Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes.

G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions with G-Quadruplex DNA.

G-quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis-indoles (3,3-diindolyl-methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which for example, show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure-activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology.

Design, synthesis, and antiprotozoal evaluation of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives.

A series of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline, and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives was designed, synthesised, and evaluated in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiprotozoal activity with IC50 values in the µM range. In addition, the in vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The quinoline 1c was identified as the most potent antimalarial candidate with a ratio of cytotoxic to antiparasitic activities of 97 against the P. falciparum CQ-sensitive strain 3D7. The quinazoline 3h was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 43 on T. brucei brucei strain. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma are possible targets of this kind of nitrogen heterocyclic compounds, we have also investigated stabilisation of the Plasmodium and Trypanosoma telomeric G-quadruplexes by our best compounds through FRET melting assays.

Self-Assembly of a Guanosine Derivative To Form Nanostructures and Transmembrane Channels.

We herein report the self-assembly of a lipophilic bromoguanosine derivative (G1) in homogeneous solution, in the solid state and in planar bilayer membranes. The self-assembly of G1, driven by H-bonding and π-π stacking interactions can form different nano-structures depending on incubation time. The G1 nanostructure is able to bind a bioactive dye like Rose Bengal. In crystal state, it shows ribbon type H-bonding pattern and exhibits birefringence in polarized light. And further, the self-assembled nanostructure of G1 can form discrete transmembrane ion channels in lipid bilayer membranes, enabling passage of potassium ions.

Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives.

Novel series of bis- and tris-pyrrolo[1,2-a]quinoxaline derivatives 1 were synthesized and tested for in vitro activity upon the intraerythrocytic stage of W2 and 3D7 Plasmodium falciparum strains. Biological results showed good antimalarial activity with IC50 in the µM range. In attempting to investigate the large broad-spectrum antiprotozoal activities of these new derivatives, their properties toward Leishmania donovani were also investigated and revealed their selective antiplasmodial profile. In parallel, the in vitro cytotoxicity of these molecules was assessed on the human HepG2 cell line. Structure-activity relationships of these new synthetic compounds are discussed here. The bis-pyrrolo[1,2-a]quinoxalines 1n and 1p were identified as the most potent antimalarial candidates with selectivity index (SI) of 40.6 on W2 strain, and 39.25 on 3D7 strain, respectively. As the telomeres of the parasite could constitute an attractive target, we investigated the possibility of targeting Plasmodium telomeres by stabilizing the Plasmodium telomeric G-quadruplexes through a FRET melting assay by our new compounds.

Design, Synthesis and Biological Evaluation of New Substituted Diquinolinyl-Pyridine Ligands as Anticancer Agents by Targeting G-Quadruplex.

G-quadruplexes (G4) are stacked non-canonical nucleic acid structures found in specific G-rich DNA or RNA sequences in the human genome. G4 structures are liable for various biological functions; transcription, translation, cell aging as well as diseases such as cancer. These structures are therefore considered as important targets for the development of anticancer agents. Small organic heterocyclic molecules are well known to target and stabilize G4 structures. In this article, we have designed and synthesized 2,6-di-(4-carbamoyl-2-quinolyl)pyridine derivatives and their ability to stabilize G4-structures have been determined through the FRET melting assay. It has been established that these ligands are selective for G4 over duplexes and show a preference for the parallel conformation. Next, telomerase inhibition ability has been assessed using three cell lines (K562, MyLa and MV-4-11) and telomerase activity is no longer detected at 0.1 µM concentration for the most potent ligand 1c. The most promising G4 ligands were also tested for antiproliferative activity against the two human myeloid leukaemia cell lines, HL60 and K562.

A DNA-inspired synthetic ion channel based on G-C base pairing.

A dinucleoside containing guanosine and cytidine at the end groups has been prepared using a modular one-pot azide-alkyne cycloaddition. Single channel analysis showed that this dinucleoside predominantly forms large channels with 2.9 nS conductance for the transport of potassium ions across a phospholipid bilayer. Transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy studies reveal that this dinucleoside can spontaneously associate through Watson-Crick canonical H-bonding and π-π stacking to form stable supramolecular nanostructures. Most importantly, the ion channel activity of this G-C dinucleoside can be inhibited using the nucleobase cytosine.

Environment-sensitive probes containing a 2,6-diethynylpyridine motif for fluorescence turn-on detection and induction of nanoarchitectures of human telomeric quadruplex.

Bis(phenylethynyl)pyridylcarboxamides with amide side chains at the para position of the NH2 group possess strong solvatochromic properties compared with the meta analogues. Fluorescence binding titrations show that these probes exhibit remarkable fluorescence turn-on responses upon interacting with the human telomeric G-quadruplex (h-TELO). Förster resonance energy transfer melting analysis shows the high selectivity of these probes for h-TELO over duplex DNA. Isothermal titration calorimetry, as well as UV/Vis and fluorescence spectroscopy studies, show that the meta analogue has a twofold binding affinity for h-TELO over the para analogue. The noncovalent interaction of these small-molecule probes with h-TELO has been used to regulate the assembly of novel supramolecular nanoarchitectures.

Triazole-tailored guanosine dinucleosides as biomimetic ion channels to modulate transmembrane potential.

A "click" ion channel platform has been established by employing a clickable guanosine azide or alkyne with covalent spacers. The resulting guanosine derivatives modulated the traffic of ions across the phospholipid bilayer, exhibiting a variation in conductance spanning three orders of magnitude (pS to nS). Förster resonance energy transfer studies of the dansyl fluorophore with the membrane binding fluorophore Nile red revealed that the dansyl fluorophore is deeply embedded in the phospholipid bilayer. Complementary cytosine can inhibit the conductance of the supramolecular guanosine channels in the phospholipid bilayers.

A small molecule peptidomimetic that binds to c-KIT1 G-quadruplex and exhibits antiproliferative properties in cancer cells.

A modular synthesis of l-proline derived peptidomimetics has been developed using the Cu(I) catalyzed Huisgen cycloaddition between an azido prolinamide with pyridine and benzene dicarboxamide containing dialkynes. Förster Resonance Energy Transfer (FRET) melting assay provided an initial indication that the pyridyl analogue can stabilize the c-KIT1 quadruplex DNA. A competitive FRET-melting assay and Fluorescent Intercalator Displacement (FID) assay suggest that the pyridyl ligand shows excellent selectivity for c-KIT1 quadruplex over duplex DNA and other investigated G-quadruplexes. Molecular docking studies indicate that the pyridyl ligand can adopt unique conformations upon binding to c-KIT1 quadruplex due to the presence of intramolecular hydrogen bonds. The pyridyl ligand can perturb cell cycle progression and induce necrotic cell death of human hepatocellular liver carcinoma HepG2 cells.

A fluorescent guanosine dinucleoside as a selective switch-on sensor for c-myc G-quadruplex DNA with potent anticancer activities.

Like likes like! A novel fluorescent C2 -symmetric guanosine-based dinucleoside has been engineered by chemical ligation of two guanosine units with a biocompatible dansyl tag. The nucleoside exhibits high selectivity for c-myc G-quadruplex DNA through fluorescence enhancement over duplex DNA and other promoter G-quadruplexes (see scheme). It stains the nucleus preferentially, arrests the cell cycle at the G2/M phase, inhibits cell growth, and induces apoptosis in A375 cancer cells.

The effect of side chain variations on quinazoline-pyrimidine G-quadruplex DNA ligands.

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

Synthesis of bis-indole carboxamides as G-quadruplex stabilizing and inducing ligands.

The design and synthesis of a series of bis-indole carboxamides with varying amine containing side chains as G-quadruplex DNA stabilising small molecules are reported. Their interactions with quadruplexes have been evaluated by means of Förster resonance energy transfer (FRET) melting analysis, UV/Vis spectroscopy, circular dichroism spectroscopy and molecular modelling studies. FRET analysis indicates that these ligands exhibit significant selectivity for quadruplex over duplex DNA, and the position of the carboxamide side chains is of paramount importance in G-quadruplex stabilisation. UV/Vis titration studies reveal that bis-indole ligands bind tightly to quadruplexes and show a three- to fivefold preference for c-kit2 over h-telo quadruplex DNA. CD studies revealed that bis-indole carboxamide with a central pyridine ring induces the formation of a single, antiparallel, conformation of the h-telo quadruplex in the presence and absence of added salt. The chirality of h-telo quadruplex was transferred to the achiral ligand (induced CD) and the formation of a preferred atropisomer was observed.

Diffusion and birefringence of bioactive dyes in a supramolecular guanosine hydrogel.

Transparent self-standing supramolecular hydrogels were readily prepared by the potassium-ion-mediated self-organization of guanosine and 8-bromoguanosine whilst the individual components precipitated within a few hours. VT-NMR spectroscopy showed that bromoguanosine was a superior gelator compared to guanosine. XRD analysis showed that gel formation was caused by stacked G-quartets. AFM analysis revealed dendritic architectures of the nanofibers in the two-component hydrogel network. DSC profiles showed that the hybrid hydrogels underwent sol-gel transition at lower temperature than the pure guanosine and bromoguanosine hydrogels. Interestingly, bioactive dyes, such as rose bengal, rhodamine-6-G, and fluorescein, could be diffused and released in a controlled manner. UV/Vis absorption and fluorescence spectroscopy and CLSM were used to investigate the diffusion behavior of dyes in the hydrogel network. These dyes exhibited strong birefringence in the gel network (0.07-0.1) as a result of the anisotropic organization.

Macrocyclization of bis-indole quinolines for selective stabilization of G-quadruplex DNA structures.

The recognition of G-quadruplex (G4) DNA structures as important regulatory elements in biological mechanisms, and the connection between G4s and the evolvement of different diseases, has sparked interest in developing small organic molecules targeting G4s. However, such compounds often lack drug-like properties and selectivity. Here, we describe the design and synthesis of a novel class of macrocyclic bis-indole quinolines based on their non-macrocyclic lead compounds. The effects of the macrocyclization on the ability to interact with G4 DNA structures were investigated using biophysical assays and molecular dynamic simulations. Overall, this revealed compounds with potent abilities to interact with and stabilize G4 structures and a clear selectivity for both G4 DNA over dsDNA and for parallel/hybrid G4 topologies, which could be attributed to the macrocyclic structure. Moreover, we obtained knowledge about the structure-activity relationship of importance for the macrocyclic design and how structural modifications could be made to construct improved macrocyclic compounds. Thus, the macrocyclization of G4 ligands can serve as a basis for the optimization of research tools to study G4 biology and potential therapeutics targeting G4-related diseases.

A site-specific self-assembled light-up rotor probe for selective recognition and stabilization of c-MYC G-quadruplex DNA.

Direct and unambiguous evidence of the formation of G-quadruplexes (G4s) in human cells have shown their implication in several key biological events and has emphasized their role as important targets for small-molecule cancer therapeutics. Here, we report on the first example of a self-assembled molecular-rotor G4-binder able to discriminate between an extensive panel of G4 and non-G4 structures and to selectively light-up (up to 64-fold), bind (nanomolar range), and stabilize the c-MYC promoter G4 DNA. In particular, association with the c-MYC G4 triggers the disassembly of its supramolecular state (disaggregation-induced emission, DIE) and induces geometrical restrictions (motion-induced change in emission, MICE) leading to a significant enhancement of its emission yield. Moreover, this optical reporter is able to selectively stabilize the c-MYC G4 and inhibit DNA synthesis. Finally, by using confocal laser-scanning microscopy (CLSM) we show the ability of this compound to localize primarily in the subnuclear G4-rich compartments of cancer cells. This work provides a benchmark for the future design and development of a new generation of smart sequence-selective supramolecular G4-binders that combine outstanding sensing and stability properties, to be utilized in anti-cancer therapy.

Asian wasp envenomation and acute renal failure: a report of two cases.

Acute renal failure is an unusual complication of wasp stings. We report two cases of renal failure after multiple wasp stings (Vespa affinis). Both patients had evidence of intravascular haemolysis, hepatic dysfunction, oligo-anuria and azotaemia and required dialysis. The first patient had severe hemolysis, rhabdomyolysis, pigment and venom nephropathy and died on the 8th day in hospital. The second patient, who recovered completely in 3 weeks time with steroid and antihistaminic therapy, had interstitial nephritis. Although acute renal failure after wasp stings is typically caused by acute tubular necrosis (ATN) in the setting of haemolysis or rhabdomyolysis, in some patients, acute renal failure may result from a direct nephrotoxic effect or acute interstitial nephritis from a hypersensitivity reaction.

Design, synthesis, and antiprotozoal evaluation of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives.

A series of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives was synthesized, and the compounds were screened in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiparasitic activity with IC50 values in the µm range. The in vitro cytotoxicity of these molecules was assessed by incubation with human HepG2 cells; for some derivatives, cytotoxicity was observed at significantly higher concentrations than antiparasitic activity. The 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline 1h was identified as the most potent antimalarial candidate with ratios of cytotoxic-to-antiparasitic activities of 107 and 39 against a chloroquine-sensitive and a chloroquine-resistant strain of P. falciparum, respectively. As the telomeres of the parasite P. falciparum are the likely target of this compound, we investigated stabilization of the Plasmodium telomeric G-quadruplexes by our phenanthroline derivatives through a FRET melting assay. The ligands 1f and 1m were noticed to be more specific for FPf8T with higher stabilization for FPf8T than for the human F21T sequence.