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

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).

  1. March 15, 2012 at 03:25

    Parkinson and Neidle perform a detailed study of GQ binding ligands in this recent work. They study the interaction of a naphthalene diimide derivative with telomeric GQ. Basically, naphthalene diimide forms a 1:1 sandwich like interaction involving pi-pi interactions. But the most interesting part is the information that they obtain by crystallography, Molecular models and FRET melting experiments. In these experiments, we can see the role of the GQ loops and the naphthalene diimide chains in this 1:1 interaction. Practically, BMSG-SH-3 fits better than BMSG-SH-4 over the GQ and reflects a better stability in accordance of the FRET melting experiments. Also, BMSG-SH-3 and BMSG-SH-4 are better growth inhibitors than BMSG-SH-5.

    Despite the good correlation in stability between crystallography, Molecular models and FRET melting experiments, this experiments reveals good information about how potent GQ-ligands can interacts without disturbing GQ topology. More over, its interesting the role of water to maintain these side chain interactions due to the increased stability of this system. In every design, there is a limit of what components we can modulate before a desired molecular interaction. I always think this when I see the important role of water in molecular interactions. I think that rigidity (double bonds) of those side chains are the key for better stability. The naphthalene diimide core works well for fitting over the GQ surface, but the side chain affects a lot the stability and the growth inhibition activity of these derivatives. So, we can think in a good loop to interact better with the phosphate groups of the loops or a rigid side chain, which its movement can be limited and then the core will fit better over the GQ surface.

    About the article, I like the presented studies; I think that the stronger part of the article is the use of these potent growth inhibitors. Yes, we have a good work explaining how the specificity and binding is correlated with stability. But, more than crystallography that don’t provide enough information of the system behavior in solution or theoretical molecular models a good 2D H NMR experiments could give more support to the presented findings. I know that they provide some references, but for these kind of study was necessary.

    About Maxier synopsis, it’s well written in terms of presenting the main points of the article with their significance. He seems more critical and mature in these topics. The picture is cool, specially the conversations, its provide good information of the article.

  2. AJS
    March 15, 2012 at 06:07

    This week’s other paper from the Neidle/Parkinson groups investigates the binding of naphthalene diimide ligands to telomeric G-quadruplex sequences. Most of the series of ligands, with varying lengths of side chains, have been reported before, but here one more member is added; they report the synthesis of this, stability data (FRET assay; added to data already reported for the other compounds), toxicity data, and, the meat of the paper, X-ray crystal structures of the ligands bound to oligomeric DNA quadruplexes. Being a rather rare direct look at how ligands interact, in contrast to the large volumes of more indirect data being produced these days (and I don’t mean to knock it – crystal structures have their limitations, too), they take advantage to discuss the structure at length and relate it to the binding ability already observed. In particular, they note that this is the first direct evidence of something long believed in the community, that positively-charged side chains interact with the negatively-charged phosphate backbone to further stabilise interactions; this may well be the key to engineering greater selectivity in future GQ-binders.

    The paper is clear, with the discussion coherent and detailed; I often praise concision in these comments, and I’m not really able to do that today, but I suspect if I managed to crystallise one of these systems I would go on a lot longer. We do not see anything shocking, but it does provide solid evidence for suppositions that perhaps have previously been referred to with a little too much certainty. I am interested in their suggestions that one route to selectivity would be to make side chains bigger, that moving further away from orthodox “druglikeness” rather than towards it might be advantageous.

    Not having read it before, I can’t say if Maxier’s got the message over to me, but it is clear and informative; I will look forward to hearing about the other work he mentions in his presentation. His picture is fine.

  3. Diana S.
    March 15, 2012 at 11:59

    This paper discusses the crystal structures formed between the telomeric DNA sequence and each of two GQ binders. The authors carefully describe the interactions in the crystal structure and rationalize the binding modes of each compound. I was surprised that, according to the authors, this paper represents the first example of something I thought was commonly assumed, ie. the interaction between positively charged molecules with the GQ phosphate groups at the grooves. The interaction seems reasonable enough and I’m pretty sure there was no real doubt on its existence. Also, I hadn’t realized that, despite all the reported examples of GQ binders, this is the first crystallographic example of a 1:1 binding mode.
    The authors comment that the larger amount of possible contacts for derivative BMSG-SH-4 ligand could be detrimental for the formation of specific higher strength interactions. I’m curious whether this is a commonly viewed feature in ligand-macromolecule (protein, aptamer, etc.) interactions, as such a general conclusion should apply to other systems. I’m more inclined to follow the explanation that the larger size of the substituents makes it harder for them to fit in the DNA groove; that the angles taken on by the substituents to fit in the groove are detrimental to the overall energy and that the entropic price is slightly larger for these longer-chained molecules. The authors also highlight the temperature factors of the ligand. I find it an interesting observation, though electron density quality/diffusion is perhaps a more commonly discussed indication of the mobility of a given group in a crystal structure. Its nice that the authors compare the conclusions they can draw from crystal structures to the FRET melting experiments, which give good support to their observations.
    Maxier’s synopsis was nice and clear, and demonstrates that he’s been doing his background research. I was pleased at the level of analysis of his comments.

  4. Mariana
    March 15, 2012 at 13:17

    Continuing wit our GQ-binder week now we have a detail structural x-ray study for two binders that show to stabilize a GQ sequence in solid state and solution. The article shows four naphthalene diimide derivatives containing as side chains different length (2, 3, 4 and 5 carbon) of charge methyl-piperazine rings. They only were able to crystallize two of them (the 3 and 4 carbon length) showing their results (x-ray, FRET melting) that these two compounds have the ideal size and length to be use as effective binders (3>4). The short version was very poor stabilizing (molecular modeling, FRET melting) as well as the longer version (poor quality x-ray, FRET melting). The article is very appealing to read with good explanations an rationales, definitely very different from Diana’s article in that regard, although both articles works toward similar topics… in this case the authors are stating to take into consideration not only the lipophilic core of the drug, but as well to consider the side change that contribute to the overall complex stability (topic that I know other research have highlighted in other articles as well as the type of functionality substituted at those side chains ends). Additional electrostatic interactions are responsible to improve the stability (positive of side chains, negative of phosphates) and can even induce selectivity for different GQs. In term of contribution as the authors said: “We report here ON A STRUCTURE-BASED APPROACH to the design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA”
    Structural details fascinates chemist, making my reading delightful….
    I like that Maxier invites to read previous work and make a relative short summary of the article. We have discuss many times in here how nice and important can be crystal structures to help in the creation of new binders, but it always good to remember also that in solution the behavior can be slightly difference maybe driving to different types of additional interactions. The good think in this article is that they can correlate pretty well their structural characterization with the behavior in solution. The picture creative, although not my favorite. I don’t like animated molecules or atoms, that scary me…

  5. PGMS
    March 15, 2012 at 17:00

    The authors Neidle and Parkinson reported a structure-based approach to the design of ligands that bind selectively to G-quadruplexes to enhance affinity and selectivity for human telomeric DNA. The naphthalene diimide and closely related scaffolds have been developed as a suitable starting-point for quadruplex binding ligands due to their chemical accessibility and large planar surfaces. Particularly, they discussed the interaction of a series of tetrasubstituted naphthalene diimide-based (ND) compounds with telomeric GQs exhibiting 1:1 interaction. Various techniques like crystallography, molecular models and FRET melting experiments were adopted to study the interactions of these NDs.

    The results symbolized the appropriate positioning of the N-methyl-piperazine group is a major determinant of both binding site affinity and direct groove interactions for future ligand designs. The ND molecules in both
    structures are stacked effectively over the terminal 3’ G-quartet surfaces, with extensive π−π interactions
    resulting in a 1:1 quadruplex/ligand stoichiometry. Increase in side-chain length increased conformational flexibility, with n = 5 resulting in poor quality diffraction due to increased internal motion and reduced crystal order.

    This article appears very complicated but good structure based approach to design ligands.
    Maxier prepared a depictive picture and the synopsis is good.

  6. Berti
    March 15, 2012 at 19:58

    Here we have an article that overlaps in many aspects with the other blog article discussed by Diana. Both of them are focused in the finding of GQ binders. Althougth they are from different research groups and have a very different style of writing. My personal opinion is that I prefer the narrative, style and artistic work of Balasubramanian.
    In this one Parkinson and Neidle further investigate how four different naphthalene diimides (ND) interacts with 22-mer intramolecular human telomeric quadruplex (Gtel22). They also present previous data (ref. 14) to complement and enrich this story. ND is known to inhibit the binding of the telomeric DNA to hPOT1 and topoisomerase IIIa. It is also known that it inhibit telomerase activity in MCF7 cells. Here they do structural studies of the ligand-GQ complex by crystallographic methods and a melting experiment of ND for n=2. Four ND ligands were studied, each having a singular linker length (n= 2, 3, 4, 5) between the naphthalene center and amino groups. Melting data show that ND of n = 3-5 could stabilize GQ-DNA. The ligand with linker n=2 was studied by molecular model, no crystal structure was reported. This was the derivative with the higher IC-50s, and lower potency as growth inhibitor. The most potent inhibitors results to be the ND ligands of n=3 and 4 follow by 5. This is evidence that the protonated N-methyl-piperazine can make electrostatic interactions with the GQ-DNA phosphates.
    They conclude that its important to choose the appropriate length in the side chain to maximize electrostatic interaction between the N-methyl-piperazine of the drug and phosphate groups of the GQ-DNA. They also found that the 1:1 complex with GQ-DNA retains the parallel topology. Are these unanticipated conclusions???
    What I like the most of his article was Figure 5. It suggest that ND is a better binder than BRACO-19 other than that I think is one more article in the GQ area.
    Maxier did a good job in his synopsis and picture. I like and understood the picture. In contrast to other blogs where the picture speaks by itself, here it was necessary to read the article in order to understand the language that these molecules are speaking in the picture. It would have look nicer and more explanatory if the naphthalene diimide ligand was show clearer.

  7. Ana Victoria
    March 15, 2012 at 20:02

    The naphthalene diimide and other similar scaffolds have been developed as a starting-point for quadruplex binding ligands because of their accesibility and large planar surfaces. The article describes the development of an improved tetra-substituted naphthalene diimide compound by a structure-based approach using crystallography. An all-parallel topology is suggested for telomeric quadruplexes when complexed with high-affinity heteroaromatic ligands. However, the study presents two crystal structures of ND complexes with the sequence of the 22-mer intramolecular G-quadruplex that has been studied previously. The design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA was reported by the use of crystal structures. The structures done were those of a 22-mer intramolecular human telomeric quadruplex and two tetra-substituted naphthalene compounds that promote parallel-stranded quadruplex topology. One of the ligands presents less mobility in the crystal complex, but on the other hand it is more stabilizing because it forms more electrostatic and hydrogen bonds with the phosphate groups on the quadruplex. The data presents the 1:1 binding of a ND compound to a human intramoleclar telomeric G-quadruplex, the role of positive charged side chains and the retention of the parallel topology. Other experiments that were used to arrive at these conclusions were FRET and molecular modeling.
    Maxier’s synopsis is very good; he summarizes the article, presents the main points and gives his own analysis. The picture reveals important aspects of the article.

  8. Marilyn
    March 15, 2012 at 22:51

    In this week article, by Neidle, Parkinson and colleagues, two tetra-substituted naphthalene derivatives, functionalized with N-methyl-piperazine side-chains were studies as binders of parallel-stranded DNA-quadruplex. In specific they were study as binders for the 3’ end of each quadruplex. They started by making a detailed analysis of the interactions between the binders and telomeric DNA in the crystal structures. They identified that the main differences between the complexes are due to the ligand mobility and the interactions with the grooves. The ligand that show less mobility also stabilized more the quadruplex. This is accomplish by making multiple electrostatic and hydrogen bond interactions with the phosphate groups from the backbone in the grooves. From FRET and inhibition studies they identified the following trend, ligands of n=3 > 4 > 5. This system is also the first example of a crystal structure of a 1:1 complex.

    The data presented here is of great relevance because as I mention in my commend in Diana’s article in order to be able to rationalized the biophysical and biological data available it is important to have structural imformation available. This is also important in order to develop drugs for targeting telomeric DNA quadruplexes. On the style of the article, it was well written and the figures were well prepared, since all of them were self-explanatory. Although the figure from the abstract, TOC, were less successful. Maxier’s synopsis was to the point and well documented.

  9. Maria
    March 15, 2012 at 22:53

    At the moment, a substantial amount of diverse compounds have been tested as OGQ ligands looking primarily for those compounds: 1) imparting the highest stabilization on OGQs; 2) that can better differentiate between ds DNA vs OGQ structures and between different types of OGQ structures/topologies; and 3) with the most promising biological properties, primarily as telomerase inhibitors for their plausible use as anticancer treatments.

    In this article Neidle, Parkinson and colleagues reported a substantial contribution towards the design of more promising naphthalene diimide (ND) derivatives to target telomeric-G-quadruplexes. The four ND derivatives that were evaluated varied only in the length of their side-chains that have N-methyl-piperazine groups at the end and the OGQ used is a 22-mer d(AG3[T2AG3]3). Considering that all the NDs have the same central core, the general trend is that the efficiency of these derivatives is governed by an optimum complementarity between the grooves and the length of the side-chains as well as the size and charge of the terminal groups on the side-chains. That is why BMSG-SH-3 and -4 seem to be the more promising candidates, however the attractive interactions between the loops and BMSG-SH-3 side-chains kind of fix it better on top of the OGQ structure. It was interesting to see that there is a particular region of the BMSG-SH-3 molecule that interacts with the GQ through water contacts (a water network). I have seen that on GQ-RNA structures water networks are a special major player on the high stabilities that GQ-RNAs have when compared to the corresponding GQ-DNA structures. The IC50 for various cancer cells was calculated with similar value for BMSG-SH-3 and -4.

    The article was very nice since the narrative was engaging, the figures were very appropriate, and the discussion section was very informative. For example they compare their system with the structure of the OGQ-BRACO-19 complex, and by the end a discussion on how increasing the molecular weight might affect the biological intake of the compound if used as drug. My recommendation for the authors is that since they have a structure-based approach they might try to do more relevant in vivo experiments like some of those discussed on the article of Balasubramanian that we will discuss this week.
    Maxier synopsis has a very good introduction and it include a discussion of the main findings complimented with a funny picture.

  10. Jean
    March 16, 2012 at 00:19

    Neidle and co-workers specialize in the synthesis and analysis of G-Quadruplex ligands and their interactions with their hosts. This article focuses on a series of naphthalene diimide derivatives that were previously shown to interact strongly with GQ structures. The crystal structures of GQ sequences interacting with the drug have been solved. As it is to be expected the large aromatic surface of the ND derivatives interacts primarily via end-stacking interactions on the 3′ end of the quadruplex(π-π stacking). However what was actually quite impressive and the highlight of the article were the interactions between the side-chains of the derivatives and the grooves of the quadruplex. Changing the length of the side chains influenced tremendously the atomic mobility (B factor) indicating strong changes in affinity. The increase in affinity is due mainly to interactions between the charged groups of the ligand and the phosphate groups of the side chain. This exemplifies the importance of the side chains and the side-chain groups since different groups may induce different quadruplex topologies.

    Maxier’s picture grasps one of the most important aspects of the article: the side-chains. This and his really good and to the point synopsis help in the understanding of the article. I am looking forward to tomorrow’s presentation as I am sure he will do a good job.

  11. Rocio Roque
    March 16, 2012 at 00:25

    In this article, Parkinson and Neidle groups investigate GQ binding ligand, naphthalene diimide. They show how four different naphthalene diimides (ND) interact with the OGQ (Gtel22). Various techniques were used; crystallography, molecular models and FRET melting experiments to conduct the studies of the ND ligand with side-chains of different lengths (2 to 5 carbons). By the melting data they were able to determine that naphthalene diimide with lenghts of 3 and 4 carbons are better binders (because of their size and length). This proving the importance of length to maximize electrostatic interactions. Also, the data presented showed the 1:1 complex (GQ-DNA) maintained its parallel topology.

    I liked the article and they way the studies were presented. Maxier synopsis was nice and his picture was very creative, conveying the important aspects of the article. I look forward to his presentation tomorrow to clarify those doubts that are still lingering in my mind.

  12. Loruhama
    March 16, 2012 at 01:48

    In this article, Parkinson and Neidle present a series of tretrasubstituded naphthalene diimide-base (ND) compounds that act as stabilizers for telomeric G-Quadruplexes. As a consequence, these molecules can be used to inhibit the activity of the protein Telomerase and also hPTO1, which is another telomeric protein. These ND molecules form complexes with the telomeric GQs in a 1:1 ratio. Some of their studies suggested an all-parallel topology for telomeric quadruplexes when completed to these types of molecules. They also did FRET melting studies, which led them to understand that the BMSG-SH-3 had a better fit than the BMSG-SH-4 because it could stabilize more the GQ according to these experiments. They also compare this two with BMSG-SH-5 in the sense that the previous two seemed to be better inhibitors.
    I really enjoyed reading this paper. The topic was very interesting and they did a very good discussion of their data. However I do agree with Maxier in the fact that they don’t correlate very well the pictures. As for Maxier’s synopsys, he did a very good job explaining the main points in the paper and giving us his own feedback on the article. I liked the picture, it was very creative.

  13. Y. Melendez
    March 16, 2012 at 05:26

    In this article, Neidle et al. talk about naphthalene diimide derivatives that showed strong interactions with G-quadruplex structures. They explored the crystal structures for sequences of the GQ’s and the drug, These ND compounds had identical side-chains that had 3 to 5 carbons linking a positivelycharged methyl-piperazine to the naphthalene diimide. They found that this group was determinant factor in the affinity of the binding site as well as direct groove interactions. The fact that these complexes observe 1:1 interactions was attributed to the aforementioned findings. The FRET data showed that all the compounds in the article have the ability to stabilize human telomeric GQ-DNA considerably, with BMSG-SH-3 being the most favorable.

    I think this was a very enjoyable article and it read pretty easily. Maxier also did a good job of understanding it and expressing the key points, as well as supplying us with the data he himself needed to understand the aspects of the paper he found confusing. I really thought he stepped it up this time and that the picture was really good and fun. I imagined the whole molecular conversation in Maxier’s voice, so I don’t know why, but it made it that much funnier.

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