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Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Proteingated, 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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ACS Chemical Neuroscience
Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-gated, Inwardly-rectifying, Potassium (GIRK) Channels Krystian A. Kozek,1,4,5 Yu Du,1,4 Swagat Sharma,3 Francis J. Prael III, 1,4 Brittany D. Spitznagel,1,4 Sujay V. Kharade,2 Jerod S. Denton,1,2 Corey R. Hopkins,3 and C. David Weaver*,1,4 1
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee, USA 3 Department of Pharmaceutical Sciences, Center for Drug Discovery, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska, USA 4 Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA 5 Vanderbilt Medical Scientist Training Program, Vanderbilt University, Nashville, Tennessee, USA 2
ABSTRACT 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 Kir3.1-4 (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 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 non-GIRK1containing 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 towards 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. 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 opioid epidemic claims tens of thousands of lives each year as people addicted to opioids overdose.1 The difficulty in combating this issue is demonstrated by the increasing number of overdose deaths, which are tied to a dramatic increase in opioid prescriptions for treatment of acute and chronic pain over the last decade.2 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 focus involves a family of inwardly-rectifying potassium (K+) channels, called GIRKs.3–5 ACS Paragon Plus Environment
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The G protein-gated, inwardly-rectifying, K+ channel (GIRK) family, also known as the Kir3 family, is part of the larger inwardly-rectifying potassium channel (Kir) family. GIRK channels function by inhibiting cellular excitability though membrane hyperpolarization. Numerous studies have revealed the importance of GIRK channel function in neural processes6 such as analgesia,7 reward,8–13 anxiety,14 memory,15 respiration,16 and seizures17 as well as in diverse functions including embryonic development,18,19 regulation of heart rate,20,21 and hormone secretion.22,23 The GIRK channel family is comprised of GIRK1, 2, 3, and 4 (Kir3.1, 3.2, 3.3, and 3.4) 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 organs and cell types has been identified.4 GIRK1 or GIRK3 homotetrameric channels are not believed to be expressed in vivo,6,24,25 while GIRK1-containing (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 system25,26 (CNS) while GIRK1 and GIRK4 subunits are expressed predominantly throughout peripheral organs, i.e. in the heart24,27 and pancreas.28 Wherever the GIRK1 subunit is expressed, GIRK1/X channels are therefore expressed; however, non-GIRK1/X channels have been reported in a few discrete brain regions.26 One example of specific GIRK expression is in dopaminergic (DA) neurons of the ventral tegmental area (VTA), which express only GIRK2 and GIRK3 subunits.29 Here, GIRK2 homotetrameric and GIRK2/3 heterotetrameric channels may play a critical role in regulating addiction and reward circuitry.8–13,30 To date, research has shown that the activity of addictive substances like ethanol9 and methamphetamine8 is affected by GIRK3 expression in DA neurons of the VTA. These data raise the intriguing possibility that modulation of non-GIRK1/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 a b 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. K+
Modulation of GIRK channel activity by a number of broadly-active ligands has been studied for many years.31 Phosphatidylinositol 4,5-bisphosphate (PIP2) is necessary for channels to open while βγ-subunits of inhibitory G proteins (Gi/o) are major endogenous intracellular channel activators. Gi/oβγ-subunits of Gi/o proteins bound to Gi/ocoupled G protein-coupled receptors (GPCR) are activated after ligand binding, and these Gi/oβγ-subunits can activate GIRK channels.32,33 Gi/o protein-coupled GPCRs include an array of receptors that are important in normal physiology and are the targets of a large number of drug-based therapies, namely subtypes of opioid, muscarinic, serotonergic, and dopaminergic receptors. GIRK channels have been shown to be targets of alcohol action34, and a discrete alcohol binding pocket on these channels has been discovered and characterized.35–37 Intracellular sodium ions (Na+) have also been shown to modulate GIRK channel activity.38 A crystal structure, solved by Wharton et al., 39 with PIP2, Gi/oβγ-subunit, and 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 cholesterol,40–42 inhibited by Gα subunits of Gi/o-coupled GPCRs,43 decreased due to PIP2 degradation by phospholipase C (PLC) after Gq-coupled GPCR activation,44 increased with phosphorylation by protein kinase A (PKA),45,46 and decreased by phosphorylation by protein kinase C (PKC).47 While GIRK channel
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Figure 1. The thallium (Tl+) flux assay enabled identification of compounds that modulated Tl+ entry pathways on the cellular membrane. (a) When GIRK homotetrameric channels are expressed in a cell, they efflux potassium (K+) under normal physiological conditions. During a Tl+ flux assay, (b) an intracellularly-loaded Tl+-sensitive dye, Thallos, enables (c) measurement of the influx of Tl+ via fluorescence (Fluo.). (d) Channel activators increase Tl+ influx, which increases 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. Recently, our lab has discovered and characterized a number of small-molecule activators of GIRK channels. While these modulators are proving highly useful as probes to investigate a variety of GIRK-expressing systems, i.e. the CNS,14,17,48–50 they show extraordinary selectivity for GIRK1/X channels.51,52 Two critical amino acids in GIRK1 govern the ability of these molecules to activate only GIRK1/X channels,14 and all efforts to generate analogs that can modulate non-GIRK1/X channels have failed. To date, we have generated and tested nearly 800 compounds based on scaffolds discovered by screening >250,000 compounds on GIRK1/2expressing HEK293 cells, and the only non-GIRK1/X-active compound we identified was abamectin, a compound closely related to ivermectin, recently described to activate GIRK channels by Su et al.53 and Chen et al.54 Abamectin and ivermectin belong to the avermectin family of compounds and have been utilized as insecticides55 and antihelminthic medications56 for decades. Although avermectins provide proof-of-concept for pharmacologic probes that activate non-GIRK1/X channels, they are far from ideal. Perhaps most damaging, avermectins are active on a wide variety of ion channels, including many Cys-loop, ligand-gated, ion channels, namely the glycine receptor chloride channel (GlyR),59 the γ-aminobutyric acid A receptor (GABAAR),60,61 the nicotinic acetylcholine receptors (nAChR),62 and the insect glutamate-gated chloride channels (GluR).63,64 Further, ivermectin has been demonstrated to activate P2X purinoceptor 4 (P2X4R).57,58 Several of these avermectin targets are co-expressed 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 > 850 gmol-1) and relatively low potency. Further, the avermectins are complex natural products,65 making synthesis and derivatization considerably more difficult than typical drug-like small molecules. Even in light of the failures of ourselves and others53,54 to discover synthetically tractable small-molecule modulators of nonGIRK1/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 non-GIRK1/X channels. RESULTS AND DISCUSSION In an attempt to discover small-molecule modulators of non-GIRK1/X channels, we performed a highthroughput screen (HTS) of a collection of ~100,000 compounds using a fluorescence-based 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 Gi/o-coupled GPCR capable of increasing the activity of GIRK2 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 partially-activated 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 whole-cell patch-clamp electrophysiology. Because the screen was conducted in HEK293 cells that express a variety of Tl+ influx pathways that may be responsible for false-positive 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 express GIRK2 channels but not NPY4R. We observed VU0529331 activity expression levels shown in untransfected HEK293 cells or cells engineered to in the absence of NPY4R, demonstrating that this phenomenon is express GIRK2, GIRK1/2, or GIRK2 and dependent on GIRK2 expression (Figure 3b). Representative examples of NPY4R. The full gel is shown in Supporting the raw Tl+ flux assay fluorescence traces used to generate concentration Information, Figure 1S. ACS Paragon Plus Environment
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Figure 3. Characterization of VU0529331 efficacy and potency using Tl+ flux. VU0529331 activated GIRK2 and GIRK1/2 channels in a concentration-dependent manner. In contrast, VU0466551 only activated GIRK1-containing channels. Potency (EC50) values were provided for active compounds. Both compounds were inactive in untransfected HEK293 cells. GIRK2 channel activity was normalized to a maximallyeffective concentration of VU0529331, while GIRK1/2 channel activity was normalized to a maximally-effective concentration of VU0466551. All data shown are representative of, at minimum, 3 independent experiments. Error bars represent the standard error of the mean (SEM).
series graphs in Figure 3 are provided in the Supporting Information, Figure S2. Next, we sought to identify whether a natively expressed Gi/ocoupled 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 bacterial exotoxin that inhibits Gi/o-coupled GPCR signaling by catalyzing ADP-ribosylation of the Gα subunit of the G proteins. Through this mechanism, PTX inhibits Gi/oβγ protein signaling to GIRK channels from any native Gi/o-coupled GPCR expressed in HEK293 cells. We conducted Tl+ flux experiments using PTX-treated 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 maximally-effective dose of hPP from activating GIRK channels indirectly (Figure 4b). Because VU0529331 was insensitive to PTX, we concluded that the activity of VU0529331 was independent of Gi/o protein signaling through any Gi/o-coupled GPCR that may be expressed in HEK293 cells. Together, these data suggest that VU0529331 is active on GIRK channels engineered into HEK293 cells. We sought to explore how broadly active this compound was on closely and distantly related K+ channels.
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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 **** b was exciting, since no other small molecules that a VHL 100 100 EC = 10.7 (10.1, 11.2) µM activate homomeric GIRK4 channels have been PTX VHL reported, to our knowledge. To study VU0529331 on EC = 12.2 (11.4, 13.0) µM PTX 75 75 GIRK1/X channels, we compared the activity of VU0529331 against VU0466551, an efficacious, 50 50 potent, and selective activator of GIRK1/X channels **** 50 25 that was previously discovered by our laboratory. We 25 verified that VU0466551 was active on GIRK1/2 0 channels with a potency of ~50 nM and inactive on 0 -6.5 -5.5 -4.5 both untransfected and GIRK2-expressing HEK293 VU0529331 hPP VU0529331 concentration, log[M] cells. We found that the efficacy of VU0529331 on -25 Figure 4. (a) Pertussis toxin (PTX) does not affect the activity of GIRK1/2 and GIRK1/4 was ~25% and ~20% of the VU0529331 on G2Y4 cells while (b) activation of GIRK2 through the Gβγmaximum efficacy of VU0466551, respectively. The subunit after GPCR activation is fully inhibited. The G2Y4 cells express raw Tl+ flux traces (Supporting Information, Figure neuropeptide Y receptor type 4 (NPY4R) in addition to any GPCRs native to HEK293 cells. NPY4R was activated using a maximally-effective S2) demonstrated that VU0529331 was capable of concentration, 200 nM, of human pancreatic polypeptide (hPP). All values generating a much greater total fluorescence in cells in (a) were normalized to the predicted maximum activity of VU0529331 VHL conditions. VU0529331 activity in (b) is normalized data from expressing just GIRK2 as compared to cells under the 30 µM value in (a). 95% confidence intervals for the EC50 expressing GIRK1/2 channels. These data suggest measurements were calculated using a 2-tailed Mann-Whitney test. **** that VU0529331 may be more effective at opening indicates p
