bret-based-g-protein-biosensors-for-measuring-g-protein-coupled

BRET-based G Protein Biosensors for Measuring G Protein-Coupled Receptor Activity in Live Cells

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors, playing a crucial role in cellular signaling by transducing ...

University of Bordeaux

Careful analysis of any new nanomedicine device or disposal should be undertaken to comprehensively characterize the new product before application, so that any unintended side effect is minimized. Because of the increasing number of nanotechnology-based drugs, we can anticipate that regulatory authorities might adapt the approval process for nanomedicine products due to safety concerns, e.g., request a more rigorous testing of the potential toxicity of nanoparticles (NPs). Currently, the use of mesoporous silica nanoparticles (MSN) as drug delivery systems is challenged by a lack of data on the toxicological profile of coated or non-coated MSN. In this context, we have carried out an extensive study documenting the influence of different functionalized MSN on the cellular internalization and in vivo behaviour. In this article, a synthesis of these works is reviewed and the perspectives are drawn. The use of magnetic MSN (FeO@MSN) allows an efficient separation of coated NPs from cell cultures with a simple magnet, leading to results regarding corona formation without experimental bias. Our interest is focused on the mechanism of interaction with model membranes, the adsorption of proteins in biological fluids, the quantification of uptake, and the effect of such NPs on the transcriptomic profile of hepatic cells that are known to be readily concerned by NPs' uptake in vivo, especially in the case of an intravenous injection.

Biosafety of Mesoporous Silica Nanoparticles.

In contrast to artificial molecules, natural photosensitizers have the benefit of excellent toxicity profiles and of life-compatible activating energy ranges. Flavins are such photosensitizers that were selected by nature in a plethora of light-triggered biochemical reactions. Flavin-rich nanoparticles could thus emerge as promising tools in photodynamic therapies and in active-targeting drug delivery. Self-assembled flavin-conjugated phospholipids improve the pharmacokinetics of natural flavins and, in the case of controlled morphologies, reduce photobleaching phenomena. The current article presents a proof of concept for the design of riboflavin-rich nanoparticles of tunable morphology from multilamellar patches to vesicular self-assemblies. Coarse-grained simulations of the self-assembling process revealed the key interactions governing the obtained nanomaterials and successfully guided the synthesis of new flavin-conjugates of predictable self-assembly. The obtained flavin-based liposomes had a 65 nm hydrodynamic diameter, were stable, and showed potential photosensitizer activity.

Flavin-Conjugated Nanobombs: Key Structural Requirements Governing Their Self-Assemblies' Morphologies.

Several biochemical and biophysical methods are available to determine ligand binding affinities between a biological target and its ligands, most of which require purification, labelling or surface immobilisation. These measurements, however, remain challenging in regards to membrane proteins, as purification processes require their extraction from their native lipid environment, which may in turn impact receptor conformation and functionality. In this study, we have developed a novel experimental procedure using microscale thermophoresis (MST) directly from cell membrane fragments, to determine different ligand binding affinities to a membrane protein, the dopamine D2 receptor (D2R). In order to achieve this, two main challenges had to be overcome: determining the concentration of dopamine D2R in the crude sample; finding ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to approximately 36.8 ± 2.6 pmol/mg. Next, the doses-responses curves allowed for the determination of K, to approximately 5.3 ± 1.7 nM, which is very close to the reported value. Important details of the experimental procedure have been detailed in this paper to allow the application of this novel method to various membrane proteins.

An original approach to measure ligand/receptor binding affinity in non-purified samples.

Structure-photosensitizing activity relationships for a series of flavin analogues were investigated with the final goal of identifying the most potent photosensitizer in these series. The main structural modifications involved the introduction of various halogen atoms in C- and/or C-positions on the isoalloxazine ring. These compounds were synthesized by reacting judiciously-functionalized anilines with alloxan. The SAR trends showed that the photosensitizing activity increased with the size of the halogen atoms, confirming the importance of the heavy-atom effect on the photosensitizer's activity. The halogens in C were more active than the di-substituted halogens, which in turn were more active than the C-substituted equivalents. However, even if the photosensitizing activity is slightly less important for the 7- compared to the 8-substituted derivatives, the 7-haloisoalloxazines are promising photosensitizers, as they present a better cellular toxicity profile than the 8-substituted analoges. The photosensitizing activity perfectly correlated with the determined fluorescence for the same compounds. Except for the dihalogeno derivatives, all the compounds were not toxic up to a 50 μM range.

Enhancing the photosensitizing activity of natural flavins: Tuning the heavy-atom effect in the isoalloxazine series.

The adipokine apelin has been directly implicated in various physiological processes during embryogenesis and human cancers. Nevertheless, the importance of the conversion of its precursor proapelin to mature apelin in tumorigenesis remains unknown. In this study, we identify Furin as the cellular proprotein convertase responsible for proapelin cleavage. We explore the therapeutic potential of targeting proapelin cleavage sites in metastatic colorectal cancer by introducing apelin-dm, a modified variant resulting from alteration in proapelin cleavage sites. Apelin-dm demonstrates efficacy in inhibiting tumor growth, promoting cell death, suppressing angiogenesis, and early colorectal liver metastasis events. Proteomic analysis reveals reciprocal regulation between apelin and apelin-dm on proteins associated with clinical outcomes in colon cancer patients. Apelin-dm emerges as a modulator of apelin receptor dynamics, influencing affinity, internalization, and repression of apelin signaling linked to various protein kinases. Pharmacokinetic and toxicity assessments confirm the specificity, safety, and stability of apelin-dm, as well as its facile hepatic metabolism. These findings position targeting proapelin cleavage as a promising therapeutic strategy against metastatic colorectal cancer, paving the way for further clinical exploration.

Article	Year
Biosafety of Mesoporous Silica Nanoparticles. Biomimetics (Basel, Switzerland)\| PubMed ID: 31105244	2018
Flavin-Conjugated Nanobombs: Key Structural Requirements Governing Their Self-Assemblies' Morphologies. Bioconjugate chemistry\| PubMed ID: 33621053	2021
An original approach to measure ligand/receptor binding affinity in non-purified samples. Scientific reports\| PubMed ID: 35354858	2022
Enhancing the photosensitizing activity of natural flavins: Tuning the heavy-atom effect in the isoalloxazine series. Bioorganic & medicinal chemistry\| PubMed ID: 36791612	2023
Repression of apelin Furin cleavage sites provides antimetastatic strategy in colorectal cancer. EMBO molecular medicine\| PubMed ID: 39962271	2025

Estelle Rascol

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