Transporter:
Na/amino acid
Sample:Plasma membraneOrigin:
CHO cellsTools:
SURFE2R 96SE

Application Note

Secondary screening assay for amino acidtransporter inhibitors on the SURFE2R 96SE
A. Pommereau and T. Licher, Sanofi, Integrated Drug Discovery, Germany.
The electrophysiology team at Nanion Technologies GmbH, Munich.
Background

Substrate specificity and affinity

Na-dependent amino acid transporters play an essen-

To investigate the substrate affinity, a neutral amino acidtial role in the uphill transport of amino acids across thewas added in different concentrations to the sensor con-plasma membrane. In this study, a novel transporter

taining the membrane sample (Figure 2). A KM of 1.75

target was used for which few specific pharmacologicalmM was determined. A comparison with other methods

tools are available. The aim of this study was to establish(FLIPR, Uptake, TEVC) revealed a high correlation of thea secondary screening assay using the direct electro-KM. Furthermore, amino acid specificity was investigated,

physiological measurement of the transporter to identify

revealing a preference of the transporter for neutral

inhibitors. Using the SURFE2R 96SE, substrate specificity wasamino acids.
A50 msboth Na and an amino acid are present.
++ Na+, + aa+ Na+, + aa (WT)

50 ms+/- Na+/- aa
Figure 1: Typical current responses of CHO cell membranes containing theamino acid transporter or WT membranes (no transporter) recorded on
the SURFE2R 96SE. Only in the presence of both Na+ and an amino acid apositive current peak occurs in membranes containing the transporter. In
the absence of either Na+ or the amino acid the transporter is not active. InWT membranes (no transporter) no current is observed in the presence ofNa+ and an amino acid.

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CD

10.8

EC50=1.75 mM
Hill=10.6
0.4
0.2
010-5 10-4 10-3 10-2 10-1
Neutral aa [M]

1.2
KM [mM]

1.0

Applied method

0.8

1.75
SURFE R
FLIPR
0.5
Radioactive uptake 1.2
Two electrode VC 1.1
2

0.6
0.4
0.2
Gly
Asp
ProGluLysArg
HisThrSer
Trp
Asn
Gln
PheAlaVal
IleCysLeu
Met+ Na , - aa+pC AA / pCNeutral aa

100 pA- Na+, + aaB
Normalized current

shows typical current responses of the transporter when

100 pA

determined and inhibitors could be identified. Figure 1

AAFigure 2: A Raw current traces of an active sensor, showing the increasing
current peak with increasing substrate concentration. B The concentration
response curve fitted with a Hill equation reveals an EC50 of 1.75 mM. C The
current peak height generated by different amino acids normalized to aneutral amino acid reveals that neutral amino acids are preferred as asubstrate. D The KM of a neutral amino acid obtained using the SURFE2R 96SE
is in good agreement with other methods.

Application Note
Secondary screening for inhibitors

Success rate and stability

Finally, the SURFE2R 96SE was used in a secondary screen-

An investigation into reproducibility revealed that in aing assay to characterize and confirm compound hits

typical experiment 98% of the sensors in a 96 well plate

generated in a primary screen. In order to first validate

met the quality criteria and generated current signals

the assay a known compound was used. A time depen-

between 200 and 500 pA. Using 10 µM of the known

dence of inhibition intensity was observed. The longer theinhibitor led to 40-50% inhibition of the signal in 94% of wells

sensor was incubated with the compound, the higherand over a series of 5 plates the overall success rate was

the percentage of inhibition. Using a workflow with a 30

approximately 90% in terms of baseline current, maximum

min incubation step resulted in an IC50 of 12.9 µM, whichcurrent, signal to noise ratio and stability of inhibition (Fig-correlates with the IC50 generated using a mass spec-ure 4). The SURFE2R 96SE is, therefore, an excellent tool fortrometry assay (MS-Assay; 12.2 µM). Using this secondary

secondary screening of compounds on transporters.

screening assay it was possible to measure 12 concentra-

A 5x10-11

tion response curves with IC50 values per day (n = 4) using
6 SURFE2R 96SE sensors. The assay was further validated

mined with the SURFE2R assay with those generated using

an MS-Assay. The correlation curve is shown in Figure 3with a correlation factor of 0.84 which confirms the valid-4x10-11Charge (As)by comparing the IC50s of 62 selected compounds deter-

ity of the SURFE2R 96 SE assay.

0.01

0.1
110Concentration [µM]

C0.01
0.1
110MS-Assay IC50 (µM)

individual wells

B

Applied method IC50 [µM]
SURFE2R

12.2MS-assay

12.9

Figure 3: A Correlation curve between IC50s obtained using the SURFE2R 96SE
and MS-Assay. The correlation factor of 0.84 indicates excellent correlation
between the two assays. B Concentration response curve reveals an IC50 =12.9 µM. C Comparison of the IC50 values show good agreement between
the two methods.

C4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.50.0181614121086420

S/N factor

0.10123# plate45low Medianhigh Median
S/N

Inhibition (%)

Inhibition [%]SURFE2R IC50 (µM)

1IC50=12.5 µM

605040302010055120451003580

2560

1540
205-51234# plate5Success rate (%)

B 70
102x10-11
1x10-11pC (pAs)

A3x10-110

Inhibition tool
Inhibition neg control
Success rate

Figure 4: Stability of current signal and inhibition. A Transported charge perwell compared within a 96 well plate. Total success rate 98%. B Current
response (charge) compared between different 96 well plates. Values
compared: Maximum signal, baseline current and signal to noise ratio. C
Total success rate and reproducibility of inhibition compared between 5plates.

Methods

Acknowledgement

The target protein was stably expressed in CHO cells

The SURFE2R 96SE assay was developed and validated byand the cell membrane was isolated and frozen inAntje Pommereau and Dr. Thomas Licher. All data sets

aliquots. On the day of the experiment the membrane

were kindly provided by Sanofi Germany.

Experimental setup

was added to a SURFE2R 96SE sensor plate. To evoke
protein specific current activity a suitable amino acidwas added to the sensor in the presence of a Na+containing assay buffer. The co-transport of the aminoacid together with Na+ generated a positive current signal.
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