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Development of a fluorescence-based technology to screen for RAGE-ligand binding inhibitors

Development of a fluorescence-based technology to screen for RAGE-ligand binding inhibitors 2016-2020
Acronym: FLUORORAGE
Project director: Ioana Popa

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:

  1. Yan SF, Ramasamy R, Schmidt AM. Receptor for AGE (RAGE) and its ligands- cast into leading roles in diabetes and the inflammatory response. J Mol Med (Berl). 2009 Mar;87(3):235-47.
  2. A. Taguchi, D. C. Blood, G. del Toro, A. Canet, D. C. Lee, W. Qu, N. Tanji, Y. Lu, E. Lalla, C. Fu, M. A. Hofmann, T. Kislinger, M. Ingram, A. Lu, H. Tanaka, O. Hori, S. Ogawa, D. M. Stern, and A. M. Schmidt, "Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases," Nature, vol. 405, no. 6784, pp. 354-360, May 2000.
  3. T. Arumugam, V. Ramachandran, and C. D. Logsdon, "Effect of cromolyn on S100P interactions with RAGE and pancreatic cancer growth and invasion in mouse models," J. Natl. Cancer Inst., vol. 98, no. 24, pp. 1806-1818, Dec. 2006.
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
  1. Obtaining of fluorescently labeled soluble RAGE and RAGE ligands, and the development of a sensitive detection system of RAGE-ligands interactions based on competitive binding assays.
  2. Automatization of the competitive binding assays in order to elaborate of complex technology suitable for high-throughput screening of compound libraries.

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