALK1 kinase activity, and can be used to treat HHT2 by inhibiting specific mutants of ALK1 through HOIP inhibitors, making HOIP inhibitors the best choice for treating specific ALK1 mutations. Potential drugs for HHT2.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of a HOIP inhibitor in the preparation of drugs for treating type II human telangiectasia (HHT2). Background technique [0002] Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disease with an incidence rate as high as 1 / 5000. Activin receptor-like kinase 1 (ALK1 Activin receptor-like kinase 1) is the most important pathogenic gene in type II human telangiectasia (HHT2). The ALK1 mutation carried by HHT2 patients often leads to the weakening of the ALK1-Smad1 / 5 signaling pathway in endothelial cells, which can easily induce arteriovenous malformations (AVMs). Capillaries are often found at the junction of arteries and veins, allowing the surrounding tissues surrounded by capillaries to obtain nutrients and oxygen. High-grade arteriovenous malformations result in a direct connection of arteries and veins, and the impact of blood flow at the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/06A61K31/277A61K31/4704A61K31/4184A61K31/548A61P9/14C12Q1/6883
CPCA61K45/06A61K31/277A61K31/4704A61K31/4184A61K31/548A61P9/14C12Q1/6883C12Q2600/156A61K2300/00
Inventor 张令强付业胜崔春萍
Owner ACADEMY OF MILITARY MEDICAL SCI
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