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

Publications de l’unité

Année de publication : 2021

Auvray M., Bolze F., Clavier G., Mahuteau-Betzer F. (2021 Jan 1)

Silafluorene as a promising core for cell-permeant, highly bright and two-photon excitable fluorescent probes for live-cell imaging

Dyes and Pigments : 187 : 109083 : DOI : https://doi.org/10.1016/j.dyepig.2020.109083 En savoir plus
Résumé

ImageJ=1.52a

In this study, we report the synthesis, linear and non-linear photophysical studies and live-cell imaging of two two-photon activatable probes based on a silafluorene core: SiFluo-V and SiFluo-L. Thanks to their quadrupolar (A-π-D-π-A) design, these probes exhibit respectively good to impressive two-photon cross-sections (from 210 GM to 2150 GM). TD-DFT calculations support the experimental evidence that SiFluo-L displays far better two-photon absorption properties than SiFluo-V. Moreover, SiFluo-L possesses all requirements for bioimaging as it is water soluble, cell-permeant and presents a low cytotoxicity (IC80 ≥ 10 μM). It labels mitochondria in live-cell imaging at low laser power with high brightness, contrast and photostability. This study demonstrates that silafluorene is a promising core to develop new two-photon fluorophores for live-cell imaging.

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Année de publication : 2020

Yu Luo, Anton Granzhan, Daniela Verga, Jean-Louis Mergny (2020 Dec 28)

FRET-MC: A fluorescence melting competition assay for studying G4 structures in vitro.

Biopolymers : Early view : bip23415 : DOI : 10.1002/bip.23415 En savoir plus
Résumé

G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRET-melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization effect (∆T ), while non-specific competitors (e.g., single- or double-stranded sequences) have little effect. Sixty-five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized.

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Année de publication : 2021

Oksana Reznichenko, Anne Cucchiarini, Valérie Gabelica, Anton Granzhan (2020 Dec 8)

Quadruplex DNA-guided ligand selection from dynamic combinatorial libraries of acylhydrazones

Organic and Biomolecular Chemistry : 19 : 379-386 : DOI : 10.1039/D0OB01908A En savoir plus
Résumé

Dynamic combinatorial libraries of acylhydrazones were prepared from diacylhydrazides and several cationic or neutral aldehydes in the presence of 5-methoxyanthranilic acid catalyst. Pull-down experiments with magnetic beads functionalized with a G-quadruplex (G4)-forming oligonucleotide led to the identification of putative ligands, which were resynthesized or emulated by close structural analogues. G4-binding properties of novel derivatives were assessed by fluorimetric titrations, mass spectrometry and thermal denaturation experiments, giving evidence of strong binding (Kd < 10 nM) for two compounds.

Quadruplex DNA-guided ligand selection from dynamic combinatorial libraries of acylhydrazones

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Année de publication : 2020

Lei He, Zhenyu Meng, Qianqian Guo, Xiangyang Wu, Marie-Paule Teulade-Fichou, Edwin Kok Lee Yeow, Fangwei Shao (2020 Nov 28)

Fluorogenic Pt complexes distinguish the quantity and folding behavior of RNA G-quadruplexes between live cancerous and healthy cells.

Chemical communications (Cambridge, England) : 56 : 14459-14462 : DOI : 10.1039/d0cc05622g En savoir plus
Résumé

Fluorogenic Pt complexes

Two Pt complexes with high quantum yields and photostability, and low cytotoxicity, were developed to track RNA G-quadruplexes (GQs) in live cells. Higher number and intensity, and longer lifetime of fluorescent foci in cancer cells than those in healthy cells suggest that the quantity and folding dynamics of RNA GQs could not only correlate to their biological functions, but be two novel biomarkers to characterize cancerous cells.

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Leduc A., Chaouni S., De marzi L., Pouzoulet F., Mégnin-Chanet F., Stephan D., Habrand J.L., Sichel F., Laurent C. (2020 Nov 24)

Biological Effects of Scattered Versus Scanned Proton Beams on Normal Tissues in Total Body Irradiated Mice: Survival, Genotoxicity, Oxidative Stress and Inflammation

Antioxidants : 9 : 1170 : DOI : 10.3390/antiox9121170 En savoir plus
Résumé

Side effects of proton therapy are poorly studied. Moreover, the differences in the method of dose delivery on normal tissues are not taken into account when proton beams are scanned instead of being scattered. We proposed here to study the effects of both modalities of proton beam delivery on blood; skin; lung and heart in a murine model. In that purpose; C57BL/6 mice were total body irradiated by 190.6 MeV proton beams either by Double Scattering (DS) or by Pencil Beam Scanning (PBS) in the plateau phase before the Bragg Peak. Mouse survival was evaluated. Blood and organs were removed three months after irradiation. Biomarkers of genotoxicity; oxidative stress and inflammation were measured. Proton irradiation was shown to increase lymphocyte micronucleus frequency; lung superoxide dismutase activity; erythrocyte and skin glutathione peroxidase activity; erythrocyte catalase activity; lung; heart and skin oxidized glutathione level; erythrocyte and lung lipid peroxidation and erythrocyte protein carbonylation even 3 months post-irradiation. When comparing both methods of proton beam delivery; mouse survival was not different. However, PBS significantly increased lymphocyte micronucleus frequency; erythrocyte glutathione peroxidase activity and heart oxidized glutathione level compared to DS. These results point out the necessity to take into account the way of delivering dose in PT as it could influence late side effects.

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Anouchka Gatin, Isabelle Billault, Patricia Duchambon, Guillaume Van der Rest, Cécile Sicard-Roselli (2020 Nov 20)

Oxidative radicals (HO or N3) induce several di-tyrosine bridge isomers at the protein scale.

Free radical biology & medicine : In Press, Corrected Proof : DOI : S0891-5849(20)31599-9 En savoir plus
Résumé

Graphical abstract

Among protein oxidative damages, di-tyrosine bridges formation has been evidenced in many neuropathological diseases. Combining oxidative radical production by gamma radiolysis with very performant chromatographic separation coupled to mass spectrometry detection, we brought into light new insights of tyrosine dimerization. Hydroxyl and azide radical tyrosine oxidation leading to di-tyrosine bridges formation was studied for different biological compounds: a full-length protein (Δ25-centrin 2), a five amino acid peptide (KTSLY) and free tyrosine. We highlighted that both radicals generate high proportion of dimers even for low doses. Surprisingly, no less than five different di-tyrosine isomers were evidenced for the protein and the peptide. For tyrosine alone, at least four distinct dimers were evidenced. These results raise some questions about their respective role in vivo and hence their relative toxicity. Also, as di-tyrosine is often used as a biomarker, a better knowledge of the type of dimer detected in vivo is now required.

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Thomas Barbot, Veronica Beswick, Cédric Montigny, Éric Quiniou, Nadège Jamin and Liliane Mouawad (2020 Nov 6)

Deciphering the mechanism of inhibition of SERCA1a by sarcolipin using molecular simulations

Frontiers in Molecular Biosciences : Accepted article : DOI : 10.3389/fmolb.2020.606254 En savoir plus
Résumé

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well understood. To decipher this mechanism, we have performed normal mode analysis in the all-atom model, with the SERCA1a-SLN complex, or the isolated SERCA1a, embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation at the atomic level for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304 and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+ low-affinity E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight into the conformational transition between the E2 and E1 states.

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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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Marchand A., Beauvineau C., Teulade-Fichou M.P., Zenobi R. (2020 Oct 14)

Competition of ligands and the 18-mer binding domain of the RHAU helicase for G-quadruplexes – orthosteric or allosteric binding mechanism?

Chemistry – A European Journal : Accepted article : DOI : 10.1002/chem.202004040 En savoir plus
Résumé

Stabilizing particular DNA and RNA structures called G-quadruplexes (G4s) using specific ligands (L) is a strategy proposed to fight cancer. However, while G4:L interactions are often investigated, whether or not ligands are able to disrupt interactions between G4s and proteins (P) remains poorly studied. Here, using native mass spectrometry, we investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. First, our results suggest that RHAU binds not only preferentially to parallel G4s but to free external G-quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu-ttpy induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug-target interactions but also how the binding to other partners is affected.

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Breton-Patient C., Naud-Martin D., Mahuteau-Betzer F., Piguel S. (2020 Oct 14)

Three-component C-H bond sulfonylation of imidazoheterocycles via visible-light organophotoredox catalysis.

European Journal of Organic Chemistry : Accepted Article : DOI : 10.1002/ejoc.202001219 En savoir plus
Résumé

10.1002-ejoc202001219

The first entirely visible-light photoredox catalyzed sulfonylation of imidazoheterocycles has been developed. This transformation demonstrates an efficient C-H functionalization for the straightforward synthesis of novel C-3 sulfonylated imidazoheterocycles from various imidazopyridines and diaryliodonium salts with different electronic and steric properties and easy handled DABCO- bis (sulfur dioxide). The reaction proceeds in moderate to good yields under mild conditions at room temperature using the inexpensive organophotocatalyst EosinY.Na 2 and shows a high functional group tolerance (37 examples).

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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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