Development of an Intracellular Quantitative Assay to Measure Compound Binding Kinetics

Charles Lay¹, Daniel Thomas², John Evans³, Kelly Gatfield³, Kristopher McCombe⁴, Alexander Phillipou³, Laurie Gordon³, Emma Jones³, Kristin Riching⁵, Mahnoor Mahmood⁶, Peter Craggs^3
1University of Nottingham, ²Arctoris, ³GlaxoSmithKline, ⁴Queen\\'s University Belfast, ⁵Promega, ⁶Cancer Research UK

Introduction/Background & aims The kinetic descriptors of drug binding, k_on and k_off, have been linked with compound efficacy. Despite this, the measurement of kinetic parameters is yet to be fully adopted in early drug development, with the effect of a molecule on a biological target typically measured by the equilibrium derived measurements of IC₅₀, EC₅₀, or K_d¹. Recent reviews have suggested that intracellular binding kinetics may vary from those measured in cell free assays, and that recombinant cell based assays for intracellular rates may provide a more accurate predictor of in vivo binding rates than existing purified protein based methodologies¹. In this study, the clinically relevant Bromo and Extra-Terminal domain (BET) protein family² was used as a model system to develop a quantitative live cell methodology to measure intracellular kinetic binding rates.

Method/Summary of work NanoLuciferase (NL) bioluminescence resonance energy transfer (NanoBRET) technology was used to create a Kinetic Intracellular Competition Assay (KICA) to measure the rates of intracellular compound binding. The NL substrate Furimizine was added to HEK293 cells transiently expressing an NL-BET protein. The cell suspension was then dispensed into microplates containing test compound and fluorolabelled pan-BET probe. Subsequently luminescence was measured using a microplate reader equipped with 450 ± 20 and >610 nm filters. Comparative cell-free rates were generated using a detergent lysed version of the BET KICA and by utilising the orthogonal purified protein based methodologies of Time Resolved Förster Resonance energy transfer (TR-FRET) kinetic Probe Competition Assay (kPCA)³ and modulation of intrinsic tryptophan fluorescence.

Results/Discussion

Table 1: The intracellular equilibrium and kinetic characterisation of labelled probe BSP-590 and test compounds. (Mean ± SEM). The cellular binding kinetics of a fluorophore conjugated Bromosporine tracer (BSP-590) to NL-BRD4 were characterised. BSP-590 was then utilised to configure a BRD4 KICA to enable the determination of intracellular forward and reverse rates for a test-set of 31 BET inhibitors. This led to the identification of exemplar molecules that were fast (JQ1), medium (Compound A) and slow (Compound B) dissociating within the context of the set.

The BRD4 KICA enabled the high throughput determination of reproducible intracellular rates which correlated well with k_on and k_off rates of compound binding in a lysed cell version of the assay, in kPCA and in an intrinsic tryptophan fluorescence modulation assay. Furthermore, the reverse rate of compound binding in the KICA assay was a stronger correlate with efficacy in an ex vivo phenotypic assay than equilibrium potency. Finally, a means for screening for kinetically selective compounds was demonstrated by comparing forward and reverse rates across the BET isoforms using KICA assays for BRD4, 3, 2 and T.

Conclusion(s) The KICA methodology enables the high throughput measurement of reproducible intracellular k_forward and k_reverse rates of unlabelled test compounds, in addition to the quantification of kinetic and equilibrium K_d values. Furthermore, when performed in tandem with a lysed cell version of the assay, KICA can provide valuable information regarding a test compound’s permeability and free intracellular accumulation.

Reference(s)

1. Vauquelin G, Charlton SJ. Long-lasting target binding and rebinding as mechanisms to prolong in vivo drug action. Br J Pharmacol. 2010;161(3):488-508.

2. Doroshow DB, Eder JP, LoRusso PM. BET inhibitors: A novel epigenetic approach. Ann Oncol. 2017;28(8):1776-1787.

3. Schiele F, Ayaz P, Fernández-Montalván A. A universal homogeneous assay for high-throughput determination of binding kinetics. Anal Biochem. 2015;468:42-49