Round Table Discussion

Thoughts on APC assay optimization -

Strengths and weaknesses of APC instruments

Prof. Clemens Möller, PhDAlbstadt-Sigmaringen University of Applied Sciencesoffice@biophysicalconsulting.dewww.clemensmoller.de

Nanion Usergroup Meeting

Sept 29, 2011

· Correlation MPC-APC

· Assay set up

· Flexibility of operation;

primary cells

2

Round Table Discussion

Controlled state of channel

Low binding of

hydrophobic compounds

Low leak currents

Control of membrane potential /

capacitance

Patch-Clampers desire to

tinker with the

experiment

PatchLiner, Port-a-Patch & SyncroPatch data are in excellent correlation to MPC

Continuous voltage control

Chips made of glass

substrateGigaseals RS

compensation

HEKA Software for experiment

and data evaluation

3

Experiments are performed under similar conditions as in MPC

Controlled state of channel

Low binding of

hydrophobic compounds

Low leak currents

Control of membrane potential /

capacitance

Patch-Clampers desire to

tinker with the

experiment

PatchLiner, Port-a-Patch & SyncroPatch data are in excellent correlation to MPC

Continuous voltage control

Chips made of glass

substrateGigaseals RS

compensation

HEKA Software for experiment

and data evaluation

4

Experiments are performed under similar conditions as in MPC

Main difference to MPC: Cells are delivered from suspension (not adherent). Pharmacology? Networks of cells?

Potential(mV)

-100 -50 0

-2

-1

0

1

Peak current(nA)

.

potential (mV)

-140 -120 -100 -80 -60 -40 -20 0 20 40

curr

en

t (n

A)

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

.

Biophysical characterization of hERG channels

5

Manual Patch-Clamp

PatchLiner

Current-voltage relationships of hERG channels correlate very well

Reference: Möller and Witchel (submitted).

6

Excellent correlation between manual and planar patch clamp

· Before employing automated (planar) patch-clamping in our programs, the devices were validated with reference and actual program compounds.

· 57 compounds within one chemical series were tested side by side on manual rigs and a planar patch clamp device (PatchLiner)

· Only 5 out of 57 compounds showed a difference in the IC50-values of ~5-fold

Correlation: Manual Patch-Clamp vs PatchLiner

Manual Patch-Clamp IC50 [µM]

0.1 1 10 100

Pat

chL

iner

IC

50

[µM

]

0.1

1

10

100

5

52

Pharmacological comparison of Electrophysiology Platforms

PatchLiner validation

Reference: Davenport et al., 2010

Key points for pharmacology: same as for MPC and HT systems

o Prepare compound solutions as freshly as possible. Observe solubility of compounds.

o Compound stock solutions required? How long are the compounds stable in DMSO, at which storage temperature?

o Store solutions with reduced vehicle (DMSO) content in glassware, for as short period as possible.

o Are currents stable under negative control conditions? Any vehicle effects?o Do currents reach steady state in presence of compounds? (No continuous

perfusion in APC; repeated cpd administration required?)o Consider pulse protocols (do the compounds exhibit preference for certain

states of the channel?) o Consider temperature effects

7

PatchLiner, Port-a-Patch & SyncroPatch data are in excellent correlation to MPC

(Many) Port-A-Patch, PatchLiner and SyncroPatch assays are easy to set up

o Operation in standard modes easy

o Very "forgiving" cell cultureo Good seal success rates can be achieved with

suboptimal cells

o But: Seal enhancer (for most cell types) appears to be required for good success rates?

8

Eccellent seal success rates

Kv1.5

HERG

1st tier profilingExample: Panel of cardiac channels on PL

Kv1.1

Kv4.3/KChIP2

NaV1.5

L-type Ca2+

Standard protocols for most cell types and channels are available. For many cells, excellent success ratescan be achieved.

Current traces from Möller et al., 2010

(Many) Port-A-Patch, PatchLiner and SyncroPatch assays are easy to set up

o The healthier the cells are, the better the seal success rate will typically beo Cell confluency ~60-80%. Can depend on cell type o Especially small / large cells? Consider different chip hole sizeo Cell density appears to be not so critical (1 x 106 – 5 x 107 cells/ml are good standard

densities, but much lower densities have worked fine for some cells)o Relatively small effect of pressure etc. settings in PatchControl software; standard

settings are often a good choice

9

Key points to consider for a good seal success rate

APC instruments complement each other

In addition, the Port-A-Patch proved very useful foro Assay development support for PatchLiner and SyncroPatcho Verification of data for compounds that showed inconsistent IC50 values on

the PatchLiner or the SyncroPatch

10

Instruments for different needs of throughput

Port-a-Patch PatchLiner SyncroPatch

APC instruments are highly flexible

11

Recordings from primary cells possible

300 nM Haloperidol

Negative control

Neurons – MAP2astrocytes – GFAP

Nuclei – DRAQ5

Neurons Cardiomyocytes

Na+

Ca2+

K+

Reference: Möller et al., 2010

Modes of operation

12

Excised patch recordings not (yet, really) possible by planar APC

"Whole-

cell"

• Most widely used for pharmacology

"Cell-

attached"

• Possible with some cells

"excised patch"

• Are you missing single channel recordings by APC? For MoA?

??

o Different features availableo Voltage clamp, current clamp (action potential recordings)o Whole cell, perforated patch (are you using this a lot?)o Intracellular solution exchangeo Fast ligand exchange (~50 ms)o Temperature control

o Interaction during experiment possible

o Patch-Clampers desire to "play around" with an experimento Also, a "screening mode" with limited user access to settings is possible

(Talk by Corinna from last years meeting)

Different features available

APC instruments are highly flexible

13

Many features available; interaction possible

Interaction during experiment possible

Thank you for your attention and input!

Andreas EbnethRainer NetzerHeike DeisemannDesireé AmmYork RudhardJohn Kemp

The whole great team, especially:Niels, Andrea, Michael,Claudia, Sonja, Timo, Ali &Cecilia

Ralf Kettenhofen

Martin Stolz

Prof. Clemens Möller, PhD | Albstadt-Sigmaringen University of Applied Sciencesclemens@biophysicalconsulting.de | www.clemensmoller.de

14

Carsten Claussen

Clemens Möller | Albstadt-Sigmaringen University of Applied Sciences

office@biophysicalconsulting.de | www.clemensmoller.de

References & Recommended Reading

15

References:

- Clemens Möller (2010). Keeping the Rhythm: hERG and beyond in Cardiovascular Safety Pharmacology. Expert Reviews Clinical

Pharmacology (Ion Channels) 3: 3. 321-329 May

- Adam J Davenport, Clemens Möller, Alexander Heifetz, Michael P Mazanetz, Richard J Law, Andreas Ebneth, Mark J Gemkow

(2010). Using Electrophysiology and in silico 3D Modelling to reduce hERG inhibition in a Histamine H3 Receptor Antagonist Program.

ASSAY and Drug Development Technologies 8: 6. 781-789 December

- Clemens Möller, Mark Slack (2010). Impact of new technologies for cellular screening along the drug value chain. Drug Discovery

Today 15: 9-10. 384-390 May

- Clemens Möller, Harry Witchel. Automated Electrophysiology Makes the Pace for Cardiac Ion Channel Safety Screening. (Submitted to

Frontiers, 2011)

Recommended reading:

Carol J Milligan, Li J, Sukumar P, Majeed Y, Dallas ML, English A, Emery P, Porter KE, Smith AM, McFadzean I, Beccano-Kelly D,

Bahnasi Y, Cheong A, Naylor J, Zeng F, Liu X, Gamper N, Jiang LH, Pearson HA, Peers C, Robertson B, Beech DJ (2009). Robotic

multiwell planar patch-clamp for native and primary mammalian cells. Nat Protoc. 2009;4(2):244-55.