Specific berenil–DNA interactions: an approach for separation of plasmid isoforms by pseudo–affinity chromatography

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the present study, pDNA binding affinities of berberine, berenil, kanamycin and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions varying the type and the salt concentration, and was performed both in absence and in presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand, showed a total retention of pDNA using 1.3 M ammonium sulphate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing salt concentration to 0.6 M and then to 0 M. These results suggest a promising application of berenil as ligand for specific pDNA supercoiled (sc) isoform purification by pseudo-affinity chromatography.C. Caramelo-Nunes acknowledges a fellowship (SFRH/BD/64918/2009) from the Portuguese Foundation for Science and Technology (FCT)

Mechanistic investigation of developmental copper chemotherapeutics

The quest for new metal-based anticancer agents, alternative to clinically established chemotherapeutics, has been motivated by deficiencies observed in current treatment regimes. Coupled with the approach of sophisticated and targeted drug design, there is a clear need for comprehending the underlying biomolecular and cellular responses of new developmental therapeutics. Reported herein is a detailed analysis of redox active developmental metallodrugs containing 1,10-phenanthroline (Phen) ligands and their action as novel cytotoxins of human cancers. This body of research describes mechanistic investigations into the oxidative nuclease activity and redox-targeting properties of new Cu(II) and Mn(II) phenanthroline chemo-types. A number of the Cu(II) complexes have been developed and examined, in collaboration with the National Cancer Institute, USA, for their ability to induce cytotoxicity within a wide variety of cancer cells. To uncover these properties, a range of molecular biology and biophysical techniques were employed including, flow cytometry, confocal microscopy, electrophoresis, and immunohistochemistry. Replacing auxiliary 1,10-phenanthroline with phenazine-type (N,Nʹ) ligands in mononuclear systems, [Cu(N,Nʹ)(Phen)]2+ , was found to enhance intercalation and oxidative DNA scission in vitro. Alternatively, incorporation of dicarboxylates (O,Oʹ) has shown to increase redox potential and stability, thereby targeting both mitochondrial and genomic DNA in human ovarian cancer cells, SKOV3. Increased nuclearity and varying rigidity was explored in dinuclear chemo-types ([Cu2(O,Oʹ)(Phen) 4 ]2+) through the addition of aliphatic and aromatic bridging dicarboxylate ligands. In combination with NCI-60 analysis, the dinulcear complexes were shown to enhance both geno- and cyto-toxic effects when compared to the mononuclear analogue, leading to an apoptotic mode of cellular death; activated through intrinsic mitochondrial machinery. Finally, exchange of the metal centre in the form of di-manganese(II) complex significantly influenced the mode of programmed cell death, activating autophagic catabolism and self-digestion

Programing strand displacement reaction pathways using small molecular DNA binders

DNA has been used in nature as carriers of heredity information for billions of years. The last four decades have witnessed the success of DNA nanotechnology, an interdisciplinary research area in which DNA is used as a synthetic engineering tool rather than a carrier of genetic information. The growth of DNA nanotechnology crosses the boundaries between physics, chemistry, biology and computer science and enables DNA to function as an electronic component, substrate, drug delivery vector and data storage unit. The hybridization of DNA strictly follows the by Watson-Crick rule; thus, DNA base pairs are the most reliable and predictable building block in the true nanometer range. New methods and designs for controlling DNA hybridization have always provided the most essential momentum for the development of DNA nanotechnology. When small molecules bind to the double helical structure of DNA, either through intercalation or minor groove binding, the stability and functionality of DNA may be significantly altered, which is a fundamental basis for many therapeutic and sensing applications. Herein, we reveal, for the first time, that small molecular DNA binders may also be used to program the reaction pathways of toehold-mediated DNA strand displacement, an elementary building block in DNA nanotechnology

Determination of in silico rules for predicting small molecule binding behavior to nucleic acids in vitro.

The vast knowledge of nucleic acids is evolving and it is now known that DNA can adopt highly complex, heterogeneous structures. Among the most intriguing are the G-quadruplex structures, which are thought to play a pivotal role in cancer pathogenesis. Efforts to find new small molecules for these and other physiologically relevant nucleic acid structures have generally been limited to isolation from natural sources or rationale synthesis of promising lead compounds. However, with the rapid growth in computational power that is increasingly becoming available, virtual screening and computational approaches are quickly becoming a reality in academia and industry as an efficient and economical way to discover new lead compounds. These computational efforts have historically almost entirely focused on proteins as targets and have neglected DNA. We present research here showing that not only can software be utilized for targeting DNA, but that selectivity metrics can be developed to predict the binding mechanism of a small molecule to a DNA target. The software Surflex and Autodock were chosen for evaluation and were demonstrated to be able to accurately reproduce the known crystal structures of several small molecules that bind by the most common nucleic acid interacting mechanisms of groove binding and intercalation. These software were further used to rationalize known affinity and selectivity data of a 67 compound library of compounds for a library of nucleic acid structures including duplex, triplex and quadruplexes. Based upon the known binding behavior of these compounds, in silica metrics were developed to classify compounds as either groove binders or intercalators. These rules were subsequently used to identify new triplex and quadruplex binding small molecules by structure and ligand-based virtual screening approaches using a virtual library consisting of millions of commercially available small molecules. The binding behavior of the newly discovered triplex and quadruplex binding compounds was empirically validated using a number of spectroscopic, fluorescent and thermodynamic equilibrium techniques. In total, this research predicted the binding behavior of these test compounds in silica and subsequently validated these findings in vitro. This research presents a novel approach to discover lead compounds that target multiple nucleic acid morphologies

Navigating the Extremes of Biological Datasets for Reliable Structural Inference and Design

Structural biologists currently confront serious challenges in the effective interpretation of experimental data due to two contradictory situations: a severe lack of structural data for certain classes of proteins, and an incredible abundance of data for other classes. The challenge with small data sets is how to extract sufficient information to draw meaningful conclusions, while the challenge with large data sets is how to curate, categorize, and search the data to allow for its meaningful interpretation and application to scientific problems. Here, we develop computational strategies to address both sparse and abundant data sets. In the category of sparse data sets, we focus our attention on the problem of transmembrane (TM) protein structure determination. As X-ray crystallography and NMR data is notoriously difficult to obtain for TM proteins, we develop a novel algorithm which uses low-resolution data from protein cross-linking or scanning mutagenesis studies to produce models of TM helix oligomers and show that our method produces models with an accuracy on par with X-ray crystallography or NMR for a test set of known TM proteins. Turning to instances of data abundance, we examine how to mine the vast stores of protein structural data in the Protein Data Bank (PDB) to aid in the design of proteins with novel binding properties. We show how the identification of an anion binding motif in an antibody structure allowed us to develop a phosphate binding module that can be used to produce novel antibodies to phosphorylated peptides - creating antibodies to 7 novel phospho-peptides to illustrate the utility of our approach. We then describe a general strategy for designing binders to a target protein epitope based upon recapitulating protein interaction geometries which are over-represented in the PDB. We follow this by using data describing the transition probabilities of amino acids to develop a novel set of degenerate codons to create more efficient gene libraries. We conclude by describing a novel, real-time, all-atom structural search engine, giving researchers the ability to quickly search known protein structures for a motif of interest and providing a new interactive paradigm of protein design

Spectroscopic Investigation into Minor Groove Binders Designed to Selectively Target DNA Sequences

Recently, there has been increasing focus toward the development of small molecules designed to target a specific sequences of double stranded DNA for therapeutic purposes1. Minor groove binding compounds have been shown to be capable of selectivity target GC sites in AT tract DNA2. In this research, binding selectivity was investigated using absorption, fluorescence and circular dichroic properties of selected DB minor groove binders in the presence of two unique DNA sequences. Further insight was gained by comparing the electrostatic potential maps and optimized structures of the compounds of interest. Using the results presented, potential selective minor groove binders can be selected for further investigation and kinetic studies

Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent

Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies

Design, synthesis and optimization of bioactive compounds: a medicinal chemistry approach

Il lungo processo di scoperta di un nuovo farmaco coinvolge diverse fasi e richiede l'integrazione di diverse discipline scientifiche e metodologie. La chimica farmaceutica, che comprende discipline come la chimica analitica, organica e computazionale, gioca un ruolo cruciale nella fase iniziale del processo di drug discovery ed è essenziale nello sviluppo di molecole con potenziale attività biologica nonché nella comprensione dei meccanismi delle malattie o delle strutture dei bersagli macromolecolari. Il mio percorso di dottorato è stato caratterizzato e fondamentale per accrescere la comprensione e sviluppare l'utilizzo di diverse tecniche, tra cui: chimica analitica, cromatografia, spettroscopia, diffrazione a raggi X, microscopia ed altre ancora; mi sono inoltre dedicata all'ottimizzazione della sintesi organica per ottenere migliori rese con reazioni più green ed economiche. In questi anni ho trattato con maggiore enfasi le seguenti tecniche: spettrometria di massa (MS), risonanza magnetica nucleare (NMR), tecniche computazionali e dicroismo circolare (CD); la mia tesi, dopo un'introduzione alla teoria e alle possibili applicazioni delle suddette, illustra come siano state utilizzate sinergicamente in diversi percorsi di ricerca. Nel progetto principale (Sezione 2), l'uso congiunto di queste tecniche nello studio delle interazioni di piccole molecole con il G-quadruplex (G4), ha evidenziato il loro potenziale nelle applicazioni antitumorali, antinfiammatorie, antivirali e neuroprotettive. Le metodologie fondamentali includono l'ESI-MS per uno screening rapido e per una prima valutazione dell’efficienza dell'interazione, l'NMR per la risoluzione tridimensionale della struttura e per approfondire la natura del legame ligando:target, il CD per convalidare i risultati e valutare la topologia del G4 e le tecniche computazionali per prevedere le interazioni presenti e per perfezionare le strutture predette. Sono stati esaminati scaffold diversi dimostrando il loro potenziale nell’interazione con diversi folding degli acidi nucleici e, in particolare con le strutture G4, suggerendo i loro possibili effetti antiproliferativi grazie all'inibizione dell'attività telomerasica mediante la stabilizzazione del G4. I due progetti secondari presentati nella Sezione 3 mostrano, rispettivamente, uno lo sviluppo di composti contenenti selenio per la mitigazione della neurodegenerazione e l'altro l'identificazione di inibitori di fosfodiesterasi (PDE) per le malattie neurodegenerative; questi progetti hanno dimostrato l'efficacia dell'integrazione delle tecniche computazionali con saggi sperimentali nella scoperta di nuovi farmaci. Il primo progetto illustra la preparazione di derivati della selenofluoxetina, enfatizzandone la capacità di mitigare lo stress ossidativo e offrire benefici neuroprotettivi. L'indagine ha coinvolto lo studio delle reazioni di questi composti con le specie reattive dell'ossigeno (ROS), sia con metodi sperimentali che computazionali, fornendo informazioni cruciali sui loro meccanismi. Il secondo progetto coinvolge l'uso di tecniche computazionali per la progettazione di nuovi inibitori di PDE utilizzando il docking molecolare e le simulazioni di dinamica molecolare (MD). I composti sono stati sottoposti ad uno screening, individuando potenziali candidati farmaci in grado di formare le interazioni significative con gli amminoacidi cruciali per il legame con le PDE. Infine le simulazioni di MD hanno ulteriormente convalidato l’effettiva stabilità dei complessi formati consolidando i risultati e i test in vitro hanno dimostrato un'elevata attività inibitoria contro la PDE9, paragonabile a quella di un noto inibitore. La sintesi e l'ottimizzazione di potenziali candidati farmaci, insieme ad una comprensione esaustiva delle interazioni bersaglio:ligando mediante l’uso di diverse tecniche strumentali, costituiscono la chiave del successo nella scoperta di nuovi farmaci.The extensive process of drug discovery encompasses multiple stages and requires a convergence of diverse scientific disciplines and methodologies. Medicinal chemistry, which includes sciences like analytical, organic, and computational chemistry, plays a significant role in the preliminary stage of drug discovery and is pivotal in developing potentially bioactive molecules and unravelling disease mechanisms or macromolecular target structures. My PhD has been characterized and essential in enhancing the understanding and development of the use of various techniques, including analytical chemistry, chromatography, spectroscopy, X-ray diffraction, microscopy, among others. Furthermore, I have dedicated myself to optimizing organic synthesis to achieve better yields through greener and more cost-effective reactions. Throughout these years, I have focused with greater emphasis on the following techniques: mass spectrometry (MS), nuclear magnetic resonance (NMR), computational techniques, and circular dichroism (CD). My thesis, after an introduction to the theory and possible applications of these techniques, illustrates how they have been synergistically employed in various research paths. In the primary project (Section 2), their collective use in studying small molecules' interactions with G-quadruplex (G4) highlighted their potential in anti-cancer, anti-inflammatory, anti-viral, and neuroprotective applications. Essential methodologies include ESI-MS for rapid screening and understanding interaction efficiency, NMR for 3D structure resolution and binding insights, CD spectroscopy to validate findings and assess G4 topology, and computational tools for predicting interactions and refining structures. Different scaffolds were investigated, revealing their potential in targeting DNA arrangements, particularly G4 structures, suggesting their anti-proliferative effects by inhibiting telomerase activity through G4 stabilization. Two ancillary projects (Section 3) showcased, respectively, one the development of seleno-containing compounds for neurodegeneration mitigation and the other the identification of inhibitors of phosphodiesterase (PDE) for neurodegenerative disorders; these projects demonstrated the efficiency of integrating computational techniques with experimental assays to streamline drug discovery. The first project presented focused on the preparation of selenofluoxetine derivatives, emphasizing their capacity to mitigate oxidative stress and offer neuroprotective effects. The investigation delved into understanding the compounds' reactions with reactive oxygen species (ROS), both in experimental and computational settings, providing crucial insights into their mechanisms. The second project showed the use of computational tools for designing novel PDE inhibitors through molecular docking, and MD simulations. After a meticulous validation of the docking method, the compounds generated through combinatorial chemistry and the molecules of an internal database underwent a screening, leading to the identification of potential hits displaying significant interactions with crucial amino acids for PDE binding. These findings allowed a preliminary structure-activity relationship (SAR) study and identified crucial design features for potential PDE inhibitors. Eventually, MD simulations further validated the stability of the formed complexes, consolidating the findings and in vitro testing demonstrated high inhibitory activity against PDE9, comparable to a known inhibitor. The synthesis and optimization of potential drug candidates, when coupled with a comprehensive understanding of target:ligand interactions through diverse techniques, form the crux of successful drug discovery. The seamless integration of these methodologies continues to be paramount in creating novel pharmacological agents, driving the need for further research to refine and expedite the drug discovery process

Investigating the DNA-Binding Interactions of Small Organic Molecules Utilizing Ultrafast Nonlinear Spectroscopy.

The DNA-binding mechanism of small organic molecules, such as DNA-targeted drugs and fluorescent nuclear dyes, is key to their performance. Therefore, understanding the DNA-binding mechanism is critical for the design and development of molecules targeted at DNA. The purpose of this present research is to investigate the DNA-binding interactions of small organic molecules by employing ultrafast nonlinear spectroscopy. While basic design principles are proposed, the DNA-binding modes of many small organic molecules cannot be unambiguously assigned based either on their structure, or through the use of many well-established spectroscopic techniques. A new methodology utilizing two-photon spectroscopy was developed to determine the DNA-binding modes of small organic molecules definitively, contrarily to other well-established spectroscopic techniques. The impact of this work is imbedded with the ultrafast nonlinear spectroscopic studies of the DNA-binding interactions of small organic molecules. The newly developed methodology demonstrated superior sensitivity at both low drug and DNA concentrations by more than order of magnitude in comparison to circular dichroism (CD). This indicates that our approach can be used to probe DNA-drug interactions at biologically relevant conditions, which is critical in drug research and development. The impact of this work also investigated the DNA-binding interactions of newly synthesized fluorescent nuclear dyes. This study has led to the emergence of structure-property relationships of DNA-binding molecules that adopt a crescent or V-shaped scaffold. The findings reveal that the structure of these fluorophores can be designed to either intercalate or groove bind with DNA by structurally modifying the electron acceptor properties of the central heterocyclic core. This is important because it allows the performance, specificity, and localization of a DNA-binding molecule to be controlled. The localization and cellular uptake of these small molecules were evaluated by conducting a series of bio-imaging studies in live HeLa cells. This work is significant because the design strategy can be applied towards the development of small molecules aimed at DNA.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133196/1/phidoan_1.pd