DOI: 10.5061/dryad.547d7wmhn
title: Discovery of a potent, Kv7.3-selective potassium channel opener from a Polynesian traditional botanical anticonvulsant

description: The voltage-gated potassium (Kv) channel formed by Kv7.2/3
 (KCNQ2/3) heteromers is a major target for anticonvulsant drug
 development. Here, we screened 1,444 extracts primarily from
 plants collected in California and the US Virgin Islands, for
 their ability to activate Kv7.2/3 but not inhibit Kv1.3, to
 select against tannic acid being the active component. We
 validated the 7 strongest hits, identified Thespesia populnea
 (miro, milo, portia tree) as the most promising, then discovered
 its primary active metabolite to be gentisic acid (GA). GA highly
 potently activated Kv7.2/3 (EC50, 2.8 nM). GA is, uniquely,
 100% selective for Kv7.3 versus other Kv7 homomers; it requires
 S5 residue Kv7.3-W265 for Kv7.2/3 activation, and it ameliorates
 pentylenetetrazole-induced seizures in mice. Also an active
 aspirin metabolite, GA provides a molecular rationale for the use
 of T. populnea as an anticonvulsant in Polynesian indigenous
 medicine and presents novel pharmacological prospects for potent,
 isoform-selective, therapeutic Kv7 channel activation.
description: <strong>Collection of plant
 samples</strong><br>We collected, between 2019 and 2022,
 aerial parts of plants under permit from Mojave National Preserve (study #
 MOJA-00321), Yosemite National Park (study # YOSE-00839), Santa Monica
 Mountains National Recreation Area (study # SAMO-00192), Muir Woods
 National Monument (study # MUWO-00035), Santa Cruz Island and Santa Rosa
 Island (study # CHIS-0023), and Boyd Deep Canyon (Indian Wells, CA)
 (permit through Philip L. Boyd Deep Canyon. Desert Research 376 Center,
 University of California, Riverside, CA) in California, US. In USVI, we
 collected from Virgin Islands National Park, St. John (study #
 VIIS-20001), Salt River Bay National Historical Park and Ecological
 Preserve, St. Croix (study # SARI-00056) and Buck Island Reef National
 Monument, St. Croix (study # BUIS-00103). Plant samples were collected in
 a manner designed to not kill the remaining plant, sealed in Ziploc bags
 (SC Johnson, Racine, WI, US), kept cold, and frozen as soon as possible.
 Other plant extracts were made from plants purchased from Crimson Sage
 Nursery (Orleans, CA) and grown in the senior author’s garden in Irvine
 CA, Mountain Rose Herbs (Eugene, OR) or Mother’s Market (Irvine, CA).
 Plant samples were stored in -20 oC freezers until extraction.
 <strong>Preparation of plant
 extracts</strong><br>Leaves and flowers were pulverized using
 a bead mill with porcelain beads in batches in 50 ml tubes (Omni
 International, Kennesaw, GA, United States), then the homogenates
 resuspended in 80% methanol/20% water (100 ml per 5 g solid) and incubated
 for 48 hours at room temperature, with occasional inversion to resuspend
 the particulate matter. We then filtered the extracts through Whatman
 filter paper #1 (Whatman, Maidstone, UK), removed the methanol using
 evaporation in a fume hood for 24-48 hours at room temperature,
 centrifuged extracts for 10 minutes at 15 <sup>o</sup>C, 4000
 RCF to remove the remaining particulate matter, followed by storage (-20
 <sup>o</sup>C). On the day of electrophysiological recording,
 we thawed the extracts and diluted them 1:50 in bath solution (see below),
 equivalent to 5 mg fresh plant matter starting material/ml, immediately
 before use. <strong>High-throughput screening for
 Kv7.2/3 activation</strong><br>Plant extracts were applied to
 human Kv7.2/3 channels expressed in HEK293 cells using a FLIPR potassium
 assay kit and a Fluorescence Imaging Plate Reader
 (FLIPRTETRA™) instrument. All chemicals used in this project were
 purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted and
 were of ACS reagent grade purity or higher. Stock solutions of the
 positive controls were prepared in dimethyl sulfoxide (DMSO) or deionized
 water, aliquoted, and stored frozen. Plant extracts were prepared in
 buffer and frozen until dilution into the assay buffers on test day. The
 ability of each plant extract to act as a Kv7.2/3 channel opener
 was evaluated in a thallium (potassium ion surrogate, Molecular Devices)
 flux assay by Charles River Laboratories (Cleveland, OH, US). The assay
 was performed with the FLIPR potassium assay kit (Molecular Devices)
 according to the manufacturer’s instructions. For dye loading, the growth
 media was removed and replaced with 20 μl of dye loading buffer for 60
 minutes at room temperature. For stimulation (agonist mode): 5x (5 μL)
 plant extract, vehicle, or control article solutions prepared in the
 stimulation buffer (K+-free buffer with 5 mM Tl+) was added to each well
 for ~5 minutes. The agonist effects of the plant extract or control
 articles on KCNQ2/3 channels were evaluated. The positive control was
 Flupirtine (9 concentrations). Data acquisition was performed via the
 ScreenWorks FLIPR control software that is supplied with the FLIPR System
 (MDS-AT). Data were analyzed using Microsoft Excel 2013 (Microsoft
 Corp., Redmond, WA). For each well, the raw kinetic data were reduced to
 the maximum or Area Under Curve fluorescence after subtracting bias and
 possibly applying the negative control correction. Reduced data were
 analyzed as follows: For each assay plate, a Z’ factor
 and Signal Window (SW) were calculated:<br>Z’ factor = (([Agonist
 Control mean] – 3 x [Agonist Control STDEV]) – ([Vehicle Control mean] + 3
 x [Vehicle Control STDEV])) / ([Agonist Control mean] – [Vehicle Control
 mean]) SW = (([Agonist Control mean] – 3 x [Agonist Control STDEV]) –
 ([Vehicle Control mean] + 3 x [Vehicle Control STDEV])) / [Agonist Control
 STDEV]<br>Where the stimulation buffer was dispensed to Vehicle
 Control wells and a high concentration of agonist positive control was
 dispensed to Agonist Control wells.<br>Concentration-response curves
 were fitted to the agonist positive control. Reduced data from test
 article wells were normalized to the vehicle and agonist control means on
 each plate and expressed as normalized percent activation: Normalized %
 Activation = ([individual we 429 ll RLU] – [Vehicle Control mean]) /
 ([Agonist Control mean] – [Vehicle Control mean]) where individual well
 RLU = the relative light units for each well to which test article
 is dispensed. A significance threshold of 3 standard deviations from the
 vehicle control mean was calculated: Significance Threshold = 3 x [Vehicle
 Control STDEV] / ([Agonist Control mean] – [Vehicle Control
 mean]) Concentration-response curves for positive agonist controls for
 each plate were also conducted. The positive control results confirmed the
 sensitivity of the test systems to agonists. The test and control samples
 were prepared in the stimulation buffer (a combination of low
 Cl439 buffer, 5 mM Tl2SO4 and water). The signal elicited in the presence
 of the positive agonist control (30 or 100 μM Flupirtine) was set to 100%
 activation and the signal from the vehicle (stimulation buffer) was set to
 0% activation. <strong>High-throughput screening
 for Kv1.3 inhibition</strong><br>Chemicals used in solution
 preparation were purchased from Sigma-Aldrich unless otherwise noted and
 were of ACS reagent grade purity or higher. Stock solutions of plant
 extracts and the positive controls were prepared in water and stored
 frozen, unless otherwise specified. Reference compound concentrations were
 prepared fresh daily by diluting stock solutions into a HEPES-buffered
 physiological saline (HB-PS) (composition in mM): NaCl, 137; KCl,
 4.0; CaCl<sub>2</sub>, 4.8; MgCl<sub>2</sub>, 1;
 HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH. To minimize run-down
 of the Kv1.3 channel currents 0.3% DMSO was added in all reference, plant
 extract and control solutions. The plant extracts (diluted to 2% and 0.2%,
 equivalent to 5 and 0.5 mg fresh plant matter starting material/ml,
 respectively, were loaded into 384-well polypropylene compound plates and
 placed in the plate well of an automated patch-clamp (APC)
 system, SyncroPatchTM 384PE (SP384PE; Nanion Technologies, Livingston, NJ)
 immediately before application to Chinese Hamster Ovary (CHO) cells (stain
 source, ATCC Manassas, VA; substrain source, Chan 456 Test Corporation,
 Cleveland, OH, US) expressing human Kv1.3. Screening was conducted by
 Charles River Laboratories. Extracellular buffer was loaded into the wells
 of the Nanion 384-well Patch Clamp (NPC-384) chips (60 μl per well). Then,
 cell suspension was pipetted into the wells (20 μL per well) of
 the NPC-384 chip. After establishment of a whole-cell patch-clamp
 configuration, membrane currents were recorded using the patch clamp
 amplifier in the SP384PE system. Plant extracts were applied to naïve
 cells (n = 3, where n = the number cells/concentration). Each
 application consisted of addition of 40 μl of 2x concentrated test article
 solution to the total 80 μl of final volume of the extracellular well of
 the NPC-384 chip. Duration of exposure to each test article concentration
 was five (5) minutes. The intracellular solution was (in mM): KCl, 70; KF,
 70; MgCl<sub>2</sub>, 2; EGTA, 2.5; HEPES, 10; pH adjusted to
 7.2 with KOH. In preparation for a recording session, the intracellular
 solution was loaded into the intracellular compartment of the
 NPC-384 chip. The extracellular solution was the HB-PS solution described
 above. Kv1.3 channel currents were elicited using test pulses with fixed
 amplitudes: depolarization pulse to +20 mV amplitude, 200 ms duration from
 the holding potential of –90 mV. The test pulses were repeated with
 frequency 0.1 Hz: 3 min before (baseline) and 5 min after test articles
 addition. Kv1.3 channel current amplitudes were measured at the peak and
 at the end of the step to +20 mV. The positive control antagonist used was
 4-aminopyridine, prepared as a 1 M stock in water; test concentrations
 were 1, 3, 10, 30, 100, 300, 1000 and 3000 μM.
 <strong>Channel subunit cRNA preparation and
 <em>Xenopus laevis</em> oocyte injection for manual
 two electrode voltage-clamp (TEVC)
 electrophysiology</strong><br>We generated cRNA transcripts
 encoding human Kv1.1 (KCNA1), Kv1.2 (KCNA2), Kv2.1 (KCNB1), Kv7.1, Kv7.2,
 Kv7.3, Kv7.4, Kv7.5 (KCNQ1-5), and KCNE3 (MiRP2) by in vitro transcription
 using the mMessage mMachine kit (Thermo Fisher Scientific, Waltham, MA,
 USA) according to manufacturer’s instructions, af 483 ter vector
 linearization, from cDNA sub-cloned into expression vectors (pTLNx, pXOOM
 and pMAX) incorporating <em>Xenopus laevis</em> β-globin 5’
 and 3’ UTRs flanking the coding region to enhance translation and cRNA
 stability. We injected defolliculated stage V and VI <em>Xenopus
 laevis</em> oocytes (Xenoocyte, Dexter, MI, USA) with the channel
 cRNAs (0.3-10 ng) and incubated the oocytes at 16 oC in ND96 oocyte
 storage solution containing penicillin and streptomycin, with daily
 washing, for 1-4 days prior to two electrode voltage-clamp (TEVC)
 recording. Mutant channel cDNAs were generated by GenScript Biotech
 (Piscataway, NJ). <strong>Two-electrode voltage
 clamp (TEVC)</strong><br>We conducted TEVC at room temperature
 with an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) and
 pClamp10 software (Molecular Devices, Sunnyvale, CA, USA) 1-4 days after
 cRNA injection. We visualized oocytes in a small-volume oocyte bath
 (Warner) using a dissection microscope for cellular electrophysiology. We
 studied the effects of plant extracts and constituents solubilized
 directly in bath solution (in mM): 96 NaCl, 4 KCl, 1
 MgCl<sub>2</sub>, 1 CaCl<sub>2</sub>, 10 HEPES (pH
 7.6). We introduced extracts or compounds into the oocyte recording bath
 by gravity perfusion at a constant flow of 1 ml per minute for 3 minutes
 prior to recording. Pipettes (1-2 MΩ resistance) were filled with 3 M KCl.
 We recorded currents in response to voltage pulses between -120 mV or -80
 mV and +40 mV at 10 mV intervals from a holding potential of -80 mV, to
 yield current-voltage relationships. We analyzed data using
 Clampfit (Molecular Devices) and Graphpad Prism software (GraphPad, San
 Diego, CA, USA), stating values as mean ± SEM. We calculated the voltage
 dependence of activation (V<sub>0.5</sub>) by measuring
 currents at a voltage pulse of -30 mV (Kv7) or -50 mV (Kv1) immediately
 following prepulse voltages between -80 mV and + 40 mV. We plotted raw or
 normalized tail currents versus prepulse voltage and fitted them with a
 single Boltzmann function. <strong>Seizure
 studies</strong><br>The mouse study was performed under an
 approved Institutional Animal Care and Use Committee protocol at the
 University of California, Irvine. Male C57Bl/6 mice at 2 months
 of<br>age were injected intraperitoneally (IP) with either vehicle
 (saline) or gentisic acid at 2, 10 or 20 mg/kg (pH7.5). After 30 minutes,
 mice were next injected IP with pentylene tetrazole (80 mg/kg) and then
 observed by a scorer blinded to the treatment, who measured the latency
 to first clonic seizure. <strong><em>In
 silico</em> docking</strong><br>We plotted and viewed
 chemical structures and electrostatic surface potential using Jmol,
 an open-source Java viewer for chemical structures in 3D:
 http://jmol.org/. For in silico ligand docking predictions of binding to
 Kv7.3, we performed unguided docking to predict potential binding sites,
 using SwissDock with CHARMM forcefields and the AlphaFold-predicted human
 Kv7.3 monomer structure. We prepared channel structures for docking using
 DockPrep in UCSF Chimera (https://www.rbvi.ucsf.edu/chimera), with which
 we also generated docking figures.
 <strong>Statistics and
 Reproducibility</strong><br>All values are expressed as mean ±
 SEM. One-way ANOVA (with Dunnett correction for multiple comparisons in
 the case of seizure studies) or t-test was applied for all tests; all
 p values were two-sided. Electrophysiological data were confirmed in at
 least two batches of oocytes. Biological replicates are defined as numbers
 of oocytes; sample sizes are given in the figure legends.
description: ## Source Data for manuscript: Discovery of a potent, Kv7.3-selective
 potassium channel opener from a Polynesian traditional botanical
 anticonvulsant. The datasets included are the original Excel files used to
 generate each panel for figures 1-8 in this manuscript. The title of each
 Excel file is labeled to directly correspond to the figure in the
 manuscript: Figure Number & panel &gt; Channel Investigated &gt;
 Condition &gt; Parameter Measured --- The types of files included within
 this repository are as follows: &gt; Plant Screen &gt; This is a database
 providing the full details of all 1444 plant extracts screened for
 activity against Kv7.2/7.3 and Kv1.3 as summarized by the dot-plot in
 Figure 1A and Figure 1B. The database contains columns providing
 identifying information for each plant where appropriate. The information
 provided in each column is as follows: Plant ID: Unique Identifier of the
 plant sample. The code corresponds to the region the sample was obtained
 and the order for that day. For example - BDC-01 means Boyd Deep Canyon,
 plant number 01. Collection Date: The date the samples were collected
 Macro Region: The precise territory/national reserve the samples were
 collected. Region Curated: The location of the territory/national reserve.
 Region Geotag: The county and state where the samples were collected.
 Genus-species: Latin name of the plant species Common Name: English name
 of the plant species \[2% extract] KCNQ2/3 Normalized % activation: Degree
 of thalium-flux mediated fluorescence \[2% extract] Kv1.3 Peak %
 inhibition (at +20 mV): Percentage inhibition of current density at +20 mV
 Plants highlighted in yellow indicate those summarized in Figure 1B. Cells
 where 'not applicable' is present indicate that either the
 information is unknown or is not pertinent to this study. &gt; IV &gt;
 These data were used to generate current-voltage relationships. Data in
 these files were measured from the peak of the prepulse current. All
 values are in microamps (uA). &gt; Gmax &gt; These data were used to
 generate conductance-voltage curves. Graphs were generated by taking
 measurements from the tail current (-30 mV) immediately following the
 prepulse current. &gt; Dose responses (Idrug/Icontrol - current density)
 &gt; These data were taken from values measured at -60 mV from the tail
 current for control and each subsequent concentration. Raw values are in
 microamps (uA). &gt; Dose responses (Idrug/Icontrol - Resting membrane
 potential) &gt; These data were taken from unclamped oocytes for control
 and each subsequent concentration. Raw values are in millivolts (mV). &gt;
 Seizure assay &gt; These data describe the scores for latency to first
 seizure in the presence of 2, 10, and 20 mg/kg of gentisic acid versus
 saline control. Mice were injected intraperitoneally (IP) 30 minutes prior
 to injection of pentylenetetrazole (80 mg/kg) to induce seizures. Raw
 values are in seconds (s). &gt; Statistics &gt; All statistical analysis
 were conducted as either paired t-test or one-way ANOVA with
 Dunnett's correction for multiple comparison. &gt; Additional
 Information &gt; Excel files with cells containing 'not
 applicable' represents those measurements where no data were
 collected. Changes made since previously published version: The file
 "Figure 3A, B, C, D - Kv7.2-7.3 Portia Tree metabolites 100 uM screen
 - IV, Gmax, EM" was updated to reflect the correct data set used to
 generate the graphs in these figure panels.