UMR9187 / U1196 – Chimie et Modélisation pour la Biologie du Cancer (CMBC)

Publications de l’unité

Année de publication : 2020

Küssau T., Van Wyk N., Johansen M.D., Alsarraf H.M.A.B., Neyret A., Hamela C., Sørensen K.K., Thygesen M.B., Beauvineau C., Kremer L., Blaise M. (2020 Nov 4)

Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus

Cells : 9 : 2410 : DOI : 10.3390/cells9112410 En savoir plus
Résumé

Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors.

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Václav Brázda, Yu Luo, Martin Bartas, Patrik Kaura, Otilia Porubiaková, Jiří Šťastný, Petr Pečinka, Daniela Verga, Violette Da Cunha, Tomio S Takahashi, Patrick Forterre, Hannu Myllykallio, Miroslav Fojta, Jean-Louis Mergny (2020 Sep 24)

G-Quadruplexes in the Archaea Domain.

Biomolecules : 10 : 1349 : DOI : 10.3390/biom10091349 En savoir plus
Résumé

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in (strikingly, more than 50% of the isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.

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Laura Mouton, Monica Ribeiro, Marc-André Mouthon, Fawzi Boumezbeur, Denis Le Bihan, Damien Ricard, François D. Boussin, Pierre Verrelle (2020 Sep 18)

Experimental and Preclinical Tools to Explore the Main Neurological Impacts of Brain Irradiation: Current Insights and Perspectives

Brain Tumors : 158 : 239-261 : DOI : 10.1007/978-1-0716-0856-2_11 En savoir plus
Résumé

Radiation therapy is a powerful tool in the treatment of primary and metastatic cancers of the brain. However, brain tissue tolerance is limited, and radiation doses must be tailored to minimize deleterious effects on the nervous system. Due to improved treatments, including radiotherapy techniques, many patients with brain tumors survive longer, but they experience late effects of radiotherapy, especially cognitive decline, for which no efficient treatment is currently available. Improving the prevention and treatment of radiation-induced neurological defects first needs to better characterize radiation injuries in brain cells and tissues. Rodent models have been widely used for this.

Here, observations from patients will be reviewed briefly as an introduction, mainly regarding clinical cognitive defects and anatomical alterations using magnetic resonance imaging (MRI). This limited descriptive clinical knowledge addresses many questions that arise in preclinical models regarding understanding the mechanism of radiation-induced brain dysfunction. From this perspective, we next present methods to characterize radiation-induced neurogenesis alterations in adult mice and then detail how MRI could be used as a powerful tool to explore these alterations.

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Sandra Cunha Silveira, Géraldine Buhagiar‑Labarchède, Rosine Onclercq‑Delic, Simon Gemble, Elias Bou Samra, Hamza Mameri, Patricia Duchambon, Christelle Machon, Jérôme Guitton & Mounira Amor‑Guéret (2020 Aug 17)

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

Scientific Reports : 10 : 13907 : DOI : 10.1038/s41598-020-70874-6 En savoir plus
Résumé

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

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Paudel B.P., Moye A.L., Assi H.A., El-Khoury R., Cohen S.B., Birrento M.L., Samosorn S., Intharapichai K., Tomlinson C.G., Teulade-Fichou M.P., González C., Beck J.L., Damha M.J., van Oijen A.M., Bryan T.M. (2020 Jul 29)

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

eLife : 9 : e56428 : DOI : 10.7554/eLife.56428 En savoir plus
Résumé

Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

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Anton Granzhan, Rodrigo Prado Martins, Robin Fåhraeus, Marc Blondel and Marie-Paule Teulade-Fichou (2020 Jun 30)

Quadruplex-interacting compounds for regulating the translation of the Epstein–Barr virus nuclear antigen 1 (EBNA1) mRNA: A new strategy to prevent and treat EBV-related cancers

Quadruplex Nucleic Acids As Targets For Medicinal Chemistry, Annual Reports in Medicinal Chemistry : Chap 8, 54 : 243-286 : DOI : 10.1016/bs.armc.2020.05.001 En savoir plus
Résumé

The Epstein–Barr (EBV) virus is linked to at least 1% of human cancers that include Burkitt’s and Hodgkin’s lymphomas, nasopharyngeal carcinoma, and 10% of gastric cancers. EBV is a latent virus that possesses a genome maintenance protein, EBNA1, which is both essential for the virus and highly antigenic. Hence, EBV has evolved a mechanism by which EBNA1 self-limits the translation of its own mRNA, thereby minimizing the production of EBNA1-derived antigenic peptides. Although not fully elucidated, this mechanism involves the Gly-Ala-rich (GAr) motif of EBNA1, encoded by a G-repeat-containing mRNA sequence able to form clusters of G-quadruplexes (G4s). This chapter summarizes recent significant advances in understanding this phenomenon. Mechanistic investigations based on yeast chemical genetics, cellular assays and in vitro experiments have shown that the host cell factor nucleolin (NCL) is involved in this limitation of EBNA1 translation through binding to the G4s of EBNA1 mRNA. This interaction can be disrupted by the benchmark G4-ligand PhenDC3 acting as a NCL competitor for binding to G4-RNA. Finally, exploration of the chemical space around PhenDC3 using combinatorial chemistry approach led to the generation of 20 compounds based on a bis(acylhydrazone) scaffold. Among these, two hits (PyDH2PhenDH2) exhibit optimized properties with regard to the disruption of NCL/G4 interaction in cells, along with lower cytotoxicity. Consequently, treatment by PyDH2 or PhenDH2 increases EBNA1 production and stimulates the GAr-restricted antigenic response. Altogether, this innovative concept of antigenic stimulation sets the basis for further identification of lead candidates that may become promising candidate drugs for treating EBV-related cancers

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Leandro H. Zucolotto Cocca, Luis M. G. Abegão, Lucas F. Sciuti, Roxane Vabre, Jonathas de Paula Siqueira, Kenji Kamada, Cleber R. Mendonca, Sandrine Piguel, and Leonardo De Boni (2020 Jun 11)

Two-Photon Emissive Dyes Based on Push–Pull Purines Derivatives: Toward the Development of New Photoluminescence Bioprobes

The Journal of Physical Chemistry C : 124 : 12185-12864 : DOI : 10.1021/acs.jpcc.0c01859 En savoir plus
Résumé

Two-Photon Emissive Dyes Based on Push–Pull Purines Derivatives: Toward the Development of New Photoluminescence BioprobesFluorescent organic molecules have received great attention due to their largest applications, for example, in DNA and RNA spectroscopies studies, development of new photoluminescence bioprobes, and applications in fluorescence spectroscopy. In specific, purine base analog molecules present high fluorescence quantum yields and significant Stokes shift. Furthermore, the addition of push–pull structures at the purine core could increase the photoluminescence properties, making candidates for photoluminescence bioprobes. To consider this, a complete spectroscopic study was performed on nine push–pull purines, distinguished by different push–pull structures. In specific, for this research, the two-photon absorption (2PA) study showed that the compounds present induced two-photon fluorescence at the therapeutic window, desired for fluorescence microscopy. The brightness property was evaluated, indicating that all chromospheres are fluorescent by a 2PA process. Additionally, ultrafast transient absorption was performed to elucidate contribution of the excited states on the 2PA spectra, and quantum chemistry calculations were performed to corroborate the experimental results.

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Mathieu E., Bernard A.S., Quévrain E., Zoumpoulaki M., Iriart S., Lung-Soong C., Lai B., Medjoubi K., Henry L., Nagarajan S., Poyer F., Scheitler A., Ivanovic-Burmazovic I., Marco S., somogyi a., Seksik P., Delsuc N., Policar C. (2020 May 29)

Intracellular location matters: rationalization of the anti-inflammatory activity of a manganese (II) superoxide dismutase mimic complex

Chem. Commun. : 56 : 7885-7888 : DOI : 10.1039/D0CC03398G En savoir plus
Résumé

Intracellular location matters

A conjugate of a Mn-based superoxide dismutase mimic with a Re-based multimodal probe was studied in a cellular model of oxidative stress. Its speciation was questioned using Re and Mn X-fluorescence. Its distribution were compared to its unconjugated analogue. Interestingly, their similar activities mirror their similar concentrations in mitochondria.

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Stéphanie Lemaître, Florent Poyer, Paul Fréneaux, Sophie Leboucher, François Doz, Nathalie Cassoux, Carole D Thomas (2020 May 1)

Low retinal toxicity of intravitreal carboplatin associated with good retinal tumor control in transgenic murine retinoblastoma.

Clinical & experimental ophthalmology : 48 : 500-511 : DOI : 10.1111/ceo.13711 En savoir plus
Résumé

Background: Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice.

Methods: Mice were divided into 9 groups: control, carboplatin 1.5 and 4μg, melphalan 0.1 and 1μg, topotecan 0.1 and 1μg, carboplatin 4μg/ topotecan 0.1μg and melphalan 1μg/ topotecan 0.1μg. The follow‐up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumor and presence of calcified tumors were the criteria for therapeutic response assessment. Ocular complications were assessed after 4 weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers.

Results: Topotecan was inactive on retinal tumors. Melphalan (1μg) led to a complete tumor control in 91.7% of eyes. Carboplatin strongly decreased the tumor burden (85.7‐93.8% of eyes without retinal tumor). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1μg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumors with a reduced retinal toxicity.

Conclusions: This preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.

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Rahima Chennoufi, Ngoc-Duong Trinh, Françoise Simon, Guillaume Bordeau, Delphine Naud-Martin, Albert Moussaron, Bertrand Cinquin, Houcine Bougherara, Béatrice Rambaud, Patrick Tauc, Céline Frochot, Marie-Paule Teulade-Fichou, Florence Mahuteau-Betzer & Eric Deprez (2020 Apr 23)

Interplay between cellular uptake, intracellular localization and the cell death mechanism in triphenylamine-mediated photoinduced cell death

Scientific Reports : 10 : 6881 : DOI : 10.1038/s41598-020-63991-9 En savoir plus
Résumé

Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.

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Michela Zuffo, Aurélie Gandolfini, Brahim Heddi, Anton Granzhan (2020 Apr 20)

Harnessing intrinsic fluorescence for typing of secondary structures of DNA

Nucleic Acids Research : 48 : e61 : DOI : 10.1093/nar/gkaa257 En savoir plus
Résumé

GA-NAR-2020

High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).

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Julie Le Bescont, Chloé Breton‐Patient et Sandrine Piguel (2020 Apr 16)

Unconventional Reactivity with DABCO-Bis(sulfur dioxide): C–H Bond Sulfenylation of Imidazopyridines

European Journal of Organic Chemistry : 2020 : 2101-2109 : DOI : 10.1002/ejoc.202000112 En savoir plus
Résumé

Exploring the unexpected reactivity of DABCO‐bis(sulfur dioxide) on various imidazo[1,2‐a ]pyridines expanded the toolbox of the sulfenylation reagent. Starting from three simple building blocks, this three‐component transformation led to various C‐3 sulfenylated substituted imidazo[1,2‐a ]pyridines in moderate to good yields.
Exploring the unexpected reactivity of DABCO‐bis(sulfur dioxide) on various imidazo[1,2‐a ]pyridines expanded the toolbox of the sulfenylation reagent. Starting from three simple building blocks, this three‐component transformation led to various C‐3 sulfenylated substituted imidazo[1,2‐a ]pyridines in moderate to good yields.

This work highlights the unexpected and unprecedented outcome of the reactivity with DABCO‐bis(sulfur dioxide). The use of this reagent led to the exclusive introduction of a sulfur atom on the C‐3 position of imidazopyridines instead of a sulfone group. The reaction methodology turned out to be robust, scalable and suitable for various imidazopyridines and aryl iodides both bearing substituents with different electronic and steric properties (38 examples). Beyond the fact that this synthetic method complements the previously reported protocols for sulfenylation reactions, this work is meant to underline the unconventional role of DABCO‐bis(sulfur dioxide).

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Ehlen A., Martin C., Miron S., Julien M., Theillet F.X., Boucherit V., Ropars V., Duchambon P., El Marjou A., Zinn Justin S., Carreira A. (2020 Apr 14)

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

Nature Communications : 11 : 1819 : DOI : 10.1038/s41467-020-15689-9 En savoir plus
Résumé

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

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Abhijit Saha, Patricia Duchambon, Vanessa Masson, Damarys Loew, Sophie Bombard, Marie-Paule Teulade-Fichou (2020 Mar 20)

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes.

Biochemistry : 59 : 1261-1272 : DOI : 10.1021/acs.biochem.9b01094 En savoir plus
Résumé

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2′-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes

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Année de publication : 2019

Katerina Duskova, Pauline Lejault, Élie Benchimol, Régis Guillot, Sébastien Britton, Anton Granzhan, David Monchaud (2019 Dec 13)

DNA junction ligands trigger DNA damage and are synthetic lethal with DNA repair inhibitors in cancer cells

Journal of the American Chemical Society : 142 : 424-435 : DOI : 10.1021/jacs.9b11150 En savoir plus
Résumé

Translocation of DNA and RNA polymerases along their duplex substrates results in DNA supercoiling. This torsional stress promotes the formation of plectonemic structures, including three-way DNA junction (TWJ), which can block DNA transactions and lead to DNA damage. While cells have evolved multiple mechanisms to prevent the accumulation of such structures, stabilizing TWJ through ad hoc ligands offer an opportunity to trigger DNA damage in cells with high level of transcription and replication, such as cancer cells. Here, we develop a series of azacryptand-based TWJ ligands, we thoroughly characterize their TWJ-interacting properties in vitro and demonstrate their capacity to trigger DNA damage in rapidly dividing human cancer cells. We also demonstrate that TWJ ligands are amenable to chemically induced synthetic lethality strategies upon association with inhibitors of DNA repair, thus paving the way towards innovative drug combinations to fight cancers.

DNA Junction Ligands Trigger DNA Damage

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