Cephradine for prevention and/or treatment of TOPK (T-LAK cell-originated protein kinase) activity abnormally increased skin inflammations by inhibition of TOPK

A technology of cephradine and activity, which is applied in the fields of new pharmacological effects of drugs and disease prevention and treatment, and can solve problems such as obvious side effects and impossibility of clinical application

Inactive Publication Date: 2016-07-06
史飞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these drugs have obvious side effects and cannot be effectively clinically applied.

Method used

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  • Cephradine for prevention and/or treatment of TOPK (T-LAK cell-originated protein kinase) activity abnormally increased skin inflammations by inhibition of TOPK
  • Cephradine for prevention and/or treatment of TOPK (T-LAK cell-originated protein kinase) activity abnormally increased skin inflammations by inhibition of TOPK
  • Cephradine for prevention and/or treatment of TOPK (T-LAK cell-originated protein kinase) activity abnormally increased skin inflammations by inhibition of TOPK

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Cephradine binds to TOPK and inhibits TOPK activity.

[0064] (A) Cephradine reagent was purchased from Sigma-Aldrich Company (StLouis, MO, USA). Human activated TOPK kinase was purchased from Millippore (Billerica, MA, USA). pGEX-GST-H2AX, lentiviralexpression, packaging vectors: TOPKshRNA, pMD2.0G and psPAX were purchased from Addgene. IL6 and TNF-αELISA assay kits were purchased from Biosen (Beijing, China). The internal reference protein was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

[0065] Molecular models and docking simulations

[0066] To estimate the interaction model of TOPK and cephradine, a TOPK structure was modeled and applied for subsequent induced fit docking. The sequence of TOPK (GI: 83305809) was downloaded from NCBI, and a BLAST similarity search was performed against the protein database (PDB) to identify homologous sequences. Among those proteins with the highest sequence identity (30%) to TOPK, a protein-ligand...

Embodiment 2

[0072] Example 2 SUV induces the downstream TOPK signaling pathway in a dose- and time-dependent manner, while cephradine inhibits this signaling pathway and also inhibits the secretion of cytokines in HaCaT cells and JB6.

[0073] (A) Cell culture and cell proliferation rate detection

[0074] Immortalized human epidermal keratinocyte cell line and mouse epidermal JB6Cl41 cell line (JB6) were purchased from ATCC, Virginia, USA. HaCaT uses DMEM as a medium (containing 100u / ml penicillin and 100u / ml streptomycin, 10% FBS), and places it in an incubator at 37°C and 5% CO2 saturated humidity. The JB6 cell line uses MEM as a medium (containing 5% FBS), and places it in an incubator at 37°C and 5% CO2 saturated humidity. After counting on a hemocytometer, the two kinds of cells were inoculated in a 6 cm culture dish at a concentration of 1×10 5 cells / ml.

[0075] Cells were seeded in 96-well culture plates at a concentration of 8×103 / ml and cultured overnight. Then, replace the ...

Embodiment 3

[0081] Example 3 Cephradine inhibits SUV-induced DNA damage by inhibiting SUV-induced (dose- and time-dependent) TOPK activation in HaCat cells and JB6 cells.

[0082] (A) Expression and purification of bacterial GST-H2AX fusion protein

[0083]Human GST-H2AX fusion protein was expressed in E. ColiBL21 bacteria. At 37°C, the bacteria grow at a wavelength of 600nm with an absorbance of 0.8-0.9, induce the production of IPTG after 2-3 hours, and then extract by centrifugation. Cell pellets were suspended in PBS. After centrifugation, the supernatant was incubated with glutathione sepharose overnight at 4°C, after which it was washed with PBS and then exposed to 50 mM glutathione. After the protein was quantified, it was electrophoresed by SDS-PAGE and visualized by Coomassie brilliant blue.

[0084] in vitro method

[0085] GST-H2AX (1μg) protein was used as substrate, and 250ng activated TOPK was used for in vitro experiments. The two were reacted at 30°C for 30 minutes in...

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Abstract

The invention discloses a new drug effect and molecular mechanism of cephradine. In the present invention, it is found that cephradine inhibits actinic dermatosis (sunburn, chronic actinic dermatitis) induced by ultraviolet rays (UV), by interacting with T-LAK cells The original protein kinase (TOPK) binds and inhibits its activity, blocking the TOPK signaling pathway. Immunopathology suggested that the expression of TOPK, P38, and JNK increased in actinic dermatosis lesions. In cell experiments, TOPK activity affects UV-induced phosphorylation of P38 and JNK. Molecular docking experiments found that cephradine can directly bind to TOPK, inhibit the activation of TOPK, inhibit the phosphorylation of p38, JNK and H2AX, and the secretion of inflammatory factors IL-6 and TNF-α. Human and animal experiments have also verified the results of cell experiments.

Description

[0001] The invention belongs to the field of new pharmacological action of medicine and disease prevention and treatment. It specifically relates to cephradine inhibiting the biological activity of T-LAK cell-derived protein kinase (TOPK), and the application of cephradine to prevent and / or treat inflammation and tumors by inhibiting the abnormal increase of TOPK activity, including ultraviolet (UV)-induced photodermatosis, Such as sunburn and chronic actinic dermatitis. Background technique [0002] Clinical epidemiological investigations have shown that long-term exposure of the human body to sunlight ultraviolet (SUV) can produce skin inflammation and skin tumors, which seriously threaten human health. Ultraviolet radiation in sunlight can be divided into three categories according to the wavelength, long-wave ultraviolet (UVA) with a wavelength of 315-400nm, medium-wave ultraviolet (UVB) with a wavelength of 280-315nm, and short-wave ultraviolet (UVB) with a wavelength of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/545A61P29/00A61P17/00A61P35/00
CPCA61K31/545
Inventor 史飞
Owner 史飞
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