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

Publications de l’unité

Année de publication : 2022

Zucolotto Cocca L.H., Pelosi A., Sciuti L.F., M. G. Abegão L., Kamada K., Piguel S., Renato Mendonça C., De Boni L. (2022 Feb 15)

Two-photon brightness of highly fluorescent imidazopyridine derivatives: Two-photon and ultrafast transient absorption studies

Journal of Molecular Liquids : 348 : 118379 : DOI : 10.1016/j.molliq.2021.118379 En savoir plus
Résumé

Imidazopyridine derivatives are unnatural purines isosteres and have been idealized and synthesized in the last decades, given their great applicability in several science areas. For instance, they may present potential to be used as fluorescent bioprobes on DNA or RNA spectroscopic studies. Furthermore, their synthesis can be performed to incorporate different peripheral structures in the central imidazopyridine scaffold, modeling and increasing the optical properties. Aiming applications as fluorescent probes as molecular liquids at the therapeutic window, thanks to the considerable emissive characteristics of this class of compound, optical spectroscopic studies considering nonlinear optical properties were performed on two distinct classes of N3-MEM-3H-imidazo[4,5-b]pyridine derivatives. The presented results exposed exceptionally high fluorescence quantum yields and two-photon absorption effects at the therapeutic window. The two-photon absorption spectra revealed a particular contribution of a higher excited state in some imidazopyridine derivatives containing naphthalene group. The excited state was confirmed by femtosecond time resolved spectroscopy. The obtained two-photon brightness shows substantial values for all compounds, with the potential to be used as fluorescent probes induced by two-photon excitation.

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Clelia Mathieu, Quentin Chamayou, Thi Thanh Hyen Luong, Delphine Naud, Florence Mahuteau-Betzer, Mouad Alami, Elias Fattal, Samir Messaoudi, Juliette Vergnaud-Gauduchon (2022 Feb 5)

Synthesis and antiproliferative activity of 6BrCaQ-TPP conjugates for targeting the mitochondrial heat shock protein TRAP1.

European journal of medicinal chemistry : 229 : 114052 : DOI : 10.1016/j.ejmech.2021.114052 En savoir plus
Résumé

A series of 6BrCaQ-C-TPP conjugates 3a-f and 5 was designed and synthesized as a novel class of TRAP1 inhibitors. Compound 3a displayed an excellent anti-proliferative activity with mean GI values at a nanomolar level in a diverse set of human cancer cells (GI = 0.008-0.30 μM) including MDA-MB231, HT-29, HCT-116, K562, and PC-3 cancer cell lines. Moreover, the best lead compound 6BrCaQ-C-TPP induces a significant mitochondrial membrane disturbance combined to a regulation of HSP and partner protein levels as a first evidence that his mechanism of action involves the TRAP-1 mitochondrial Hsp90 machinery.

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Sounderya Nagarajan, Florent Poyer, Laura Fourmois, Delphine Naud-Martin, Kadda Medjoubi, Andrea Somogyi, Gabrielle Schanne, Lucas Henry, Nicolas Delsuc, Clotilde Policar, Helene C Bertrand, Florence Mahuteau-Betzer (2022 Jan 25)

Cellular detection of a mitochondria targeted brominated vinyl triphenylamine optical probe (TP-Br) by X-ray fluorescence microscopy.

Chemistry (Weinheim an der Bergstrasse, Germany) : Accepted Article : DOI : 10.1002/chem.202104424 En savoir plus
Résumé

Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine  TP-2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP-2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP-RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP-Br probe retaining good photophysical properties and mitochondria labeling in live cells. The influence of calcium channels in the uptake of TP-Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP-Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.

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Florence Mahuteau-Betzer, Marie Auvray, Frédéric Bolze, Delphine Naud-Martin, Matthieu Poulain, Margaux Bossuat, Gilles Clavier (2022 Jan 21)

On the road for more efficient biocompatible two-photon excitable fluorophores.

Chemistry (Weinheim an der Bergstrasse, Germany) : Accepted Article : DOI : 10.1002/chem.202104378 En savoir plus
Résumé

Red to NIR absorption and emission wavelengths are key requirements for intravital bioimaging. One of the way to reach such excitation wavelengths is to use two-photon excitation. Unfortunately, there is still a lack of two-photon excitable fluorophores that are both efficient and biocompatible. Thus, we design a series of biocompatible quadrupolar dyes in order to study their ability to be used for live-cell imaging, and in particular for two-photon microscopy. Hence, we report the synthesis of 5 probes based on different donor cores (phenoxazine, acridane, phenazasiline and phenothiazine) and the study of their linear and non-linear photophysical properties. TD-DFT calculations were performed and were able to highlight the structure-property relationship of this series. All these studies highlight the great potential of three of these biocompatible dyes for two-photon microscopy, as they both exhibit high two-photon cross-sections (up to 3 650 GM) and emit orange to red light. This potential was confirmed through live-cell two-photon microscopy experiments, leading to images with very high brightness and contrast.

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Gatin A., Duchambon P., Rest G.v.d., Billault I., Sicard-Roselli C. (2022 Jan 21)

Protein Dimerization via Tyr Residues: Highlight of a Slow Process with Co-Existence of Numerous Intermediates and Final Products

International Journal of Molecular Sciences : 23 : 1174 : DOI : 10.3390/ijms23031174 En savoir plus
Résumé

Protein dimerization via tyrosine residues is a crucial process in response to an oxidative attack, which has been identified in many ageing-related pathologies. Recently, it has been found that for isolated tyrosine amino acid, dimerization occurs through three types of tyrosine–tyrosine crosslinks and leads to at least four final products. Herein, considering two protected tyrosine residues, tyrosine-containing peptides and finally proteins, we investigate the dimerization behavior of tyrosine when embedded in a peptidic sequence. After azide radical oxidation and by combining UPLC-MS and H/D exchange analyzes, we were able to evidence: (i) the slow kinetics of Michael Addition Dimers (MAD) formation, i.e., more than 48 h; (ii) the co-existence of intermediates and final cyclized dimer products; and (iii) the probable involvement of amide functions to achieve Michael additions even in proteins. This raises the question of the possible in vivo existence of both intermediates and final entities as well as their toxicity and the potential consequences on protein structure and/or function.

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Pelosi A.G., Zucolotto Cocca L.H., Abegão L.M., Sciuti L.F., Piguel S., De Boni L., Mendonça C.R. (2022 Jan 1)

Influence of electron-withdrawing groups in two-photon absorption of imidazopyridines derivatives

Dyes and Pigments : 198 : 109972 : DOI : 10.1016/j.dyepig.2021.109972 En savoir plus
Résumé

This work investigates the influence of different electron-withdrawing groups (Cl, Br, fluorobenzonitrile), on the two-photon absorption cross-section of six imidazo[4,5-b]pyridine derivatives. The two-photon absorption cross-section spectra were obtained by ultra-fast Z-scan technique from 470 nm up to 700 nm. The Sum-Over-States approach was applied to model the two-photon absorption spectra, using a three-level energy system. Photophysical properties, such as transition dipole moment, the difference of permanent dipole moments, and anisotropy coefficient were determined through the analysis of one-photon absorption spectra, solvatochromism, and fluorescence anisotropy, respectively. Besides, the excited state absorption spectra were measured through ultra-fast transient absorption, allowing the excited state lifetime and spectral position determination. Two-photon absorption cross-sections of about 160 GM were observed when two electron-withdrawing groups are linked to the imidazo[4,5-b]pyridine core, elucidating a path to achieve high optical nonlinearities in imidazopyridine derivatives. Furthermore, a increase in the two-photon cross-section was observed when chloride is linked at the C-6 position (90 GM) instead of the C-5 position (50 GM), which is related to the proximity of a higher excited state.

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Année de publication : 2021

Masson T., Landras Guetta C., Laigre E., Cucchiarini A., Duchambon P., Teulade-Fichou M.P., Verga D. (2021 Dec 7)

BrdU immuno-tagged G-quadruplex ligands: a new ligand-guided immunofluorescence approach for tracking G-quadruplexes in cells

Nucleic Acids Research : 49 : 12644–12660 : DOI : 10.1093/nar/gkab1166 En savoir plus
Résumé


Graphical abstract

G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag. However, this approach is limited by a low-labeling degree impeding precise visualization in specific subcellular regions. Herein, we present a new visualization strategy based on the immuno-recognition of 5-bromo-2′-deoxyuridine (5-BrdU) modified G4 ligands, functionalized prior- or post-G4-target binding by CuAAC. Remarkably, recognition of the tag by antibodies leads to the detection of the modified ligands exclusively when bound to a G4 target both in vitro, as shown by ELISA, and in cells, thereby providing a highly efficient G4-ligand Guided Immunofluorescence Staining (G4-GIS) approach. The obtained signal amplification revealed well-defined fluorescent foci located in the perinuclear space and RNase treatment revealed the preferential binding to G4-RNA. Furthermore, ligand treatment affected significantly BG4 foci formation in cells. Our work headed to the development of a new imaging approach combining the advantages of immunostaining and G4-recognition by G4 ligands leading to visualization of G4/ligands species in cells with unrivaled precision and sensitivity.

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Nils-Jørgen Knudsen Dal, Martin Speth, Kerstin Johann, Matthias Barz, Claire Beauvineau, Jens Wohlmann, Federico Fenaroli, Brigitte Gicquel, Gareth Griffiths, Noelia Alonso-Rodriguez (2021 Nov 29)

The zebrafish embryo as an in vivo model for screening nanoparticle-formulated lipophilic anti-tuberculosis compounds.

Disease models & mechanisms : Online ahead of print : DOI : 10.1242/dmm.049147 En savoir plus
Résumé

With the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening (HTS) campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models such as mice is time-consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model, to evaluate the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified to possess anti-tuberculosis activity in vitro. To aid solubilization compounds were formulated in biocompatible polymeric micelles (PM). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited to pinpoint promising compounds for further development.

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Alice J-L Zheng, Aikaterini Thermou, Pedro Guixens Gallardo, Laurence Malbert-Colas, Chrysoula Daskalogianni, Nathan Vaudiau, Petter Brohagen, Anton Granzhan, Marc Blondel, Marie-Paule Teulade-Fichou, Rodrigo Prado Martins, Robin Fahraeus (2021 Nov 17)

The different activities of RNA G-quadruplex structures are controlled by flanking sequences.

Life science alliance : 5 : e202101232 : DOI : 10.26508/lsa.202101232 En savoir plus
Résumé

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

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Piguel S., Le Bescont J., Mouawad L., Boddaert T., Bombard S. (2021 Nov 1)

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

ChemPhotoChem : 5 : 989-994 : 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.

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Jaime Franco Pinto, Alexandra Fillion, Patricia Duchambon, Sophie Bombard, Anton Granzhan (2021 Oct 9)

Acridine–O6-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity

European Journal of Medicinal Chemistry : 227 : 113909 : DOI : 10.1016/j.ejmech.2021.113909 En savoir plus
Résumé

Acridine–O6-benzylguanine hybrids
O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a–c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 μM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1–2 μM), similarly to acridine 8 (GI50 = 0.6 μM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2–0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.

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Joanna Zell, Katerina Duskova, Leïla Chouh, Madeleine Bossaert, Nicolas Chéron, Anton Granzhan, Sébastien Britton, David Monchaud (2021 Sep 22)

Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells

Nucleic Acids Research : 49 : 10275–10288 : DOI : 10.1093/nar/gkab796 En savoir plus
Résumé

Dual targeting of higher-order

DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.

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Aleksandr S. Oshchepkov, Oksana Reznichenko, Dan Xu, Boris S. Morozov, Anton Granzhan, Evgeny A. Kataev (2021 Sep 22)

Dye-functionalized Phosphate-binding Macrocycles: From Nucleotide to G-quadruplex Recognition and “turn-on” Fluorescence Sensing

Chemical Communications : 57 : 10632-10635 : DOI : 10.1039/D1CC04096K En savoir plus
Résumé

PBM-Dye strategy

A novel strategy to design “turn-on” fluorescent receptors for G-quadruplexes of DNA is presented, which relies on the connection of phosphate binding macrocycles (PBM) with naphthalimide dyes. A new PBM-dye family was synthesized and evaluated in terms of binding and detection of nucleotides and DNA G-quadruplexes of different topologies.

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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

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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 Aug 1)

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

Molecular oncology : 15 : 1995-2010 : 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.

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