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Design and Synthesis of G-quadruplex DNA Binding Ligands Using Click Chemistry

Chauhan, Ajay (2016) Design and Synthesis of G-quadruplex DNA Binding Ligands Using Click Chemistry. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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    Abstract

    Genomic DNA is most often regarded as a double stranded molecule in which the two self-complementary DNA strands are held together by Watson–Crick base pairing. In this work, we have used Click Chemistry to develop prolinamide derivatives (Chapter 1) and binapthyl amines (Chapter 2) as novel G-quadruplex binding ligands. Two new bis-prolinamide derivatives containing pyridine and benzene dicarboxamide central units were prepared using ‘Click’ chemistry. Pyridine analogue was found to be a more promising c-KIT1 G-quadruplex binding ligand than the benzene analogue, suggesting that Hbonding plays a key role in controlling the conformation of the pyridyl ligand for the selective recognition. FRET melting analysis showed high selectivity of the pyridyl ligand for quadruplexes over duplex DNA. The FID assay revealed that the pyridyl ligand binds with high affinity to c-KIT1 and displays moderate to high c-KIT1 specificity over c-MYC, h-TELO and c- KIT2 quadruplexes. In addition, the pyridyl ligand effectively inhibited the cell growth and induced cell death of HepG2 cancer cells. These results demonstrate that conformationally constrained peptidomimetic ligands can be engineered for achieving high selectivity for a particular G-quadruplex. Further chemical modifications in the scaffold has been described in Chapter 1B and 1C for developing more selective G-quadruplex binding ligands. The interaction of prolinamide derivatives ligands Pro-3 and Pro-4 with duplex and quadruplex DNA sequences has been investigated. These studies demonstrate that ligands can selectively bind and stabilize G-quadruplex structures. Ligand Pro-4 containing paraprolinamide units is found to be the most promising c-MYC G-quadruplex binding ligand then meta analogue. Furthermore, it is interesting to mention that by simply substituting the amine containing side chains at the 4th position of the pyridine ring the selectivity of these prolinamides for quadruplxes could be modulated, while ligand Pro-2 (Chapter 1 A) shows selectivity for c- KIT1 quadruplex the ligand Pro-4 shows high sepecificity for the c-MYC quadruplex DNA. Ligand Pro-4 shows significant cytotoxicity towards A549 cells and down-regulates c-MYC expression in cancer cells. These results collectively suggest that the ligands are potent anticancer agent and can be used as model compounds for development of anticancer drugs. The interaction of new prolinamide derivatives with a variety of DNA sequences has been investigated. These studies demonstrate that prolinamide derivatives can selectively bind and stabilize G-quadruplex structures. Ligand Pro-8 containing three para-prolinamide units is found to be a promising c-MYC G-quadruplex binding ligand as compared to other bis- and trisprolinamide derivatives used in this study. Since proline residues are present in numerous bioactive peptides, this class of molecules may exhibit biological activities. Ligand Pro-8 shows cytotoxicity towards HepG2 cells and it is able to down-regulate c-MYC expression in cancer cells. AFM studies indicate that Pro-8 can direct the formation of c-MYC G-quadruplex higher order nanostructures, which may be useful for the development of novel devices, with medical and nanotechnology applications. We have designed and synthesized two novel derivatives of binaphthyl amine using Buchwald reaction as the key step. BNP 1 was synthesized using a sequence of reactions including amide coupling of diacid while BNP 2 was synthesized by copper (I)-catalyzed click chemistry using azide–alkyne Huisgen cycloaddition. Binding properties of these ligands were evaluated towards four distinct G-quadruplexes found in the promoter regions of the protooncogenes of c-MYC, k-RAS, c-KIT1 and c-KIT2. These results reveal that both the ligands show binding preferences for the quadruplexes over the duplex DNA. BNP 2 binds to c-MYC quadruplex with higher affinity compared to BNP 1. Cellular assays showed that BNP 2 is able to induce DNA damage, leading to cell senescence. In addition, BNP 1 causes the arrest of cell growth at Sub G1 while BNP 2 shows arrest at Sub G1 and G2/M phase and leads the cells towards apoptosis. Fluorescence microscopic images showed the cell permeability and localization of the ligands in the nucleus region of HeLa cells indicating that the ligands may bind to the cellular DNA and drive the cell towards apoptosis by stabilizing the G-quadruplex. Taken together this observation indicates that both ligands exhibited cytotoxic affect in HeLa cells while BNP 2 shows higher cytotoxicity compared to BNP 1. Therefore, in future modification of amine side chain of binaphthyl ligands with triazole-rings can be done in order to increase binding affinities as well as discrimination between different quadruplex sequences for anticancer therapeutics.

    Item Type: Thesis (PhD)
    Additional Information: Supervisor: Dr. Jyotirmayee Dash and Dr. Balaram Mukhopadhyay
    Uncontrolled Keywords: Bis-prolinamide Derivatives; c-MYC G Quadruplex; Click Chemistry; Fluorescent Binaphthyl Amines; G-quadruplex; G-quadruplex DNA Binding Ligands; Prolinamide Ligands; Tris-prolinamide Derivatives
    Subjects: Q Science > QD Chemistry
    Divisions: Department of Chemical Sciences
    Depositing User: IISER Kolkata Librarian
    Date Deposited: 29 Aug 2016 12:46
    Last Modified: 29 Aug 2016 12:46
    URI: http://eprints.iiserkol.ac.in/id/eprint/490

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