WO2008043851A1

WO2008043851A1 - A non-homogeneous quencher free assay method for the noradrenaline transporter

Info

Publication number: WO2008043851A1
Application number: PCT/EP2007/060916
Authority: WO
Inventors: Anders Haunso
Original assignee: N.V. Organon
Priority date: 2006-10-13
Filing date: 2007-10-12
Publication date: 2008-04-17

Abstract

The present invention relates to a method of screening for an inhibitor of the noradrenaline transporter utilising a non-homogeneous whole cell assay method for measuring noradrenaline transporter activity as a function of 4-(4-dimethylaminostyryl)-N-methylpyridinium accumulation in the absence of a quencher.

Description

A non-homogeneous quencher free assay method for the noradrenaline transporter.

The present invention relates to a non-homogeneous whole cell assay method of measuring noradrenaline transporter activity. The present invention further relates to a rapid, non-homogeneous, whole cell assay method of screening for an inhibitor of said transporter.

The release of neurotransmitters within the nervous system plays a pivotal role in signal transduction and affects the responses to a number of physiological and sensory stimuli. Neurotransmitter uptake in turn prevents over-stimulation of synaptic receptors and attenuates neuronal signalling via rapid neurotransmitter clearance \ The monoamine transporters belong to the Na7CI^" dependent family of transporters, are central to the processing of information in the nervous system and are associated with numerous neurological disorders making them important targets for central nervous system drug discovery ²'³. The noradrenaline transporter (NET) is a member of the monoamine transporters and is a target for drugs useful for the treatment or prevention of depression, pain, obsessive-compulsive disorder, attention-deficit hyperactivity disorder and posttraumatic stress disorder as well as drugs of abuse, such as cocaine and amphetamines ³.

Despite the important role played by the NET in numerous disease states of the central nervous system, there is a lack of non-radioactive, non-homogeneous approaches to identify inhibitors of said transporter. Methods used to identify inhibitors of the NET typically involve non-homogeneous equilibrium binding experiments or functional uptake assays, which rely on either a radiolabeled uptake inhibitor or neurotransmitter respectively ²'^4"8.

Recent reports indicate that development of homogeneous in vitro assays based on fluorescent dyes rather than radiolabeled approaches are feasible for members of the NaVcr dependent family of transporters ⁹'¹⁰. Furthermore, a novel homogeneous uptake assay for monoamine transporters using 4-(4-dimethylaminostyryl)-Λ/-methylpyridinium (ASP⁺) was recently described in a published patent application (US 2004/115703) and related publication¹² in which the use of quencher (such as Trypan Blue) enabled the study of transporter localisation, activity and regulation of noradrenaline transport and dopamine transport. In our hands, use of Trypan Blue as a quencher adversely affected the potency of ASP⁺ to a degree where we were unable to replicate the assay method. As fluorescent-based assays are generally homogeneous in nature, it was surprising that by avoiding the use of a quencher, we were able to develop a robust and reproducible non-homogeneous whole cell based assay method for measuring noradrenaline transport. Furthermore our method was found not to be applicable to assays utilising other monoamine transporters such as the dopamine or serotonin transporters. The end-point readout carried out in non-homogeneous assays such as the classical functional uptake assays relying on radiolabeled ³H-noradrenaline (³H-NA) have proven very effective in identifying novel chemical entities inhibiting the noradrenaline transporter. Thus a new method combining the advantages of fluorescent dyes over ³H-NA with the proven screening feasibility of non-homogeneous approaches to identifying inhibitors of the noradrenaline transporter is highly desirable.

In a first aspect, the present invention provides a non-homogeneous whole cell assay method of screening for an inhibitor of the noradrenaline transporter comprising: (a) establishing a whole cell expressing an exogeneous noradrenaline transporter; (b) treating said cell with a putative inhibitor of said transporter;

(c) introducing an assay buffer containing 4-(4-dimethylaminostyryl)-Λ/-methylpyridinium and

(d) measuring activity of said transporter as a function of 4-(4-dimethylaminostyryl)-/V- methylpyridinium accumulation, characterised in that following step (c), cells are washed to remove 4-(4- dimethylaminostyryl)-Λ/-methylpyridinium not taken up by the cells, thereby determining the effect of the compound to be tested as an inhibitor by the whole cell accumulation of 4-(4-dimethylaminostyryl)-Λ/-methylpyridinium in the absence of background fluorescence and that step (d) is carried out in the absence of a quencher.

The term whole cell, as used herein, encompasses all types of eukaryotic cell or vesicular entitity thereof capable of expressing the transporter. For example, Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO) cells or Human Embryonic Kidney (HEK) cells. The term whole cell, as used herein, further includes neuronal cells and derived vesicular entities such as synaptosomes.

The term exogeneous noradrenaline transporter, as used herein, encompasses all mammalian orthologues of the noradrenaline transporter that are either stably or transiently expressed in said whole cell. Examples of such an exogeneous noradrenaline transporter include the human noradrenaline transporter (hNET) and the rat noradrenaline transporter (rNET). The skilled person will appreciate that the non-homogeneous quencher free assay method described herein, may also be used to monitor the activity of endogenously expressed noradrenaline transporter expressed in neurones and derived vesicular entities such as synaptosomes. Furthermore, it is also conceivable that the accumulation of ASP⁺ may also be utilized as a peripheral biomarker for noradrenaline activity in human platelets, lymphocytes or other cells derived from bone marrow.

A variety of assay buffers are compatible with the method of the present invention, for example, Hanks' Balanced Saline Solution, Phosphate-Buffered Saline (PBS) solution or Krebs-Ringer's (KRH) buffer.

The term quencher refers to a molecular entity that deactivates (quenches) an excited state of another molecular entity, either by energy transfer, electron transfer, or by a chemical mechanism as defined by IUPAC (see compendium of chemical terminology 2^nd ed. 1997).

The skilled person will appreciate that the purpose of measuring said transporter activity as a function Of ASP⁺ accumulation can include characterizing the kinetics, affinity, uptake of neurotransmitter, retention or accumulation of neurotransmitter or other substrates, regulation by phosphorylation or other biochemical modifications.

The non-homogeneous whole cell assay method can be performed in a variety of in vitro formats, for example, in multi-well plates, in particular in 96-well plates or 384-well plates, thereby enabling the parallel screening of hundreds or thousands of compounds.

Measuring said transporter activity as a function of ASP⁺ accumulation, by the non- homogeneous whole cell assay method of the present invention can be achieved by standard measurement methods well known in the art, for example, by using a fluorescent plate reader.

In a further embodiment of the present invention the non-homogeneous whole cell assay method for identifying inhibitors for the noradrenaline transporter can be used to determine inhibitor potency. Furthermore, the non-homogeneous whole cell assay can be automated thereby providing a high throughput screening method for the identification of chemical entity inhibitors of said transporter. Such an automated method can involve addition of reagents/components of the assay using robotic fluid delivery, analysis of multiple samples in multi-well formats using a fluorescent plate reader as well as other automation methods known in the art. In a further aspect, the present invention provides a noradrenaline transporter inhibitor identified by the non-homogeneous whole cell assay method as claimed and described herein. More particularly, said inhibitor is a small molecule chemical entity which mediates its effect either as a substrate for said transporter or as a modulator or blocker of said transporter. In a further embodiment said inhibitor is a novel chemical entity.

In a further aspect, the present invention relates to use of a noradrenaline transporter inhibitor identified according to the non-homogeneous whole cell assay method of the present invention for the manufacture of a medicament for the treatment or prevention of nervous system disorders. Nervous system disorders falling within the ambit of said use include anxiety, depression, schizophrenia, bipolar disorder, cognitive enhancement, attention deficit hyperactivity disorder or pain.

The invention is further illustrated by the following examples.

Example 1 - A non-homogeneous quencher free method for determining ASP⁺ uptake in Madin-Darby canine kidney cells expressing the human noradrenaline transporter.

Cell culture: A Madin-Darby canine kidney cell line stably overexpressing the human noradrenaline transporter (MDCK-hNET) was obtained from the laboratory of Dr. Susan Amara ¹³. The cell line was propagated according to standard cell culture techniques using Minimum Essential Media (MEM) (Invitrogen, UK) containing 10% Fetaclone Il (HyClone, USA) and 1 % non-essential amino acid solution (Invitrogen, UK).

General assay procedure for accumulation assays: Uptake assays were essentially carried out as described by Pacholczyk and colleagues ⁷. Uptake assays were performed using Hanks' balanced saline solution supplemented with CaCI₂ (1.3 mM), MgSO₄ (0.8 mM), NaHCO₃ (4.2 mM), ascorbic acid (1 mM) and pargyline (0.02 mM) pH7.4 (HBSS). ASP⁺ was prepared as a 1 mM stock solution in HBSS and stored at 4°C for no longer than 7 days. NET modulators were solubilized in 100% anhydrous dimethyl sulfoxide (DMSO; Sigma-Aldrich, UK) and further diluted in HBSS. On the day of the assay, medium was removed from the cells by aspiration and they were washed twice in HBSS. Cells were pre-incubated in the presence of various concentrations of NET modulator and uptake assays were initiated by addition of either ³H-NA or ASP⁺. All assays were terminated by two rapid washes with ice-cold HBSS. Competition experiments were conducted with triplicate determinations for each point and concentration-responses were generated using a Biomek 2000 (Beckman Coulter, UK). In 96-well plates, all NET modulators and the selective serotonin re-uptake inhibitor, citalopram, were tested as 6-point full-log concentration-responses whereas in 384-well plates, 12-point half-log concentration-responses were carried out. In all uptake experiments the final concentration of DMSO was kept constant at 0.1% throughout the assays.

Radiolabeled accumulation assay: Cells for the radiolabeled uptake assays were seeded in Costar 96-well white-walled, clear bottomed plates (Corning, USA) at a density of 40000 cells per well. Pre-incubations with NET modulators were for 5 minutes at 37°C and incubation with ³H-NA (1 :100 mixture of cold and hot noradrenaline, final concentration 20 nM) for 10 minutes at 37°C. Following assay termination, cells were solubilized in 100 μl_ MicroScint-20 scintillation mixture (Fisher Scientific, UK) and the ³H-NA accumulation determined by liquid scintillation spectrometry with a MicroBeta Trilux (PerkinElmer, USA).

96-well plate format of non-homogeneous quencher free ASP⁺ accumulation assay: Pharmacological characterization of psychotropic compounds performed using ASP⁺ as substrate were carried out in Costar 96-well black-walled, clear bottomed plates (Corning, USA). Cells were seed at a density of 10000 cells per well. Pre-incubation with psychoactive compounds was for 5 minutes at 37°C and incubation with ASP⁺ (final concentration 500 nM) was for 10 minutes at 37°C. Following assay termination, the fluorescent signal (excitation 475 nM; emission 605 nM; cut-off 590 nM) was monitored using a FLEXstation in 'end-point' record mode (Molecular Devices, UK).

384-well plate format of non-homogeneous quencher free ASP⁺ accumulation assays: Non-homogeneous, quencher free fluorescent accumulation assays were carried out in a fully automated 384-well plate format using Costar 384-well black-walled, clear bottomed plates (Corning, USA). Cells were seeded at a density of 7000 cells per well. Preincubation with psychoactive compounds was for 10 minutes at room temperature and incubation with ASP⁺ (final concentration 1500 nM) was for 30 minutes at room temperature. Following assay termination, emitted fluorescence (excitation 488 nM; emission filter 605 ± 50 nM) was monitored using Molecular Devices' fluorometric imaging plate reader (FLIPR) (Molecular Devices, UK) with a cooled charge coupled device camera. The exposure length of the camera was set to 0.4 seconds and the camera gain set to 80. A bespoke emission filter with an optimal wavelength of 605 nm and a bandpass of 50 nm was purchased from Glen Spectra (Glen Spectra UK).

Data analysis: All data were analysed using GraphPad Prism 4.00 (GraphPad, USA). Specific uptake was calculated by subtracting values obtained for non-specific uptake and all data normalized using total and non-specific uptake values obtained from triplicates in each plate. Non-specific uptake was defined in the presence of 1 μM nisoxetine. Nonlinear regression on data for saturation curves of ASP⁺ accumulation were analysed according to the one-site Michaelis-Menten equation to obtain the kinetic parameters K_m and V_MAX- IC₅₀ values obtained from non-linear regression on concentration-response curves were used to calculate K, values using the Cheng-Prusoff equation for competitive inhibitors to correct for substrate concentration ¹⁴. All K, data are presented as the geometric mean ± S. E. M. of at least three independent experiments carried out in triplicates. All pK, correlations were conducted by Pearson correlations and the screening window coefficients, Z'-factors, were computed as defined by Zhang and colleagues ¹⁵.

Experiment 2 - Results

ASP⁺ is a potent modulator of the noradrenaline transporter. In the present Example, the observation¹¹'¹² that the fluorescent molecule ASP⁺ is a potent modulator of ³H- noradrenaline (³H-NA) uptake by the noradrenaline transporter with a potency (K, value) of 775 ± 186 nM is confirmed (Figure 1A).

Accumulation of ASP⁺ by MDCK cells stably overexpressing hNET can be monitored without the use of a quencher such as Trypan Blue. Kinetic parameters, K_m and V_MAX, for accumulation of ASP⁺ were determined as 834 ± 1 14 nM and 11.4 ± 0.4 rfu/sec for assays carried out at 37°C and 639 ± 71 nM and 4.3 ± 0.1 rfu/sec for assays carried out at room temperature (Figure 1 B), respectively.

To further validate the use of ASP⁺ as a substrate suitable to create a non-homogeneous, quencher free uptake assay for hNET, pharmacological characterization of several psychotropic compounds was carried out. Representative concentration-response curves for inhibition of uptake of either ³H-NA or ASP⁺ by the potent and selective inhibitory of NET, nisoxetine, are illustrated in Figure 2. Pharmacological characterization described for several psychoactive compounds both in the classical ³H-NA uptake assay and a novel non-homogeneous, quencher free fluorescent-based accumulation assay showed that both the overall profile of the concentration-response curves and inhibition potencies were comparable between the two assays (Table 1 ).

The correlation in pharmacology between the ³H-NA and the non-homogeneous, quencher free ASP⁺ accumulation assays was further substantiated when statistical analyses on inhibition potencies obtained in the ³H-NA and 96-well plate based fluorescent (non-homogeneous, quencher free) accumulation assays were shown to be highly correlated (see Figure 3; r = 0.96, p<0.0001 ), overall suggesting analogous pharmacology of the psychoactive compounds in both assay formats.

To validate that the non-homogeneous quencher free accumulation assay is fully amenable to automation and suitable for high throughput screening (HTS), the assay was reconfigured and all incubations were carried out at room temperature to facilitate automation. To ensure that the pharmacology of the automated non-homogeneous quencher free NET uptake assays was comparable to assays performed at 37°C, five potent modulators (atomoxetine, desipramine, maprotiline, mazindol and nisoxetine) of the noradrenaline transporter were profiled and compared to data obtained both in the radioactive- and non-homogeneous quencher free accumulation assays performed at 37⁰C (see Table 1 ). Furthermore, MDCK-hNET cells were challenged either with 1 μM nisoxetine or vehicle and the fluorescence signal measured using Molecular Devices' fluorometric imaging plate reader (FLIPR). Highly reproducible differences between vehicle and nisoxetine treated cells were obtained when analysing the signal window in the automated 384-well based accumulation assay. Z'-values of signal from nisoxetine- or vehicle-treated cells of 397 ± 23 % and a Z'-value of 0.64 ± 0.02 (see Figure 4) was obtained.

³H-NA ASP⁺ (96-well) ASP⁺ (384-well)

Drug Name Ki (riM) S.E.M. Ki (riM) S.E.M. Ki (riM) S.E.M.

Atomoxetine 7.9 1.8 2.2 0.5 2.1 0.1

Chlorpromazine 513.6 278.1 139.2 32.0

Citalopram <10000 <6000

Desipramine 4.9 1.1 9.2 3.6 2.6 0.1 lmipramine 166.2 35.0 52.3 2.6

Maprotiline 101.3 10.1 36.4 5.4 56.5 6.3

Mazindol 10.2 2.3 4.1 1.0 6.0 0.5

Nisoxetine 8.2 1.6 3.8 0.4 5.1 0.8

Paroxetine 324.8 74.8 383.3 32.3

Reboxetine (S,S) 10.2 2.0 5.1 1.1

Venlafaxine 1 172.0 386.3 662.2 230.8

Table 1 : K, values for inhibition of ³H-noradrenaline (³H-NA) or ASP⁺ uptake by psychoactive compounds. IC₅₀ values for inhibition of ³H-NA or ASP⁺ (96-well) uptake were obtained by non-linear regression on concentration-response curves and converted to K, values using the Cheng-Prusoff equation for competitive inhibitors to correct for substrate concentration. Data are geometric mean ± S.E.M. of at least three independent experiments carried out in triplicates.

Discussion

This study demonstrates that ASP⁺ accumulation by the noradrenaline transporter can be measured without the need for a quencher. Characterizing the properties of ASP⁺ in modulating the uptake of ³H-noradrenaline (³H-NA) by hNET confirmed that it is a potent modulator of the noradrenaline transporter ¹¹'¹². ASP⁺ successfully competed with ³H-NA for uptake and was itself taken up by the hNET overexpressing MDCK cell line with high potency (see Fig 1A). An excellent correlation between potency for ASP⁺ in the ³H-NA uptake assay and the kinetic parameter, K_m, generated in the saturation experiments was obtained, both of which corresponded favourably to previously reported values ¹¹'¹². The V_MAX values obtained for ASP⁺ uptake in the non-homogeneous assay described here were approximately three times higher than those reported by Mason and colleagues ¹¹ The higher maximal uptake rate may be due to differences in the cell line used to overexpress the transporter (HEK293 v MDCK), differences in transporter expression levels within the cell lines or the change from a homogeneous to a non-homogeneous format.

Pharmacological characterization of several psychoactive compounds both in the classical ³H-NA uptake assay and a novel non-homogeneous fluorescent-based uptake assay showed that both the overall profile of the concentration-response curves and inhibition potencies were comparable between the two assays (see Figure 2 and Table 1 ). Furthermore, the potencies obtained in both radiolabeled and fluorescent-based uptake assays (non-homogeneous, quencher free) corresponded favourably with published literature values ⁷ '^{16 Λ1}.

Statistical analyses on NET inhibition potencies obtained in the ³H-NA and 96-well plate based fluorescent uptake assays also demonstrated that pK, values were highly correlated. This would suggest that the pharmacological profile of psychoactive compounds modifying NET activity in classical ³H-NA and the non-homogeneous fluorescent-based uptake assay described herein correlates well. Furthermore, this would suggest a similar mechanism of action for assays based on ³H-NA and ASP⁺ uptake in the absence of a quencher.

In order to automate the fluorescence-based uptake assay to make it amenable for HTS, the 384-well based format was performed at room temperature using high concentration Of ASP⁺ to increase the signal window. Uptake by the noradrenaline transporter has been shown to be temperature dependent both when using ASP^{+ 11}'¹⁶ and in studies using ³H- NA ¹⁶'¹⁸. To ensure that the pharmacology of the automated fluorescent-based NET accumulation assays carried out at room temperature was comparable to assays performed at 37°C, five potent modulators of the noradrenaline transporter were profiled and compared to data obtained both in the radioactive- and fluorescent-based uptake assays performed at 37°C. When using the Cheng-Prusoff equation to correct for substrate concentration, potency of the selective NET modulators in the automated uptake assay compared favourably both to published uptake values for the noradrenaline transporter and to data obtained in the assays performed at 37°C ⁷'¹⁷. This would indicate that the lower maximal uptake rate observed for hNET at room temperature does not affect the potencies of psychoactive compounds and that pharmacological characterization can be carried out both at room temperature and at 37°C. Highly reproducible differences between vehicle and nisoxetine treated cells were obtained when analysing the signal window in the automated 384-well based uptake assay and Z'-values of 0.64 were obtained. References

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Figure legends:

Figure 1. (A) Concentration-response curve for inhibition of ³H-NA uptake by ASP⁺ into

MDCK-hNET cells. Non-linear regression on uptake data produced a K, = 775 ± 186 nM for inhibition of ³H-NA uptake by ASP+ (B). Saturation curves of ASP+ uptake into MDCK- hNET cells at 37°C (T) and room temperature (■: RT). Saturation experiments were carried out for 10 or 30 minutes at 37°C or RT, respectively. For uptake assays carried out at 37°C, K_m and V_MAX values of 834 ± 1 14 nM and 1 1.4 ± 0.4 rfu/sec were obtained. For uptake assays carried out at RT, K_m and V_MAX values of 639 ± 71 nM and 4.3 ± 0.1 rfu/sec were obtained. All data shown are the results of three separate experiments performed in triplicates (A) or quadruplicates (B) ± S. E. M.

Figure 2: Concentration-response curves for inhibition of uptake by nisoxetine. Plotted are inhibition by nisoxetine of ³H-NA (•) or ASP⁺ uptake in the 96-well (T ) or 384-well format (D). K, values for inhibition of uptake were obtained by non-linear regression on concentration-response curves. Nisoxetine blocked uptake of ³H-NA with a K, value of 8.2 ± 1.6 nM and uptake of ASP+ in the 96-well or 384-well format with K, values of 3.8 ± 0.4 nM and 5.1 ± 0.8 nM, respectively. Curves represent data from at least three independent experiments carried out in triplicates.

Figure 3: Correlation between inhibition of ³H-NA and ASP⁺ uptake in MDCK-hNET cells by psychoactive compounds. Plotted are pK, values for inhibition of fluorescent ASP⁺ (ordinate axis) or ³H-NA (abscissa axis) uptake by the psychotropic compounds outlined in Table 1 , except citalopram which was inactive in both uptake assays. The line represents the linear regression on data. Statistical analysis of the correlation produced a Pearson correlation coefficient r= 0.96, and a two-tailed p value of p<0.0001.

Figure 4: Assay signal window and Z'-parameter evaluation for 384-well assay set-up. MDCK-hNET cells were incubated with 1.5 μM ASP⁺ in the presence of vehicle (A ) or 1 μM Nisoxetine (■) and fluorescent signal measured using a FLIPR (left ordinate axis). Results represent measurements of 6 individual 384-well plates tested on the same day. The right ordinate axis show the variation in corresponding Z'-parameter (T ).

Claims

1. A non-homogeneous whole cell assay method of screening for an inhibitor of the noradrenaline transporter comprising: (a) establishing a whole cell expressing an exogeneous noradrenaline transporter;

(b) treating said cell with a putative inhibitor of said transporter;

(d) measuring activity of said transporter as a function of 4-(4-dimethylaminostyryl)-/V- methylpyridinium accumulation, characterised in that, following step (c), cells are washed to remove 4-(4- dimethylaminostyryl)-Λ/-methylpyridinium not taken up by the cells, thereby determining the effect of the compound to be tested as an inhibitor by the whole cell accumulation of

4-(4-dimethylaminostyryl)-Λ/-methylpyridinium in the absence of background fluorescence and that step (d) is carried out in the absence of a quencher.

2. The non-homogeneous whole cell assay method according to claim 1 , wherein measuring activity at said transporter utilises a fluorescent plate reader.

3. The non-homogeneous whole cell assay method according to claim 1 or claim 2, wherein the whole cell assay is performed in a multi-well plate.

4. The non-homogeneous whole cell assay method according to claim 3, wherein the multi-well plate is a 96-well or 384-well plate.

5. The non-homogeneous whole cell assay method according to claim 4, wherein steps (b) to (d) are automated.