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A method for screening drugs for the treatment of steatohepatitis by targeting dimerization of apoptosis signal-regulated kinase 1 N-terminus

A technology of steatohepatitis and apoptosis signaling, applied in the biological field, can solve problems such as damage to the function of the digestive system, reduce human immunity, and heavy burden, and achieve the effect of promoting development

Active Publication Date: 2020-01-07
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can further damage the function of the digestive system, reduce human immunity, weaken detoxification function, affect hormone metabolism, etc., seriously affect people's health and quality of life, and also bring a heavy burden to society

Method used

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  • A method for screening drugs for the treatment of steatohepatitis by targeting dimerization of apoptosis signal-regulated kinase 1 N-terminus
  • A method for screening drugs for the treatment of steatohepatitis by targeting dimerization of apoptosis signal-regulated kinase 1 N-terminus
  • A method for screening drugs for the treatment of steatohepatitis by targeting dimerization of apoptosis signal-regulated kinase 1 N-terminus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] [Example 1] Screening for polypeptides that can inhibit the N-terminal dimerization of ASK1

[0135] Stable expression of ASK1 N The HEK-293T plasmids for dimerization detection are divided into 5 groups, which are marked as A, B, C, D, and E respectively. In group A, only the pG5luc plasmid is added without the psi-flag-Peptide plasmid when the target polypeptide is transiently transfected. , Groups B, C, D, and E were transfected with psi-flag-Peptide1, 2, 3, 4 plasmids and pG5luc plasmids into HEK-293T cells respectively, that is, the five groups were:

[0136] A: HEK-293T cells (pACT-ASK1 N +pBIND-ASK1 N )+pG5luc

[0137] B: HEK-293T cells (pACT-ASK1 N +pBIND-ASK1 N )+psi-flag-Peptide1+pG5luc

[0138] C: HEK-293T cells (pACT-ASK1 N +pBIND-ASK1 N )+psi-flag-Peptide2+pG5luc

[0139] D: HEK-293T cells (pACT-ASK1 N +pBIND-ASK1 N )+psi-flag-Peptide3+pG5luc

[0140] E: HEK-293T cells (pACT-ASK1 N +pBIND-ASK1 N )+psi-flag-Peptide4+pG5luc

[0141] After addin...

Embodiment 2

[0143] [Example 2] Co-immunoprecipitation verification of the inhibitory effect of polypeptides on the N-terminal dimerization of ASK1

[0144] HEK-293T cells were divided into 7 groups, coded as A, B, C, D, E, F, and G. Cells in group A and group B were only transfected with HA-ASK1 N or Myc-ASK1 N Plasmid, cells in groups C, D, E, F, and G were simultaneously transfected with HA-ASK1 N and Myc-ASK1 N Two plasmids, positive cells were screened after 48 hours. The resulting cells are as follows:

[0145] Group A: HEK-293T cells (HA-ASK1 N )

[0146] Group B: HEK-293T cells (Myc-ASK1 N )

[0147] C, D, E, F, G groups: HEK-293T cells (HA-ASK1 N +Myc-ASK1 N )

[0148] Afterwards, the transient transfection of the target polypeptide plasmid was carried out. Only the same amount of transfection solution without the target polypeptide plasmid was added to the cells in groups A, B, and C, and the cells in groups D, E, F, and G were respectively added with psi-flag-Peptide1 ...

Embodiment 3

[0159] [Example 3] Inhibiting the N-terminal dimerization of ASK1 can inhibit the ASK1-JNK1 signaling pathway

[0160]Western blot analysis was used to detect the effect of inhibiting the N-terminal dimerization of ASK1 on the intracellular JNK1 signaling pathway. Required primary antibody information: p-ASK1 (Cell Signaling Technology, #3765), ASK1 (GeneTex, #GTX107921), p-MKK7 (Aviva Systems Biology, #OAAF05547), MKK7 (Cell Signaling Technology, #4172), p-JNK1 (NOVUS, #NB100-82009), JNK1 (Abcam, #ab199380), Flag (Sigma, #F3165); required secondary antibody information: HRP AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson, #111-035 -003), Biotin AffiniPure Goat Anti-Mouse IgG (H+L) (Abbkine, A21210).

[0161] L02 cells were divided into 5 groups and cultured in culture dishes, numbered A, B, C, D, E, and cultured at 37°C until the cell density was 70%. Cells in group A were transfected with psi-flag plasmid as a control, and cells in group B, C, and D were cultured in culture ...

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Abstract

The invention discloses an application of apoptosis signal-regulating kinase 1 (ASK1) in screening drugs for preventing, relieving and / or treating steatohepatitis by taking N-terminal dimerization of ASK1 as a target. The invention further discloses a method for screening drugs for preventing, relieving and / or treating steatohepatitis by taking N-terminal dimerization of ASK1 as the target. The invention firstly discloses that the N-terminal dimerization of ASK1 has important significance on the phosphorylation activation of ASK1 and the signal path of ASK1-JNK, has an important regulation role in fatty degeneration of liver cells, and can promote the fatty degeneration development of the liver. By taking N-terminal dimerization of ASK1 as the target and detecting whether candidate substances inhibit the N-terminal dimerization of ASK1, novel drugs for preventing, relieving and / or treating steatohepatitis can be screened.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to the establishment of a screening model targeting the N-terminal dimerization of apoptosis signal-regulated kinase 1 (ASK1, Apoptosis signal-regulated kinase 1), and screening The obtained ASK1 N-terminal dimerization inhibitor is applied to the preparation of drugs for preventing, alleviating and / or treating steatohepatitis. Background technique [0002] Apoptosis signal-regulated kinase 1 (ASK1, Apoptosis signal-regulated kinase 1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. ASK1 protein contains 1375 amino acid residues and has a molecular weight of about 160kDa [1] . The protein structure of ASK1 is divided into five parts, of which amino acids 46-277 at the N-terminal are the thioredoxin (Trx) binding region, amino acids 297-324 are the N-terminal coiled-coil domain, and amino acids 384-655 are the TRAF binding regio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/861C12N5/071
CPCA01K67/0275A01K2217/20A01K2267/03C12N5/067C12N15/85C12N15/86C12N2501/727C12N2710/10043C12N2800/107
Inventor 李红良王丕晓
Owner WUHAN UNIV
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