CE07 - Chimie moléculaire

Catalytic Manganese-based anti-oxidants: an integrated approach from chemical design to bio-activity in cells – CATMAN

Submission summary

Superoxide dismutases (SOD) are very efficient redox metalloproteins, which protect the cell from oxidative stress. We have considered manganese SOD (MnSOD) as a model for the design of new Mn-based catalytic antioxidants (MnAOs). They will be studied directly in a cellular context, cells being considered here as new reactors involving intricate reaction media. MnAOs have been shown to have an anti-inflammatory bio-activity, but the link with their anti-superoxide activity remains elusive. The characterization of the MnAOs in a cellular context and the deep understanding of the mechanisms underlying their bio-activity will be both addressed in the blossoming field of inorganic cellular and biological chemistry.
This project will involve several steps. First, we will design new MnAOs, with improved properties (stability, inertia, targeting) (WP1A). These compounds will be first characterized outside of any cellular context (thermodynamics, kinetics of the dismutation of superoxide) (WP1B). The cellular study will focus on three questions: who (what is the speciation or nature of the MnAOs in cells?), how many (what is the cell-penetration, the amount in organelles?) and where (how is the MnAO distributed in cells?) (WP2B). The cellular model consists of intestinal epithelial cells on which LPS stimulation induces a strong inflammation mediated by oxidative stress. We will set up a specific methodology to characterize the effects of LPS and MnOAs on protein expression levels and on the redox state of the cell evaluated through the level of oxidized and reduced proteome-wide cysteines (WP2C-D), with innovative bioinformatics tools (WP2E). This innovative approach in quantitative redoxomics will allow us, by a kinetic study, to investigate in this model the links between oxidative stress and inflammation, or between anti-superoxide and anti-inflammatory activities of MnAOs. It will shed new light on the metabolic pathways altered by LPS and restored back to the basal state by MnAOs. This will enable us to select markers for a metabolic high-content screening (HCS) (WP3B-C), requiring miniaturization of cell culture and relevant assays (WP3A). This approach goes through the comparison of (a) basal cells (no induced oxidative stress, no inflammation) and (b) LPS stimulated cells (c) LPS stimulated treated with MnAO. In other terms, as in a color scale, the metabolic screening consists of questioning the proximity of (c) to (a) or (b) in a multi-dimensional space delimited by choice of the markers.
What are the main challenges in this project? This project aims at performing inorganic chemistry inside cells and is thus in line with emergent studies dealing with the control and characterization of small metal complexes in cells. This very active new field in inorganic chemical biology requires us to translate into living cells the chemical knowledge we have acquired in the chemist’s round-bottom flasks. Enhancing cell penetration, controlling the targeting of MnAO to specific organelles, knowing and mastering their speciation in cells are real challenges. Physico-chemical techniques to quantify and map metal cations at the sub-cellular level are now emerging: we will use innovative techniques, such as X-fluorescence, for direct sub-cellular mapping of Mn. We will seek a correlation between the intracellular content in MnAOs, their speciation and location on the one hand, and their biological activity on the other. This approach will provide guidelines for the rational improvement of MnAOs with an intracellular activity.
The project in inorganic biological chemistry, dealing with bio-inspired catalysts design, evaluation and characterization in cells, using subcellular imaging, and quantitative redoxomics and HCS, will be developed by a consortium with multidisciplinary expertise.

Project coordination

Clotilde Policar (Laboratoire des biomolécules)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

ICPB INSTITUT CURIE - SECT DE RECHERCHE
LBM Laboratoire des biomolécules
SMBP Spectrométrie de Masse Biologique et Protéomique

Help of the ANR 383,881 euros
Beginning and duration of the scientific project: March 2021 - 48 Months

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