5' to 3' Unfolding Directionality of DNA Secondary Structures by Replication Protein A: G-QUADRUPLEXES AND DUPLEXES.

The replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5' toward 3'. We explain the 5' to 3' unfolding directionality in terms of the 5' to 3' oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding.

G-quadruplexes unfolding by RHAU helicase.

G-quadruplexes (G4) are RNA and DNA secondary structures formed by the stacking of guanine quartets in guanine rich sequences. Quadruplex-prone motifs may be found in key genomic regions such as telomeres, ribosomal DNA, transcriptional activators and regulators or oncogene promoters. A number of proteins involved in various biological processes are able to interact with G4s. Among them, proteins dedicated to nucleic acids unwinding such as WRN, BLM, FANCJ or PIF1, can unfold G4 structures. Mutations of these helicases are linked to genome instability and to increases in cancer risks. Here, we present a high-throughput fluorescence-based reliable, inexpensive and fast assay to study G4/RHAU interaction. RHAU is an RNA helicase known as the major source of G4 resolution in HeLa cells. Our assay allows to monitor the unfolding properties of RHAU towards DNA and RNA quadruplexes in parallel and to screen for the optimal conditions for its activity. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.

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.

A Flexible Terpyridine Derivative Interacts Specifically with G-Quadruplexes.

G-quadruplexes formed by nucleic acids are implicated in pathologies ranging from cancers to neurodegenerative diseases. We evaluated interactions of 29 bi- and terpyridine derivatives with G-quadruplexes and duplexes. FRET-melting, circular dichroism, and (1) H NMR spectroscopy showed that one terpyridine derivative interacted very selectively with G-quadruplexes. This G-quadruplex ligand inhibited helicase activity and should influence G-quadruplex-related biological processes.

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.

Design, Synthesis, and Evaluation of 2,9-Bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline Derivatives as G-Quadruplex Ligands.

Genomic sequences able to form guanine quadruplexes (G4) are found in oncogene promoters, in telomeres, and in 5'- and 3'-untranslated regions as well as introns of messenger RNAs. These regions are potential targets for drugs designed to treat cancer. Herein, we present the design and syntheses of ten new phenanthroline derivatives and characterization of their interactions with G4-forming oligonucleotides. We evaluated ligand-induced stabilization and specificity and selectivity of ligands for various G4 conformations using FRET-melting experiments. We investigated the interaction of compound 1 a (2,9-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-1,10-phenanthroline), which combined the greatest stabilizing effect and specificity for G4, with human telomeric sequences using FRET, circular dichroism, and ESI-MS. In addition, we showed that compound 1 a interferes with the G4 helicase activity of Saccharomyces cerevisiae Pif1. Interestingly, compound 1 a was significantly more cytotoxic toward two human leukemic cell lines than to normal human blood mononuclear cells. These novel phenanthroline derivatives will be a starting point for further development and optimization of potent G4 ligands that have potential as anticancer agents.