Development of an Intracellular Quantitative Assay to Measure Compound Binding Kinetics
In this webinar, you will learn how:
- A reproducible NanoBRET® technology-based assay for intracellular compound kinetics was optimized for the BET protein family
- This assay was used to identify long, medium and short residence compounds, with rank order confirmed in lysate, TR-FRET and intrinsic tryptophan fluorescence modulation assays
- The utility of the assay to identify kinetically selective compounds was demonstrated by screening compounds against all four BET proteins.
Summary
Contemporary drug discovery typically quantifies the effect of a molecule on a biological target using the equilibrium derived measurements of IC50, EC50, or KD. Kinetic descriptors of drug binding are frequently linked with the effectiveness of a molecule in modulating a disease phenotype, however these parameters are yet to be fully adopted in early drug development. Whilst techniques have been demonstrated for quantifying binding rates to extracellular targets there are no methodologies capable of making high-throughput reproducible measurements of intracellular binding rates in live cells. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) can be used to measure interactions between fluorophore-conjugated probes and luciferase fused target proteins. In this study, an intracellular NanoBRET® competition assay is demonstrated that is capable of quantifying cellular kinetic rates of compound binding to Nanoluciferase (NL)-fused bromodomain and extra-terminal (BET) proteins. Long, medium and short residence compounds are identified within the compound test-set, with exemplars where this is driven by minor changes in compound structure. Comparative rates are generated using a lysate NanoBRET® assay and by utilising orthogonal recombinant protein-based methodologies, including TR-FRET and stopped-flow rapid-mixing intrinsic tryptophan fluorescence modulation. Finally, a screen of known pan-BET inhibitors was used to demonstrate the value of this approach in the investigation of kinetic selectivity between closely related proteins.
Speakers
Daniel Thomas, PhD
Head of Discovery Biology
Arctoris
Dan Thomas is Head of Discovery Biology at Arctoris where he is responsible for the implementation of a comprehensive scientific strategy designed to deliver the best in data quality. Bringing together a world-class team of engineers and drug discovery experts, Arctoris has developed the world’s first fully automated drug discovery platform, enabling researchers globally to configure experiments remotely and have them executed in Arctoris’ robotic laboratory facility. Together with several of the world’s leading AI drug discovery companies, Arctoris generates research data at unprecedented quality and speed, building a unique data resource for machine learning applications. Combining the latest in laboratory techniques with advanced robotics and AI, Arctoris brings drug discovery into the digital age.
Charles Lay
PhD Student
University of Nottingham and GSK
Charles is a BBSRC and GlaxoSmithKline (GSK) funded iCASE PhD student at the University of Nottingham. As part of an undergraduate degree in Biochemistry at the University of Exeter he spent a year in industry at GSK. During which time he participated in the development of a novel NanoBRET® technology-based cellular assay to measure compound binding kinetics to intra-cellular proteins. He also gained experience developing and running NanoBRET® ligand binding, competition, protein:protein interaction (PPI) and biosensor assays for intracellular proteins. Charles’s PhD project involves the pharmacological characterisation of the IL-23 receptor. Over course of his PhD he has generated fluorescent probes and tagged receptor expression constructs to create NanoBRET® and NanoBiT® assays to examine IL-23 receptor assembly, ligand binding and activation.