Developing a robust screening assay for acid …...Developing a robust screening assay for acid sensing ion channel 1a using Biacore systems Hamno Kadar, Markku Hämäläinen, and

Developing a robust screening assay for acid sensing ion channel 1a using Biacore™ systems Hamno Kadar, Markku Hämäläinen, and Ewa Pol GE Healthcare Bio-Sciences, Björkgatan 30, SE-75184 Uppsala, Sweden

GE and GE monogram are trademarks of General Electric Company. Biacore is trademark of General Electric Company or one of its subsidiaries. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.© 2014 General Electric Company – All rights reserved. First published in May. 2014.GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK.

www.gelifesciences.com Correspondence should be addressed to: [email protected]

Acknowledgements: We thank F. Hoffmann-La Roche Ltd., Basel, Switzerland, for ASIC1a, Psalmotoxin and 47 LMW compounds.

29-1051-60 AA 05/2014

Membrane proteins are important drug targets

Goal: Test binding activity of membrane protein that was captured directly from cell extract, during the time that is needed for screen, by repetitive injection of reference/control compound over time in the absence of LMW compounds.

Goal: Select potential hits

Single concentration (100 µM) screen against target membrane proteins

Goal: Select high affi nity binders

Concentration series (6 to 100 µM) screen against target membrane protein

Goal: Cleaning the library from sticky compounds prior to binding level screen.

Identify an appropriate target for prescreen by performing screen (at least in duplicate) against capturing antibodies, dummy proteins, sensor surfaces, and liposomes, and comparing observed binding pattern with stickiness to membrane proteins.

• Easy capture directly from cell extract using tag, with < 5% background on reference and active surfaces (when tested with membrane protein-negative cell extract).

• Binding activity was retained during ~ 24 h, as shown by similar sensorgram shapes, affi nity (~ 2 nM) and response levels of Psalmotoxin.

• Deviating affi nity of Psalmotoxin to ASIC1a at time “0” is likely to depend on conformational change of ASIC1a induced by the fi rst binding occurrence.

• Control sample (here: Psalmotoxin) gives important information about the binding activity of membrane protein throughout the screen, and the “adjustment for controls” software functionality allows compensation for a decay of binding responses over time.

• Reinjection of fresh membrane protein and regeneration of surface was not needed after the stickiness cleaning.

• Screen was performed in duplicate and on two sensor surfaces, CM4 and CM5, showing a good correlation between responses on two types of sensor surface.

• 26 compounds, with binding responses above cut-off, were selected to affinity screen.

Preferably, the binding level and affinity screens should be performed with a reference protein being the target with an impaired binding site of interest (blocking, mutation), to exclude nonspecific binders.

Apparent affinity ≤ 50 µM (half value of the highest concentration used).Uncertain estimated affinity > 50 µM. Repeat affinity measurements with concentrations > 2 × K

D,estimated.Uncertain estimated affinity » 50 µM. Fit was performed using a modified steady-state model, in which the maximal response of a compound was calculated from the response of control sample and “locked”.

Binding of Psalmotoxin ASIC1a

12 out of 26 compounds were the confirmed ASIC1a binders Control samples

Screen on CM4 and CM5 sensor surfaces

Membrane protein screen process: A structured way to find high-quality leads

Experimental steps

The problem: “sticky” LMW compounds inactivate ASIC1a

Maximize information per experiment in a Biacore T200

How to identify sticky compounds?• Irregular shape of residual binder with slow or no dissociation.

• Stability report point gives the fi rst indication of stickiness.

What do the compounds stick to?Sensor surfaces? → Test the binding to sensor surfaces CM5, CM4, and L1

Capture molecules? → Test the binding to an anti-C9 Ab (1D4) and anti-His Ab

Membrane protein? → Test the binding to a control membrane protein, from GPCR family

Any protein? → Test the binding to “dummy” proteins, p38α MAP kinase and thrombin

Liposomes? → Test the binding to POPC and POPC:POPS liposomes

Use a set of 47 compounds, molecular weight (Mr) ~ 300 to 700

ReferencesDawson, R. J. P. et al. Nature Communications 3, article number 936 (2012). Rich, R. L. et al. Anal. Biochem. 409(2), 267–272 (2011).Navratilova, I. et al. Anal. Biochem. 355, 132–139 (2006).Navratilova, I. et al. Anal. Biochem. 339, 271–281 (2005).Rich, R. L. et al. Anal. Biochem. 386, 98–104, (2009).Karlsson, Å. F. et al. J. Pharm. Sci. 94, 25–37 (2005).

Conclusions• A single capture of solubilized ASIC1a from cell extract gave a

stable surface for ~ 20 h with minimal background.

• A prescreen against dummy proteins can clean a library from sticky compounds and secure the binding activity of membrane protein throughout the entire screen.

• High sensitivity detection together with steady-state models that can handle suboptimal concentrations and allow early prioritization of the LMW compounds in Biacore T200.

”Binding” report point – to fi nd potential binders”Stability” report points – to fi nd potential sticky compounds

Tag

Tag

Tag

+Detergentmicelles

Mixedmicelles

Liposomes

Anti-tag capturing antibody

Sensor surface CM5 or CM4

LMW compounds

Ion channel

Solubilization Capture Binding analysis

GPCRAcid sensing ion channel 1a (ASIC1a)

Memory, pain, taste, fear Immune system

Cysteine-cysteine chemokine receptor 5 (CCR5)

Membrane protein in lipid bilayer Confirm activityScreen compounds

Solubilized membrane protein is captured directley from cell extract

Tag

Tag

Tag

+Detergentmicelles

Mixedmicelles

Liposomes

Anti-tag capturing antibody

Sensor surface CM5 or CM4

LMW compounds

Ion channel

Solubilization Capture Binding analysis

GPCRAcid sensing ion channel 1a (ASIC1a)

Memory, pain, taste, fear Immune system

Cysteine-cysteine chemokine receptor 5 (CCR5)

Membrane protein in lipid bilayer Confirm activityScreen compounds

Solubilized membrane protein is captured directley from cell extract

Nonsticky Sticky Sticky

Resp

onse

(0=b

asel

ine)

RU

Time (s)30 60 90 120 150 180

55

45

35

25

15

5

-5 Resp

onse

(0=b

asel

ine)

RU

Time (s)50 50 100 150 200

290

230

170

110

50

-10

Binding

Binding

Stability

Stability

Resp

onse

(0=b

asel

ine)

RU

Time (s)50 50 100 150 200

90

70

50

30

10

-10

Binding

Stability

Screen against sensor surfaces, ”dummy” and capturing proteins, and target membrane protein

Screen against membrane proteins and reference (capturing) proteins

Screen against several types of liposomes and reference

Nonspecific binding/stickiness to sensor surface

fc1

Stickiness or specific binding to dummy protein

fc2

Nonspecific binding/stickiness to capturing antibody

fc3

Stickiness and specific binding to membrane protein

fc4


fc1


fc2


fc3


fc4

Capturing antibody

Membrane protein

thrombin or p38α MAP kinase

anti-His Ab 1D4 Ab

C9-tagged GPCR


fc1

Binding/stickiness to liposomes

fc2


fc3

Binding/stickiness to liposomes

fc4

POPC:POPSPOPC

Sensor Lsurface CM4 or CM

5Sensor Lsurface CM

4 or CM5

Sensor surface L1

Histidine-taggedASIC1a

ASIC1a (-) ~100 RU

ASIC1a (+) ~3000 RU

+

Resp

onse

(0=c

aptu

re_b

asel

ine)

RU

Time (s)

2000 400 800 1000 1200600 1400

12 000

10 000

8000

6000

4000

2000

0

-2000

fc1 fc2

anti-His Ab

Histidine-taggedASIC1a

Psalmotoxin

ASIC1a capture

Sensorgram

Resp

onse

(RU

)

Time (s)

Bindingresponses

100 300 500 700 900

1350

1300

1250

1200

1150

1100

2–4 h1–time3–8 h4–12 h5–16 h6–20 h7–24 h

Binding response plot

Rela

tive

resp

onse

, bin

ding

(RU

)

Cycle

Bindingresponses

200 40 60 80 100 120 140 160 180

120

100

80

60

40

20

0

-20

PsalmotoxinBuffer

Aligned and normalized sensorgrams

Resp

onse

(RU

)

Time (s)100 300 500 700 900

100

60

20

-20

1–time 0

1

Time 0 4 h 8 h 12 h 16 h 20 h 24 h

2 3 4 5 6 7

Resp

onse

(0=b

asel

ine)

RU

Time (s)30 60 90 120 150 180

55

45

35

25

15

5

-5

Binding

Stability

evaluate ”stability”report point

Controls, binding

Rela

tive

resp

onse

,bi

ndin

g (R

U)

Sample

180

130

80

30

-200 20 40 60 80 100 120 140 160

Controls, adjusted binding

Rela

tive

resp

onse

,bi

ndin

g. a

djus

ted

(RU

)

Sample

120100

80604020

0-20

0 20 40 60 80 100 120 140 160

Controls on CM5Controls on CM4

Positive controls

Negative controls

Controls on CM5Controls on CM4

ASIC1a screen

Rela

tive

resp

onse

, bin

ding

(RU

)

Sample

90

70

50

30

10

-10

Cut off = Ave. of negative controls + 6×SDCM4CM5

Resp

onse

(0=b

asel

ine)

RU

Time (s)30 60 90 120 150 180

55

45

35

25

15

5

-5

Binding

Stability

evaluate ”binding”report point

Resp

onse

(RU

)

Time (s)-20 0 20 40 60 80

20

15

10

5

0

-5

plot ”binding” report points against concentrations

Compounds sticky to membrane proteins (shown in red, in table) were found to be sticky to other proteins tested and to sensor surfaces. Different stickiness pattern was observed to liposomes.

Absence of ASIC1a in knock-out mouse affects a number physiological responses, such as memory, taste and emotions of fear and pain.

Thrombin

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

70

50

30

10

-10

21

37

34

1D4 antibody

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

90

70

50

30

10

-10

21

37

34

p38α MAP kinase

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

20

15

10

5

0

-5

2137

2634

anti-His antibody

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

6

5

4

3

2

1

0

-1

2137

37Sensor surface CM4

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

8

6

4

2

0

-2

Sensor surface L1

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

40

30

20

10

0

-10

21

37

4

Sensor surface CM5

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

25

20

15

10

5

0

-5

37

393–7

POPC:POPS liposomes

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

160

120

80

40

0

42

45

POPC liposomes

Rela

tive

resp

onse

, sta

bilit

y (R

U)

Sample

140

100

60

20

-20

42

39 45

3–7

Proteins Sensor surface Liposomes

ASIC1a 21

Rela

tive

resp

onse

,st

abili

ty (R

U)

Sample

100

60

20

-20

37

34

GPCR (used as a reference membrane protein)21

Rela

tive

resp

onse

,st

abili

ty (R

U)

Sample

140

100

60

20

-20

37

34

-50 0

50 100 150 200 250

40 60 80 100 140 180

RU

(s)Time

21 34 37

-40 0 40 80 120

40 60 80 100 140 180

RU

(s)Time

213437

ASIC1a 21

Rela

tive

resp

onse

,st

abili

ty (R

U)

Sample

100

60

20

-20

37

34

GPCR (used as a reference membrane protein)21

Rela

tive

resp

onse

,st

abili

ty (R

U)

Sample

140

100

60

20

-20

37

34

-50 0

50 100 150 200 250

40 60 80 100 140 180

RU

(s)Time

21 34 37

-40 0 40 80 120

40 60 80 100 140 180

RU

(s)Time

213437

Capture from cell extract and confirmation of binding activity

Affinity screen Binding level screen

“Stickiness” screen

Documents

Developing a robust screening assay for acid …...Developing a robust screening assay for acid sensing ion channel 1a using Biacore systems Hamno Kadar, Markku Hämäläinen, and