RESUMO
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 photodamage, while allowing imaging about 10 times deeper than with a confocal microscope. 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-9ylmethanol (BCN) with a good to impressive kinetic rate constant (up to 1.1 × 103 M-1 s-1) and emit in the red window with moderate to high turn-on ratios. TDDFT allowed the rationalization of both the kinetic and fluorogenic performance of the different probes. The best candidate displays a 13.8-fold turn-on measured by quantifying fluorescence intensities in live cells under one-photon excitation, whereas a value of 3 is sufficient for high contrast live-cell imaging. In addition, live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe since an 8.0-fold turn-on 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.
RESUMO
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.
Assuntos
Corantes Fluorescentes , Fótons , Microscopia de Fluorescência , Mitocôndrias , Imagem Óptica , Raios XRESUMO
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 3650â 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.
Assuntos
Corantes Fluorescentes , Fótons , Diagnóstico por Imagem , IonóforosRESUMO
Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial 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, TPn -2Bzim, TP1+ -2Bzim, TN-2Bzim) differ in 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 TP1+ -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.
Assuntos
MitocôndriasRESUMO
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.
Assuntos
Aminas/toxicidade , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Espaço Intracelular/metabolismo , Luz , Aminas/química , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Quadruplex G/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Platina/química , Proteínas Proto-Oncogênicas c-myc/química , Anticarcinógenos/química , Anticarcinógenos/uso terapêutico , Cisplatino/efeitos adversos , Cisplatino/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Ligantes , Conformação de Ácido Nucleico/efeitos dos fármacos , Piridinas/químicaRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Fluoresceína-5-Isotiocianato/química , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Mutação de Sentido Incorreto , Peptídeos/química , Peptídeos/metabolismoRESUMO
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 ß-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.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Pirimidinas/farmacologia , Pirróis/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Colchicina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Pirimidinas/química , Pirróis/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this study, we aimed to decipher the natural resistance mechanisms of mycobacteria against novel compounds isolated by whole-cell-based high-throughput screening (HTS). We identified active compounds using Mycobacterium aurum. Further analyses were performed to determine the resistance mechanism of M. smegmatis against one hit, 3-bromo-N-(5-nitrothiazol-2-yl)-4-propoxybenzamide (3), which turned out to be an analog of the drug nitazoxanide (1). We found that the repression of the gene nfnB coding for the nitroreductase NfnB was responsible for the natural resistance of M. smegmatis against 3. The overexpression of nfnB resulted in sensitivity of M. smegmatis to 3. This compound must be metabolized into hydroxylamine intermediate for exhibiting antibacterial activity. Thus, we describe, for the first time, the activity of a mycobacterial nitroreductase against 1 analogs, highlighting the differences in the metabolism of nitro compounds among mycobacterial species and emphasizing the potential of nitro drugs as antibacterials in various bacterial species.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Nitrorredutases/metabolismo , Tiazóis/química , Tiazóis/farmacologia , Regulação para Baixo , Farmacorresistência Bacteriana , Humanos , Mycobacterium/efeitos dos fármacos , Mycobacterium/enzimologia , Mycobacterium/genética , Infecções por Mycobacterium/tratamento farmacológico , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium smegmatis/genética , Nitrocompostos , Nitrorredutases/genéticaRESUMO
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 kinaseâ 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclinâ 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/cyclinâ D.
Assuntos
Ciclina D/química , Ciclina D/metabolismo , Quinase 4 Dependente de Ciclina/química , Quinase 4 Dependente de Ciclina/metabolismo , Corantes Fluorescentes/química , Maleimidas/química , Tiazolidinas/química , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Maleimidas/síntese química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Tiazolidinas/síntese químicaRESUMO
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.
Assuntos
Telomerase/biossíntese , Acridinas/química , Acridinas/farmacologia , Técnicas Genéticas , Células HEK293 , Humanos , Niacinamida/análogos & derivados , RNA/química , RNA/metabolismo , Dobramento de RNA , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Telomerase/antagonistas & inibidores , Telomerase/química , Telomerase/genética , Telomerase/metabolismo , Tiazóis , Trissacarídeos/química , Trissacarídeos/farmacologiaRESUMO
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.
Assuntos
Acridinas/química , DNA/análise , Corantes Fluorescentes/química , Compostos de Piridínio/química , Técnicas Biossensoriais , DNA/química , DNA/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Células HT29 , Ensaios de Triagem em Larga Escala , Humanos , Bibliotecas de Moléculas Pequenas , Espectrometria de FluorescênciaRESUMO
Aurora kinases play an essential role in mitotic progression and are potentially druggable targets in cancer therapy. We identified benzo[e]pyridoindoles (BePI) as powerful aurora kinase inhibitors. Their efficiency was demonstrated both in enzymatic inhibition studies and in cell culture assays. New BePI molecules were synthesized, and a structure-activity relationship study was conducted with the aim of improving the activity and solubility of the lead compound. Tetracyclic BePI derivatives are characterized by a particular curved shape, and the presence of an oxo group on the pyridine ring was found to be required for aurora kinase B inhibition. New hydrosoluble benzo[e]pyridoindolones were subsequently designed, and their efficacy was tested by a combination of cell-cycle analysis and time-lapse experiments in live cells. The most active BePI derivative, 13 b, inhibited the cell cycle, drove cells to polyploidy, and eventually induced apoptosis. It exhibited high antiproliferative activity in HeLa cells with an IC(50) value of 63 nM. Relative to compounds tested in clinical trials, this antiproliferative potency places 13 b among the top 10 aurora kinase inhibitors. Our results justify further in vivo evaluation in preclinical animal models of cancer.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Indóis/química , Indóis/farmacologia , Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Aurora Quinase B , Aurora Quinases , Ciclo Celular/efeitos dos fármacos , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
This paper describes the conception, synthesis, and characterization of new tetrapyrrolic chromophores bearing glycodendrimeric moieties inducing a potential increase of tumor targeting by a cluster effect. Two families of monoglycodendrimeric photosensitizers bearing three glycosyl units were designed, prepared with an acceptable overall efficiency and characterized by NMR, UV-visible, and fluorescence spectroscopies. The polarity and log P were evaluated by HPLC and the stir-flask method, respectively. The in vitro photoefficiency against two human tumor cell lines was assessed. The presence of the glycodendrimeric group does not appear to increase the tumor in vitro targeting.