The receptor for advanced glycation end-products (RAGE) and its ligands are important players in pathological conditions such as diabetes, neurodegenerative diseases, and cancers. RAGE is a cell surface molecule of the immunoglobulin superfamily. Alternatively spliced variants lacking either only the intracellular domain or both the intracellular and the transmembrane domains are also expressed in some tissues.
The receptor for advanced glycation end-products (RAGE) and its ligands are important players in pathological conditions such as diabetes, neurodegenerative diseases, and cancers. RAGE is a cell surface molecule of the immunoglobulin superfamily. Alternatively spliced variants lacking either only the intracellular domain or both the intracellular and the transmembrane domains are also expressed in some tissues. RAGE functions in development and inflammation. It interacts with multiple ligands including members of the S100 protein family, high mobility group box-1 protein (HMGB1), and advanced glycation end products. It has been shown that the activation of RAGE triggers a positive feedback loop that stimulates the expression of both the receptor and some of its ligands, thus amplifying their deleterious effects (1-3). Therefore, the targeting of the specific interactions of RAGE with its ligands is a potential method to control these diseases. Toward this aim the project proposes an in vitro assay to screen for new molecules that block the interaction of the receptor with its pathological ligands.
The project aims to deliver purified unlabeled and fluorescently-labeled soluble RAGE and several of its ligands, a protocol for competitive binding assays based on the obtained products, and furthermore, an automated and validated technology which provides a rapid and easy way for screening of libraries of compounds.
References:
Project Team: | Florin Pastrama, PhD Tech. Emilia Ardelean |
Project Leader: | Dr. Ioana Popa Institute of Biochemistry Protein Folding Department Splaiul Independentei 296, Bucuresti Email: ipopa@biochim.ro |
Scientific Results:
Our project aimed at the development of a method for rapid and sensitive detection of RAGE-ligand interactions. We obtained various recombinant variants of soluble RAGE and some of its ligands. Using these proteins we developed, optimized and validated two fluorescence based methods. One method relies on the direct measurement of the fluorescent ligand binding to pre-adsorbed sRAGE in microplates, whereas in the other method a FRET signal is generated in solution upon RAGE, indirectly fluorescently labeled, interacted with a fluorescent ligand. These methods are suitable for high-throughput screening of compounds, potential inhibitors of RAGE-ligand binding.
Dissemination:
Poster
Title:"New Tools for Discovering Inhibitors of the Receptor for Advanced Glycation End Products"
Authors: Carmen A. Tanase, Florin Pastrama, and Ioana L. Popa* (*corresponding author)
Meeting: International Annual Meeting of the Romanian Society of Biochemistry and Molecular Biology, June 8-9 2017, Timisoara, Romania
Abstract published in New Front. Chem. (2017) Vol.26, Iss.2, S2_P11
Poster
Title:"High-throughput assay for discovery of new inhibitors of S100-RAGE binding"
Authors: Ioana L. Popa*, Carmen A. Tanase, Florin Pastrama (*corresponding author)
Meeting:International Annual Meeting of the Romanian Society of Biochemistry and Molecular Biology, September 5-7 2018, Bucharest, Romania
Poster
Title:"Homogenous TR-FRET-based high-throughput screening assay to identify inhibitors of RAGE-ligand interaction"
Authors: Carmen A. Tanase, Florin Pastrama, and Ioana L. Popa* (*corresponding author)
Meeting: International Annual Meeting of the Romanian Society of Biochemistry and Molecular Biology, September 5-7 2018, Bucharest, Romania