Combinatorial
Chemistry & High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 12, Number 1, January 2009
Contents
Ion Channels: Applications in Ion Channel Drug Discovery
Guest Editor: Douglas S. Krafte
Editorial Pp. 1
Evolution of the Human Ion Channel Set Pp. 2-23
Timothy J. Jegla, Christian M. Zmasek, Serge
Batalov and Surendra K. Nayak
[Abstract]
[Full
text article]
[PMID: 19149488 PubMed - indexed for MEDLINE]
Port-a-Patch and Patchliner: High Fidelity
Electrophysiology for Secondary Screening and Safety
Pharmacology Pp. 24-37
Cecilia Farre, Alison Haythornthwaite, Claudia Haarmann, Sonja
Stoelzle, Mohamed Kreir, Michael George, Andrea Brüggemann
and Niels Fertig
[Abstract]
[Full
text article] [PMID:
19149489 PubMed - indexed for MEDLINE]
High Throughput Electrophysiology with
Xenopus Oocytes Pp. 38-50
Roger L. Papke and Cathy Smith-Maxwell
[Abstract]
[Full
text article] [PMID:
19149490 PubMed - indexed for MEDLINE]
Automated Planar Electrode Electrophysiology
in Drug Discovery: Examples of the Use of QPatch in Basic
Characterization and High Content Screening on Nav,
KCa2.3, and Kv11.1
Channels Pp. 51-63
Mads P.G. Korsgaard, Dorte Strøbæk
and Palle Christophersen
[Abstract]
[Full
text article]
[PMID:
19149491 PubMed - indexed for MEDLINE]
HCN Channels as Targets for Drug Discovery
Pp. 64-72
Michael P. Maher, Nyan-Tsz Wu, Hong-Qing
Guo, Adrienne E. Dubin, Sandra R. Chaplan and Alan
D. Wickenden
[Abstract]
[Full
text article]
[PMID:
19149492 PubMed - indexed for MEDLINE]
The Chiptip: A Novel Tool for Automated
Patch Clamp Pp. 73-77
Albrecht Lepple-Wienhues, Dirck Lassen,
Alexander Hümmer, Uwe Czubayko, Martina Knirsch and
Andjelko Golubovic
[Abstract]
[Full
text article] [PMID:
19149493 PubMed - indexed for MEDLINE]
QPatch: The Missing Link Between HTS
and Ion Channel Drug Discovery Pp. 78-95
Chris Mathes, Søren Friis, Michael
Finley and Yi Liu
[Abstract]
[Full
text article] [PMID:
19149494 PubMed - indexed for MEDLINE]
Use of Planar Array Electrophysiology
for the Development of Robust Ion Channel Cell Lines Pp.
96-106
Jeffrey J. Clare, Mao Xiang Chen, David
L. Downie, Derek J. Trezise and Andrew J. Powell
[Abstract]
[Full
text article] [PMID:
19149495 PubMed - indexed for MEDLINE]
Sodium Channel Inhibitor Drug Discovery
Using Automated High Throughput Electrophysiology Platforms
Pp. 107-122
Neil Castle, David Printzenhoff, Shannon
Zellmer, Brett Antonio, Alan Wickenden and Christopher
Silvia
[Abstract]
[Full
text article] [PMID:
19149496 PubMed - indexed for MEDLINE]
Meet
the Guest Editor Pp. 123
Abstracts
[Back to top]
Editorial:
Ion channel proteins are fascinating molecules that
play key roles in many physiological processes. In fact, no
mammalian cell type has been identified which does not express
a complement of ion channel proteins. However, while all cell
types express ion channels, the specific role these channels
play can vary significantly. In excitable cells such as neurons,
skeletal muscle and heart, ion channels act as molecular switches
to either trigger excitation or reset the membrane potential
to a baseline, resting level depending on which type of ion
channel is activated. In non-excitable cells, channels can
play other roles such as regulation of fluid movement, salt
secretion or resorption, and triggering of signal transduction
cascades that eventually lead to gene transcription.
Over the past several years, there has been an explosion of
data elucidating the role of specific channels in genetic
disorders due to mutations which typically affect gating and/or
expression. These ‘channelopathies’ further validate
the role various ion channels play in maintaining normal physiological
processes. The genetic data also suggest that modulation of
ion channel function is a good means to intervene in pathophysiological
processes and restore normal function. One good example of
the genetics leading to a greater understanding of the role
for a particular channel is a series of mutations in Kv7 channels
that cause a neonatal seizure disorder [1]. The genetic data
highlight the key role of these channels in regulating CNS
excitability, and since decreases in channel expression lead
to seizures activation of Kv7 channels may be a useful therapeutic
approach to treating epilepsy [2]. Many other examples in
a variety of diseases or disorders also exist including pain,
myotonias and arrhythmia [3-5]. Channelopathies as a whole
have been nicely reviewed by Ashcroft [6].
Given the key role ion channels play in health and disease
and the wealth of new data validating various ion channel
subtypes as possible therapeutic drug discovery targets, there
has been something of a resurgence in ion channel drug discovery.
Facilitating this resurgence have been advances in the technology
available to academic and industrial scientists, which can
be applied to investigate ion channel pharmacology [4]. In
this volume, we have included reviews and original research
by a number of investigators who are at the forefront in the
development and/or application of new technologies for the
characterization of ion channel function and pharmacology.
The volume begins with an excellent review on the ion channel
genome, which describes the landscape with respect to potential
molecular targets for drug discovery, relevant comparisons
across species and thoughts regarding the potential for overlapping
pharmacology among gene families. The remainder of the volume
describes several of the most widely applied technologies
available to the research community including planar patch
clamp technology, advanced semi-automated micropipette applications,
use of voltage-sensitive dyes, and high-throughput Xenopus
oocyte recordings. In addition, a number of the articles focus
on real-world experiences from pharmaceutical/biotech investigators
applying several of these technologies in an industrial setting.
While all available methods and/or instrumentation can not
be covered in a single volume, the articles cover many of
the most widely used approaches and the investigators report
their experiences and suggest how best to make progress using
these technologies in a research setting and/or a drug screening
environment. We hope the readers find the articles informative
and helpful in advancing their own ion channel research and
drug discovery programs.
REFERENCES
[1] Wang, H.S.; Pan, Z.; Shi, W.; Brown, B.S.; Wymore, R.S.;
Cohen, I.S.; Dixon, J.E.; McKinnon, D. Science, 1998,
282, 1890.
[2] Wickenden, A.D.; Roeloffs, R.; McNaughton-Smith, G.; Rigdon,
G.C. Expert Opin. Ther. Patents, 2004,
14, 457.
[3] Drenth, J.P.; Waxman, S.G. J. Clin. Invest.,
2007, 117, 3603.
[4] Ryan, A.M.; Matthews, E.; Hanna, M.G. Curr. Opin.
Neurol., 2007, 20, 558.
[5] Brugada, J.; Brugada, R.; Brugada, P. Herz,
2007, 32, 185.
[6] Ashcroft, F.A. Nature, 2006,
440, 440.
[7] Priest, B.T.; Swenson, A.M.; McManus, O.B. Curr. Pharm.
Des., 2007, 13, 2325.
Douglas S. Krafte
Biology & Scientific Affairs
Icagen Inc.
P.O. Box 14487
Research Triangle Park
NC 27709
USA
E-mail: DKRAFTE@icagen.com
[Back to top]
[PMID:
19149488 PubMed - indexed for MEDLINE]
Evolution of the Human Ion Channel Set
Timothy J. Jegla, Christian M. Zmasek, Serge
Batalov and Surendra K. Nayak
[Full
text article]
Ion channels are intimately involved in virtually every
physiological process of consequence in humans. Their importance
is underscored by the identification of numerous “channelopathies”,
human diseases caused by ion channel mutations. Ion Channels
have consequently been viewed as fertile ground for drug discovery
and, indeed, they represent one of the largest target classes
for current medicines. The future prospects of ion channels
as a target class are tied to the functional characterization
of the human ion channel set on a genomic scale. The focus
of this review is to describe the molecular diversity and
conservation of human ion channels. The human genome contains
at least 232 genes that encode the poreforming subunits of
plasma membrane ion channels. Comparative genome analysis
shows that most human ion channel gene families have their
origins in the earliest metazoans but the human genes are
largely derived from duplications that took place in the vertebrate
lineage. The mouse and human ion channel gene sets are virtually
identical, but differ significantly from fish channel sets.
Genome comparisons highlight a number of highly conserved
channel families that do not yet have specifically defined
functional roles in vivo. These channel families
are likely to have non-redundant functions in metazoans and
represent some of the best new opportunities for channel target
prospecting. Furthermore, genome-wide patterns of sequence
conservation can now be used to refine strategies for the
identification of gene-specific channel probes.
[Back to top]
[PMID:
19149489 PubMed - indexed for MEDLINE]
Port-a-Patch and Patchliner: High Fidelity Electrophysiology
for Secondary Screening and Safety Pharmacology
Cecilia Farre, Alison Haythornthwaite, Claudia
Haarmann, Sonja Stoelzle, Mohamed Kreir, Michael George, Andrea
Brüggemann and Niels Fertig
[Full
text article]
Ion channel dysfunction is known to underlie several
acute and chronic disorders and, therefore, ion channels have
gained increased interest as drug targets. During the past
decade, ion channel screening platforms have surfaced that
enable high throughput drug screening from a more functional
perspective. These two factors taken together have further
inspired the development of more refined screening platforms,
such as the automated patch clamp platforms described in this
article.
Approximately six years ago, Nanion introduced its entry level
device for automated patch clamping - the Port-a-Patch. With
this device, Nanion offers the world’s smallest patch-clamp
workstation, whilst greatly simplifying the experimental procedures.
This makes the patch clamp technique accessible to researchers
and technicians regardless of previous experience in electrophysiology.
The same flexibility and high data quality is achieved in
a fully automated manner with the Patchliner, Nanion’s
higher throughput patch clamp workstation. The system utilizes
a robotic liquid handling environment for fully automated
application of solutions, cells and compounds. The NPC-16
chips come in a sophisticated, yet simplistic, microfluidic
cartridge, which allow for fast and precise perfusion. In
this way, full concentration response curves are easily obtained.
The Port-a-Patch and Patchliner workstations from Nanion are
valuable tools for target validation, secondary screening
and safety pharmacology (for example hERG and Nav1.5 safety
screening). They are widely used in drug development efforts
by biotechnological and pharmaceutical companies, as well
as in basic and applied bio-physical research within academia.
[Back to top]
[PMID:
19149490 PubMed - indexed for MEDLINE]
High Throughput Electrophysiology with Xenopus Oocytes
Roger L. Papke and Cathy Smith-Maxwell
[Full
text article]
Voltage-clamp techniques are typically used to study
the plasma membrane proteins, such as ion channels and transporters
that control bioelectrical signals. Many of these proteins
have been cloned and can now be studied as potential targets
for drug development. The two approaches most commonly used
for heterologous expression of cloned ion channels and transporters
involve either transfection of the genes into small cells
grown in tissue culture or the injection of the genetic material
into larger cells. The standard large cells used for the expression
of cloned cDNA or synthetic RNA are the egg progenitor cells
(oocytes) of the African frog, Xenopus laevis. Until
recently, cellular electrophysiology was performed manually
by a single operator, one cell at a time. However, methods
of high throughput electrophysiology have been developed which
are automated and permit data acquisition and analysis from
multiple cells in parallel. These methods are breaking a bottleneck
in drug discovery, useful in some cases for primary screening
as well as for thorough characterization of new drugs. Increasing
throughput of high-quality functional data greatly augments
the efficiency of academic research and pharmaceutical drug
development. Some examples of studies that benefit most from
high throughput electrophysiology include pharmaceutical screening
of targeted compound libraries, secondary screening of identified
compounds for subtype selectivity, screening mutants of ligand-gated
channels for changes in receptor function, scanning mutagenesis
of protein segments, and mutant-cycle analysis. We describe
here the main features and potential applications of OpusXpress,
an efficient commercially available system for automated recording
from Xenopus oocytes. We show some types of data
that have been gathered by this system and review realized
and potential applications.
[Back to top]
[PMID:
19149491 PubMed - indexed for MEDLINE]
Automated Planar Electrode Electrophysiology in Drug Discovery:
Examples of the Use of QPatch in Basic Characterization and
High Content Screening on Nav,
KCa2.3, and Kv11.1
Channels
Mads P.G. Korsgaard, Dorte Strøbæk
and Palle Christophersen
[Full
text article]
Planar chip technology has strongly facilitated the progress
towards fully automated electrophysiological systems that,
in contrast to the traditional patch clamp technology, have
the capability of parallel compound testing. The throughput
has been increased from testing below 10 compounds per day
to a realized capacity approaching high throughput levels.
Many pharmaceutical companies have implemented automated planar
chip electrophysiology in their drug discovery process, particularly
at the levels of lead optimization, secondary screening and
safety testing, whereas primary screening is generally not
performed. In this review, we briefly discuss the technology
and give examples from selected NeuroSearch ion channel programs,
where one of the systems, the QPatch, has been evaluated for
use in lead optimization and primary screening campaigns,
where high information content was a requirement.
[Back to top]
[PMID:
19149492 PubMed - indexed for MEDLINE]
HCN Channels as Targets for Drug Discovery
Michael P. Maher, Nyan-Tsz Wu, Hong-Qing
Guo, Adrienne E. Dubin, Sandra R. Chaplan and Alan
D. Wickenden
[Full
text article]
Hyperpolarization- and Cyclic
Nucleotide-gated (HCN) channels are
a family of six transmembrane domain, single pore-loop, hyperpolarization
activated, non-selective cation channels. The HCN family consists
of four members (HCN1-4). HCN channels represent the molecular
correlates of Ih
(also known as ‘funny’ If
and ‘queer’ Iq),
a hyperpolarization-activated current best known for its role
in controlling heart rate and in the regulation of neuronal
resting membrane potential and excitability. A significant
body of molecular and pharmacological evidence is now emerging
to support a role for these channels in the function of sensory
neurons and pain sensation, particularly pain associated with
nerve or tissue injury. As such, HCN channels may represent
valid targets for novel analgesic agents. This evidence will
be reviewed in this article. We will then summarize our efforts
to develop and validate methods for screening for novel HCN
channel blockers.
[Back to top]
[PMID:
19149493 PubMed - indexed for MEDLINE]
The Chiptip: A Novel Tool for Automated Patch Clamp
Albrecht Lepple-Wienhues, Dirck Lassen,
Alexander Hümmer, Uwe Czubayko, Martina Knirsch and
Andjelko Golubovic
[Full
text article]
To facilitate automated patch clamp measurements of ion
channels in cells, the development of an all-glass Chiptip
pipette is reported that may be combined with the previously
described Flip-the-Tip technology. A single measurement requires
less than 50 cells, and the addition of drugs for screening
can be limited to very low volumes down to 1 µL. This
apparatus is suitable for the study small cells, subcellular
organelles and bacteria.
[Back to top]
[PMID:
19149494 PubMed - indexed for MEDLINE]
QPatch: The Missing Link Between HTS and Ion Channel Drug
Discovery
Chris Mathes, Søren Friis, Michael
Finley and Yi Liu
[Full
text article]
The conventional patch clamp has long been considered
the best approach for studying ion channel function and pharmacology.
However, its low throughput has been a major hurdle to overcome
for ion channel drug discovery. The recent emergence of higher
throughput, automated patch clamp technology begins to break
this bottleneck by providing medicinal chemists with high-quality,
information-rich data in a more timely fashion. As such, these
technologies have the potential to bridge a critical missing
link between high-throughput primary screening and meaningful
ion channel drug discovery programs. One of these technologies,
the QPatch automated patch clamp system developed by Sophion
Bioscience, records whole-cell ion channel currents from 16
or 48 individual cells in a parallel fashion. Here, we review
the general applicability of the QPatch to studying a wide
variety of ion channel types (voltage-/ligand-gated cationic/anionic
channels) in various expression systems. The success rate
of gigaseals, formation of the whole-cell configuration and
usable cells ranged from 40-80%, depending on a number of
factors including the cell line used, ion channel expressed,
assay development or optimization time and expression level
in these studies. We present detailed analyses of the QPatch
features and results in case studies in which secondary screening
assays were successfully developed for a voltage-gated calcium
channel and a ligand-gated TRP channel. The increase in throughput
compared to conventional patch clamp with the same cells was
approximately 10-fold. We conclude that the QPatch, combining
high data quality and speed with user friendliness and suitability
for a wide array of ion channels, resides on the cutting edge
of automated patch clamp technology and plays a pivotal role
in expediting ion channel drug discovery.
[Back to top]
[PMID:
19149495 PubMed - indexed for MEDLINE]
Use of Planar Array Electrophysiology for the Development
of Robust Ion Channel Cell Lines
Jeffrey J. Clare, Mao Xiang Chen, David
L. Downie, Derek J. Trezise and Andrew J. Powell
[Full
text article]
The tractability of ion channels as drug targets has
been significantly improved by the advent of planar array
electrophysiology platforms which have dramatically increased
the capacity for electrophysiological profiling of lead series
compounds. However, the data quality and throughput obtained
with these platforms is critically dependent on the robustness
of the expression reagent being used. The generation of high
quality, recombinant cell lines is therefore a key step in
the early phase of ion channel drug discovery and this can
present significant challenges due to the diversity and organisational
complexity of many channel types. This article focuses on
several complex and difficult to express ion channels and
illustrates how improved stable cell lines can be obtained
by integration of planar array electrophysiology systems into
the cell line generation process per se. By embedding
this approach at multiple stages (e.g., during development
of the expression strategy, during screening and validation
of clonal lines, and during characterisation of the final
cell line), the cycle time and success rate in obtaining robust
expression of complex multi-subunit channels can be significantly
improved. We also review how recent advances in this technology
(e.g., population patch clamp) have further widened the versatility
and applicability of this approach.
[Back to top]
[PMID:
19149496 PubMed - indexed for MEDLINE]
Sodium Channel Inhibitor Drug Discovery Using Automated High
Throughput Electrophysiology Platforms
Neil Castle, David Printzenhoff, Shannon
Zellmer, Brett Antonio, Alan Wickenden and Christopher
Silvia
[Full
text article]
Voltage dependent sodium channels are widely recognized
as valuable targets for the development of therapeutic interventions
for neuroexcitatory disorders such as epilepsy and pain as
well as cardiac arrhythmias. An ongoing challenge for sodium
channel drug discovery is the ability to readily evaluate
state dependent interactions, which are known to underlie
inhibition by many clinically used local anesthetic, antiepileptic
and antiarrhythmic sodium channel blockers. While patch-clamp
electrophysiology is still considered the most effective way
of measuring ion channel function and pharmacology, it does
not have the throughput to be useful in early stages of drug
discovery in which there is often a need to evaluate many
thousands to hundreds of thousands of compounds. Fortunately
over the past five years, there has been significant progress
in developing much higher throughput electrophysiology platforms
like the PatchXpressTM and
Ion WorksTM, which are now
widely used in drug discovery. This review highlights the
strengths and weaknesses of these two high throughput devices
for use in sodium channel inhibitor drug discovery programs.
Overall, the PatchXpressTM
and IonWorksTM electrophysiology
platforms have individual strengths that make them complementary
to each other. Both platforms are capable of measuring state
dependent modulation of sodium channels. IonWorksTM
has the throughput to allow for effective screening of libraries
of tens of thousands of compounds whereas the PatchXpressTM
has more flexibility to provide quantitative voltage clamp,
which is useful in structure activity evaluations for the
hit-to-lead and lead optimization stages of sodium channel
drug discovery.
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