Category: Assay Development and Screening

1165-A - Development of a Quantitative Intracellular NanoBRET Assay for Compound Binding Kinetics Demonstrated Within Bromodomains

Monday, February 5, 2018
2:00 PM - 3:00 PM

Development of a Quantitative Intracellular NanoBRET Assay for Compound Binding Kinetics Demonstrated Within Bromodomains


The early optimisation of residence time and association rate have been recognised as important factors in drug design. Recent advances in biophysical and biochemical techniques are enabling increased understanding of drugs at isolated proteins, however there is still a need for a high throughput, quantitative kinetic assay that can assess binding kinetics within cells. Such an assay would allow a more physiologically relevant predictor of in vivo pharmacokinetic/pharmacodynamic disconnects and bridge the gap between biochemical and in vivo studies. Here we outline a methodology for a novel NanoBRET based intracellular kinetic assay that delivers target engagement and quantitative cellular observed kinetic rates. In order to demonstrate the applicability of this assay format to an intracellular protein of interest, we utilised an exemplar from the bromodomain containing class of nuclear proteins. We carried out a screen for slowly dissociating molecules at this bromodomain target to identify inhibitors with differential binding kinetics, identifying several long residence inhibitors from the GSK compound collection. Finally, we have also demonstrated the applicability of this approach by developing NanoBRET Tracer assays for related bromodomain containing proteins. We identified a mildly kinetically selective molecule within similar bromodomains and assessed the kinetic drivers of potency difference across the domains.

Charles S. Lay

Undergraduate
University of Exeter
Tunbridge Wells, England, United Kingdom

Charles Lay: Undergraduate at the University of Exeter reading biochemistry (Final Year).
In the 3rd year of my degree I spent a year in industry working at GlaxoSmithKline, Stevenage, UK (2016/2017). At GSK I worked within the Screening Profiling and Mechanistic department developing and running assays for the profiling of new chemical entities. During my year I specialised in binding kinetics working to develop a cellular kinetic assay using NanoBRET technology.