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The work of this thesis focuses on the targeting of G-quadruplexes (G4s), wherein several specific and potential ligands were designed, synthesized and characterized for its structural and biological activity. G4s are nucleic acid secondary structures that may form in single-stranded guanine (G)-rich sequences under physiological conditions. Four Guanines (Gs) bind via Hoogsteen-type hydrogen bonds base pairing to yield G-quartets, which in turn stack on top of each other to form the G4. G4s are highly polymorphic, both in terms of strand stoichiometry (forming both inter and intramolecular structures) and strand orientation/topology. The presence of K+ cations specifically supports G4 formation and stability. In the human genome G4 DNA motifs have been found in telomeres, G-rich micro and mini-satellites, up-stream to oncogene promoters and within the ribosomal DNA (rDNA). Human G4 DNA motifs are over-expressed in recombinogenic regions, which are associated with genomic damage in cancer cells.
In the present work, we focus on lead identification with specificity towards the c-MYC promoter G4s. Drug discovery is a highly time consuming and costly process. Lead identification and development are key steps in the drug discovery program. Studies have suggested that a large number of commercially available drugs exhibit deep structural similarity to the lead compounds from which they were developed. Quality lead identification in terms of compounds with high potency and selectivity, favorable physicochemical parameters and in vitro Absorption Distribution Metabolism and Excretion (ADME) parameters are the foremost requirements for the success of the drug discovery process. We herein describe the fragment-based drug design approach for the development of pyrrolidine-substituted 5-nitroindole derivatives as a new class of G4 ligands that exhibit high affinity and selectivity for the c-MYC promoter G-quadruplex. This chapter focuses on the methodology explored whilst finding a suitable hit and its optimization with fragment expansion strategies which undergo efficient G4 binding.
To target G4 DNA, screenings of numerous heterocycles have been reported including indoles, 7-azaindoles, 1H-indazol-3-yl, benzothiazole, imidazo[1,5-a]pyridine, 2,6- diaminopyrimidin-4-ol, 1H-pyrazolo[4,3 d]pyrimidin-7-amine, morpholino, bis-indoles, 2-hydroxynaphthalene-1,4-dione, 1,4-dihydroxyanthracene-9,10-dione, benzofuran and piperonal derived from several alkaloids. In this part of the thesis, we set out to identify new binders targeting the c-MYC G-quadruplex starting from the indole fragment. Several synthetic strategies are reported to optimize and generate best hits starting from 5-nitro indole derivatives by introducing the secondary cationic linked pyrrolidine side chain. Interestingly, all improved versions of G4-indole fragments 5, 7 and 12 contain this 5-nitro functionality, which may aid in the electrostatic binding and contributes to hydrogen binding interactions of the ligands to G4 DNA. In-silico drug design, biological and biophysical analyses illustrated that the substituted 5-nitro indoles scaffolds show preferential affinity towards the c-MYC promoter G-quadruplex compared to other G-quadruplexes and double stranded DNA. In vitro cellular studies confirm that the substituted indole scaffolds downregulate c-MYC expression in cancer cells and have the potential to induce cell cycle arrest in the G0/G1 phase. NMR analysis suggests that 5, 7, and 12 interacts in a fast exchange regime with the terminal G-quartets (5’ and 3’end) in a 2:1 stoichiometry.
To further optimize the fragment generated in chapter II, a novel series of triazole linked indole derivatives as a potential G quadruplex stabilizers have been described in chapter III. The potential ligands can be obtained through an efficient, convergent, synthetic route in moderate to good yields. The synthesized triazole linked indole derivatives are selective towards c- MYC G4-DNA vs. duplex-DNA. The planarity of the aromatic core and its ability to occupy more surface area by stacking over the G4 greatly affect the ability of the compounds to stabilize the G4. Further biophysical and biological studies revealed that the triazole linked nitro indoles are more promising than the amino indole derivatives.
Additionally, the importance of the nitro functional group has been justified by molecular docking studies, where hydrogen-bonding interactions were observed in between the nitro group and the G4 base pairs of the G-quadruplex. In biological findings, most of the synthesized triazole linked nitro indoles has found to be effective against human carcinoma (cervical) HeLa cell lines. Furthermore, western blot and cell cycle analysis confirms that the novel triazole linked 5-nitro indole derivatives (9b) could down-regulate c-MYC oncogene expression in cancer cells via stabilizing its promoter quadruplex structure, arresting cell cycle in G0/G1 phase. NMR analysis suggests that 9b interacts in slow exchange regime with the terminal G-quartets (5’ and 3’-end).
In chapter IV of the thesis, we have developed the synthetic strategies to generate more potent G4 ligands via Knoevenagel condensation. To investigate novel and selective G4 ligands for cancer chemotherapy, we designed and synthesized a series of azaindolin-2-one derivatives (11, 14, 15, 16 and 22) by attaching cationic pyrrolidine side chains and introducing a fluorine atom into the aromatic chromophore (Fig. 3). Fluorine atoms, with high electronegativity and small size, often exhibit unique properties in functional molecules. The electron-withdrawing effect of fluorine could reduce the electron density of the aromatic chromophore, which might favor a stronger interaction with the electron-rich π-system of the G-quartet. In addition, the introduction of fluorine atoms into small molecules might improve lipophilicity and thus the bioavailability. Fluorescent indicator displacement assay (FID) assays suggests that the synthesized azaindolin-2-one derivatives are selective towards c-MYC G4-DNA vs. duplex-DNA and showed potent anticancer activity against human carcinoma (cervical) HeLa cell lines. They down-regulate c-MYC expression in cancer cells via stabilizing its promoter quadruplex structure, arresting cell cycle in G0/G1 phase. Furthermore, NMR spectroscopy suggests that azaindolin-2-one conjugate interacts with terminal G-quartets as well as with the nearby G-rich tract (G13-G14-G15 and G8-G9-G10) of c-MYC quadruplex in intermediate exchange regime.
This dissertation contains two chapters. Each chapter covers a unique topic within RNA sci-ence and is divided in two sub sections, part A and B. Each chapter contains an introduction.
Chapter 1 gives an insight into challenges encountered during sample design and preparation for single molecule Förster energy transfer (smFRET) spectroscopy and offers a solution via a newly establishedestablished workflow to obtain accurate smFRET constructs. Following this workflow, a FRET network could be generated, which allowed a detailed structural dynamics study on H/ACA RNP during catalysis with smFRET spectroscopy. This led to detailed mech-anistic insights into H/ACA RNPs dynamics during catalysis.
Chapter 2 deals with RNA synthetic biology whereby a novel eclectic design strategy for RNA of interest (ROI) release platform is presented, which allows to release a diverse ROI se-quences with single nucleotide precision triggered by an external stimulus. This design strat-egy was used to establish a ROI release system and its powerful performance in in vitro and in vivo applications was shown.
This dissertation contains two chapters. Each chapter covers a unique topic within RNA science and is divided in two sub sections, part A and B. Each chapter contains an introduction.
Chapter 1 gives an insight into challenges encountered during sample design and preparation for single molecule Förster energy transfer (smFRET) spectroscopy and offers a solution via a newly establishedestablished workflow to obtain accurate smFRET constructs. Following this workflow, a FRET network could be generated, which allowed a detailed structural dynamics study on H/ACA RNP during catalysis with smFRET spectroscopy. This led to detailed mechanistic insights into H/ACA RNPs dynamics during catalysis.
Chapter 2 deals with RNA synthetic biology whereby a novel eclectic design strategy for RNA of interest (ROI) release platform is presented, which allows to release a diverse ROI sequences with single nucleotide precision triggered by an external stimulus. This design strategy was used to establish a ROI release system and its powerful performance in in vitro and in vivo applications was shown.
This doctoral thesis deals with the structural and dynamical NMR characterization of biomolecules, covering a broad range of proteins, from small peptides to large GPCRs proteins. This work consists of two projects, which are presented in chapter II and III. Chapter II is focused on the structural screening of peptides and small proteins ranging from 14 to 71 amino acids, while chapter III describes the structure and light dynamics of the disease relevant rhodopsin G90D mutant. The main method used to investigate both types of proteins is NMR spectroscopy. Both chapters comprise individual general introduction, materials and methods, results and discussion sections, and a final conclusion paragraph.
‘Chapter I: Methodological aspects of protein NMR spectroscopy’ presents an overview of different NMR methods developed for the rapid characterization of protein structure and dynamics. Multidimensional NMR, which is routinely used in structural biology, is indispensable for protein structure determination in solution. However, detailed information with resolution at the atomic level is time consuming and requires weeks of expensive measurement time, followed by the manual data analysis. Therefore, the development of time-saving NMR techniques is highly required for screening studies of a large amount of proteins, and can be also helpful for studying unstable biomolecules, as their short lifetime often restricts the experimental procedure.
This chapter covers the two main approaches to accelerate a multidimensional NMR experiment: fast-pulsing techniques that aim to reduce the duration of an individual measurement, and non-uniform sampling technique (NUS), which was developed to reduce the overall number of increments in virtual time domains. A combination of both approaches, fast-pulsing and non-uniform sampling, allows speeding up the measurement time by 2-3 orders of magnitude. Furthermore, recently developed software called TA (targeted acquisition) combines various time-saving approaches, including fast-pulsing, non-uniform sampling and targeted acquisition. Targeted acquisition algorithm records a set of multidimensional NMR spectra in semi-interleaved incremental mode. This provides the ability to monitor the quality of the recorded spectra in real-time and therefore enables the completion of the experiments after the desired quality is achieved. Using this approach will greatly reduce the measurement time without losing important structural information. The implemented automated FLYA assignment further contributes to the rapid and simplified readout of the chemical shift assignment progress of the TA program. During this doctoral dissertation, the scientific collaboration with the TA software developer Prof. Vladislav Orekhov (Sweden) took place, and resulted in the successful establishing of this new NMR technology in the Schwalbe laboratory. TA is now routinely applied in Prof. Schwalbe group for the structure elucidation of small proteins.
‘Chapter II: Rapid NMR and biophysical characterization of small proteins’ describes the structural analysis of peptides and small proteins, which were recently identified within the framework of the Priority Program (SPP 2002). Due to technical limitations in detections of small systems and strict assumptions concerning the smallest size of the gene that can be translated, small open reading frames (sORFs) were excluded from the automated gene annotation for a very long time. Thanks to the newly developed computational and experimental approaches, the ability to identify and detect the small proteins consisting of less than approximately 70 amino acids sparked a growing scientific interest by microbiologist. In the past years, hundreds of new short protein sequences were discovered. Although some peptides were found to be involved in diverse essential biological processes, the functional elucidation of a large number of recently discovered peptides and small proteins remains a challenging task. It is well established that the structure of proteins is often linked to their function. However, the size of small constructs often restricts the possible diversity of secondary structure elements that might be adopted by a protein. Furthermore, as was shown for intrinsic discorded proteins (IDPs), the absence of a well-defined three-dimensional structure does not necessarily mean lack of function. Moreover, peptides, which are initially unstructured in the isolated form can fold in a stable structured conformation upon interaction with their biological partners. Solution state NMR spectroscopy is perfectly amenable for the structural characterization of systems of this size. It provides a rapid readout about the conformational state of small peptides unambiguously, distinguishing between folded, molten globule and unstructured conformations.
During this doctoral thesis the workflow protocol for fast screening of peptides and small proteins was established and applied to 20 candidates ranging from 14 to 71 amino acids, which were identified and selected by six microbiological groups, all members of the Priority Program on small proteins (SPP2002) funded by the German research foundation (DFG). The screening protocol includes sample preparation and biochemical characterization. Peptides containing less than 30 amino acids were synthesized by solid phase synthesis (SPPS), while small proteins containing more than 30 amino acids were heterologously expressed in E. coli.
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