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Application of isobavachalcone in preparation of therapeutic drug for acute myeloid leukemia (AML)

A technology of acute myeloid cells and psoralen B, which is applied in the fields of chemical industry and medicine, and can solve the problems that the application of psoralen B has not been reported.

Inactive Publication Date: 2018-01-19
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] But there is no report about the application of psoralen B in anti-acute myeloid leukemia

Method used

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  • Application of isobavachalcone in preparation of therapeutic drug for acute myeloid leukemia (AML)
  • Application of isobavachalcone in preparation of therapeutic drug for acute myeloid leukemia (AML)
  • Application of isobavachalcone in preparation of therapeutic drug for acute myeloid leukemia (AML)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Expression and Purification of Human Recombinant hDHODH

[0037] Transform E.coli BL21(DE3) competent cells with the recombinant plasmid pET-19b-hDHODH with correct sequencing, spread it on the LB plate containing ampicillin and culture it, pick a single clone and inoculate it in a 2× Cultivate overnight in YT medium on a shaker at 37°C and 230 rpm. After overnight culture, the bacteria were inoculated in 8×1L TB medium containing 100g / mL ampicillin at a ratio of 1:200 and expanded at 37°C and 230rpm. When the OD value of the bacteria reaches 0.8-1, IPTG is added to the medium to make the final concentration of IPTG 0.5mM, and the expression of the fermentation broth is induced overnight at 16°C.

[0038] After overnight culture, the induced cells were collected by centrifugation at 4°C and 4000rpm, the cells were washed once with a buffer solution of 50mM HEPES pH8.0, 400mM NaCl, the cell pellet was collected by centrifugation, and the cells were stored at -...

Embodiment 2

[0041] Example 2 Screening of human recombinant hDHODH inhibitors

[0042] The purified hDHODH protein was diluted to 10 μM with an activity test solution, which was 50 mM HEPES pH 8.0, 150 mM KCl, 0.1% Triton X-100. Add Coenzyme Q 0 and DCIP, so that the final concentrations were 100 μM and 120 μM. After mixing well, put it into a 96-well plate, incubate at room temperature for 5 minutes, then add the substrate DHO to start the reaction, and the final concentration of DHO is 500 μM. Use a PE microplate reader to detect the absorbance at 600 nm, read it every 30 s for 6 min, and calculate the inhibition rate.

[0043] figure 2 It is the linear relationship between psoralen B and hDHODH activity, and psoralen B has strong inhibitory effect on hDHODH activity; figure 2 Among them, the abscissa is the psoralen B concentration (μM), and the ordinate is the enzyme activity inhibition rate Inhibition (%); the compounds are psoralen B and the positive control A771726 respective...

Embodiment 3

[0044] Example 3 Study on thermal stability of psoralen B to human recombinant hDHODH

[0045] The hDHODH protein was diluted to 5 μM with the test solution, and the activity test solution was 25 mM HEPES, 150 mM NaCl, pH7.5. Add the fluorescent dye Sypro orange to a final concentration of 5X. After mixing, add 40 μL per well into a 96-well plate, and then add the compound at a pre-set concentration for incubation. Use Bio-Rad RT-PCR to determine the protein Tm value.

[0046] image 3 After incubating psoralen B and hDHODH protein at concentrations of 2:1 and 10:1, respectively, the melting temperature of the protein was measured. The results showed that psoralen B could significantly increase the melting temperature of hDHODH protein. Under the conditions of these two ratios, the dissolution temperature increased by 12°C and 14.4°C respectively, which indicated that psoralen B could directly combine with hDHODH protein.

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Abstract

The invention relates to the field of chemical engineering and medicines, and particularly relates to application of isobavachalcone in preparation of therapeutic drug for acute myeloid leukemia (AML). The invention provides application of inner mitochondrial membrane protein hDHODH taken as an action target in screening of natural products. The isobavachalcone can specifically inhibit hDHODH activity in vitro and in vivo and can induce the apoptosis and differentiation of cells of the acute myeloid leukemia by acting on hDHODH, and the apoptosis and differentiation effects of AML cells induced by the isobavachalcone depend on hDHODH activity. The isobavachalcone is taken as a regulatory factor of the apoptosis and differentiation approach of the cells of targeted hDHODH to become an extremely effective anti-AML therapeutic drug. The drug for treating AML takes the inner mitochondrial membrane protein hDHODH as an action target.

Description

technical field [0001] The invention relates to the fields of chemical industry and medicine, in particular to the application of psoralen B in the preparation of acute myeloid leukemia (AML) therapeutic medicine. Background technique [0002] Acute myeloid leukemia (AML), a malignant disease of myeloid hematopoietic stem / progenitor cells, is characterized by abnormal growth and differentiation of hematopoietic stem cells (HSCs), during which immature bone marrow precursors Cells (myeloid cells) accumulate in the bone marrow and peripheral blood. The disease has a high degree of malignancy. According to epidemiological statistics, the natural incidence rate of leukemia in my country is 3 / 100,000 to 4 / 100,000, and it shows an increasing trend. There are currently 4 million leukemia patients in the country, and about 40,000 new leukemia patients are added every year, of which more than 20,000 are children, and most of them are children aged 2 to 9. Therefore, AML seriously e...

Claims

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Application Information

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IPC IPC(8): A61K31/12A61P35/02
Inventor 黄瑾黄莹卢伟强付志敏吴荡梁宏
Owner EAST CHINA UNIV OF SCI & TECH
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