Sophie Bombard

A platinum-quinacridine hybrid as a G-quadruplex ligand

A novel platinum–quinacridine hybrid, comprising a monofunctional Pt moiety and a G-quadruplex ligand (mono-para-quinacridine or MPQ), has been synthesized and shown to interact with quadruplex DNA via a dual noncovalent/covalent binding mode. Denaturing gel electrophoresis was used to separate the various platination products of 22AG (an oligonucleotide that mimics the human telomeric repeat) by Pt-MPQ, and it was shown that two platinated adducts are highly stable quadruplex structures. Dimethylsulfate/piperidine treatment and 3′-exonuclease digestion of the isolated adducts allowed us to precisely determine the platination pattern of 22AG by Pt-MPQ, which displays three main sites G2, G10 and G22. Data presented herein support the hypothesis that Pt-MPQ traps preferentially the antiparallel structure of the 22AG quadruplex. Finally, the kinetics of Pt-MPQ platination using a construct containing both quadruplex DNA and a duplex DNA parts provide the first insights into the Pt-MPQ preference for quadruplex DNA over duplex DNA.

Antitumor trans-N-heterocyclic carbene-amine-Pt(II) complexes: synthesis of dinuclear species and exploratory investigations of DNA binding and cytotoxicity mechanisms.

A series of bimetallic [(NHC)PtX2]2(diamine) complexes have been prepared as a new chemotype for potential anticancer agents. These complexes display an uncommon set of structural features as far as they combine two bifunctional, trans-configured platinum centers. They display cytotoxic activities in the micromolar range on many cancerous cell lines and do not cross-react with cisplatin in A2780/DDP cell lines. They bind slowly to double-stranded DNAs, giving monoadducts as the major products. Pathways for cellular toxicity have been investigated for both mono- and bimetallic trans-(NHC)PtX2(amine) complexes. It has been highlighted that, unlike cisplatin, these complexes do not induce cell cycle arrest. They trigger apoptosis in A2780 cells by a pathway involving translocation of apoptosis-inducing factor and caspase 12 to the nucleus. Moreover, bimetallic complexes may induce necrosis.

Nanoparticle‐Mediated Delivery of Bleomycin

The occurrence of side effects induced by poor distribution of antitumor agents is still an important problem in cancer treatment. The challenge consists of both improving the tumor bioavailability of drugs and confining them as closely as possible to their biological targets. The use of nanoparticles as vectors for drug delivery has been intensively documented during the last two decades. Some of them (ironand gold-based nanoparticles, quantum dots) are at the same time agents for imaging, thus allowing the simultaneous follow-up of the treatment efficiency. They can also take part in the treatment: magnetic nanoparticles can be used for hyperthermia, while gold-based nanoparticles can be used for photothermal therapy. The use of nanometric vectors brings some answers to the problem of bioavailability. Considering their large surface-tovolume ratio, they offer the possibility of transporting major quantities of drugs. Thanks to passive and active targeting, they ensure limited harmful systemic distribution. Indeed, taking into account the enhanced permeation and retention (EPR) effects, suitable nanoparticles can carry drugs into solid tumors. Moreover, the grafting of targeting moieties, such as antibodies or folic acid, onto the particles surface allows active targeting, thus decreasing the interaction with healthy cells. The efficiency of treatments is also limited because of intracellular drug resistance mechanisms: only a small amount of an agent reaches its biological target. Significant improvement in drug efficiency could be obtained through the development of therapeutic strategies to pass drugs across the biological barriers. From this point of view, nanoparticles appear to be good candidates for drug delivery, because they are internalized in cells mainly by endocytosis pathways. Nevertheless, nanoparticles suffer from two major limitations: the alteration of their surface in biological media and their in vivo stealth. To cope with these two main problems, hybrid systems have been designed, which combine inorganic cores with organic or inorganic shells. Therapeutic molecules can either be inserted into the shell or grafted onto it. One should also mention the association with polymers such as polyethylene glycol that are able to ensure both stealth and in vivo stability. The strategy we propose herein is based on the use of multifunctional core–shell nanoparticles made of gFe2O3@SiO2-PEG-NH2 (PEG = polyethylene glycol), which allow covalent anchoring of biomolecules. Compared with a nanometric system incorporating drugs, the grafting of an antineoplastic agent at the surface allows a drastic reduction in the uncontrolled release of the agent and thus in side effects in healthy tissues. We have chosen in this work to study the grafting of bleomycin-A5 (BLM-A5), an anticancer drug that chelates metals such as Fe and catalyzes the formation of single-stranded (ss) or double-stranded (ds) DNA lesions in the presence of oxygen. The therapeutic efficiency of bleomycin (BLM) is severely limited because of its side effects, notably strong pulmonary toxicity. Dispensing the drug at lower doses near the biological target could lead to its wider use in oncology. Moreover, the delivery of this drug has been poorly studied, mainly using micrometric systems, such as glass beads or polyvinylpyridine microgels. The nanoplatforms we propose are core–shell magnetic nanoparticles (CSMNs) obtained using a procedure developed by our group (Figure 1A). The core consists of citrate-coated maghemite nanoparticles (g-Fe2O3, diameter around 7 nm). The shell is a layer of silica, twice functionalized by PEG chains, to hide the nanoparticles from the reticuloendothelial system (RES), and by amino groups to ensure BLM-A5 anchoring. Following van Blaaderen s method, fluorescent CSMNs can be synthesized by addition of rhodamine isothiocyanate-derived 3-aminopropyltriethoxysilane (APTS) during the coating of the magnetic cores by silica. These core–shell particles are characterized by a mean hydrodynamic diameter of 40 nm, and transmission electron microscopy (TEM) images display spherical particles with a mean physical diameter of 35 nm (Figure 1B). These particles are positively charged (+ 14 mV), and the surface density of amino groups can be tuned by varying the APTS to 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEOS) ratio. Such particles, with different surface densities of amino groups, have been reported previously and have been fully characterized using capillary zone electrophoresis. The magnetic properties of the maghemite cores, which contain from two to three g-Fe2O3 nanoparticles, are not modified by encapsulation. These CSMNs can be dispersed in 150 mm 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH 7.4 or in other media with high concentrations of salts and/or proteins, while maintaining colloidal stability at room temperature for at least six months. BLM-A5 (Figure 1C) was covalently anchored onto these platforms [*] T. Georgelin, Dr. J.-M. Siaugue, Prof. V. Cabuil Physicochimie des Electrolytes, Collo des et Sciences Analytiques (PECSA), UMR 7195 UPMC-CNRS-ESPCI-ENSCP Universit Pierre et Marie Curie Univ Paris 06, CC 51 4 place Jussieu, 75252 Paris Cedex 05 (France) Fax: (+ 33)1-4427-3228 E-mail: jean-michel.siaugue@upmc.fr Homepage: http://www.pecsa.upmc.fr

Interactions of Pt‐ttpy with G‐Quadruplexes Originating from Promoter Region of the c‐myc Gene Deciphered by NMR and Gel Electrophoresis Analysis

This study provides insights into the interactions of Pt-ttpy, that is, a metallo-organic heterocycle-comprising platinum(II) complex of terpyridine, and G-quadruplexes adopted by G-rich DNA from the transcriptional regulatory element of the c-myc gene, a well-known attractive target for artificial modulation of oncogene expression. A previously noted drug-like potential of Pt-ttpy relies on its antiproliferative activity on cancer cells and its increased selectivity for G-quadruplex binding attributed to the combination of distinct interacting modes. The predominant interaction between the herein used models of a parallel G-quadruplex exhibiting short propeller-type loops and Pt-ttpy occurs through stacking to the outer G-quartets. The presence of adenine versus thymine residue at the 5-end overhanging region allows the coordinative binding of Pt-ttpy to the G-quadruplex structure. Interestingly, Pt-ttpy triggers the formation of the G-quadruplex even in the absence of cations. Furthermore, NMR-based characterisation revealed common structural features of Pt-ttpy-G-quadruplex complexes in the presence and absence of cations, which indicate that cations may be expelled from the cores of the corresponding structures.

Photo-cross-linking probes for trapping G-quadruplex DNA.

We have developed a straightforward synthetic pathway to a set of six photoactivatable G-quadruplex ligands with a validated G4-binding motif (the bisquinolinium pyridodicarboxamide PDC-360A) tethered through various spacers to two different photo-cross-linking groups: benzophenone and an aryl azide. The high quadruplex-versus-duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well-known G-quadruplexes (human telomeric G4 and oncogene promoter c-myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G-quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c-myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.

Biological activity of enantiomeric complexes [PtCl 2 L 2 ] (L 2 is aromatic bisphosphanes and aromatic diamines)

Enantiomeric complexes of formula [PtCl2L2] [L2xa0isxa0(R)-(+)-BINAP and (S)-(−)-BINAP, where BINAPxa0isxa02,2′-bis(diphenylphosphane)-1,1′-binaphthyl, and (R)-(+)-DABN and (S)-(−)-DABN, where DABNxa0isxa01,1′-binaphthyl-2,2′-diamine], were tested for their cytotoxic activity against three cancer cell lines and for their ability to bind to the human telomeric sequence folded in the G-quadruplex structure. Similar experiments were carried out on prototypal complexes cisplatin and cis-[PtCl2(PPh3)2] for comparison. Platinum complexes containing phosphanes proved less cytotoxic to cancer cell lines and less likely to interact with the nucleobases of the G-quadruplex than those containing amines; in both cases the S-(−) isomer was more active than the R-(+) counterpart. More specifically, whereas all the platinum complexes were able to platinate the G-quadruplex structure from the human telomeric repeat, the extent and sites of platination depended on the nature of the ligands. Complexes containing (bulky) phosphanes interacted only with the adenines of the loops, whereas those containing the less sterically demanding amines interacted with adenines and some guanines of the G-quartet.

Aminoglycoside–Quinacridine Conjugates: Towards Recognition of the P6.1 Element of Telomerase RNA

A modular synthesis has been developed which allows easy and rapid attachment of one or two aminoglycoside units to a quinacridine intercalator, thereby leading to monomeric and dimeric conjugates. Melting temperature (Tm) experiments show that the tobramycin dimeric conjugate TD1 exhibits strong binding to the P6.1 element of human telomerase RNA. By contrast, tobramycin alone is much less efficient and the monomeric compound TM1 elicits a poor binding ability. Monitoring of the interaction by an electrophoretic mobility shift assay shows a 1:1 stoichiometry for the binding of the dimeric compound to the hairpin structure and confirms the lower affinity for a control duplex. Protection experiments with RNase T1 indicate interaction of the drug both in the stem and in the loop of the hairpin. Taken together, the data suggest a binding of TD1 inside the hairpin at the stem‐loop junction. The same trends are observed with paromomycin and kanamycin analogues but with a lower affinity.

Loss of the Malignant Phenotype of Human Neuroblastoma Cells by a Catalytically Inactive Dominant-Negative hTERT Mutant

Telomerase, a ribonucleoprotein complex mainly composed of the reverse transcriptase catalytic subunit (human telomerase reverse transcriptase, hTERT) and the RNA component (hTR), is a key enzyme of cancer progression. That aggressive stage 4-neuroblastoma expressed high levels of telomerase activity, whereas favorable tumors had no or little telomerase expression and activity, prompted us to investigate the role of this enzyme in this tumor model of altered proliferation, neuronal differentiation, and apoptosis. A human MYCN-amplified neuroblastoma cell line (IGR-N-91) was engineered to stably express either the normal hTERT protein (WT-hTERT) or a catalytically inactive dominant-negative mutant of this protein (DN-hTERT). We showed that DN-hTERT expression inhibited the endogenous hTERT in the malignant neuroblasts without telomere shortening nor loss of in vitro proliferative capacity. Importantly, DN-hTERT expression induced major changes in cell morphology of neuroblasts that switched them from a neuronal to a substrate adherent phenotype, which was more prone to apoptosis and lost their tumorigenic properties in nude mice. These biologic effects arose from modifications in the expression of genes involved in both apoptosis and neuroblastoma biology. Taken together these results highlighted the functional relevance of noncanonical functions of hTERT in the determination of neuroblast cell fate. Therefore, our results envision new therapeutic strategies for metastatic neuroblastoma therapeutic management. Mol Cancer Ther; 11(11); 2384–93. ©2012 AACR.

Platination of telomeric DNA by cisplatin disrupts recognition by TRF2 and TRF1

Telomeres, the nucleoprotein complexes located at the ends of chromosomes, are involved in chromosome protection and genome stability. Telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) are the two telomeric proteins that bind to duplex telomeric DNA through interactions between their C-terminal domain and several guanines of the telomeric tract. Since the antitumour drug cisplatin binds preferentially to two adjacent guanines, we have investigated whether cisplatin adducts could affect the binding of TRF1 and TRF2 to telomeric DNA and the property of TRF2 to stimulate telomeric invasion, a process that is thought to participate in the formation of the t-loop. We show that the binding of TRF1 and TRF2 to telomeric sequences selectively modified by one GG chelate of cisplatin is markedly affected by cisplatin but that the effect is more drastic for TRF2 than for TRF1 (3–5-fold more sensitivity for TRF2 than for TRF1). We also report that platinum adducts cause a decrease in TRF2-dependent stimulation of telomeric invasion in vitro. Finally, in accordance with in vitro data, analysis of telomeric composition after cisplatin treatment reveals that 60% of TRF2 dissociate from telomeres.

Identification of human telomerase assembly inhibitors enabled by a novel method to produce hTERT

Telomerase is the enzyme that maintains the length of telomeres. It is minimally constituted of two components: a core reverse transcriptase protein (hTERT) and an RNA (hTR). Despite its significance as an almost universal cancer target, the understanding of the structure of telomerase and the optimization of specific inhibitors have been hampered by the limited amount of enzyme available. Here, we present a breakthrough method to produce unprecedented amounts of recombinant hTERT and to reconstitute human telomerase with purified components. This system provides a decisive tool to identify regulators of the assembly of this ribonucleoprotein complex. It also enables the large-scale screening of small-molecules capable to interfere with telomerase assembly. Indeed, it has allowed us to identify a compound that inhibits telomerase activity when added prior to the assembly of the enzyme, while it has no effect on an already assembled telomerase. Therefore, the novel system presented here may accelerate the understanding of human telomerase assembly and facilitate the discovery of potent and mechanistically unique inhibitors.