Abstract

[Background]  Recently, it has become clear that
drug-target residence time, in addition to affinity, often drives
pharmacodynamic activity and disease efficacy in vivo. In the aim of determining the dissociation properties of
test compounds, the kinase field has traditionally utilized purified proteins
in various competitive probe displacement assay formats. Using such detection
formats, kinetic parameters of drug-target binding can be quantified using analytical
approaches described by Motulsky and Mahan. While quantitative, such approaches
may fail to correlate with cellular efficacy due to reliance on pure proteins
or protein fragments. Furthermore, these methods are generally not amenable to
high-throughput analysis for exploring intracellular drug-target residence time.
Thus, a simple and quantitative method to evaluate the residence time of
compounds inside living cells is highly desirable.

[Method]  The NanoBRET™ Target Engagement Intracellular
Kinase Assay was employed to investigate the kinetics of target engagement for
various test compounds in living cells. This method utilizes BRET
(Bioluminescence Resonance Energy Transfer) in cells by molecular proximity of
the NanoBRET™ Tracer to the NanoLuc® luciferase-fused kinase. Quantitative
determination of both of apparent affinity as well as binding kinetics for unmodified
test compounds can be achieved by competitive displacement of the Tracer. Full-length
BTK fused with the NanoLuc® luciferase was transiently transfected in HEK293
cells and the cells were incubated at 37°C, 5% CO₂ overnight. Transfected
cells were harvested and suspended in 1 ml of Assay Medium in a conical tube. After
incubation with test compound for 2 hours, the cells were washed with Assay
Medium to remove unbound compound. Dosing of test compounds was an
approximately IC_80-90 concentration unless otherwise specified. The
cell suspension was dispensed into a 96-well plate and the NanoBRET™ Tracer K-4
or K-5 was added to the wells following the addition of the Nano-Glo® Substrate
Solution. BRET was measured repeatedly with the Glomax® Discover Multimode
Microplate Reader. Data were fitted to the one-phase association equation or
the kinetic equation developed by Malany to obtain the dissociation rate constants
of test compounds.

[Results]  The kinetic constants
using tracer K-4 or K-5 were successfully quantitated in live cells using
NanoBRET™. Though the kinetic constants as determined by simple k_obs
fitting using tracer K-4 or K-5 were different, the constants as determined
using the Malany approach for each tracer were in gratifying agreement. The results
determined using the Malany equation revealed suitable differentiation of
residence time for the irreversible and reversible compounds. As expected,
longer residence time was observed for the irreversible/covalent inhibitors. Some
reversible inhibitors also showed protracted residence time, offering
opportunities for durable target inhibition in cells.

[Conclusion]  We propose the method by which the data in the
compound wash-out experiments are fitted to the kinetic equation of Malany as a
simple and quantitative method to determine intracellular residence time of
kinase inhibitors.