András Dávid Tóth1, Dániel Garger1, András Balla1,2, Gábor Turu1,2, László Hunyady1,2
1 Department of Physiology, Semmelweis University, Budapest
2 MTA-SE Laboratory of Molecular Physiology, Hungarian Academy of Sciences and Semmelweis University, Budapest
Introduction: The α1A-adrenergic receptor (α1AAR) is a key regulator of blood pressure. Therefore, it may be an excellent target of therapeutic intervention using novel, better-performing drug molecules.
Aims: The purpose of our research was to identify new, previously unknown α1AAR ligands.
Method: We have developed a competitive ligand binding assay to identify receptor ligands. The essence of our method is that we detect a bioluminescence resonance energy transfer (BRET) signal between the receptor-fused bioluminescent luciferase and a fluorescently labeled receptor ligand. This signal can be prevented by any unlabeled compound which binds to the orthosteric binding site, indicating that the test compound is a receptor ligand. In addition to identification of new ligands, we also intended to set up our method for high throughput screening.
Results: The excellent signal to noise ratio of our competitive ligand binding assay was statistically demonstrated by Z' value calculation. The ligand library tested by this method was constructed using a novel in silico analysis developed by our research group. The machine learning algorithm, based on the molecular properties of the compounds, selected 479 possible α1AAR ligands from a library of 123,000 molecules. As controls of the algorithm, another 180 compounds were randomly assigned. Ligands inducing at least 30% displacement were considered as hits. In our screening, 15.6% (76) of the algorithm-chosen compounds were found to be α1AAR ligands and only 2.8% (5) from the control set.
Conclusion: In summary, we have identified 81, previously unknown α1AAR ligands with our new ligand binding assay, which ligands may be potential blood pressure regulating drugs in the future. Our results also show that the effectiveness of ligand screening can be significantly increased by in silico molecular analysis.
SUPPORTED BY THE ÚNKP_17-3-III-SE-23 NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY OF HUMAN CAPACITIES.
Doctoral School: Molecular Medicine, Semmelweis University
Program: Cellular Physiology
Supervisors: László Hunyady, Gábor Turu