CN112159327A - Compound for inhibiting proliferation of human cancer cells in nude mice - Google Patents

Compound for inhibiting proliferation of human cancer cells in nude mice Download PDF

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CN112159327A
CN112159327A CN202011171491.8A CN202011171491A CN112159327A CN 112159327 A CN112159327 A CN 112159327A CN 202011171491 A CN202011171491 A CN 202011171491A CN 112159327 A CN112159327 A CN 112159327A
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compound
protein
cancer cells
proliferation
combined
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章晓波
许晨曦
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China Ocean Mineral Resources R & D Association (china's Ocean Affairs Administration)
Zhejiang University ZJU
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China Ocean Mineral Resources R & D Association (china's Ocean Affairs Administration)
Zhejiang University ZJU
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Abstract

The invention discloses a compound for inhibiting the proliferation of human cancer cells in a nude mouse. The compound is 2- [ (4-hydroxyphenyl) amino ] phenol, is a benzoquinone compound, and is obtained by screening a deep-sea hydrothermal oral thermophile strain; the compound can change the conformation of a p53 protein after being combined with the p53 protein, and the p53 protein flows out before being combined with the unbound p53 protein through sephadex G50 screening; the derivative of the compound can inhibit the growth of tumor cells with p53 gene mutation; after the hydroxyl groups in the derivatives of the compound are all replaced, the growth inhibition effect on tumor cells disappears; the compound can be combined with human recombinant p53 protein. The invention relates to a method for screening and obtaining a compound capable of being combined with mutant p53 protein by using a molecular sieve method, wherein the compound can inhibit the proliferation of cancer cells with p53 gene mutation in a nude mouse and has no obvious influence on immune cells of the mouse.

Description

Compound for inhibiting proliferation of human cancer cells in nude mice
Technical Field
The invention relates to a compound which is crudely extracted from deep-sea hydrothermal oral thermophile, in particular to a compound for inhibiting the proliferation of human cancer cells in nude mice.
Background
Compounds act intracellularly, generally by interacting with a particular protein, by affecting the function of the protein, and then further affecting cell growth. However, it has been a challenge to find small molecule compounds that can affect the target protein.
The p53 protein is used as a transcription factor and is a guardian of genome, and when the genome is damaged, the transcription activity of the p53 protein is activated, so that the cell cycle is blocked and the apoptosis of the cell is caused. According to the existing statistics, point mutation is generated in p53 in 50% of human cancers, so that the function of p53 protein is influenced. And related documents report that the restoring activity of the mutant p53 protein can remarkably prevent the occurrence and the development of cancers.
The traditional screening of the p53 protein activating factor is mostly from the cell level, a cell line with p53 protein mutation is constructed, and then a compound which can affect a p53 mutant cell line is screened from an existing compound library, but the compound does not affect a p53 wild-type cell line. Through this screening method, several compounds capable of activating mutant p53 proteins have been discovered. Some of these compounds function by directly binding to the mutated p53 protein, thereby increasing the stability of the p53 protein, while others can indirectly activate the mutated p53 protein by altering the protein that interacts with the p53 protein. Because p53 protein has a high probability of mutation in cancer, finding a protein that specifically binds to mutant p53 protein and restores its wild-type function would provide a beneficial concept for cancer therapy.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a compound for inhibiting the proliferation of human cancer cells in a nude mouse, and the compound is obtained by screening a target protein binding compound from deep-sea thermoorale thermophile by a sephadex method, can inhibit the growth of p53 gene mutant human breast cancer cells in the nude mouse, and is proved by a derivative experiment that the hydroxyl group of the compound plays an important role in inhibiting the growth of the tumor cells, and finally, a toxicological experiment shows that the compound has no obvious toxic or side effect on the mouse under a normal dosage condition.
The technical scheme adopted by the invention is as follows:
the compound for inhibiting the proliferation of human cancer cells in a nude mouse is 2- [ (4-hydroxyphenyl) amino ] phenol (2- [ (4-hydroxybenzyl) amino ] phenol, HBAP for short), and is a benzoquinone compound, and the compound is obtained by screening a deep-sea thermostomatal thermophile strain.
The compound can change the conformation of the p53 protein after being combined with the p53 protein, and the p53 protein flows out before being combined with the unbound p53 protein through sephadex G50 screening.
Derivatives of the compounds HBAP1, HBAP2, HBAP3 and HBAP4 can inhibit the growth of tumor cells with p53 gene mutation; after the hydroxyl groups in the derivatives of the compounds have been replaced, the growth inhibitory effect on tumor cells is lost.
The compound HBAP combined with the recombinant p53 protein is fed according to the proportion of one ten thousandth of the weight of the mice, the weight and the blood cell number of the mice have no obvious change, which indicates that the compound HBAP has no obvious toxic and side effect on the mice under normal dosage.
The compound can be combined with human recombinant p53 protein.
The specific method for binding the compound with the human recombinant p53 protein comprises the following steps:
step a) extracting a compound generated by Geobacillus sp.E263 infected by bacteriophage GVE2 to obtain a thermophilic bacteria crude compound;
step b) purification of p53 protein;
step c) obtaining recombinant His label p53 protein;
step d) obtaining the compound of the thermophilic bacteria crude body compound combined with the recombinant p53 protein.
The step a) is specifically as follows: geobacillus sp.E263 is cultured in TTMM (1 liter culture medium containing 4 g of peptone, 2 g of yeast extract, 1 g of sodium chloride and 1 g of magnesium chloride) culture medium at 60 ℃ for 24 hours, and then the bacterial liquid is centrifuged and the supernatant is discarded to obtain thalli; incubating the extract containing 80% methanol with the thallus for 24 hours, filtering to obtain filtrate, continuously extracting for three times, combining the three extracts, and filtering; finally evaporating the filtered extract liquor to be in a semisolid state by a rotary evaporator, and dissolving the extract liquor by dimethyl sulfoxide (DMSO) to obtain a thermophilic bacteria crude compound;
the step b) is specifically as follows: obtaining a human p53 gene from a human cell cDNA library through PCR amplification, obtaining a p53 gene fragment with a wild type and a mutant type of R280K through a point mutation method, connecting the p53 gene fragment to a prokaryotic expression vector pET 28a after double enzyme digestion, and transforming to a prokaryotic expression strain BL21DE3 after correct sequencing.
The step c) is specifically as follows: placing the prokaryotic expression strain BL21DE3 in the step b) in an LB culture medium, culturing at 37 ℃ until OD600 is about 0.6, adding IPTG (isopropyl thiogalactoside) to enable the final concentration of the prokaryotic expression strain in the culture solution to be 0.5mM, and then continuously culturing at 37 ℃ for 4 hours; centrifuging the cultured bacterial liquid to obtain bacterial cells containing p53 protein; resuspending the cells with Hepes buffer (pH 8.0) containing 20mM imidazole, then disrupting the cells by ultrasonication, centrifuging, collecting the supernatant, binding the supernatant to Ni-NTA agarose medium, incubating at 4 ℃ for 3 hours, washing the agarose medium with 50ml of p53 protein binding buffer, and eluting the medium with Hepes buffer with 300mM imidazole concentration to obtain recombinant His-tagged p53 protein;
the step d) is specifically as follows: incubating the thermophilic coarse compound obtained in the step a) and the recombinant protein obtained in the step c) at the temperature of 25 ℃ for 1 hour, passing through a sephadex G50 (sephadex G50) packing material, and collecting effluent of a protein peak under the wavelength of 280nm, wherein after the target protein p53 is combined with the thermophilic coarse compound, the structure of the target protein may be changed, so that the target protein flows out in the sephadex G50 gel, and a compound combined with the target protein can be obtained by monitoring the protein outflow peak; adding methanol solution with the final concentration of 80% into the effluent liquid for extraction, centrifuging after the extraction is finished to remove denatured protein, and performing rotary evaporation and concentration on supernate to obtain the compound combined with the recombinant p53 protein.
And identifying the obtained compound by using a GC-MS (gas chromatography-mass spectrometer), and comparing the compound with a standard mass spectrum database to obtain a compound structure combined with the recombinant p53 protein.
The invention has the beneficial effects that:
the invention relates to a method for screening a compound capable of being combined with mutant p53 protein by using a molecular sieve method, wherein the compound can inhibit the proliferation of cancer cells with p53 gene mutation in a nude mouse at an effective concentration of 10mg/mL, and has no obvious influence on immune cells of the mouse.
Description of the drawings:
FIG. 1 shows the prokaryotic expression of wild-type His-tagged and R280K-mutated p53 protein, the expression of which was analyzed by SDS-PAGE electrophoresis.
FIG. 2 shows the isolation of recombinant wild-type p53 protein from compound after incubation with molecular sieves, sephadex G50, and the absorbance at 280nm for GST wild-type protein alone and compound separately over molecular sieves.
FIG. 3 shows that recombinant R280K mutant p53 protein was isolated by molecular sieve sephadex G50 after incubation with compound, and recombinant mutant p53 protein and compound were separately passed through molecular sieve with absorbance at 280nm on the ordinate.
FIG. 4 shows that recombinant p53 protein was isolated by molecular sieve sephadex G50 after incubation with compound, and recombinant p53 protein and compound were separately passed through molecular sieve with absorbance at 280nm on the ordinate.
FIG. 5 is the structure of four derivative compounds capable of binding to the mutant R280Kp53 protein.
FIG. 6 is a synthetic pathway for three derivatives.
FIG. 7 is a graph showing the effect of four derivatives on the intracellular transcriptional activity of mutant p53 protein.
FIG. 8 is a graph of the effect of a compound of the present invention and corn oil on the body weight of mice after feeding the mice.
FIG. 9 shows the change in the number of white blood cells and red blood cells in mice fed with the compound of the present invention and corn oil, (a) shows the change in the number of white blood cells in mice, and (b) shows the change in the number of red blood cells in mice.
Detailed Description
The invention is further illustrated by the following figures and examples.
The compound for inhibiting the proliferation of human cancer cells in a nude mouse is 2- [ (4-hydroxyphenyl) amino ] phenol (2- [ (4-hydroxybenzyl) amino ] phenol, abbreviated as HBAP), the HBAP comprises two hydroxyl groups which can play an important role in the functions of the compound, and four derivatives of the HBAP are synthesized, namely, one of the two hydroxyl groups is respectively replaced by a chlorine atom (HBAP-1 and HBAP-2), the two hydroxyl groups are both replaced by chlorine atoms (HBAP-3) and the two hydroxyl groups are both converted into hydroxymethyl groups (HBAP-4), and the specific structural formula is shown in figure 5.
The synthetic pathways for the three derivatives are shown in figure 6.
FIG. 7 is a graph showing the effect of four derivatives on the intracellular transcriptional activity of mutant p53 protein. Breast cancer cells (MDA-MB-468) carrying mutant p53R273H were treated with 4 derivatives of HBAP, respectively, and the transcriptional activity of the mutant p53 protein was analyzed by a dual fluorescence reporter assay. The results of the experiments showed that the compound did not activate the transcriptional activity of mutant p53 protein when both hydroxyl groups were substituted with chlorine atom or methyl group, but did not significantly affect the activity of the compound when only one of the two hydroxyl groups was substituted with chlorine atom. These experimental results indicate that two hydroxyl groups of HBAP, which are the active sites of HBAP, play a crucial role in transcriptional activity of HBAP activation mutant p 53.
The compound is screened from deep-sea hydrothermal oral thermophiles by the following method:
a) extracting the compound produced by the thermophilic bacterium Geobacillus sp.E263 infected by the bacteriophage GVE2 to obtain the thermophilic bacterium crude compound. Geobacillus sp.E263 is cultured in TTMM (1 liter culture medium containing peptone 4 g, yeast extract 2 g, sodium chloride 1 g, magnesium chloride 1 g) at 60 ℃ for 24 hours, and then the bacterial liquid is centrifuged to discard the supernatant to obtain the thallus. The extract containing 80% methanol was incubated with the cells for 24 hours, and then the filtrate was filtered, extracted three times consecutively, and the three extracts were combined and then filtered. Finally, the filtered extract is evaporated to a semisolid state by a rotary evaporator and dissolved by DMSO (dimethyl sulfoxide) to obtain a thermophilic bacteria crude compound.
b) purifying p53 protein, obtaining human p53 gene from human cell cDNA library by PCR method, obtaining p53 gene fragment with wild type and mutant type R280K by point mutation method, connecting the gene to prokaryotic expression vector pET 28a after double enzyme digestion, and transforming into prokaryotic expression strain BLDE3 after sequencing is correct.
c) Culturing the prokaryotic expression strain BL21DE3 in the step b) in an LB culture medium at 37 ℃ until OD600 is about 0.6, adding IPTG (isopropyl thiogalactoside) to make the final concentration in the culture solution be 0.5mM, and then continuing culturing at 37 ℃ for 4 hours. The cultured cell suspension was centrifuged to obtain bacterial cells containing p53 protein. The cells were resuspended in Hepes buffer (pH 8.0) containing 20 mmole/l imidazole, disrupted by sonication, centrifuged, and the supernatant was bound to Ni-NTA agarose medium, incubated at 4 ℃ for 3 hours, the agarose medium was washed with 50ml of p53 protein binding buffer, and the medium was eluted with Hepes buffer at 300 mmole/l imidazole to give recombinant His-tagged p53 protein.
d) Incubating the compound obtained in the step a) and the recombinant protein obtained in the step c) at the temperature of 25 ℃ for 1 hour, passing through a sephadex G50 (sephadex G50) packing material, collecting effluent of a protein peak at the wavelength of 280nm, and after the target protein is combined with the target compound, possibly changing the structure of the target protein to enable the target protein to flow out in sephadex G50 gel, so that the target protein combined compound can be screened by monitoring the protein outflow peak. Adding methanol solution with the final concentration of 80% into the effluent liquid for extraction, centrifuging after the extraction is finished to remove denatured protein, and performing rotary evaporation and concentration on supernate to obtain the compound combined with the recombinant p53 protein. And identifying the obtained compound by using a GC-MS (gas chromatography-mass spectrometer), and comparing the compound with a standard mass spectrum database to obtain a compound structure combined with the recombinant p53 protein.
The experimental results are as follows:
wild type and mutant p53 recombinant bacteria are adopted to induce protein expression in a large quantity, and then the wild type and mutant p53 proteins are obtained by purifying with GST tag protein purification medium respectively, and the expression results of the proteins analyzed by SDS-PAGE electrophoresis are shown in figure 1.
To screen compounds from the compounds produced by the interaction of GVE2 with Geobacillus sp.E263 for specific binding to mutant p53 protein, Geobacillus sp.E263 was infected with GVE2, followed by extraction of the compounds, incubation of the extracted compounds with purified recombinant wild-type p53 protein and mutant p53 protein, respectively, followed by passing the incubations through molecular sieves (size exclusion) and determining the OD of the effluent280(absorbance of protein at 280 nm) Compounds that bind to p53 protein were screened as shown in FIG. 2. After incubation of GST with the extracted compound, the time for its efflux product through the molecular sieve was the same as the time for GST-pure protein to pass through the molecular sieve, indicating that there was no compound that bound to the GST protein in the compound produced by interaction of GVE2 with Geobacillus sp.ej263.
After the recombinant R280K mutant p53 protein is incubated with a compound, the compound is separated by a molecular sieve sephadexG50, and the recombinant mutant p53 protein is neutralizedThe compounds were separately passed through a molecular sieve with the ordinate showing the absorbance at 280nm as shown in FIG. 3. Fusion protein GST-p53R280KAfter incubation with the extracted compound, the time of efflux through the molecular sieves is comparable to that of the pure fusion protein GST-p53R280KThe time of molecular sieve is obviously advanced, which indicates that among the compounds generated by the interaction of GVE2 and Geobacillus sp.E263, the compound is combined with mutant p53 protein (p 53)R280KProtein).
After the recombinant p53 protein and the compound are incubated, the protein and the compound are separated by a molecular sieve sephadex G50, and the recombinant p53 protein and the compound are respectively separated by the molecular sieve, wherein the ordinate of the absorbance value at 280nm is shown in figure 4. After incubation of the GST-p53 fusion protein with the extracted compound, the efflux time through the molecular sieve was the same as the time for GST-p53 fusion protein to pass through the molecular sieve, indicating that no compound binds to the wild-type p53 protein.
The anti-tumor capability and toxicological experiment method of the derivative is as follows:
one or both of the hydroxyl groups were replaced, respectively, and the effect of each derivative on the transcriptional activity of the mutated p53 gene in breast cancer cells was then examined.
10 mice weighing about 20 g were divided into two groups, and then the mice were fed with 2mg of each compound for 14 days continuously, the change in the weight of the mice was monitored every day during the feeding period, and the change in the number of blood cells of the mice was detected after the feeding was completed.
As shown in fig. 8, the body weights of the mice in the experimental and control groups did not change significantly after two weeks of continuous feeding, thus indicating that the compound had no toxic side effects on the mice at low doses.
As shown in FIG. 9, the number of leukocytes in mice was maintained at 10 for both HBAP compound-fed and corn oil-fed control groups3Mu.l, the number of erythrocytes maintained at 106Normal levels of/. mu.l, and therefore no significant toxic side effects on the immune system of the mice.

Claims (9)

1. A compound for inhibiting the proliferation of human cancer cells in a nude mouse, comprising: the compound is 2- [ (4-hydroxyphenyl) amino ] phenol, is a benzoquinone compound, and is obtained by screening a deep-sea hydrothermal oral thermophile strain.
2. The compound of claim 1, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the compound can change the conformation of the p53 protein after being combined with the p53 protein, and the p53 protein flows out before being combined with the unbound p53 protein through sephadex G50 screening.
3. The compound of claim 1, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: derivatives of the compounds HBAP1, HBAP2, HBAP3 and HBAP4 can inhibit the growth of tumor cells with p53 gene mutation; after the hydroxyl groups in the derivatives of the compounds have been replaced, the growth inhibitory effect on tumor cells is lost.
4. The compound of claim 1, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the compound can be combined with human recombinant p53 protein.
5. The compound of claim 1, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the specific method for binding the compound with the human recombinant p53 protein comprises the following steps:
step a) extracting a compound generated by Geobacillus sp.E263 infected by bacteriophage GVE2 to obtain a thermophilic bacteria crude compound;
step b) purification of p53 protein;
step c) obtaining recombinant His label p53 protein;
step d) obtaining the compound of the thermophilic bacteria crude body compound combined with the recombinant p53 protein.
6. The compound of claim 5, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the step a) is specifically as follows: geobacillus sp.E263 is cultured in a TTMM medium at 60 ℃ for 24 hours, and then the bacterial liquid is centrifuged and the supernatant is discarded to obtain thalli; incubating the extract containing 80% methanol with the thallus for 24 hours, filtering to obtain filtrate, continuously extracting for three times, combining the three extracts, and filtering; finally, the filtered extract is evaporated to a semisolid state by a rotary evaporator and dissolved by dimethyl sulfoxide (DMSO) to obtain a thermophilic bacteria crude compound.
7. The compound of claim 5, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the step b) is specifically as follows: obtaining a human p53 gene from a human cell cDNA library through PCR amplification, obtaining a p53 gene fragment with a wild type and a mutant type of R280K through a point mutation method, connecting the p53 gene fragment to a prokaryotic expression vector pET 28a after double enzyme digestion, and transforming to a prokaryotic expression strain BL21DE3 after correct sequencing.
8. The compound of claim 5, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the step c) is specifically as follows: placing the prokaryotic expression strain BL21DE3 in the step b) in an LB culture medium, culturing at 37 ℃ until OD600 is about 0.6, adding IPTG (isopropyl thiogalactoside) to enable the final concentration of the prokaryotic expression strain in the culture solution to be 0.5mM, and then continuously culturing at 37 ℃ for 4 hours; centrifuging the cultured bacterial liquid to obtain bacterial cells containing p53 protein; the cells were resuspended in Hepes buffer (pH 8.0) containing 20mM imidazole, disrupted by sonication, centrifuged, and the supernatant was bound to Ni-NTA agarose medium, incubated at 4 ℃ for 3 hours, the agarose medium was washed with 50ml of p53 protein binding buffer, and the medium was eluted with Hepes buffer at 300mM imidazole concentration to give recombinant His-tagged p53 protein.
9. The compound of claim 5, wherein the compound inhibits the proliferation of human cancer cells in a nude mouse, and wherein: the step d) is specifically as follows: incubating the thermophilic bacteria crude compound obtained in the step a) and the recombinant protein obtained in the step c) for 1 hour at the temperature of 25 ℃, passing through a sephadex G50 (sephadex G50) packing material, and collecting the effluent of a protein peak under the wavelength of 280nm, wherein after the target protein p53 is combined with the thermophilic bacteria crude compound, the structure of the target protein is changed, so that the target protein flows out in the sephadex G50 gel, and the target protein combined compound can be obtained by screening through monitoring the protein outflow peak; adding methanol solution with the final concentration of 80% into the effluent liquid for extraction, centrifuging after the extraction is finished to remove denatured protein, and performing rotary evaporation and concentration on supernate to obtain the compound combined with the recombinant p53 protein.
CN202011171491.8A 2020-10-28 2020-10-28 Compound for inhibiting proliferation of human cancer cells in nude mice Withdrawn CN112159327A (en)

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