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

Publications de l’unité

Année de publication : 2021

Jérémie Mitteaux, Pauline Lejault, Filip Wojciechowski, Alexandra Joubert, Julien Boudon, Nicolas Desbois, Claude P. Gros, Robert H. E. Hudson, Jean-Baptiste Boulé, Anton Granzhan, David Monchaud (2021 Aug 4)

Identifying G-Quadruplex-DNA-Disrupting Small Molecules

Journal of the American Chemical Society : 143 : 12567–12577 : DOI : 10.1021/jacs.1c04426 En savoir plus
Résumé

The quest for small molecules that strongly bind to G-quadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV–vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4-disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.

G4-unfold

Replier
Marc Lavigne, Olivier Helynck, Pascal Rigolet, Rofia Boudria-Souilah, Mireille Nowakowski, Bruno Baron, Sébastien Brülé, Sylviane Hoos, Bertrand Raynal, Lionel Guittat, Claire Beauvineau, Stéphane Petres, Anton Granzhan, Jean Guillon, Geneviève Pratviel, Marie-Paule Teulade-Fichou, Patrick England, Jean-Louis Mergny, Hélène Munier-Lehmann (2021 Jul 7)

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction.

Nucleic Acids Research : 49 : 7695–7712 : DOI : 10.1093/nar/gkab571 En savoir plus
Résumé

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

Replier
Piguel S., Le Bescont J., Mouawad L., Boddaert T., Bombard S. (2021 Jun 29)

Photoactivatable small-molecule inhibitors for light-controlled TAM kinase activity

ChemPhotoChem : Accepted Author Manuscript : DOI : 10.1002/cptc.202100131 En savoir plus
Résumé

The TAM kinase family arises as a promising therapeutical target for cancer therapy, auto-immune, and viral diseases. In this study, we report the first photoactivatable caged inhibitors of Tyro3 and Mer. This strategy enables spatial and temporal control of the biological activity of the inhibitor upon irradiation with UV light. We describe the design, the synthesis, the photocleavage properties, and the inhibitory activity of four Tyro3 and Mer photoactivatable small molecules. The proof of concept on the TAM kinase family was achieved in vitro , since irradiation by UV light restored the full inhibitory activity of two prodrugs.

Replier
Samar Ali, Emilia Puig Lombardi, Deepanjan Ghosh, Tao Jia, Géraldine Vitry, Lina Saker, Joël Poupon, Marie-Paule Teulade-Fichou, Alain Nicolas, Arturo Londono-Vallejo, Sophie Bombard (2021 May 22)

Pt-ttpy, a G-quadruplex binding platinum complex, induces telomere dysfunction and G-rich regions DNA damage.

Metallomics : integrated biometal science : 13 : mfab029 : DOI : 10.1093/mtomcs/mfab029 En savoir plus
Résumé

PMID-34021581

Pt-ttpy (tolyl terpyridin-Pt complex) covalently binds to G-quadruplex (G4) structures in vitro and to telomeres in cellulo via its Pt moiety. Here, we identified its targets in the human genome, in comparison to Pt-tpy, its derivative without G4 affinity, and cisplatin. Pt-ttpy, but not Pt-tpy, induces the release of the shelterin protein TRF2 from telomeres concomitantly to the formation of DNA damage foci at telomeres but also at other chromosomal locations. γ-H2AX chromatin immunoprecipitation (ChIP-seq) after treatment with Pt-ttpy or cisplatin revealed accumulation in G- and A-rich tandemly repeated sequences, but not particularly in potential G4 forming sequences. Collectively, Pt-ttpy presents dual targeting efficiency on DNA, by inducing telomere dysfunction and genomic DNA damage at specific loci.

Replier
Laura Fourmois, Florent Poyer, Aude Sourdon, Delphine Naud-Martin, Sounderya Nagarajan, Rahima Chennoufi, Eric Deprez, Marie-Paule Teulade-Fichou, Florence Mahuteau-Betzer (2021 May 19)

Modulation of cellular fate of vinyl triarylamines through structural fine tuning: to stay or not to stay in the mitochondria?

Chembiochem : a European journal of chemical biology : Accepted Article : DOI : 10.1002/cbic.202100168 En savoir plus
Résumé

Mitochondria is involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been driven to design mitochondria-targeted fluorophores for monitoring the mitochondria status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues ( TP-1Bzim, TP n -2Bzim, TP 1+ -2Bzim, TN-2Bzim ) differ by the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP 1+ -2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨ m variations.

Replier
Abegão L.M., Santos F.A., Piguel S., Rodrigues J.J., Mendonça C.R., De Boni L. (2021 Apr 15)

The ability of 2,5-disubstituted oxazole dyes derivatives to generate two-photon upconversion photoluminescence and its brightness evaluation

Journal of Photochemistry and Photobiology A: Chemistry : 411 : 113214 : DOI : 10.1016/j.jphotochem.2021.113214 En savoir plus
Résumé

S1010603021000873-ga1_lrgThe brightness study of emissive compounds is one of the fundamental spectroscopic characterizations. In this work, we assessed the brightness values of eight 2,5-disubstituted oxazole dyes derivatives by combining linear and nonlinear spectroscopic parameters. The range of the brightness values obtained is from 0.26 GM to 15.15 GM. The highest value belongs to compound 14 m, which, compared to previously investigated compounds of similar π-conjugation length, is at least two times higher. Brightness values were determined in the spectral region between 700 nm–720 nm, revealing this class of dyes’ potential to be used as photoluminescence bioprobes excited by two-photons.

Replier
Yu Luo, Anton Granzhan, Daniela Verga, Jean-Louis Mergny (2021 Apr 1)

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

Biopolymers : 112 : e23415 : 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 (∆Tm), 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.

FRET-MC

Replier
Elisa Le Boiteux, Franck Court, Pierre-Olivier Guichet, Catherine Vaurs-Barrière, Isabelle Vaillant, Emmanuel Chautard, Pierre Verrelle, Bruno M Costa, Lucie Karayan-Tapon, Anne Fogli, Philippe Arnaud (2021 Mar 15)

Widespread overexpression from the four DNA hypermethylated HOX clusters in aggressive (IDHwt) glioma is associated with H3K27me3 depletion and alternative promoter usage.

Molecular oncology : Accepted article : DOI : 10.1002/1878-0261.12944 En savoir plus
Résumé

In human, the 39 coding HOX genes and 18 referenced non-coding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression-, DNA methylation- and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative « master » HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.

Replier
Alexandre Leduc, Samia Chaouni, Frédéric Pouzoulet, Ludovic De Marzi, Frédérique Megnin-Chanet, Erwan Corre, Dinu Stefan, Jean-Louis Habrand, François Sichel, Carine Laurent (2021 Mar 13)

Differential normal skin transcriptomic response in total body irradiated mice exposed to scattered versus scanned proton beams.

Scientific reports : 11 : 5876 : DOI : 10.1038/s41598-021-85394-0 En savoir plus
Résumé

Proton therapy allows to avoid excess radiation dose on normal tissues. However, there are some limitations. Indeed, passive delivery of proton beams results in an increase in the lateral dose upstream of the tumor and active scanning leads to strong differences in dose delivery. This study aims to assess possible differences in the transcriptomic response of skin in C57BL/6 mice after TBI irradiation by active or passive proton beams at the dose of 6 Gy compared to unirradiated mice. In that purpose, total RNA was extracted from skin samples 3 months after irradiation and RNA-Seq was performed. Results showed that active and passive delivery lead to completely different transcription profiles. Indeed, 140 and 167 genes were differentially expressed after active and passive scanning compared to unirradiated, respectively, with only one common gene corresponding to RIKEN cDNA 9930021J03. Moreover, protein-protein interactions performed by STRING analysis showed that 31 and 25 genes are functionally related after active and passive delivery, respectively, with no common gene between both types of proton delivery. Analysis showed that active scanning led to the regulation of genes involved in skin development which was not the case with passive delivery. Moreover, 14 ncRNA were differentially regulated after active scanning against none for passive delivery. Active scanning led to 49 potential mRNA-ncRNA pairs with one ncRNA mainly involved, Gm44383 which is a miRNA. The 43 genes potentially regulated by the miRNA Gm44393 confirmed an important role of active scanning on skin keratin pathway. Our results demonstrated that there are differences in skin gene expression still 3 months after proton irradiation versus unirradiated mouse skin. And strong differences do exist in late skin gene expression between scattered or scanned proton beams. Further investigations are strongly needed to understand this discrepancy and to improve treatments by proton therapy.

Replier
Sylvie Gory-Fauré, Rebecca Powell, Julie Jonckheere, Fabien Lanté, Eric Denarier, Leticia Peris, Chi Hung Nguyen, Alain Buisson, Laurence Lafanechère, Annie Andrieux (2021 Mar 12)

Pyr1-Mediated Pharmacological Inhibition of LIM Kinase Restores Synaptic Plasticity and Normal Behavior in a Mouse Model of Schizophrenia.

Frontiers in pharmacology : 12 : 627995 : DOI : 10.3389/fphar.2021.627995 En savoir plus
Résumé

The search for effective treatments for neuropsychiatric disorders is ongoing, with progress being made as brain structure and neuronal function become clearer. The central roles played by microtubules (MT) and actin in synaptic transmission and plasticity suggest that the cytoskeleton and its modulators could be relevant targets for the development of new molecules to treat psychiatric diseases. In this context, LIM Kinase – which regulates both the actin and MT cytoskeleton especially in dendritic spines, the post-synaptic compartment of the synapse – might be a good target. In this study, we analyzed the consequences of blocking LIMK1 pharmacologically using Pyr1. We investigated synaptic plasticity defects and behavioral disorders in MAP6 KO mice, an animal model useful for the study of psychiatric disorders, particularly schizophrenia. Our results show that Pyr1 can modulate MT dynamics in neurons. In MAP6 KO mice, chronic LIMK inhibition by long-term treatment with Pyr1 can restore normal dendritic spine density and also improves long-term potentiation, both of which are altered in these mice. Pyr1 treatment improved synaptic plasticity, and also reduced social withdrawal and depressive/anxiety-like behavior in MAP6 KO mice. Overall, the results of this study validate the hypothesis that modulation of LIMK activity could represent a new therapeutic strategy for neuropsychiatric diseases.

Replier
Auvray M., Bolze F., Clavier G., Mahuteau-Betzer F. (2021 Mar 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 : 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.

Replier
Thomas Barbot, Veronica Beswick, Cédric Montigny, Éric Quiniou, Nadège Jamin and Liliane Mouawad (2021 Feb 4)

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

Frontiers in Molecular Biosciences : 7 : 606254 : 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.

Replier
Oksana Reznichenko, Anne Cucchiarini, Valérie Gabelica, Anton Granzhan (2021 Jan 14)

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

Replier
Marchand A., Beauvineau C., Teulade-Fichou M.P., Zenobi R. (2021 Jan 13)

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 : 23 : 1113-1121 : DOI : 10.1002/chem.202004040 En savoir plus
Résumé

Stabilizing the DNA and RNA structures known as G‐quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G‐quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G‐quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also 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 (ttpy=4′‐tolyl‐2,2′:6′,2′′‐terpyridine) 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.

Replier
Anouchka Gatin, Isabelle Billault, Patricia Duchambon, Guillaume Van der Rest, Cécile Sicard-Roselli (2021 Jan 3)

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

Free radical biology & medicine : 162 : 461-470 : DOI : 10.1016/j.freeradbiomed.2020.10.324 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.

Replier