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). It is currently recognized that the binding rate constants of drug-target interactions may bring additional prognostic value in scenarios, in which there are no stable ligand concentrations, as is typical for in vivo systems (Swinney, 2004; Copeland et al., 2006; Lu and Tonge, 2010; Guo et al., 2014; Hoffmann et al., 2015). In particular the target residence time, which is the reciprocal of the dissociation rate constant of a bound ligand, is thought to be an important metric for drug optimization. A long residence time is, for example, related to an insurmountable mode of antagonism when endogenous agonist concentrations are transiently increased, as is observed in the case of neuronal signaling (Vauquelin et al., 2012). Additionally, compounds with a long residence time show prolonged drug-target occupancies beyond the point at which pharmacologically active drug concentrations are present in the blood (Ramsey et al., 2011; Bradshaw 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). In analogy with insurmountable antagonism observed in neuronal signaling, it has been described that histamine levels after allergen challenge are only transiently increased, implying that an insurmountable mode of antagonism could effectively block high concentrations of histamine (Petersen et al., 1996). Moreover, in an ex vivo organ bath experiment in which antagonists were continuously removed, it was observed that the long residence time compounds azelastine and GSK1004723 both retained a long inhibition of the histamine-induced, H₁R mediated bronchial contraction (Slack et al., 2011a,b). In vivo, the clinically used anthistamine levocetirizine with a long residence time, also 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.

The H₁R is a prototypic member of the therapeutically relevant family of G protein-coupled receptors (GPCRs). The kinetic binding rate constants of unlabeled ligands for GPCRs are often measured using radioligand or fluorescent binding techniques (Schuetz et al., 2017). In these experiments, the effect of the unlabeled ligand on the binding of the labeled ligand is measured over time and kinetic binding rate constants are determined. Such experiments are often done using cell membranes as a source of the receptor. In this study, we developed methods to measure the kinetics of H₁R antagonism upon depletion of the free concentration antihistamine, by measuring recovery of the histamine-induced response over time in a physiologically relevant cell system. To this end, a fluorescent based calcium mobilization assay and a label-free, dynamic mass redistribution (DMR) based assay were evaluated for the measurement of histamine-induced responses in human HeLa cells, cervical cancer cells known to endogenously express low levels of the H₁R (Govoni et al., 2003; Das et al., 2007). Using these assay formats, it is shown that the receptor recovery time is correlated to the residence time of antihistamines, hence, this parameter might therefore have predictive value for the in vivo efficacy of such ligands. The described orthogonal assays will also be very relevant for future GPCR drug discovery projects, as both calcium signaling, as well as DMR-responses, can be measured for a large number of GPCRs (Charlton and Vauquelin, 2010; Schröder et al., 2010).

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). As such, this cell line was selected as a physiological relevant test model for the proposed kinetic receptor recovery studies. Expression levels of the H₁R were quantified in HeLa cells as well as in transiently transfected HEK293T cells using a radioligand saturation binding experiment (Table 1). Increasing concentrations of the H₁R antagonist [³H]mepyramine were incubated with cell homogenates of the respective cell line for 4 h at 37°C. [³H]mepyramine displayed comparable affinity for H₁R on HeLa cells and HEK293T cells with pK_d values of 8.5 ± 0.2 and 8.1 ± 0.1, respectively. A very low window of specific [³H]mepyramine binding was observed for the HeLa cell line, in sharp contrast to the HEK293T cells, which transiently expressed the human H₁R at >100-fold higher expression levels (Table 1). The observed expression of the H₁R on HeLa cells was in the same order of magnitude as described in literature (55–130 fmol/mg protein) (Govoni et al., 2003; Das et al., 2007).

A set H₁R antihistamines was selected based on the diversity in chemotype and expected diversity in receptor residence times (Supplementary Table 1) (Gillard et al., 2002; Bosma et al., 2016). First, the equilibrium (i.e., steady-state) binding of antihistamines to the H₁R was characterized using radioligand binding experiments. To determine the binding affinity (pK_i) of antihistamines for the H₁R, a homogenate of HEK293T cells transiently expressing the H₁R was co-incubated with a single concentration [³H]mepyramine (3–6 nM) and increasing concentrations of unlabeled antihistamines for 4 h at 37°C. Examples of four [³H]mepyramine displacement curves for the prototypical H₁R antihistamines mepyramine, doxepin, levocetirizine, and olopatadine are depicted in Figure 1. More than 2 log unit differences in pK_i values were observed between antihistamines (Table 2) and the respective K_i-values were comparable to those described previously (Nonaka et al., 1998; Wieland et al., 1999; Gillard et al., 2002; Kuhne et al., 2016).

Subsequently, the kinetic binding rate constants of antihistamines were determined in [³H]mepyramine competitive association experiments. Antihistamines were therefore co-incubated with [³H]mepyramine and a homogenate of HEK293T cells, transiently expressing the human H₁R, for 0–81 min at 25°C (Figure 2 and Table 2). In such competitive association binding experiments a typical overshoot pattern in the binding of the radioligand is observed when it has a higher k_off than the unlabeled ligand at the receptor (Motulsky and Mahan, 1984). In the presence of unlabeled mepyramine (Figure 2A) there is no overshoot pattern in the binding of the radioligand to the H₁R, indicating that the ligand k_off at the H₁R is similar (or higher) for mepyramine as compared to [³H]mepyramine. In the presence of doxepin (Figure 2B), levocetirizine (Figure 2C), and olopatadine (Figure 2D) an overshoot in [³H]mepyramine binding to the H₁R is observed which reflects the relative low k_off of these unlabeled ligands at the H₁R compared to the k_off of [³H]mepyramine. The kinetic binding rate constants (k_on and k_off) are determined from these kinetic binding traces using the Motulsky–Mahan model (Motulsky and Mahan, 1984). The representative antihistamines mepyramine, doxepin, levocetirizine and olopatadine, depicted in Figures 2A–D, illustrate the range in k_off values that were obtained from competitive association experiments. From the kinetic binding constants, the respective binding affinity for the H₁R could be calculated [pK_d,calc = -log(k_off/k_on)]. Additionally, the residence time was calculated (RT = 1/k_off), which is a measure for the length of H₁R engagement by the antihistamine. The binding characteristics of all tested ligands are reported in Table 2.

Based on the kinetic and equilibrium [³H]mepyramine binding experiments, it is shown that the tested set of antihistamines display >100-fold differences in K_i, binding rates and residence times for their binding to the H₁R (Table 2). The >35-fold differences in residence time at the H₁R between levocetirizine (RT = 140 min), desloratadine (RT = 160 min) and mepyramine (RT = 3.9 min) agreed with previously reported data showing a 50 to 140-fold longer residence time for levocetirizine and desloratadine compared to mepyramine (RT, mepyramine: 0.8–8 min) (Gillard et al., 2002; Gillard and Chatelain, 2006; Schiele et al., 2015). Interestingly, the differences in residence time at the H₁R between antihistamines were not reflected by the respective differences in the pK_i. For example, levocetirizine has a similar H₁R binding affinity but much longer residence time at the H₁R as compared to mepyramine (Table 2).

The H₁R is known to activate Gαα_q, leading to increased intracellular calcium levels via activation of phospholipase C (Jacob et al., 1988; Smit et al., 1992). Indeed, in HeLa cells, an histamine-induced dose dependent increase in the calcium mobilization can be measured as an increase in the fluorescence of the calcium-sensitive Fluo4 NW dye (λ_excitation 494 nm and λ_emission 516 nm). As can be seen in Figure 3A, a peak increase in fluorescence is obtained within seconds after stimulation of HeLa cells with histamine. The fluorescence signals then decrease to a steady-state level for at least 30 s. The H₁R response was quantified both by the histamine-induced peak response or the AUC observed between 0 and 30 s after stimulating with histamine. Plotting the log concentration histamine versus the relative calcium mobilization leads to overlapping histamine-dose-response curves. Analyzing both curves with a four-parameter sigmoidal fit resulted in similar pEC₅₀ values of 6.1 ± 0.1 and a Hill slope of 1.1 ± 0.1 (Figure 3B).

Pre-incubated long-residence-time antagonists can display insurmountable antagonism of receptor signaling if agonist-induced responses are measured before the establishment of a binding equilibrium between agonist, antagonist and the receptor (Kenakin et al., 2006). The slow dissociation of the antagonist from the receptor will reduce the number of receptors available for agonist-induced activation, which is reflected by a decreased E_max that might be preceded by an initial rightward shift of the dose response curve in the presence of a receptor reserve (Kenakin et al., 2006; Riddy et al., 2015). It was therefore tested whether insurmountable antagonism of histamine-induced calcium mobilization could be observed when cells were pre-incubated with the antihistamines mepyramine, doxepin, levocetirizine, and olopatadine. This series of H₁R antagonists reflects a wide range in ligand residence times at the H₁R (Table 2). HeLa cells were pre-incubated with various concentrations of antagonists overnight and cells were subsequently stimulated with increasing concentrations histamine (Figure 4). For all four inhibitors the maximal histamine-induced response in HeLa cells was reduced compared to vehicle treated cells. For mepyramine, the H₁R antagonist with a short residence time at the H₁R, some decrease in the E_max was observed next to a rightward shift of the histamine dose-response curves (Figure 4A). For doxepin (Figure 4B), levocetirizine (Figure 4C), and olopatadine (Figure 4D), a full insurmountable inhibition of the histamine-induced calcium mobilization was observed. The insurmountable antagonism was marked by a clear drop in the E_max for each subsequent increase in antagonist concentration up to a (near) complete block of any histamine-induced effect at the highest concentrations. These data are in line with the expectations for H₁R antagonists with a long residence time, but this assay format does not easily allow one to obtain quantitative data on the kinetic properties of the tested H₁R antagonists. Moreover, the insurmountable antagonism by doxepin could not be differentiated from the insurmountable antagonism of levocetirizine and olopatadine, despite the 6 to 8-fold difference in residence time that was measured in radioligand binding experiments (Table 2).

An alternative assay format was therefore evaluated in which the relative blockade of the receptor was quantified over time in order to discriminate antihistamines with relatively long drug target engagement times (Figure 5A). HeLa cells were incubated overnight with a 10-fold K_i concentration of the various antagonists. Cell were subsequently washed and stimulated with 10^-5 M histamine at different time points to probe the responsiveness of the H₁R (Figure 5B). After pre-incubation and subsequent washout of mepyramine from the HeLa cells, histamine rapidly (within 40 min) induces a similar level of calcium mobilization as observed in cell pre-incubated with only buffer. In contrast, pre-incubation with doxepin resulted in a delayed recovery of the histamine induced calcium mobilization and the recovery of histamine responsiveness was even more delayed in cells pre-incubated with levocetirizine and olopatadine. Since the free receptor population after the wash step dependents on the dissociation of the antagonist, the functional response of histamine was expected to recover with rates that would mirror the k_off of the tested H₁R antagonists. The receptor recovery over time after washing away unbound antagonist was fitted to a one-phase association model (Eq. 3; Figure 5C). This one-phase exponential model is the same as the dissociation model used for describing drug-target dissociation and was observed to fit the functional response over time reasonably well. As a measure for the absolute time-scale in which recovery rate takes place, the k_rec was transformed into the receptor recovery time (RecT) by taking the reciprocal of the k_rec. This is analogous to the way in which residence time is calculated from the k_off values for the dissociation of ligand binding. The histamine induced response after olopatadine incubation did not approach a steady-state within the time-frame of the experiment. To make an estimate of the receptor recovery rate for olopatadine, the steady-state was constrained to the average histamine induced calcium levels measured in vehicle treated cells. Additionally, after pre-treatment with levocetirizine the functional H₁R did not fully recovery within the time frame of the experiment either, but the average steady-state recovery (%), reflected by the Y_max, suggests proper fitting of the curves. The determined k_rec, RecT and steady-state recovery was quantified for all antihistamines (Table 3). As can be seen, analyses allowed for good discrimination between antihistamines over a 40-fold range in k_rec and RecT. However, full recovery of histamine induced calcium mobilization was already obtained after 1 or 2 data points following a pre-incubation with, e.g., VUF14454 and VUF14493. The kinetic resolution of this assay cannot readily discriminate between antihistamines with very fast recovery rates of the receptor responses.

As an orthogonal assay to measure the histamine-H₁R induced response in HeLa cells, DMR was employed. In this assay format cells are grown on RWG biosensors. This allows to detect changes in cellular mass distribution (e.g., translocation of proteins, cytoskeleton rearrangements) close to the biosensor surface (Fang et al., 2006, 2008). DMR was shown to be an effective way to quantify histamine induced signaling mediated by the H₁R (Lieb et al., 2016). Upon stimulating HeLa cells with increasing concentrations of histamine, DMR response reached a maximum after approximately 5 min (Figure 6A), as previously reported for Gα_q mediated signaling by other GPCRs (Fang et al., 2008). After fitting the data with a four-parameter sigmoidal fit a pEC₅₀ of 6.1 was obtained from both curves with a Hill slope of 0.88 (AUC) and 0.92 (peak response) (Figure 6B).

Co-incubating HeLa cells with histamine at a concentration of approximately EC₈₀ and increasing concentrations of antihistamines reduced the DMR response concentration-dependently (Figure 7) with the following pIC₅₀ values determined from AUC analysis (mean ± SD of 2 experiments): mepyramine 8.1 ± 0.2, doxepin 8.0 ± 0.4, olopatadine 7.0 ± 0.1, and levocetirizine 6.3 ± 0.2. It was observed for all inhibitors that the DMR-signal was not fully reduced to background level at the highest concentration. From the full kinetic traces it was observed that the peak response from co-incubated cells stabilized at different levels compared to untreated cells (Supplementary Figure 2). Incubation of cells with antagonists alone resulted in a negligible DMR response, comparable to the background traces (data not shown).

The RecT of the histamine-induced DMR response after removal of unbound antihistamines was subsequently quantified. HeLa cells were incubated with a 10x K_i concentration of each of the antagonists to ensure high occupancy of the receptor. On the following day cells were washed with assay buffer and antagonists thereby removed. When the DMR signal was detected directly after washing, a drift was observed due to the adaptation of cells to room temperature and DMSO concentration of the assay buffer. The signal stabilized after approximately 1 h (data not shown). Therefore, cells were stimulated with increasing concentrations of histamine earliest 1 h after antagonist washout. The DMR response was recorded for 30 min and baseline corrected for each individual well. The relative suppression of the histamine E_max was estimated by comparing the peak response induced at a saturating histamine concentration (63 μM) of cells pre-incubated with or without the respective antihistamine (Table 4). The maximal response of histamine was not suppressed by pre-incubation with the fast-dissociating antihistamine mepyramine (Figure 8A). In contrast, the slow-dissociating antihistamines levocetirizine and olopatadine (Figures 8C,D), suppressed the E_max up to 2 h after removing unbound antihistamines. Pre-incubation with the medium-slow dissociating doxepin suppressed E_max, but to a lower extent than olopatadine and levocetirizine (Figure 8B).

Insurmountable antagonism was pronounced for olopatadine, levocetirizine, desloratadine, doxepin, acrivastine and VUF14506. Triprolidine also showed insurmountable antagonism, but this was the same at 1 and 2 h after washing away unbound antagonist, indicating that binding equilibrium had been reached. As such, the observed level of insurmountable antagonism is most likely not caused by prolonged drug-target occupancy, but might be due to a yet unidentified mechanism of action of triprolidine affecting the DMR response in HeLa cells. The receptor recovery rate after preincubation with each of the antihistamines was estimated from the relative effect of a saturating concentration histamine, obtained at 1 and 2 h after removal of unbound antihistamines (see section “Materials and Methods”).

A comparison between the receptor recovery rates and kinetic dissociation rate constants of the set of antihistamines from Tables 2–4 are depicted in Figure 9 and Supplementary Table 1. The ln k_off of the antihistamines correlated with the ln k_rec of the histamine-induced DMR (not shown) and calcium mobilization upon washout of these antihistamines (Figure 9A). Moreover, the ln k_rec as determined by both methods correlated to each other also very well (Figure 9B). Although the receptor recovery times and residence times seem to be related, the absolute values of the various metrics are not in the same range. In general, recovery time of the DMR response seem to take longer than for the calcium mobilization (Tables 3, 4 and Supplementary Table 1).

Drug-target residence time has received a lot of attention over the last decade and has now been suggested as a way to better predict drug-efficacy in vivo in an early stage of drug development (Swinney, 2004; Copeland et al., 2006; Lu and Tonge, 2010; Guo et al., 2014; Hoffmann et al., 2015). It has been postulated that a long drug-target residence time can prolong the in vivo effects of such drugs compared to drugs with a shorter residence time. Moreover long-residence antagonists might exert insurmountable antagonism of the receptor, which is independent on the concentration agonist and can therefore be much stronger than competitive antagonism (Kenakin et al., 2006; Vauquelin et al., 2012). However, whether a long drug-target residence time is beneficial for the in vivo efficacy, is dependent on the specific biology of the drug-target (Schuetz et al., 2017). For example, a long drug-target residence time only leads to a prolonged receptor occupancy in vivo if the receptor turnover is low (Hong et al., 2008; Bradshaw et al., 2015). Moreover, an insurmountable mode of antagonism is also highly dependent on the cellular context, like e.g., the expression level of the target-receptor (Kenakin et al., 2006). For several GPCR-targets the importance of a prolonged drug-occupancy has however, been advocated and the residence time of GPCR ligands are therefore currently explored as a way to increase the therapeutic window (Guo et al., 2014).

The determination of drug-target residence time for GPCR-ligands is usually performed by radioligand binding experiments, or more recently, by fluorescent ligand binding experiments (Schiele et al., 2015; Bosma et al., 2016; Schuetz et al., 2017). The residence time of unlabeled ligands can be quantified by measuring their competitive effects on the probe binding-kinetics, using the Motulsky–Mahan model (Motulsky and Mahan, 1984). Although the residence time values obtained in these experiments describe the kinetics of GPCR binding at the molecular level, these parameters do not necessarily directly reflect the functional effects in a physiological relevant setting. In order to bridge the gap between GPCR binding studies on membrane preparations and the in vivo evaluation of the action of long residence time GPCR ligands, we evaluated the kinetics of GPCR antagonism for a set of H₁R antagonists with different ligand binding kinetics in two cell-based assays. The potential success of such a strategy is exemplified for the Neurokinin 1 Receptor, for which it was shown that prolonged antagonism after a washout of unbound antagonists, corresponded with the respective therapeutic window of these antagonists in vivo (Lindström et al., 2007). Moreover, the functional effects observed for the set of H₁R antagonists were compared to the observed residence times, as determined in [³H]mepyramine competition binding assays.

In this study, we employed two GPCR assays that can be performed without altering the genetic cellular context and which can be broadly applied to measure GPCR signaling. The calcium mobilization assay has been widely used to measure signaling of GPCRs that couple to the Gα_q/11 protein, but also GPCRs that couple to Gα_i subunits can still induce a calcium mobilization response via released βγ-subunits (Charlton and Vauquelin, 2010). The DMR-response relies on the morphological changes of the cell, which has been linked to all canonical GPCR-signaling cascades (Schröder et al., 2010). HeLa cells, endogenously expressing the H₁R at low, but physiological relevant densities, were used in both assays.

Stimulation of HeLa cells with histamine induced both a robust calcium-mobilization (Figure 3), as well as DMR-response (Figure 6) in the HeLa cells with similar potencies (pEC₅₀ 6.1–6.2). The rise in intracellular calcium concentrations was clearly more rapid (within seconds, Figure 3), than the induction of DMR response (within 5–10 min, Figure 6), in line with previous reports (Smit et al., 1992; Lieb et al., 2016). The calcium response was effectively antagonized by the tested H₁R antagonists, but whereas co-incubation with the short residence time antagonist mepyramine resulted in rightward shifts of the histamine dose response curves with some depression of the maximal response, the long residence time antagonists like doxepin, levocetirizine, and olopatadine (Figure 4) showed substantial insurmountable antagonism. The slow dissociation of these long residence time antagonists from the H₁R strongly suggests a temporal insurmountable mode of antagonism by engaging to the orthosteric binding site. An allosteric binding mechanism might theoretically also explain the depression of the maximal histamine response (Kenakin et al., 2006). Yet, this seems unlikely in view of the overlapping binding sites of the tested antagonists and histamine, as experimentally determined by x-ray crystallography (Shimamura et al., 2011), site-directed mutagenesis studies (Nonaka et al., 1998; Wieland et al., 1999; Gillard et al., 2002; Cordova-Sintjago et al., 2012) and molecular modeling experiments (Shimamura et al., 2011; Cordova-Sintjago et al., 2012; Kooistra et al., 2013). Recently, insurmountable antagonism of the muscarinic acetylcholine receptor and orexin-2 receptor was used to calculate k_off rate constants of antagonists (Mould et al., 2014; Riddy et al., 2015). The observed insurmountable antagonism of the histamine induced calcium response (Figure 4) could potentially also be used to discriminate antihistamines based on their relative k_off at the H₁R. However, the used hemi-equilibrium model (Kenakin et al., 2006) is only applicable in the case of a partial insurmountable effect, as was observed for mepyramine at the highest concentrations (Figure 4A). Since saturating concentrations doxepin (Figure 4B), levocetirizine (Figure 4C), and olopatadine (Figure 4D) completely block the response to histamine, it is not possible to distinguish these antihistamines by this means.

As alternative assay-format, the functional recovery time (RecT) of the H₁R responsiveness was measured after washing away pre-bound antihistamines. In this study, 40-fold differences in the RecT were observed for the tested H₁R antihistamines, including fivefold (DMR) to eightfold (calcium mobilization) differences in RecT between doxepin, levocetirizine, and olopatadine. Hence, the functional recovery time allows the discrimination of antihistamines with a broad range of H₁R residence times. For both the calcium mobilization and the DMR-assay, a similar trend in the k_rec of the H₁R was obtained after pre-incubating with the various antihistamines (Figure 9B). Moreover, the measured k_rec values nicely correlated with the k_off values of the respective H₁R antihistamines, as measured by [³H]mepyramine competition binding kinetics (Figure 9A). Our data suggests that the drug-target residence time indeed prolongs the antagonism by H₁R-antihistamines following washout and hence a rapid and strong reduction of its free concentration ligand. Moreover, our observations are also in line with the prolonged therapeutic window in vivo that has been described for some long residence time antihistamines (Schoepke et al., 2013).

The presented methods effectively determine the length of receptor antagonism using a functional readout that is broadly applicable to GPCR research. The measured non-equilibrium antagonism (RecT), which e.g., reflects the level of insurmountable antagonism (Figure 4), is proposed to affect the in vivo efficacy of drugs (Kapur and Seeman, 2001; Vauquelin et al., 2012). Insurmountable antagonism is described to be affected by the transduction coefficient of the functional receptor response and is therefore dependent on the cellular context (Kenakin et al., 2006). Since the described methods are applicable to cell lines endogenously expressing the target receptor, RecT does not only reflect the relative residence time, it has the potential to reflect which antagonists have a long enough residence time to impose prolonged non-equilibrium antagonism. Recently, a different functional readout of the H₁R was employed to measure the relative residence time of antihistamines on the H₁R (Bosma et al., 2016). This BRET based β-arrestin2 recruitment assay allows a continuous readout of the H₁R response that was stable in time, improving therefore the throughput in which the relative residence time was determined. As a downside, this method requires genetic manipulation of cells by introducing a tagged H₁R and tagged β-arrestin2, resulting therefore in, e.g., non-physiologically relevant expression levels.

One limitation of the washout experiments is the possibility that unbound antihistamines are not completely removed by the wash step. An incomplete recovery of receptor signaling was, e.g., observed after pre-incubation with doxepin and desloratadine (Figure 5B and Table 3). During the washout, only the unbound ligands are removed and the dissociation of H₁R-bound ligands might provide enough ligands after the initial washout to result in residual receptor occupancy. However, this would only happen if the binding affinity (pK_i) is high enough to allow binding at these low ligand concentrations, especially as HeLa cells express low numbers of H₁Rs. Interestingly, doxepin and desloratadine have the highest pK_i values of all the antihistamines that were tested in this study (Table 2). Another confounding factor in the assay might be the potential partitioning of H₁R ligands in the cell or membranes, resulting in ligands accumulation and H₁R rebinding after washout of free ligands. Yet, this issue has also been described to affect the determination of residence times via the kinetic competition binding method (Sykes et al., 2014).

The kinetic information in the DMR washout experiments was low and to prevent over-interpretation of the data, the k_rec values were only estimated when there was a significant difference in the histamine induced DMR response between 1 and 2 h after washout of the antihistamines. As a drawback, this could exclude compounds which have a very low k_rec value, but this seems unlikely for all ligands that were excluded here, since in all cases a >70% recovery of the histamine induced effect was observed already 1 h after the wash step. For the evaluated H₁R ligands the calculated k_rec values obtained in the DRM assay correlate well with the values obtained in the orthogonal calcium-mobilization assay, despite the fact that the kinetic resolution of the measurements was much lower.

In this study we describe two orthogonal, functional kinetic assays for GPCR ligands, that can be performed with cells that have endogenous expression of the receptor without the need for any genetic manipulation. The obtained RecT values for H₁R antagonists correlate well with their H₁R residence times as determined by radioligand binding. Hence, the use of RecT as a drug metric for the kinetics of antagonism might be valuable to steer the lead optimization of GPCR, as the described generic assays can potentially be used to measure the kinetics of antagonism of ligands for a large variety of GPCRs. Moreover, the assays can additionally be employed with clinically relevant cell lines (e.g., primary cells).

Participated in research design: RB, GW, PG, HV, SG, and RL. Conducted experiments: RB, GW, and IJ. Performed data analysis: RB, GW, LV, and IJ. Wrote or contributed to the writing of the manuscript: RB, GW, LV, IJ, PG, HV, SG, and RL.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.