Structure-activity relationships, pharmacokinetics, and pharmacodynamics_ of the Kir6.2/Sur1-specific channel opener VU0071063

Article abstract of DOI:10.1124/jpet.119.257204

Glucose-stimulated insulin secretion from pancreatic b-cells is controlled by ATP-regulated potassium (K_ATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The K_ATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular K_ATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular K_ATP channels. VU0071063 induces hyperpolarization of b-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

Full text of DOI:10.1124/jpet.119.257204

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Structure-activity relationships, pharmacokinetics, and pharmacodynamics of the Kir6.2/SUR1-
specific channel opener, VU0071063
Sujay V. Kharade, Juan Vicente Sanchez-Andres, Mark G. Fulton, Elaine L. Shelton, Anna L. Blobaum,
Darren W. Engers, Christopher S. Hofmann, Prasanna K. Dadi, Louise Lantier, David A. Jacobson, Craig
W. Lindsley, and Jerod S. Denton
Department of Anesthesiology, Vanderbilt University Medical Center (S.V.K., J.S.D.)
Department of Medicine, Jaume I University, Castellon de la Plana, Spain (J.V.S.A.)
Department of Chemistry, Vanderbilt University (M.G.F., C.W.L.)
Department of Pediatrics, Vanderbilt University Medical Center (E.L.S.)
Department of Pharmacology, Vanderbilt University (M.G.F., D.W.E., A.L.B., C.S.H., C.W.L., J.S.D.)
Department of Molecular Physiology and Biophysics, Vanderbilt University (P.K.D., D.A.J.)
Mouse Metabolic Phenotyping Core, Vanderbilt University (L.L.)
Vanderbilt Center for Neuroscience Drug Discovery (D.W.E., A.L.B., C.W.L.)
S.V.K. and J.V.S.A. contributed equally to this work
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Running title: Kir6.2/SUR1 activator VU0071063
Corresponding author:
Jerod S. Denton, Ph.D.
T4208 Medical Center North
1161 21st Avenue South
Nashville, TN 37232
jerod.s.denton@vumc.org
Number of text pages: 34
Number of tables: 3
Number of figures: 7
Number of references: 59
Number of words in Abstract: 209
Number of words in Introduction: 696
Number of words in Discussion: 1432
Abbreviations: area under the curve, AUC; predicted hepatic clearance, CLHEP; maximal plasma
concentration, Cmax; Confidence Interval, CI; ATP-regulated potassium channel, KATP; fraction unbound,
fu; glucose-stimulated insulin secretion, GSIS; inward rectifier potassium channel, Kir; sulfonylurea
receptor (SUR); mg/kg, mpk; plasma brain levels, PBL; Time of maximal plasma concentration, Tmax
.
Section assignments: Drug discovery and translational medicine; Endocrine and diabetes.
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Abstract
Glucose-stimulated insulin secretion from pancreatic beta-cells is controlled by ATP-regulated potassium
(KATP) channels comprised of Kir6.2 and SUR1 subunits. The KATP channel opener diazoxide is FDA
approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular
KATP channels and other ion channels. The development of more specific openers would provide critically
needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we
characterize a novel-scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-
throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that
are essential for VU0071063-dependent opening Kir6.2/SUR1. VU0071063 has no effects on
heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open
vascular KATP channels. VU0071063 induces hyperpolarization of beta-cell membrane potential and
inhibits insulin secretion with greater potency than diazoxide. VU0071063 exhibits metabolic and
pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration
of VU0071063 inhibits glucose-stimulated insulin secretion and glucose lowering in mice. Taken together,
these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide
and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential
of Kir6.2/SUR1 expressed in the pancreas and brain.
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Introduction
ATP-regulated potassium (KATP) channels are key determinants of pancreatic beta-cell
electrophysiology, insulin secretion, and blood glucose homeostasis (Nichols, 2006). Elevations in blood
glucose following a meal lead to the uptake and metabolism of glucose in pancreatic beta-cells, an increase
in the ratio of intracellular ATP/ADP (Nilsson et al., 1996), and ATP-mediated inhibition of KATP channel
activity. KATP channel inhibition, in turn, leads to membrane potential (Vm) depolarization, action potential
generation, opening of voltage-gated calcium (Ca2+) channels and influx of Ca2+, and fusion of insulin-
containing granules with the plasma membrane (Nichols et al., 2007). The regulated secretion of insulin
leads to the uptake, utilization, and storage of glucose by insulin-responsive target organs and return of
blood glucose to pre-prandial levels.
Beta-cell KATP channels are hetero-octameric assemblies of four pore-forming inward rectifier
potassium (Kir) channel Kir6.2 (KCNJ11) subunits and four regulatory sulfonylurea receptor 1 (SUR1;
ABBC8) subunits (Aguilar-Bryan et al., 1992; Aguilar-Bryan et al., 1995; Inagaki et al., 1995b; Martin et
al., 2017; Shyng and Nichols, 1997). Kir6.2/SUR1 channels are also expressed in various brain regions
(Karschin et al., 1997; Thomzig et al., 2005), where their physiological roles are not as well understood.
The SUR2 (ABBC9) splice variants, SUR2A and SUR2B, assemble with Kir6.1 (and Kir6.2) in different
types of muscle cells. Whereas vascular smooth muscle cells express Kir6.1/SUR2B channels
predominantly, cardiac myocytes express Kir6.2/SUR2A (atria) and Kir6.1/SUR2B (ventricles)(Aziz et
al., 2014; Flagg et al., 2010; Li et al., 2013; Quayle et al., 1997). The assembly of unique KATP channel
subtypes enables them to carry out specific functions in diverse cell types.
Different KATP channels subtypes exhibit unique pharmacological properties that are imbued by
the SUR subunit (D'Hahan et al., 1999a; Inagaki et al., 1995a; Inagaki et al., 1996; Moreau et al., 2000;
Schwanstecher et al., 1998). This differential pharmacology creates therapeutic opportunities for
modulating the activity of KATP channel activity in specific tissues. Beta-cell Kir6.2/SUR1 channels have
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targeted clinically for the treatment of Type 2 Diabetes since World War II, when the first generation of
inhibitory sulfonylurea drugs were discovered to stimulate insulin secretion and lower blood glucose
(Loubatieres-Mariani, 2007). Sulfonylurea and glinide drugs with improved potency and selectivity for
Kir6.2/SUR1 have been developed in subsequent years and are still in clinical use today (Kharade et al.,
2016).
The KATP channel opener diazoxide (Proglycem) was approved in 1976 by the Food and Drug
Administration (FDA) for treatment of disorders of excessive insulin secretion and associated
hypoglycemia. Diazoxide’s hyperglycemic actions are mediated through opening of pancreatic
Kir6.2/SUR1 channels, hyperpolarization of beta-cell Vm, inhibition of Ca2+ entry, and suppression of
insulin secretion. Diazoxide also lowers blood pressure due to its promiscuous activity on vascular smooth
muscle KATP channels (Standen et al., 1989). Reports of pulmonary hypertension led the FDA to issue a
warning against its use in infants for treating hypoglycemia. In addition, diazoxide can cause shortness of
breath, tachycardia, chest pain, blurred vision, and swelling, among other side effects. These are likely
mediated through the known pleiotropic effects of diazoxide on voltage gated ion channels, mitochondria,
endothelial cells, and cardiac function (Coetzee, 2013). Considerable efforts have been made to develop
diazoxide analogs that are more specific for Kir6.2/SUR1 to avoid these off-target effects (Alemzadeh et
al., 2004; Carr et al., 2003; Dabrowski et al., 2003; Hansen, 2006; Rasmussen et al., 2000; Zdravkovic et
al., 2005).
We previously reported the discovery of VU0071063, a Kir6.2/SUR1 opener that is structurally
unrelated to diazoxide (Raphemot et al., 2014). It was discovered in selectivity screens of inhibitors of
Aedes aegypti (mosquito) Kir1 channels. VU0071063 activates heterologously expressed Kir6.2/SUR1
channels with a half-maximal efficacy concentration (EC50) of 7 µM, acts directly on Kir6.2/SUR1
channels in excised membrane patches, and is selective for SUR1-containing KATP channels over
Kir6.1/SUR2A, Kir6.2/SUR2A, Kv2.1, and other K+ channels. In contrast to diazoxide, VU0071063 does
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not inhibit mitochondrial Complex II. VU0071063 inhibits high glucose-stimulated Ca2+ influx in mouse
pancreatic beta-cells, consistent with activation of native Kir6.2/SUR1 channels. Here, we expanded the
characterization of VU0071063 by studying its structure-activity relationships (SAR), pharmacokinetics,
and pharmacodynamics. We report that VU0071063 is more potent than diazoxide in vitro, does not cause
vasodilation, and inhibits glucose-stimulated insulin secretion (GSIS) in vitro and in vivo.
Materials and Methods
Reagents
Initially, small quantities of VU0071063 was purchased from Calbiochem, and larger quantities
were prepared following the experimental procedure below. Reagents and solvents were purchased from
Sigma-Aldrich or other commercial sources.
Chemical synthesis
Synthesis of VU0071063 analogs was carried about by alkylation of commercially available
theophylline (1) with various benzyl bromides (2). Benzyl bromides that weren’t commercially available
were synthesized by reduction of the benzaldehyde to the alcohol then conversion to the bromide via
Appel reaction or by radical bromination of the methylbenzene. Generally, 1 equivalent of theophylline
and benzyl bromide were dissolved in 1 ml of DMF in a 2 dram screw cap vial at room temperature. Then,
2 equivalents of potassium carbonate were added and the reactions were allowed to stir overnight. The
product was precipitated by addition of 2 mL of water and an additional hour of stirring, then isolation via
vacuum filtration. Impure products were purified by normal phase column chromatography. Products were
isolated in 5-85% yield. Guanine based compounds were synthesized from guanosine using the method
above, except DMSO was used in place of DMF. After overnight stirring, the sugar was cleaved by
addition of concentrated HCl and an hour of stirring, then isolation via vacuum filtration. For VU0071063,
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theophylline (50 mg, 0.278 mmol) and 4-tert-butylbenzyl bromide (63.038mg, 0.278 mmol) were mixed
in a 2 gram vial. To this mixture was added DMF (1 ml, 0.278M) and then potassium carbonate (77.831
mg, 0.555 mmol). The reaction was stirred overnight, forming a white precipitate, to which 1 ml water
was added to induce further precipitation. The white solid was collected via vacuum filtration, washed
with water and dried under vacuum to yield VU0071063 (60mg, 66%) as a white solid. HMBC analysis
1
confirmed N7 alkylation. H NMR (400 MHz, CDCl3) δ (ppm): 7.55 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H),
13
7.26 (d, J = 8.4 Hz, 2H), 5.47 (s, 2H), 3.58 (s, 3H), 3.41 (s, 3H), 1.29 (s, 9H); C NMR (100.6 MHz,
CDCl3) δ (ppm): 155.4, 151.8, 151.7, 148.9, 140.9, 132.5, 127.9, 126.1, 107.1, 50.1, 34.7, 31.3, 29.8, 28.1;
high-resolution mass spectrometry (TOF, ES+): calculated for C18H22N4O2, 326.1743; found, 326.1746.
Quantitative thallium flux assays of Kir6.2/SUR1 and Kir6.1/SUR2B activity
Thallium flux assays of KATP channel activity were performed essentially as described previously
(Raphemot et al., 2014). Briefly, stably transfected T-REx-HEK-293 cells expressing Kir6.2/SUR1 or
Kir6.1/SUR2B were cultured in 384-well plates (20,000 cells/20 μL per well; black-walled, clear-
bottomed PureCoat amine-coated plates; BD bioscience, Bedford, MA) with a platting media containing
Dulbecco’s modified Eagle’s medium containing 10% dialyzed fetal bovine serum, 1µg/mL G-418,
50ꢀU/mL penicillin, 50ꢀμg/mL streptomycin, 5ꢀμg/mL blasticidin S, and 250ꢀμg/mL hygromycin, and were
induced overnight with 1ꢀμg/mL tetracycline to express Kir6.2/SUR1 or Kir6.1/SUR2B channels. On the
day of the experiment, the cell culture medium was replaced with dye-loading solution containing assay
buffer (Hank’s balanced salt solution with 20 mM HEPES, pH 7.3), 0.01% (v/v) Pluronic F-127 (Life
Technologies), and 1.2 μM of the thallium-sensitive dye Thallos-AM (WaveFront Biosciences, Franklin,
TN). Following 1-hour incubation at room temperature, the dye-loading solution was washed from the
plates and replaced with 20 μl/well of assay buffer. The plates were transferred to a Panoptic kinetic
imaging plate reader (Wavefront Bioscience, Franklin, TN, USA) where 20 μL/well of test compounds
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dissolved in assay buffer was added to wells. After an 8-min incubation period, a baseline recording was
collected at 1 Hz for 10 seconds (excitation 470 ± 20 nm, emission 540 ± 30 nm) followed by addition of
10 μl/well thallium stimulus buffer (125 mM NaHCO3, 1.8 mM CaSO4, 1 mM MgSO4, 5 mM glucose,
1.8 mM Tl2SO4, and 10 mM HEPES, pH 7.4) and data collection for 4 mins. All compounds were tested
at concentrations to obtain 11-point three-fold dilution CRCs. The data acquisition and analysis were
performed using Waveguide (VU-HTS center) and Microsoft excel. EC50 values were determined by
fitting the Hill equation using variable-slope nonlinear regression analyses performed with GraphPad
Prism version 5.01 (GraphPad Software, San Diego, CA).
Animal studies
The rat studies performed at the Jaume I University, the animal housing, and all protocols were
approved and in accordance with the institutional animal care guidelines, Spanish animal protection law,
and conform to Directive 2010/63 EU of the European Parliament. All animal procedures performed at
Vanderbilt were approved by the Vanderbilt Animal Care and Use Committee.
Pressure myography
Ductus arteriosus (DA) vessels, which are enriched in Kir6.1/SUR2B channels (Shelton et al.,
2014; Yarboro et al., 2018), were isolated from term-gestation fetal mice and mounted on glass pipette
tips submerged in microvessel perfusion chambers as previously reported (Hooper et al., 2016; Pfaltzgraff
et al., 2014; Reese et al., 2009). Chambers were placed on inverted microscopes equipped with IonOptix
digital image capture and dimensioning software. Vessels were pressurized in a stepwise manner to fetal
mouse mean arterial pressure (20 mmHg) using a column of Krebs buffer. Vessels were then challenged
with 50 mM KCl to test for reactivity and non-contractile vessels were excluded from further study. For
dose-response studies, vessels were treated with increasing concentrations (1 nM – 1 mM) of VU0071063
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or pinacidil (Sigma-Aldrich). Lumen diameter measurements were allowed to plateau (20-60 minutes)
before addition of the next concentration of drug. At the conclusion of each experiment, vessels were
challenged with 50 mM KCl to confirm viability. An N of 8 vessels from multiple litters was used for
each experimental protocol. Changes in vessel diameter were expressed as a percent change in lumen
diameter compared to baseline diameter at resting tone. Dose-response relationships were illustrated
using point-fit curves. Myography studies are expressed as means + SEM. GraphPad Prism version 6.0
was used to analyze the data. Dose-response curves were compared using two-way ANOVA followed by
a Tukey’s multiple comparisons test. A multiplicity adjusted P value <0.05 was taken to be indicative of
statistical significance.
Mouse islet electrophysiology
The Vm of beta-cells was recorded using sharp microelectrodes from dissected islets of Langerhans
obtained from albino mice, killed by cervical dislocation, as previously described (Sanchez-Andres et al.,
1988). The modified Krebs solution used for the isolation had the following composition (in mM): 120
NaCl, 25, NaHCO3, 5 KCl, 2.6 CaCl2 and 1 MgCl2, and was equilibrated with a gas mixture containing
95% 02-5% CO2 at 37 °C (pH 7 4). An Axoprobe microelectrode amplifier (Axon Instruments, Foster
City, CA) was used for performing electrophysiological recordings. Borosilicate microelectrodes (o.d.,
2.0 mm; i.d., 1.0 mm; Sutter Instruments, Novato, CA, USA) were pulled with a Narishige PE2 puller
(Narishige, Japan) and filled with 3 M potassium citrate and 50 mM KCl. Data were acquired at 1 kHz
frequency sampling using Clampex software (v10.6, Molecular Devices, Sunnyvale, CA, USA) through
an acquisition card (Digidata 1500, Molecular Devices, Sunnyvale, CA, USA), and stored on computer
hard disk for further analysis using ClampFit (v10.6, Molecular Devices, Sunnyvale, CA, USA).
Islet cells were impaled in the presence of 10 mM glucose in modified Krebs solution to identify
cells exhibiting robust oscillatory electrical behavior typical of beta-cells. Beta-cells were further
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identified by the characteristic membrane hyperpolarization and cessation of electrical activity upon
perfusion of modified Krebs solution without glucose. The minimal criteria for cell viability and quality
were: at least -40 mV membrane potential during the silent phases of oscillations, 10 mV of depolarization
in response to 20 mM glucose addition, and 10 mV action potential amplitude. Current clamp were
performed as follows. Islets were first exposed to 20 mM glucose and then 0 mM glucose to establish
maximal (100%) and minimal (0%) KATP channel inhibition, respectively (Gopel et al., 1999). Increasing
concentrations of VU0071063 and diazoxide were then added to 20 mM glucose-containing buffer to
induce membrane hyperpolarization. Recordings were made for 2-3 mins after compound addition to
allow the cells to reach a steady-state. The Vm recorded in the absence of glucose was subtracted from that
recorded in each drug dose, and this difference was normalized to membrane potential recorded in 20 mM
glucose. A similar subtraction procedure was used to determine if the KATP channel inhibitor tolbutamide
(50 µM) could reverse the membrane potential hyperpolarization induced by 20 µM VU0071063. The
tolbutamide effect was calculated as the percentage of the Vm recorded in the presence of 20 mM glucose.
Statistically significant differences were determined by 2-way ANOVA with Tukey’s or Sidak’s multiple
comparison post-hoc test.
Drug metabolism and pharmacokinetics
Detailed methods for determining VU0071063 drug metabolism and pharmacokinetic (DMPK)
properties are described in Supplemental Methods.
In vitro insulin secretion assays
Islets were isolated from pancreata of 8- to 10-week-old C57BL/6 mice, by digesting the pancreas
with collagenase P (Roche) and performing density gradient centrifugation as previously described (Roe
et al., 1994). Islets were cultured for 16 hours in RPMI 1640 (11875 Gibco) medium supplemented with
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15% fetal bovine serum, 100-IU mL−1 penicillin, and 100-mg mL−1 streptomycin before secretion studies.
Islets were maintained in a humidified incubator at 37°C under an atmosphere of 95% air–5% CO2. After
a 16-hour recovery, mouse islets were incubated with DMEM containing 5.6 mM glucose for 1 hour
followed by secretion. For insulin secretion measurements, 15 islets were incubated in tubes at 37°C
containing either 2 mM (low) or14 mM (high) glucose in DMEM with or without the test compound for
1 hour. The supernatant was collected, and insulin content was analyzed using an RIA-based detection by
the Vanderbilt Hormone Core.
In vivo glucose tolerance tests
All procedures were performed in the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC;
http://www.vmmpc.org) and were approved by the Vanderbilt Animal Care and Use Committee. Male
C57BL/6 mice were purchased from Envigo at 8 weeks of age and studied at 10-12 weeks of age. Housing
was temperature (23 ⁰C) and humidity controlled on a 12-h light:dark schedule with mice given free access
to food (Harlan Teklad LM-485, no. 7912) and water. Mice were injected intraperitoneally (at t = -15)
with either vehicle (0.5% methyl cellulose + 10% tween 80 in water), indicated dose of diazoxide, or
VU0071063 followed by 1 g/kg of glucose (at t=0, 15 mins after drug dose). Blood samples (5 µl for
glucose or 50 µl for insulin) were collected from the tail vein. Blood glucose was measured at = -15, 0,
5, 10, 15, 20, 30, 45, 60, 90, and 120 min. Samples to measure plasma insulin were taken at 0, 10, 30, 60
and 120 min.
Results
VU0071063 SAR
Optimization efforts for the VU0071063 scaffold were initially aimed at improving potency
toward Kir6.2/SUR1 channels heterologously expressed in HEK293 cells (Fig. 1; Supplemental Table
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S1). Thallium flux assays were used to quantitate channel activation by synthetic analogs. Parallel
synthesis with a varied set of benzyl bromides afforded 29 compounds, none of which improved upon
VU0071063. Moving the tert-butyl group to the 3 position (3a) resulted in a 3-fold loss in potency and 2-
fold loss in efficacy, while the 2 substitution (3b) lost activity entirely. Modest activity was observed in
analogs with trifluoromethoxy substitution at the 3 position (3u), while moving the trifluoromethoxy
group to the 4 position (3t) was 1.5-fold less potent than VU0071063. The most potent alkyl-substituted
analog was the 4-isopropyl (3c), indicating that the steric bulk of tert-butyl was necessary for optimal
interaction with the channel protein. Due to the steep SAR encountered on the eastern portion of the
molecule, efforts were next focused on changes to the western ring. Removal of the N1 methyl group while
keeping the 4-tert-butyl benzyl (3ae) did not prove efficacious. Shifting back to the eastern portion,
substitution of a tertiary amine or hydroxyl (3ac-ad) on the benzyl ring was introduced to increase
hydrogen bond interactions. These were synthesized by action of methyl magnesium bromide on the nitrile
and ketone, respectively, but these analogs were inactive. Changing the scaffold to guanine (3am-3an) in
the hope of increasing the polarity of the ring system showed that the theophylline backbone was necessary
for activity.
VU0071063 does not actívate heterologously expressed Kir6.1/SUR2B or relax ductus arteriosis vessel
tone
We evaluated the activity of VU0071063 on Kir6.2/SUR1 and Kir6.1/SUR2B channels
heterologously expressed in HEK-293 cells, as well as on native Kir6.1/SUR2B expressed in ductus
arteriosis vessels. Theywere chosen for these studies because they are enriched in vascular KATP channels
comprised of Kir6.1 and SUR2B (Shelton et al., 2014; Yarboro et al., 2018). As shown in Fig. 2A,
VU0071063 and diazoxide dose-dependently opened Kir6.2/SUR1 with EC50 values of 7.44 µM (95%
Confidence Interval [CI]- 6.98 to 7.90 µM) and 78.42 µM (95% CI- 74.13 to 83.20 µM), whereas DMSO
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(solvent control) and pinacidil did not. Both pinacidil (EC50 = 6.50 µM; 95% CI- 5.781 to 7.33 µM) and
diazoxide (no fit) activated Kir6.1/SUR2B channels whereas VU0071063 (or DMSO) did not (Fig. 2B).
Thus, VU0071063 is selective for Kir6.2/SUR1 over Kir6.1/SUR2B channels. Next, term-gestation mouse
ductus arteriosis vessels were challenged with increasing concentrations of the SUR2-specific opener
pinacidil (positive control), diazoxide, or VU0071063 (1 nM – 1 mM). As expected, exposure of DA
vessels to pinacidil led to a dose-dependent vasodilation by activating vascular KATP channels. Diazoxide
also induced relaxation at higher concentrations. However, VU0071063 had no significant effect on DA
tone (Fig. 2C). Taken together, these data point to the ability of VU0071063 to discriminate between
Kir6.2/SUR1 and Kir6.1/SUR2B channels.
VU0071063 inhibits beta-cell excitability in mouse islets
We next determined if VU0071063 modulates native KATP channel activity in a manner consistent
with its pharmacological properties defined using heterologously expressed Kir6.2/SUR1 (Raphemot et
al., 2014). In those studies, the EC50 for activation of Kir6.2/SUR1 expressed in HEK-293 cells was
approximately 7 µM. In the present study, beta-cell Vm was used as a proxy for KATP channel activity
since Vm is largely determined by the activity of resident KATP channels in these cells (Nichols, 2006;
Gopel, et al., 1999). Sharp microelectrodes were used to measure the Vm from excised mouse islets. Beta-
cells were distinguished from other cell types by their responses to glucose (see Materials Methods). As
shown in Fig. 3A-B., increasing bath glucose from 0 mM to 20 mM led to a mean ± SEM depolarization
of 27.7 ± 1.7 mV (n=12) mV and action potential generation. In the continuous presence of 20 mM
glucose, both diazoxide and VU0071063 led to a dose-dependent hyperpolarization of Vm and cessation
of action potentials. Importantly, however, VU0071063 inhibited beta-cell excitability at lower doses than
diazoxide. To ensure that the effects of VU0071063 on Vm were mediated through KATP, we confirmed
that tolbutamide could reverse the effects of VU0071063 (Fig. 3D).
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VU0071063 inhibits glucose-stimulated insulin secretion in vitro
We previously reported that VU0071063 inhibits glucose-induced Ca2+ influx in isolated mouse
beta cells, prompting us to examine in the present study if VU0071063 can inhibit insulin secretion in a
KATP channel-dependent manner. Isolated mouse islets were treated with either 2 mM (low) or 14 mM
(high) glucose alone or together with 10 µM VU0071063 or 10 µM diazoxide for 60 mins. The inactive
VU0071063 analog 34MT (Fig. 4A) was used as a negative control to confirm that any effects of
VU0071063 on insulin secretion are mediated through KATP channels. As shown in Fig. 4B, high glucose
significantly stimulated insulin secretion (35.6 ±8.1 ng/ml) above that observed under low glucose
conditions (1.6 ±0.3 ng/ml). As expected, 10 µM diazoxide significantly reduced the glucose-stimulated
insulin secretion (21.14 ± 1.6 ng/ml). Importantly, the same concentration of VU0071063 virtually
abolished the glucose-stimulated insulin secretion (4.01 ±0.84 ng/ml). Consistent with VU0071063-
dependent inhibition of insulin secretion being mediated through activation of KATP, 34MT had no effect
on GSIS.
VU0071063 exhibits favorable drug metabolism and pharmacokinetic properties in mice
VU0071063 possesses favorable physiochemical properties and displays an attractive drug
metabolism and pharmacokinetic profile in both rat and mouse (Table 1). VU0071063 is a low molecular
weight compound (mw = 326 g/mol) with low lipophilicity (log P = 2.64) and is Lipinski compliant. In
vitro, VU0071063 shows good free fraction in both rat and mouse plasma (rat fu = 0.048, mouse fu =
0.133), good fraction unbound in rat and mouse brain homogenate (rat fu = 0.023, mouse fu = 0.024) and
moderate predicted hepatic clearance in rat (CLhep = 49.6 mL/min/kg) and moderate to high in mouse
(CLhep = 73.2 mL/min/kg). VU0071063 achieved high brain exposure in both rat (Kp = 8.06, Kp,uu = 3.86)
and mouse (Kp = 2.22, Kp,uu = 0.4). In the case of the rat, it is possible that there is active uptake into the
brain via a transporter mechanism. When dosed at 30 mg/kg intraperitoneally in mice, VU0071063
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achieved a Tmax at 0.25 hr, a Cmax exposure of 6.8 µM and a plasma AUC(0-4 hr) of 11.4 µM, providing
sufficient exposure to support in vivo studies (Fig. 5).
VU0071063 inhibits glucose-stimulated insulin secretion in vivo
The drug metabolism and pharmacokinetic properties of VU0071063 suggested that it could be a
useful tool compound for activating Kir6.2/SUR1 channels in vivo. To determine if VU0071063 could
engage beta-cell KATP channels in vivo, we measured its effects on blood glucose and insulin levels in
glucose tolerance tests in mice. Based on the Tmax of VU0071063 (Fig. 5), mice were injected IP at t = -
15 min with either vehicle (0.5% methyl cellulose + 10% tween 80 in water), 50 mg/kg diazoxide, or 50
or 100 mg/kg VU0071063, followed by IP administration of glucose (1g/kg) 15 mins later (t=0 min).
Blood samples were collected periodically from the tail vein for determination of blood glucose and
insulin concentrations. As expected, IP administration of glucose led to a significant increase of both blood
glucose (Fig. 6A2) and insulin (Fig. 6B2) levels in vehicle-treated animals at the 60-min time point. Blood
glucose in vehicle-treated animals exhibited a biphasic response over the 120-min collection period and
began to wane toward the end of the experiment due to pancreatic secretion of insulin (Fig. 6A3). Both
diazoxide and VU0071063 when administered at a dose of 50 mg/kg led to a significant increase in blood
glucose at 60 min, but only the diazoxide effect was sustained at 120 min. The relatively short
pharmacodynamic response of VU0071063 likely reflects its rapid clearance from the plasma following
administration (Fig. 5). Diazoxide or VU0071063 (50 mg/kg) significantly reduced plasma insulin
concentration at the 60-min and 120-min time points, however insulin levels began to rebound at 120-min
compared to the 60-min time point in diazoxide-treated mice. The effect of 50 mg/kg VU0071063 on
insulin levels was sustained through 120 min. Increasing the dose of VU0071063 to 100 mg/kg
significantly lowered insulin and increased glucose as compared to 50 mg/kg diazoxide.
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Discussion
The major findings of this study are: 1) there is a very steep SAR around the VU0071063
chemotype; 2) VU0071063 does not cause vasodilation of DA vessels, indicating a lack of activity on
Kir6.1/SUR2B channels; 3) VU0071063 inhibits insulin secretion from isolated beta-cells by
hyperpolarizing Vm, and does so with greater potency than diazoxide; 4) VU0071063 exhibits favorable
exposure in the plasma and brain following IP administration in mice; and 5) IP administration of
VU0071063 inhibits glucose-stimulated insulin secretion in a manner that is similar to diazoxide. Taken
together with our previous study (Raphemot et al., 2014), this work indicates that VU0071063 is superior
in its potency and selectivity to diazoxide and should be a useful tool compound for teasing out specific
roles of Kir6.2/SUR1 channels in vitro and in vivo.
To our knowledge, VU0071063 is the only Kir6.2/SUR1-specific opener that is not a synthetic
derivative of diazoxide. Novo Nordisk reported on several diazoxide derivatives that exhibit improved
selectivity for Kir6.2/SUR1 over vascular KATP channels. They developed these compounds with the goal
of improving glucose responsiveness in type 1 and 2 diabetic patients by inducing so-called “beta-cell
rest”, a concept that inhibiting the electrical activity of beta-cells will protect beta-cell mass and improve
glucose-stimulated insulin secretion. Two of the more widely studied compounds from this work are
NNC-55-0118 (6-chloro-1,1-dioxo-N-propan-2-yl-4H-thieno[3,2-e][1,2,4]thiadiazin-3-amine and NN414
(6-chloro-N-(1-methylcyclopropyl)-1,1-dioxo-4H-thieno[3,2-e][1,2,4]thiadiazin-3-amine)(Table
2).
Both compounds open Kir6.2/SUR1 channels heterologously expressed in HEK-293 cells with sub-
micromolar potency and exhibit no activity toward Kir6.2/SUR2A or Kir6.2/SUR2B expressed in
Xenopus oocytes (Dabrowski et al., 2003). Diazoxide and NNC-55-0118 (both at 100 mg/kg/day by oral
gavage) showed partial and comparable (i.e. ~30%) efficacy in protecting beta-cell mass in diabetic rats
(Rasmussen et al., 2000). NN414 also showed favorable in vitro and in vivo efficacy in protecting beta-
cell mass and function (Alemzadeh et al., 2004; Carr et al., 2003; Maedler et al., 2004; Ritzel et al., 2004;
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Skak et al., 2004) and eventually progressed to human trials. In a limited study of 24 patients with type 2
diabetes treated with NN414 for 7 days, no improvements in glycemic control were observed (Zdravkovic
et al., 2007).
As noted earlier, VU0071063 is structurally distinct from NN414 and NNC-55-0118, prompting
us to expand on the SAR reported previously by our group (Raphemot et al., 2014). The bicyclic system
of VU0071063 is identical to that of theophylline, which prompted us originally to test if theophylline
itself could activate Kir6.2/SUR1. However, theophylline at a concentration of 250 µM had no effect on
Kir6.2/SUR1 activity, indicating the eastern arm of VU0071063 (Table 2) attached to theophylline is
essential for opening the channel. Here, we found that the SAR of the eastern arm was very steep, as
migrating the tert-butyl group from the 4-position around the ring led to a step-wise loss of potency toward
Kir6.2/SUR1. Replacing the 4’-tert butyl group with several other moieties also led to a loss of activity,
suggesting that the steric bulk of tert-butyl is important for optimal activity.
We previously reported that VU0071063 is selective for SUR1 over SUR2A (Raphemot et al.,
2014). Thus, VU0071063 can open either Kir6.1 or Kir6.2 pores when expressed with SUR1, but not
when they are expressed with SUR2A. This indicates that the VU0071063 binding site is likely localized
in SUR1. This pharmacological profile predicted that VU0071063 should be able to modulate pancreatic
and brain (see below) KATP channel activity without having untoward effects on vascular physiology
mediated through Kir6.1/SUR2B channels. Indeed, in the present study, we showed that whereas the
SUR2-selective opener pinacidil causes vasodilation of DA vessels, VU0071063 had no significant effect
at a concentration that fully inhibits glucose-stimulated insulin secretion in isolated beta cells. As noted
earlier, diazoxide can induce vasodilation and lower blood pressure via off-target effects on vascular KATP
channels. Opening of vascular KATP channels can be mediated through direct actions on the channel or
through inhibition of mitochondrial Complex II and oxidative phosphorylation (Grimmsmann and
Rustenbeck, 1998). The subsequent reduction of cytosolic ATP concentrations could lead to disinhibition
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of KATP channel activity and enhancement of diazoxide sensitivity (D'Hahan et al., 1999b). The lack of
activity of VU0071063 on mitochondrial Complex II (Raphemot et al., 2014) likely increases its
selectivity toward pancreatic and brain over vascular KATP channels.
With emerging data implicating Kir6.2/SUR1 as a therapeutic target for the treatment of various
neurological diseases, there is a growing need for the development of brain-penetrant openers for
exploring the integrative physiology and druggability Kir6.2/SUR1 in the brain. There is surprisingly little
data on the broader expression patterns of different KATP channel subunit proteins in the CNS. Kir6.2 and
SUR1 mRNA expression appears to be widespread and overlapping in the rodent brain, with higher levels
of expression in the hippocampus, neocortex, olfactory bulb, cerebellum, midbrain, and brainstem
(Karschin et al., 1997). In contrast, Kir6.1 appears to be weakly expressed in the brain (Thomzig et al.,
2005). The SUR2 splice variants SUR2A and SUR2B exhibit distinct expression patterns, with SUR2A
mRNA expression highest in neurons and SUR2B mRNA expression highest in certain neuronal
populations, astrocytes, and oligodendrocytes (Zhou et al., 2012). With few exceptions, the myriad
functions of molecularly defined KATP channels in the brain are poorly understood.
In comparison, KATP channels expressed in the hippocampus have been studied extensively and
contribute to neuronal protection during ischemia (Sun et al., 2006) and memory processes (Betourne et
al., 2009). They are composed primarily of Kir6.2 and SUR1 subunits (Betourne et al., 2009; Sun et al.,
2006; Tanner et al., 2011), are opened by diazoxide, and are blocked by the SUR1-preferring inhibitor
tolbutamide (Matsumoto et al., 2002; Ohno-Shosaku and Yamamoto, 1992; Zawar and Neumcke, 2000).
Hippocampal KATP channels may provide a mechanistic link between hyperglycemia in type 2 diabetes
and the development of Alzheimer’s disease (AD)(Crane et al., 2013; Huang et al., 2014; Ott et al., 1999).
Holtzman and colleagues reported that elevation of peripheral blood glucose in a mouse model of AD led
to a corresponding increase in hippocampal interstitial fluid glucose, lactate (a proxy of neuronal activity),
and amyloid-β concentrations (Macauley et al., 2015). Importantly, microdialysis of glibenclamide or
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diazoxide into the hippocampus enhanced and inhibited, respectively, hyperglycemia-induced increase in
amyloid-β. These and other data (Moriguchi et al., 2018; Salgado-Puga et al., 2017) suggest that
hippocampal Kir6.2/SUR1 channels may represent a novel therapeutic target for AD. To our knowledge,
the ability of NNC-55-0118 and NN414 to cross the blood-brain-barrier (BBB) has not been reported.
However, VU0071063 readily crosses the BBB following peripheral administration and could be useful
for evaluating the therapeutic potential of Kir6.2/SUR1 channels for treating AD.
The data presented here and previously (Raphemot et al., 2014) show that VU0071063 can be used
to manipulate beta-cell physiology both in vitro and in vivo. In isolated cells, VU0071063 dose-
dependently and reversibly hyperpolarizes the beta-cell membrane potential, which, in turn, inhibits
glucose-stimulated Ca2+ entry and insulin secretion. The actions of VU0071063 on the beta-cell membrane
potential are reversed by tolbutamide, and glucose-stimulated insulin secretion is unaffected by the
inactive analog 34MT, indicating that the effects are mediated through Kir6.2/SUR1. VU0071063 is more
potent (this study) and faster acting (Raphemot et al., 2014) than diazoxide and might therefore represent
a better in vitro tool compound for opening Kir6.2/SUR1. VU0071063 exhibits favorable drug metabolism
and pharmacokinetic properties that enable it to engage KATP channels in vivo. Following IP administration
at a dose of 30 mg/kg, VU0071063 reaches peak plasma concentration of over 2,000 ng/mL at 15 minutes
and declines over the following 4 hours. Brain VU0071063 levels reach concentrations twice that of
plasma VU0071063 at 15 minutes post-administration and then decline with a similar time course.
Diazoxide and VU0071063 were similarly efficacious at inhibiting glucose-stimulated insulin secretion
and raising blood glucose over a 2-hour period. Opening of hypothalamic KATP channels with diazoxide
has been reported to inhibit peripheral gluconeogenesis in rodents (Pocai et al., 2005) and humans
(Kishore et al., 2011). It will be of interest in future studies to determine if activation of brain KATP
channels with VU0071063 contributes to glucose homeostasis in vivo.
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In conclusion, VU0071063 is a new tool compound for opening Kir6.2/SUR1 channels in vitro
and in vivo. VU0071063 is specific for SUR1-containing KATP channels, opens native pancreatic KATP
channels in vitro and in vivo, and inhibits glucose-stimulated calcium signaling and insulin secretion from
beta cells. To our knowledge, VU0071063 is the only Kir6.2/SUR1-specific opener to be discovered in a
molecular target-based high-throughput screening campaign. Unlike the other Kir6.2/SUR1-specific
channel activators, NN414 and NNC-55-0118, VU0071063 is structurally unrelated to diazoxide. The
discovery of VU0071063 suggests that structurally diverse activators of Kir6.2/SUR1 channels can be
discovered with HTS in the future.
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Acknowledgments: We wish to acknowledge the assistance of the Vanderbilt Mouse Metabolic
Phenotyping Center (DK059637) and the Vanderbilt Hormone Assay and Analytical Core (DK059637,
DK020593). We thank Dr. Colin Nichols (Washington University) for the SUR2B cDNA.
Authorship Contributions:
Participated in research design: Kharade, Sanchez-Andres, Fulton, Shelton, Engers, Hofmann, Dadi,
Lantier, Jacobson, Lindsley, Denton
Conducted experiments: Kharade, Sanchez-Andres, Fulton, Shelton, Engers, Hofmann, Dadi
Contributed new reagents or analytical tools: Fulton, Engers, Lindsley
Performed data analysis: Kharade, Sanchez-Andres, Fulton, Lantier, Shelton, Hofmann
Wrote or contributed to the writing of the manuscript: Kharade, Sanchez-Andres, Shelton, Lantier,
Lindsley, Denton
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Footnotes: This work was supported in part by the National Institutes of Health National Institute of
Diabetes and Digestive and Kidney Diseases [Grant R01 DK082884; R01 DK115620]; National Institutes
of Health National Heart, Lung, and Blood Institute [Grant R21 HL132805]; American Diabetes
Association [Grant 1-17-IBS-024]; American Heart Association [Grant AHA15SDG25280015]
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