Characterization of 12 GnRH peptide agonists – a kinetic perspective

Abstract

Background and Purpose

Drug‐target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin‐releasing hormone (GnRH) receptor for the treatment of hormone‐dependent diseases. Surprisingly, the kinetic receptor‐binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor‐binding kinetics of 12 GnRH peptide agonists, including many marketed drugs.

Experimental Approach

A novel radioligand‐binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [¹²⁵I]‐triptorelin. In addition to radioligand‐binding studies, a homogeneous time‐resolved FRET Tag‐lite™ method was developed as an alternative assay for the same purpose.

Key Results

Two novel competition association assays were successfully developed and applied to determine the kinetic receptor‐binding characteristics of 12 high‐affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin).

Conclusions and Implications

Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor.

Abbreviations

CHOhGnRH

CHO cells stably expressing the human GnRH receptor

GnRH

gonadotropin‐releasing hormone

k₁

the association rate constant of the radioligand

k₂

the dissociation rate constant of the radioligand

k₃

the association rate constant of the unlabelled ligand

k₄

the dissociation rate constant of the unlabelled ligand

RT

residence time

TR‐FRET

time‐resolved FRET

Tables of Links

TARGETS
GnRH receptors

LIGANDS
125I‐triptorelin	Goserelin	Nafarelin
Buserelin	Leuprorelin	Triptorelin
GnRH

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).

Introduction

Drug‐target residence time is emerging as an important parameter in the drug discovery process. Multiple studies provide evidence that the binding kinetics of drug‐target interactions rather than the typical equilibrium binding parameters are important for in vivo efficacy (Swinney, 2004; Copeland et al., 2006; Tummino and Copeland, 2008; Zhang and Monsma, 2009). Several marketed drugs in the field of GPCRs have retrospectively been shown to display slow receptor dissociation rates or, in other words, long receptor residence times (Guo et al., 2014). For instance, the histamine H₁ receptor antagonist desloratidine was found to have a long residence time, which could explain its high potency and 24 h duration of action observed in clinical studies (Anthes et al., 2002). Another example is the insurmountable antagonist for the angiotensin AT₁ receptor, telmisartan. The authors deemed the insurmountability and therefore improved efficacy of telmisartan to be partly due to its very slow dissociation from AT₁ receptors (Maillard et al., 2002).

The hypothalamic neuropeptide gonadotropin‐releasing hormone (GnRH) is a central mediator of reproductive functions. This decapeptide binds to a class A GPCR, namely, the GnRH receptor located mainly on pituitary gonadotrophs. Along with the pituitary, GnRH receptors are expressed in reproductive tissues, both normal and malignant, such as those of the prostate and mammary gland (Kakar et al., 1994; von Alten et al., 2006; Angelucci et al., 2009; Aguilar‐Rojas et al., 2012). Upon receptor activation, the gonadotropins luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) are synthesized and secreted from gonadotrophic cells. LH and FSH consecutively induce follicle stimulation and ovulation in women and promote steroidogenesis in both men and women (Stojilkovic et al., 1994).

The pulsatile release of GnRH from the hypothalamus is essential for the maintenance of ovarian function. Sustained exposure of GnRH receptors to GnRH or GnRH analogues leads to activation, commonly named ‘flare’, followed by desensitization of GnRH receptor‐mediated gonadotropin secretion. Accordingly, blockade by antagonists and desensitization of GnRH receptor‐mediated gonadotropin secretion both ultimately reduce circulating levels of gonadotropins and gonadal steroids (Belchetz et al., 1978; Lahlou et al., 1987). This so‐called chemical castration underlies the therapeutic use of GnRH analogues to treat sex hormone‐dependent diseases (Depalo et al., 2012; Labrie, 2014; Leone Roberti Maggiore et al., 2014).

Consequently, considerable efforts have been put towards the development of agonists and antagonists targeting the GnRH receptor (Karten and Rivier, 1986; Sealfon et al., 1997; Millar et al., 2004; Heitman and IJzerman, 2008; Millar and Newton, 2013). Only a few studies have examined the receptor‐binding kinetics of GnRH ligands. A paper of Heise et al. (2007) described a scintillation proximity assay to qualitatively distinguish between fast and slowly dissociating antagonists for the GnRH receptor. The authors demonstrated that slow dissociation rates were responsible for large discrepancies between a ligand's K_i value determined at 30 min versus 10 h, and they suggested using the K_i ratio as a screening method to select slowly dissociating compounds. Two other studies focused on a quantitative determination of receptor‐binding kinetics of small molecule GnRH antagonists. Here, a direct correlation between the insurmountability and slow dissociation rates of these antagonists was shown (Sullivan et al., 2006; Kohout et al., 2007).

Currently, multiple peptide GnRH analogues have been approved for the treatment of advanced prostatic cancer, endometriosis, in vitro fertilization and more (Depalo et al., 2012; Romero et al., 2012; Aydiner et al., 2013; Goericke‐Pesch et al., 2013; Lewis et al., 2013; Labrie, 2014; Leone Roberti Maggiore et al., 2014). Remarkably, the receptor‐binding kinetics of peptide GnRH receptor ligands have never been reported. Therefore, we developed a novel radioligand‐binding competition association assay that allowed us to determine the kinetic binding parameters and focused on 12 GnRH peptide agonists, including many marketed drugs (Table 1). In addition, we compared these kinetic parameters with those from a newly developed homogeneous time‐resolved fluorescent assay. Both assays may improve future drug discovery targeting the GnRH receptors by incorporating kinetic receptor‐binding parameters into current research and development trajectories.

Table 1.

Amino acid sequences of the 12 GnRH peptide agonists examined

	1	2	3	4	5	6	7	8	9	10
GnRH	pGlu*	His	Trp	Ser	Tyr	Gly	Leu	Arg	Pro	Gly–NH2
Triptorelin	pGlu*	His	Trp	Ser	Tyr	d‐Trp	Leu	Arg	Pro	Gly–NH2
[d‐Ala6]‐GnRH	pGlu*	His	Trp	Ser	Tyr	d‐Ala	Leu	Arg	Pro	Gly–NH2
[d‐Lys6]‐GnRH	pGlu*	His	Trp	Ser	Tyr	d‐Lys	Leu	Arg	Pro	Gly‐NH2
Fertirelin	pGlu*	His	Trp	Ser	Tyr	Gly	Leu	Arg	Pro	NHEt *
Alarelin	pGlu*	His	Trp	Ser	Tyr	d‐Ala	Leu	Arg	Pro	NHEt *
Deslorelin	pGlu*	His	Trp	Ser	Tyr	d‐Trp	Leu	Arg	Pro	NHEt *
Leuprorelin	pGlu*	His	Trp	Ser	Tyr	d‐Leu	Leu	Arg	Pro	NHEt *
Nafarelin	pGlu*	His	Trp	Ser	Tyr	D2Nal	Leu	Arg	Pro	Gly–NH2
Buserelin	pGlu*	His	Trp	Ser	Tyr	Ser‐tBu *	Leu	Arg	Pro	NHEt *
Goserelin	pGlu*	His	Trp	Ser	Tyr	Ser‐tBu *	Leu	Arg	Pro	azaGly–NH2 *
Histerelin	pGlu*	His	Trp	Ser	Tyr	His(Bzl) *	Leu	Arg	Pro	NHEt *

The differences between the peptides are expressed in bold

^* pGlu, pyroglutamic acid; D2Nal, (2‐naphthyl)‐d‐alanine; Ser‐tBu, serine‐tert‐butyl; His(Bzl), N‐benzyl‐l‐histidine; azaGly–NH₂, aza‐glycine amine; NHEt, ethylamide

Methods

Cell culture

For radioligand‐binding assays, CHOhGnRH cells were grown in Ham's F12/DMEM (1:1) medium supplemented with 10% normal bovine serum, 2 mM glutamine, penicillin (100 IU·mL⁻¹), streptomycin (100 µg·mL⁻¹) and G418 (200 µg·mL⁻¹) at 37°C in 5% CO₂.

For time‐resolved FRET (TR‐FRET) experiments, 1 mL (7 * 10⁶ cells·mL⁻¹) of Tag‐lite GnRH cells were thawed, washed with 15 mL ice‐cold Tag‐lite buffer (TLB), resuspended in 5 mL TLB and immediately used.

Membrane preparation

CHOhGnRH cells were scraped from the plates in 5 mL PBS, collected and centrifuged at 700× gfor 5 min. Derived pellets were pooled and resuspended in 50 mM Tris HCl buffer pH 7.4 at 25°C supplemented with 2 mM MgCl₂ and subsequently homogenized with an UltraThurrax (Heidolph Instruments, Schwabach, Germany). The cytosolic fraction and membranes were separated by centrifugation at 100 000× g in an Optima LE‐80 K ultracentrifuge (Beckman Coulter, Fullterton, CA, USA) for 20 min at 4°C. The pellet was resuspended, and centrifugation was repeated. The obtained pellet was resuspended; membranes were aliquoted and stored at −80°C. Membrane protein concentrations were determined using the bicinchoninic acid method with BSA as a standard (Smith et al., 1985).

Radioligand equilibrium assays

Displacement experiments were performed as previously reported (Heitman et al., 2008). In short, membrane aliquots containing 15–20 µg protein were incubated in a total volume of 100 μL assay buffer (25 mM Tris HCl, pH 7.4 at 25°C, supplemented with 2 mM MgCl₂, 0.1% (w v^‐1) BSA) at 25°C for 2 h. Ten concentrations of competing ligand were used in the presence of 30.000 c.p.m. (~0.1 nM) [¹²⁵I]‐triptorelin. At this concentration, total radioligand binding did not exceed 10% of that added to prevent ligand depletion. Non‐specific binding was determined in the presence of an excess amount of GnRH (1 μM). The reaction was terminated by the addition of 1 mL ice‐cold wash buffer (25 mM Tris HCl, pH 7.4 at 25°C, supplemented with 2 mM MgCl₂ and 0.05% (w v^‐1) BSA). Separation of bound from free radioligand was performed by rapid filtration through Whatman GF/B filters saturated with 0.25% polyethylene imine using a Brandel harvester. Filters were subsequently washed three times with 2 mL ice‐cold wash buffer. Filter bound radioactivity was determined using a γ‐counter (Wizard 1470, PerkinElmer).

Radioligand kinetic association and dissociation assays

Association experiments were carried out by incubating membrane aliquots containing 15–20 µg protein in a total volume of 100 μL assay buffer at 25°C with 30.000 c.p.m. (~0.1 nM) [¹²⁵I]‐triptorelin. The amount of radioligand bound to the receptor was determined at different time intervals for a total incubation time of 120 min.

Dissociation experiments were performed by pre‐incubating membrane aliquots containing 15–20 µg protein in a total volume of 100 μL assay buffer at 25°C for 45 min with 30.000 c.p.m. (~0.1 nM) [¹²⁵I]‐triptorelin. After pre‐incubation, dissociation was initiated by addition of an excess amount of GnRH (1 μM) in a total volume of 2.5 μL. The amount of radioligand still bound to the receptor was measured at various time intervals for a total incubation time of 120 min. The reaction was stopped, and samples were harvested as described under Radioligand Equilibrium Assays.

Radioligand kinetic competition association assays

The binding kinetics of unlabelled ligands were quantified using the competition association assay based on the method by Motulsky and Mahan (1984). During optimization, three different concentrations of unlabelled triptorelin were tested; 0.3‐fold, 1‐fold and 3‐fold its K_i value. The kinetic parameters of all other unlabelled ligands were determined at a concentration of onefold their K_i, unless stated otherwise. The competition association assay was initiated by adding membrane aliquots containing 15–20 µg protein in a total volume of 100 μL assay buffer at 25°C with 50.000 c.p.m. (~0.15 nM) [¹²⁵I]‐triptorelin in the absence or presence of competing ligand. Of note, total radioligand binding did not exceed 10% of that added at this concentration to prevent ligand depletion. The amount of radioligand bound to the receptor was determined at different time intervals for a total incubation time of 120 min. The reaction was stopped, and samples were harvested as described under Radioligand Equilibrium Assays.

TR‐FRET probe equilibrium assays

Unless otherwise specified, TR‐FRET measurements were carried out using the conditions described in Schiele et al. (2014). To determine the equilibrium affinity of the fluorescent probe, 5 μL Tag‐lite GnRH cells (1400 cells·μL⁻¹) were incubated for 1 h, to ensure signal stability, with increasing probe concentrations (ranging from 0 to 100 nM: Supporting Information Fig. S1) in a final volume of 10 μL. In parallel, a non‐specific binding control was carried out in the presence of an excess amount of buserelin (100 μM). Binding signals were measured in a PHERAstar FS plate reader BMG Labtech (Offenburg, Germany) by exciting the Tb donor with five laser flashes at a wavelength of 337 nm and recording acceptor and donor emission fluorescence channels (A and B channels), at wavelengths of 520 and 490 nm respectively.

TR‐FRET equilibrium probe competition assays

‘Ready‐to‐use’ assay plates containing serial dilutions of the test agonists were prepared as described in Schiele et al. (2014). Subsequently, 5 μL Tag‐lite GnRH cells (1400 cells·μL⁻¹) and 50 nM probe were added to the competitors and incubated at room temperature for 1 h, to ensure signal stability, in a final volume of 10 μL. Non‐specific binding (‘low signal’) controls contained an excess amount of buserelin (100 μM), whereas in ‘high signal’ controls, the test compounds were replaced by DMSO. Binding signals were recorded as described under TR‐FRET Probe Equilibrium Assays.

TR‐FRET kinetic probe association and dissociation assays

Measurements were carried out in quadruplicate and in a final volume of 15 μL per well. First, a 5‐point, twofold serial dilution of fluorescent probe (Supporting Information Fig. 2) was pre‐dispensed on black 384‐well low volume plates (Greiner Bio‐one (Frickenhausen, Germany)), and the PHERAstar FS injection system's syringes (previously washed with NaOH/H₂O) were primed either with 1500 μL solution of Tag‐lite GnRH cells (1000 cells·μL⁻¹) or with 200 μM buserelin. Then, 4 μL of cells were quickly added to the probe with the first syringe, and the association traces were recorded as described under TR‐FRET Probe Equilibrium Assays, with kinetic intervals of 26 s. After 30 min, fluorescent probe dissociation was initiated by addition of 5 μL of an excess of unlabelled buserelin (final concentration 67 μM) with the second syringe, and the traces were recorded with kinetic intervals of 300 s in the same fashion. Alternatively, a 1‐point measurement was performed with 5 μL of probe (final concentration 25 nM) and 5 μL of Tag‐lite GnRH cells (1400 cells·μL⁻¹), and association was recorded with kinetic intervals of 120 s. After 24 min, dissociation was initiated and recorded with the same kinetic interval.

TR‐FRET kinetic probe titration assays

First, 5 μL of increasing concentrations of fluorescent probe were dispensed into 384‐well plates; the injection system of the PHERAstar FS plate reader was primed with Tag‐lite GnRH cells as described under TR‐FRET Probe Equilibrium Assays. Then, 5 μL of cell solution was added with the syringe, and the TR‐FRET signals corresponding to probe association were recorded as described under TR‐FRET Probe Equilibrium Assays.

TR‐FRET kinetic probe competition assays

The basic principle of this assay is explained in Schiele et al. (2014). Before each experiment, 6 μL of fluorescent probe (final concentration 15 nM) was dispensed to the ‘assay‐ready’ plates containing 100 nL of compound dilutions using a Multidrop Combi Thermo Fischer Scientific (Dreieich, Germany), and the injection system of the PHERAstar FS plate reader was washed with NaOH/H₂0 and primed with Tag‐lite GnRH cells. Finally, the assay plates were introduced into the instrument, 4 μL of cells (1000 cells·μL⁻¹) were rapidly dispensed with the syringe to each well and the TR‐FRET signals corresponding to the competitive binding of probe and test compounds were recorded as described under TR‐FRET Probe Equilibrium Assays with kinetic intervals of 78 s.

Data analysis

All experimental data were analysed using the nonlinear regression curve‐fitting programme GraphPad Prism v. 5.00 (GraphPad Software Inc., San Diego, CA, USA). Further details on the handling of TR‐FRET data are available in Schiele et al. (2014).

For radioligand‐binding assays, the previously reported K_D value of 0.35 nM for [¹²⁵I]‐triptorelin (Heitman et al., 2008) was used to convert IC₅₀ values obtained from competition curve analysis into K_i values with the help of the Cheng–Prusoff equation (Cheng and Prusoff, 1973):

$$K_i={\mathrm{I}\mathrm{C}}_{50}/\left(1+\left[\text{radioligand}\right]/K_D\right)$$

Likewise, a K_D value of 0.8 nM obtained for the ‘red’‐labelled buserelin by fitting the data from the TR‐FRET probe equilibrium binding assay (Supporting Information Fig. S1) to the model ‘One site – Specific binding’ was used to convert IC₅₀ values from TR‐FRET experiments to K_i values.

The observed association rates (k _obs) derived from both assays were obtained by fitting association data using one‐phase exponential association. The dissociation rates were obtained by fitting dissociation data to a one‐phase exponential decay model. The k _obs values were converted into association rate (k _on) values using the following equation:

$$k_{\text{on}}=\left(k_{\mathrm{o}\mathrm{b}\mathrm{s}}-k_{\text{off}}\right)/\left[\text{radioligand}\right]$$

The association and dissociation rates were used to calculate the kinetic K_D using the following equation:

$$K_D=k_{\text{off}}/k_{\text{on}}$$

To further validate probe affinity and kinetic rate constants, association data from kinetic probe titration experiments were fitted to the ‘Association kinetics – two or more concentrations of hot’ model (Supporting Information Fig. S2). The k _on obtained from these experiments and K_D from equilibrium binding were used to calculate k _off as described previously.

Association and dissociation rates for unlabelled ligands were calculated by fitting the data of the competition association assay using kinetics of competitive binding (Motulsky and Mahan, 1984):

$$\begin{array}{c}{K}_A=k_1\left[L\right]\cdot {10}^{-9}+k_2\\ K_B=k_3\left[I\right]\cdot {10}^{-9}+k_4\\ S=\sqrt{{\left(K_A-K_B\right)}^2+4\cdot k_1\cdot k_3\cdot L\cdot I\cdot {10}^{-18}}\\ K_F=0.5\left(K_A+K_B+S\right)\\ K_S=0.5\left(K_A+K_B-S\right)\\ Q=\frac{B_{\max }\cdot k_1\cdot L\cdot {10}^{-9}}{K_F-K_S}\\ Y=Q\cdot \left(\frac{k_4\cdot \left(K_F-K_S\right)}{K_F\cdot K_S}+\frac{k_4-K_F}{K_F}{e}^{\left(-K_F\cdot X\right)}-\frac{k_4-K_S}{K_S}{e}^{\left(-K_S\cdot X\right)}\right)\end{array}$$

where k ₁ is the k _on of the radioligand (M⁻¹·min⁻¹), k ₂ is the k _off of the radioligand (min⁻¹), L is the radioligand concentration (nM), I is the concentration of the unlabelled competitor (nM), X is the time (min) and Y is the specific binding of the radioligand (DPM). During a competition association, these parameters are set, obtaining k ₁ from the control curve without competitor and k ₂ from previously performed dissociation assays described under Radioligand Association and Dissociation Assays. With that, the k ₃, k ₄ and B _max can be calculated, where k ₃ represents the k _on (M⁻¹·min⁻¹) of the unlabelled ligand, k ₄ stands for the k _off of the unlabelled ligand and B _max equals the total binding (DPM). All competition association data were globally fitted.

In case of kinetic probe competition assays (kPCA), the kinetics of the competitive binding model was enhanced with a mathematical term describing a mono‐exponential decay that accounts for signal drift (Schiele et al., 2014). As stated for the radioligand‐binding assays, the kinetic rate constants of the fluorescent probe (k ₁ and k ₂) were determined in separate experiments and set constant in kPCA data analysis.

The RT was calculated as in the following equation:

$$\mathrm{R}\mathrm{T}=1/k_{\text{off}}$$

All values obtained from radioligand‐binding assays are means of at least three independent experiments performed in duplicate, unless stated otherwise. Values obtained from TR‐FRET assays are means of two independent experiments performed in quadruplicate, unless stated otherwise.

Reagents and peptides

Deslorelin and fertirelin (Table 1) were obtained from Genway Biotech Inc. (San Diego, CA, USA) and American Peptide Company (Sunnyvale, CA, USA) respectively. All other peptide analogues (Table 1) and BSA were purchased from Sigma‐Aldrich Chemie B.V. (Zwijndrecht, The Netherlands). Bicinchoninic acid protein assay reagent was obtained from Pierce Chemical Company (Rockford, IL, USA). [¹²⁵I]‐triptorelin (specific activity 2200 Ci·mmol⁻¹) was purchased from PerkinElmer (Groningen, The Netherlands). CHO cells stably expressing the human GnRH receptor (from now on CHOhGnRH cells) were kindly provided by MSD (Oss, The Netherlands). Tag‐lite™ HEK293 cells containing a stably overexpressed human GnRH receptor labelled with Tb (from now on Tag‐lite GnRH cells) were obtained as frozen stocks from Cisbio (Codolet, France). A buserelin‐derived tracer, labelled at the sixth position with a red emitting fluorophore, and (TLB) were also purchased from Cisbio. All other chemicals and cell culture materials were obtained from standard commercial sources.

Results

Determination of the association and dissociation rate constants of [¹²⁵I]‐triptorelin

The binding properties of [¹²⁵I]‐triptorelin to CHOhGnRH membranes were quantified with traditional kinetic radioligand‐binding assays. Association and dissociation experiments provided k _on and k _off values of 0.4 ± 0.1 nM⁻¹·min⁻¹ and 0.05 ± 0.0004 min⁻¹ respectively (Figure 1A and Table 2). From these data, the equilibrium dissociation constant (kinetic K_D) was calculated, which had a value of 0.2 nM.

Figure 1.

Association and dissociation kinetics of [¹²⁵I]‐triptorelin (A,B) or fluorescent probe (C) at the hGnRH receptor. Representative graphs from one experiment performed in duplicate (see Tables 2 and 3 for kinetic parameters).

Table 2.

Comparison of the affinity, dissociation constants and kinetic parameters of reference agonist triptorelin obtained with different radioligand‐binding assays

Assay	pKD b and (KD (nM))	pKi and (Ki (nM))	k on (nM−1·min−1)	k off (min−1)
Displacement	NA	9.6 ± 0.09 (0.27)	NA	NA
Association and dissociation	9.9 ± 0.11 (0.13)	NA	0.40 ± 0.12	0.050 ± 0.0004
Competition associationa	9.7 ± 0.12 (0.22)	NA	0.12 ± 0.014	0.026 ± 0.008

Values are means ± SEM of three separate experiments performed in duplicate

NA, not applicable

^aThe binding kinetics of unlabelled triptorelin were determined by addition of 0.3‐fold, 1‐fold and 3‐fold its K_i value

^b K_D = k _off/k _on

Determination of the association and dissociation rate constants of the fluorescently labelled buserelin derivative probe

A fluorescently labelled buserelin derivative was used as a probe in all TR‐FRET assays. The kinetic parameters of the fluorescent tracer were determined by performing association and dissociation experiments. Experiments yielded a k _on and k _off of 0.008 ± 0.001 nM⁻¹·min⁻¹ and 0.01 ± 0.001 min⁻¹ respectively (Figure 1B, Table 3 and Supporting Information Fig. 2). The kinetic K_D value calculated from these experiments was 1.2 nM.

Table 3.

Comparison of the affinity, dissociation constants and kinetic parameters of the fluorescent buserelin probe obtained with different TR‐FRET assays

Assay	pKD and (KD (nM))	pKi and (Ki (nM))	k on (nM−1·min−1)	k off (min−1)b
Equilibrium association	9.1 ± 0.8 (0.8)	NA	NA	NA
Association and dissociationa	8.9 ± 0.9 (1.2)	NA	0.008 ± 0.001	0.010 ± 0.001
Multiple association and dissociation	8.7 ± 0.06 (2.1)	NA	0.008 ± 0.001	0.016 ± 0.002

Values are means ± SEM of three separate experiments

NA, not applicable

^aThe dissociation kinetics of fluorescently labelled buserelin derivative were determined by addition of 10 μM buserelin

^b k _off = K_D(equilibrium)/k _on

Determination of the binding affinity of hGnRH receptor agonists with [¹²⁵I]‐triptorelin

Equilibrium radioligand‐binding assays were performed to assess the ability of 12 GnRH analogues to displace [¹²⁵I]‐triptorelin from CHOhGnRH cell membranes. All ligands were able to fully displace [¹²⁵I]‐triptorelin in a concentration‐dependent manner (Figure 2A and Table 4). All peptides had a Hill coefficient close to unity in the [¹²⁵I]‐triptorelin displacement assay (data not shown), which indicated a competitive mode of inhibition with regard to the radioligand. Of all tested ligands, nafarelin had the highest affinity for the hGnRH receptor with a K_i value of 0.06 nM, and GnRH had the lowest affinity of 13 nM. All other ligands had affinities in the low to sub‐nanomolar range.

Figure 2.

Displacement of [¹²⁵I]‐triptorelin (A) or fluorescent probe (B) from the hGnRH receptor by the 12 peptide agonists. Representative graphs from one experiment performed in duplicate (see Table 4 for affinity values).

Table 4.

Binding parameters of GnRH peptide agonists derived from radioligand binding and TR‐FRET experiments

	Radioligand binding		TR‐FRET
Agonist	pKi and (Ki (nM))	pKD and (KD (nM))	pKi and (Ki (nM))	pKD and (KD (nM))
GnRH	7.9 ± 0.05 (13)	8.5 ± 0.08 (2.9)	8.4 ± 0.6 (4.0)	7.7 ± 0.03 (22)
Triptorelin	9.6 ± 0.09 (0.3)	9.7 ± 0.1 (0.2)	9.5 ± 0.2 (0.4)	9.5 ± 0.03 (0.4)
[d‐Ala6]‐GnRH	9.0 ± 0.05 (0.8)	9.1 ± 0.09 (0.8)	8.6 ± 0.4 (2.3)	8.9 ± 0.02 (1.3)
[d‐Lys6]‐GnRH	8.3 ± 0.1 (5.2)	8.4 ± 0.2 (3.7)#	7.8 ± 0.3 (16)	7.8 ± 0.01 (15)
Fertirelin	9.2 ± 0.05 (0.7)	9.1 ± 0.08 (0.8)#	9.0 ± 0.3 (1.0)	9.0 ± 0.02 (0.9)
Alarelin	9.4 ± 0.1 (0.5)	9.8 ± 0.1 (0.2)	9.0 ± 0.3 (0.9)	9.4 ± 0.03 (0.4)
Deslorelin	10 ± 0.1 (0.1)	9.9 ± 0.1 (0.1)#	8.6 ± 0.6 (0.8)	9.4 ± 0.04 (0.4)
Leuprorelin	9.5 ± 0.09 (0.3)	9.8 ± 0.1 (0.2)	9.7 ± 0.3 (0.2)	8.9 ± 0.03 (1.2)
Nafarelin	10 ± 0.06 (0.06)	10.6 ± 0.1 (0.03)	9.8 ± 0.3 (0.2)	9.7 ± 0.06 (0.2)
Buserelin	9.9 ± 0.05 (0.1)	10.4 ± 0.2 (0.04)	9.4 ± 0.2 (0.4)	9.5 ± 0.04 (0.3)
Goserelin	8.8 ± 0.06 (1.6)	9.0 ± 0.08 (1.1)	9.1 ± 0.3 (0.9)	8.6 ± 0.02 (2.7)
Histerelin	9.8 ± 0.2 (0.2)	10 ± 0.08 (0.04)	8.7 ± 0.5 (1.9)	9.0 ± 0.04 (1.0)

Values are means ± SEM of at least three separate experiments performed in duplicate

^# Values are means ± SEM of two separate experiments performed in duplicate

K_D = k _off/k _on

Determination of the binding affinity of hGnRH receptor agonists with TR‐FRET

The binding affinity of 12 agonists was also determined using a fluorescently labelled buserelin derivative as a tracer and Tag‐lite GnRH cells in a TR‐FRET assay. In accordance with the radioligand‐binding results, all agonists were able to fully displace the fluorescent tracer from the hGnRH receptor in a concentration‐dependent manner (Figure 2B and Table 4). The data were in fair agreement with the affinities determined in the radioligand displacement assay despite the inherent differences between the two assays (r ² = 0.5 and P < 0.05) (Figure 4C).

Figure 4.

Correlation between affinities (pK_i) and dissociation constants (pK_D) derived from (A) radioligand binding (r ² = 0.9, P < 0.0001) and (B) TR‐FRET experiments (r ² = 0.5, P < 0.05). (C) Correlation between affinities (pK_i) derived from radioligand binding and homogeneous time‐resolved fluorescent experiments (r ² = 0.5, P < 0.05). (D) Correlation between dissociation constants (pK_D) derived from radioligand binding and TR‐FRET experiments (r ² = 0.8, P < 0.001). In all cases, pK_i values were obtained from equilibrium displacement studies, and pK_D values were determined with competition association experiments.

Validation and optimization of the competition association assay with [¹²⁵I]‐triptorelin

With the k _on and k _off values of [¹²⁵I]‐triptorelin obtained from traditional association and dissociation experiments, the k _on (k ₃) and k _off (k ₄) values of unlabelled triptorelin could be determined by fitting the kinetic parameters into the model of ‘kinetics of competitive binding’ as described under Methods. Three different concentrations of unlabelled triptorelin were tested and presented a shared k _on (k ₃) and k _off (k ₄) value of 0.1 ± 0.01 nM⁻¹·min⁻¹ and 0.03 ± 0.008 min⁻¹ respectively (Figure 3A). These values were in good agreement with the association and dissociation rates obtained with traditional kinetic experiments (Table 2). Additionally, a comparison of the affinity (0.3 nM) and dissociation constants (0.1 and 0.2 nM), acquired from equilibrium and kinetic experiments, respectively, further confirmed the competition association assay as a valid tool to determine the binding kinetics of unlabelled ligands at the hGnRH receptor (Table 2).

Figure 3.

Competition association experiment with [¹²⁵I]‐triptorelin in the absence or presence of 0.3, 1 or 3 * K_i value of unlabelled triptorelin (A) or 1 * K_i value of buserelin, leuprorelin or GnRH (B). Representative graphs from one experiment performed in duplicate (See Table 2 for kinetic parameters).

To improve the throughput of this assay, one concentration of competitor was selected that yielded an assay window discernable from both the baseline and control curve (i.e. specific binding approximately 40–60%). In this case, a concentration of competitor equal to onefold its K_i value presented the best assay window. Analysis of this single concentration showed similar kinetic rates for triptorelin in comparison with the three‐concentration method, which were statistically indifferent (data not shown; P > 0.05). Thus, this one‐concentration method was used for subsequent determination of the binding kinetics of other unlabelled hGnRH receptor peptide agonists.

Determination of the receptor‐binding kinetics of unlabelled hGnRH receptor agonists with [¹²⁵I]‐triptorelin

By use of the one‐concentration competition association assay, the binding kinetics of 11 other unlabelled hGnRH receptor agonists were quantified (Figure 3B and Table 5). Juxtaposing affinities (K_i values) and dissociation constants (K_D values) acquired from equilibrium and kinetic experiments resulted in a high correlation (r ² = 0.9, P < 0.0001). Firstly, this further confirmed that the competition association assay was a valid tool to determine the binding kinetics of unlabelled ligands for the hGnRH receptor (Figure 4A) and, secondly, proved that equilibrium was reached for all agonists in the displacement experiments. The dissociation rates ranged from 0.2 ± 0.03 min⁻¹ for goserelin to 0.01 ± 0.003 min⁻¹ for buserelin, a variance of roughly 20‐fold. Interestingly, three distinctive association patterns were obtained from the competition association assays (Figure 3B). Firstly, an ‘overshoot’ in [¹²⁵I]‐triptorelin association was observed for slowly dissociating compounds, such as buserelin. Secondly, we noticed a shallow increase in [¹²⁵I]‐triptorelin association for rapidly dissociating compounds, such as GnRH, and lastly, no difference was observed in the shape of the [¹²⁵I]‐triptorelin association curve for equally fast‐dissociating compounds, such as leuprorelin. The observed differences in dissociation kinetics were all in comparison with those of the radioligand [¹²⁵I]‐triptorelin (Figure 3B). Association rates ranged from 0.8 ± 0.2 nM⁻¹·min⁻¹ for nafarelin to 0.02 ± 0.004 nM⁻¹·min⁻¹ for fertirelin, a span of approximately 35‐fold.

Table 5.

Kinetic receptor‐binding parameters of GnRH peptide agonists derived from radioligand‐binding competition association assays and kPCA TR‐FRET experiments

	Radioligand binding			TR‐FRET
Agonist	k on (nM−1·min−1)a	k off (min−1)a	RT (min)c	k on (nM−1·min−1)b	k off (min−1)b	RT (min)c
GnRH	0.06 ± 0.01	0.2 ± 0.02	6.3 ± 0.6	0.02 ± 0.01	0.44 ± 0.3	2.3 ± 1.6
Triptorelin	0.1 ± 0.01	0.03 ± 0.008	39 ± 12	0.1 ± 0.02	0.05 ± 0.008	21 ± 3.7
[d‐Ala6]‐GnRH	0.08 ± 0.01	0.07 ± 0.01	15 ± 3.1	0.05 ± 0.007	0.07 ± 0.01	14 ± 2.3
[d‐Lys6]‐GnRH	0.04 ± 0.02#	0.1 ± 0.04#	7.7 ± 2.3#	0.02 ± 0.01	0.25 ± 0.17	4 ± 2.6
Fertirelin	0.02 ± 0.004#	0.02 ± 0.001#	56 ± 3.1#	0.07 ± 0.009	0.06 ± 0.01	15 ± 2.4
Alarelin	0.09 ± 0.02	0.01 ± 0.002	77 ± 12	0.09 ± 0.009	0.03 ± 0.005	31 ± 4.8
Deslorelin	0.07 ± 0.01#	0.01 ± 0.002#	100 ± 20#	0.05 ± 0.005	0.02 ±0.004	44 ± 6.9
Leuprorelin	0.2 ± 0.04	0.03± 0.005	36 ± 6.4	0.03 ± 0.004	0.04 ± 0.006	26 ± 4.4
Nafarelin	0.8 ± 0.2	0.02 ± 0.003	50 ± 7.5	0.1 ± 0.02	0.03 ± 0.006	39 ± 9.8
Buserelin	0.2 ± 0.06	0.009 ± 0.003	111 ± 37	0.05 ± 0.004	0.02 ± 0.003	61 ± 10
Goserelin	0.2 ± 0.002	0.2 ± 0.03	5.6 ± 0.8	0.03 ± 0.005	0.08 ± 0.01	13 ± 2.4
Histerelin	0.3 ± 0.04	0.01 ± 0.002	83 ± 14	0.02 ± 0.002	0.02 ± 0.004	50 ± 8.8

Values are means ± SEM of at least three separate experiments performed in duplicate

^# Values are means ± SEM of two separate experiments performed in duplicate

^a k _on and k _off of unlabelled GnRH agonists were determined at onefold K_i concentrations

^b k _on and k _off of unlabelled GnRH agonists were determined at 0.5, 5, 50 and 500 nM

^c RT = 1/k _off

Determination of the receptor‐binding kinetics of unlabelled hGnRH receptor agonists with a fluorescently labelled buserelin derivative

The kinetic parameters of the 12 GnRH agonists were also determined with TR‐FRET experiments (Figure 5, Table 5 and Supporting Information Fig. 3). Association rates ranged from 0.1 ± 0.02 nM⁻¹·min⁻¹ for triptorelin to 0.02 ± 0.002 nM⁻¹·min⁻¹ for histerelin. Buserelin was again one of the slowest dissociating agonists with a dissociation rate of 0.02 ± 0.003 min⁻¹, while GnRH had the fastest dissociation rate of 0.4 ± 0.03 min⁻¹. The dissociation constants (K_D) calculated from k _on and k _off values were consistent with the affinities determined in homogeneous time‐resolved fluorescent displacement assays (Figure 4B) as well as with the K_D values obtained from the radioligand‐binding studies (Figure 4D). Dissociation rate constants (k _off) were in good agreement with the data obtained from radioligand‐binding experiments (r ² = 0.7, P < 0.0005) (Figure 6A), while the association rates (k _on) presented no correlation (r ² = 0.03, P = 0.6) (Figure 6B).

Figure 5.

Competition association experiment with fluorescent probe in the absence or presence of increasing concentrations of buserelin (A) or one‐concentration of unlabelled agonist that showed around 50% displacement (B). Representative graphs from one experiment performed in quadruplicate.

Figure 6.

Correlation between the kinetic parameters obtained from radioligand‐binding assays and kPCA TR‐FRET experiments. (A) Dissociation rate (k _off) (r ² = 0.7, P < 0.0005); (B) association rate (k _on) (r ² = 0.03, P = 0.6).

Discussion and conclusions

Over the years, several studies have indicated that long duration of action is an important feature contributing to improved efficacy of drugs designed to treat chronic illness. Moreover, increased target residence time offers the potential for a once‐daily dosage form that increases patient compliance, which is crucial for the management of diseases (Smith et al., 1996; Tashkin, 2005; Copeland et al., 2006; Dowling and Charlton, 2006; Vauquelin and Van Liefde, 2006; Swinney, 2009; Zhang and Monsma, 2009; Guo et al., 2014).

The GnRH receptor is the target of multiple marketed peptide agonists, classified as functional antagonists, used to treat hormone‐dependent diseases. Available patient information for the most commonly prescribed GnRH analogues suggests that the pharmacokinetic/pharmacodynamics profiles are very similar. Hence, knowledge of the in vitro binding kinetics could give extra insights into these well‐known drugs. However, the potential impact of variable‐binding kinetics of these GnRH peptide derivatives on clinical efficacy has not been investigated. Aside from agonists, a few studies have detailed the effect of slow dissociation kinetics of antagonists for the GnRH receptor to decrease the maximal response of an agonist (insurmountability) in vitro and to improve and prolong efficacy in vivo. A study of Kohout and coworkers (2007) addressed the insurmountability of a small molecule GnRH antagonist, TAK‐013. The authors examined the differences in antagonistic and kinetic properties of TAK‐013 for hGnRH receptors, mouse GnRH receptors and mutated mouse GnRH receptors and found a good correlation between the degree of insurmountability in in vitro functional assays and the dissociation rate from the receptor. Therefore, they proposed slow receptor dissociation kinetics to be accountable for the mechanism of insurmountability of TAK‐013. Similar findings were published (Sullivan et al., 2006) for another series of small molecule antagonists, that is, uracils. Slowly dissociating ligands displayed insurmountable antagonism, whereas faster dissociating ligands proved to be surmountable antagonists. To determine the dissociation rates of these uracil series of antagonists, the competition association method (Motulsky and Mahan, 1984) was used with a proprietary small molecule radioligand as a tracer. Such a competition association assay has recently been applied to determine the receptor kinetics of ligands for several different GPCRs such as the adenosine A_2A receptor (Guo et al., 2012), the muscarinic M₃ receptor (Sykes et al., 2009), the chemokine receptor CCR2 (Zweemer et al., 2013) and the histamine H₁ and H₃ receptor (Slack et al., 2011). We were able, for the first time, to determine the kinetic parameters of 12 GnRH peptide agonists, including many marketed drugs.

Two different techniques were applied, namely, radioligand‐binding studies and kPCA with a TR‐FRET read‐out. For the former, a comparison of the radioligand's kinetic parameters obtained from traditional radioligand‐binding experiments showed a good consistency with the kinetic parameters for triptorelin derived from the competition association assay (Table 2). Moreover, the kinetics of the 11 remaining GnRH agonists presented a good correlation between the kinetically derived K_D and the affinity obtained from equilibrium radioligand‐binding studies (Figure 4). Secondly, we also conducted these experiments with a fluorescently labelled buserelin probe in a TR‐FRET assay. This technology has already been used for examining equilibrium GPCR ligand binding (Degorce et al., 2009; Zhang and Xie, 2012), and more recently, it was used to characterize the binding kinetics of the histamine H₁ receptor (Schiele et al., 2014).

Comparing affinities and kinetic K_D values from both the radioligand‐binding and TR‐FRET assays yielded significant correlations demonstrating a good reproducibility between both techniques. The two distinct assays also proved to be very amenable to the determination of the kinetic receptor‐binding parameters of (peptide) GnRH agonists. Dissociation rates, and thus residence times, between assays were in good accordance with P‐values of <0.0005, while the association rates were in less agreement between techniques. It should be noted that the experimental differences between both assays are considerable, which may have consequences for the kinetic parameters derived in the two assays. For example, the radioligand‐binding studies were manually dispensed while the TR‐FRET assays were performed using automated dispensing devices. It has been reported that compound handling can be an important source of assay variability (Gubler et al., 2013). In addition, the kinetic binding parameters were determined using a one‐concentration method for the radioligand‐binding experiments, whereas the kPCA studies used five different concentrations. Another notable difference is that in the radioligand‐binding studies, CHOhGnRH membranes were used, whereas the TR‐FRET assays were performed with Tag‐lite HEK293 GnRH cells. Packeu et al. (2008) discussed the differences in membrane interactions of membrane preparations and whole cells and their effects on binding kinetics for the D_2L‐dopamine receptors. Moreover, the authors found slower dissociation rates from intact cells in comparison with membrane preparations, and they proposed that an intact cellular environment could play a role in stabilizing the D_2L‐dopamine receptors in a particular conformation. A similar reasoning might be applicable to the GnRH receptors, although in our case, the receptor appears in a way that slows down the association rates of the peptides (Table 5). It may also be that the peptides simply have more difficulty in reaching the receptor on intact cells than on membrane fragments.

It might be argued that the assay temperature of 25°C is not representative for binding kinetics observed in vivo. For example, Sakai (1991) examined the effect of temperature on the dissociation of [¹²⁵I]‐prolactin from the rabbit mammary gland prolactin receptor. They found a linear relationship between the dissociation rate and temperature with an increased dissociation rate at higher temperatures. Another study (Treherne and Young, 1988) also showed that the dissociation of [³H]‐QMDP from the histamine H₁ receptor was temperature‐dependent, which was also true for the association rate but to a lesser extent. Arrhenius plots for both the association rate and dissociation rate of [³H]‐QMDP were linear between 6 and 37°C. It should be noted that, although these studies show a linear increase in dissociation rates with higher temperatures, the slope of this increase could be very different between targets and their ligands. Taken together, this indicates that the kinetic ranking of ligands for the same receptor can be expected to stay the same over different temperatures. Therefore, even though all our experiments were performed at 25°C, the results are still of great value for translation to in vivo outcomes.

Numerous peptide GnRH derivatives have been synthesized and studied for their so‐called structure–affinity relationships, with the aim to improve their affinity, potency and/or metabolic stability (Fujino et al., 1972; Monahan et al., 1973; Karten and Rivier, 1986; Sealfon et al., 1997; Hovelmann et al., 2002; Millar et al., 2004) In summary, it was established that the NH₂‐terminal domain (pGlu–His–Trp–Ser) of GnRH is important for receptor binding and activation with Trp³ as a critical residue. In addition, the COOH‐terminal domain (Pro–Gly–NH₂) is crucial for receptor binding where substitution of Pro⁹ or removal of NH₂ results in very low affinity unless the COOH‐terminal tail is substituted for an ethylamide, which also improves metabolic stability. In contrast, the central domain of the peptide is less conserved, and studies show that exchange of Tyr⁵, Leu⁷ or Arg⁸ is mostly well tolerated. The most beneficial substitution is that of Gly⁶ with a d‐amino acid, which provides a more favourable conformation and in turn results in increased potency. d‐amino acids at the sixth position of the peptide are therefore incorporated in all the GnRH analogues marketed. The amino acid sequences of the 12 GnRH peptides tested in this study are identical with the exception of the sixth amino acid and the carboxylic tail (Table 1). A tentative structure–kinetic relationship could be established for the carboxylic tail (i.e. substitution of the glycine amide for an ethylamide). For instance, a comparison of triptorelin and deslorelin showed 2/3‐fold changes in affinity (Table 4), which was also observed in residence time. This ethylamide‐induced improvement in residence time was also true to a bigger extent for goserelin and buserelin, where the affinity was improved 2‐fold or 16‐fold (TR‐FRET and radioligand binding, respectively), while the residence time was more significantly affected, witnessed by a fivefold increase in the kPCA TR‐FRET experiments and a 20‐fold increase in the radioligand‐binding studies. This shows that shortening the carboxylic tail of the peptide slightly increases the affinity but results in a more significant improvement in residence time. Interestingly, three decades ago, it was already speculated that buserelin has a longer residence time. In these studies, the authors proposed that the high potency and long duration of action of buserelin in vivo was a result of prolonged GnRH receptors binding (Yeo et al., 1981; Koiter et al., 1984; Koiter et al., 1986). Along similar lines, Flanagan and coworkers (1998) discussed slower dissociation rates of GnRH agonists with a more hydrophobic amino acid at position 6. However, no mechanism or kinetic binding data were reported at that time.

Previously published mutagenesis studies further strengthen our hypothesis indicating the importance of the ethylamide at the carboxylic tail. Davidson and coworkers (1995) showed that the Asn^2.65(102) residue located near the extracellular end of TM2 plays a role in ligand binding, specifically with the carboxylic tail of GnRH analogues. Mutations to alanine at this position significantly decreased the potency of GnRH analogues with Gly¹⁰–NH₂ but had a lesser effect on GnRH analogues with an ethylamide tail (Davidson et al., 1996; Hoffmann et al., 2000; Millar et al., 2004). It may be hypothesized that substitution of Gly¹⁰–NH₂ with an ethylamide moiety creates less steric hindrance and increases hydrophobicity, thereby improving the fit of the agonist and thus elongating its residence time on the receptor.

In conclusion, two novel competition association assays were successfully developed and applied to determine the kinetic binding characteristics of 12 peptide agonists, including many marketed drugs targeting the GnRH receptor. All agonists proved to have high affinity for the GnRH receptor, whereas significant differences were observed in their binding kinetics. These findings provide new insights and tools for the development of improved drugs targeting the GnRH receptor by incorporating optimized kinetic binding parameters. They also suggest that bringing this knowledge on kinetics to the clinic may help in improving or adjusting treatment protocols with better patient outcomes.

Author contributions

I. N., F. S., V. G., K. N‐R., A. E. F‐M., A. P. I. and L. H. H. participated in research design. I. N., F. S. and V. G. conducted experiments. I. N., F. S. and V. G. performed data analysis. I. N., L. H. H., A.P. I., F. S., V. G. and A. E. F‐M. wrote or contributed to the writing of the manuscript.

Conflict of interest

None.

Supporting information

Figure 1. Saturation equilibrium binding of fluorescent buserelin probe to Tag‐lite™ GnRH cells (IC₅₀ = 5.9 nM, r² = 0.99). Representative graph from one experiment performed in duplicate.

Figure 2. Association and dissociation kinetics of five concentrations of fluorescent buserelin probe to Tag‐lite™ GnRH cells. Representative graph from one experiment performed in duplicate.

Figure 3. kPCA traces of the GnRH peptide agonists analysed. Four concentrations (0.5, 5, 50 and 500 nM) were examined. Representative graphs from one experiment performed in duplicate.

Supporting info item

Supporting info item

Supporting info item

Nederpelt, I. , Georgi, V. , Schiele, F. , Nowak‐Reppel, K. , Fernández‐Montalván, A. E. , IJzerman, A. P. , and Heitman, L. H. (2016) Characterization of 12 GnRH peptide agonists – a kinetic perspective. British Journal of Pharmacology, 173: 128–141. doi: 10.1111/bph.13342.