Study On The Mechanism Of Action And Antitumor Activity Of Thiazole Orange Derivatives As G-quadruplex DNA Probes

Nucleic acids are important genetic material in living organisms and play many important roles in life activities.In addition to double-helical DNA structures,nucleic acids also have various advanced structures such as triple-stranded,crossed-cross-stranded,G-quadruplexes and other atypical structures.These structures often have certain unique functions in the regulation of cellular activity,but most of the biological mechanisms associated with such atypical nucleic acid structures are not fully understood.In vivo real-time identification of any higher order DNA structures,such as G-quadruplex DNA,by monitoring their structural dynamics and studying their biological activity in living cells is a challenging task.The presence of a G-quadruplex forming sequence at the end of human telomeres and the formation of a stable telomeric G-quadruplex structure leads to reduced telomerase activity,which is highly expressed in tumor cells and is the main regulatory protein for their immortalization.The c-MYC oncoprotein is involved in the regulation of many physiological processes such as cell cycle and apoptosis.The c-MYC G-quadruplex DNA found in its promoter region can control the expression of c-MYC oncoprotein,which is expected to become a potential drug target.Currently,There are numerous ligands developed for these two G-quadruplex structures,and some progress has been made in the research of antitumor cell proliferation and regulatory mechanism.However,these ligands still have some disadvantages,such as insufficient specificity,poor medicinal properties,poor membrane permeability,insufficient uptake capacity and unable to trace G-quadruplex DNA in real time.Based on this background,this thesis has developed a series of thiazole orange derivatives and explored the interactions between thiazole orange derivatives and Telomere / c-MYC G-quadruplex structure and the related antitumour mechanism in detail from spectroscopy,biochemistry and molecular biology.The research for this thesis includes:(1)The fluorescence recognition properties of thiazole orange derivatives A1 to A10 and B1 to B4 for various nucleic acids were studied.(2)The binding ability of A3,B2 and B3 to various nucleic acids using CD spectroscopy,CD melting point,UV spectroscopy and isothermal calorimetric titration.(3)Study of the binding pattern of A3 to telomeric G-quadruplex DNA using 1H MNR titration and fluorescence changes of single point mutant nucleic acids.(4)The ability of A3 to trap and stabilize telomeric G-quadruplex DNA has been studied in vitro and in cells.(5)Study of the toxic effects of A3 on various cell types using the MTT method.(6)Using q RT-PCR to study the effect of A3 on the transcript levels of telomerase related genes.(7)Investigating the effects of A3 on telomerase activity,telomere length and senescence in cancer cells.(8)Binding patterns of B2 and B3 to telomeric / c-MYC G-quadruplex DNA using 1H MNR titration and computer molecular simulation.(9)Study of the ability of B2 and B3 to selectively fluorescently image G-quadruplex DNA in cells.(10)Study of the toxic effects of B2 and B3 on various cell types using the MTT method.(11)Using q RT-PCR to study the effect of B2 and B3 on the transcript levels of c-MYC and telomerase-related genes in cancer cells.(12)Study of the effects of B2 and B3 on the expression of c-MYC,telomerase,apoptotic pathway and DNA damage pathway related proteins using Western Blot technique.(13)The effect of B2 and B3 on the ability to stabilize the structure of the c-MYC G-quadruplex and the transcription of the c-MYC gene was investigated in exon-specific experiments.(14)Tumour activity inhibition by B2 and B3 studied in mice.(15)The blood drug metabolism of B2 and B3 in animals was investigated by LS-MS.(16)Studying the ability of B3 to target and tracer tumours in animals.The results show that derivative A3 has a high specificity and excellent binding ability to telomeric G-quadruplex DNA,interacting with it mainly through ?-?-terminal stacking and lateral loops near the 5' end.A3 has good fluorescence stability,can specifically recognize and image telomere G-quadruplex DNA in living cells,can down regulate the transcription of h TERC and h TERT and other genes that play a key role in regulating telomerase activity,so as to hinder telomere function and reduce telomerase activity in cancer cells,and induce cancer cell aging and apoptosis.When evaluating the anticancer activity of ligands,A3 has significantly higher toxicity to a variety of cancer cells than normal cells.In addition,the results show that the structural difference between derivatives B2 and B3(substitution of dimethylamino group to diphenylamine group)will lead to the selectivity for different G-quadruplexes,in which B2 is selective for telomere G-quadruplexes and B3 is selective for c-MYC G-quadruplexes.Further study found that aside from the fact that B3 interacts with the side-loop hydrogen bond near the 5' end of the G-quadruplex through the same ?-? stacking interaction as B2,the non-polar diphenylamine substituent at the end of the B3 structure may play a key role in the selective recognition of the c-MYC G-quadruplex DNA structure.Both ligands have specific fluorescent recognition signals for G-quadruplex DNA in living cells.In addition,B2 and B3 have high oncotoxicity and both downregulate the expression of c-MYC,h TERT and h TERC genes in cancer cells,with B2 having the most significant downregulation of h TERT expression.In contrast,B3 had the most significant down-regulation effect on c-MYC expression and a relatively weak effect on h TERT expression.B2 and B3 are able to induce acute cellular senescence and apoptosis in cancer cells,possibly due to inhibition of c-MYC expression and telomerase activity,leading to telomere dysfunction,induction of DNA damage accumulation and proliferation arrest in cancer cells.Exon-specific assays verified that B3 represses c-MYC gene transcription by stabilizing c-MYC G-quadruplex DNA.Animal experiments show that the antitumour activity of B3 was superior to that of B2,and pharmacokinetic studies showed a better retention effect of B3 in animals,this may be an important reason why B3 has better ability to inhibit tumor growth.Taken together,these results may provide meaningful insights into the design of anticancer drugs that target G-quadruplex structures with high specificity.