Understanding the Recognition of Mixed Sequence DNA through Minor Groove Binding Compounds

The broad range of diseases controlled by transcription factors (TFs) and their potential abilities to modulate gene expression have led to an emerging interest in the development of small molecules that target TF-DNA complexes. Still, there is only a limited number of types of designed small molecules that show strong and sequence-specific binding to DNA along with good cellular uptake properties for therapeutic use. Most of the successful nuclear stains or therapeutic agents that bind non-covalently in the minor groove of DNA are AT specific. Synthesis of novel compounds to better target the mixed AT/GC base pair (bp) sequences with a broad range of applications like targeting TF is a daunting task. Our novel heterocyclic cation, DB2277, contains the aza-benzimidazole group (aza-BI) that specifically recognizes the single GC bp interspersed between AT bp sequences in the minor groove of DNA. NMR spectroscopy revealed the presence of major and minor binding species in the DB2277 complex with AAAGTTT type of DNA. NMR exchange dynamics have shown that the exchange between major and minor species is much faster than the compound’s dissociation from the complex, as determined using surface plasmon resonance (SPR). To understand the molecular basis of recognition of mixed bp sequences and to acquire ideas to design new sequence-specific compounds, structural information of the DB2277-DNA complex is essential. Experimental structure of the unique and selective binding orientation of DB2277 with “AAGATA” binding site of DNA has been obtained using high-resolution NMR and molecular dynamics (MD) simulations, which suggests the involvement of two specific and strong H-bonds in recognition of the central GC bp. Extended MD calculations have shown dynamic water-mediated H-bond contacts between amidine of DB2277 and the bases at the floor of the minor groove and 180° rotations of the phenyl linked to a flexible linker (OCH2) in a bound compound for the first time. Therefore, designing additional compounds with the ability to recognize a vast array of biologically important DNA sequences is essential for extending the use of new heterocyclic compounds in therapeutic applications in the future

Targeting Duplex and Higher Order Nucleic Acids Using Neomycin / Neomycin-Hoechst 33258 Conjugates

Small molecules have provided means to combat many diseases caused by pathogens. In the last six decades, advances in the structural and biological understanding of nucleic acid functions have led to rational drug design programs in order to solve current therapeutic challenges. The work described in this dissertation addresses discovery of aminoglycosides as novel binders to G-quadruplex nucleic acids. Understanding of effect of linker length on the B-DNA binding of a series of Hoechst 33258 derivatives have been provided. Novel Hoechst 33258 based monobenzimidazoles have been synthesized and their biological properties have been compared with the bisbenzimidazoles. The bisbenzimidazoles have been designed to be useful towards click chemistry applications. These clickable Hoechst 33258 derivatives were then used to prepare neomycin-Hoechst 33258 conjugates with varied linker spacing between them. The binding studies of these novel neomycin-Hoechst 33258 conjugates show intercalative binding of bisbenzimidazole moiety of the ligand to an RNA duplex. Finally, the novel neomycin-Hoechst 33258 conjugates have been screened against G-quadruplex forming promoter sequences and biophysical characterization to its binding has been provided. Overall, these studies have led to synthesis of novel small molecules that bind to various nucleic acids and their new binding modes have been discovered. The studies presented here are expected to help in the design of novel therapeutics

DNA-binding Small Molecules as Drug Agents that Interfere with Transcription Factors: the Development, the Potential and the Future

DNA-minor groove binding small molecules have been extensively developed to achieve higher binding affinity and specificity. Polyamides are a class of small molecules that can be programmed to target any predetermined DNA sequence. The development of hairpin polyamides along with introduction of β-alanine substituents, has greatly enhanced the DNA binding properties of these molecules. Yet the correlation between β-insert and binding properties remains unclear. On the other hand, the design of small-size, fluorescent hybrid polyamides has facilitated cell studies due to their ease of observation. There is a strong need to expand the DNA recognition sites of such molecules and extend their biological applications. This dissertation has explored the systematic design and synthesis of eight-ring hairpin polyamides as well as the modified Pyr-AzaHx hybrid polyamides. Comprehensive biophysical and biochemical tools were employed to evaluate their binding properties. The effects of β-alanine and N-terminal cationic groups on hairpin polyamides-DNA binding have been discussed. The binding properties of modified Pyr-AzaHx polyamides were explored. Altogether, the work provided fundamental guidance for the prediction of binding properties of similar molecules as well as strategies for the design of more competitive molecules. Transcription factors bind to specific DNA sequences in the major groove and regulate gene expression. Abnormal expression of transcription factors is involved in the development of many serious diseases. Precise control of gene expression by targeting transcription factors can be an alternative therapeutic approach. Polyamides bind to DNA with affinities comparable to proteins, empowering them with the ability to interfere with transcription factors at specific DNA binding site and consequently altering the gene expression level. In this dissertation, the effect of polyamides on the binding of transcription factor PU.1 was studied. Abnormal expression of PU.1 is involved in the development of acute myeloid leukemia (AML). A positive correlation was established between eight-ring polyamide binding affinity and inhibition efficacy for PU.1. A non-inhibitor polyamide FH1024 was identified and the mechanism of action among polyamide, DNA and PU.1 was explored. The studies showed strong evidence of the capability of polyamides serving as drug agents. This work also established solid basis for the further cell studies

Biophysical Properties of DNA Minor Groove Binding by Heterocyclic Cations of Varying Structures

Small heterocyclic cations that bind to DNA are interesting systems to study due to their structural diversity, pharmaceutical potential, and characteristic target recognition patterns. Clinically, such compounds offer an attractive therapeutic approach as inhibitors of protein-DNA interactions implicated in disease. Due to their typical intrinsic fluorescence, these compounds also have potential as convenient biotechnological probes for studying DNA. Finally, from a biophysics perspective, an intricate understanding of the factors driving DNA binding by these compounds can extend our understanding of DNA targeting more broadly. In this thesis, the electrostatics and hydration properties of DNA binding by eight of these heterocyclic cations in complex with various DNA sequences are investigated

Synthesis and biological evaluation of 2-(2'-hydroxyphenyl) benzoxazole analogs of UK-1 and G-quadruplex selectivity of perylene diimide compounds: /

A great number of pharmaceutical drugs target nucleic acids. However, drug-DNA interactions can be region non-specific and lead to undesired side effects. Understanding the mechanisms that regulate drug-DNA binding can help in the design of potent and selective therapeutic agents with fewer deleterious side effects. The present investigation explores the metal-mediated DNA binding of a group of 2-(2-hydroxyphenyl)benzoxazole (HPB) ligands and the aggregation dependant G-quadruplex selectivity of a series of perylene tetracarboxylic acid diimides (PTCDI) compounds. HPB ligands are simplified analogs of the bis-benzoxazole natural product UK-1. This compound is able to inhibit cell growth of various tumor cell lines, bind divalent cations, and interact with DNA in a metal dependant fashion. The HPB moiety present in UK-1 was identified as relevant for its metal ion binding and biological properties. For this work, novel HPB ligands were synthesized with different substitutions at the C4 or C7 position. Their ability to bind metal ions and DNA was evaluated and their cytotoxicity was assessed in multiple cancer cell lines. The ligands bound to Cu²⁺ with the highest affinity among metals studied. Consequently, Cu²⁺ promoted the most dramatic increase in DNA binding and affected the ligand's cellular cytotoxicity. The second project focused on targeting four-stranded structures called G-quadruplexes, which can form in G-rich nucleic acid sequences. Compounds that stabilize these structures may inhibit nucleic acid-processing enzymes such as telomerase and potentially act as anti-cancer agents. PIPER is a PTCDI that is particularly selective for G-quadruplex DNA versus duplex DNA under conditions in which it forms aggregates. This work investigated ligand aggregation in a series of PIPER analogs with different structural features under high and low salt buffers, changes in pH, metal binding and temperature changes. A negatively charged analog was determined to form metal-mediated aggregates while novel thermophilic mediated aggregation was discovered for an analog with methoxyethoxymethyl groups. The ability of these ligands to bind different DNA structures was evaluated under aggregating and non-aggregating conditions. This study supports the idea that ligand aggregation increases their quadruplex selectivity and decreases double-stranded DNA binding.Chemistry and BiochemistryChemistr

Drug-targetting of duplex and quadruplex DNA

This thesis investigates structural and dynamic properties of drug recognition mechanisms to duplex and quadruplex DNA using primarily high field NMR techniques and molecular dynamics simulations. The mechanism of co-operative binding of Hoechst 33258 to the DNA minor groove of duplexes that contain two binding sites such as d(CTTTTGCAAAAG)2, d(GAAAAGCTTTC)2 and d(CTTTTGGCCAAAAG)2 has been studied. NMR and other titration techniques have evidenced co-operative binding and no detection of an intermediate 1:1 complex. High-resolution NMR structure determination showed no evidence of direct contact between Hoechst 33258 molecules or DNA structure deformation that would facilitate co-operativity, Molecular dynamics simulations based on NMR data, allowed us to calculate thermodynamic quantities of the two binding events, and lead us to conclude that ligand binding can induce changes in DNA conformational flexibility in sites of the structure distant from the binding site and result in more favourable second ligand binding. The results highlight the general importance of flexibility in determining the properties of ligand-DNA interactions. The relative importance of ligand isohelicity and phasing in DNA minor groove has been investigated by studying the structure and dynamics of the 1:1 complex of Hoechst IO-d(GCAAATTTGC)2. The results suggest that DNA sequence-dependent structure and flexibility have significant role for the strong binding of Hoechst 10 to the duplex. The formation, stability, structure and dynamics of the d(TTAGGGT)4 quadruplex structure, which contains the human telomeric repeat TTAGGG, have been studied. Characteristic features of the quadruplex structure were determined and this information was used for understanding drug-quadruplex interactions. The complex of the fluorinated polycyclic methylacridinium cation RHPS4, lead compound for telomerase inhibition, with the d(TTAGGGT)4 quadruplex structure has been investigated. RHPS4 forms a stable G-quadruplex complex by endstacking externally to the a-tetrads of the Apa and Gp'T steps. This study presents detailed properties of the complex and provides further information for lead optimisation studies

Drug-targetting of duplex and quadruplex DNA

This thesis investigates structural and dynamic properties of drug recognition mechanisms to duplex and quadruplex DNA using primarily high field NMR techniques and molecular dynamics simulations. The mechanism of co-operative binding of Hoechst 33258 to the DNA minor groove of duplexes that contain two binding sites such as d(CTTTTGCAAAAG)2, d(GAAAAGCTTTC)2 and d(CTTTTGGCCAAAAG)2 has been studied. NMR and other titration techniques have evidenced co-operative binding and no detection of an intermediate 1:1 complex. High-resolution NMR structure determination showed no evidence of direct contact between Hoechst 33258 molecules or DNA structure deformation that would facilitate co-operativity, Molecular dynamics simulations based on NMR data, allowed us to calculate thermodynamic quantities of the two binding events, and lead us to conclude that ligand binding can induce changes in DNA conformational flexibility in sites of the structure distant from the binding site and result in more favourable second ligand binding. The results highlight the general importance of flexibility in determining the properties of ligand-DNA interactions. The relative importance of ligand isohelicity and phasing in DNA minor groove has been investigated by studying the structure and dynamics of the 1:1 complex of Hoechst IO-d(GCAAATTTGC)2. The results suggest that DNA sequence-dependent structure and flexibility have significant role for the strong binding of Hoechst 10 to the duplex. The formation, stability, structure and dynamics of the d(TTAGGGT)4 quadruplex structure, which contains the human telomeric repeat TTAGGG, have been studied. Characteristic features of the quadruplex structure were determined and this information was used for understanding drug-quadruplex interactions. The complex of the fluorinated polycyclic methylacridinium cation RHPS4, lead compound for telomerase inhibition, with the d(TTAGGGT)4 quadruplex structure has been investigated. RHPS4 forms a stable G-quadruplex complex by endstacking externally to the a-tetrads of the Apa and Gp'T steps. This study presents detailed properties of the complex and provides further information for lead optimisation studies

Novel Neomycin Conjugates for Multi-Recognition of Nucleic Acids

Neomycin is an aminoglycoside antibiotic known for its high affinity for RNA duplex structures. Recent developments from our labs have indicated that aminoglycoside binding is not limited to RNA, but to nucleic acids that, like RNA, adopt conformations similar to A-form. Our group sought to further expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Envisioning a dual groove-binding mode, we have extended the potential recognition process to include a third, intercalative moiety. Furthermore, we observe remarkable recognition of such conjugates with RNA duplex. Spectroscopic studies such as UV melting, differential scanning calorimetry, isothermal fluorescence titrations, and circular dichroism together illustrate the multi-recognition properties of the novel neomycin-based conjugates

Heterocyclic Diamidines Induce Sequence Dependent Topological Changes in DNA; A Study Using Gel Electrophoresis

Diamidines are a class of compounds that target the minor groove of DNA and have antiparasitic and antimicrobial properties. Their mechanism of action has not been fully elucidated, but may include changes in DNA topology. In this study we have investigated such changes using methods of gel electrophoresis including ligation ladders and cyclization assays. We found that topology changes were sequence dependent. Compounds typically caused non-anomalously migrating ATATA sequences to migrate as if they were bent, while A5 sequences that normally migrated anomalously became less so in the presence of certain diamidines. Select compounds induced changes in cyclization efficiency that were also sequence dependent; DB75 significantly abolished cyclization in A5 containing sequences but enhanced it in sequences containing ATATA sites

A Commemorative Issue in Honor of Professor Nick Hadjiliadis: Metal Complex Interactions with Nucleic Acids and/or DNA

This Special Issue of the International Journal of Molecular Science comprises a comprehensive study on “Metal Complex Interactions with Nucleic Acids and/or DNA”. This Special Issue has been inspired by the important contribution of Prof. Nick Hadjiliadis to the field of palladium or/and platinum/nucleic acid interactions. It covers a selection of recent research and review articles in the field of metal complex interactions with nucleic acids and/or DNA. Moreover, this Special Issue on "Metal Complexes Interactions with Nucleic Acids and/or DNA" provides an overview of this increasingly diverse field, presenting recent developments and the latest research with particular emphasis on metal-based drugs and metal ion toxicity