Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC50 values of 18–100 nM in a standard TRAP assay.

G-quadruplex Formation Enhances Splicing Efficiency Of Pax9 Intron 1.

G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.

G-quadruplex ligands for cancer therapy

DNA may fold into a diversity of structures and topologies such as duplexes and triplexes. Some specific guanine-rich DNA sequences may even fold into a higher order structures denominated guanine G-quadruplexes (G4). These G-quadruplex forming sequences have shown biological interest since were found in telomeres and in promoter region of oncogenes. Thus, these G4 forming sequences have been explored as therapeutic targets for cancer therapy, since G4 formation was demonstrated to inhibit RNA-polymerase and telomerase activity. However, the G4 structures are transient and are only formed under specific conditions. Hence the main objective of this work is to develop new G4-specific ligands which may potentially find applications in the therapeutic area. Several potential G4-binding ligands were synthesized and characterized. The synthesis of these compounds consisted on a procedure based on van Leusen chemistry and a cross-coupling reaction through C-H activation, affording phenanthroline compounds (Phen-1, 50%; Phen-2, 20%), phenyl (Iso-1, 61%; Iso-2, 21%; Ter-1, 85%; Ter-2, 35%), and quinolyl (Quin-1, 85%; Quin-2, 45%) compounds. Screening assays for selecting the potential G4 compounds were performed by FRET-melting, G4-FID, CD-melting and DSF. Qualitative biophysical studies were performed by fluorescence and CD spectroscopy. Two high-specific G-quadruplex ligands, Phen-1 and Phen-2, were found to effectively bind telomeric and c-myc G4 structures. Phen-1 was found to stabilize parallel telomeric 22AG and c-myc sequence by 4.1 and 4.3 ˚C, respectively. Phen-2 also displayed high affinity towards 22AG (

Design and Synthesis of Acridine-Based Macrocyclic G-Quadruplex DNA Ligands

DNA sequences that are rich in the guanine nucleic base possess the ability to fold into higher order structures called G-quadruplexes. These higher level structures are formed as a result of two sets of four guanine bases hydrogen-bonding together in a planar arrangement called a guanine quartet. Guanine quartets subsequently stack upon each other to form quadruplexes. G-quadruplexes are mainly localized in telomeres as well as in oncogene promoters. One unique and promising therapeutic approach against cancer involves targeting and stabilizing G-quadruplexes with small molecules, generally in order to suppress oncogene expression and telomerase enzyme activity; the latter has been found to contribute to “out-of control” cell growth in ca. 80-85% of all cancer cells and primary tumours while being absent in normal somatic cells. In this work, we present efforts towards designing and synthesizing acridine-based macrocycles (Mh) and (Mb) with the purpose of providing potential G4 ligands that are suited for selective binding to G4 vs. duplex DNA, and stabilize G-quadruplex structures. Two ligands described in this study include an acridine core which provides an aromatic surface capable of π-π interactions with the surface of G-quadruplexes. The successful synthesis of 4,5-diaminoacridine is described in chapter 2, as an essential fragment of the macrocycles (Mh) and (Mb). In order to investigate the synthetic method for macrocyclization, model compounds composing almost half of the designed macrocycles were explored. As discussed in chapter 3, the synthesis of the model compound for (Mb) turned out to be challenging. However, as a step towards the synthesis of (Mh), the synthesis of the hydrogen-containing model compound, which is almost half of the desired macrocycle (Mh) was achieved in our group and proved to be promising.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...

Supramolecular functional assemblies with photo or chemical addressability

Supramolecular self-assembly represents a key technology for the spontaneous construction of nanoarchitectures and for the fabrication of materials with enhanced physical and chemical properties. In addition, a significant asset of supramolecular self-assemblies rests on their reversible formation, thanks to the kinetic lability of their non-covalent interactions. This dynamic nature can be exploited for the development of “self-healing” and “smart” materials towards the tuning of their functional properties upon various external factors. One particular intriguing objective in the field is to reach a high level of control over the shape and size of the supramolecular architectures, in order to produce well-defined functional nanostructures by rational design. In this direction, many investigations have been pursued toward the construction of self-assembled objects from numerous low-molecular weight scaffolds, for instance by exploiting multiple directional hydrogen-bonding interactions. In particular, nucleobases have been used as supramolecular synthons as a result of their efficiency to code for non-covalent interaction motifs. Among nucleobases, guanine represents the most versatile one, because of its different H-bond donor and acceptor sites which display self-complementary patterns of interactions. Interestingly, and depending on the environmental conditions, guanosine derivatives can form various types of structures. Most of the supramolecular architectures reported in this Thesis from guanosine derivatives require the presence of a cation which stabilizes, via dipole-ion interactions, the macrocyclic G-quartet that can, in turn, stack in columnar G-quadruplex arrangements. In addition, in absence of cations, guanosine can polymerize via hydrogen bonding to give a variety of supramolecular networks including linear ribbons. This complex supramolecular behavior confers to the guanine-guanine interactions their upper interest among all the homonucleobases studied. They have been subjected to intense investigations in various areas ranging from structural biology and medicinal chemistry – guanine-rich sequences are abundant in telomeric ends of chromosomes and promoter regions of DNA, and are capable of forming G-quartet based structures– to material science and nanotechnology. This Thesis, organized into five Chapters, describes mainly some recent advances in the form and function provided by self-assembly of guanine based systems. More generally, Chapter 4 will focus on the construction of supramolecular self-assemblies whose self-assembling process and self-assembled architectures can be controlled by light as external stimulus. Chapter 1 will describe some of the many recent studies of G-quartets in the general area of nanoscience. Natural G- quadruplexes can be useful motifs to build new structures and biomaterials such as self-assembled nanomachines, biosensors, therapeutic aptamer and catalysts. In Chapters 2-4 it is pointed out the core concept held in this PhD Thesis, i.e. the supramolecular organization of lipophilic guanosine derivatives with photo or chemical addressability. Chapter 2 will mainly focus on the use of cation-templated guanosine derivatives as a potential scaffold for designing functional materials with tailored physical properties, showing a new way to control the bottom-up realization of well-defined nanoarchitectures. In section 2.6.7, the self-assembly properties of compound 28a may be considered an example of open-shell moieties ordered by a supramolecular guanosine architecture showing a new (magnetic) property. Chapter 3 will report on ribbon-like structures, supramolecular architectures formed by guanosine derivatives that may be of interest for the fabrication of molecular nanowires within the framework of future molecular electronic applications. In section 3.4 we investigate the supramolecular polymerizations of derivatives dG 1 and G 30 by light scattering technique and TEM experiments. The obtained data reveal the presence of several levels of organization due to the hierarchical self-assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures. The elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties. Chapter 4 will describe photoresponsive self-assembling systems. Numerous research examples have demonstrated that the use of photochromic molecules in supramolecular self-assemblies is the most reasonable method to noninvasively manipulate their degree of aggregation and supramolecular architectures. In section 4.4 we report on the photocontrolled self-assembly of modified guanosine nucleobase E-42: by the introduction of a photoactive moiety at C8 it is possible to operate a photocontrol over the self-assembly of the molecule, where the existence of G-quartets can be alternately switched on and off. In section 4.5 we focus on the use of cyclodextrins as photoresponsive host-guest assemblies: αCD–azobenzene conjugates 47-48 (section 4.5.3) are synthesized in order to obtain a photoresponsive system exhibiting a fine photocontrollable degree of aggregation and self-assembled architecture. Finally, Chapter 5 contains the experimental protocols used for the research described in Chapters 2-4.

Design, synthesis and biological evaluation of substituted naphthalene diimides as anticancer agents

It has been proved that naphthalene diimide (NDI) derivatives display anticancer properties as intercalators and G-quadruplex-binding ligands, leading to DNA damage, senescence and down-regulation of oncogene expression. This thesis deals with the design and synthesis of disubstituted and tetrasubstituted NDI derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development. Disubstituted NDI compounds have been designed with the aim to provide potential multitarget directed ligands (MTDLs), in order to create molecules able to simultaneously interact with some of the different targets involved in this pathology. The most active compound, displayed antiproliferative activity in submicromolar range, especially against colon and prostate cancer cell lines, the ability to bind duplex and quadruplex DNA, to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Tetrasubstituted NDI compounds have been designed as G-quadruplex-binding ligands endowed with anticancer activity. In order to improve the cellular uptake of the lead compound, the N-methylpiperazine moiety have been replaced with different aromatic systems and methoxypropyl groups. The most interesting compound was 1d, which was able to interact with the G-quadruplexes both telomeric and in HSP90 promoter region, and it has been co-crystallized with the human telomeric G-quadruplex, to directly verify its ability to bind this kind of structure, and also to investigate its binding mode. All the morpholino substituted compounds show antiproliferative activity in submicromolar values mainly in pancreatic and lung cancer cell lines, and they show an improved biological profile in comparison with that of the lead compound. In conclusion, both these studies, may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

R-loops and G4-structures: from DNA damage to innate immune response gene activation

Non-B DNA structures like R-loops and G-quadruplexes play a pivotal role in several cellular vital processes like DNA transcription regulation. Misregulation of said non-canonical DNA structures can often lead to genome instability, DNA damage, and, eventually, to the activation of an innate immune response. For such reasons they have been studied as adjuvants in anticancer therapies. Here we studied drugs targeting R-loops (Top1 poisons) and G4s (hydrazone derivatives) in order to observe their effects in terms of DNA damage induction and, subsequently, activation of innate immune response. We studied how non-cytotoxic doses of ampthotecin and LMP-776 impact on genome instability, are capable to induce DNA damage and micronuclei, and, eventually lead to an innate immune gene response via the cGAS/STING pathway. G-quadruplexes are another ubiquitous, non-canonical DNA structure, more abundant in telomeric regions, demonstrating a marked relation with the impairment of telomerase and the regulation of DNA replication and transcription. Furthermore, we investigated the properties of new-synthesized molecules belonging to the highly promising class of hydrazone derivatives, in terms of cytotoxicity, ability to stabilize G4-structures, induce DNA damage, and activate interferon-B production. Both Top1 poisons and G4-stabilizers possess several features that can be very useful in clinical applications, in light of their ability to stimulate innate immune response factors and exert a certain cell-killing power, plus they offer a broad and diverse range of treatment options in order to face a variety of patient treatment needs. It is for these very reasons that it is of uttermost importance that further studies are conducted on these compounds, in order to synthesize new and increasingly powerful and flexible ones, with fewer side effects to customize therapies on specific cancers’ and patients’ features.

Innovative photoresponsive materials & supramolecular structures

This thesis focuses on two main topics: photoresponsive azobenzene-based polymers and supramolecular systems generated by the self-assembly of lipophilic guanosines. In the first chapters describe innovative photoresponsive devices and materials capable of performing multiple roles in the field of soft robotics and energy conversion. Chapter 2 describes a device obtained by coupling a photoresponsive liquid-crystalline network and a piezoelectric polymer to convert visible light into electricity. Chapter 3 deals with a material that can assume different shapes when triggered by three different stimuli in different environments. Chapter 4 reports a highly performing artificial muscle that contracts when irradiated. The last two chapters report on supramolecular structures generated from functionalized guanosines dissolved in organic solvents. Chapter 6 illustrates the self-assembly into G-quadruplexes of 8- and 5’-functionalized guanosines in the absence of templating ions. Chapter 7 describes the supramolecular structure generated by the assembly of a lipophilic guanosine in the presence of silver cations. Chapter 6 is reproduced from an already published paper, while the other chapters are going to be submitted to different journals in a couple of months.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

Granulocyte Colony-stimulating Factor (g-csf) Positive Effects On Muscle Fiber Degeneration And Gait Recovery After Nerve Lesion In Mdx Mice.

G-CSF has been shown to decrease inflammatory processes and to act positively on the process of peripheral nerve regeneration during the course of muscular dystrophy. The aims of this study were to investigate the effects of treatment of G-CSF during sciatic nerve regeneration and histological analysis in the soleus muscle in MDX mice. Six-week-old male MDX mice underwent left sciatic nerve crush and were G-CSF treated at 7 days prior to and 21 days after crush. Ten and twenty-one days after surgery, the mice were euthanized, and the sciatic nerves were processed for immunohistochemistry (anti-p75(NTR) and anti-neurofilament) and transmission electron microscopy. The soleus muscles were dissected out and processed for H&E staining and subsequent morphologic analysis. Motor function analyses were performed at 7 days prior to and 21 days after sciatic crush using the CatWalk system and the sciatic nerve index. Both groups treated with G-CSF showed increased p75(NTR) and neurofilament expression after sciatic crush. G-CSF treatment decreased the number of degenerated and regenerated muscle fibers, thereby increasing the number of normal muscle fibers. The reduction in p75(NTR) and neurofilament indicates a decreased regenerative capacity in MDX mice following a lesion to a peripheral nerve. The reduction in motor function in the crushed group compared with the control groups may reflect the cycles of muscle degeneration/regeneration that occur postnatally. Thus, G-CSF treatment increases motor function in MDX mice. Nevertheless, the decrease in baseline motor function in these mice is not reversed completely by G-CSF.

Padrões de distribuição geográfica das espécies de Schefflera J. R. Forst. & G. Forst. (Araliaceae) do Brasil extra-amazônico

Uma análise da distribuição geográfica de Schefflera no Brasil extra-amazônico foi realizada com base em mapas atualizados plotando as ocorrências conhecidas das 26 espécies do gênero encontradas nessa grande área: S. angustissima (Marchal) Frodin, S. aurata Fiaschi, S. botumirimensis Fiaschi & Pirani, S. burchellii (Seem.) Frodin & Fiaschi, S. calva (Cham.) Frodin & Fiaschi, S. capixaba Fiaschi, S. cephalantha (Harms) Frodin, S. cordata (Taub.) Frodin & Fiaschi, S. distractiflora (Harms) Frodin, S. fruticosa Fiaschi & Pirani, S. gardneri (Seem.) Frodin & Fiaschi, S. glaziovii (Taub.) Frodin & Fiaschi, S. grandigemma Fiaschi, S. kollmannii Fiaschi, S. longipetiolata (Pohl ex DC.) Frodin & Fiaschi, S. lucumoides (Decne. & Planch. ex Marchal) Frodin & Fiaschi, S. macrocarpa (Cham. & Schltdl.) Frodin, S. malmei (Harms) Frodin, S. morototoni (Aubl.) Maguire, Steyermark & Frodin, S. racemifera Fiaschi & Frodin, S. ruschiana Fiaschi & Pirani, S. selloi (Marchal) Frodin & Fiaschi, S. succinea Frodin & Fiaschi, S. villosissima Fiaschi & Pirani, S. vinosa (Cham. & Schltdl.) Frodin & Fiaschi e S. aff. varisiana Frodin. Dois centros de endemismo associados com áreas de altitude elevada foram reconhecidos: Cadeia do Espinhaço em Minas Gerais e florestas montanas do Estado do Espírito Santo. Os padrões de distribuição geográfica ilustrados são discutidos com base em dados obtidos para outros grupos de angiospermas e em estudos fitogeográficos das principais fitocórias do Brasil extra-amazônico. São apresentadas também hipóteses acerca de prováveis relações filogenéticas entre alguns táxons, visando à busca de possíveis correlações entre estas e a biogeografia do grupo.