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Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamages, while allowing to image about 10 times deeper than with a confocal. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. These probes react with bicyclo[6.1.0]no-4-yn-9yl)methanol (BCN) with good to impressive kinetic rate constant (up to 1.1x103 M-1.s-1) and emit in the red window with moderate to high turn-on. TDDFT allowed to rationalize both the kinetic and fluorogenic performance of the different probes. The best candidate displays a turn-on of 13.8-fold measured by fluorescence intensities quantification in living cells under one-photon excitation, whereas a value of 3 is enough for live-cell imaging with high contrast. Moreover, live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe as a turn-on of 8.0-fold was measured under two-photon excitation. Finally, the high two-photon brightness of the clicked adduct (>300 GM) allows the use of a weak laser power compatible with in vivo imaging.

Identifying new molecular targets for novel anticancer treatments is a major challenge in clinical cancer research. We have shown that cytidine deaminase (CDA) expression is downregulated in about 60% of cancer cells and tissues. In this study, we aimed to develop a new anticancer treatment specifically inhibiting the growth of CDA-deficient tumor cells. High-throughput screening of a chemical library led to the identification of a naphthol derivative, X55, targeting CDA-deficient tumor cells preferentially, without affecting the growth of non-tumoral cells regardless of CDA expression status. Metabolomic profiling revealed that CDA-deficient HeLa cells differed markedly from control HeLa cells. X55 treatment had a moderate effect on control cells, but greatly disturbed the metabolome of CDA-deficient HeLa cells, worsening the deregulation of many metabolites. In particular, the levels of the three oncometabolites, fumarate, succinate and 2-hydroxyglutarate, were significantly lower in CDA-depleted cells, and this decrease in levels was exacerbated by X55 treatment, revealing an unexpected link between CDA deficiency, mitochondrial function and X55 response. Finally, we identified strong downregulation of MAPT (encoding Tau, a microtubule associated protein) expression as a reliable predictive marker for tumor cell X55 sensitivity.

International audience; 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.

International audience; In the search for new fluorescent triphenylamine (TP) derivatives, we studied the influence of the position and substitution of diverse heterocyclic substituents. A library of 10 fluorescent triphenylamines bearing either oxazoles or thiazoles and pyridiniums, substituted at different positions has been developed. The approach is based on a convergent C-H activation reaction between pyridineoxazoles or pyridine-thiazoles and di-iodo triphenylamine. We showed that the nature and substitution pattern of the 5-membered-(oxazole, thiazole) or 6-membered heterocycle (pyridine) has a strong influence on their fluorescence properties and on their localization in living cells as they stain either the nucleus or mitochondria.

International audience; 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 labelling 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

A series of 6BrCaQ-C

International audience; 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 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.

Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.

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.

International audience; 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

International audience; 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 che-moresistant 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 −1) treatment significantly reduced the metastatic invasiveness of human cancer cells engrafts on chicken chorioallantoic membrane. In nude mice, 0.5 or 1 mg.kg −1 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 −1 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.

International audience; The p53 tumour suppressor and guardian of the genome undergoes missense mutations that lead to functional inactivation in 50 % of human cancers. These mutations occur mostly in the DNA‐binding domain of the protein, and several of these result in conformational changes that lead to amyloid‐like protein aggregation. Herein, we describe a fluorescent biosensor that reports on the R248Q mutant of p53 in vitro and in living cells, engineered through conjugation of an environmentally sensitive probe onto a peptide derived from the primary aggregation segment of p53. This biosensor was characterised both in vitro and by means of fluorescence microscopy following facilitated delivery into cultured cells. It is shown that this biosensor preferentially reports on the p53 R248Q mutant in the PC9 lung cancer cell line compared with other lung cancer cell lines harbouring either wild‐type or no p53.

Understanding the intricate steps of protein kinase regulation requires characterization of protein-protein interactions between the catalytic subunit, its regulatory partners and the substrate. Fluorescent probes are useful tools with which to study such interactions and to gain insight into their affinities and specificities. Solvatochromic probes, which display changes in their fluorescence emission in response to changes in the polarity of the medium, are particularly attractive. Here we describe conjugation of a switchable fluorescent dye, TP-2Rho, to peptide and protein derivatives of cyclin-dependent kinase 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclin D1 and a peptide substrate. We demonstrate the sensitivity of TP-2Rho in relation to of those other dyes used for monitoring peptide-protein and protein-protein interactions. Moreover, we show that TP-Rho-labelled peptides can be introduced into living cells to probe endogenous CDK4/cyclin D.

In this work, six π-conjugated oxazole compounds dissolved in dichloromethane were characterized with linear and nonlinear optical measurements. Z-scan with femtosecond laser pulses was employed to determine the two-photon absorption (TPA) spectra. Other photophysical parameters, such as absorbance, solvatochromism, lifetime fluorescence, and fluorescence anisotropy, were evaluated with linear optical techniques. The experimental TPA cross section spectra were adjusted by the sum-over-states (SOS) model, by which important parameters such as transition dipole moments and broadening parameters were determined. To better understand the TPA spectra of the oxazole compounds, quantum-chemical calculations using the response function formalism and the density functional theory level of theory were performed. Using the results provided by the quantum-chemical calculations and the broadening parameters estimated through the application of the SOS model, the TPA spectra were simulated by the superposition (summation) of individual homogeneous Lorentzian absorption profiles.

The French National Compound Library (Chimiotheque Nationale) has been created in 2003 and is the federation of local collections. It contains more than 56 000 small molecules and natural compounds synthesised or isolated in different laboratories over the past years. This explains the diversity of the collection. The strength of this initiative is the ability to connect chemists and biologists for the development of hits. This development involves the synthesis of analogues or/and chemical tools to find new targets. These collaborations lead to the identification of new chemical probes. These probes able to modulate a biological function are essential to study biological pathways. They can also be useful for therapeutic applications. This article will describe the major achievements and perspectives of the French Chemical Library.

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.

We report a new turn-off fluorescent probe, PhenDV, for the identification of high affinity quadruplex (G4) DNA ligands. This push-pull fluorophore displays a high fluorescence quantum yield in water (ΦF = 0.21) and is a selective and strong quadruplex DNA binder. We describe its use as a fluorescent indicator for the G4 Fluorescent Intercalator Displacement (FID) assay as its fluorescence is strongly quenched when bound to G4 DNA and fully restored when displaced by ligand. This probe improves the sensitivity of the G4-FID assay, as the read out relies on increased fluorescence instead of quenching observed with classical on/off probes.

Cyclin-dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP-binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T-loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP-competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T-loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T-loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.

An intriguing conversion of 3-bromo-2H-coumarins to 3-(benzofuran-2-yl)-2H-coumarins under palladium catalysis is reported. The process involves, from only one single starting material, three transformations and two bond formations in one pot: C–C bond formation via C–H activation and C–O bond formation through 2H-coumarin-to-benzofuran ring contraction under palladium catalysis. Moreover, the photophysical properties of all synthesized compounds were studied.

International audience; Despite the emergence of targeted therapies and immunotherapy, chemotherapy remains the gold-standard for the treatment of most patients with solid malignancies. Spindle poisons that interfere with microtubule dynamics are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of chemoresistance highlight the need for identifying alternative agents. We performed a high throughput cell-based screening and selected a pyrrolopyrimidine molecule (named PP-13). In the present study, we evaluated its anticancer properties in vitro and in vivo. We showed that PP-13 exerted cytotoxic effects on various cancer cells, including those resistant to current targeted therapies and chemotherapies. PP-13 induced a transient mitotic blockade by interfering with both mitotic spindle organization and microtubule dynamics and finally led to mitotic slippage, aneuploidy and direct apoptotic death. PP-13 was identified as a microtubule-targeting agent that binds directly to the colchicine site in β-tubulin. Interestingly, PP-13 overcame the multidrug-resistant cancer cell phenotype and significantly reduced tumour growth and metastatic invasiveness without any noticeable toxicity for the chicken embryo in vivo. Overall, PP-13 appears to be a novel synthetic microtubule inhibitor with interesting anticancer properties and could be further investigated as a potent alternative for the management of malignancies including chemoresistant ones. In the last two decades, advances in the understanding of carcinogenesis have revolutionized the management of cancer patients with the development of targeted therapies and immunotherapy. Identification of genetic alterations in subsets of cancers leading to different subcellular signals of tumour growth and progression elicited the clinical use of oncogene-targeted therapies for patients harbouring such anomalies 1-4. In lung cancers with EGFR-activating mutations, anti-EGFR therapies, including gefitinib, erlotinib, and afatinib, have been shown to improve progression-free survival and were approved as first-line options 5. However, despite the initial response, resistance mechanisms almost inevitably ensue and limit the long-term potency of targeted therapies 6, 7. Moreover, efforts are still needed for the management of patients without targetable oncogenic driver

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.

Incorporation of π-conjugated spacers at the 2 or 5 position of a 2,5-disubstitutedaryloxazole led to new series of fluorescent dyes. They show emissions from visible to 700 nm along with significant Stokes shift up to 208 nm and a strong solvatochromic fluorescence. These compounds are easily accessible in one step through direct C–H bond functionalization.

Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760–860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.

Herein we report on the synthesis and DNA recognition properties of a series of three N-phenyl carbazole-based light-up probes initially designed for two-photon absorption. The vinylic derivatives (Cbz-2Py, Cbz-3Py) display strong fluorescence enhancement when bound to various duplex- and quadruplex-forming oligonucleotides whereas the oxazole derivative is not fluorescent in DNA. Determination of affinity constants by fluorimetric titrations evidenced that Cbz-2Py has a clear preference for AT-rich duplex structures. Circular Dichroism (CD) measurements confirmed the sequence-dependent binding of this compound and suggest insertion in the minor groove as shown by a strong induced CD (ICD) signal and further supported by molecular modeling. Altogether the data indicate that duplex vs quadruplex selectivity of the dyes is strongly dependent on the sequence of the duplex. Finally, the dyes exhibit high two-photon absorption cross-sections (up to 540GM in glycerol) and allow a fine and bright staining of nuclear DNA with low background fluorescence as shown by one and two-photon confocal microscopy imaging of fixed cells.

International audience; In mammals, nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades mRNAs harboring a premature termination codon, in order to prevent the synthesis of truncated proteins. To gain insight into NMD mechanism, we identified NMDI 1 (for nonsense-mediated mRNA decay inhibitor 1) as a small molecule inhibitor of the NMD pathway. We characterized the mode of action of this compound and demonstrated that it acts upstream of hUPF1. NMDI 1 induced the loss of interactions between hSMG5 and hUPF1 and the stabilization of hyperphosphorylated isoforms of hUPF1. Incubation of cells with NMDI 1 allowed us to demonstrate that NMD factors and mRNAs subject to NMD transit through processing bodies (P-bodies) as it is the case in yeast. The results suggest a model in which mRNA and NMD factors are sequentially recruited to P-bodies.

A straightforward access to numerous novel substituted dihydrobenzo- and dihydronaphthoacridines is described using a unique molecular platform in two key steps. A large range of carbon-based substituents such as aromatic, vinyl, alkynyl fragments through Pd-catalysed couplings has been installed. The molecular diversity is extended to the introduction of aza-heterocycles and further authorizes the installation of alkylamino chains by means of Cu-promoted C–N bond formation. Possible access to quinolinium salts is also described. The methodology revealed convenient preparation of a wide panel of molecules that display various rigidity/flexibility and lipophilic/hydrophilic balances. Finally, the influence of structural modulations on the photophysical properties of these novel architectures is also studied. It is noteworthy that styryl and alkynyl derivatives are emissive in water (ϕF up to 12%).

The French National Compound Library (Chimiothèque Nationale) has been created in 2003 and is the federation of local collections. It contains more than 56 000 small molecules and natural compounds synthesised or isolated in different laboratories over the past years. This explains the diversity of the collection. The strength of this initiative is the ability to connect chemists and biologists for the development of hits. This development involves the synthesis of analogues or/and chemical tools to find new targets. These collaborations lead to the identification of new chemical probes. These probes able to modulate a biological function are essential to study biological pathways. They can also be useful for therapeutic applications. This article will describe the major achievements and perspectives of the French Chemical Library.

Telomerase is the enzyme that maintains the length of telomeres. It is minimally constituted of two components: a core reverse transcriptase protein (hTERT) and an RNA (hTR). Despite its significance as an almost universal cancer target, the understanding of the structure of telomerase and the optimization of specific inhibitors have been hampered by the limited amount of enzyme available. Here, we present a breakthrough method to produce unprecedented amounts of recombinant hTERT and to reconstitute human telomerase with purified components. This system provides a decisive tool to identify regulators of the assembly of this ribonucleoprotein complex. It also enables the large-scale screening of small-molecules capable to interfere with telomerase assembly. Indeed, it has allowed us to identify a compound that inhibits telomerase activity when added prior to the assembly of the enzyme, while it has no effect on an already assembled telomerase. Therefore, the novel system presented here may accelerate the understanding of human telomerase assembly and facilitate the discovery of potent and mechanistically unique inhibitors.

International audience; Background: Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis. Results: Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies. Conclusions: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.

International audience; On the basis of our previous work on DNA fluorophores derived from vinylpyridinium-triphenylamine, we explored the structure space around the electron-rich triphenylamine (TP) core by changing the vinyl bond to an oxazole ring. As 2,5-diaryloxazoles are known to be highly fluorescent and efficient two photon absorbers, we synthesized analogues with two different connections of the oxazole to the triphenylamine core: TP-Ox2Py and TP-Ox5Py sets. Since the benzimidazolium group was proven to be more effective in the TP series than the pyridinium, we also synthesized a TP-Ox5Bzim set. The TP-Ox5Py series retains the TP-Py properties: on/off behavior on DNA, good two-photon cross-section and bright staining of nuclear DNA by microscopy under both one or two-photon excitation. On the other hand, the TP-Ox2Py series does not display fluorescence upon binding to DNA. The TP-Ox5Bzim set is fluorescent even in the absence of DNA and displays lower affinity than the corresponding TP-Ox5Py. CD experiments and docking were performed to understand these different behaviors.

Triphenylamines are on/off fluorescent DNA minor groove binders, allowing nuclear staining of fixed cells. By contrast, they accumulate in the cytoplasm of living cells and efficiently trigger cell apoptosis upon prolonged visible light irradiation. This process occurs concomitantly with their subcellular re-localization to the nucleus, enabling fluorescence imaging of apoptosis.

'Keteniminium triflates' (=N-alk-1-enylideneaminium trifluoromethanesulfonates; [MeN(Ts)CH=C= NR1(R-2)]+TfO-) generated in situ from N-alpha-tosylsarcosinamides MeN(Ts)CH2CONR1(R-2) unexpectedly react with cyclopenta-1,3-diene and cyclohexa-1,3-diene to give Diets-Alder reactions across the C=C bond of the cumulene. The use of N-alpha-tosylsarcosinamides derived from chiral pyrrolidines allows the direct preparation of trinorbornenone derivative 6 in high enantiomer purity.

Evaluation of an hypothesis regarding the biomimetic synthesis of manzamine alkaloids from the stepwise condensation of malonate, aldehydes and primary amine derivatives is presented. The first part of this study was concentrated on the Knoevenagel reaction, with various aldehydes, of glutaconaldehydes 7 and their ester analogues 17 and 23, in order to prepare stable derivatives of dienes 8. This reaction, previously reported to work only with salicylaldehyde and glutaconic esters, appeared to be difficult to control in the case of the glutaconaldehyde derivative 7a, but was successful when starting from esters 17 and 23. This result allowed the preparation of new dienes 21, 22a−c, and 25. The second part of this paper reports preliminary results concerning the reactivity of diene 21 towards primary amines and aldehyde equivalents. Dienals such as 21 were found to react with primary amines to afford 1,2-dihydropyridines such as 26 or 27. Diene 21 was also shown to give diastereoisomeric cycloadducts 30 or 32 with enamines or oxazolidine derivatives. The cycloadducts 30 were finally shown to react with n-butylamine in acidic medium to give rearranged cyclopentenes 36 and 37. Although the synthesis of derivatives such as 9 or 10, corresponding to the AB ring system of manzamine A, by this approach have yet to be successful, the reported results reveal some new aspects of this multicomponent chemistry, some features of which are related to polyketide-type condensations. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

A two-step sequence for the asymmetric vicinal acylation of olefins by a [2+2+1] strategy is reported. The key reaction is a [2+2] cycloaddition of an olefin to a chiral keteniminium salt derived from N-tosylsarcosinamide. This is followed by a regioselective Baeyer-Villiger oxidation of the resulting cyclobutanone to yield a lactol derivative that is equivalent to the product of addition of a carboxyl and a carbonyl group to the olefin. N-Tosylsarcosinamides derived from prolinol methyl ether and 2,5-dimethylpyrrolidine gave the best yields and diastereoselectivities. Five- and six-membered cycloolefins only gave cis products as expected. With seven- and eight-membered rings and cis 1,2-disubstituted acyclic olefins, partial or complete epimerisation of the cis to the trans adducts was observed. Facial selectivities were generally good except for terminal olefins. The oxidation step proceeded in high yields to give crystalline compounds which could usually be obtained in enantiopure form by simple recrystallisation.

alpha-N-Methyl-N-tosyl cyclobutanones 2 which had been previously prepared in good yields and high enantiomeric excesses from olefins and chiral keteniminium salts have been converted into the corresponding oxiranes 3 by reaction with dimethylsulfonium methylid. The stereochemistry of this reaction was found to be dependent on several factors which have been analyzed. Treatment of these oxiranes with a stoichiometric amount of lithium iodide in refluxing tetrahydrofuran gave excellent yields of monocyclic or fused cyclopentenones 4 resulting from a P-elimination of N-methyl-N-tosylamide from a primarily formed cyclopentanone. The ring-expansion was totally selective but for oxiranes attached to a bicyclo[4.2.0]octanone system. In all cases, the enantiomeric purities of the starting cyclobutanones were preserved throughout the sequence which thus represents a useful [2+2+1] strategy for the cyclopentannulation of olefins. (C) 2002 Elsevier Science Ltd. All rights reserved.

An efficient microwave-assisted palladium/copper co-mediated direct arylation of oxazoles with aryl bromides under ligandless conditions has been developed. The method is functional group tolerant and provides rapid access to medicinally relevant compounds in good yields. Coupled to the van Leusen oxazole ring synthesis, this methodology is illustrated by an expedient two-step synthesis of the four 2,5-diaryloxazole alkaloids texamine, texaline, balsoxin, and O-Me-halfordinol from commercially available starting materials.

The preparation of new thienyl-, oxazolyl-, and pyridyl-fused benzo[c]fluorenones has been developed from a unique and easily available common precursor. Starting from bromotetralone, a short sequence allowed the construction of various heterocycles followed by Suzuki coupling and final acid-promoted formation of the central cyclopentanone unit. The flexible strategy allowed the selective preparation of fused penta- to hepta-cyclic architectures. Fully aromatic targets have been obtained through the application of an oxidation process. The influence of structural modulations on the photophysical properties are described.

International audience; We report on the design and synthesis of two-photon fluorescent triphenylamines bearing two or three vinyl branches terminated by a N-methyl benzimidazolium moiety. The new compounds (TP-2Bzim, TP-3Bzim) are light-up fluorescent DNA probes with a long wavelength emission (>580 nm). Compared to their pyridinium models, the TP-Bzim dyes exhibit a remarkable improvement of both their DNA affinity and fluorescence quantum yield, especially for the two-branch derivative (TP-2Bzim: ΦF = 0.54, Ka = 107 M–1), resulting in a large fluorescence emission turn-on ratio of up to 140. Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ = 1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time. This is attributed to a tight fit of the molecule inside the minor groove of AT-rich DNA which induces geometrical rearrangements in the dye ground state as supported by circular dichroism and molecular modeling data. Consequently, TP-2bzim displays an exceptional two-photon molecular brightness (δ×ΦF = 583 GM), a value unrivalled for a small biofluorophore. These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant, and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of “stemness”. These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.

On the basis of our previous work on vinyl-triphenylamine derived DNA fluorophores we explored the structure space around this core by coupling it to diverse cationic, anionic and zwitterionic groups in the aim of targeting different classes of biomolecules. In parallel core modifications were performed to optimize the photophysical properties (quantum yield, two-photon absorption). The resulting water soluble π-conjugated molecules are called TP dyes and display an exceptional combination of optical properties: high two-photon absorption cross-section, high photostability, no self-quenching, and switchable fluorescence emission when bound to a biopolymer matrix. The linear and nonlinear optical properties of the TP dyes were studied in vitro in presence of DNA and in presence of a model protein (human serum albumin) using complementary absorption and fluorescence spectroscopy characterization tools. Structure modifications enabled to switch from DNA probes (cationic TP-pyridinium series) to protein probes (anionic TP-rhodanine series) without affecting the optical properties. Finally most TP compounds appear cell-permeant and show an intracellular localization consistent with their in vitro target specificity.

International audience; In this paper we describe the synthesis of vinyl-diazine triphenylamines. These compounds exhibit strong green-yellow fluorescence in dichloromethane solution, important emission solvatochromism and acidochromism. Regioselective N-alkylation of these dyes provides cationic compounds that exhibit affinity for double-stranded DNA. Binding to the biopolymer results in a strong bathochromic shift and increase of the emission intensity.