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Exploring the periodic table with supramolecular G-quadruplexes

Exploring the periodic table with supramolecular G-quadruplexes.

via Exploring the periodic table with supramolecular G-quadruplexes.

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Structural studies of naphthalene diimide ligands with telomeric G-Quadruplex DNA

March 15, 2012 13 comments

Structural Basis for Telomeric G-Quadruplex Targeting by Naphthalene Diimide Ligands

Gavin W. Collie, Rossella Promontorio, Sonja M. Hampel, Marialuisa Micco, Stephen Neidle*, and Gary N. Parkinson*

J. Am. Chem. Soc., 2012, 134 (5), 2723; DOI: 10.1021/ja2102423

A synopsis by Maxier Acosta

Previously Neidle had reported a series on naphthalene diimide (ND) oligo G-quadruplex (OGQ) ligands with side-chains (n) of 3-5 carbons with N-methyl-piperazine end groups. They showed experimentally how it inhibited binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibited telomerase activity in MCF7 cells via the stabilization of OGQs (DOI: 10.1016/j.bmcl.2010.09.066). Now, in collaboration with Parkinson, they report the crystalline structure of each one of those naphthalene ligands with the addition of a two-carbons side-chain.

They first give an overview of the tendencies of the overall parallel OGQ (Gtel22) with each ND ligand. With the telomere sequence d(AGGG[TTAGGG]3) they highlight the stacking of two OGQs making a dimer interacting from the 5’ terminal G-quartet. But the ratio between the ND and each OGQ is 1:1. Taking this in consideration, when each ND is bound to the quadruplexes, they force the topology of the loops into parallel strands as first proposed in DOI: 10.1016/j.bmcl.2010.09.066. While going more into detail, stability studies via FRET and inhibition studies where done for each ND. In the case of the ND with a two-carbons side-chain, it didn’t enhanced by much the stability of the Gtel22 due to the inappropriate side-chain length to enable effective interactions (in the OGQ groove) between the protonated N-methyl-piperazine and the DNA backbone phosphates. Although the n=5 ND OGQ complex showed poor quality in its crystal diffraction, it was still higher than that corresponding to n=2. For the n=4 ND, the side-chains were too long to fit well into the grooves as indicated by the disorder of the chains leading to a decrease of strong specific contacts, yet it was still more stabilizing than n=5 ND. For n=3 ND, it was observed that the cation-phosphate interactions were specifically coordinated, making it the best ligand of the small library presented in the paper. The structural features for these ND ligands correlated well with the inhibition of two types of cancer cells (MCF7 and A549).

In the discussion they summarized the data in three major topics: (1) the 1:1 binding of ND and OGQs; (2) the importance of the electrostatic side-chain interaction with the groove; and (3) the retention of the parallel topology of the Gtel22. Also, as might be expected for scientists from a pharmacy school they maintain their focus on how biologically relevant these binders could be for anticancer treatments.

In general, I thought that this was a good OGQ-binder structural article. I know that our systems are difficult to crystallize, yet this type of studies can help us to understand them to a new level so we could also start talking about potential inhibitors among other things. In terms of the organization of the paper, I found confusing the fact that they do not address explicitly some of the figures. In the discussion it was not that clear for me why the NDs induced the parallel topology; so, for that I encourage you guys to read the reference that I mentioned at the beginning, which has additional useful experimental data that may help anyone in the same situation. Other than this, I wish I had seen all of the ND side-chains interactions with the groove (some of them are in the supplementary information).

Raiders of the lost G-quadruplexes …in the human genome

March 14, 2012 15 comments

Small-molecule–induced DNA damage identifies alternative DNA structures in human genes

Raphaël Rodriguez, Kyle M Miller, Josep V Forment, Charles R Bradshaw, Mehran Nikan, Sébastien Britton, Tobias Oelschlaegel, Blerta Xhemalce, Shankar Balasubramanian* & Stephen P. Jackson*

Nature Chemical Biology 8, 301–310 (2012) doi: 10.1038/nchembio.780

A synopsis by Diana Silva Brenes

The authors of this week’s paper play detective to find out -with great detail- what exactly happens to a human cell when it’s treated with the versatile, potent GQ-binder, pyridostatin. Using a combination of biomolecular assays, the authors manage to give strong support for the in vivo formation of GQ-DNA in human cells, and show their role in the activity of the new drug.

Pyridostatin is shown to induce damage to cellular DNA, stumping their proliferation. This happens because cellular checkpoints, which revise DNA before continuing the cellular division cycle, detect the damage and signal to the cell that something is wrong. The cell stops in its tracks to try to correct the problem before it continues the cycle. The drug, however, isn’t too toxic and most cells can survive long-term exposition to it without undergoing apoptosis. Interestingly, inhibition of the checkpoints restores cell proliferation.

Many of the results rely on detecting the presence of γH2AX (a protein that indicates double strand breaks in DNA) as a way to follow damage done to DNA. In cells treated with pyridostatin, γH2AX is present during the DNA transcription and replication processes, pointing at damage to DNA occurring during both stages.

Next, the authors wanted to localize where in the DNA is pyridostatin taking effect. Fluorescence labeling of γH2AX and the telomeres (marked by the labeling of a telomere binding protein) didn’t show co-localization. It was, thus, necessary to modify the drug to add direct fluorescence labeling. Addition of an alkyne group to the drug allowed an in cellulo click reaction with an azide containing fluorescent dye. After making sure that the modified pyridostatin did not affect drug activity, staining of pyridostatin was performed and fluorescent spots (foci) were compared with the a fluorescently labeled human helicase reputed to bind and resolve GQ-DNA during replication. Good co-localization was observed, suggesting that pyridostatin was localized mostly at putative GQ-DNA sites. In another experiment they showed that addition of pyridostatin before of after “freezing” the cellular processes in formaldehyde gave almost identical results, suggesting that GQ structures are pre-folded even without addition of pyridostatin.

They then performed ChIP sequencing to try to figure out which genes (aka, DNA segment) were targeted by pyridostatin. They found several specific genes (mostly away from the telomeres) that sustained pyridostatin induced damage to DNA, and all of them had above average putative GQ sequences. However, not all areas enriched in putative GQ sequences were affected, suggesting that there are other important requirements for interaction.

A particularly affected gene was SRC as confirmed by checking for loss of its corresponding mRNA transcription activity. Out of 25 putative GQ sequences estimated for this gene, 23 of them could be observed to form QGs in vitro using CD and NMR spectra.

The effect of pyridostatin on the bioactivity of SRC was also evaluated. SRC is important for wound healing and motility of cells. Cells treated with pyridostatin displayed a reduced ability to heal. As a control, cells treated with another DNA-damaging drug (DOX), didn’t affect healing, proving that the deficiency was not due merely to DNA damage.

It was previously shown that pyridostatin binds to GQs with enough strength to resist polymerases. It is hypothesized that damage to DNA by pyridostatin is due to mechanical forces breaking the DNA during the cell’s attempt to transcribe or replicate DNA. The findings of this paper support the potential drugability of GQs in cells.

The data reported by this paper is really important for the field of GQ binders and raises large hopes for the future of the field. Being able to use GQ to recognize and regulate specific genes is a dream come true in drug design, and the authors present strong data as to the viability of this approach. As a chemist, it’s difficult to get used to the rather indirect type of evidence that supports these findings, making it hard for me to comment on this paper’s methods. However, the controls and the analyses they did appear to be adequate. Overall, I find the results in this paper to be really important to anyone in the GQ field.

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