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

Publications de l’unité

Année de publication : 2019

Coralie Caron, Xuan N T Duong, Régis Guillot, Sophie Bombard, Anton Granzhan (2019 Feb 6)

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage, DNA Cleavage Inhibition, and Formation of Ligand-DNA Adducts.

Chemistry - A European Journal : 25 : 1949–1962 : DOI : 10.1002/chem.201805555 En savoir plus
Résumé

GA-ChemEurJ-2018-2Ligands interacting with abasic (AP) sites in DNA may generate roadblocks in base-excision DNA repair (BER) due to indirect inhibition of DNA repair enzymes (e.g., APE1) and/or formation of toxic byproducts, resulting from ligand-induced strand cleavage or covalent cross-links. Herein, we prepared and systematically studied a series of 12 putative AP-site ligands, sharing the common naphthalenophane scaffold but endowed with a variety of substituents. Our results demonstrate that most naphthalenophanes bind to AP-sites in DNA and inhibit the APE1-induced hydrolysis of the latter in vitro. Remarkably, their APE1 inhibitory activity, as characterized by IC50 and Ki values, can be directly related to their affinity and selectivity to AP-sites, assessed from the fluorescence-melting experiments. On the other hand, the molecular design of naphthalenophanes has crucial influence on their intrinsic AP-site cleavage activity (i.e., ligand-catalyzed β- and β,δ-elimination reactions at the AP site), as illustrated by the compounds either having an exceptionally high AP-site cleavage activity (e.g., 2,7 BisNP-S, 125-fold more efficacious than spermine) or totally devoid of this activity (four compounds). Finally, we reveal the unprecedented formation of a stable covalent DNA adduct upon reaction of one ligand (2,7-BisNP-NH) with its own product of AP-site cleavage.

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Pauline Gilson, Morgane Couvet, Laetitia Vanwonterghem, Maxime Henry, Julien Vollaire, Vladimir Baulin, Marco Werner, Anna Orlowska, Véronique Josserand, Florence Mahuteau-Betzer, Laurence Lafanechère, Jean-Luc Coll, Benoit Busser, Amandine Hurbin (2019 Feb 1)

The pyrrolopyrimidine colchicine-binding site agent PP-13 reduces the metastatic dissemination of invasive cancer cells in vitro and in vivo.

Biochemical pharmacology : 160 : 1-13 : DOI : S0006-2952(18)30503-3 En savoir plus
Résumé

Standard chemotherapies that interfere with microtubule dynamics are a chemotherapeutic option used for the patients with advanced malignancies that invariably relapse after targeted therapies. However, major efforts are needed to reduce their toxicity, optimize their efficacy, and reduce cancer chemoresistance to these agents. We previously identified a pyrrolo[2,3d]pyrimidine-based microtubule-depolymerizing agent (PP-13) that binds to the colchicine site of β-tubulin and exhibits anticancer properties in solid human cancer cells, including chemoresistant subtypes. Here, we investigated the therapeutic potential of PP-13 in vitro and in vivo. PP-13 induced a mitotic blockade and apoptosis in several cancer cells cultured in two-dimensions or three-dimensions spheroids, in conjunction with reduced cell proliferation. Capillary-like tube formation assays using HUVECs showed that PP-13 displayed antiangiogenic properties. It also inhibited cancer cell motility and invasion, in in vitro wound-healing and transwell migration assays. Low concentration PP-13 (130 nmol.L) treatment significantly reduced the metastatic invasiveness of human cancer cells engrafts on chicken chorioallantoic membrane. In nude mice, 0.5 or 1 mg.kg PP-13 intraperitoneally administered three-times a week reduced the sizes of paclitaxel-refractory orthotopic breast tumors, delayed the progression of metastasis, and decreased the global metastatic load compared to 0.5 mg.kg paclitaxel or vehicle alone. PP-13 did not show any apparent early adverse effect in vivo. These data suggest that PP-13 is a promising alternative to standard chemotherapy in antimitotic drug-refractory tumors, especially through its impact on metastasis.

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Delphine Naud-Martin, Corinne Landras-Guetta, Daniela Verga, Deepanjan Ghosh, Sylvain Achelle, Florence Mahuteau-Betzer, Sophie Bombard, Marie-Paule Teulade-Fichou (2019 Jan 26)

Selectivity of Terpyridine Platinum Anticancer Drugs for G-quadruplex DNA.

Molecules (Basel, Switzerland) : 24 : 404 : DOI : 10.3390/molecules24030404 En savoir plus
Résumé

Guanine-rich DNA can form four-stranded structures called G-quadruplexes (G4s) that can regulate many biological processes. Metal complexes have shown high affinity and selectivity toward the quadruplex structure. Here, we report the comparison of a panel of platinum (II) complexes for quadruplex DNA selective recognition by exploring the aromatic core around terpyridine derivatives. Their affinity and selectivity towards G4 structures of various topologies have been evaluated by FRET-melting (Fluorescence Resonance Energy Transfert-melting) and Fluorescent Intercalator Displacement (FID) assays, the latter performed by using three different fluorescent probes (Thiazole Orange (TO), TO-PRO-3, and PhenDV). Their ability to bind covalently to the c-myc G4 structure in vitro and their cytotoxicity potential in two ovarian cancerous cell lines were established. Our results show that the aromatic surface of the metallic ligands governs, in vitro, their affinity, their selectivity for the G4 over the duplex structures, and platination efficiency. However, the structural modifications do not allow significant discrimination among the different G4 topologies. Moreover, all compounds were tested on ovarian cancer cell lines and normal cell lines and were all able to overcome cisplatin resistance highlighting their interest as new anticancer drugs.

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M Schmidt-Cernohorska, I Zhernov, E Steib, M Le Guennec, R Achek, S Borgers, D Demurtas, L Mouawad, Z Lansky, V Hamel, P Guichard (2019 Jan 19)

Flagellar microtubule doublet assembly in vitro reveals a regulatory role of tubulin C-terminal tails.

Science (New York, N.Y.) : 363 : 285-288 : DOI : 10.1126/science.aav2567 En savoir plus
Résumé

Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism governing their formation is unknown. We used a cell-free assay to demonstrate a crucial inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly. Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with binding protein partners. The dynamics of B-microtubule nucleation and its distinctive isotropic elongation was elucidated by using live imaging. Thus, inherent interaction properties of tubulin provide a structural basis driving flagellar MTD assembly.

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Michela Zuffo, Xiao Xie, Anton Granzhan (2018 Dec 6)

Strength in Numbers: Development of a Fluorescence Sensor Array for Secondary Structures of DNA.

Chemistry - A European Journal : 25 : 1812–1818 : DOI : 10.1002/chem.201805422 En savoir plus
Résumé

GA-ChemEurJ-2018-3High-throughput assessment of secondary structures adopted by DNA oligonucleotides is currently hampered by the lack of suitable biophysical methods. Fluorescent sensors hold great potential in this respect; however, the moderate selectivity that they display for one DNA conformation over the others constitutes a major drawback to the development of accurate assays. Moreover, the use of single sensors impedes a comprehensive classification of the tested sequences. Herein, we propose to overcome these limitations through the development of a fluorescence sensor array constituted by easily accessible, commercial dyes. Multivariate analysis of the emission data matrix produced by the array allows to explore the conformational preferences of DNA sequences of interest, either by calculating the probability of group membership in the six predefined structural categories (three G-quadruplex groups, double-stranded, and two groups of single-stranded forms), or by revealing their particular structural features. The assay enables rapid screening of synthetic DNA oligonucleotides in a 96-wells plate format.

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Année de publication : 2018

Prado Martins R., Findakly S., Daskalogianni C., Teulade-Fichou M.P., Blondel M., Fåhraeus R. (2018 Nov 29)

In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules

Molecules : 23 : 3124 : DOI : 10.3390/molecules23123124 En savoir plus
Résumé

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

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Verga D., Nguyen C.H., Dakir M., Coll J.L., Teulade-Fichou M.P., Molla A. (2018 Nov 20)

Polyheteroaryl Oxazole/Pyridine-based compounds selected in vitro as G-quadruplex ligands inhibit Rock kinase and exhibit antiproliferative activity

Journal of Medicinal Chemistry : 61 : 10502-10518 : DOI : 10.1021/acs.jmedchem.8b01023 En savoir plus
Résumé

Heptaheteroaryl compounds comprised of oxazole and pyridine units (TOxaPy) are quadruplex DNA (G4)-interactive compounds. Herein, we report on the synthesis of parent compounds bearing either amino side chains (TOxaPy-1-5) or featuring an isomeric oxazole-pyridine central connectivity (iso-TOxapy, iso-TOxapy 1-3) or a bipyridine core (iso-TOxabiPy). The new isomeric series showed significant G4-binding activity in vitro and remarkably 3 compounds (iso-TOxaPy, iso-TOxaPy-1, iso-TOxabiPy) exhibited high antiproliferative activity towards a tumor panel of cancer cell lines. However, these compounds do not behave as typical G-quadruplex binders and the kinase profiling assay revealed that the best antiproliferative molecule iso-TOxaPy selectively inhibited Rock-2. The targeting of Rock kinase was confirmed in cells by the dephosphorylation of Rock-2 substrates, the decrease of stress fibers and peripheral focal adhesions, as well as the induction of long neurite-like extensions. Remarkably two of these molecules were able to inhibit the growth of cells organized as spheroids.

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Hee-Sheung Lee, Mar Carmena, Mikhail Liskovykh, Emma Peat, Jung-Hyun Kim, Mitsuo Oshimura, Hiroshi Masumoto, Marie-Paule Teulade-Fichou, Yves Pommier, William C Earnshaw, Vladimir Larionov, Natalay Kouprina (2018 Nov 1)

Systematic Analysis of Compounds Specifically Targeting Telomeres and Telomerase for Clinical Implications in Cancer Therapy.

Cancer research : 78 : 6282-6296 : DOI : 10.1158/0008-5472.CAN-18-0894 En savoir plus
Résumé

The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the EGFP transgene: compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. Our assay allowed quantification of chromosome loss by routine flow cytometry. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, two compounds -Cu-ttpy and Pt-ttpy- induced a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis as well as UFB (Ultrafine Bridges). Chromosome loss after Pt-ttpy or Cu-ttpy treatment correlated with the induction of telomere-associated DNA damage. Overall, this platform enables identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.

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M Lupu, Ph Maillard, J Mispelter, F Poyer, C D Thomas (2018 Nov 1)

A glycoporphyrin story: from chemistry to PDT treatment of cancer mouse models.

Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology : 17 : 1599-1611 : DOI : 10.1039/c8pp00123e En savoir plus
Résumé

Photodynamic therapy (PDT) represents a non-toxic and non-mutagenic antitumor therapy. The photosensitizer’s (PS) chemo-physical properties are essential for the therapy, being responsible for the biological effects induced in the targeted tissues. In this study, we present the synthesis and development of some glycoconjugated porphyrins based on lectin-type receptor interaction. They were tested in vitro for finally choosing the most effective chemical structure for an optimum antitumor outcome. The most effective photosensitizer is substituted by three diethylene glycol α-D-mannosyl groups. In vivo studies allow firstly the determination of some characteristics of the biological processes triggered by the initial photochemical activation. Secondly, they make it possible to improve the therapeutic protocol in the function of the structural architecture of the targeted tumor tissue.

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Annalisa Patriarca, Charles Fouillade, Michel Auger, Frédéric Martin, Frédéric Pouzoulet, Catherine Nauraye, Sophie Heinrich, Vincent Favaudon, Samuel Meyroneinc, Rémi Dendale, Alejandro Mazal, Philip Poortmans, Pierre Verrelle, Ludovic De Marzi (2018 Nov 1)

Experimental set-up for FLASH proton irradiation of small animals using a clinical system

International Journal of Radiation Oncology • Biology • Physics : 102 : 619-626 : DOI : 10.1016/j.ijrobp.2018.06.403 En savoir plus
Résumé

Purpose

Recent in vivo investigations have shown that short pulses (FLASH) of electrons are less harmful to healthy tissues, but just as efficient as conventional dose-rate radiation to inhibit tumor growth. In view of the potential clinical value of FLASH and the availability of modern proton therapy infrastructures to achieve this goal, we herein describe a series of technological developments required to investigate the biology of FLASH irradiation, using a commercially available clinical proton therapy system.

Methods and materials

Numerical simulations and experimental dosimetric characterization of a modified clinical proton beamline, upstream from the isocenter were performed with Monte Carlo toolkit and different detectors. A single scattering system was optimized together with a ridge filter and a high current monitoring system. In addition, a submillimetric set-up protocol based on image-guidance using a digital camera and an animal positioning system was also developed.

Results

The dosimetric properties of the resulting beam and monitoring system were characterized: linearity with dose rate and homogeneity for a 12×12 mm2 field size were assessed. Dose rates exceeding 40 Gy/s at energies between 138 and 198 MeV were obtained, enabling uniform irradiation for radiobiology investigations on small animals in a modified clinical proton beam line.

Conclusion

This approach will enable us to conduct FLASH proton therapy experiments on small animals, specifically for mouse lung irradiation. Dose rates exceeding 40 Gy/s were achieved, which was not possible with the conventional clinical mode of the existing beamline.

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Tom Baladi, Jessy Aziz, Florent Dufour, Valentina Abet, Véronique Stoven, François Radvanyi, Florent Poyer, Ting-Di Wu, Jean-Luc Guerquin-Kern, Isabelle Bernard-Pierrot, Sergio Marco Garrido, Sandrine Piguel (2018 Nov 1)

Design, synthesis, biological evaluation and cellular imaging of imidazo[4,5-b]pyridine derivatives as potent and selective TAM inhibitors.

Bioorganic & medicinal chemistry : 26 : 5510-5530 : DOI : 10.1016/j.bmc.2018.09.031 En savoir plus
Résumé

The TAM kinase family arises as a new effective and attractive therapeutic target for cancer therapy, autoimmune and viral diseases. A series of 2,6-disubstituted imidazo[4,5-b]pyridines were designed, synthesized and identified as highly potent TAM inhibitors. Despite remarkable structural similarities within the TAM family, compounds 28 and 25 demonstrated high activity and selectivity in vitro against AXL and MER, with IC value of 0.77 nM and 9 nM respectively and a 120- to 900-fold selectivity. We also observed an unexpected nuclear localization for compound 10Bb, thanks to nanoSIMS technology, which could be correlated to the absence of cytotoxicity on three different cancer cell lines being sensitive to TAM inhibition.

design,synthesis

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Abhijit Saha, Sophie Bombard, Anton Granzhan, Marie-Paule Teulade-Fichou (2018 Oct 27)

Probing of G-Quadruplex Structures via Ligand-Sensitized Photochemical Reactions in BrU-Substituted DNA.

Scientific Reports : 8 : 15814 : DOI : 10.1038/s41598-018-34141-z En savoir plus
Résumé

We studied photochemical reactions of BrU-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both ligands bind to and stabilize G4-DNA structures without altering their folding topology, as demonstrated by FRET-melting experiments, fluorimetric titrations and CD spectroscopy. Notably, the bis-pyrene derivative (M-2PY) behaves as a significantly more affine and selective G4 ligand, compared with its mono-pyrene counterpart (M-1PY) and control compounds. Upon short UVA irradiation (365 nm) both ligands, in particular M-2PY, efficiently sensitize photoreactions at BrU residues incorporated in G4 structures and give rise to two kinds of photoproducts, namely DNA strand cleavage and covalent ligand-DNA photoadducts. Remarkably, the photoinduced strand cleavage is observed exclusively with G4 structures presenting BrU residues in lateral or diagonal loops, but not with parallel G4-DNA structures presenting only propeller loops. In contrast, the formation of fluorescent photoadducts is observed with all BrU-substituted G4-DNA substrates, with M-2PY giving significantly higher yields (up to 27%) than M-1PY. Both ligand-sensitized photoreactions are specific to BrU-modified G4-DNA structures with respect to double-stranded or stem-loop substrates. Thus, ligand-sensitized photoreactions with BrU-substituted G4-DNA may be exploited (i) as a photochemical probe, allowing « photofootprinting » of G4 folding topologies in vitro and (ii) for covalent trapping of G4 structures as photoadducts with pyrene-substituted ligands.

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Filippo Doria, Valentina Pirota, Michele Petenzi, Marie-Paule Teulade-Fichou, Daniela Verga, Mauro Freccero (2018 Aug 30)

Oxadiazole/Pyridine-Based Ligands: A Structural Tuning for Enhancing G-Quadruplex Binding.

Molecules (Basel, Switzerland) : 23 : 2162 : DOI : 10.3390/molecules23092162 En savoir plus
Résumé

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure⁻activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.

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Xiao Xie, Oksana Reznichenko, Ludovic Chaput, Pascal Martin, Marie-Paule Teulade-Fichou, Anton Granzhan (2018 Aug 27)

Topology-Selective Fluorescent « Light-Up » Probes for G-Quadruplex DNA Based on Photoinduced Electron Transfer.

Chemistry - A European Journal : 24 : 12638-12651 : DOI : 10.1002/chem.201801701 En savoir plus
Résumé

GA-ChemEurJ-2018-1Six novel probes were prepared by covalent attachment of a G4-DNA ligand (PDC) to various coumarin or pyrene fluorophores. In the absence of DNA, the fluorescence of all probes is quenched due to intramolecular photoinduced electron transfer (PET) evidenced by photophysical and electrochemical studies, molecular modeling and DFT calculations. All probes demonstrate similarly high thermal stabilization of various G4-DNA substrates belonging to different folding topologies, as assessed by fluorescence melting experiments; however, their fluorimetric response is strongly heterogeneous with respect to structures of the probes and G4-DNA targets. Thus, the probes containing the 7-diethylaminocoumarin fluorophore demonstrate significant fluorescence enhancement in the presence of G4-DNA, with the strongest « light-up » response (20- to 180-fold) observed for antiparallel G4 structures as well as for hybrid G4 structures, formed by the variants of human telomeric sequence and capable of a conformation change to the antiparallel isoform. These results shed light on the influence of the linker and electronic properties of fluorophores on the efficiency of G4-DNA « light-up » probes operating via PET.

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Donia Essaid, Ali Tfayli, Philippe Maillard, Christophe Sandt, Véronique Rosilio, Arlette Baillet-Guffroy, Athena Kasselouri (2018 Jul 21)

Retinoblastoma membrane models and their interactions with porphyrin photosensitisers: An infrared microspectroscopy study.

Chemistry and physics of lipids : 215 : 34-45 : DOI : 10.1016/j.chemphyslip.2018.07.003 En savoir plus
Résumé

Fourier Transform Infrared (FTIR) microspectroscopy was used to highlight the interactions between two photosensitisers (PS) of different geometries, TPPmOH4 and a glycoconjugated analogous, TPPDegMan, and lipid bilayers modelling retinoblastoma cell membranes. Retinoblastoma is a rare disease occurring in young infants, for whom conservative treatments may present harmful side-effects. Photodynamic therapy (PDT) is expected to induce less side-effects, as the photosensitiser is only activated when the tumour is illuminated.

Since efficiency of the treatment relies on photosensitiser penetration in cancer cells, bilayers with three lipid compositions – pure SOPC, SOPC/SOPE/SOPS/Chol (56:23:11:10) and SOPC/SOPE/SOPS/Chol/CL (42:32:9:8:6) – were used as plasma and mitochondria model membranes. FTIR spectra showed that the interaction of the PSs with the lipid bilayers impacted the lipid organization of the latter, causing significant spectral variations. Both studied photosensitisers inserted at the level of lipid hydrophobic chains, increasing chain fluidity and disorder. This was confirmed by surface pressure measurements. Photosensitisers – TPPmOH4 more than TPPDegMan – also interacted with the polar region of the bilayer, forming hydrogen bonds with phosphate groups that induced major shifts of phosphate absorption bands. This difference in PS interaction with moieties in the polar region was more pronounced with the models with complex lipid composition.

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