Sara N. Richter

Quinone Methides Tethered to Naphthalene Diimides as Selective G-Quadruplex Alkylating Agents

We have developed novel G-quadruplex (G-4) ligand/alkylating hybrid structures, tethering the naphthalene diimide moiety to quaternary ammonium salts of Mannich bases, as quinone-methide precursors, activatable by mild thermal digestion (40 degrees C). The bis-substituted naphthalene diimides were efficiently synthesized, and their reactivity as activatable bis-alkylating agents was investigated in the presence of thiols and amines in aqueous buffered solutions. The electrophilic intermediate, quinone-methide, involved in the alkylation process was trapped, in the presence of ethyl vinyl ether, in a hetero Diels-Alder [4 + 2] cycloaddition reaction, yielding a substituted 2-ethoxychroman. The DNA recognition and alkylation properties of these new derivatives were investigated by gel electrophoresis, circular dichroism, and enzymatic assays. The alkylation process occurred preferentially on the G-4 structure in comparison to other DNA conformations. By dissecting reversible recognition and alkylation events, we found that the reversible process is a prerequisite to DNA alkylation, which in turn reinforces the G-quadruplex structural rearrangement.

Naphthalene diimide scaffolds with dual reversible and covalent interaction properties towards G-quadruplex

Selective recognition and alkylation of G-quadruplex oligonucleotides has been achieved by substituted naphathalene diimides (NDIs) conjugated to engineered phenol moieties by alkyl-amido spacers with tunable length and conformational mobility. FRET-melting assays, circular dichroism titrations and gel electrophoresis analysis have been carried out to evaluate both reversible stabilization and alkylation of the G-quadruplex. The NDIs conjugated to a quinone methide precursor (NDI-QMP) and a phenol moiety by the shortest alkyl-amido spacer exhibited a planar and fairly rigid geometry (modelled by DFT computation). They were the best irreversible and reversible G-quadruplex binders, respectively. The above NDI-QMP was able to alkylate the telomeric G-quadruplex DNA in the nanomolar range and resulted 100-1000 times more selective on G-quadruplex versus single- and double-stranded oligonucleotides. This compound was also the most cytotoxic against a lung carcinoma cell line.

Photogeneration and Reactivity of Naphthoquinone Methides as Purine Selective DNA Alkylating Agents

A one-step protecting-group-free synthesis of both 6-hydroxy-naphthalene-2-carbaldehyde and the bifunctional binaphthalenyl derivative afforded 6-hydroxymethylnaphthalen-2-ol, 6-methylaminomethyl-naphthalen-2-ol, [(2-hydroxy-3-naphthyl)methyl]trimethyl ammonium iodide, and a small library of bifunctional binol analogues in good yields. Irradiation of naphthol quaternary ammonium salt and binol-derivatives (X = OH, NHR, NMe(3)(+), OCOCH(3), and L-proline) at 310 and 360 nm resulted in the photogeneration of the 2,6-naphthoquinone-6-methide (NQM) and binol quinone methide analogues (BQMs) by a water-mediated excited-state proton transfer (ESPT). The hydration, the mono- and bis-alkylation reactions of morpholine and 2-ethanethiol, as N and S prototype nucleophiles, by the transient NQM (λ(max) 310, 330 nm) and BQMs (λ(max) 360 nm) were investigated in water by product distribution analysis and laser flash photolysis (LFP). Both the photogeneration and the reactivity of NQM and BQMs exhibited striking differences. BQMs were at least 2 orders of magnitude more reactive than NQM, and they were generated much more efficiently from a greater variety of photoprecursors including the hydroxymethyl, quaternary ammonium salt and several binol-amino acids. On the contrary, the only efficient precursor of NQM was the quaternary ammonium salt. All water-soluble BQM precursors were further investigated for their ability to alkylate and cross-link plasmid DNA and oligonucleotides by gel electrophoresis: the BQMs were more efficient than the isomeric o-BQM (binol quinone methide analogue of 2,3-naphthoquinone-3-methide). Sequence analysis by gel electrophoresis, HPLC, and MS showed that the alkylation occurred at purines, with a preference for guanine. In particular, a BQM was able to alkylate N7 of guanines resulting in depurination at the oligonucleotide level, and ribose loss at the nucleotide level. The photoreactivity of BQM precursors translated into photocytotoxic and cytotoxic effects on two human cancer cell lines: in particular, one compound showed promising selectivity index on both cell lines.

A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter.

G-Quadruplexes, noncanonical nucleic acid structures, act as silencers in the promoter regions of human genes; putative G-quadruplex forming sequences are also present in promoters of other mammals, yeasts, and prokaryotes. Here we show that also the HIV-1 LTR promoter exploits G-quadruplex-mediated transcriptional regulation with striking similarities to eukaryotic promoters and that treatment with a G-quadruplex ligand inhibits HIV-1 infectivity. Computational analysis on 953 HIV-1 strains substantiated a highly conserved G-rich sequence corresponding to Sp1 and NF-κB binding sites. Biophysical/biochemical analysis proved that two mutually exclusive parallel-like intramolecular G-quadruplexes, stabilized by small molecule ligands, primarily fold in this region. Mutations disrupting G-quadruplex formation enhanced HIV promoter activity in cells, whereas treatment with a G-quadruplex ligand impaired promoter activity and displayed antiviral effects. These findings disclose the possibility of inhibiting the HIV-1 LTR promoter by G-quadruplex-interacting small molecules, providing a new pathway to development of anti-HIV-1 drugs with unprecedented mechanism of action.

Targeting Loop Adenines in G-Quadruplex by a Selective Oxirane

Caught in the oxirane: Naphthalene diimides conjugated to a quinone methide and an oxirane have been synthesized and investigated as selective DNA G-quadruplex alkylating agents. The oxirane derivative generates a stable adduct with a G-quadruplex and shows selective alkylation of the loop adenines, as illustrated.

KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011: massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units

BackgroundKlebsiella pneumoniae carbapenemases (KPCs) producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams, therefore worryingly limiting antimicrobial treatment options. Analysis of circulating bacterial strains and KPC alleles may help understanding the route of KPC dissemination and therefore help containing the infection.MethodsKPC-producing Klebsiella pneumoniae dissemination in two 1580- and 300- bed hospitals in Padua, Italy, from initial outbreak in 2009 to late 2011 was analysed. Molecular and clinical epidemiology, including bacterial strains, KPC-encoding plasmid sequences and associated resistance genes, involved hospital wards and relocation of patients were described. Routine antimicrobial susceptibility testing and MIC of carbapenems on clinical isolates were performed. Detection of resistance genes was obtained by PCR and sequencing. MLST, PFGE and ERIC were used for molecular genotyping. Plasmid analysis was obtained by digestion with restriction enzymes and deep sequencing.ResultsKPC-positive clinical samples were isolated from nearly 200 patients. In the initial outbreak intensive care units were almost exclusively involved, while medical, surgical and long-term wards were successively massively concerned. Analysis of KPC alleles, plasmids and bacterial sequence types (STs) indicated that during the initial outbreak KPC-3 in ST258 and KPC-2 in ST147 were each confined in one of the two surveilled hospitals. While KPC-2 dissemination was effectively contained, KPC-3 in ST258 cross-spreading was observed. The simultaneous presence of two carbapenemases, VIM-1 and KPC-2, in the same isolate was also observed in three patients. Total sequencing of plasmid content of two KPC-3 strains showed novel association of resistance plasmids.ConclusionsThe acquired molecular epidemiology demonstrated that 1) both acquisitions from outward sources and patient relocation within the hospitals were responsible for the observed spreading; 2) KPC-3-encoding Klebsiella pneumoniae ST258 prevailed over other strains. In addition, the described massive transfer of KPC-mediated resistance to non-intensive care units may anticipate spreading of resistance to the non-hospitalized population. Therefore, genotypic analysis alongside phenotypic identification of carbapenemase producers, also at the carriage state, is advisable to prevent and contain further carbapenemase resistance dissemination.

Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands

Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK(a) 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC(50) of 300 nM.

Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity

G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment.

The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex: Implications for the antiviral activity of a G-quadruplex ligand.

Abstract Guanine-rich nucleic acids can fold into G-quadruplexes, secondary structures implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. The remarkably high guanine content of the Herpes Simplex Virus-1 (HSV-1) genome prompted us to investigate both the presence of G-quadruplex forming sequences in the viral genome and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with a novel mechanism of action. Using biophysical, molecular biology and antiviral assays, we showed that the HSV-1 genome displays multiple clusters of repeated sequences that form very stable G-quadruplexes. These sequences are mainly located in the inverted repeats of the HSV-1 genome. Treatment of HSV-1 infected cells with the G-quadruplex ligand BRACO-19 induced inhibition of virus production. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. This work, presents the first evidence of extended G-quadruplex sites in key regions of the HSV-1 genome, indicates the possibility to block viral DNA replication by G-quadruplex-ligand and therefore provides a proof of concept for the use of G-quadruplex ligands as new anti-herpetic therapeutic options.

Antimicrobial Treatment and Containment Measures for an Extremely Drug-Resistant Klebsiella pneumoniae ST101 Isolate Carrying pKPN101-IT, a Novel Fully Sequenced blaKPC-2 Plasmid

ABSTRACT An extremely drug-resistant Klebsiella pneumoniae isolate, sequence type ST101, was isolated from a patient in Italy. We describe antibiotic treatment, measures to clear and contain the infection, and a complete sequence analysis of a novel large plasmid, pKPN101-IT, harboring the bla KPC-2 gene and arising from the threatening recombination of different worldwide-distributed backbones.