9 research outputs found
A multicomponent reaction platform towards multimodal near-infrared BODIPY dyes for STED and fluorescence lifetime imaging
We report a platform combining multicomponent reaction synthesis and automated cell-based
screening to develop biocompatible NIR-BODIPY fluorophores. From a library of over 60 fluorophores,
we optimised compound NIRBD-62c as a multimodal probe with suitable properties for STED superresolution
and fluorescence lifetime imaging. Furthermore, we employed NIRBD-62c for imaging
trafficking inside cells and to examine how pharmacological inhibitors can alter the vesicular traffic
between intracellular compartments and the plasma membrane.Fundacion Seneca 21124/SF/19
MCIN/AEI PID2020-114256RB-I00Spanish GovernmentEuropean Commission PID2019-107991RB-I00
European Research Council (ERC)
European Commission 771443Royal Society of LondonEuropean Commission RG16028
Self-Assembled Lanthanide Antenna Glutathione Sensor for the Study of Immune Cells
This work was supported by grants CTQ2017-85658-R, BFU2015-67284-R, and PID2019-104366RB-C22 funded by MCIN/AEI/10.13039/501100011033/FEDER "Una manera de hacer Europa"; grant PID2020-114256RB-I00 funded by MCIN/AEI/10.13039/501100011033; grant A-FQM-386-UGR20 funded by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, and the CSIC grant 201580E073. Funding for open access charge: Universidad de Granada/CBUA.The small molecule 8-methoxy-2-oxo-1,2,4,5-
tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves
as a reactive non-fluorescent Michael acceptor, which after reaction
with thiols becomes fluorescent, and an efficient Eu3+ antenna,
after self-assembling with this cation in water. This behavior makes
2b a highly selective GSH biosensor, which has demonstrated high
potential for studies in murine and human cells of the immune
system (CD4+ T, CD8+ T, and B cells) using flow cytometry. GSH
can be monitored by the fluorescence of the product of addition to
2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was
able to capture baseline differences in GSH intracellular levels
among murine and human CD4+ T, CD8+ T, and B cells. We also
successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of
CD4+ naiv̈ e T cells into regulatory T cells (TREG).MCIN/AEI/FEDER "Una manera de hacer Europa" CTQ2017-85658-R
BFU2015-67284-R
PID2019-104366RB-C22MCIN/AEI PID2020-114256RB-I00FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades A-FQM-386-UGR20CSIC grant 201580E073Universidad de Granada/CBU
New Red-Emitting Chloride-Sensitive Fluorescent Protein with Biological Uses
This work was financially supported by CTQ2017-85685-R, CTQ2017-85454-C2-1-P, and CTQ2017-86125-P (MICIU/AEI/ERDF). J.M.P. and J.D.V. thank UEQ for funding. J.V.P. is supported by an FPU fellowship (FPU17/04749). D.A. was supported by the Italian Cystic Fibrosis Foundation (grant FFC#3/2019) and by Fondazione Cassa Rurale Trento Rovereto (ref 2018.256). The authors acknowledge the Universidad of Granada (Spain) cell culture and microscopy central facilities (CIC-UGR).A new chloride-sensitive red fluorescent protein
derived from Entacmaea quadricolor is described. We found that
mBeRFP exhibited moderate sensitivity to chloride and, via sitedirected
mutagenesis (S94V and R205Y), we increased the
chloride affinity by more than an order of magnitude (kd = 106
± 6 mM) at physiological pH. In addition, cis−trans isomerization
of the chromophore produces a dual emission band with different
chloride sensitivities, which allowed us to develop a ratiometric
methodology to measure intracellular chloride concentrations.MICIU/AEI/ERDF CTQ2017-85685-R
CTQ2017-85454-C2-1-P
CTQ2017-86125-PUEQSpanish Government FPU17/04749Ministry of Health, ItalyItalian Cystic Fibrosis Research Foundation 3/2019Fondazione Cassa Rurale Trento Rovereto 2018.25
A multicomponent reaction platform towards multimodal near-infrared BODIPY dyes for STED and fluorescence lifetime imaging
We report a platform combining multicomponent reaction synthesis and automated cell-based screening to develop biocompatible NIR-BODIPY fluorophores. From a library of over 60 fluorophores, we optimised compound NIRBD-62c as a multimodal probe with suitable properties for STED super-resolution and fluorescence lifetime imaging. Furthermore, we employed NIRBD-62c for imaging trafficking inside cells and to examine how pharmacological inhibitors can alter the vesicular traffic between intracellular compartments and the plasma membrane
A solvatofluorochromic silicon-substituted xanthene dye useful in bioimaging
In this work, we have performed an in-depth study of the photophysics and solvatofluorochromism of a red-emitting Si-xanthene dye, an analog of Tokyo Magenta (TM) historically developed by Egawa et al. (Chem. Commun. 2011, 47, 4162–4164). The results show a strong dependency of the emission properties of 2-Me-4-OMe-TM on the polarity of the solvent. For instance, the dye exhibited an increase in its fluorescence lifetime with the decrease in solvent polarity. Therefore, in this work, this spectral behavior has been used as a new approach for determining the intracellular microenvironment polarity through the measurement of its fluorescence lifetime by Fluorescence Lifetime Imaging Microscopy (FLIM). Our experiments confirmed the ability of the dye to detect changes in polarity between different intracellular compartments.This work was funded by grants CTQ2017-85658-R, CTQ2014-55474-C2-2-R (Spanish Ministry of Economy and Competitiveness; Agencia Estatal de Investigacion, AEI; and European Regional Development Fund, ERDF), QM2012-790 (Junta de Andalucía), and a grant from the Fundación Botín
Photophysical study of new fluorescent probes and their application in biological systems of biomedical interest
El principal tema de investigación de esta Tesis es el estudio de nuevas y diferentes
sondas y su aplicación en procesos biológicos a través de la innovadora técnica de microscopía
de fluorescencia como es la microscopía de imagen de tiempo de vida de fluorescencia (FLIM).
Las técnicas de fluorescencia, y en particular la microscopía, representan un método no invasivo
caracterizado por su baja toxicidad, proporcionando un gran número de ventajas entre las que
se incluyen un alto nivel de sensibilidad, especificidad y un amplio rango de concentración. Este
trabajo puede dividirse en el estudio de dos tipos diferentes de fluoróforos, las sondas sensibles
al medio ambiente, incluyendo los colorantes solvatocrómicos y fluorogénicos, y las sondas de
larga duración, como los biosensores basados en lantánidos, con sus aplicaciones biomédicas.
En el primer grupo, hemos incluido dos sondas diferentes; una es un derivado de
xanteno modificado con silicio (2-4-TM), que presenta unas fuertes propiedades
solvatocrómicas capaces de detectar cambios en la polaridad de su entorno. Durante el estudio
de sus propiedades solvatocrómicas descubrimos una unión de adsorción natural de este
derivado de xanteno modificado con silicio a macroestructuras. La combinación de estas
propiedades nos permitió detectar en tiempo real los cambios en la polaridad del entorno
alrededor del colorante durante las etapas iniciales del proceso de agregación del β-amiloide
(Aβ), utilizando la espectroscopia de fluorescencia en estado estacionario y resuelta en el tiempo
y técnicas avanzadas de obtención de imágenes de fluorescencia como FLIM. Mediante el uso
de FLIM como un excelente enfoque para el estudio de la agregación amiloidogénica como
método de detección no invasivo, pudimos establecer una escala de polaridad para distinguir la
hidrofobicidad de los agregados y diferenciar entre los diferentes tipos de agregados Aβ-42
preamiloides. Las primeras etapas del proceso de agregación del péptido β-amiloide (Aβ) son de
especial interés para comprender el origen de múltiples trastornos neurodegenerativos, como
la enfermedad de Alzheimer, un importante problema de salud pública que afecta a millones de
personas en todo el mundo y que sigue aumentando de forma espectacular.
El segundo fluoróforo incluido en las sondas sensibles al medio consiste en la sonda
basada en emisión inducida por agregación (AIE, por sus siglas en inglés), denominada PEMC.
Tras estudiar sus propiedades solvatocrómicas, descubrimos que presentaba preferencias por
medios no polares, exhibiendo AIE en condiciones específicas por inmovilización. Esto nos
permitió estudiar su tasa de incorporación espontánea en las células mediante imágenes de
tiempo de vida de fluorescencia y observar su patrón intracelular producido por la AIE.
Curiosamente, las fuertes diferencias en la intensidad y el tiempo de vida de fluorescencia de
los diferentes compartimentos intracelulares facilitaron un aislamiento selectivo para el estudio detallado de orgánulos específicos, como el citoplasma, las mitocondrias y las estructuras
periféricas de F-actina en la membrana plasmática. Dado que los orgánulos de las células
eucariotas desempeñan un papel fundamental en la función celular, la importancia de visualizar
y controlar la morfología y los cambios de actividad de determinados orgánulos proporciona
información muy útil a nivel subcelular y molecular en el diagnóstico y la terapéutica de
enfermedades.
En lo que respecta a las sondas de larga duración, hemos realizado el estudio de un
biosensor basado en lantánidos, que consiste en una pequeña molécula que se comporta como
un aceptor de Michael reactivo no fluorescente, que al reaccionar con tioles se vuelve
fluorescente, y una antena eficiente de Eu3+, que tras autoensamblarse con este catión en agua
representa un logro muy innovador y químicamente interesante en la detección de biotioles, y
específicamente de glutatión (GSH). El comportamiento de nuestro biosensor altamente
selectivo de GSH mostró un alto potencial para estudios en células murinas y humanas del
sistema inmune (células T CD4+, T CD8+ y B) por citometría de flujo, siendo capaz de capturar las
diferencias basales en sus niveles de GSH intracelular. Nuestro biosensor también fue capaz de
monitorizar con éxito los cambios intracelulares de GSH asociados a las variaciones metabólicas
que rigen la inducción de las células T naïve CD4+ en células T reguladoras (Treg).The main research topic of this Thesis is the study of new different probes and their
application in biological processes through the innovative fluorescence microscopy technique as
fluorescence lifetime imaging microscopy (FLIM). Fluorescence techniques, and particularly
microscopy, represent a non-invasive method characterized by their low toxicity, providing a
number of advantages including a high level of sensitivity, specificity and wide concentration
range. This work can be divided into the study of two different types of fluorophores,
environment-sensitive probes, including solvatochromic and fluorogenic dyes, and long-lifetime
probes, such as lanthanide-based biosensors, with their biomedical applications.
In the first group, we have included two different probes; one is a silicon-modified
xanthene derivative (2-4-TM), that present a strong solvatochromic properties able to detect
changes in the environment polarity. We studied the solvatochromic properties and discover a
natural adsorption binding of this silicon-modified xanthene derivative to macrostructures. The
combination of these properties allowed us to detect in real time changes in the environment
polarity around the dye during the initial stages of the β-amyloid (Aβ) aggregation process, using steady-state, time resolved fluorescence spectroscopy and advanced fluorescence imaging
techniques such as FLIM. By using FLIM as an excellent approach to study amyloidogenic
aggregation as non-invasive detection method, we were able to establish a polarity scale to
distinguish the hydrophobicity of the aggregates and differentiate between different types of
pre-amyloid Aβ-42 aggregates. The early stages of the β-amyloid (Aβ) peptide aggregation
process are of particular interest in understanding the origin of multiple neurodegenerative
disorders, such as Alzheimer’s disease, an important public health problem affecting millions of
people worldwide, and one that continues to increase dramatically.
The second fluorophore included in the environment-sensitive probes is the
aggregation-induced emission (AIE)-based probe, named PEMC. After study its solvatochromic
properties, we found that presents preferences for nonpolar media, exhibiting AIE under specific
conditions by immobilization. This allowed us to study the rate of its spontaneous incorporation
into cells by fluorescence lifetime imaging and to observe its intracellular pattern produced by
the AIE. Interestingly, the strong differences in fluorescence intensity and fluorescence lifetime
of the different intracellular compartments facilitated selective isolation for detailed study of
specific organelles, such as cytoplasm, mitochondria and peripheral F-actin structures in the
plasma membrane. Since organelles in eukaryotic cells play a key role in cellular function, the
importance of visualizing and monitoring the morphology and activity changes of specific
organelles provide very useful information at the subcellular and molecular level that opens up
opportunities for use in disease diagnosis and therapy.
Respecting long-lifetime probes, we have studied and lanthanide based biothiol sensor,
consisting of a small molecule that behaves as a reactive non-fluorescent Michael acceptor,
which upon reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after selfassembling
with this cation in water represents a very innovative and chemically interesting
achievement to detect biothiols, and specifically glutathione (GSH). The behaviour of our highly
GSH-selective biosensor showed a high potential for studies in murine and human cells of the
immune system (CD4+ T, CD8+ T, and B cells) by flow cytometry, being able to capture their
baseline differences in intracellular GSH levels. Our biosensor was also successfully monitored
intracellular changes in GSH associated with the metabolic variations governing the induction of
CD4+ naïve T cells into regulatory T cells (Treg).Tesis Univ. Granada
Synthesis, photophysics, and solvatochromic studies of an aggregated-induced-emission luminogen useful in bioimaging
Biological samples are a complex and heterogeneous matrix where different macromolecules with different physicochemical parameters cohabit in reduced spaces. The introduction of fluorophores into these samples, such as in the interior of cells, can produce changes in the fluorescence emission properties of these dyes, caused by the specific physicochemical properties of cells. This effect can be especially intense with solvatofluorochromic dyes, where changes in the polarity environment surrounding the dye can drastically change the fluorescence emission. In this article, we studied the photophysical behavior of a new dye and confirmed the aggregation-induced emission (AIE) phenomenon with different approaches, such as by using different solvent proportions, increasing the viscosity, forming micelles, and adding bovine serum albumin (BSA), through analysis of the absorption and steady-state and time-resolved fluorescence. Our results show the preferences of the dye for nonpolar media, exhibiting AIE under specific conditions through immobilization. Additionally, this approach offers the possibility of easily determining the critical micelle concentration (CMC). Finally, we studied the rate of spontaneous incorporation of the dye into cells by fluorescence lifetime imaging and observed the intracellular pattern produced by the AIE. Interestingly, different intracellular compartments present strong differences in fluorescence intensity and fluorescence lifetime. We used this difference to isolate different intracellular regions to selectively study these regions. Interestingly, the fluorescence lifetime shows a strong difference in different intracellular compartments, facilitating selective isolation for a detailed study of specific organelles.</p
Self-Assembled Lanthanide Antenna Glutathione Sensor for the Study of Immune Cells
The small molecule 8-methoxy-2-oxo-1,2,4,5-
tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves
as a reactive non-fluorescent Michael acceptor, which after reaction
with thiols becomes fluorescent, and an efficient Eu3+ antenna,
after self-assembling with this cation in water. This behavior makes
2b a highly selective GSH biosensor, which has demonstrated high
potential for studies in murine and human cells of the immune
system (CD4+ T, CD8 + T, and B cells) using flow cytometry. GSH
can be monitored by the fluorescence of the product of addition to
2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was
able to capture baseline differences in GSH intracellular levels
among murine and human CD4 + T, CD8 + T, and B cells. We also
successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of
CD4+ naï ve T cells into regulatory T cells (TREG ).This work was supported by grants CTQ2017-85658-R,
BFU2015-67284-R, and PID2019-104366RB-C22 funded by
MCIN/AEI/10.13039/501100011033/FEDER “Una manera
de hacer Europa”; grant PID2020-114256RB-I00 funded by
MCIN/AEI/10.13039/501100011033; grant A-FQM-386-
UGR20 funded by FEDER/Junta de Andalucía-Consejería de
Transformación Económica, Industria, Conocimiento y Universidades, and the CSIC grant 201580E073. Funding for
open access charge: Universidad de Granada/CBUA.Peer reviewe
New bioisosteric sulphur-containing choline kinase inhibitors with a tracked mode of action
: Since the identification of human choline kinase as a protein target against cancer progression, many compounds have been designed to inhibit its function and reduce the biosynthesis of phosphatidylcholine. Herein, we propose a series of bioisosteric inhibitors that are based on the introduction of sulphur and feature improved activity and lipophilic/hydrophilic balance. The evaluation of the inhibitory and of the antiproliferative properties of the PL (dithioethane) and FP (disulphide) libraries led to the identification of PL 48, PL 55 and PL 69 as the most active compounds of the series. Docking analysis using FLAP suggests that for hits to leads, binding mostly involves an interaction with the Mg2+ cofactor, or its destabilization. The most active compounds of the two series are capable of inducing apoptosis following the mitochondrial pathway and to significantly reduce the expression of anti-apoptotic proteins such as the Mcl-1. The fluorescence properties of the compounds of the PL library allowed the tracking of their mode of action, while PAINS (Pan Assays Interference Structures) filtration databases suggest the lack of any unspecific biological response