New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.
One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol.
Department of Biosciences Applied to Pharmacy, Faculty of Pharmaceutical Sciences from Ribeirao Preto, University of Sao Paulo, P.O. Box: 14040-903, Ribeirao Preto, SP, Brazil.
Larissa
L
Pernomian
Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences from Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
Mayara Santos
MS
Gomes
Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Josimar Dornelas
JD
Moreira
Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences from Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
Carlos Henrique Tomich de Paula
CHTP
da Silva
Department of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy, University of Granada, Granada. Spain.
Joaquin Maria Campos
JMC
Rosa
Department of Biosciences Applied to Pharmacy, Faculty of Pharmaceutical Sciences from Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
Cristina Ribeiro de Barros
CRB
Cardoso
D055808:Drug Discovery / Q000639:trends*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D010600:Pharmacology / Q000639:trends*; D011485:Protein Binding; D011868:Radioisotopes / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism
D008024:Ligands; D011868:Radioisotopes; D043562:Receptors, G-Protein-Coupled; D008156:Luciferases
G protein-coupled receptors (GPCR); NanoLuc luciferase (Nluc); Nluc-based BRET (NanoBRET); bioluminescence
resonance energy transfer (BRET); drug discovery; ligand binding affinity; radioligand binding assay; real-time proximity
assays