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International audience; 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 ΔΨm variations.

The proof of concept for two-photon activated photodynamic therapy has already been achieved for cancer treatment but the efficiency of this approach still heavily relies on the availability of photosensitizers combining high two-photon absorption and biocompatibility. In this line we recently reported on a series of porphyrin-triphenylamine hybrids which exhibit high singlet oxygen production quantum yield as well as high two-photon absorption cross-sections but with a very poor cellular internalization. We present herein new photosensitizers of the same porphyrin-triphenylamine hybrid series but bearing cationic charges which led to strongly enhanced water solubility and thus cellular penetration. In addition the new compounds have been found localized in mitochondria that are preferential target organelles for photodynamic therapy. Altogether the strongly improved properties of the new series combined with their specific mitochondrial localization lead to a significantly enhanced two-photon activated photodynamic therapy efficiency.

Platinum(II) tolylterpyridine (ttpy) complexes, have been shown to generate monofunctional adducts with various G-quadruplex DNA forming sequences resulting from an efficient π-stacking mode on the top of the quadruplex structure. To further explore the potential of this series with regard to quadruplex recognition, classical photocrosslinking groups (benzophenone, tetraphenylazide) have been grafted on the tolylterpyridine ligand moiety, thereby generating two new derivatives Pt-ttpy-Bn and Pt-ttpy-N3. Evaluation of their non-covalent binding for G-quadruplex DNA has been performed by FRET-melting and FID assays using two G-quadruplex matrices i.e. the telomeric sequence 22AG and the oncogene promotor sequence c-myc, which revealed high affinity and improved selectivity as compared to the parent compound. Subsequently the capacity of the compounds to establish one or two anchorage points (one by platination, one by photoinduced crosslinking) with the quadruplexes has been studied by gel electrophoresis with and without photoactivation. Interestingly both compounds do platinate the quadruplexes studied with high selectivity as the platination yield is poorly affected by the presence of duplex competitor. By contrast, only the azido derivative Pt-ttpy-N3 was found to form a second covalent bond within the G-quadruplexes upon photoactivation indicating a higher confinement of the crosslinking moiety in this case. Finally the two compounds exhibit poor cytotoxicity in the dark on two cancer cell lines (A2780 and HT29), whereas that of the benzophenone derivative is significantly enhanced upon irradiation. Altogether the two new compounds represent novel prototypes usable for trapping G4 DNA alone or eventually G4-DNA protein interactions in complex in vitro systems or in cells.

International audience; BackgroundRetinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice.MethodsMice were divided into nine groups: control, carboplatin 1.5 and 4 μg, melphalan 0.1 and 1 μg, topotecan 0.1 and 1 μg, carboplatin 4 μg/topotecan 0.1 μg and melphalan 1 μg/topotecan 0.1 μg. The follow‐up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumour and presence of calcified tumours were the criteria for therapeutic response assessment. Ocular complications were assessed after four weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers.ResultsTopotecan was inactive on retinal tumours. Melphalan (1 μg) led to a complete tumour control in 91.7% of eyes. Carboplatin strongly decreased the tumour burden (85.7‐93.8% of eyes without retinal tumour). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1 μg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumours with a reduced retinal toxicity.ConclusionsThis preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.

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 IC50 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.

International audience; A new family of amphiphilic glycoconjugated porphyrin dimers usable in two-photon photodynamic therapy was designed, prepared and characterized. The physicochemical and biological data were presented and discussed. Their two-photon absorption cross section was determined, revealing quite good performances of the compounds (d % 300 GM at 880 nm for 2 and 3 and d % 1000 GM at 840 nm for 5 and 6). Despite their promising photophysical properties, porphyrin dimers are penalized by their low solubility and an insufficient penetration into tumour cells. Incorporation into nanocarriers such as lipid vesicles could be profitable, provided that the positioning and degree of freedom of the saccharide moieties are optimal to target the receptors of the cells of interest.

Bis-naphthalene macrocycles, which bind with high affinity and selectivity to abasic sites in DNA, efficiently inhibit their cleavage by APE1 (IC50 = 55-60 nM in the kinetic assay with a model THF substrate). These results demonstrate that substrate masking by non-covalent abasic-site ligands is an efficient strategy for inhibition of APE1.

G-quadruplex(es) (G4) are non-canonical nucleic acid structures found in guanine-rich sequences. They can be targeted with small molecules (G4-ligands) acting as reporters, for tracking both in vitro and in cells. We explored the cellular localisation of PhenDC3, one of the most powerful G4 ligands, by synthesising two clickable azide and alkyne derivatives (PhenDC3-alk, PhenDC3-az) and labelling them in situ with the corresponding Cy5 click partners. A careful comparison of the results obtained for the copper-based CuAAC and copper-free SPAAC methodologies in fixed cells implicated Cu(I) /alkyne intermediates in the non-specific localisation of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labelling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localise drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.

Purpose Because retinoblastoma therapies have many adverse effects, new approaches must be developed and evaluated on animal models. We describe orthotopic xenograft models of retinoblastoma using different strains of mice, suitable for this purpose. Methods Human retinoblastoma tumors were established on immunodeficient mice by subcutaneous engraftment of tumors from enucleated eyes. The orthotopic model was obtained by subretinal injections of suspension cells into the right eye of immunodeficient (Swiss-nude, severe combined immunodeficiency [SCID]) and immunocompetent mice (C57BL/6N, B6Albino). In vivo tumor growth was monitored by fundus and spectral-domain optical coherence tomography (SD-OCT) imaging and compared with histology. Results Retinal and vitreal tumor growth was achieved both in immunocompetent and immunodeficient strains after the subretinal injection of tumor cells. The best tumor engraftment rate was obtained in the SCID mice (68.8%). No tumor growth was observed in the C57BL/6N strain. Chronic retinal detachment may occur in most strains after the subretinal injection, in particular the Swiss-nude strain, which exhibits retinal degeneration. Conclusions The setting up of an orthotopic mouse model depends mainly on the choice of the engrafted cells (cell lines or patient-derived xenografts) but it can also depend on the xenografted mouse strain. Severe combined immunodeficiency mice (an immunodeficient strain) achieved the best tumor engraftment rate (68.8%). However, intraocular tumor growth was also satisfactory (50%) in the immunocompetent strain B6Albino, and this strain will allow to exploit the immune response after a tumor treatment. Both of these strains may therefore be recommended when setting up orthotopic retinoblastoma xenografts.

The identification of DNA sensors is still a challenge since no DNA probe possesses all the photophysical properties required for live-cell imaging: high fluorescence yield, red emission, permeability, no photobleaching and no cytotoxicity. We describe the preparation of a distyryl dye library and its evaluation on a panel of nucleic acids with various structures (duplex DNA, quadruplex DNA and RNA). The screening involved measuring the modification of the fluorescence properties of the dyes with or without nucleic acids on a microplate reader, and allowed the identification of selective quadruplex DNA ligands with good affinities. Using this screening method we discovered a new bright red-emitting DNA stain, Acri-2,7-Py, for fixed cells. In living cells, the staining was not nuclear and photodamage generated through illumination induced cellular death. These processes require further studies to determine the relevance of Acri-2,7-Py in photodynamic therapy.

Background: Porphyrins are a group of heterocyclic macrocycle organic compounds. They have shown good efficiency in the photodynamic treatment of tumors in the last twenty years. The photodynamic therapy could be an alternative treatment for retinoblastoma using a photosensitizer with low dark toxicity and low mutagenic properties unlike the conventional chemotherapies. The objectives of this study were to develop a photosensitizer in our lab which is a diethylene glycol mannoconjugated tetraphenyl porphyrin (TPP, MW: 1413.6) and to determine the optimal interval time for photodynamic therapy using the concentration of TPP in eye and blood samples of mice. Materials and method: Transgenic mice developed bilateral retinoblastoma from the retina since 4 weeks of age to 16 weeks of age (tumors fill the ocular globe and the mice will be sacrificed for ethic purpose). The evaluation of the biodistribution of our TPP was realized to know the best drug-light interval to perform the photodynamic therapy. For this purpose, an intraperitoneal injection of 200 µl at 5.25 mg/ml in PEG 400/physiological serum (1:1, v/v) was performed on the LHbetaTag transgenic mice. At different time intervals (8h, 24h, 40h, 48h, and 72h) following injection, the mice were sacrificed and the eyes and blood were collected. TPP in eye samples were extracted in methanol and the level of TPP was measured by ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) analysis (0.5-100 ng/mL). The level of TTP in blood samples was quantified after protein precipitation by UPLC-MS/MS method (5.00-1000 ng/mL). Results: We observed a time dependency accumulation of TPP in eyes with observed Tmax=48 hours. TPP serum concentrations decreased by the same manner up to 48 h. The determination of drug-light interval allowed us to perform the light delivery when the TPP level is highest (48 h) in the ocular tumor tissue (retinoblastoma). Conclusions: Our TPP had a good in vitro photocytotoxicity at very low doses and showed a good efficacy in vivo with subcutaneous patient derived retinoblastoma xenografts in immunodeficient mice. The knowledge of Tmax and this interval is essential to realize the photodynamic therapy with the better efficiency, the final goal being to eradicate the retinoblastoma from the retina. Citation Format: Florent Poyer, Samuel Hugeut, Stéphanie Lemaitre, Olivier Madar, Carole D Thomas, Philippe Maillard, Marie-Paule Teulade-Fichou, Nathalie Cassoux, François Doz, Keyvan Rezai. Evaluation of porphyrin biodistribution for use in photodynamic therapy of retinoblastoma in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3213.

International audience; A porphyrin monosubstituted by three triethyleneglycol chains grafted on a pentaerythritol skeleton was designed to display an optimized amphiphilicity for an enhanced cellular uptake and thus to exert enhanced photocytotoxicity. This porphyrin proved to be an excellent photosensitiser with submicromolar IC50.

International audience; In order to avoid side effects at the time of cancer eradication to the patients, the selectivity of treatments has become of strategic importance. In the case of photodynamic therapy (PDT), two-photon absorption combined with active targeting of tumors could allow both spatial and chemical selectivity. In this context, we present the synthesis, spectroscopic, and biological properties of a series of porphyrin-triphenylamine hybrids with excellent singlet oxygen production capacities and good two-photon absorption.

We have developed a straightforward synthetic pathway to a set of six photoactivatable G-quadruplex ligands with a validated G4-binding motif (the bisquinolinium pyridodicarboxamide PDC-360A) tethered through various spacers to two different photo-cross-linking groups: benzophenone and an aryl azide. The high quadruplex-versus-duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well-known G-quadruplexes (human telomeric G4 and oncogene promoter c-myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G-quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c-myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.

The design of a dendrimeric-like diglycerol-tetrasubstituted Zn ( II ) phthalocyanine resulted in a remarkably water-soluble compound due to the presence of 16 hydroxyls. Several parameters relevant to evaluate the photodynamic efficiency of a potentiel photosensitizer such as: aggregation behavior, fluorescence properties, singlet oxygen generation, binding to a carrier protein model (Bovine Serum Albumin) and partition coefficient have been measured. Biocompatibility was demonstrated by dark cytotoxicity in in vitro experiments. The absence of phototoxicity can be explained by an elevated hydrophilicity. All the collected data have confirmed that this new substitution pattern is promising to be used on phthalocyanines aiming at being photodynamic therapy agents.