CN109771415B - Small molecule inhibitor containing benzodioxole and application of small molecule inhibitor in inhibition of Ornithine Decarboxylase (ODC) - Google Patents
Small molecule inhibitor containing benzodioxole and application of small molecule inhibitor in inhibition of Ornithine Decarboxylase (ODC) Download PDFInfo
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Abstract
The invention provides a small molecule inhibitor containing benzodioxole, which is N' - ({ 6-nitro-1, 3-benzodioxol-5-yl } methylene) spiro [2.3]Hexane-1-carbohydrazide of the formula:
the application of the benzodioxole-containing small-molecule inhibitor in inhibiting Ornithine Decarboxylase (ODC), the application in preparing a medicament for treating tumors and the application in preparing a medicament for treating pathogenic microorganism infection have obvious effects.
Description
Technical Field
The invention relates to a small molecule inhibitor of Ornithine Decarboxylase (ODC) and application thereof, in particular to a small molecule inhibitor of human Ornithine decarboxylase and application thereof in inhibiting and killing tumor cells.
Background
Proteins are one of the major components of living organisms and are the main substances that perform various vital activities. Among various proteins, proteases are essential for life activities, and almost all biochemical reaction processes in organisms are catalyzed by proteases. The activity of various proteases in organisms has strict regulation mechanism, and once the regulation mechanism has problems, the corresponding diseases can be caused if the activity of the proteases is too high, too low or completely inactivated. Therefore, the regulation and control of the activity of the protease by the medicament to recover and maintain the protease at a normal level have very important theoretical significance and practical significance. The structure-based drug design is a very important means for designing a protein-targeted drug.
Polyamines (polyamines) are positively charged cationic small molecules produced from amino acid metabolism, which are present in all organisms and essential for cell growth, differentiation, survival and normal biological functions. The multiple positive charge nature of polyamines allows them to regulate a very wide range of biological processes including chromosome structure formation, DNA synthesis and stabilization, DNA replication, transcription and translation, protein phosphorylation, ribosome production, regulation of ion channels and membrane surface receptors, free radical scavenging, etc. by electrostatic interaction with negatively charged biological macromolecules (DNA, RNA, proteins, cell membranes, etc.). There are many natural polyamines. In mammals, there are three naturally occurring species, putrescine (putrescine), spermidine (speramine), spermine (speramine), which are essential for the normal growth and development of mammals. Since polyamines have important biological functions, their intracellular levels are tightly regulated. In rapidly proliferating cells, such as tumor cells, polyamine levels and ODC expression levels can also rise and become deregulated. The increase of polyamine level is accompanied by the acceleration of cell proliferation, the decrease of apoptosis, the increase of expression level of tumor infiltration and metastasis related genes and the like. Therefore, the regulation and control of polyamine become an important means in tumor treatment and drug development.
The initial substrate for polyamine metabolism is ornithine (ornithine), which is the reaction product of arginine catalyzed by arginase (arginase) in the urea cycle (urea cycle). ODC is the first enzyme in the polyamine synthesis pathway, catalyzing the reaction from ornithine (ornithline) to putrescine, a step which is also a rate limiting step in the polyamine synthesis pathway. Therefore, synthesizing ODC inhibitor, inhibiting putrescine generation, is a tumor treatment approach which is very much concerned at present. Also, because pathogenic microorganisms also require normal polyamine levels, ODC inhibitors have also become important targets for pathogenic microorganisms (e.g., trypanosoma brucei causing africana trypanosomiasis).
Currently, the ODC inhibitor DFMO (α -difluoromethylornithine) has been used clinically to assist cancer chemotherapy. But the binding capacity of the inhibitor and ODC is weak, the action concentration is high, and the toxic and side effects are very large due to the suicide inhibitor forming covalent bonds with ODC. Therefore, there is a great need to develop novel ODC inhibitors having better effects.
Disclosure of Invention
The invention aims to provide a novel small molecule inhibitor aiming at ODC through computer-assisted high-throughput drug screening, which is applied to an ornithine decarboxylase inhibitor and can be possibly used for preparing drugs for treating tumors and pathogenic microorganism infection. The method specifically comprises the following steps:
a micromolecule inhibitor containing benzodioxole has a structural formula as follows:
the benzodioxole-containing small molecule inhibitor is applied to inhibiting Ornithine Decarboxylase (ODC).
The application of the benzodioxole-containing small-molecule inhibitor in preparing the medicine for treating tumor is adopted.
The method for using the benzodioxole-containing small molecule inhibitor for inhibiting Ornithine Decarboxylase (ODC) comprises the following steps:
1) construction of ODC prokaryotic expression plasmid
Inserting the gene sequence of ODC into pET28a plasmid through BamH I and Xho I enzyme cutting sites to construct pET28a-hODC plasmid, and carrying out DNA sequencing verification;
2) expression of ODC protein
Passing the pET28a-hODC plasmid constructed in the step 1) through CaCl2A method of transforming into E.coli BL21 strain, screening by kanamycin, then inoculating the strain grown on Luria-Bertani (LB) plate containing kanamycin into LB liquid medium containing kanamycin, culturing at 37 ℃ and 250rpm to logarithmic growth phase, further adding isopropyl thiogalactoside (IPTG) to 0.5mM, inducing expression at 28 ℃ for 4 hours, and finally, centrifuging to collect the bacteria;
3) purification of ODC proteins
Resuspending the bacteria collected in the step 2) by using a lysate, then carrying out cell lysis by an ultrasonic method, centrifuging the lysate at 4 ℃ at 12000 r/min, and then keeping a supernatant; finally, the supernatant is combined and purified by using Ni-NTA His label protein combined filler to obtain human ODC protein, and the elution buffer solution of the ODC protein is 50mM Tris/HCl, pH8.0, 300mM NaCl,1mM DTT and 100mM imidazole (imidazole);
4) activity assay of ODC proteins
Adding 400uL of substrate reaction mixture and 50ug of ODC protein into an EP tube, uniformly mixing, and placing the EP tube in a water bath at 37 ℃ for 30 min; adding 400uL 10% trichloroacetic acid to terminate the reaction, centrifuging at room temperature for 5min at 5000rpm, then taking out 100uL of supernatant, mixing with 200uL 4mol/L NaOH solution, adding 400uL n-amyl alcohol, fully mixing, centrifuging at 2000rpm for 5min, then transferring 200uL of supernatant into a new EP tube, adding 200uL0.1mol/LpH sodium tetraborate of 8.0, uniformly mixing, adding 200uL10mmol/L trinitrobenzene sulfonic acid, fully mixing, adding 400uL DMSO, fully mixing for 1min, and centrifuging at 3000rpm for 5 min; and finally, taking out the supernatant to a 96-well plate, and detecting the light absorption value at 426nm by using an enzyme-labeling instrument to obtain the OD value without adding the enzyme.
5) Detection of inhibitory Activity of inhibitor on ODC protein
According to the method described in step 4), after adding 400uL of substrate reaction mixture, a small molecule inhibitor of ornithine decarboxylase was added immediately, and the subsequent operation was the same as in step 4);
ODC inhibition was calculated by the following formula:
the average OD value of the control group added with the small molecule inhibitor-the average OD value of the control group not added with the small molecule inhibitor, wherein the inhibitor added in the step 5) of the control group is the DFMO inhibitor;
the experimental difference is the average OD value of the experimental group added with the small-molecule inhibitor-the average OD value of the experimental group not added with the small-molecule inhibitor;
ODC inhibition rate ═ [ (control difference-experimental difference)/control difference ] × 100%.
The lysis solution in the step 3) is a mixed solution of 50mM Tris/HCl, pH8.0, 300mM NaCl,1mM DTT,1mM PMSF and 5mM imidazole (imidazole).
The substrate reaction mixture in step 4) was prepared by dissolving 17.57ul β -mercaptoethanol, 55.84mg 1.5mM disodium EDTA dihydrochloride, 75nM stock of pyridoxal phosphate (PLP), 2mM ornithine hydrochloride in 150mM Phosphate Buffered Saline (PBS) pH 7.1.
The ornithine decarboxylase is human ornithine decarboxylase, non-human ornithine decarboxylase or a protein highly homologous to putrescine substrate and pyridoxal phosphate binding site of the human ornithine decarboxylase.
According to the scheme, firstly, a Pocket protein drug Pocket analysis software is utilized, the putrescine substrate and the PLP ligand binding Pocket of the human ODC are analyzed on the basis of the crystal structure of the human ODC, and a theoretical model of the protein Pocket is generated. Then, using a protein docking program DOCK to DOCK 19 ten thousand small molecules in the SPECS small molecule drug library to the protein pocket model, and screening out small molecules which at least contain 15 non-hydrogen heavy atoms, at least form 2 hydrogen bonds, at least 1 hydrophobic center and have a docking score of not more than-10. Then, the small molecules are further butted into the protein pocket in sequence by utilizing a protein-small molecule butting program Autodock again, further butting calculation is carried out, and finally, the small molecules with the butting score lower than-7 are selected.
The invention also provides a composition, which contains an effective amount of the small molecule inhibitor or the analogue thereof for inhibiting the ornithine decarboxylase and a pharmaceutically applicable carrier. Preferably, the composition is a pharmaceutical composition comprising a therapeutically effective amount of a small molecule inhibitor or analog thereof provided by the present invention and a pharmaceutically acceptable carrier. More preferably the pharmaceutical composition is a pharmaceutical composition for the treatment or prevention of a disease responsive to inhibition of Ornithine Decarboxylase (ODC), preferably human Ornithine Decarboxylase (ODC); also comprises a small molecule inhibitor or an analogue thereof which contains an effective amount for preventing and treating diseases responding to the inhibition of the ornithine decarboxylase and a pharmaceutically applicable carrier.
The small molecule inhibitors or analogs thereof of the invention and the compositions described above are useful for inhibiting Ornithine Decarboxylase (ODC), preferably human Ornithine Decarboxylase (ODC), wherein the inhibition is therapeutic or non-therapeutic. Preferably, the small molecule inhibitor or the analogue thereof of the present invention is used for preparing a medicament for inhibiting Ornithine Decarboxylase (ODC), preferably human-derived Ornithine Decarboxylase (ODC). The invention therefore also provides a method of inhibiting ornithine decarboxylase activity comprising administering to a subject in need of ornithine decarboxylase inhibition (ODC) an effective amount of a small molecule inhibitor of the invention or an analog thereof or a composition as described above, wherein said inhibition is of therapeutic or non-therapeutic interest.
The invention also provides a method of treating a disease responsive to inhibition of ornithine decarboxylase comprising administering to a subject in need of such treatment or prevention a prophylactically or therapeutically effective amount of a small molecule inhibitor of the invention, or an analog thereof, that inhibits ornithine decarboxylase, preferably human Ornithine Decarboxylase (ODC), or a composition described above.
The small molecule inhibitor or the analogue thereof of the invention can be used for preparing a medicament or a pharmaceutical composition for treating diseases responding to the inhibition of ornithine decarboxylase, wherein the Ornithine Decarboxylase (ODC) or the analogue thereof inhibits the activity of the ornithine decarboxylase, and the diseases comprise tumors or the infection of pathogenic microorganisms, preferably the tumors. Protozoal infections are neoplastic diseases or diseases infected by pathogenic microorganisms in response to inhibition of ornithine decarboxylase.
Drawings
FIG. 1 is a graph showing the inhibitory effect of the small molecule inhibitor of example 1 on human ornithine decarboxylase.
FIG. 2 shows the effect of the inhibitor on the killing of tumor cells measured by MTT method in example 1.
Detailed Description
Example 1
The related small molecule inhibitors are: n' - ({ 6-nitro-1, 3-benzodioxol-5-yl } methylene) spiro [2.3] hexane-1-carbohydrazide, the formula is shown in the figure.
The activity detection method comprises the following steps:
1. construction of human ODC prokaryotic expression plasmid
The gene sequence of the human ODC is inserted into pET28a plasmid through BamH I and Xho I restriction enzyme sites to construct pET28a-hODC plasmid, and DNA sequencing verifies.
Gene sequence of human ODC:
atgaacaactttggtaatgaagagtttgactgccacttcctcgatgaaggttttactgccaaggacattctggaccagaaaattaatgaagtttcttcttctgatgataaggatgccttctatgtggcagacctgggagacattctaaagaaacatctgaggtggttaaaagctctccctcgtgtcacccccttttatgcagtcaaatgtaatgatagcaaagccatcgtgaagacccttgctgctaccgggacaggatttgactgtgctagcaagactgaaatacagttggtgcagagtctgggggtgcctccagagaggattatctatgcaaatccttgtaaacaagtatctcaaattaagtatgctgctaataatggagtccagatgatgacttttgatagtgaagttgagttgatgaaagttgccagagcacatcccaaagcaaagttggttttgcggattgccactgatgattccaaagcagtctgtcgtctcagtgtgaaattcggtgccacgctcagaaccagcaggctccttttggaacgggcgaaagagctaaatatcgatgttgttggtgtcagcttccatgtaggaagcggctgtaccgatcctgagaccttcgtgcaggcaatctctgatgcccgctgtgtttttgacatgggggctgaggttggtttcagcatgtatctgcttgatattggcggtggctttcctggatctgaggatgtgaaacttaaatttgaagagatcaccggcgtaatcaacccagcgttggacaaatactttccgtcagactctggagtgagaatcatagctgagcccggcagatactatgttgcatcagctttcacgcttgcagttaatatcattgccaagaaaattgtattaaaggaacagacgggctctgatgacgaagatgagtcgagtgagcagacctttatgtattatgtgaatgatggcgtctatggatcatttaattgcatactctatgaccacgcacatgtaaagccccttctgcaaaagagacctaaaccagatgagaagtattattcatccagcatatggggaccaacatgtgatggcctcgatcggattgttgagcgctgtgacctgcctgaaatgcatgtgggtgattggatgctctttgaaaacatgggcgcttacactgttgctgctgcctctacgttcaatggcttccagaggccgacgatctactatgtgatgtcagggcctgcgtggcaactcatgcagcaattccagaaccccgacttcccacccgaagtagaggaacaggatgccagcaccctgcctgtgtcttgtgcctgggagagtgggatgaaacgccacagagcagcctgtgcttcggctagtattaatgtgtag
we used the above-mentioned human ODC sequence with one base change from the sequence in the database (http:// www.ncbi.nlm.nih.gov/nuccore/NM-002539.1) (box labeled C is T in the database sequence and the corresponding amino acid is changed from arginine to cysteine), but without affecting its activity.
2. Expression of human ODC protein
The pET28a-hODC plasmid was put through CaCl2The method was carried out by transforming into E.coli strain BL21 and screening with kanamycin. The strain grown on Luria-Bertani (LB) plate containing kanamycin was inoculated into LB liquid medium containing kanamycin, cultured at 37 ℃ at 250rpm to logarithmic growth phase, then IPTG (isopropyl thiogalactoside) was added to 0.5mM, and expression was induced at 28 ℃ for 4 hours. Finally, the bacteria were collected by centrifugation.
3. Purification of human ODC protein
The bacteria collected above were resuspended in a lysis solution (50mM Tris/HCl, pH8.0,300 mM NaCl,1mM DTT,1mM PMSF, 5mM Imidazole.), and then subjected to cell lysis by sonication. The lysate was then centrifuged at 12000 rpm at 4 ℃ and the supernatant was retained. The supernatant was bound and purified using Ni-NTA His-tagged protein binding filler, elution buffer for human ODC protein 50mM Tris/HCl, pH8.0,300 mM NaCl,1mM DTT,100mM imidazole.
4. Activity detection of human ODC protein
In a 1.5mL EP tube, 400uL of substrate reaction mixture (17.57 uL β -mercaptoethanol dissolved in 150mM PBS (pH 7.1), 55.84mg 1.5mM EDTA dibasic sodium, 75nM PLP stock, 2mM ornithine hydrochloride) was added. Then, 50ug of ODC protein was added. After mixing, the EP tube was placed in a 37 ℃ water bath for 30 min. The reaction was then stopped by adding 400uL of 10% trichloroacetic acid. Centrifuge at 5000rpm for 5min at room temperature. 100uL of the supernatant was taken out, mixed with 200uL of 4mol/L NaOH solution, and then added with 400uL of n-pentanol, and mixed well. After centrifugation at 2000rpm for 5min, 200uL of the supernatant was transferred to a new EP tube, and 200uL of sodium tetraborate (0.1mol/L, pH8.0) was added and mixed thoroughly. Then 200uL of trinitrobenzenesulfonic acid (10mmol/L) was added and mixed well. Then 400uL of DMSO was added and mixed well for 1 min. Centrifuge at 3000rpm for 5 min. And taking out supernatant liquid to a 96-well plate, and detecting the light absorption value at 426nm by using a microplate reader.
5. Detection of inhibitory Activity of inhibitor against human ODC protein
In the activity assay procedure described above, 400uL of substrate reaction mixture was added followed by addition of the small molecule drug and mixing. The same applies to the subsequent steps.
ODC inhibition rate was calculated by the following formula
Mean OD value of enzyme-added control group-mean OD value of enzyme-not-added control group
Mean OD value of enzyme-added experimental group-mean OD value of enzyme-not-added experimental group
Inhibition rate ═ control difference-experimental difference)/control difference × 100%
The inhibitory effect of the small molecule inhibitor obtained by the above method is shown in FIG. 1, and it can be seen from the figure that the drug inhibits the ODC activity at a concentration of 50% or more, and the inhibitory activity thereof is equivalent to 2.5mM DFMO.
6. MTT method is adopted to detect killing effect of inhibitor on tumor cells
MTT method is adopted to detect killing effect of inhibitor on tumor cells
A549 cells were seeded at a density of 2000 cells/well in 96-well cell culture plates in RPMI-1640, 10% calf serum, 1% chain/penicillin, volume 100 uL/well. After 24 hours of incubation at 37 ℃ in a cell culture chamber, 100uL of small molecule drug or medium diluted with RPMI-1640 was added. After further culturing for 60 hours, the medium was aspirated, and after adding 100uL of MTT (final concentration: 250ug/mL), the culture was continued for 4 hours. The culture was then aspirated off, 150uL DMSO was added, and the mixture was shaken on a shaker at low speed for 15min to dissolve the crystals sufficiently. Finally, the absorbance at 490nm was read using a microplate reader.
The killing effect of the inhibitor on tumor cells measured by the MTT method obtained as described above is shown in FIG. 2, and it can be seen from the graph that the drug is capable of inhibiting and killing tumor cells at a concentration of 50% or more of inhibition, but the drug is weak at a concentration of 50% or less of inhibition.
Claims (1)
1. The application of a benzodioxole-containing small molecule inhibitor in preparing a medicament for treating A549 tumor responding to the inhibition of ornithine decarboxylase ODC is disclosed, wherein the small molecule inhibitor has the structural formula:
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