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Establishment Of A Screening Method For Inhibitors To The Nucleic Acid Cytosine Deaminase APOBEC3B And Its Application

Posted on:2022-11-11Degree:MasterType:Thesis
Country:ChinaCandidate:Y H ZhangFull Text:PDF
GTID:2480306782953159Subject:Organic Chemical Industry
Abstract/Summary:PDF Full Text Request
Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like(APOBEC)DNA cytidine deaminases have nucleic acid editing activity and edit exogenous nucleic acid,which convert cytosine to uracil in pathogen-related single-stranded DNAs.A lethal G-A hypermutation was introduced into the synthetic viral coding strand DNA,which inhibited viral gene infection.At the same time,aberrant activation of APOBEC enzymes in tumour cells leads to hypermutations that drive heterogeneity and clonal evolution,which results in tumour progression and treatment adaptation.Some APOBEC3(A3)family members(mainly those able to enter the nucleus)have been reported to be responsible for DNA damage and mutagenesis,while APOBEC3B(A3B)is the only family member that is constitutively localized to the nucleus.Therefore,the high expression of A3B is the main reason for the drug resistance of various tumors and the poor clinical prognosis of cancer treatment.It is expected to become a potential drug target to combat drug-resistant tumor.Li et al.first proposed a biosensor for detecting the enzymatic activity of A3 based on the fluorescence resonance energy transfer(FRET)effect and the involvement of uracil DNA glycosylase(UDG).The biosensor can sensitively and stably detect the enzymatic activity of APOBEC3.It is able to screen out effective AP3 inhibitors and has been used in high-throughput screening.However,due to the participation of UDG enzyme in this biosensor,additional counter assay is required to exclude the interference of UDG enzyme inhibitor.In addition,the strong reaction conditions required by this biosensor will increase the probability of screening false positive inhibitors.Therefore,we hope to improve the A3activity detection biosensor to avoid false positive results.Since there are few reports on A3B activity inhibitors,we will further use the newly developed sensing system to find effective A3B small molecule inhibitors,so as to provide a valuable reference for the design and structural modification of A3B activity inhibitors.The main research contents of this thesis are:(1)Construction of recombinant A3BCTDprotein expression strain then induction,expression and purification of A3BCTD in vitro;(2)Design of a biosensor for detecting A3 activity based on FRET effect and restriction endonuclease Bsp H?;(3)TBE-PAGE to verify the feasibility of the biosensor and optimize the detection conditions of the biosensor;(4)To explore the application of the biosensor,including the detection of intracellular APOBEC deaminase content,high-throughput screening and activity evaluation of A3B inhibitors;(5)To explore the potential of a series of flavonoids to become A3B inhibitors;(6)To explore the specific binding mode of small molecule inhibitors to A3BCTD by molecular docking;(7)Verification of the binding site and binding ability of the small molecule inhibitor to A3BCTD by gene site-directed mutagenesis and isothermal calorimetric titration.The results showed that(1)we successfully established a recombinant A3BCTD protein expression strain and obtained purified A3BCTD protein.The product was verified as A3BCTDprotein with deamination activity by SDS-PAGE,Western blot and TBE-PAGE.(2)The designed sensor for A3 activity detection based on FRET effect and Bsp H?was confirmed to be feasible by TBE-PAGE.During the optimization of the detection conditions,it was found that he buffer contains 50 m M potassium ions and 10 m M magnesium ions provided the best performance on the detection of A3BCTD activity with the widest linear range(0-1100 n M),good stability and low limit od detection(14 n M).(3)In terms of application of the biosensor,the results show that the biosensor we established can be used to detect the content of APOBEC deaminase in cell lysates,and can be combined with laboratory automation workstations to achieve high-throughput screening of A3B inhibitors.(4)A series of flavonoids in our laboratory were screened,and it was found that icariin I and phloretin had A3B inhibitory activity,with IC50 values of 32.9?M and 7.4?M for A3BCTD,respectively.(5)The results of molecular modelling showed that these small molecules can stably bind in the catalytic cleft of A3BCTD and the binding sites were further confirmed by gene site-directed mutagenesis and isothermal calorimetry experiments.In addition,we also carried out a structure-activity analysis on the results obtained in this thesis to provide a reference for the follow-up work.
Keywords/Search Tags:APOBEC3B, biosensors, inhibitor screening, anti-tumor
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