Tumor-specific Multi-Operating Radiotracers targeting Telomerase And G-quadruplex – TuMOR TAG

The very first prototype of quadruplex-selective compounds (termed G-quadruplex ligands) was reported in 1997 by the groups of S. Neidle (School of pharmacy, London, UK) and L. Hurley (University of Arizona, USA). Since then, hundreds of chemical synthesis programs have been launched with the goal of identifying new and efficient quadruplex ligands. While the diversity of chemical motifs used to construct G-quadruplex ligands is somewhat hard to gauge, given the myriad reported studies, it is however evident that the overwhelming majority of designed compounds directly thrive from the erstwhile used duplex-DNA intercalators pool. The logic behind using duplex-DNA interacting motifs might seem quite counterintuitive, since the duplex recognition is the major unwanted interaction for valuable quadruplex ligands. This approach has been undoubtedly successful, with dozens of promising candidates reported each year. However, in this ANR project, we decided to follow an alternative road: instead of structurally fine-tuning duplex-DNA intercalators, why not simply learn from Nature? Nature indeed devises an astute strategy to make quadruplex architectures quite stable: the self-recognition and self-assembly of G-quartets. The relationship between the number of constitutive G-quartet layers and the quadruplex stability being straightforward (the more, the better), we decided herein to design, synthesize and study synthetic G-quartets as very first prototypes of nature-inspired quadruplex ligands.

During this 3-year program, we have succeeded in providing the proof-of-concept that synthetic G-quartets can indeed be valuable G-quadruplex ligands, both in terms of affinity and selectivity for their DNA target. DOTASQ and PorphySQ were the very first prototypes of nature-inspired ligands, interacting with quadruplexes according to a “like-likes-like” association between synthetic (ligand) and native quartets (quadruplex). A second generation of biomimetic ligands was subsequently designed, with compounds named PNA-DOTASQ and PNA-PorphySQ, which differs from the first generation in that they display primary amine side-chains that leverage a quite unique quadruplex-promoted G-quartet folding that triggers the overall quadruplex-affinity of the ligand. PNA-DOTASQ and PNA-PorphySQ, in addition to being a shining example of bioinspired quadruplex interacting compound, were also the very first prototypes of smart G-quadruplex ligands. Finally, all these compounds were also used as cornerstones for nanobiotechnological applications, thereby further and definitively enlightening the versatility of synthetic G-quartets.

The results that have been obtained all along this 3-year TuMOR TAG research program are not only interesting per se but also pioneering and highly promising notably for the perspectives they open. Indeed, before this work, quadruplex ligands were designed according to a traditional approach that has proved to be yet effective but also highly risked since based on an empiric and a posteriori analysis of obtained results; herein, the approach is definitively different since we have not only designed and studied synthetic G-quartets as the very first prototypes of bioinspired quadruplex ligands (a promise of enticing selectivity for their targets) but also opened up some very interesting prospects for the emergence of novel generations of ligands that will display even enhanced properties (like PNA-DOTASQ for instance, which was a fully innovative prototype of “smart quadruplex ligand”). Only the skills and creativity of researchers will prevent the raise of ever more effective and multitasking prototypes; as such, we are currently investing efforts on the use of biomimetic ligands as imaging agents (notably smart fluorescent probes) and let us wager that many future studies on biomimetic quadruples ligands will rapidly emerge and nurture optimism about the outlook for synthetic G-quartet applications.

The results obtained in the course of this project have been well-perceived by the scientific community, as demonstrated by the number and quality of the publications they have lead to (13 articles in international, high impact factor (IF) journals, including J. Am. Chem. Soc. 2011 & 2013 (IF=10.67), Chem. Commun. 2011 & 2013 (IF=6.38), Chem. Eur. J. 2011 & 2013 (IF=5.83), Nucleic Acids Res. 2012 (IF=8.28), ChemBioChem 2012 (IF=3.74), Org. Biomol. Chem. 2012 (IF=3.57), Biochimie 2012 (IF=3.14), ChemMedChem 2014 (IF=3.07), Inorg. Chem. 2014 (IF=4.59) and Nanoscale 2014 (IF=6.23), average IF 6.03)). We have also been involved in different book projects, including one chapter dealing with the bionantechnological applications of synthetic G-quartets in « DNA in Supramolecular Chemistry and Nanotechnology », E. Stulz, G.H. Clever (Eds.), Wiley-VCH: Weinheim, 2014 and two chapters related to our nature-inspired strategy to target quadruplexes in « Biological relevance and therapeutic applications of DNA and RNA quadruplexes », D. Monchaud (Ed.), Future Science: London, 2014, of which I had the honor to have been selected as an Editor.

The present research project aims at developing a new generation of molecular imaging agents. This multidisciplinary project stands at the interface of chemistry, biophysics and biology and will more precisely lead to the development and the applications of new cancer-specific radiotracers. To achieve this goal, we will design small synthetic molecules functionalized with radionuclide complexes that will be detected via nuclear medical imaging; these readily accessible molecules will be designed and developed to interact specifically with biological targets, over-expressed in tumorous context (as compared to normal cells). Our approach will thus be stepwise, and organized as follows:

a) Our first series of molecules will target telomerase: this enzyme, whose discoverers recently won the Nobel Prize in Medicine, is greatly responsible for the immortalization of cancer cells; interestingly, it is over-expressed in a vast majority of cancer types (~85% of tested cell lines) whereas it is silent in normal cells (and basically expressed in germinal cell lines). The telomerase is thus a high-value molecular marker for carcinogenesis, since very specific. We will thus design and synthesize molecules able to selectively target a short RNA sequence that is accessible in the active site of the enzyme, via what could be termed an ‘antisense’ strategy; to this end, we will develop DNA chimers, like PNA (for peptidic nucleic acids) or GPNA (for guanidine-based PNA), known to be more readily synthetically accessible than their DNA counterparts, robust in physiological media and able to form highly stable chimeric (G)PNA/RNA duplexes. These peptides will be subsequently equipped with polyazamacrocycles (like cyclen or cyclam), able to strongly chelate a radionuclide (like 111In for example) that will be useful for the detection via nuclear medical imaging.

b) Our second series of molecules will not only target telomerase but concomitantly the telomerase enzyme AND its substrate: as indicated by its name, the substrate of telomerase is telomere, a nucleoproteic assembly that ends the eukaryotic chromosomes. This assembly is comprised of a constellation of proteins associated to the telomeric DNA, which is more particularly targeted by the telomerase. Given that this DNA is comprised of a single-stranded G-rich DNA (known as G-overhang), it may possess the ability to fold into a high-order DNA structure called G-quadruplex-DNA. We will thus further equip previously prepared (G)PNA – polyazamacrocycles conjugates with molecules able to interact strongly and efficiently with quadruplex-DNA (termed “quadruplex ligands”). By doing this, we will greatly enhance the efficiency of our approach, increasing both the specificity of the molecule/target interactions and the residence time in cells, thereby improving the detection signal.

c) Our third series of molecules will target only G-quadruplex-DNA: indeed, it is now widely agreed that G-quadruplexes play a significant role in key biological processes and that they can be targeted for therapeutic intervention; however, while their in vitro existence is thoroughly documented, their in vivo existence is still debated, because of a lack of molecular tools that will enable their localization in vivo. Therefore, on the basis of the knowledge acquired during the previously described investigations, we will develop a series of G-quadruplex specific radionuclide complexes, which will enable us to “image” G-quadruplex in situ, and thus to contribute to address the issue of their in vivo existence.