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Article: Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for Nav1.5 peak current in a high throughput screening environment

Rotordam, Maria Giustina / Obergrussberger, Alison / Brinkwirth, Nina / Takasuna, Kiyoshi / Becker, Nadine / Horváth, András / Goetze, Tom A. / Rapedius, Markus / Furukawa, Hatsue / Hasegawa, Yuka / Oka, Takayuki / Fertig, Niels / Stoelzle-Feix, Sonja

Journal of pharmacological and toxicological methods. 2021 Nov., Dec., v. 112

2021

Abstract: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC₅₀s of compounds on NaV1.5 peak ...

Abstract	For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC₅₀s of compounds on NaV1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices.APC instruments SyncroPatch 384 and Patchliner were used to record NaV1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (−80 mV or − 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC₅₀ values on hNaV1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures.The IC₅₀s of 18 compounds on the NaV1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC₅₀s but voltage protocol and holding potential did cause differences in IC₅₀ values. Temperature can affect Vₕₐₗf of inactivation and also compound potency. A compound incubation time of 5–6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect.We conclude that holding potential, voltage protocol and temperature can affect IC₅₀ values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record NaV1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC₅₀s.
Keywords	automation ; drugs ; electric potential difference ; risk ; temperature
Language	English
Dates of publication	2021-11
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1105919-9
ISSN	1873-488X ; 1056-8719
ISSN (online)	1873-488X
ISSN	1056-8719
DOI	10.1016/j.vascn.2021.107125
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Article ; Online: Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for Nav1.5 peak current in a high throughput screening environment.

Rotordam, Maria Giustina / Obergrussberger, Alison / Brinkwirth, Nina / Takasuna, Kiyoshi / Becker, Nadine / Horváth, András / Goetze, Tom A / Rapedius, Markus / Furukawa, Hatsue / Hasegawa, Yuka / Oka, Takayuki / Fertig, Niels / Stoelzle-Feix, Sonja

Journal of pharmacological and toxicological methods

2021 Volume 112, Page(s) 107125

Abstract: Introduction: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including Na: Methods: APC instruments SyncroPatch 384 and Patchliner were used to record Na: ... ...

Abstract	Introduction: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including Na Methods: APC instruments SyncroPatch 384 and Patchliner were used to record Na Results: The IC Discussion: We conclude that holding potential, voltage protocol and temperature can affect IC
MeSH term(s)	Animals ; CHO Cells ; Cardiac Conduction System Disease/diagnosis ; Cricetinae ; Cricetulus ; Drug Discovery ; High-Throughput Screening Assays ; NAV1.5 Voltage-Gated Sodium Channel ; Patch-Clamp Techniques
Chemical Substances	NAV1.5 Voltage-Gated Sodium Channel
Language	English
Publishing date	2021-09-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	1105919-9
ISSN	1873-488X ; 1056-8719
ISSN (online)	1873-488X
ISSN	1056-8719
DOI	10.1016/j.vascn.2021.107125
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Article: There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments.

Rapedius, Markus / Obergrussberger, Alison / Humphries, Edward S A / Scholz, Stephanie / Rinke-Weiss, Ilka / Goetze, Tom A / Brinkwirth, Nina / Rotordam, Maria Giustina / Strassmaier, Tim / Randolph, Aaron / Friis, Søren / Liutkute, Aiste / Seibertz, Fitzwilliam / Voigt, Niels / Fertig, Niels

Frontiers in molecular neuroscience

2022 Volume 15, Page(s) 982316

Abstract: Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used ... ...

Abstract	Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (Na
Language	English
Publishing date	2022-08-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2452967-9
ISSN	1662-5099
ISSN	1662-5099
DOI	10.3389/fnmol.2022.982316
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Article ; Online: The suitability of high throughput automated patch clamp for physiological applications.

Obergrussberger, Alison / Rinke-Weiß, Ilka / Goetze, Tom A / Rapedius, Markus / Brinkwirth, Nina / Becker, Nadine / Rotordam, Maria Giustina / Hutchison, Laura / Madau, Paola / Pau, Davide / Dalrymple, David / Braun, Nina / Friis, Søren / Pless, Stephan A / Fertig, Niels

The Journal of physiology

2021 Volume 600, Issue 2, Page(s) 277–297

Abstract: Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large ... ...

Abstract	Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on Na
MeSH term(s)	Drug Discovery ; High-Throughput Screening Assays ; Ion Channels ; Myocytes, Cardiac ; Patch-Clamp Techniques
Chemical Substances	Ion Channels
Language	English
Publishing date	2021-10-08
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3115-x
ISSN	1469-7793 ; 0022-3751
ISSN (online)	1469-7793
ISSN	0022-3751
DOI	10.1113/JP282107
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Article ; Online: Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore.

Seyedmohammad, Saeed / Fuentealba, Natalia Alveal / Marriott, Robert A J / Goetze, Tom A / Edwardson, J Michael / Barrera, Nelson P / Venter, Henrietta

Bioscience reports

2016 Volume 36, Issue 2

Abstract: Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an ... ...

Abstract	Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel.
MeSH term(s)	Bacterial Proteins/chemistry ; Cation Transport Proteins/chemistry ; Models, Molecular ; Protein Domains ; Protein Structure, Quaternary ; Pseudomonas aeruginosa/chemistry
Chemical Substances	Bacterial Proteins ; Cation Transport Proteins
Language	English
Publishing date	2016
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	764946-0
ISSN	1573-4935 ; 0144-8463
ISSN (online)	1573-4935
ISSN	0144-8463
DOI	10.1042/BSR20160046
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Article ; Online: α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors adopt different subunit arrangements.

Balasuriya, Dilshan / Goetze, Tom A / Barrera, Nelson P / Stewart, Andrew P / Suzuki, Yuki / Edwardson, J Michael

The Journal of biological chemistry

2013 Volume 288, Issue 30, Page(s) 21987–21998

Abstract: Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers ... ...

Abstract	Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.
MeSH term(s)	Amino Acid Sequence ; Animals ; Cell Line ; Fluorescent Antibody Technique ; Humans ; Microscopy, Atomic Force ; Models, Molecular ; Molecular Sequence Data ; Protein Multimerization ; Protein Subunits/chemistry ; Protein Subunits/genetics ; Protein Subunits/metabolism ; Rats ; Receptors, AMPA/chemistry ; Receptors, AMPA/genetics ; Receptors, AMPA/metabolism ; Receptors, N-Methyl-D-Aspartate/chemistry ; Receptors, N-Methyl-D-Aspartate/genetics ; Receptors, N-Methyl-D-Aspartate/metabolism ; Transfection
Chemical Substances	NMDA receptor A1 ; Protein Subunits ; Receptors, AMPA ; Receptors, N-Methyl-D-Aspartate ; glutamate receptor ionotropic, AMPA 2 (P6W5IXV8V9) ; glutamate receptor ionotropic, AMPA 1 (TFZ3H25BS1) ; N-methyl D-aspartate receptor subtype 2A (VH92ICR8HX)
Language	English
Publishing date	2013-06-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M113.469205
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Article: A general procedure to select calibration drugs for lab-specific validation and calibration of proarrhythmia risk prediction models: An illustrative example using the CiPA model

Journal of pharmacological and toxicological methods. 2020 Sept., v. 105

2020

Abstract: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to ... ...

Abstract	In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet.The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction.The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation).This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.
Keywords	calibration ; data collection ; model validation ; models ; prediction ; risk ; toxicology
Language	English
Dates of publication	2020-09
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1105919-9
ISSN	1873-488X ; 1056-8719
ISSN (online)	1873-488X
ISSN	1056-8719
DOI	10.1016/j.vascn.2020.106890
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Article ; Online: An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery.

Obergrussberger, Alison / Goetze, Tom A / Brinkwirth, Nina / Becker, Nadine / Friis, Søren / Rapedius, Markus / Haarmann, Claudia / Rinke-Weiß, Ilka / Stölzle-Feix, Sonja / Brüggemann, Andrea / George, Michael / Fertig, Niels

Expert opinion on drug discovery

2018 Volume 13, Issue 3, Page(s) 269–277

Abstract: Introduction: Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the ... ...

Abstract	Introduction: Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the same device without the need for months of training and experience. APC devices are being developed to increase throughput and flexibility. Areas covered: Experimental options such as temperature control, internal solution exchange and current clamp have been available on some APC devices for some time, and are being introduced on other devices. A comprehensive review of the literature pertaining to these features for the Patchliner, QPatch and Qube and data for these features for the SyncroPatch 384/768PE, is given. In addition, novel features such as dynamic clamp on the Patchliner and light stimulation of action potentials using channelrhodosin-2 is discussed. Expert opinion: APC devices will continue to play an important role in drug discovery. The instruments will be continually developed to meet the needs of HTS laboratories and for basic research. The use of stem cells and recordings in current clamp mode will increase, as will the development of complex add-ons such as dynamic clamp and optical stimulation on high throughput devices.
MeSH term(s)	Animals ; Drug Design ; Drug Discovery/methods ; High-Throughput Screening Assays/methods ; Humans ; Ion Channels/metabolism ; Patch-Clamp Techniques/methods
Chemical Substances	Ion Channels
Language	English
Publishing date	2018-01-17
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	2259618-5
ISSN	1746-045X ; 1746-0441
ISSN (online)	1746-045X
ISSN	1746-0441
DOI	10.1080/17460441.2018.1428555
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Article ; Online: A general procedure to select calibration drugs for lab-specific validation and calibration of proarrhythmia risk prediction models: An illustrative example using the CiPA model.

Journal of pharmacological and toxicological methods

2020 Volume 105, Page(s) 106890

Abstract: Introduction: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development ... ...

Abstract	Introduction: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet. Methods: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction. Results: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation). Discussion: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.
MeSH term(s)	Arrhythmias, Cardiac/chemically induced ; Biological Assay/methods ; Calibration ; Drug Evaluation, Preclinical/methods ; Drug-Related Side Effects and Adverse Reactions/prevention & control ; Electrocardiography/methods ; Humans ; Myocytes, Cardiac/drug effects ; Pharmaceutical Preparations/administration & dosage
Chemical Substances	Pharmaceutical Preparations
Language	English
Publishing date	2020-06-21
Publishing country	United States
Document type	Journal Article
ZDB-ID	1105919-9
ISSN	1873-488X ; 1056-8719
ISSN (online)	1873-488X
ISSN	1056-8719
DOI	10.1016/j.vascn.2020.106890
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Article ; Online: Direct evidence for functional TRPV1/TRPA1 heteromers.

Fischer, Michael J M / Balasuriya, Dilshan / Jeggle, Pia / Goetze, Tom A / McNaughton, Peter A / Reeh, Peter W / Edwardson, J Michael

Pflugers Archiv : European journal of physiology

2014 Volume 466, Issue 12, Page(s) 2229–2241

Abstract: Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient ...

Abstract	Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.
MeSH term(s)	Binding Sites ; Calcium Channels/chemistry ; Calcium Channels/metabolism ; Capsaicin/pharmacology ; HEK293 Cells ; Humans ; Ion Channel Gating ; Nerve Tissue Proteins/agonists ; Nerve Tissue Proteins/chemistry ; Nerve Tissue Proteins/metabolism ; Protein Binding ; Protein Multimerization ; TRPA1 Cation Channel ; TRPV Cation Channels/agonists ; TRPV Cation Channels/chemistry ; TRPV Cation Channels/metabolism ; Transient Receptor Potential Channels/agonists ; Transient Receptor Potential Channels/chemistry ; Transient Receptor Potential Channels/metabolism
Chemical Substances	Calcium Channels ; Nerve Tissue Proteins ; TRPA1 Cation Channel ; TRPA1 protein, human ; TRPV Cation Channels ; TRPV1 protein, human ; Transient Receptor Potential Channels ; Capsaicin (S07O44R1ZM)
Language	English
Publishing date	2014-03-19
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	6380-0
ISSN	1432-2013 ; 0031-6768
ISSN (online)	1432-2013
ISSN	0031-6768
DOI	10.1007/s00424-014-1497-z
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