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2.
Artigo em Inglês | MEDLINE | ID: mdl-34487611

RESUMO

Pharmacological inactivation of antitumor drugs toward healthy cells is a critical factor in prodrug development. Typically, pharmaceutical chemists graft temporary moieties to existing antitumor drugs to reduce their pharmacological activity. Here, we report a platform able to generate the cytotoxic agent by intramolecular cyclization. Using phenanthridines as cytotoxic model compounds, we designed ring-opened biaryl precursors that generated the phenanthridines through bioorthogonal irreversible imination. This reaction was triggered by reactive oxygen species, commonly overproduced in cancer cells, able to convert a vinyl boronate ester function into a ketone that subsequently reacted with a pendant aniline. An inactive precursor was shown to engender a cytotoxic phenanthridine against KB cancer cells. Moreover, the kinetic of cyclization of this prodrug was extremely rapid inside living cells of KB cancer spheroids so as to circumvent drug action.

3.
Methods Mol Biol ; 2350: 253-265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331290

RESUMO

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.


Assuntos
Corantes Fluorescentes , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência/métodos , Estrutura Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Peixe-Zebra
4.
Methods Mol Biol ; 2350: 191-227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331287

RESUMO

Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.


Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Óptica/métodos , Algoritmos , Animais , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Luz , Modelos Teóricos , Coloração e Rotulagem
5.
ACS Sens ; 6(3): 1157-1165, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33565309

RESUMO

Composed of a reversibly photoswitchable unit allosterically linked to a sensing module, reversibly photoswitchable sensors (rs-sensors) represent a new and attractive strategy to quantitatively read-out analyte concentrations. However, their kinetic response to illumination is complex, and much attention is required from the design to the application steps. Here, we exploit a generic kinetic model of rs-sensors which enables us to point to key thermokinetic parameters, such as dissociation constants and kinetic rates for exchange toward the analyte, and cross-sections for photoswitching. The application of the model allows to evaluate the robustness of the analyzed parameters and to introduce a methodology for their reliable use. Model and methodology have been experimentally tested on a newly reported calcium sensor based on a reversibly photoswitchable green fluorescent protein allosterically linked to a calcium-sensing module integrating calmodulin and an RS20 peptide.


Assuntos
Cálcio , Calmodulina , Proteínas de Fluorescência Verde , Cinética , Proteínas Luminescentes/metabolismo
6.
ACS Omega ; 5(30): 19312-19313, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32775935

RESUMO

[This corrects the article DOI: 10.1021/acsomega.0c00957.].

7.
ACS Omega ; 5(25): 15105-15114, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32637783

RESUMO

Noninvasiveness, minimal handling, and immediate response are favorable features of fluorescence readout for high-throughput phenotyping of labeled plants.Yet, remote fluorescence imaging may suffer from an autofluorescent background and artificial or natural ambient light. In this work, the latter limitations are overcome by adopting reversibly photoswitchable fluorescent proteins (RSFPs) as labels and Speed OPIOM (out-of-phase imaging after optical modulation), a fluorescence imaging protocol exploiting dynamic contrast. Speed OPIOM can efficiently distinguish the RSFP signal from autofluorescence and other spectrally interfering fluorescent reporters like GFP. It can quantitatively assess gene expressions, even when they are weak. It is as quantitative, sensitive, and robust in dark and bright light conditions. Eventually, it can be used to nondestructively record abiotic stress responses like water or iron limitations in real time at the level of individual plants and even of specific organs. Such Speed OPIOM validation could find numerous applications to identify plant lines in selection programs, design plants as environmental sensors, or ecologically monitor transgenic plants in the environment.

8.
Methods Enzymol ; 624: 1-23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31370925

RESUMO

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.


Assuntos
Optogenética/métodos , Compostos Policíclicos/química , Receptores de Estrogênio/genética , Ativação Transcricional , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Expressão Gênica , Luz , Processos Fotoquímicos , Receptores de Estrogênio/química , Peixe-Zebra/embriologia
9.
Nat Chem ; 11(9): 797-805, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31383980

RESUMO

The well-established oxidative addition-reductive elimination pathway is the most followed one in transition metal-catalysed cross-coupling reactions. While readily occurring with a series of transition metals, gold(I) complexes have shown some reluctance to undergo oxidative addition unless special sets of ligands on gold(I), reagents or reaction conditions are used. Here we show that under visible-light irradiation, an iridium photocatalyst triggers-via triplet sensitization-the oxidative addition of an alkynyl iodide onto a vinylgold(I) intermediate to deliver C(sp)2-C(sp) coupling products after reductive elimination. Mechanistic and modelling studies support that an energy-transfer event takes place, rather than a redox pathway. This particular mode of activation in gold homogenous catalysis was applied in several dual catalytic processes. Alkynylbenzofuran derivatives were obtained from o-alkynylphenols and iodoalkynes in the presence of catalytic gold(I) and iridium(III) complexes under blue light-emitting diode irradiation.

10.
Light Sci Appl ; 7: 97, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30510693

RESUMO

Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging.

11.
Phys Chem Chem Phys ; 20(37): 23998-24010, 2018 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-30215648

RESUMO

In order to design a dynamic titration method, we propose a theoretical model harnessing the kinetic properties of the complexation of the titrated species with a titrating photoswitchable reagent. Forced oscillations of illumination are imposed and concentration oscillations of the targeted species are deduced from the equations of chemical kinetics. We determine analytical expressions of the resonance conditions on the control parameters, angular frequency, mean light intensity, and total concentration of the photoswitchable reagent, which optimize the out-of-phase amplitude of concentration oscillations. A user-friendly protocol of dynamic titration is proposed.

12.
Org Lett ; 20(16): 4950-4953, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30070483

RESUMO

An original and mild synthetic route for the preparation of novel azafluorenones and derivatives via a ruthenium-mediated [2 + 2 + 2] cycloaddition of α,ω-diynes and cyanamides has been developed. This atom-economical catalytic process demonstrated remarkable regioselectivities to access fluorescent azafluorenone derivatives. The photophysical properties of azafluorenone derivatives have been evaluated, and photoluminescence phenomena at solid and liquid states have been highlighted.

13.
Chem Commun (Camb) ; 54(49): 6396-6399, 2018 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-29872786

RESUMO

Spatiotemporal control of molecular distribution is much in demand in many fields of chemistry. To address this goal, we exploit a low molecular weight branched self-immolative architecture, which acts as a triggerable chemically encoded timer for autonomous sequential release of two chemicals. Using a light-activated model liberating two distinct fluorophores, we generated a tunable spatially contrasted molecular distribution.

14.
Bioconjug Chem ; 29(6): 1823-1828, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29791141

RESUMO

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.


Assuntos
Compostos de Benzilideno/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/metabolismo , Rodanina/análogos & derivados , Compostos de Benzilideno/análise , Membrana Celular/química , Corantes Fluorescentes/análise , Células HEK293 , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/análise , Microscopia de Fluorescência/métodos , Transporte Proteico , Rodanina/análise , Rodanina/metabolismo
15.
Chembiochem ; 19(12): 1232-1238, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29341391

RESUMO

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.


Assuntos
Optogenética/métodos , Compostos Policíclicos/metabolismo , Receptores de Estrogênio/genética , Animais , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Compostos Policíclicos/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
Nat Commun ; 8(1): 2173, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29242600

RESUMO

The Peer Review File associated with this Article was updated shortly after publication to redact from the authors' point-by-point response a description of unpublished work describing how Speed OPIOM may in future be used to facilitate discrimination between FRET and direct excitation.

17.
Chem Sci ; 8(8): 5598-5605, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28970939

RESUMO

Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14 kDa protein tag giving a bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene rhodanine (HMBR). Here, we report a collection of fluorogens enabling tuning of the fluorescence color of FAST from green-yellow to orange and red. Beyond allowing the multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST's reversible labeling. This unprecedented behavior allows for selective detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color cross-correlation, opening up exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.

19.
Nat Commun ; 8(1): 969, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29042541

RESUMO

We present speed out-of-phase imaging after optical modulation (OPIOM), which exploits reversible photoswitchable fluorophores as fluorescent labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal. Speed OPIOM can extract the fluorescence emission from a targeted label in the presence of spectrally interfering fluorophores and autofluorescence. Up to four fluorescent proteins exhibiting a similar green fluorescence have been distinguished in cells either sequentially or in parallel. Speed OPIOM is compatible with imaging biological processes in real time in live cells. Finally speed OPIOM is not limited to microscopy but is relevant for remote imaging as well, in particular, under ambient light. Thus, speed OPIOM has proved to enable fast and quantitative live microscopic and remote-multiplexed fluorescence imaging of biological samples while filtering out noise, interfering fluorophores, as well as ambient light.Generally, fluorescence imaging needs to be done in a dark environment using molecules with spectrally separated emissions. Here, Quérard et al. develop a protocol for high-speed imaging and remote sensing of spectrally overlapping reversible photoswitchable fluorophores in ambient light.


Assuntos
Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Brassicaceae/genética , Desenho de Equipamento , Corantes Fluorescentes/análise , Análise de Fourier , Proteínas de Fluorescência Verde/análise , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Dispositivos Lab-On-A-Chip , Imagem Óptica/instrumentação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética
20.
Sci Rep ; 7(1): 12316, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28951577

RESUMO

Fluorogen-binding tags, which activate the fluorescence of a specific chromophore (so-called fluorogen) upon reversible binding, have recently been proposed as a way of reducing photobleaching via fluorogen renewal. However, no generic methodology has been proposed to systematically analyze the photodamage of the fluorogen and the protein tag. Using Y-FAST (Yellow Fluorescence-activating and Absorption-Shifting Tag) as a case study we propose here a generic experimental and theoretical approach to assess how fluorogen renewal reduces the apparent photobleaching rate of a fluorogen-binding tag. Y-FAST has its apparent photobleaching rate greatly reduced by fluorogen renewal and its photostability is mainly limited by oxidation of specific residues in the protein scaffold by reactive oxygen species generated by the bound fluorogen. This study sets the groundwork for the optimization of fluorogenic systems, helping guide rational improvements to their photostability.

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