Johans Fakhoury

Rolling Circle Amplification-Templated DNA Nanotubes Show Increased Stability and Cell Penetration Ability

DNA nanotubes hold promise as scaffolds for protein organization, as templates of nanowires and photonic systems, and as drug delivery vehicles. We present a new DNA-economic strategy for the construction of DNA nanotubes with a backbone produced by rolling circle amplification (RCA), which results in increased stability and templated length. These nanotubes are more resistant to nuclease degradation, capable of entering human cervical cancer (HeLa) cells with significantly increased uptake over double-stranded DNA, and are amenable to encapsulation and release behavior. As such, they represent a potentially unique platform for the development of cell probes, drug delivery, and imaging tools.

Platinum(II) phenanthroimidazoles for targeting telomeric G-quadruplexes.

A rationally designed progression of phenanthroimidazole platinum(II) complexes were examined for their ability to target telomere‐derived intramolecular G‐quadruplex DNA. Through the use of circular dichroism, fluorescence displacement assays, and molecular modeling we show that these complexes template and stabilize G‐quadruplexes from sequences based on the human telomeric repeat (TTAGGG)n. The greatest stabilization was observed for the p‐chlorophenyl derivative 6 (G4DC50=0.31 μM). We also show that the G‐quadruplex binding complexes are able to inhibit telomerase activity through a modified telomerase repeat amplification protocol (TRAP‐LIG assay). Preliminary cell studies show that complex 6 is preferentially cytotoxic toward cancer over normal cell lines, indicating its potential use in cancer therapy.

Sequence-responsive unzipping DNA cubes with tunable cellular uptake profiles

Here, we demonstrate a new approach for the design and assembly of a dynamic DNA cube with an addressable cellular uptake profile. This cube can be selectively unzipped from a 3D to a flat two-dimensional structure in the presence of a specific nucleic acid sequence. Selective opening is demonstrated in vitro using a synthetic RNA marker unique to the LNCaP human prostate cancer cell line. A robust uptake in LNCaP cells, HeLa cells (human cervical cancer) and primary B-lymphocytes isolated from the blood of chronic lymphocytic leukemia (CLL) patients is observed using fluorescence-activated cell sorting (FACS), confocal microscopy and a new cluster analysis algorithm combined with image cross-correlation spectroscopy. The DNA cube was modified with hydrophobic and hydrophilic dendritic chains that were found to coat its exterior. The dynamic unzipping properties of these modified cubes were retained, and assessment of cellular uptake shows that the hydrophobic chains help with the rapid uptake of the constructs while the hydrophilic chains become advantageous for long term internalization.

Harnessing Telomerase in Cancer Therapeutics

Telomerase is an attractive target for anti-cancer therapeutics due to its requirement for cellular immortalization and expression in greater than 85% of human neoplasms. Though initially promising, strategies that inhibit telomerase with either small molecules or antisense oligonucleotides have a major limitation, namely the lag time required for telomere shortening before cellular effects are attained. As alternative approaches, immunotherapy and gene therapy have been tailored to exploit, rather than antagonize telomerase expression and/or activity. Immunotherapy requires the presence of the catalytic subunit of telomerase, hTERT, to elicit an immune response directed towards hTERT peptide-presenting cells. hTERT promoter-driven gene therapy and mutant telomerase RNA (hTR) gene therapy depend on the innate telomerase activity of cancer cells to drive the expression of pro-apoptotic genes and to synthesize mutated DNA sequences onto telomeres, respectively. In addition, we will discuss telomestatin, a G-quadruplex binding ligand that may exert anti-proliferative effects independently of telomere shortening. In this review, the progress, advantages, and limitations of these strategies in the ongoing effort to develop clinically relevant telomerase-based cancer therapeutics will be examined.

A Platinum(II) Phenylphenanthroimidazole with an Extended Side‐Chain Exhibits Slow Dissociation from a c‐Kit G‐Quadruplex Motif

A series of three platinum(II) phenanthroimidazoles each containing a protonable side-chain appended from the phenyl moiety through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) were evaluated for their capacities to bind to human telomere, c-Myc, and c-Kit derived G-quadruplexes. The side-chain has been optimized to enable a multivalent binding mode to G-quadruplex motifs, which would potentially result in selective targeting. Molecular modeling, high-throughput fluorescence intercalator displacement (HT-FID) assays, and surface plasmon resonance (SPR) studies demonstrate that complex 2 exhibits significantly slower dissociation rates compared to platinum phenanthroimidazoles without side-chains and other reported G-quadruplex binders. Complex 2 showed little cytotoxicity in HeLa and A172 cancer cell lines, consistent with the fact that it does not follow a telomere-targeting pathway. Preliminary mRNA analysis shows that 2 specifically interacts with the ckit promoter region. Overall, this study validates 2 as a useful molecular probe for c-Kit related cancer pathways.

Telomeric function of mammalian telomerases at short telomeres.

Telomerase synthesizes telomeric sequences and is minimally composed of a reverse transcriptase (RT) known as TERT and an RNA known as TR. We reconstituted heterologous mouse (m) and human (h) TERT-TR complexes and chimeric mTERT-hTERT-hTR complexes in vitro and in immortalized human alternative lengthening of telomere (ALT) cells. Our data suggest that species-specific determinants of activity, processivity and telomere function map not only to the TR but also to the TERT component. The presence of hTERT-hTR, but not heterologous TERT-TR complexes or chimeric mTERT-hTERT-hTR complexes, significantly reduced the percentage of chromosomes without telomeric signals in ALT cells. Moreover, heterologous and chimeric complexes were defective in recruitment to telomeres. Our results suggest a requirement for several hTERT domains and interaction with multiple proteins for proper recruitment of telomerase to the shortest telomeres in human ALT cells. Late-passage mTERT−/− mouse embryonic stem (ES) cells ectopically expressing hTERT or mTERT harboured fewer chromosome ends without telomeric signals and end-to-end fusions than typically observed in late-passage mTERT−/− ES cells. The ability of hTERT to function at mouse telomeres and the inability of mTERT to function at human telomeres suggest that mechanisms regulating the recruitment and activity of hTERT at mouse telomeres might be less stringent than the mechanisms regulating mTERT at human telomeres.

Nucleobase peptide amphiphiles

A new class of peptide materials is introduced, integrating orthogonal aspects of peptide, nucleoside, and amphiphile chemistry. In solution, species such as rod-like or helical micelles are formed, which can lead to nanoribbons under lateral or longitudinal hierarchical growth regimes. Gelation of a wide range of solvents can be induced, including water and aqueous buffer, providing new avenues for nucleobase-specific electrophoresis, oligonucleotide delivery and bioactive cell growth media.

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.

Cellular Studies of an Aminoglycoside Potentiator Reveal a New Inhibitor of Aminoglycoside Resistance

Aminoglycosides are a group of broad‐spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside‐modifying enzymes has, however, dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators that work by inhibiting aminoglycoside N‐6′‐acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among these, prodrugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here a series of cellular studies to investigate the activity and mechanism of action of these prodrugs further. Our results reveal a new aminoglycoside resistance inhibitor, as well as the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability, and potentiation of various aminoglycosides, against both Gram‐positive and Gram‐negative bacteria, make them interesting compounds for the development of new drugs.