Abstract
The pharmacodynamics of drug-candidates is often optimized by metrics that describe target binding (Kd or Ki value) or target modulation (IC50). However, these metrics are determined at equilibrium conditions, and consequently information regarding the onset and offset of target engagement and modulation is lost. Drug-target residence time is a measure for the lifetime of the drug-target complex, which has recently been receiving considerable interest, as target residence time is shown to have prognostic value for the in vivo efficacy of several drugs. In this study, we have investigated the relation between the increased residence time of antihistamines at the histamine H1 receptor (H1R) and the duration of effective target-inhibition by these antagonists. Hela cells, endogenously expressing low levels of the H1R, were incubated with a series of antihistamines and dissociation was initiated by washing away the unbound antihistamines. Using a calcium-sensitive fluorescent dye and a label free, dynamic mass redistribution based assay, functional recovery of the H1R responsiveness was measured by stimulating the cells with histamine over time, and the recovery was quantified as the receptor recovery time. Using these assays, we determined that the receptor recovery time for a set of antihistamines differed more than 40-fold and was highly correlated to their H1R residence times, as determined with competitive radioligand binding experiments to the H1R in a cell homogenate. Thus, the receptor recovery time is proposed as a cell-based and physiologically relevant metric for the lead optimization of G protein-coupled receptor antagonists, like the H1R antagonists. Both, label-free or real-time, classical signaling assays allow an efficient and physiologically relevant determination of kinetic properties of drug molecules.
Highlights
Despite the wide utilization of binding affinity and related drug binding metrics in drug discovery, it is increasingly debated whether these values provide sufficient information to allow prediction of in vivo efficacy (Waring et al, 2015)
A long residence time (>1 h) was, e.g., observed for several clinically used antihistamines that bind to the histamine H1 receptor (H1R), an important drug target for the treatment of, e.g., allergic rhinitis (Anthes et al, 2002; Gillard et al, 2002; Slack et al, 2011b; Bosma et al, 2016)
Data was fitted using a sigmoidal four-parameter fit and EC50 values were determined. It is well-known that the human adenocarcinoma HeLa cell line endogenously expresses functional histamine H1Rs, coupled to the mobilization of calcium and changes in cell morphology (Smit et al, 1992; Yu et al, 2006)
Summary
Despite the wide utilization of binding affinity and related drug binding metrics in drug discovery, it is increasingly debated whether these values provide sufficient information to allow prediction of in vivo efficacy (Waring et al, 2015). The clinically used anthistamine levocetirizine with a long residence time, shows hysteresis of efficacy after depletion of free levocetirizine concentrations in the blood (Gillard et al, 2002; Schoepke et al, 2013). Together, these data suggest that residence time is an important predictor of in vivo efficacy of H1R antihistamines
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