Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels

Article abstract of DOI:10.1021/acschemneuro.8b00287

G protein-gated, inwardly rectifying, potassium (GIRK) channels are important regulators of cellular excitability throughout the body. GIRK channels are heterotetrameric and homotetrameric combinations of the K_ir3.1-4 (GIRK1-4) subunits. Different subunit combinations are expressed throughout the central nervous system (CNS) and the periphery, and most of these combinations contain a GIRK1 subunit. For example, the predominance of GIRK channels in the CNS are composed of GIRK1 and GIRK2 subunits, while the GIRK channels in cardiac atrial myocytes are made up mostly of GIRK1 and GIRK4 subunits. Although the vast majority of GIRK channels contain a GIRK1 subunit, discrete populations of cells that express non-GIRK1-containing GIRK (non-GIRK1/X) channels do exist. For instance, dopaminergic neurons in the ventral tegmental area of the brain, associated with addiction and reward, do not express the GIRK1 subunit. Targeting these non-GIRK1/X channels with subunit-selective pharmacological probes could lead to important insights into how GIRK channels are involved in reward and addiction. Such insights may, in turn, reveal therapeutic opportunities for the treatment or prevention of addiction. Previously, our laboratory discovered small molecules that can specifically modulate the activity of GIRK1-containing GIRK channels. However, efforts to generate compounds active on non-GIRK1/X channels from these scaffolds have been unsuccessful. Recently, ivermectin was shown to modulate non-GIRK1/X channels, and historically, ivermectin is known to modulate a wide variety of neuronal channels and receptors. Further, ivermectin is a complex natural product, which makes it a challenging starting point for development of more selective, effective, and potent compounds. Thus, while ivermectin provides proof-of-concept as a non-GIRK1/X channel activator, it is of limited utility. Therefore, we sought to discover a synthetic small molecule that would serve as a starting point for the development of non-GIRK1/X channel modulators. To accomplish this, we used a high-throughput thallium flux assay to screen a 100 000-compound library in search of activators of homomeric GIRK2 channels. Using this approach, we discovered VU0529331, the first synthetic small molecule reported to activate non-GIRK1/X channels, to our knowledge. This discovery represents the first step toward developing potent and selective non-GIRK1/X channel probes. Such molecules will help elucidate the role of GIRK channels in addiction, potentially establishing a foundation for future development of therapies utilizing targeted GIRK channel modulation.

Full text of DOI:10.1021/acschemneuro.8b00287

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Article
Discovery and Characterization of VU0529331, a Synthetic
Small-Molecule Activator of Homomeric G Protein-
gated, Inwardly-rectifying, Potassium (GIRK) Channels
Krystian A Kozek, Yu Du, Swagat Sharma, Francis J Prael, Brittany D Spitznagel,
Sujay V. Kharade, Jerod S. Denton, Corey R. Hopkins, and C. David Weaver
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00287 • Publication Date (Web): 23 Aug 2018
Downloaded from http://pubs.acs.org on August 29, 2018
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Page 1 of 20
ACS Chemical Neuroscience
Discovery and Characterization of VU0529331, a Synthetic SmallꢀMolecule Activator of Homomeric G
Proteinꢀgated, Inwardlyꢀrectifying, Potassium (GIRK) Channels
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Krystian A. Kozek,
Yu Du, Swagat Sharma, Francis J. Prael III,
Brittany D. Spitznagel, Sujay V.
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,1,4
Kharade, Jerod S. Denton, Corey R. Hopkins, and C. David Weaver*
1
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA
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Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee, USA
Department of Pharmaceutical Sciences, Center for Drug Discovery, College of Pharmacy, University of
Nebraska Medical Center, Omaha, Nebraska, USA
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Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA
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Vanderbilt Medical Scientist Training Program, Vanderbilt University, Nashville, Tennessee, USA
ABSTRACT
G proteinꢀgated, inwardlyꢀrectifying, potassium (GIRK) channels are important regulators of cellular excitability
throughout the body. GIRK channels are heterotetrameric and homotetrameric combinations of the K 3.1ꢀ4
ir
(
(
GIRK1ꢀ4) subunits. Different subunit combinations are expressed throughout the central nervous system
CNS) and the periphery, and most of these combinations contain a GIRK1 subunit. For example, the
predominance of GIRK channels in the CNS are comprised of GIRK1 and GIRK2 subunits, while the GIRK
channels in cardiac atrial myocytes are made up mostly of GIRK1 and GIRK4 subunits. Although the vast
majority of GIRK channels contain a GIRK1 subunit, discrete populations of cells that express nonꢀGIRK1ꢀ
containing GIRK (nonꢀGIRK1/X) channels do exist. For instance, dopaminergic neurons in the ventral
tegmental area of the brain, associated with addiction and reward, do not express the GIRK1 subunit.
Targeting these nonꢀGIRK1/X channels with subunitꢀselective pharmacological probes could lead to important
insights into how GIRK channels are involved in reward and addiction. Such insights may, in turn, reveal
therapeutic opportunities for the treatment or prevention of addiction. Previously, our laboratory discovered
small molecules that can specifically modulate the activity of GIRK1ꢀcontaining GIRK channels. However,
efforts to generate compounds active on nonꢀGIRK1/X channels from these scaffolds have been unsuccessful.
Recently, ivermectin was shown to modulate nonꢀGIRK1/X channels, and historically, ivermectin is known to
modulate a wide variety of neuronal channels and receptors. Further, ivermectin is a complex natural product,
which makes it a challenging starting point for development of more selective, effective, and potent
compounds. Thus, while ivermectin provides proofꢀofꢀconcept as a nonꢀGIRK1/X channel activator, it is of
limited utility. Therefore, we sought to discover a synthetic small molecule that would serve as a starting point
for the development of nonꢀGIRK1/X channel modulators. To accomplish this, we used a highꢀthroughput
thallium flux assay to screen a 100,000ꢀcompound library in search of activators of homomeric GIRK2
channels. Using this approach, we discovered VU0529331, the first synthetic small molecule reported to
activate nonꢀGIRK1/X channels, to our knowledge. This discovery represents the first step towards developing
potent and selective nonꢀGIRK1/x channel probes. Such molecules will help elucidate the role of GIRK
channels in addiction, potentially establishing a foundation for future development of therapies utilizing targeted
GIRK channel modulation.
KEYWORDS
GIRK channel, Kir3, GIRK2, ion channel, modulator, activator, small molecule, high-throughput screening,
thallium flux assay, whole-cell patch-clamp electrophysiology
INTRODUCTION
Opioids are some of the most effective pain medications, but they are also extremely addictive. The nationwide
1
opioid epidemic claims tens of thousands of lives each year as people addicted to opioids overdose. The
difficulty in combating this issue is demonstrated by the increasing number of overdose deaths, which are tied
2
to a dramatic increase in opioid prescriptions for treatment of acute and chronic pain over the last decade.
Research into understanding the underlying mechanisms of opioid addiction has also become increasingly
intense, aiming to illuminate new paths for the prevention and treatment of opioid addiction. One such area of
+
3–5
focus involves a family of inwardlyꢀrectifying potassium (K ) channels, called GIRKs.
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The G proteinꢀgated, inwardlyꢀrectifying, K channel (GIRK) family, also known as the K 3 family, is part of the
ir
larger inwardlyꢀrectifying potassium channel (K ) family. GIRK channels function by inhibiting cellular
excitability though membrane hyperpolarization. Numerous studies have revealed the importance of GIRK
channel function in neural processes such as analgesia, reward,
seizures as well as in diverse functions including embryonic development,
ir
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8–13
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anxiety, memory, respiration, and
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18,19
20,21
regulation of heart rate,
and
22,23
hormone secretion.
The GIRK channel family is comprised of GIRK1, 2, 3, and 4 (K 3.1, 3.2, 3.3, and 3.4)
ir
subunits that are encoded by the KCNJ3, 6, 9, and 5 genes, respectively. These individual subunits form
homotetrameric and heterotetrameric channels. Differential GIRK channel expression between various human
4
organs and cell types has been identified. GIRK1 or GIRK3 homotetrameric channels are not believed to be
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expressed in vivo,
while GIRK1ꢀcontaining (GIRK1/X) heterotetrameric channels are believed to be most
commonly found GIRK channels throughout the body. GIRK1, GIRK2, and GIRK3 subunits are widely
expressed throughout the central nervous system
predominantly throughout peripheral organs, i.e. in the heart
is expressed, GIRK1/X channels are therefore expressed; however, nonꢀGIRK1/X channels have been
reported in a few discrete brain regions. One example of specific GIRK expression is in dopaminergic (DA)
neurons of the ventral tegmental area (VTA), which express only GIRK2 and GIRK3 subunits. Here, GIRK2
homotetrameric and GIRK2/3 heterotetrameric channels may play a critical role in regulating addiction and
25,26
(CNS) while GIRK1 and GIRK4 subunits are expressed
24,27
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and pancreas. Wherever the GIRK1 subunit
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–13,30
9
reward circuitry.
To date, research has shown that the activity of addictive substances like ethanol and
8
methamphetamine is affected by GIRK3 expression in DA neurons of the VTA. These data raise the intriguing
possibility that modulation of nonꢀGIRK1/X channels in the VTA may decrease DA neuron activity and provide
a mechanism for decreasing drug abuse. While our knowledge of GIRK channel involvement in regulating key
neuronal circuitry in reward and addiction is still in its
infancy, improving our understanding of the role of GIRK
channels in drug abuse may provide new targets for
development of medicines to help combat the
increasingly deadly opioid epidemic.
a
b
K+
Modulation of GIRK channel activity by a number of
broadlyꢀactive ligands has been studied for many years.
31
Phosphatidylinositol 4,5ꢀbisphosphate (PIP ) is necessary
Thallos
2
for channels to open while βγꢀsubunits of inhibitory G
proteins (G ) are major endogenous intracellular channel
activators. G βγꢀsubunits of Gi/o proteins bound to Gi/oꢀ
proteinꢀcoupled receptors (GPCR) are
activated after ligand binding, and these G βγꢀsubunits
i/o
Fluorescence
i/o
coupled
G
c
d
i/o
32,33
Tl+
Tl+
can activate GIRK channels.
Gi/o proteinꢀcoupled
Activator
GPCRs include an array of receptors that are important in
normal physiology and are the targets of a large number
of drugꢀbased therapies, namely subtypes of opioid,
muscarinic, serotonergic, and dopaminergic receptors.
GIRK channels have been shown to be targets of alcohol
action , and a discrete alcohol binding pocket on these
channels has been discovered and characterized.
Intracellular sodium ions (Na ) have also been shown to
modulate GIRK channel activity. A crystal structure,
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35–37
+
3
8
Thallos
Fluo.
Thallos
Fluo.
39
solved by Wharton et al., with PIP , G βγꢀsubunit, and
2
i/o
+
Na bound to homomeric GIRK2 revealed the binding
pockets of these ligands. The activity of GIRK channels
has also been demonstrated to be sensitive to
+
Figure 1. The thallium (Tl ) flux assay enabled identification of
compounds that modulated Tl entry pathways on the cellular
4
0–42
+
cholesterol,
GPCRs,
inhibited by Gα subunits of G ꢀcoupled
decreased due to PIP2 degradation by
(PLC) after G ꢀcoupled GPCR
i/o
43
membrane. (a) When GIRK homotetrameric channels are
+
expressed in a cell, they efflux potassium (K ) under normal
+
phospholipase
C
physiological conditions+. During
a
Tl flux assay, (b) an
q
4
4
intracellularlyꢀloaded Tl ꢀsensitive+ dye, Thallos, enables (c)
measurement of the influx of Tl via fluorescence (Fluo.). (d)
activation, increased with phosphorylation by protein
4
5,46
kinase A (PKA),
protein kinase
and decreased by phosphorylation by
+
Channel activators increase Tl
influx, which increases
47
C
(PKC).
While GIRK channel
fluorescence and enables compound identification.
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modulation by this broad array of ligands and mechanisms has been thoroughly studied, none of these entities
are selective modulators of GIRK channels.
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Recently, our lab has discovered and characterized a number of smallꢀmolecule activators of GIRK channels.
While these modulators are proving highly useful as probes to investigate a variety of GIRKꢀexpressing
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4,17,48–50
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1
systems, i.e. the CNS,
they show extraordinary selectivity for GIRK1/X channels.
Two critical amino
4
acids in GIRK1 govern the ability of these molecules to activate only GIRK1/X channels, and all efforts to
generate analogs that can modulate nonꢀGIRK1/X channels have failed. To date, we have generated and
tested nearly 800 compounds based on scaffolds discovered by screening >250,000 compounds on GIRK1/2ꢀ
expressing HEK293 cells, and the only nonꢀGIRK1/Xꢀactive compound we identified was abamectin, a
compound closely related to ivermectin, recently described to activate GIRK channels by Su et al. and Chen
et al. Abamectin and ivermectin belong to the avermectin family of compounds and have been utilized as
insecticides and antihelminthic medications for decades. Although avermectins provide proofꢀofꢀconcept for
pharmacologic probes that activate nonꢀGIRK1/X channels, they are far from ideal. Perhaps most damaging,
avermectins are active on a wide variety of ion channels, including many Cysꢀloop, ligandꢀgated, ion channels,
namely the glycine receptor chloride channel (GlyR), the γꢀaminobutyric acid A receptor (GABA R),
nicotinic acetylcholine receptors (nAChR), and the insect glutamateꢀgated chloride channels (GluR).
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the
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Further, ivermectin has been demonstrated to activate P2X purinoceptor 4 (P2X R).
Several of these
4
avermectin targets are coꢀexpressed in the same neuronal populations as GIRK channels. This severely limits
avermectins’ utility as probes to study the role of GIRK channels in the VTA and other brain and peripheral
tissues. These issues of poor selectivity are compounded by the compounds’ high molecular weight (MW >
ꢀ1
65
8
50 gmol ) and relatively low potency. Further, the avermectins are complex natural products, making
synthesis and derivatization considerably more difficult than typical drugꢀlike small molecules. Even in light of
53,54
the failures of ourselves and others
to discover synthetically tractable smallꢀmolecule modulators of nonꢀ
GIRK1/X channels, we conducted a ~100,000 compound screen exclusively targeting homomeric GIRK2
channels. Herein, we describe the discovery and characterization of VU0529331, a synthetic small molecule
capable of activating nonꢀGIRK1/X channels.
RESULTS AND DISCUSSION
In an attempt to discover smallꢀmolecule modulators of nonꢀGIRK1/X channels, we performed a highꢀ
+
throughput screen (HTS) of a collection of ~100,000 compounds using a fluorescenceꢀbased thallium (Tl ) flux
assay (Figure 1). This screen was conducted on HEK293 cells engineered to express GIRK2 and the
neuropeptide Y receptor type 4 (NPY4R), a G ꢀcoupled GPCR capable of increasing the activity of GIRK2
i/o
channels in the presence of its agonist, human pancreatic polypeptide (hPP). Henceforth, this HEK293 cell line
expressing GIRK2 and NPY4R will be referred to as G2Y4 cells. We confirmed GIRK2 expression by western
blot (Figure 2, full membrane shown in Supporting Information, Figure S1) and NPY4R expression using a
+
functional assay, through which we confirmed hPP activity in G2Y4 cells using Tl flux, similar to the
experiment reported in Figure 4b. During the HTS, we screened a partiallyꢀactivated GIRK2 channel using an
EC30 concentration of hPP. We chose this approach in order to enable discovery of inhibitors, activators, or
potentiators of GIRK2 channels.
Using this screening approach, we discovered VU0529331 (Figure 3a). We characterized the ability of
+
VU0529331 to activate GIRK channels using Tl flux assays and wholeꢀcell patchꢀclamp electrophysiology.
+
Because the screen was conducted in HEK293 cells that express a variety of Tl influx pathways that may be
responsible for falseꢀpositive hits, we conducted studies to determine which
+
proteins were responsible for VU0529331’s ability to increase Tl influx.
+
First, we observed that VU0529331 does not increase Tl influx in
untransfected HEK293 cells (Figure 3d), suggesting that VU0529331 was
dependent on the expression of GIRK channels and/or NPY4R for activity.
To investigate whether VU0529331 was dependent on NPY4R expression
+
for activity, we conducted Tl flux assays using HEK293 cells engineered to Figure 2. GIRK1 and GIRK2 protein
expression levels shown in untransfected
HEK293 cells or cells engineered to
express GIRK2, GIRK1/2, or GIRK2 and
express GIRK2 channels but not NPY4R. We observed VU0529331 activity
in the absence of NPY4R, demonstrating that this phenomenon is
dependent on GIRK2 expression (Figure 3b). Representative examples of NPY4R. The full gel is shown in Supporting
+
Information, Figure 1S.
the raw Tl flux assay fluorescence traces used to generate concentration
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a
b
series graphs in Figure 3 are provided in the
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Supporting Information, Figure S2. Next, we
sought to identify whether a natively expressed Gi/oꢀ
coupled GPCR in HEK293 cell lines was the target of
VU0529331 and, thus, indirectly responsible for the
increase in GIRK activity downstream of the GPCR.
To investigate this possibility, we tested VU0529331
in G2Y4 cells treated with pertussis toxin (PTX), a
VU0529331
VU0466551
1
00 EC50 = 5.1 (4.7, 5.4) ꢁM
100
75
50
25
0
7
5
bacterial exotoxin that inhibits G ꢀcoupled GPCR
50
i/o
signaling by catalyzing ADPꢀribosylation of the Gα
subunit of the G proteins. Through this mechanism,
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PTX inhibits G βγ protein signaling to GIRK channels
i/o
ꢀ
7
ꢀ6
ꢀ5
ꢀ8.5
ꢀ7
ꢀ5.5
from any native G ꢀcoupled GPCR expressed in
i/o
c
d
EC50 = 5.2 (4.3, 6.3) ꢁM
EC50 = 49 (45, 53) nM
+
100
100
75
HEK293 cells. We conducted Tl flux experiments
7
5
0
using PTXꢀtreated G2Y4 cells and found that PTX
treatment induced little to no difference in the activity
of VU0529331 (Figure 4a). In contrast, PTX
treatment dramatically inhibited a maximallyꢀeffective
dose of hPP from activating GIRK channels indirectly
(Figure 4b). Because VU0529331 was insensitive to
PTX, we concluded that the activity of VU0529331
5
100
50
100
25
25
75
7
5
2
5
0
0
ꢀ7
0
50
ꢀ8.5
ꢀ6
ꢀ6
ꢀ5
ꢀ5
ꢀ7
ꢀ5.5
ꢀ5.5
5
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was independent of G protein signaling through any
0
0
i/o
ꢀ
7
ꢀ8.5
ꢀ7
G ꢀcoupled GPCR that may be expressed in
i/o
VU0529331 (log[M])
VU0466551 (log[M])
HEK293 cells. Together, these data suggest that
VU0529331 is active on GIRK channels engineered
+
Figure 3. Characterization of VU0529331 efficacy and potency using Tl
flux. VU0529331 activated GIRK2 and GIRK1/2 channels in
a
concentrationꢀdependent manner. In contrast, VU0466551 only activated into HEK293 cells. We sought to explore how broadly
GIRK1ꢀcontaining channels. Potency (EC50) values were provided for
active this compound was on closely and distantly
active compounds. Both compounds were inactive in untransfected
HEK293 cells. GIRK2 channel activity was normalized to a maximallyꢀ
effective concentration of VU0529331, while GIRK1/2 channel activity was
normalized to a maximallyꢀeffective concentration of VU0466551. All data
shown are representative of, at minimum, 3 independent experiments.
Error bars represent the standard error of the mean (SEM).
+
related K channels.
We tested whether VU0529331 activity was specific
to homomeric GIRK2 channels or whether
VU0529331 was capable of activating other GIRK
+
channels as well. Using Tl flux assays, we observed that VU0529331 was able to activate GIRK1/2 (Figure
3c), GIRK1/4, and GIRK4 channels (Table 1) expressed in HEK293 cells. This finding is not unexpected
considering that GIRK subunits are highly homologous, GIRK4 being the most homologous member of the Kir3
family with respect to GIRK2. However, this finding
was exciting, since no other small molecules that
activate homomeric GIRK4 channels have been
reported, to our knowledge. To study VU0529331 on
GIRK1/X channels, we compared the activity of
VU0529331 against VU0466551, an efficacious,
potent, and selective activator of GIRK1/X channels
that was previously discovered by our laboratory. We
verified that VU0466551 was active on GIRK1/2
channels with a potency of ~50 nM and inactive on
both untransfected and GIRK2ꢀexpressing HEK293
cells. We found that the efficacy of VU0529331 on
GIRK1/2 and GIRK1/4 was ~25% and ~20% of the
maximum efficacy of VU0466551, respectively. The subunit after GPCR activation is fully inhibited. The G2Y4 cells express
raw Tl flux traces (Supporting Information, Figure
S2) demonstrated that VU0529331 was capable of
generating a much greater total fluorescence in cells in (a) were normalized to the predicted maximum activity of VU0529331
expressing just GIRK2 as compared to cells
expressing GIRK1/2 channels. These data suggest
that VU0529331 may be more effective at opening
homotetrameric GIRK2 channels than GIRK1/2 or
*
***
a
b
VHL
100 EC50= 10.7 (10.1, 11.2) ꢁM
100
PTX
VHL
PTX
EC50= 12.2 (11.4, 13.0) ꢁM
75
75
5
0
50
25
0
50
****
25
0
ꢀ
6.5
ꢀ5.5
ꢀ4.5
VU0529331 hPP
VU0529331 concentration, log[M]
ꢀ25
Figure 4. (a) Pertussis toxin (PTX) does not affect the activity of
VU0529331 on G2Y4 cells while (b) activation of GIRK2 through the Gβγꢀ
+
neuropeptide Y receptor type 4 (NPY4R) in addition to any GPCRs native
to HEK293 cells. NPY4R was activated using a maximallyꢀeffective
concentration, 200 nM, of human pancreatic polypeptide (hPP). All values
under VHL conditions. VU0529331 activity in (b) is normalized data from
the 30 ꢁM value in (a). 95% confidence intervals for the EC50
measurements were calculated using a 2ꢀtailed MannꢀWhitney test. ****
indicates p<0.0001. Error bars represent the standard error of the mean
(SEM).
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ACS Chemical Neuroscience
Efficacy, %
(95% CI)
Potency, ꢁM
(95% CI)
GIRK1/4 heterotetrameric channels. Overall, these results suggest
that VU0529331 is a modestly selective, nonꢀGIRK1/X channel
activator in addition to being the first synthetic small molecule
known to activate both GIRK2 and GIRK4 homomeric channels.
Target
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
GIRK2
AD100
5.1 (4.7, 5.4)
5.2 (4.3, 6.3)
4.8 (4.2, 5.4)
22.3 (21.1, 23.7)
15.7 (13.8 ,17.8)
1.23 (1.21,1.25)
Inactive
GIRK1/2
GIRK1/4
GIRK4
B25.9 (+/− 2.4)
B18.1 (+/− 1.1)
AD100
To further investigate the selectivity of VU0529331 within several
+
families of K channels, we tested the ability of VU0529331 to
K 6.1/SUR2a
C1418 (+/− 108)
C127(+/− 2)
Inactive
ir
modulate the activity of a variety of K channels as well as more
ir
K 6.1/SUR2b
+
ir
distantly related K channels (Supporting Information, Figure
S4). Using Tl flux assays on HEK293 cells engineered to express
K 6.2/SUR1
+
ir
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
K 4.1
Inactive
Inactive
ir
the channels of interest, we found that VU0529331 was inactive on
K 2.1
Inactive
Inactive
K 2.1, K 4.1, α1β2 MaxiK, α1β4 MaxiK, K 2.1, and Slack channels.
ir
i
r
i
r
v
Slack
Inactive
Inactive
We did discover that VU0529331 activated K 6.1/SUR2a and
ir
K 6.1/SUR2b channels. This finding suggests that K 6.1 and the
K 2.1
Inactive
Inactive
ir
ir
v
Kir3 channel family share some, thusꢀfar, unappreciated
pharmacological homology. K 6 and K 3 are closely related K
α1β2 MaxiK
α1β4 MaxiK
α1 GlyR
Inactive
Inactive
ir
ir
ir
Inactive
Inactive
families, and future experiments involving siteꢀdirected
mutagenesis and chimeric channels will aim to elucidate the amino
acids involved in VU0529331 binding and activity. In addition to
Inactive
Inactive
Untransfected
Inactive
Inactive
+
Table 1. VU0529331 was tested on an array of targets
expressed in HEK293 cells. VU0529331 activated
GIRK2, GIRK1/2, GIRK1/4, GIRK4, Kir6.1/SUR2a, and
testing VU0529331 selectivity amongst K channels, we also
59
investigated whether VU0529331 modulated homomeric α1 GlyR,
K
ir6.1/SUR2b channels. The compound was inactive on one of the many Cysꢀloop receptors activated and potentiated by
A
all other listed targets.B Normalized to maximum
ivermectin. To test the activity of VU0529331 on α1 GlyR, we
expressed α1 GlyR in a HEK293 cell line engineered to express
VU0529331 activityC;
Normalized to maximum
VU0466551 activity; Normalized to 250 ꢁM pinacidil
D
ꢀ
activity; Confidence interval (95% CI) cannot be
both SuperClomeleon, a genetically encoded chloride ion (Cl )
calculated.
68
+
ꢀ
sensor,
and the K ꢀCl transporter
a
GIRK2
b
GIRK1/2
member 5 (KCC2), which is essential for
****
** ****
*
***
*** ***
ꢀ
**
the generation of a Cl gradient that favors
n.s.
***
*
n.s.
n.s.
n.s.
ꢀ
69
mV
ꢀ110 ꢀ90
n.s.
mV
inward Cl flux.
While ivermectin
*
*
ꢀ110 ꢀ90 ꢀ70***
ꢀ30 ꢀ10
ꢀ25
10
demonstrated both activation of α1 GlyR
and potentiation of glycine on α1 GlyR,
VU0529331 neither activated α1 GlyR nor
potentiated the activity of glycine on α1
GlyR (Supporting Information, Figure
ꢀ30
ꢀ10
10
*
***
****
ꢀ
25
****
****
***
*
***
****
****
ꢀ75
ꢀ75
*
ꢀ125
S3).
The
selectivity
results
for
ꢀ125
VU0529331, including efficacies and
potencies, are listed in Table 1.
ꢀ175
ꢀ175
2
V
0
H
m
L
M K+ w/ 0.25% DMSO
d
After establishing that the ability of
VU0529331 to increase Tl flux in GIRKꢀ
expressing HEK293 cells was mediated
by GIRK channels, we then explored the
GIRKꢀactivating properties of VU0529331
Current Density at ꢀ10 mV
+
2
V
0
U
m
0
M
5
K
2
+
9
w
331
/ 8 mM VU331 in 0.25% DMSO
3
3
2
1
0
p = 0.015
c
Untransfected HEK293
using
wholeꢀcell
patchꢀclamp
ꢀ25
mV
electrophysiology. First, we investigated
the effect of VU0529331 on the currentꢀ
voltage relationship (IV) of GIRK
channels. Here, we utilized the same
engineered HEK293 cells expressing
GIRK2 and GIRK1/2 channels used for
ꢀ110 ꢀ90 ꢀ70
ꢀ50 ꢀ30 ꢀ10
25
10
ꢀ
GIRK1/2 GIRK2
Target
Figure 5. 80 ꢁM VU0529331 significantly increased both inward and outward currents in
HEK293 cells expressing (a) GIRK2 and (b) GIRK1/2 channels. VU0529331 did not evoke
currents in (c) untransfected HEK293 cells. (d) The increase in current density for GIRK1/2
+
Tl flux assays. Currents were recorded in
1
1
ꢁ
2 increments of 10 mV, from −100 mV to and GIRK2 cells at −10 mV was significantly different. For each cell type in (a), (b), and (c),
the conditions were compared using
a 2ꢀway ANOVA with Bonferroni’s multiple
0 mV, in the absence or presence of 80
M VU0529331, which was a maximallyꢀ
comparisons test. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The fold differences
+
in (d) were compared using a Welch’s tꢀtest. Recordings were generated in 20 mM K
effective concentration. Representative external buffer solution containing 0.25% (v/v) DMSO (VHL). Error bars indicate standard
error around the mean (SEM).
traces of the recordings used to generate
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these IVs are shown in Supporting Information,
Figure S5. IVs generated in GIRK2 (Figure 5a) and
a
b
GIRK2 Rectification Ratio
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
.5
0.4
.3
GIRK1/2 (Figure 5b) cell lines were consistent with
+
inwardlyꢀrectified
K
channels.
Recordings
demonstrated that inward and outward GIRK2 and
GIRK1/2 currents were increased in the presence of
VU0529331, while we did not record any changes
in the currents of untransfected HEK293 cells
0
0.2
0.1
(
Figure 5c). The magnitude of activation was
greater for GIRK2 channels than GIRK1/2 channels
when comparing the physiologicallyꢀrelevant
outward currents (Figure 5d), further supporting the
view that VU0529331 is a better activator of GIRK2
channels than GIRK1/2 channels. For both
channels, inward rectification was maintained in the
presence of VU0529331, and the rectification ratio
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.0
VHL VU0529331
Condition
Figure 6. 80 ꢁM VU0529331 had no effect on the rectification ratio of
HEK293 cells expressing (a) GIRK2 and (b) GIRK1/2 channels. Wholeꢀcell
patchꢀclamp electrophysiology recordings were generated under 20 mM K
external buffer solution containing 0.25% (v/v) DMSO (VHL). A Wilcoxon
matchedꢀpairs signed rank test analysis indicated that there was not a
+
(
RR = (−1)*Current Densityꢀ100mV/Current Densityꢀ statistically significant difference between the two conditions in (a) or (b).
30mV)
was not significantly altered by VU0529331
a
GIRK2
b
Untransfected HEK293
(Figure 6). Additionally, no significant change in the
reversal potential was observed (GIRK295%CI: (−43.9,
−
GIRK1/295%CI: (−42.9, −42.0) mV vs (−44.1, −41.9)
mVVU0529331) indicating that K selectivity was unaffected.
Together, these data indicate that VU0529331 does not
affect the inward rectification or the K selectivity of
GIRK channels.
0
5
5.0
2.5
3
3 ꢂM VU331
3
3 ꢂM VU331
10
3
10
3
40.8) mVVHL vs (−44.4, −42.8) mVVU0529331
;
ꢀ
0
.1% DMSO
0
.1% DMSO
VHL
+
ꢀ
ꢀ
10
15
0.0
ꢀ2.5
ꢀ5.0
+
ꢀ20
0
50
100
150
0
50
100
150
Time (sec)
Time (sec)
To further investigate the activity of VU0529331, we
recorded currents with the membrane potential clamped
at −60 mV. Increasing concentrations of VU0529331
incrementally increased inward GIRK2 and GIRK1/2
currents in engineered HEK293 cells (Figure 7a,d)
while failing to induce changes in currents in
untransfected HEK293 cells (Figure 7b). Application of
an extracellular solution containing 2 mM barium (Ba )
blocked nearly all basal and VU0529331ꢀevoked GIRK
currents (Figure 7c,e). These data corroborated the
c
GIRK2
3
3 ꢂM
2 mM Ba2+
2 mM Ba2+
.1% DMSO
VU331
4
0
0
ꢀ
4
8
ꢀ
2
+
ꢀ
12
0
25
50
75
100
125
150
175
200
Time (sec)
+
results of the Tl flux data and demonstrated that
VU0529331 does act on and increase currents through
homotetrameric and heterotetrameric GIRK channels.
d
GIRK1/2
e
GIRK1/2
3
3 ꢂM
VU331
0
ꢀ5
7
mM Ba2+
2
33 ꢂM VU331
1
0
0.1% DMSO
Overall,
these
electrophysiology
experiments
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
10
15
20
25
30
0
ꢀ7
demonstrated that VU0529331 is capable of increasing
the activity of GIRK channels while maintaining the K
selectivity and inward rectification inherent to these
channels.
0.1% DMSO
+
ꢀ14
0
50
100
Time (sec)
150
0
25 50 75 100 125
Time (sec)
Although VU0529331 represents an exciting opportunity
for smallꢀmoleculeꢀbased modulation of nonꢀGIRK1/X
Figure 7. Wholeꢀcell patchꢀclamp electrophysiology using either
untransfected HEK293 cells or cells engineered to express GIRK channels, the measured potency at GIRK2 channels of
channels. Currents were held at −60 mV while cells were bathed in 20
+
~5 ꢁM is not ideal. We sought to identify analogs of
VU0529331 with improved potency by studying the
structureꢀactivity relationship (SAR) between analogs
and GIRK channels. To this end, we purchased 43
mM K external buffer. Exposure of (a) GIRK2 cells or (d) GIRK1/2 cells
to escalating VU0529331 (VU331) concentrations showed
concentrationꢀdependent increases in inward current density. (b)
Untransfected HEK293 cells did not show any changes in currents after
VU331 exposure. Both (c) GIRK2 and (e) GIRK1/2 currents evoked by
maximallyꢀactive VU331 concentrations were blocked by exposure to analogs (Supporting Information, Figure S6) and
2+
extracellular solutions containing 2 mM barium (Ba ), the nonꢀselective
synthesized 4 additional analogs with substitutions that
were not available for purchase (Supporting
+
inwardlyꢀrectifying K channel inhibitor.
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Information, Figure S7). The synthetic strategy is illustrated in Supporting Information, Scheme S1. By
testing this limited analog library on GIRK2 and GIRK1/2 channels, we found that none of the modifications to
the parent structure of VU0529331 were tolerated. Although this limited number of analogs did not yield any
improvement in the activity of VU0529331, further medicinal chemistry efforts may result in improvements in
both potency and efficacy. However, these efforts are beyond the scope of the present study.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
While GIRK physiology has been extensively studied for the past two decades, development of molecular
probes to study nonꢀGIRK1/X channels has proven a difficult endeavor. To date, two other screens
independently identified ivermectin as an activator of nonꢀGIRK1/X channels. While an exciting discovery,
ivermectin is a large, macrocyclic natural product with multiple known important CNS targets that evoke a
variety of known in vivo effects. More specifically, ivermectin’s activation and potentiation of Cysꢀloop
receptors, such as GlyR and GABA R receptors, severely limit ivermectin’s utility, especially since GABA R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
A
and GIRK channels are often coꢀlocalized in the same neurons throughout the CNS. Thus, efforts to utilize
ivermectin as a GIRK probe are quite limited. Therefore, as a GIRK channel probe, VU0529331 fills an
important and unique pharmacologic gap. Although the activity of VU0529331 on K 6.1/SUR2a and
ir
K 6.1/SUR2b is not ideal, the lack of K 6.1 expression in neurons mitigates some of the concern. Additionally,
ir
ir
70,71
the K 6.2/SUR1 channel, which is expressed in neurons,
was not activated by VU0529331, further
ir
7
0
mitigating concerns. However, K 6.1 is expressed in astrocytes and, even though SUR expression in
ir
7
2
astrocytes has not been explored, this target should be considered when VU0529331 is used ex vivo (e.g.
brain slice preparations). Nevertheless, we are eager to capitalize on the similarity between K 3 and K 6 to
ir
ir
explore the amino acids important in VU0529331 activity on these families. Such work may reveal
opportunities to develop more potent and selective compounds for both K 3 and K 6.1 channels, alike.
ir
ir
CONCLUSIONS
In summary, we have discovered and characterized VU0529331, the first smallꢀmolecule GIRKꢀchannel
activator capable of activating nonꢀGIRK1/X channels. Although this compound activated all the GIRK
channels we tested, we demonstrated that its activity was greater on nonꢀGIRK1/X channels. Further,
+
VU0529331 did not change the K selectivity or rectification of GIRK channels. Neither VU0529331 nor a
limited number of its analogs possessed sufficient potency for ex vivo brain slice or in vivo studies; however,
VU0529331 provides evidence that a synthetic small molecule can activate nonꢀGIRK1/X channels. This
molecule presents a starting point for the development of analogs with improved potency, selectivity, and
favorable ADME properties. Such improved analogs will enable ex vivo brain slice and in vivo studies, which
have the potential to help better our understanding of the role of GIRK channels in addiction and reward.
Additionally, such molecules may indicate that modulation of nonꢀGIRK1/X channels with pharmaceuticals
could be an important new mechanism through which to help combat the nationwide opioid epidemic and other
forms of addiction.
METHODS
Cell Line Generation and Cell Culture
HEK293 cells expressing both human GIRK1 and human GIRK2 subunits were generated by transfecting a
pCMV6ꢀAꢀBSD vector (Origene, Rockville, MD) containing KCNJ3 and a pCMV6ꢀAꢀpuro vector (Origene,
Rockville, MD) with KCNJ6 into lowꢀpassage HEK293 cells (CRLꢀ1573, ATCC, Manassas, VA). The FuGENE
6
(Promega, Madison, WI) transfection reagent and protocol, together with OptiꢀMEM (Thermo Fisher
Scientific, Waltham, MA), were utilized in all transfections herein. Similarly, HEK293 cells expressing the
human GIRK2 subunit were generated by transfecting only the KCNJ6ꢀcontaining vector. Further, GIRK2 cells
were transfected with a vector encoding human NPY4R, generously provided by the Dr. Annette Beckꢀ
73,74
Sickinger laboratory,
to create the G2Y4 cells used for HTS and PTX experiments. To study GIRK4
channels, HEK293 cells were transientlyꢀtransfected with a pCMV6ꢀAꢀNeo vector (Origene, Rockville, MD)
containing human KCNJ5. HEK293 cells stablyꢀexpressing human α1β2 MaxiK and α1β4 MaxiK channels
were generated by transfecting a pCMV6ꢀAꢀpuro vector (Origene, Rockville, MD) with human KCNMA1 and a
pcDNA3.1(+)/zeo vector (Genscript, Piscataway, NJ) with either human KCNMB2 or human KCNMB4,
respectively. HEK293 cells expressing human Slack channels were generated by transfecting a pCMV6ꢀEntry
vector (Origene, Rockville, MD) containing the KCNT1 gene. HEK293 cells expressing human K 2.1 channels
v
were generated by transfecting a vector encoding the KCNB1 gene, generously provided by the Dr. Jennifer
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76
Kearney laboratory. HEK293 cells expressing both human K 6.1 and rat SUR2a were generated by
ir
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
transfecting a pBUDCE4.1 vector (Invitrogen, Carlsbad, CA) containing KCNJ8 and ABCC9 isoform A.
HEK293 cells expressing both human K 6.1 and rat SUR2b were generated by transfecting a pcDNA5/TO
ir
vector (Origene, Rockville, MD) containing KCNJ8 and a pcDNA3.1 vector, generously provided by the Dr.
Colin Nichols laboratory, containing ABCC9 isoform B. Stablyꢀtransfected monoclonal TꢀRExꢀHEK293 cell lines
77
78
79
expressing human K 2.1, K 4.1, or K 6.2/SUR1 under a tetracyclineꢀinducible promoter were previously
ir
ir
ir
generated and graciously provided by the laboratory of Dr. Jerod S. Denton. HEK293 cells stablyꢀexpressing
SuperClomeleon and KCC2 were generated in a TꢀRExꢀHEK293 background by transducing a pLenti CMV
Puro DEST vector containing SuperClomeleon and transfecting a pcDNA4/TO vector containing human KCC2
isoform B. To study α1 GlyR, this cell line was then transientlyꢀtransfected with a pCMV6ꢀAꢀhygro vector
containing human GLRA1. All stablyꢀtransfected cells were maintained under antibiotic selection over their
lifetime.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Monoclonal cell lines were generated by limitingꢀdilution cloning, where polyclonal populations of cells were
distributed in 384ꢀwell amineꢀcoated plates at <0.8 cells per well and cultured till colonies grew to encompass
at least a quarter of a well. These colonies were further tested and selected for expansion as described
80
previously. Selected clones were expanded for use in experiments and for cryopreservation. Cell culture
medium consisted of Minimal Essential Medium, Alpha Medium (Corning, Corning, NY) supplemented with
10% (v/v) heatꢀinactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and GlutaGro (Corning,
Corning, NY). Medium was supplemented with antibiotics, as appropriate, to maintain selection pressure.
+
For use in Tl flux experiments, monoclonal HEK293 cells engineered to express proteins of interest were
grown in cell culture medium on tissueꢀcultureꢀtreated and vented T75, T150, or T300 flasks (TPP,
Trasadingen, Switzerland) in a humidified incubator at 37°C and 5% CO . These cells were grown to reach
2
90% confluence on the day before experimentation. Experiments with GIRK4 and α1 GlyR channels were
conducted in transientlyꢀtransfected cells, which were not processed for monoclonal selection. To generate
transientlyꢀexpressing cell lines for experiments, cells were transfected when flasks were 40% confluent, and
the flasks were incubated overnight to reach 90% confluence in a humidified incubator at 37°C and 5% CO2.
To prepare cells for transfer into 384ꢀwell plates, culture medium was removed from flasks, flasks were treated
using TrypLE Express (Thermo Fisher Scientific, Waltham, MA) for 5 min to dislodge cells, and singleꢀcell
suspensions were created through trituration. Cells were resuspended in antibioticꢀcontaining medium at 1,000
cells/ꢁL. 1 ꢁg/mL of tetracycline (SigmaꢀAldrich, St. Louis, MO) was added to suspensions where cells
expressed a protein under a tetracyclineꢀinducible promoter. 20 ꢁL of cellular suspensions were plated in
®
Corning PureCoat™, amineꢀcoated, 384ꢀwell microtiter plates (cell plates; cat#354719, Corning, Corning, NY)
to achieve a cell density of 20,000 cells/well. Cell plates were incubated overnight in a humidified incubator at
37°C and 5% CO2.
+
Thallium (Tl ) Flux Experiments
The Tl flux assays were conducted largely as previously described,
+
81,82
and below we detail the specifics.
Compounds for primary screening were managed by the Vanderbilt Highꢀthroughput Screening Facility and
were received on the day of screening as 80 nL aliquots of 10 mM compound stocks dissolved in dimethyl
sulfoxide (DMSO). With 1 compound/well, 320 compounds were formatted in 384ꢀwell, roundꢀbottom,
polypropylene microplates (compound plates; Greiner, Monroe, NC) and diluted to 40 ꢁL with assay buffer
comprising Hanks Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA) and 20 mM HEPES pH 7.3
(
Corning, Corning, NY). This generated compound solutions at concentrations twice the final concentration
because solutions were diluted 2ꢀfold during the assay to a final concentration of 10 ꢁM. For commercially
purchased VU0529331 and its available analogs, dry compounds were ordered through AldrichMarketSelect
(
SigmaꢀAldrich, St. Louis, MO) and dissolved in DMSO to generate 10ꢀ100 mM stocks. Specifically,
VU0529331 was stored in DMSO at 33.3 mM. For an experiment, 10 ꢁL aliquots in 384ꢀwell, Echo qualified,
low dead volume microplates (Labcyte, Sunnyvale, CA) were reformatted using an Echo555 plate reformatter
(Labcyte, Sunnyvale, CA) to generate 10ꢀconcentration, 3ꢀfold dilution series in compound plates. Compounds
were similarly diluted with assay buffer to twice the final concentration. To help solubilize compounds,
compound plates with diluted compounds were sealed using a PlateLoc Thermal Microplate Sealer (Agilent,
Santa Clara, CA), briefly ultrasonicated in a bath sonicator (ColeꢀParmer, Vernon Hills, IL), and shaken
vigorously using a Teleshake (Inheco, Martinsried, Germany) for at least 30 min. Compounds were used within
an hour of preparation, and the final DMSO concentrations in all assays were ≤0.25% (v/v).
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Cells for experiments were prepared 1 day before screening, as described in the Methods section above. For
experiments involving PTX treatment, medium in cell plates was replaced with fresh medium containing 800
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ng/mL PTX (Tocris, Bristol, United Kingdom) and incubated in a humidified incubator at 37°C and 5% CO for 8
2
hours prior to dyeꢀloading. Dye was loaded into cells for 1 hour, directly prior to imaging. For this, culture
+
medium was replaced with 20 ꢁL/well of dyeꢀloading buffer comprising assay buffer with 0.6 ꢁM Tl ꢀsensitive
dye ThallosꢀAM. ThallosꢀAM was stored dry in 20 ꢁg aliquots and resuspended with 30 ꢁL of DMSO (Sigmaꢀ
Aldrich, St. Louis, MO) containing 6.7% (w/v) Pluronic Fꢀ127 (SigmaꢀAldrich, St. Louis, MO) immediately prior
to dilution. After incubating cells in dyeꢀloading buffer for 1 hour at room temperature (RT), the buffer was
replaced with assay buffer and loaded onto the WaveFront Biosciences Panoptic (Franklin, TN). Data were
acquired at 5 Hz (excitation 482 ± 35 nm, emission 536 ± 40 nm) for 10 s, at which time 20 ꢁL/well of test
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compounds was added and allowed to equilibrate for 120 s. Next, 10 ꢁL/well of Tl stimulus buffer (125 mM
NaHCO , 1.8 mM CaSO , 1 MgSO , 5 mM glucose, 4 mM Tl SO , and 20 mM HEPES pH 7.3) was added.
3
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2
4
Imaging was concluded after an additional 120 s. When preparing hPP for testing, 100 ꢁM hPP stock solutions
+
were diluted to 5ꢀfold over the desired final concentration in Tl stimulus buffer additionally containing 0.0025%
fatty acid free, low endotoxin, lyophilized, powdered bovine serum albumin (SigmaꢀAldrich, St. Louis, MO).
During the screen, a final concentration of 0.7 nM hPP was used to achieve approximately 30% GIRK channel
activity through the NPY4R, and 200 nM hPP was a maximallyꢀeffective concentration. To better observe
activity of certain channels, assay parameters were varied as follows. MaxiKꢀexpressing cells were tested
+
2+
using a Tl stimulus buffer containing 3 ꢁM ionomycin, which elevated intracellular Ca and promoted channel
+
activity to approximately EC . K 2.1ꢀexpressing cells were tested using a modified Tl stimulus buffer (87.5
30
v
mM NaHCO , 37.5 mM KHCO , 1.8 mM CaSO , 1 MgSO , 5 mM glucose, 4 mM Tl SO , and 20 mM HEPES
3
3
4
4
2
4
+
pH 7.3) containing an elevated K concentration to promote channel activity at approximately EC .
30
+
Tl flux assay data was collected using WaveGuide (WaveFront Biosciences, Franklin, TN) and analyzed using
Excel (Microsoft, Redmond, WA). Data reported are the averages of at least 3 independent experiments.
Compound activity was extracted from normalized and controlꢀsubtracted fluorescence waves generated over
2
8
50 s of imaging. The wave for each well was normalized by dividing by the average fluorescence from the first
s of imaging (F/F ), which accounted for differences in cell number and dye loading. Next, each wave was
0
controlꢀsubtracted, and the change in wave amplitude (∆A) due to stimulus buffer addition was calculated. At
the time of stimulus buffer addition (T ), the ∆A of active wells increased dramatically, as demonstrated in
s
Supporting Information, Figure S2. ∆A was calculated as the difference in amplitude between time points
T −5 s and T +X s, where X ranges between 2 and 15 s depending on the particular cell line. For each cell line,
s
s
this time point was chosen to provide a high signalꢀtoꢀnoise ratio while measuring nearest the linear portion of
+
the initial Tl influx. These values are inversely proportional to the overall measured activity of a specific
channel. Specifically, X=15 for untransfected HEK293, GIRK2, K 6.1/SUR2a, K 6.1/SUR2b, K 6.2/SUR1, α1β2
ir
ir
ir
MaxiK, and α1β4 MaxiK cells, X=5 for GIRK1/4, Slack, K 2.1, K 2.1, and K 4.1 cells, and X=2 for GIRK1/2
v
ir
ir
cells. ∆A values for each compound at different concentrations were used to generate fits to a fourꢀparameter
logistic equation in XLfit (IDBS, Guildford, Surrey, United Kingdom). Statistical analyses were performed using
Prism 7 (GraphPad, La Jolla, CA).
+
As described in Results and Discussion, we utilized the Tl flux assay in the HTS format to screen partiallyꢀ
activated G2Y4 cells for activators of GIRK2 channels. Potential activators, or “hits”, were chosen if the
compound ranked in the top 0.5% of molecules that displayed the greatest ∆A relative to vehicle controls.
Compounds were then characterized through a series of assays against a variety of engineered cell types, as
described in the Results and Discussion section, and led to the discovery of VU0529331. Although here we
have reported our efforts towards identifying a novel activator from this HTS, our screen did also yield
+
hundreds of hits that inhibited Tl influx in our system. However, due to our focus on activators, we have yet to
further explore the activity and selectivity of these compounds.
Screened Compound Library
The compound collection that we screened for this work was composed of the following libraries: Vanderbilt
Discovery Collection, which is selected from the Life Chemicals collection for HTS. The compounds in this
collection were chosen by Vanderbilt medicinal and computational chemists to provide leadꢀlike motifs,
minimum panꢀassay interference, and maximum diversity; NIH Clinical Collection I and II, which are small
molecules that have history of use in human clinical trials; NCI Focused Natural Product Collection, which is
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comprised of pure compounds acquired by the NCI from Analytical and MerLion; Cayman Lipid Library, which
is a broad variety of bioactive lipids; Ion Channel Library, which is a collection from Life Chemicals targeted to
ion channels compiled using 2D fingerprint similarity methodology; Epigenetics Collection, which is a group of
small molecule modulators with biological activity for use in epigenetic research; Marnett Collection, which
contains NSAID derivatives that contain cyclooxygenase inhibitors, PPARγ activators, and apoptosis inducers;
and the Enzo Kinase Inhibitor Library, which is the ScreenꢀWell™ Kinase Inhibitor Library containing 80 known
kinase inhibitors of wellꢀdefined activity.
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Western Blot
Untransfected and engineered GIRK1/2, GIRK2, and G2Y4ꢀexpressing HEK293 cell lines were grown to 100%
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confluence in tissue culture treated 150 cm dishes. The growth medium was aspirated and plates were twice
washed with 5 mL phosphateꢀbuffered saline (PBS) pH 7.4 (Thermo Fisher Scientific, Waltham, MA). Cells
were scraped from the dish and triturated. Cell suspensions were then centrifuged at 500 g for 5 min at RT.
Next, the supernatant solution was removed, and the pellet was resuspended in 1 mL of PBS and centrifuged
once more. After removal of the supernatant solution, cells were resuspended in 1 mL of RIPA buffer (Sigmaꢀ
Aldrich, St. Louis, MO) containing Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA)
and stored on ice. Suspensions were then ultrasonicated in 4 brief 5 s pulses and allowed to cool on ice for 15
min before centrifugation at 6,000 g for 10 min in a 4°C room. The supernatant was recovered, and the protein
concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham,
MA) with a SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA). Sample were diluted
to contain 30 ꢁg of protein in 25 ꢁL of Laemmli Sample Buffer (BioꢀRad, Hercules, CA) containing 355 mM βꢀ
mercaptoethanol (SigmaꢀAldrich, St. Louis, MO). Samples were heated at 55 °C for 10 min before loading, and
the gel was run according to the NuPage BisꢀTris/MOPS protocol using NuPage buffers and gel (Invitrogen,
Carlsbad, CA). 25 ꢁL/well of each sample was loaded onto a precast NuPAGE 4ꢀ12% BisꢀTris Gel and
separated for 50 min at 200 V in NuPAGE MOPS SDS Running Buffer. The gel was transferred onto Odyssey
Nitrocellulose Membrane (LIꢀCOR Biosciences, Lincoln, NE) for 1 h at 30 V in NuPAGE Transfer Buffer. The
membrane was rinsed using Trisꢀbuffered Saline (TBS; Corning, Corning, NY) before blocking with 25 mL of
TBS containing 5% (w/v) Blotting Grade Blocker Nonꢀfat Dry Milk (BioꢀRad, Hercules, CA) at RT for 1 hour. For
the remainder of the membrane treatments, 0.05% Tween 20 was added to the milkꢀcontaining TBS buffer and
will be referred to as TT20. Next, the membrane was incubated overnight at 4°C in TT20 containing a 1:2,000
dilution of a monoclonal IgG2b mouse antiꢀGIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a
1:1,000 dilution of monoclonal IgG rabbit antiꢀGIRK2 antibody (cat#25797, Cell Signaling Technology,
Danvers, MA). On the next day, the membrane was washed 3 times with 5 mL of TT20 for 5 min each before
incubating for 1 h at RT in TT20 containing a 1:20,000 dilution of fluorescent IRDye 800CW donkey antiꢀrabbit
antibody (LIꢀCOR Biosciences, Lincoln, NE) and a 1:20,000 dilution of fluorescent IRDye 680LT donkey antiꢀ
mouse antibody (LIꢀCOR Biosciences, Lincoln, NE). Afterwards, the membrane was washed 3 times in 5 mL of
TT20 for 5 minutes before a final rinse with TBS. Fluorescence from secondary antibodies was detected using
the Odyssey CLx (LIꢀCOR Biosciences, Lincoln, NE).
ꢀ
SuperClomeleon Chloride (Cl ) Influx Assay of CysꢀLoop Receptor Activity
Two days before an experiment, TꢀRExꢀHEK293 cells stably expressing SuperClomeleon and KCC2 were
grown to 40% confluency in multiple T25 flasks (Corning, Corning, NY). Half of these flasks were transfected
with α1 GlyR using FuGENE 6, and all the flasks were incubated overnight in a humidified incubator at 37°C
and 5% CO . One day before an experiment, cell medium was treated with 1 ꢁg/mL tetracycline to enable
2
KCC2 expression and cells were transferred onto 384ꢀwell cell plates as described above at 20,000 cells/well.
Each cell plate contained at least 1 transfected and 1 untransfected cell type. On the day of an experiment,
one compound plate containing VU0529331 and ivermectin (SigmaꢀAldrich, St. Louis, MO) at 2ꢀfold the
desired final compound concentrations was generated as described above. A second compound plate
containing 5ꢀfold the desired final glycine concentrations was prepared by hand. Compound plates were used
within an hour of preparation, and the final DMSO concentrations in all conditions assayed was 0.25% (v/v). At
20 minutes prior to imaging, culture medium on cell plates was replaced with 20 ꢁL/well of assay buffer. At the
time of imaging, the experiment was loaded into the WaveFront Biosciences Panoptic, and data were acquired
ꢀ
at 1 Hz with alternating filters set to capture both the Cl ꢀinsensitive cyan fluorescent protein (CFP) signal (440
ꢀ
±
40 nm excitation; 480 ± 17 nm emission filters) and the Cl ꢀsensing yellow fluorescent protein (YFP) signal
(
440 ± 40 nm excitation; 536 ± 40 nm emission). After 10 s of imaging, 20 ꢁL/well from the first compound plate
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ACS Chemical Neuroscience
was added to the cell plate and imaged for 120 s. Next, 10 ꢁL/well from the second compound plate was
added, and imaging was concluded after an additional 120 s. Fluorescence data was collected using
WaveFront Biosciences WaveGuide and analyzed using Microsoft Excel. Statistical analyses were performed
using GraphPad Prism 7. Data reported are the averages of at least 3 independent experiments. The potential
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ꢀ
of a compound to affect the movement of Cl across the cell membrane was calculated from normalized and
controlꢀsubtracted measurements of the area under the curve (AUC) of the FRET ratio (FCFP/FYFP) generated
ꢀ
throughout imaging. In this way, increased signal correlated with increased intracellular Cl concentrations.
Wholeꢀcell Patchꢀclamp Electrophysiology
Serial dilutions of compounds were done by hand in DMSO, and DMSO stocks were diluted into a buffer
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consisting of 20 mM KCl, 140 mM, NaCl, 0.5 mM CaCl , 2 mM MgCl , 10 mM glucose, and 10 mM HEPES (pH
2
2
7.4; 315 mOsm), named the 20K extracellular solution (KES). Cells were prepared by triturating flasks of
engineered cells (cultured and dissociated as described above) to create singleꢀcell suspensions. Cell
suspensions were sparsely plated on 35 mm cell culture dishes (cat#83.3900, SARSTEDT, Nümbrecht,
Germany) and incubated overnight in a humidified incubator at 37°C and 5% CO . Before patching, culture
2
medium was removed and each dish was washed twice with 1 mL of assay buffer (Hanks Balanced Salt
Solution and 20 mM HEPES pH 7.3) before being filled with 1 mL of KES. Borosilicate glass electrodes (3ꢀ7
Mꢂ resistance) were filled with intracellular solution containing 130 mM KCl, 20 mM NaCl, 5 mM EGTA, 5.46
mM MgCl , and 10 mM HEPES (pH 7.4; 305 mOsm). Wholeꢀcell current recordings were conducted in KES
2
using a system comprised of an EPC10 amplifier (HEKA Elektronik, Ludwigshafen am Rhein, Germany), an
MPꢀ285 micromanipulator (Sutter Instruments, Novato, CA), and an Axiovert 200 microscope (Zeiss,
Oberkochen, Germany). HEKA PatchMaster software was used to control the amplifier, enabling
semiautomatic signal compensation for electrode capacitance, cellular capacitance, and series resistance.
Currents were filtered using the 6ꢀpole Bessel prefilter with 10 kHz bandwidth, and signals were digitized at 20
kHz. Cells were exposed to solutions using a homemade, 8ꢀchannel, local superfusion manifold with manual
2+
solution switching. Sensitivity to Ba was measured by applying KES, +/− compound, containing 2 mM BaCl2
during recordings. Currentꢀvoltage (IV) relationships were measured with cells held at −40 mV before switching
between −100 mV and 10 mV in 10 mV steps lasting 250 ms and separated by 500 ms. Current density at
~
200 ms into each pulse for each step was extracted and averaged for 3 consecutive pulse protocols
conducted on an individual cell at each condition. Current density was calculated by dividing current
amplitudes by each cell’s cellular capacitance. Data presented are averages of IVs generated for at least 3
different cells per channel. Data was analyzed using Microsoft Excel. Statistical analyses were performed
using GraphPad Prism 7.
Compound Synthesis
Nꢀ(tertꢀbutoxycarbonyl)ꢀNꢀ(propꢀ2ꢀynꢀ1ꢀyl)glycine (2) : NaH (2.8 g, 2.5 equiv., 60% in mineral oil) was
added to a solution of Bocꢀglycine (1, 5 g, 1.0 eq.) in DMF (100 mL) at 0°C. The suspension was stirred for 1
hr, followed by addition of propargyl bromide (4.95 mL, 1.8 eq., 80% in toluene) at 0°C. The reaction was
allowed to react up to RT overtime and stirred overnight. After completion product was extracted between ethyl
acetate and cold HClꢀwater (pH 4). Organic layer was separated, dried over sodium sulfate, filtered,
1
concentrated and used as such. (Yieldꢀ4.8 g, 80%). H NMR (499 MHz, Chloroformꢀd) δ 4.29 – 4.09 (m, 4H),
2.29 (t, J = 2.5 Hz, 1H), 1.48 (d, J = 20.4 Hz, 9H).
Nꢀ(3ꢀbromopropꢀ2ꢀynꢀ1ꢀyl)ꢀNꢀ(tertꢀbutoxycarbonyl)glycine (3) : To a solution of KOH (2.2 g, 7 eq.) in water
40 mL) at 0 °C was added bromine (0.319 mL, 1.1 eq.). After 30 min, a solution of 2 (1.2 g) in MeOH (15 mL)
(
was added dropwise over 2 min and reaction was stirred for 1 h at 0°C. The solution was diluted with water
and washed with ethyl acetate. Aqueous layer was acidified with HCl (pH 4) and product was extracted with
ethyl acetate. Organic layer was separated, dried over sodium sulfate, filtered, concentrated and used as such.
1
(Yield = 1.24 g, 75%). H NMR (499 MHz, Chloroformꢀd) δ 4.29 – 4.08 (m, 4H), 1.56 – 1.41 (m, 9H).
tertꢀbutyl(3ꢀbromopropꢀ2ꢀynꢀ1ꢀyl)(2ꢀ(2ꢀ(3,4ꢀdimethylphenyl)hydrazineyl)ꢀ2ꢀoxoethyl)carbamate (4): To a
stirred solution of 3 (1 g, 1 eq.), (3,4ꢀdimethylphenyl)hydrazine hydrochloride (1.2 g, 1 eq.), TEA (1.2 mL, 2.5
eq.) in DCM (10 mL) at 0°C was added propylphosphonic anhydride solution ≥50 wt. % in ethyl acetate (2.5
mL, 1.2 eq.). The reaction was stirred at RT for 12 h. Product formed was partitioned between DCM and water.
Organic layer was separated, dried over sodium sulfate, filtered, concentrated, and used as such. (Yield = 1.4
+
+
g crude). Analytical LCMS, single peak (254 nm), R = 2.69 min; MS (ESI ) m/z = 432.1 [M + Na] .
T
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tertꢀbutyl(E)ꢀ(3ꢀbromopropꢀ2ꢀynꢀ1ꢀyl)(2ꢀchloroꢀ2ꢀ(2ꢀ(3,4ꢀ
dimethylphenyl)hydrazineylidene)ethyl)carbamate (5): Resin bound tripehnyl phosphine (6.0 g, 3 eq.) was
added to a stirred solution of 4 (1 g, 1 eq.) and CCl (2 mL, 3 eq.) in CAN (20 mL). Reaction was stirred
4
overnight. Reaction was filtered, filtrate was evaporated and purified by Normal phase flash chromatography
(
0ꢀ50% Ethyl acetate:Hexane). (Yield = 0.31 g, 30%). Analytical LCMS, single peak (254 nm), R = 3.38 min;
T
+
+ 1
MS (ESI ) m/z = 450.0 [M + Na] . H NMR (499 MHz, DMSOꢀd ) δ 9.27 (s, 1H), 7.00 – 6.93 (m, 2H), 6.88 (dd,
6
J = 8.2, 2.3 Hz, 1H), 4.28 (s, 2H), 4.12 (dd, J = 14.5, 6.1 Hz, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 1.45 – 1.36 (m,
9H).
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tertꢀbutyl3ꢀbromoꢀ2ꢀ(3,4ꢀdimethylphenyl)ꢀ2,6ꢀdihydropyrrolo[3,4ꢀc]pyrazoleꢀ5(4H)ꢀcarboxylate (6) : A
solution of compound 5 (0.78 g, 1 eq.) and TEA (0.76 mL, 3 eq.) in toluene (15 mL) was refluxed at 110 °C for
3 hr. Water was added to the reaction mixture, and extracted with ethyl acetate. Organic layer was separated,
dried over sodium sulfate, filtered, concentrated and purified by flash chromatography (0ꢀ50% Ethyl
+
acetate:Hexane). (Yield = 0.49 g, 70%). Analytical LCMS, single peak (254 nm), R = 3.27 min; MS (ESI ) m/z
T
+
1
= 392.0 [M + H] . H NMR (499 MHz, DMSOꢀd ) δ 7.30 (d, J = 8.1 Hz, 2H), 7.22 (dt, J = 8.2, 2.0 Hz, 1H), 4.46
6
(
d, J = 11.3 Hz, 2H), 4.38 (d, J = 17.0 Hz, 2H), 2.29 (s, 6H), 1.47 (d, J = 1.5 Hz, 9H).
3
ꢀbromoꢀ2ꢀ(3,4ꢀdimethylphenyl)ꢀ2,4,5,6ꢀtetrahydropyrrolo[3,4ꢀc]pyrazole (7) : A solution of compound 6
0.45 g) and TFA (1 mL) in DCM 5 mL was stirred overnight and concentrated. The residue was dissolved in
DCM and washed with saturated aqueous NaHCO The organic phase was dried over MgSO , filtered and
(
3.
4
concentrated to afford desired product. (Yield = 0.32 g, 95%). Analytical LCMS, single peak (254 nm), R =
T
+
+ 1
1
.88 min; MS (ESI ) m/z = 292.0 [M + H] . H NMR (499 MHz, DMSOꢀd ) δ 7.28 (dd, J = 5.3, 2.8 Hz, 2H), 7.21
6
(
dd, J = 8.0, 2.3 Hz, 1H), 3.88 (s, 2H), 3.83 (s, 2H), 2.29 (s, 6H).
(
3ꢀbromoꢀ2ꢀ(3,4ꢀdimethylphenyl)ꢀ2,6ꢀdihydropyrrolo[3,4ꢀc]pyrazolꢀ5(4H)ꢀyl)(pyrazinꢀ2ꢀyl)methanone (8) :
pyrazineꢀ2ꢀcarbonyl chloride (0.17 g, 1.1 eq.) was added to a stirred solution of compound 7 (0.32 g, 1 eq.) and
TEA (0.45 mL, 3 eq.) in DCM (8 mL) at 0°C. Reaction was stirred at RT for 2 hr. Product was partitioned
between water and ethyl acetate. Organic layer was dried over sodium sulfate, filtered, concentrated, and
purified by flash chromatography 0ꢀ80% Ethyl acetate:Hexane. (Yield = 0.38 g, 87%). Analytical LCMS, single
+
+ 1
peak (254 nm), R = 2.6 min; MS (ESI ) m/z = 398.0 [M + H] . H NMR (499 MHz, Chloroformꢀd) δ 9.30 (d, J =
T
6.4 Hz, 1H), 8.73 (dd, J = 5.0, 2.4 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 6.5 Hz, 1H), 7.26 (s, 2H), 5.18
(s, 1H), 5.08 (s, 1H), 4.97 (s, 1H), 4.87 (s, 1H), 2.35 (s, 6H). C NMR (126 MHz, CDCl ) chemical shifts of the
major rotamer are reported δ 164.46, 155.84, 153.87, 146.42, 145.93, 142.24, 137.67, 137.49, 129.96, 126.76,
1
3
3
123.03, 121.86, 119.70, 48.83, 47.27, 19.84, 19.56.
(
2ꢀ(3,4ꢀdimethylphenyl)ꢀ3ꢀ(pyridinꢀ4ꢀyl)ꢀ2,6ꢀdihydropyrrolo[3,4ꢀc]pyrazolꢀ5(4H)ꢀyl)(pyrazinꢀ2ꢀ
yl)methanone (9) : A solution of compound 8 (50 mg, 1 eq.), pyridinꢀ4ꢀylboronic acid (30 mg, 2 eq.), K PO (80
3
4
mg, 3 eq.) in dioxane (1.5 mL) was degassed with argon for 10 min. Tetrakis (7.21 mg, 0.05 eq.) was added to
the mixture and reaction was subjected to microwave irradiation at 140 °C for 2 hours. Water was added to
crude, and product was extracted with ethyl acetate. Organic layer was dried over sodium sulfate, filtered,
concentrated, and purified by reverse phase PrepꢀHPLC (0ꢀ100% ACN:water). Analytical LCMS, single peak
+
+ 1
(
254 nm), R = 2.08 min; MS (ESI ) m/z = 397.1 [M + H] . H NMR (499 MHz, DMSOꢀd ) δ 9.08 (dd, J = 17.3,
T
6
1
8
=
.4 Hz, 1H), 8.83 (dd, J = 6.1, 2.5 Hz, 1H), 8.77 (ddd, J = 8.2, 2.6, 1.4 Hz, 1H), 8.59 – 8.56 (m, 1H), 8.54 –
.51 (m, 1H), 7.20 (dt, J = 8.3, 3.3 Hz, 3H), 7.13 – 7.11 (m, 1H), 6.96 (ddd, J = 8.4, 6.5, 2.2 Hz, 1H), 5.07 (d, J
23.8 Hz, 2H), 4.96 (s, 1H), 4.84 (s, 1H), 2.27 – 2.21 (m, 6H). C NMR (126 MHz, DMSO) chemical shifts of
1
3
the major rotamer are reported δ 164.86, 155.48, 150.59, 150.56, 148.94, 146.62, 145.66, 143.51, 138.23,
37.93, 133.70, 130.56, 126.78, 123.40, 122.69, 122.64, 122.20, 120.04, 48.32, 46.95, 19.78, 19.50.
1
(2ꢀ(3,4ꢀdimethylphenyl)ꢀ3ꢀ(pyridinꢀ3ꢀyl)ꢀ2,6ꢀdihydropyrrolo[3,4ꢀc]pyrazolꢀ5(4H)ꢀyl)(pyrazinꢀ2ꢀ
yl)methanone (10) : Same procedure as compound 9. Analytical LCMS, single peak (254 nm), R = 2.2 min;
T
+
+ 1
MS (ESI ) m/z = 397.1 [M + H] . H NMR (499 MHz, DMSOꢀd ) δ 9.08 (dd, J = 15.6, 1.5 Hz, 1H), 8.83 (dd, J =
6
10.4, 2.5 Hz, 1H), 8.77 (dt, J = 14.6, 1.9 Hz, 1H), 8.57 – 8.49 (m, 1H), 8.44 (dd, J = 27.6, 2.2 Hz, 1H), 7.60
(
ddt, J = 53.3, 8.2, 2.0 Hz, 1H), 7.40 (ddd, J = 25.3, 8.0, 4.8 Hz, 1H), 7.22 – 7.14 (m, 2H), 6.94 (dt, J = 8.1, 2.3
13
Hz, 1H), 5.06 (d, J = 15.8 Hz, 2H), 4.93 (s, 1H), 4.84 (s, 1H), 2.23 (d, J = 17.8 Hz, 7H). C NMR (126 MHz,
DMSO) chemical shifts of the major rotamer are reported δ 165.08, 153.36, 149.62, 149.02, 146.55, 145.64,
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43.48, 138.12, 137.06, 136.04, 133.11, 130.47, 126.84, 125.98, 124.12, 123.24, 121.52, 119.25, 48.24,
6.76, 19.78, 19.45.
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9
1
1
1
1
1
1
1
1
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1
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2ꢀ(3,4ꢀdimethylphenyl)ꢀ2,4,5,6ꢀtetrahydropyrrolo[3,4ꢀc]pyrazole (11) : To a solution of compound 8 (60 mg,
1
eq.) in THF (4 mL) at –78°C was added nꢀBuLi (0.15 mL, 2.0 eq., 2.5 M in hexane), the reaction was stirred
at –78°C for 30 min, and quenched with water. Product was extracted with ethyl acetate. Organic layer was
dried over sodium sulfate, filtered, concentrated and used as such. Analytical LCMS, single peak (254 nm), R
=
T
+
+
2.08 min; MS (ESI ) m/z = 214.1 [M + H] .
(
2ꢀ(3,4ꢀdimethylphenyl)ꢀ2,6ꢀdihydropyrrolo[3,4ꢀc]pyrazolꢀ5(4H)ꢀyl)(pyrazinꢀ2ꢀyl)methanone
(12)
:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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8
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0
pyrazineꢀ2ꢀcarbonyl chloride (0.22 mg, 1.1 eq.) was added to a stirred solution of compound 11 (30 mg, 1 eq.)
and TEA (58 ꢁL, 3 eq.) in DCM (1 mL) at 0°C. Reaction was stirred at RT for 2 hours. Product was partitioned
between water and ethyl acetate. Organic layer was dried over sodium sulfate, filtered, concentrated and
purified by reverse phase PrepꢀHPLC. (Yield = 25 mg, 55%). Analytical LCMS, single peak (254 nm), R = 2.08
min; MS (ESI ) m/z = 214.1 [M + H] . H NMR (499 MHz, Chloroformꢀd) δ 9.29 (dd, J = 4.8, 1.5 Hz, 1H), 8.74 –
8.70 (m, 1H), 8.63 (dt, J = 8.0, 1.8 Hz, 1H), 7.69 (d, J = 43.4 Hz, 1H), 7.48 (dd, J = 8.2, 2.3 Hz, 1H), 7.36 (dd, J
T
+
+ 1
=
1
1
4
8.2, 2.3 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 5.14 (d, J = 22.7 Hz, 2H), 4.95 (d, J = 21.7 Hz, 2H), 2.33 (d, J =
13
7.1 Hz, 6H). C NMR (126 MHz, CDCl ) chemical shifts of the major rotamer are reported δ 164.53, 156.22,
54.18, 146.40, 145.85, 142.28, 138.44, 138.02, 135.21, 130.44, 121.05, 120.61, 120.21, 118.77, 116.52,
8.60, 46.84, 19.97, 19.31.
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ASSOCIATED CONTENT
Supporting Information
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Additional figures illustrating the complete western blot, thallium flux fluorescence traces, raw electrophysiology
traces, potassium channel dendrogram, FRET assay results, VU0529331 analog structures, and synthetic
strategy scheme are provided in the supporting information. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
Address: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232ꢀ6600,
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USA. Eꢀmail: david.weaver@vanderbilt.edu. Phone: 615ꢀ936ꢀ6461.
ORCID
Krystian A. Kozek: 0000ꢀ0002ꢀ6951ꢀ9404
Corey R. Hopkins: 0000ꢀ0003ꢀ4958ꢀ1697
Author Contributions
C.D.W. conceived and designed the overall study. K.A.K. conducted the highꢀthroughput screen with the help
of B.D.S., F.J.P., and Y.D. C.D.W. generated the stable GIRK1/2 cell line. Y.D. generated the stable GIRK2,
GIRK2/NPY4R, and GIRK1/4 cell lines. B.D.S. generated the stable Slack, α1β2 MaxiK, and α1β4 MaxiK cell
lines. F.J.P. generated the stable KCC2/SuperClomeleon cell line. C.R.H. designed the target compounds and
S.S. performed the chemical synthesis. K.A.K. conducted the thallium flux experiments, generated the western
blot, and performed the electrophysiology experiments. K.A.K. and F.J.P. conducted the FRET assays. All
authors commented on the manuscript. K.A.K. and C.D.W. coꢀwrote the manuscript.
Funding
Funding for this project was provided by the National Institute of Mental Health (NIMH) of the National
Institutes of Health under the award number 5R21MH099363. This research was supported by funds from the
Vanderbilt University Pharmacology Department and Vanderbilt Institute of Chemical Biology (C.D.W.).
Additional funds were available through University of Nebraska Medical Center startꢀup (C.R.H.). Research
funding was also provided through the PhRMA Foundation’s Paul Calabresi Medical Student Research
Fellowship (K.A.K.). Funding for the WaveFront Biosciences Panoptic kinetic imaging plate reader was
provided by the Office of The Director (OD) of the National Institutes of Health under the award number
1S10OD021734. The Panoptic is housed and managed within the Vanderbilt Highꢀthroughput Screening Core
Facility, an institutionally supported core. Research reported in this publication was supported by the National
Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award numbers
T32GM007347 (K.A.K.), T32GM07628 (B.D.S.), and T32GM065086 (F.J.P.). The content of this manuscript is
solely the responsibility of the authors and does not necessarily represent the official views of the National
Institutes of Health.
Notes
C.D.W. is an owner of WaveFront Biosciences (Franklin, TN), the manufacturer of Panoptic plate readers.
C.D.W. also receives royalties from the sale of Thallos.
ACKNOWLEDGMENTS
We thank the laboratory of Prof. Dr. Annette G. BeckꢀSickinger for their generous supply of the NPY4R
plasmid and the human pancreatic polypeptide. We thank the laboratory of Dr. Colin G. Nichols for their
generous supply of the SUR2b plasmid. We thank the laboratory of Dr. Jennifer A. Kearney for their generous
supply of the K 2.1 plasmid. We thank Dr. Connie Cepko for her generous gift of pCAGꢀGFP (Addgene
V
plasmid # 11150). We are grateful to Dr. Brendan F. Dutter for synthesizing ThallosꢀAM for our laboratory and
for his comments on the manuscript. The NIH Clinical Collection is provided through the National Institutes of
Health Molecular Libraries Roadmap Initiative, and the Marnett library was donated by Dr. Lawrence J.
Marnett. We thank Dr. Paige N. Vinson for her descriptions of the HTS libraries. All the libraries utilized in this
work were distributed by the Vanderbilt Highꢀthroughput Screening (HTS) Core Facility. Many experiments
were performed in the HTS Core with assistance provided by Corbin Whitwell. The HTS Core receives support
from the Vanderbilt Institute of Chemical Biology and the Vanderbilt Ingram Cancer Center (P30 CA68485).
ABBREVIATIONS USED
2+
AUC, area under the curve; Ba , barium ion; CFP, cyan fluorescent protein; CNS, central nervous system; DA,
dopaminergic; DCM, dichloromethane; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EC , 30% of
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maximum efficacy; EC , potency; FRET, fluorescence resonance energy transfer; G2Y4, GIRK2 and NPY4Rꢀ
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expressing HEK293 cells; GABA R, γꢀaminobutyric acid A receptor; G , inhibitory G protein; GIRK, G proteinꢀ
A
i/o
gated inwardlyꢀrectifying potassium channel; GIRK1/X, GIRK1ꢀsubunitꢀcontaining; GlyR, glycine receptor
chloride channel; GPCR, G proteinꢀcoupled receptor; hPP, human pancreatic polypeptide; HTS, highꢀ
+
throughput screen; IV, currentꢀvoltage; K , potassium ion; KCC2, potassiumꢀchloride transporter member 5;
kDa, kilodalton; KES, 20 mM potassium ion extracellular solution; K , inwardlyꢀrectifying potassium channel
ir
+
family; LCMS, liquid chromatographyꢀmass spectrometry; MS, mass spectrometry; Na , sodium ion; NMR,
nuclear magnetic resonance; NPY4R, neuropeptide Y4 receptor; PBS, phosphateꢀbuffered saline; PIP2,
phosphatidylinositol 4,5ꢀbisphosphate; PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C;
+
RT, room temperature; TEA, triethanolamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tl , thallium ion;
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VTA, ventral tegmental area; VU331, VU0529331; YFP, yellow fluorescent protein
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