# Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors

## TL;DR

This paper studies new drug compounds that inhibit a specific transporter protein, showing they bind strongly and for a long time.

## Contribution

The study introduces a new series of spirobenzo-oxazinepiperidinone derivatives with high affinity and long residence time at hENT1.

## Key findings

- 28 out of 29 compounds showed high affinity and a residence time of 87 minutes at hENT1.
- Molecular moieties were found to separately govern affinity and binding kinetics.
- Binding kinetics are shown to be crucial for drug efficacy and safety at transport proteins.

## Abstract

Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand’s duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds’ chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.

The online version contains supplementary material available at 10.1007/s11302-023-09948-9.