New positive Ca2+-activated K+ channel gating modulators with selectivity for KCa3.1

Article abstract of DOI:10.1124/mol.114.093286

Small-conductance (K_Ca2) and intermediate-conductance (K _Ca3.1) calcium-activated K⁺ channels are voltage-independent and share a common calcium/calmodulin-mediated gating mechanism. Existing positive gating modulators like EBIO, NS309, or SKA-31 activate both K_Ca2 and K_Ca3.1 channels with similar potency or, as in the case of CyPPA and NS13001, selectively activate K _Ca2.2 and K_Ca2.3 channels. We performed a structure-activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 toward K_Ca3.1 selectivity. We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), which displays 123-fold selectivity for K_Ca3.1 (EC₅₀ 111 ± 27 nM) over K_Ca2.3 (EC₅₀ 13.7 ± 6.9 μM), and SKA-121 (5-methylnaphtho [2,1-d]oxazol-2-amine), which displays 41-fold selectivity for K_Ca3.1 (EC₅₀ 109 nM ± 14 nM) over K_Ca2.3 (EC₅₀ 4.4 ± 1.6 μM). Both compounds are 200- to 400-fold selective over representative K_V (K_V1.3, K_V2.1, K_V3.1, and K_V11.1), Na_V (Na_V1.2, Na_V1.4, Na_V 1.5, and Na_V 1.7), as well as Ca_V1.2 channels. SKA-121 is a typical positive-gating modulator and shifts the calcium-concentration response curve of K_Ca3.1 to the left. In blood pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) significantly lowered mean arterial blood pressure in normotensive and hypertensive wild-type but not in KCa3.1^-/- mice. SKA-111, which was found in pharmacokinetic experiments to have a much longer half-life and to be much more brain penetrant than SKA-121, not only lowered blood pressure but also drastically reduced heart rate, presumably through cardiac and neuronal KCa2 activation when dosed at 100 mg/kg. In conclusion, with SKA-121, we generated a KCa3.1-specific positive gating modulator suitable for further exploring the therapeutical potential of K_Ca3.1 activation. Copyright

Full text of DOI:10.1124/mol.114.093286

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1521-0111/86/3/342–357$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.114.093286
Mol Pharmacol 86:342–357, September 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
New Positive Ca21-Activated K1 Channel Gating
Modulators with Selectivity for KCa3.1 s
Nichole Coleman, Brandon M. Brown, Aida Oliván-Viguera, Vikrant Singh,
Marilyn M. Olmstead, Marta Sofia Valero, Ralf Köhler, and Heike Wulff
Department of Pharmacology (N.C., B.M.B., V.S., H.W.), School of Medicine, and Department of Chemistry (M.M.O.), University
of California, Davis, California; Aragon Institute of Health Sciences, Instituto de Investigación Sanitaria, Fundación Agencia
Aragonesa para la Investigación y el Desarrollo, Zaragoza, Spain (A.O.-V., R.K.); and Grupo de Investigación del Medio Ambiente
del Centro de Estudios Superiores, Faculty of Health Sciences, Universidad San Jorge, Villanueva de Gállego, Spain (M.S.V.)
Received April 14, 2014; accepted June 23, 2014
ABSTRACT
Small-conductance (KCa2) and intermediate-conductance (KCa3.1) KV (KV1.3, KV2.1, KV3.1, and KV11.1), NaV (NaV1.2, NaV1.4,
calcium-activated K1 channels are voltage-independent and NaV1.5, and NaV1.7), as well as CaV1.2 channels. SKA-121 is
share a common calcium/calmodulin-mediated gating mecha- a typical positive-gating modulator and shifts the calcium-
nism. Existing positive gating modulators like EBIO, NS309, or concentration response curve of KCa3.1 to the left. In blood
SKA-31 activate both KCa2 and KCa3.1 channels with similar pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) sig-
potency or, as in the case of CyPPA and NS13001, selectively nificantly lowered mean arterial blood pressure in normotensive
activate KCa2.2 and KCa2.3 channels. We performed a structure- and hypertensive wild-type but not in KCa3.12/2 mice. SKA-111,
activity relationship (SAR) study with the aim of optimizing the which was found in pharmacokinetic experiments to have a much
benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity. longer half-life and to be much more brain penetrant than SKA-121,
We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), not only lowered blood pressure but also drastically reduced
which displays 123-fold selectivity for KCa3.1 (EC50 111 6 27 nM) heart rate, presumably through cardiac and neuronal KCa2 ac-
over KCa2.3 (EC50 13.7 6 6.9 mM), and SKA-121 (5-methylnaphtho tivation when dosed at 100 mg/kg. In conclusion, with SKA-121,
[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 we generated a KCa3.1-specific positive gating modulator suit-
(EC50 109 nM 6 14 nM) over KCa2.3 (EC50 4.4 6 1.6 mM). Both able for further exploring the therapeutical potential of KCa3.1
compounds are 200- to 400-fold selective over representative activation.
Introduction
The human genome contains four voltage-independent
This work was supported by the CounterACT Program, National Insti-
Ca21-activated K1 channels: the three small-conductance KCa
2
tutes of Health (NIH) Office of the Director [Grant U54NS079202] and Na-
tional Institute of Neurological Disorders and Stroke [R21NS072585], the Deutsche
Forschungsgemeinschaft [Grant KO1899/11-1], the Danish Hjerteforening,
and the Fondo de Investigación Sanitaria [Red HERACLES RD12/0042/0014].
N.C. was supported by an NIH National Heart, Lung and Blood Institute T32
Training Program in Basic and Translational Cardiovascular Science [Grant
T32HL086350]. B.M.B. was supported by an NIH National Institute of General
Medical Sciences–funded Pharmacology Training Program [Grant T32GM099608].
N.C. and B.M.B. contributed equally to this work. R.K. and H.W. are co-senior
authors.
channels: KCa2.1 (5 KCNN1, SK1), KCa2.2 (5 KCNN2, SK2),
and KCa2.3 (5 KCNN3, SK3), as well as the intermediate-
conductance KCa3.1 (5 KCNN4, IK1, SK4) (Kohler et al., 1996;
Joiner et al., 1997; Wei et al., 2005). Their lack of voltage de-
pendence enables these channels to remain open at negative
membrane potentials and to hyperpolarize the membrane to-
ward values near the K1 equilibrium potential of 289 mV.
The work forms part of the Ph.D. thesis of Nichole Coleman in fulfillment of
the degree requirements of the University of California, Davis.
dx.doi.org/10.1124/mol.114.093286.
K
Ca3.1 and KCa2 channels are accordingly expressed in cells
that need to be able to hyperpolarize to regulate Ca21 influx
through inward rectifier Ca21 channels, pass on hyperpolarization
s
This article has supplemental material available at molpharm.aspetjournals.
org.
ABBREVIATIONS: BK, bradykinin; CaM, calmodulin; CamBD, calmodulin binding domain; CAS, Chemical Abstracts Service; CM-TMF, N-{7-[1-(4-
chloro-2-methylphenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N’-methoxy-formamidine; CyPPA, cyclohexyl-[2-(3,5-dimethyl-pyrazole-1-yl-)
6-methyl-pyrimidin-4-yl]-amine; DMSO, dimethylsulfoxide; EBIO, 1-ethylbenzimidazolin-2-one; EDH, endothelium-derived hyperpolarization; HEK,
human embryonic kidney; HR, heart rate; HRMS, high-resolution mass spectrometry; KCa, Ca21-activated K1 channel; KCa3.1, intermediate-
conductance Ca21-activated K1 channel; KV, voltage-gated K1 channel; LC, liquid chromatography; MAP, mean arterial blood pressure; m.p.,
melting point; MS, mass spectrometry; NMR, nuclear magnetic resonance; NS309, 6,7-dichloro-1H-indole-2,3-dione 3 oxime; PCA, porcine
coronary arteries; SK, small-conductance KCa channel; SKA-31, naphtho[1,2-d]thiazol-2-ylamine; SKA-111, 5-methylnaphtho[1,2-d]thiazol-2-
amine; SKA-121, 5-methylnaphtho[2,1-d]oxazol-2-amine; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; UCL1684, 6,10-diaza-3
(1,3)8,(1,4)-dibenzena-1,5(1,4)-diquinolinacyclodecaphane; U46619, (Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-
6-yl]hept-5-enoic acid; UPLC, ultraperformance liquid chromatography.
342

Positive Gating Modulation of KCa3.1
343
through gap junctions, or regulate firing frequency by preventing hyperpolarization (EDH) response (Grgic et al., 2009; Dalsgaard
an untimely or premature action potential initiation (Adelman et al., 2010; Edwards et al., 2010; Köhler et al., 2010), SKA-31
et al., 2012; Wulff and Köhler, 2013). Pharmacologic ac- was used as a pharmacologic tool to explore the role of KCa
tivation of KCa channels has therefore been suggested for the channels in blood pressure regulation. While mice deficient in
treatment of various diseases. Whereas KCa2 activators can
KCa3.1 and/or KCa2.3 exhibit impaired EDH responses and an
potentially reduce neuronal excitability in central nervous increased mean arterial blood pressure (MAP) (Brahler et al.,
system disorders like epilepsy and ataxia, KCa3.1 activators 2009), pharmacologic KCa channel activation with SKA-31 lowered
could be useful as endothelial targeted antihypertensives blood pressure in both mice and dogs (Sankaranarayanan
and to enhance fluid secretion in the airways in cystic fibrosis et al., 2009; Damkjaer et al., 2012; Radtke et al., 2013). In dogs,
(Wulff and Zhorov, 2008; Balut et al., 2012; Wulff and Köhler, i.v. injection of 2 mg/kg SKA-31 produced an immediate and
2013).
strong (230 mmHg) but short-lived reduction in blood pressure
All four KCa2/3 channels are voltage-independent and share (Damkjaer et al., 2012). In mice, SKA-31 doses of 10–30 mg/kg
a Ca21/calmodulin-mediated gating mechanism (Xia et al., have been reported to lower blood pressure more prolonged
1998; Fanger et al., 1999). Calmodulin (CaM), which can be (∼30 mmHg) for 60–90 minutes) (Sankaranarayanan et al.,
regarded as a b-subunit for these channels, is constitutively 2009; Köhler, 2012). Significant blood pressure–lowering ef-
bound to a CaM-binding domain (CaMBD) in the intracellular fects with SKA-31 doses of 30 mg/kg have been further ob-
C-terminus. On Ca21 binding to CaM, the channels activate in served in models of hypertension like angiotensin II–infused
a highly coordinated fashion with an extremely steep Hill (Sankaranarayanan et al., 2009) and connexin 40–deficient
equation and EC50 values in the range of 250–900 nM (Wei mice (Radtke et al., 2013), which exhibit severe chronic renin-
et al., 2005). KCa2/3 activators modulate this gating process dependent hypertension. However, the responses typically
and have therefore been termed positive gating modulators. lasted only about 1 hour. Higher doses of SKA-31 (100 mg/kg)
The oldest positive modulator of KCa2/3 channels is the induced a stronger and longer-lasting response, accompanied
benzimidazolone EBIO (Devor et al., 1996), which activates by significant bradycardia. This reduction in heart rate
KCa3.1 with an EC50 of ∼30 mM and all three KCa2 channels (HR) was probably due to a centrally mediated decrease in
with EC50s around 300 mM (Wulff and Köhler, 2013). Two sympathetic drive through activation of neuronal KCa2 chan-
structurally similar, but more potent molecules, are the oxime nels by the brain penetrant SKA-31, as well as possible direct
NS309 (Strobaek et al., 2004) and the benzothiazole SKA-31 effects on KCa2 channels in cardiac pacemaker tissue (Radtke
(Sankaranarayanan et al., 2009). Whereas NS309 is exqui- et al., 2013). Another side effect that might prohibit the use
sitely potent (EC50 for KCa3.1 ∼20 nM; EC50 for KCa2 channels of KCa2 activators as antihypertensives is a possible impair-
∼600 nM), it unfortunately has an extremely short in vivo ment of learning and memory because of the role neuronal
half-life and inhibits KV11.1 (hERG) at a concentration of 1 mM
KCa2 channels play in in synaptic plasticity and long-term
(Strobaek et al., 2004). SKA-31 is 10 times less potent than potentiation (Blank et al., 2003; Adelman et al., 2012). To
NS309 but has become a relatively widely used in vivo tool avoid these KCa2 channel–mediated side effects, it therefore
compound to activate both KCa3.1 and/or KCa2 channels be- seems highly desirable to identify selective KCa3.1 activators
cause of its long half-life of 12 hours in rats (Sankaranarayanan that could be used as pharmacological tools to further dissect
et al., 2009). In contrast to these benzimidazole/benzothiazole- the in vivo role of KCa3.1 in blood pressure control and to
type KCa activators, all of which show only a modest 5- to 10-fold help determine whether KCa3.1 activators could eventually
selectivity for KCa3.1 and do not distinguish at all between be developed into a new class of endothelial-targeted anti-
the three KCa2 channels, CyPPA, and its derivative NS13001, hypertensives. We therefore here explored whether we could
have quite different selectivity profiles. Both compounds ac- further modify the benzothiazole SKA-31 and develop a
tivate KCa2.3 and KCa2.2 but are completely inactive on
KCa3.1-selective small-molecule activator. SKA-121, a com-
KCa2.1 and KCa3.1 (Hougaard et al., 2007; Kasumu et al., 2012). pound generated through an isosteric replacement ap-
GW542573X and (-)-CM-TMPF, in contrast, are selective for proach, activates KCa3.1 with an EC50 of 111 nM, exhibits
KCa2.1 (Hougaard et al., 2009, 2012). So while there are some 40- to 80-fold selectivity over the three KCa2 channels and
compounds that allow for the selective pharmacologic activa- lowers blood pressure in mice as determined by telemetry
tion of KCa2 channels, there currently is no selective KCa3.1 without exerting KCa2 channel–mediated effects on HR. We
activator.
propose SKA-121 as a new KCa3.1 selective pharmacolog-
We previously used riluzole, a drug for the treatment of ical tool compound despite its relatively short half-life in
amyotrophic lateral sclerosis, as a template for the design of mice.
SKA-31. Riluzole is a “dirty” compound that exerts multiple
pharmacological activities, the most prominent of which are
inhibition of voltage-gated sodium (NaV) channels at concen-
Materials and Methods
trations of 1–50 mM (Debono et al., 1993; Duprat et al., 2000)
and activation of KCa2/3 channels with EC50s of 10–20 mM
(Grunnet et al., 2001). Through directed derivatization of
riluzole, we managed to significantly reduce NaV channel
blocking effects and increase activity on KCa2/3 channels.
Whereas SKA-31 affects NaV channels only at concentrations
of 25 mM or higher, it activates KCa2.3 with an EC50 of 3 mM
and KCa3.1 with an EC50 of 260 nM. Since both KCa3.1 and
Commercially Available Compounds
2-Amino-4-(1-naphthyl)thiazole (SKA-75, CAS no. 56503-96-9),
2-amino-4-(2-naphthyl)thiazole (SKA-76), Chemical Abstracts Service
(CAS) no. 21331-43-1), 2,3,3-trimethyl-3H-benzo[g]indole (SKA-92,
CAS no. 74470-85-2), 2-methylnaphtho[2,3-d]oxazole (SKA-104, CAS
no. 20686-66-2), and 2-methylnaphtho[2,1-d]oxazole (SKA-103,
CAS no. 85-15-4) were purchased from Alfa Aesar (Pelham, NH);
2-methylnaphtho[1,2-d]thiazole (SKA-74, 2682-45-3) was purchased
KCa2.3 are expressed in vascular endothelium and have been
shown to be involved in the so-called endothelium-derived from Sigma (St. Louis, MO).

344
Coleman et al.
were purified and separated by flash chromatography over silica gel
(cyclohexane-EtOAc, 1:1).
Chemical Synthesis
Compounds that were not commercially available were synthesized
in our laboratory by the general methods described below. Compounds
reported previously were characterized by melting point, proton nu-
clear magnetic resonance (1H NMR) and 13 carbon (13C) NMR to con-
firm their chemical identity. New chemical entities were additionally
characterized by high-resolution mass spectrometry (HRMS) and a fully
interpreted 13C NMR.
General Method I. Preparation of Thiazoles. Thiourea (17 mmol)
was added to a solution of substituted ketones (6 mmol) in 30 ml of
absolute ethanol. The mixture was refluxed for 8 hours, which resulted
in 2-aminothiazole hydrobromide salts. The 2-aminothiazole was obtained
by treating the hydrobromide salt with 2M NaOH (5 ml) and extract-
ing with ethyl acetate. The crude residue was concentrated, recon-
stituted in a methanol-water mixture (99:1), treated with charcoal, and
recrystallized.
General Method II. Alternative Preparation of Benzothiazoles.
Thiourea (17 mmol) was added to a solution of substituted ketones
(6 mmol) in 30 ml of absolute ethanol. The mixture was refluxed for
8 hours, which resulted in 2-aminothiazole hydrobromide salts. The free
2-aminothiazole was obtained by treating the hydrobromide salt with 2 M
NaOH (5 ml) and extracting with ethyl acetate. The crude residue was
concentrated, reconstituted in a methanol-water mixture (99:1), treated
with charcoal, and recrystallized. The resulting 2-aminothiazole,
2-iodoxybenzoic acid and tetrabutylammonium tribromide were com-
bined in ethyl acetate and stirred at room temperature (RT) for 10 hours.
8H-Indeno[1,2-d]thiazol-2-amine (SKA-69). SKA-69 was prepared
from 1-indanone (1 g, 4.7 mmol) according to general method I. The
product was isolated as brown crystals (563 mg, 63%); melting point (m.p.) 5
212oC (CAS no. 85787-95-7). 1H NMR [500 MHz, dimethylsulfoxide
(DMSO)-d6, d]: 7.45 (doublet (d), J 5 7.4 Hz, 1H, 4-H), 7.37 (d, J 5 7.4 Hz,
1H, 7-H), 7.28 (t, J 5 7.4 Hz, 1H, 5-H), 7.17–7.11 (m, 3H, 6-H, and NH2), 3.68
(s, 2H, CH2). 13C NMR (125 MHz: DMSO-d6, d):173.74, 146.09, 138.04,
128.58, 126.68, 126.60, 125.47, 118.2, and 32.85.
4,5-Dihydronaphtho[1,2-d]thiazol-2-amine (SKA-70). SKA-70
was prepared from 1-tetralone (1 g, 6.84 mmol) according to general
method I. The product was isolated as white crystals (906 mg, 64%);
m.p. 5 135oC (CAS no. 34176-49-3). 1H NMR (500 MHz, DMSO-d6, d):
7.52 (d, J 5 7.3 Hz, 1H, 9-H), 7.2327.14 (m, 2H, 7-H and 6-H), 7.10
(t, J 5 7.3 Hz, 1H, 8-H), 6.93 (s, 2H, NH2), 2.93 (t, J 5 7.8 Hz, 2H, 5-H),
2.76 (t, J 5 7.8 Hz, 2H, 4-H). 13C NMR (125 MHz: DMSO-d6, d):
167.28, 145.01, 134.99, 132.39, 128.37, 127.31, 126.87, 122.83, 118.25,
29.18, and 21.79.
6-Fluoro-8H-indeno[1,2-d]thiazol-2-amine (SKA-71). SKA-71 was
prepared from 5-fluoro-1-indanone (1.84 g, 12.4 mmol) according to
general method I. The product was isolated as lavender crystals (934 mg,
40%); m.p. 5 217oC dec (CAS no. 1025800-52-5). 1H NMR (500 MHz,
DMSO-d6, d): 7.33 (doublet of triplets (dt), J 5 7.9, 3.8 Hz, 2H, 4-H, and
7-H), 7.16 (s, 2H, NH2), 7.10 (doublet of doublet of doublets (ddd),
J 5 10.2, 8.4, 2.5 Hz, 1H, 5-H), 3.70 (s, 2H, CH2). 13C NMR (125 MHz:
DMSO-d6, d): 182.98, 160.95, 139.84, 128.3, 126.68, 126.53, 124.01,
The reaction mixture was filtered through a pad of Celite, and the filtrate 114.03, 112.77, and 32.72.
5-Chloronaphtho[1,2-d]thiazol-2-amine (SKA-72). SKA-72 was
was diluted with saturated Na2S2O3 and extracted with ethyl acetate.
The combined organic layers were dried with anhydrous sodium sulfate
(anhydrous Na2SO4), concentrated, and then purified via flash chroma-
tography (cyclohexane-EtOAc, 1:1).
prepared from 1-amino-4-chloronaphthalene (1.6 g, 8.8 mmol) accord-
ing to general method III. The product was isolated as lavender crystals
(1.27 g, 60%); m.p. 5 253oC (CAS no. 1369250-74-7). 1H NMR (500 MHz,
DMSO-d6, d): 8.41 (m, 1H, 9-H), 8.14 (m, 1H, 6-H), 8.07 (s, 1H, 4-H), 7.75
(s, 2H, NH2), 7.63 (m, 2H, 8-H, and 7-H). 13C NMR (125 MHz: DMSO-d6, d):
168.17, 147.65, 128.19, 126.46, 126.40, 126.22, 124.82, 124.14, 123.94,
122.07, and 119.66.
4,5-Dihydroacenaphtho[5,4-d]thiazol-8-amine (SKA-73). SKA-73
was prepared from 1,2-dihydroacenaphthylen-5-amine (0.4 g, 2 mmol)
according to general method III. The product was isolated a brown solid
(400 mg, 30%); m.p. 5 257°C (CAS. 108954-84-3). 1H NMR (500 MHz,
DMSO-d6, d): 7.87 (d, J 5 8.2 Hz, 1H, 9-H), 7.56 (singlet (s), 1H, 4-H),
7.45 (t, J 5 7.5 Hz, 1H, 8-H), 7.30 – 7.20 (m, 3H, 7-H and NH2), 3.37 (d,
J 5 12.2 Hz, 4H, 4-H, and 5-H). 13C NMR (125 MHz: DMSO-d6, d): 167.17,
146.65, 139.05, 128.19, 128.46, 124.40, 119.66, 119,24, 113.08, 31.25, and
29.92. (Note: We are following the NMR numbering designation of
2-aminobenzothiazoles, not dihydroacenaphthothiazoles.)
General Method III. Preparation of Benzothiazoles. Benzoyl
chloride was added drop wise to a stirred solution of NH4SCN in
acetone and stirred at 50°C for 2 hours. Next, a solution of substituted
naphthylamines in acetone was added drop wise, and the mixture was
stirred at 50oC for 24 hours. The reaction mixture was diluted with
water; the precipitated crystals collected by filtration and washed
with water. The crystals were then suspended in 2M NaOH (50 ml),
refluxed for 1 hour, and poured into cold water and filtered. The crude
crystals of the resulting thiourea were dissolved in acetic acid, to
which benzyl trimethylammonium tribromide was added and allowed
to react overnight. Ethyl ether (Et2O) was added and the precipitate of
the resulting product-HBr salt was collected by filtration and washed
with Et2O. The salt was then treated with 1M NaOH to liberate the
free base, which was recrystallized in methanol.
7,8-Dihydro-6H-indeno[4,5-d]thiazol-2-amine (SKA-81). SKA-81
was prepared from 4-aminoindan (500 mg, 3.7 mmol) according to
general method III. The product was isolated as a white solid (200 mg,
30%); m.p. 5 195°C. 1H NMR (500 MHz, DMSO-d6, d): 7.38–7.37 (d,
J 5 7.75 Hz, 1H, 5-H), 7.25 (s, 2H, NH2), 6.90 (d, J 5 7.8 Hz, 1H, 4-H),
3.00 (t, J 5 7.3, 2H, 8-H), 2.91 (t, J 5 7.3 Hz, 2H, 6-H), 2.10–2.04 (q,
J 5 7.3 Hz, 2H, 7-H). 13C NMR (125 MHz, DMSO-d6, d): 167.37 (2-C),
149.90 (3-C), 141.97 (4-C), 133.02 (6-C), 129.00 (8-C), 119.08 (5-C),
117.58 (4-C), 33.42 (6-C), 31.55 (8-C), 25.62 (7-C). HRMS (ESI): cal-
culated: 191.0637; found: 191.0638.
5-Bromonaphtho[1,2-d]thiazol-2-amine (SKA-87). SKA-87 was
prepared from 1-amino-4-bromonaphthalene (1.6 g, 8.8 mmol) accord-
ing to general method III. The product was isolated as silver crys-
talline rods (500 mg, 45%); m.p. 5 253°C (CAS. 412312-09-5). 1H NMR
(800 MHz, DMSO-d6, d): 8.40 (d, J 5 8.6 Hz, 1H, 9-H), 8.24 (s, 1H, 4-H),
8.10 (d, J 5 8.1 Hz, 1H, 6-H), 7.78 (s, 2H, NH2), 7.65–7.60 (multiplet (m),
2H, 7-H, and 6-H). 13C NMR (125 MHz, DMSO-d6, d): 168.67, 148.68,
129.71, 127.10, 127.00, 126.89, 126.88, 125.90, 124.61, 123.50, and 112.73.
Naphtho[1,2-d]oxazol-2-amine (SKA-102). Solid 1-amino-2-naphthol
hydrochloride (0.80 g, 4 mmol) was suspended in 20 ml of dichloromethane,
treated with triethylamine (0.6 ml, 20 mmol) and cyanogen bromide
(3M BrCN in dichloromethane; 3 ml, 6.2 mmol), and allowed to react
General Method IV. Preparation of 2-Aminonaphthooxazoles.
To procure the intermediate 2-hydroxy-naphthalenones, substituted
ketones (1 g, 6.2 mmol) were added to a stirred mix of water (25 ml),
acetonitrile (25 ml) trifluoroactic acid (6 ml, 7 mmol), iodobenzene (0.7 ml,
6 mmol), and oxone (11 g, 37 mmol). The resulting solution was re-
fluxed for 1 hour, and the progress of the reaction was monitored by
thin layer chromatography (TLC). The reaction was then allowed to
cool to RT and filtered. The mixture was extracted with ethyl acetate
(3 Â 30 ml) and lastly neutralized with saturated NaHCO3 (3 Â 30 ml).
The combined organic phase was washed with brine (30 ml), dried with
anhydrous Na2SO4, filtered, and concentrated. The residue was purified
by flash chromatography over silica gel (cyclohexane-EtOAc, 3:1) to
give substituted 2-hydroxy-naphthalenones (Rf 5 0.20). The prepara-
tion of 2-aminonaphthooxazole began by adding cyanamide (2 mmol)
to a stirred solution of substituted 2-hydroxy-naphthalenone (1.5 mmol),
water (20 ml), and acetonitrile (10 ml). The resulting mixture was
refluxed for 15 hours, and progression of the reaction was monitored
by TLC. The reaction was allowed to cool to RT. The mixture was
extracted with ethyl acetate (3 Â 30 ml) to give a mixture of isomers.
The combined organic phase was washed with brine (30 ml), dried
with anhydrous Na2SO4, filtered, and concentrated. The isomers

Positive Gating Modulation of KCa3.1
345
overnight yielding naphtho[1,2-d]oxazol-2-amine HBr. To isolate the
8-Fluoronaphtho[1,2-d]thiazol-2-amine (SKA-112). SKA-112
free amine, the HBr salt was suspended in ethyl acetate and free-based was prepared from 7-fluoro-1-tetralone (0.5 g, 3 mmol) according to
with ammonium hydroxide (NH4OH). The solid residue was dissolved general procedure II. The product was isolated a clear oil (10 mg, 3%).
in a diethyl ether-ethyl acetate, treated with charcoal, and recrystal- 1H NMR (500 MHz, DMSO-d6, d): 8.00 (dd, J 5 5.74, 9.02 Hz, 1H, 7-H),
7.90 (dd, J 5 2.67, 10.46 Hz, 1H, 9-H), 7.79 (d, J 5 8.61 Hz, 1H, 5-H),
7.67 (s, 2H, NH2), 7.60 (d, J 5 8.64 Hz, 1H, 4-H), 7.37 (triplet of
doublets (td), J 5 2.73, 8.83 Hz, 1H, 6-H). 13C NMR (125 MHz, DMSO, d):
168.40 (2-C), 161.60 (8-CF), 159.67 (39-C), 148.183 (6-C), 131.73
(69-C), 129.52 (19-C), 126.84 (99-C), 121.27 (7-C), 119.43 (4-C), 115.54
(5-C), and 107.29 (9-C). HRMS (ESI): calculated: 219.0387; found:
219.0383.
lized from diethyl ether-ethyl acetate (10:1), resulting in a purple solid
(100 mg, 45%); m.p. 5 194°C (CAS. 858432-45-8). 1H NMR (800 MHz,
DMSO-d6, d): 8.27 (d, J 5 8.3 Hz, 1H, 9-H), 7.9 (d, 1H, J 5 8.16 Hz, 6-H),
7.60 (d, J 5 8.88 Hz, 1H, 5-H), 7.58 (triplet (t), J 5 7.92 Hz, 1H, 8-H),
7.53 (d, J 5 8.72 Hz, 1H, 4-H), 7.47 (t, J 5 8.01 Hz, 1H, 8-H). 13C NMR
(200 MHz, DMSO-d6, d): 163.25, 144.14, 138.53, 130.95, 128.80, 125.97,
124.72, 124.49, 121.99, 120.29, and 110.32.
5-Methyl-4,5-dihydronaphtho[1,2-d]thiazol-2-amine (SKA-
113). SKA-113 was prepared from 4-methyl-1-tetralone (2 g, 11 mmol)
according to general procedure I. The product was isolated as white
crystals (250 mg, 20%); m.p. 5 109°C dec (CAS no. 896156-31-3). 1H NMR
(500 MHz, DMSO-d6, d): 7.56 (d, J 5 6.4 Hz, 1H, 9-H), 7.40 (s, 2H, NH2),
7.21 (m, 3H, 6-H, 7-H, and 8-H), 3.12 (h, J 5 6.8 Hz, 1H, 5-H), 2.76 (ddd,
J 5 175.4, 16.2, 6.6 Hz, 2H, 4-CH2), 1.23 (d, J 5 6.9 Hz, 3H, 5-CH3).
13C NMR (125 MHz, DMSO-d6, d): 167.85, 152.42, 140.02, 127.69,
127.37, 127.29, 122.97, 116.67, 99.85, 33.48, 29.20, and 21.45.
6-Methoxy-4,5-dihydronaphtho[1,2-d]thiazol-2-amine (SKA-
114). SKA-114 was prepared from 5-methoxy-1-tetralone (2 g, 11 mmol)
according to general procedure I. The product was isolated as brown
crystals (1.5 g 57%); m.p. 5 200°C (CAS no. 489430-53-7). 1H NMR (500
MHz, DMSO-d6, d): 7.23–7.13 (m, 2H, 9-H, and 8-H), 6.91 (s, 2H, NH2),
6.84 (d, J 5 7.59 Hz, 1H, 7-H), 3.79 (s, 3H, OCH3), 2.89 (t, J 5 8.09 Hz,
2H, 4-CH2), 2.73 (t, J 5 8.07 Hz, 2H, 5-CH2). 13C NMR (125 MHz,
DMSO-d6, d): 167.04, 156.69, 144.90, 133.25, 127.75, 122.02, 118.20,
115.94, 110.01, 56.10, 21.50, and 21.18.
5-Methoxynaphtho[1,2-d]thiazol-2-amine (SKA-117). SKA-117
was prepared from 1-amino-4-methoxynaphthalene (0.1 g, 0.51 mmol)
according to general method III. The product was isolated as lavender
crystals (16 mg, 14%); m.p. 5 213°C (CAS. 1368289-59-1). 1H NMR
(500 MHz, DMSO-d6, d): 8.87 (d, J 5 8.25 Hz, 1H, 9-H), 8.68 (d, J 5 8.3 Hz,
1H, 6-H), 8.02 (t, J 5 7.4 Hz, 1H, 7-H), 7.95 (t, J 5 7.4 Hz, 1H, 8-H),
7.56 (s, 1H, 4-H), 7.11 (s, 2H, NH2), 4.49 (s, 3H, OCH3). 13C NMR
(125 MHz, DMSO-d6, d): 168.11, 155.81, 148.63, 127.48, 126.6, 126.56,
123.94, 119.16, 116.51, 114.98, 104.55, and 56.26.
5-Methylnaphtho[1,2-d]oxazol-2-amine (SKA-120). SKA-120
was prepared from 4-methyl-1-tetralone according to general pro-
cedure IV. The product was isolated as light brown crystals (50 mg,
4%); m.p. 5 209°C; Rf 5 0.38 (cyclohexane-EtOAc, 1:1). 1H NMR (800 MHz,
CDCl3, d): 8.28 (d, J 5 8.3 Hz, 1H, 9-H), 8.03 (d, J 5 8.4 Hz, 1H, 6-H),
7.58 (t, J 5 7.4 Hz, 1H, 8-H), 7.52 (t, J 5 7.5 Hz, 1H, 7-H), 7.38 (s, 1H,
4-H), 5.39 (bs, 2H, NH2), and 2.74 (s, 3H, CH3). 13C NMR (200 MHz,
CDCl3, d): 160.37 (2-C), 152.05 (19-C), 135.20 (39-C), 130.07 (99-C), 129.14
(69-C), 126.12 (8-C), 125.33 (5-C), 124.91 (6-C), 124.77 (79-C), 122.38 (9-C),
110.56 (4-C), 19.93 (5-CH3). 1H,13C-HSQC (800 MHz, CDCl3, cross-peaks d):
8.28/122.38, 8.03/124.91, 7.58/126.12, 7.51/124.77, 7.38/110.57, and
2.80/19.93. HRMS (ESI): calculated: 199.0866; found: 199.0864.
5-Methylnaphtho[2,1-d]oxazol-2-amine (SKA-121). SKA-121
was prepared from 4-methyl-1-tetralone according to general pro-
cedure IV. The product was isolated as brown crystals (50 mg, 4%);
m.p. 5 186°C dec; Rf 5 0.28 (cyclohexane-EtOAc, 1:1). 1H NMR (800 MHz,
CDCl3, d): 8.03 (d, J 5 8.5 Hz, 1H, 9-H), 8.01 (d, J 5 8.3 Hz, 1H, 6-H), 7.56
(t, J 5 7.5 Hz, 1H, 7-H), 7.45 (t, J 5 6.0 Hz, 1H, 8-H), 7.43 (s, 1H, 4-H), 5.33
(bs, 2H, NH2), 2.73 (s, 3H, CH3). 13C NMR (200 MHz, CDCl3, d): 160.80
(2-C), 141.72 (1’-C), 137.24 (39-C), 128.85 (5-C), 119.28 (6-C), 125.21 (9-C),
126.39 (7-C), 123.92 (8-C), 131.34 (69-C), 119.77 (99-C), 117.17 (4-C),
19.58 (5-CH3). 1H,13C-HSQC (800 MHz, CDCl3, cross-peaks d):
8.03/125.21, 8.01/119.28, 7.56/126.39, 7.45/123.92, 7.43/117.17, and
2.73/19.58. HRMS (ESI): calculated: 199.0866; found: 199.0864.
5-Fluoronaphtho[1,2-d]thiazol-2-amine (SKA-106). SKA-106 was
prepared from 4-fluoronaphthalen-1-amine (1 g, 6 mmol) according to
general method III. The product was isolated as a clear oil (210 mg,
20%). 1H NMR (500 MHz, acetone-d6, d): 8.48 (d, J 5 8.1 Hz, 1H, 9-H),
8.06 (d, J 5 8.2 Hz, 1H, 6-H), 7.62 (m, 3H, 8-H, 7-H, and 4-H), 6.91
(s, 2H, NH2). 13C NMR (125 MHz, acetone-d6, d): 167.23 (2-C), 157.95 (5-C),
145.12 (39-C), 128.33 (6-C), 125.17 (19-C), 126.94 (7-C), 125.80 (9-C), 125.01
(8-C) 124.29 (99-H), 116.06 (69-C), 103.59 (4-C). HRMS (ESI): calculated:
219.0387; found: 219.0383.
2-Aminonaphtho[1,2-d]thiazole-5-carbonitrile (SKA-107). SKA-
107 was prepared from 4-amino-1-naphthalenecarbonitrile (1 g, 6 mmol)
according to general method III. The product was isolated as a brown
solid (121 mg, 45%); m.p. 5 265°C. 1H NMR (500 MHz, acetone-d6, d):
8.62 (d, J 5 8.22 Hz, 1H, 9-H), 8.42 (s, 1H, 4-H), 8.20 (d, J 5 8.34 Hz, 1H,
6-H), 7.78 (t, J 5 7.04 Hz, 1H, 7-H), 7.73 (t, J 5 7.25 Hz, 1H, 8-H), 7.48
(s, 2H, NH2).13C NMR (200 MHz, DMSO-d6, d): 171.82 (2-C), 153.14
(39-C), 131.15 (6-C), 128.32 (4-C), 127.55 (9-C), 127.39 (8-C), 125.25 (7-C),
124.96 (69-C), 124.84 (19-C), 124.69 (99-C), 118.94 (CN), and 99.96 (5-C).
HRMS (ESI): calculated: 226.0433; found: 226.0432.
6,8-Dimethyl-4,5-dihydronaphtho[1,2-d]thiazol-2-amine
(SKA-108). SKA-108 was prepared from 5,7-dimethyl-1-tetralone
(2 g, 11 mmol) according to general method I. The product was isolated
as pink crystals (300 mg, 16%); m.p. 5 159°C. 1H NMR (500 MHz,
acetone-d6, d): 7.40 (s, 1H, 9-H), 6.83 (s, 1H, 7-H), 6.17 (s, 2H, NH2),
2.91 (t, J 5 7.87 Hz, 2H, 4-H), 2.81 (t, J 5 7.56 Hz, 2H, 5-H), 2.27
(s, 3H, 8-CH3), 2.25 (s, 3H, 6-CH3). 13C NMR (200 MHz, DMSO-d6, d):
166.86 (2-C), 145.13 (39-C), 135.11 (8-C), 135.09 (6-C), 131.86 (69-H),
129.81 (7-H), 129.41 (99-C), 121.69 (9-C), 117.36 (19-C), 28.8 (4-CH2),
24.57 (5-CH2), 21.37 (8-CH3), and 19.88 (6-CH3). HRMS (ESI): cal-
culated: 231.0950; found: 231.0949.
6,8-Dimethylnaphtho[1,2-d]thiazol-2-amine (SKA-109). SKA-109
was prepared from 5,7-dimethyl-1-tetralone (2 g, 11 mmol) according to
general method II. The product was isolated as pink crystals (15 mg,
0.6%); m.p. 5 157°C. 1H NMR (500 MHz, DMSO-d6, d): 8.02 (s, 1H, 9-H),
7.73 (d, J 5 8.81 Hz, 1H, 4-H), 7.59 (d, J 5 8.84 Hz, 1H, 5-H), 7.53 (s, 2H,
NH2), 7.17 (s, 1H, 7-H), 2.61 (s, 3H, 8-CH3), 2.45 (s, 3H, 6-CH3).
13C NMR (125 MHz, DMSO-d6, d): 167.84 (2-C), 148.80 (39-C), 135.61
(8-C), 134.92 (6-C), 128.70 (7-C), 126.90 (9-C), 121.68 (69-C), 118.78
(19-C), 117.57 (4-C), 100.61 (99-C), 99.85 (5-C), 22.21 (8-CH3), and 20.14
(6-CH3). HRMS (ESI): calculated: 222.0794; found: 222.0794.
Thieno[29,39:5,6]benzo[1,2-d]thiazol-2-amine (SKA-110). SKA-
110 was prepared from 6,7-dihydro-4-benzo[b]thiophenone (0.5 g, 3 mmol)
according to general procedure II. The product was isolated as a white
solid (26 mg, 3.8%), m.p. 5 161°C (CAS. 35711-03-6). 1H NMR (800 MHz,
DMSO-d6, d): 7.71 (d, J 5 5.36 Hz, 1H, 7-H), 7.68 – 7.62 (m, 4H, 4-H, 5-H,
and NH2), 7.58 (d, J 5 5.41 Hz, 1H, 6-H). 13C NMR (125 MHz, DMSO-d6, d):
168.26, 147.81, 137.83, 131.22, 126.97, 125.72, 121.95, 117.97, and
115.51.
5-Methylnaphtho[1,2-d]thiazol-2-amine (SKA-111). SKA-111
was prepared from 4-methyl-1-tetralone (1 g, 11 mmol) according to
general procedure II. The product was isolated as yellow crystals
(100 mg, 16%); m.p. 5 209°C (CAS. 1369170-24-0). 1H NMR (800 MHz,
DMSO-d6, d): 8.96 (d, J 5 7.86 Hz, 1H, 9-H), 8.46 (d, J 5 7.99 Hz, 1H,
6-H), 8.06 (s, 1H, 4-H), 7.98 (dt, J 5 6.83, 13.83 Hz, 2H, 7-H and 8-H),
7.16 (s, 2H, NH2), 3.14 (s, 3H, CH3).13C NMR (125 MHz, DMSO-d6, d):
Crystal Structure Determinations
The SKA-120 and SKA-121 crystals selected for data collection
166.73, 147.60, 131.54, 127.88, 127.33, 126.02, 125.58, 125.27, 124.75, were mounted in the 90-K nitrogen cold stream provided by a CRYO
124.70, 119.39, and 19.01. Industries of America (Manchester, NH) low-temperature apparatus

346
Coleman et al.
on the goniometer head of a Bruker D8 diffractometer equipped with
from 2120 mV to 40 mV applied every 10 seconds, and the -fold
an ApexII CCD detector (Bruker, Fremont, CA). Data were collected increase of slope conductance at 280 mV by drug taken as a measure
with the use of Mo Ka radiation (l 5 0.71073 Å). The structures were
of channel activation. KV2.1, KV1.3, and KV3.1 currents were recorded
in normal Ringer solution with a Ca21-free KF-based pipette solution
solved by direct methods (SHELXS-97) and refined by full-matrix
least-squares on F2 (SHELXL-2013). All nonhydrogen atoms were re- as previously described (Schmitz et al., 2005). HERG (KV11.1) cur-
fined with anisotropic displacement parameters. For a description of
the method, see Sheldrick (2008).
rents were recorded with a two-step pulse from 280 mV first, to 20 mV
for 1 second, and then to 250 mV for a 1-second reduction of both peak
and tail current by the drug. NaV1.7 currents were recorded with
30-millisecond pulses from 290 mV to 10 mV every 10 seconds with
a CsF-based pipette solution and normal Ringer as an external so-
lution. CaV1.2 currents were elicited by 100-millisecond depolariz-
ing pulses from 280 to 20 mV every 10 seconds with a CsCl-based
pipette solution and an external solution containing 30 mM BaCl2.
Blockade of both Na1 and Ca21 currents was determined as reduc-
tion of the current minimum.
Crystal Data SKA-120. C12H10N2O, F.w. 5 198.22, brown plate,
dimensions 0.18 Â 0.34 Â 0.60 mm, monoclinic, P21/n, a 5 14.2110(9)
Å, b 5 3.8854(3) Å, c 5 17.5101(11) Å, b 5 107.537(2)°, V 5 921.89(11)
Å3, Z 5 4, R1 [1518 reflections with I . 2s(I)] 5 0.0307, wR2 (all 1671
data) 5 0.0900, 176 parameters, 0 restraints.
Crystal Data SKA-121. C12H10N2O, F.w. 5 198.22, brown plate,
monoclinic, P21/n, a 5 8.0532(12) Å, b 5 21.377(3) Å, c 5 11.5094(17)
Å, b 5 107.823(2)°, V 5 1886.3(5) Å3, Z 5 8, R1 [2571 reflections with
I . 2s(I)] 5 0.0376, wR2 (all 3413 data) 5 0.0949, 335 parameters,
0 restraints.
For automated electrophysiology experiments, cells were grown to
∼70% confluency, rinsed in sterile phosphate-buffered saline containing
0.02% EDTA, and lifted with 2 ml of TrypLE Express (Gibco, Grand
Island, NY) for ∼2 minutes. When cells were rounded but not detached,
they were dislodged by gentle tapping, suspended in Dulbecco’s modified
Eagle’s medium, centrifuged, and resuspended in 1 ml of external
solution; placed into the Qfuge tube; and resuspended in 150–200 ml of
extracellular solution after one additional spin on the QPatch. Whole-
cell patch-clamp experiments were then carried out using disposable
16-channel planar patch chip plates (QPlates; patch-hole diameter
approximately 1 mm, resistance 2.00 6 0.02 MV). Cell positioning and
sealing parameters were set as follows: positioning pressure 270 mbar,
resistance increase for success 750%, minimum-seal resistance 0.1 GV,
holding potential 280 mV, and holding pressure 220 mbar. To avoid
the rejection of cells with large KCa3.1 currents, the minimum seal
resistance for whole-cell requirement was lowered to 0.001 GV. Access
was obtained with the following sequence: 1) suction pulses in 29-mbar
increments from 2250 mbar to 2453 mbar; 2) a suction ramp of an
amplitude of 2450 mbar; 3) 400-mV voltage zaps of 1-milliseconds’
duration (10Â). After establishment of the whole-cell configuration,
cells were held at 280 mV and KCa3.1, KCa2.1, or KCa2.2 currents
elicited by a voltage protocol that held at 280 mV for 20 ms, stepped
to 2120 mV for 20 milliseconds, ramped from 2120 to 40 mV in
200 milliseconds, and then stepped back to 2120 mV for 20 milliseconds.
This pulse protocol was applied every 10 seconds. KCa1.1 currents were
elicited by 160-ms voltage ramps from 280 to 80 mV applied every
10 seconds (500 nM free Ca21), and channel modulation was measured
as a change in mean current amplitude. NaV1.2 currents from N1E-115
cells, NaV1.4, and NaV1.5 currents from stably transfected HEK cells
were recorded with 20-millisecond pulses from 290 mV to 0 mV every
10 seconds with a KF-based internal solution and normal Ringer as an
external solution. Current slopes (in ampere per sec) were measured
using the Sophion QPatch software and exported to Microsoft Excel
and Origin 7.0 (OriginLab Corp., Northampton, MA) for analysis.
Increases or decreases of slopes between 285 and 265 mV were used
to calculate KCa2/3 activation. Data fitting to the Hill equation to
obtain EC50 and IC50 values were performed with Origin 7.0. Data are
expressed as mean 6 S.D.
CCDC 9966657 and 996658 contains the supplemental crystallo-
graphic data for this article. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
Cells, Cell Lines, and Clones
Human embryonic kidney cells (HEK-293) stably expressing
hKCa2.1, rKCa2.2, and hKCa3.1 were obtained from Khaled Houamed
(University of Chicago, IL) in 2002 and have been maintained in the
Wulff Laboratory at the University of California since then. The
cloning of hKCa2.3 (19 CAG repeats) and hKCa3.1 was previously
described (Wulff et al., 2000). The hKCa2.3 clone was later stably
expressed in COS-7 cells at Aurora Biosciences Corp. (San Diego, CA).
Cell lines stably expressing other mammalian ion channels were gifts
from several sources: hKCa1.1 in HEK-293 cells (Andrew Tinker, Uni-
versity College London); hKV2.1 in HEK293 cells (James Trimmer,
University of California Davis, Davis, CA); KV11.1 (HERG) in HEK-293
cells (Craig January, University of Wisconsin, Madison); hNaV1.4 in
HEK-293 cells (Frank Lehmann-Horn, University of Ulm, Germany);
hNaV1.5 in HEK-293 cells (Christopher Lossin, University of California
Davis); and hCaV1.2 in HEK-293 cells (Franz Hofmann, Munich,
Germany). L929 cells stably expressing mKV1.3 and mKV3.1 have been
previously described (Grissmer et al., 1994); N1E-115 neuroblastoma
cells (expressing mNaV1.2) were obtained from ATCC; division ar-
rested CHO cells expressing hNaV1.7 were purchased from ChanTest
(Cleveland, OH).
Electrophysiology
Experiments were conducted either manually with an EPC-10
amplifier (HEKA, Lambrecht/Pfalz, Germany) or on a QPatch-16 auto-
mated electrophysiology platform (Sophion Biosciences, Ballerup,
Denmark). For manual experiments, COS-7, HEK-293, or L929 cells
were trypsinized, plated onto poly-L-lysine–coated coverslips, and
typically recorded from between 20 minutes and 4 hours after plating.
Patch pipettes were pulled from soda lime glass (micro-hematocrit
tubes, Kimble Chase, Rochester, NY) and had resistances of 2-3 MV.
For measurements of KCa channels expressed in HEK-293 cells (KCa2.1,
The inside-out experiments shown in Fig. 4 were performed on the
K
Ca3.1-stable HEK 293 cell line. Symmetrical K1 was used to obtain
larger currents. The extracellular solutions contained (in mM) the
following: 154 KCl, 10 HEPES (pH 5 7.4), 2 CaCl2, and 1 MgCl2.
Solutions on the intracellular side contained (in mM) the following:
154 KCl, 10 HEPES (pH 5 7.2), 10 EGTA, 1.75 MgCl2, and CaCl2 to
yield calculated free Ca21-concentrations of 0.05, 0.1, 0.25, 0.3, 0.5,
1.0, and 10.0 mM. Cells were clamped to a holding potential of at 0 mV,
and KCa currents were elicited by 200-millisecond voltage ramps from
280 to 80 mV applied every 10 seconds.
K
Ca2.2, and KCa3.1), we used normal Ringer solution as external with
an internal pipette solution containing (in mM): 140 KCl, 1.75 MgCl2,
10 HEPES, 10 EGTA, and 7.4 CaCl2 (500 nM free Ca21) or 6 CaCl2
(250 nM free Ca21), pH 7.2, 290–310 mOsm. Free Ca21 concentrations
were calculated with MaxChelator assuming a temperature of 25°C,
a pH of 7.2, and an ionic strength of 160 mM. To reduce contaminating
currents from native chloride channels in COS-7 cells, KCa2.3 cur-
rents were recorded with an internal pipette solution containing (in
mM) the following: 145 K1 aspartate, 2 MgCl2, 10 HEPES, 10 EGTA,
For all electrophysiology experiments, solutions of benzothiazoles
and 7.4 CaCl2 (500 nM free Ca21), pH 7.2, 290–310 mOsm. Na1 as- and benzooxazoles were always freshly prepared from 1 mM or 10 mM
partate Ringer was used as an external solution (in mM): 160 Na1
aspartate, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, 290–310 mOsm.
stock solutions in DMSO during the experiment. The final DMSO
concentration never exceeded 1%. For automated assays, glass vial
Both KCa2 and KCa3.1 currents were elicited by 200-ms voltage ramps inserts (Sophion Biosciences) were filled with 350–400 ml of compound

Positive Gating Modulation of KCa3.1
347
solution and placed into the glass insert base plate for use in the
QPatch assay right before starting the QPatch.
Institutional Animal Care and Use Committee. For i.v. application,
SKA-111 and SKA-121 were dissolved at 5 mg/ml in a mixture of 10%
CremophorEL (Sigma-Aldrich, St. Louis, MO) and 90% phosphate-
buffered saline and then injected at 10 mg/kg into the tail vein (n 5 8
mice per compound). Another group of mice (n 5 8) received SKA-121
orally. At various time points after the injection, blood was collected
into EDTA blood sample collection tubes either from the saphenous
vein or by cardiac puncture under deep isoflurane anesthesia. After
the cardiac puncture, mice were sacrificed by cutting the heart, and
then the brain was removed. Individual mice were typically used
for three times points (two blood collections from the saphenous vein
plus the terminal blood collection). Plasma was separated by centri-
fugation, and plasma and brain samples were stored at 280°C pend-
ing analysis. Brain samples were homogenized in 1 ml of H2O with
a Brinkman Kinematica PT 1600E homogenizer, and the protein was
precipitated with 1 ml of acetonitrile. The samples were then centri-
fuged at 3000 rpm, and supernatants concentrated to 1 ml. Plasma
and homogenized brain samples were purified using C18 solid-phase
extraction cartridges (ThermoFisher Scientific, Waltham, MA) pre-
conditioned with acetonitrile, followed by 1 ml of water. The loaded
column was washed with 2 ml of water. SKA-121 was eluted with 3 ml
of acetonitrile. SKA-111 was eluted with 3 ml of methanol containing
1% NH4OH. Eluted fractions were dried under nitrogen and re-
constituted in acetonitrile. LC/MS analysis was performed with a
Waters Acquity UPLC (Waters, New York, NY) equipped with a
Acquity UPLC BEH 1.7 mm RP-18 column (Waters, New York, NY)
interfaced to a TSQ Quantum Access Max mass spectrometer (MS)
(ThermoFisher Scientific, Waltham, MA). The isocratic mobile phase
consisted of 80% acetonitrile and 20% water, both containing 0.1%
formic acid with a flow rate of 0.25 ml per minute. Under these
conditions SKA-111 had a retention time of 0.83 minute and SKA-121
a retention time of 0.96 minute. Using electrospray ionization, MS
and selective reaction monitoring (capillary temperature 300°C, cap-
illary voltage 4000 V, collision energy -34 eV, positive ion mode),
SKA-121 was quantified by its base peak of 128.14 m/z and its con-
centration was calculated with a 5-point calibration curve from 100 nM
to 10 mM. SKA-111 (capillary temperature 325°C, capillary voltage
4000 V, collision energy 228eV, positive ion mode) was quantified by its
base peak of 200.045 m/z and its concentration was calculated with
a six-point calibration curve from 100 nM to 20 mM.
Isometric Myography on Porcine Coronary Arteries
Porcine coronary arteries (PCAs) were carefully dissected from
hearts kindly provided by the local abattoir (Mercazaragoza, Zaragoza,
Spain), cleaned of fat and connective tissue, and cut into 3- to 4-mm
rings. Rings were mounted on hooks connected to an isometric force
transducer (Pioden UF1, Graham Bell House, Canterbury, UK) and
prestretched to an initial tension of 1 g. Composition Krebs’ buffer (in mM)
was as follows: NaCl 120, NaHCO3 24.5, CaCl2 2.4, KCl 4.7, MgSO4 1.2,
KH2PO4 1, and glucose 5.6, pH 7.4, at 37°C and equilibrated with
95% O2/5% CO2. Changes in tension were registered using a Mac
Laboratory System/8e program (AD Instruments Inc., Milford, MA).
The buffer contained the NO-synthase blocker, Nv-nitro-L-arginine
(L-NNA, 300 mM), and the cyclooxygenase blocker indomethacin (10 mM)
to measure EDH-type relaxation. After three washes, rings were pre-
contracted with the thromboxane analog U46619 (0.2 mM). Thereafter
rings were exposed to bradykinin (BK, 1 mM) in combination with
vehicle DMSO, SKA-111 (1 mM), SKA-111 plus TRAM-34 (1 mM),
SKA-111 plus TRAM-34 plus UCL-1684 (1 mM), SKA-121 (1 mM),
SKA-121 plus TRAM-34, or SKA-121 plus TRAM-34 plus UCL-1684.
After washout, rings were contracted with a high KCl buffer (60 mM)
to determine maximal contraction. TRAM-34 was synthesized as pre-
viously described (Wulff et al., 2000). UCL1684 and U46619 were
purchased from Tocris (Wiesbaden-Nordenstadt, Germany). Data anal-
ysis was as follows: EDH-type relaxations were determined as per-
centage of change of U46619 contraction and are shown relative to the
totally relaxed state (without U46619).
Telemetry
The experiments were in accordance with the Animal Research
Reporting In Vivo Experiments guidelines and approved by the
Institutional Animal Care and Use Committee of the IACS. Surgical
implantation of TA11PA-C10 pressure transducers (Data Sciences
International, St. Paul, MN) into the left carotid artery and telemetry
were performed as described previously (Brahler et al., 2009). Four
female wild-type (22 6 1 g) and four female KCa3.12/2 (27 6 2 g) mice
were used in the present study. After surgery, mice were allowed to
recover for 10 days before compounds or vehicle were injected and
telemetry data were collected. After a washout phase of at least
48 hours after a first injection, animals were reused for injections of a
higher dose of the SKA-111, SKA-121, or vehicle. Thereafter, mice were
treated with 50 mg/ml Nv-nitro-L-arginine methyl ester (L-NAME,
Sigma-Aldrich DK) in the drinking water. This L-NAME treatment
over 2 days increased MAP by 10 6 2 mm Hg in wild-type mice.
Injection of compounds started on day 3 of L-NAME treatment.
Preparation and injection of SKA-111 and SKA-121 were as follows:
Appropriate amounts of SKA-111 and SKA-121 were dissolved in
warmed peanut oil (SKA-111) or in a mixture of peanut oil/DMSO (9:1 v/v,
both from Sigma-Aldrich DK) to give a dose of 30 or 100 mg/kg. Maximal
injection volume was 6600 ml. SKA-111 solution, well-stirred suspen-
sion (SKA-121), or vehicle was injected i.p. during hour 3 of the dark
phase. Mice were subjected to isoflurane anesthesia to minimize stress
and pain during compound application. Telemetry data were col-
lected and analyzed after the mice fully recovered from anesthesia
(20 minutes after injection). Telemetry data were recorded over 1 minute
every 10 minutes over 24 hours and averaged. Data were analyzed using
Data Sciences International software.
The percentage of plasma protein binding for SKA-111 and SKA-
121 was determined by ultrafiltration. Rat plasma (500 ml) was spiked
with 10 mM of compound in 1% DMSO, and the sample was loaded
onto a Microcon YM-30 Centrifugal Filter (Millipore Corp., Bedford,
MA) and centrifuged at 13,500g for 30 minutes at RT. The retentate
was collected by inverting the filter into an Eppendorf tube and
spinning at 13,500g for 15 minutes. The retentate then underwent
sample preparation as per the previously described procedure for
SKA-111 or SKA-121. Plasma protein binding was found to be to be
59 6 2% (n 5 3) for SKA-111 and 81 6 4% (n 5 2) for SKA-121.
Results
SAR Study Aiming to Obtain Selectivity for KCa3.1
with SKA-31 as a Template. As described in the Introduc-
tion, the limited ability of existing benzimidazole/benzothiazole-
type KCa2/3 activators such as SKA-31 to differentiate between
KCa2 and KCa3.1 channels made it desirable to determine
whether additional structural modification would increase se-
lectivity for KCa3.1. Toward this goal, we synthesized a small
focused library of 2-aminothiazoles, 2-aminobenzothiazoles or
2-aminonapthooxazoles (Fig. 1). Substituted 2-aminothiazoles
were prepared by a one-step Hantzsch thiazole synthesis (Goblyos
et al., 2005) from the appropriate substituted 1-tetralone,
thiourea, and iodine (Method I in Fig. 1). This method al-
Pharmacokinetics
Twelve-week-old male C57Bl/6J mice were purchased from Charles
River Laboratories (Wilmington, MA) and housed in microisolator
cages with rodent chow and autoclaved water ad libitum. All
experiments were in accordance with National Institutes of Health
guidelines and approved by the University of California, Davis lowed us to obtain both “open” 2-aminothiazoles as well as to

348
Coleman et al.
Fig. 1. General scheme for the synthesis of thiazoles, 2-aminonaphtho[1,2-d]thiazoles, and 2-aminonaphtho[1,2-d]oxazoles.
replace the central aromatic ring of SKA-31 with aliphatic (EBIO, riluzole, SKA-31) and found that the QPatch results
rings. Fully aromatic 2-aminobenzothiazoles could then be were virtually identical to the IC50 and EC50 values obtained
produced by aromatizing with 2-iodoxybenzoic acid (Method by manual patch clamp in our hands (Jenkins et al., 2013). We
II in Fig. 1). An alternative route to 5-position substituted here made use of this assay and determined EC50 values for
2-aminobenzothiazole was the classic Hugerschoff benzothiazole
KCa3.1 activation using HEK-293 cells stably expressing
synthesis (Jordan et al., 2003), in which appropriately sub- human KCa3.1. Activities on human KCa2.3 were determined
stituted amines were transformed into the corresponding by manual electrophysiology since we currently only have
thioureas and then subsequently reacted with benzyltrimethyl
KCa2.3 available in COS-7 cells, which are difficult to handle
ammonium tribromide to deliver bromine in stoichiometric on the QPatch. For both channels we used 250 nM of free
amounts as an alternative to liquid bromine (Method III in [Ca21]i since positive gating-modulators like SKA-31 typically
Fig. 1). Lastly, naphthooxazoles were prepared by first oxidizing increase KCa currents at this Ca21 concentration roughly
4-methyl-1-tetralone with in situ formed bis(trifluoroacetoxy)iodo] 30-fold, creating a large assay window (Sankaranarayanan
benzene and then adding cyanamide (Schuart and Müller, 1973) et al., 2009; Jenkins et al., 2013).
to the intermediately produced 4-methylnaphthalene-1,2-dione
(Method IV in Fig. 1).
Removal of the continuous conjugation by opening of the
napthothiazole system (SKA-75 and SKA-76) of SKA-31 or re-
The compounds synthesized by these methods as well as placement of the internal aromatic ring with either a cyclohexyl
five commercially available compounds were tested for their (SKA-70, SKA-108, SKA-113, SKA-114) or a cyclopentyl ring
KCa2.3- and KCa3.1-activating activity using either manual or (SKA-69, SKA-71) in general reduced both potency and selec-
automated whole-cell patch-clamp. Our group previously tivity irrespective of whether the compounds bore any sub-
described the establishment of a QPatch assay for KCa3.1 stituents or not (Fig. 2, blue compounds). Since these results
modulators. In this study, we benchmarked data obtained on demonstrated that aromaticity of the internal ring was re-
the QPatch against manual patch-clamp electrophysiology by quired for both KCa2.3 and KCa3.1 activation, we went back to
determining the potency of several commonly used KCa3.1 benzothiazoles (Fig. 2 green compounds) and next explored sub-
inhibitors (TRAM-34, NS6180, charybdotoxin) and activators stitutions on the napthothiazole system of SKA-31. Introduction

Positive Gating Modulation of KCa3.1
349
Fig. 2. Chemical structures and EC50 values for KCa2.3 and KCa3.1 activation. All compounds were tested at least three times at concentrations of 4 to 5,
and EC50 values were determined by fitting the Hill equation to the increase in slope conductance between 280 and 265 mV.
of substituents in the 5-position had varying effects: chloride in a compound that was too insoluble to be tested. Introduc-
(SKA-72), which is both electron withdrawing and lipophilic, tion of a methyl group in the 5-position, which is less lipophilic
increased potency on both KCa2.3 (EC50 335 nM) and KCa3.1 than chloride but has a positive inductive effect on the ring
(EC50 110 nM) but basically abolished any selectivity between system, slightly increased potency for KCa3.1 (EC50 111 nM)
the two channels. Fluoride in the 5-position (SKA-106) reduced in comparison with SKA-31 and dramatically increased
potency roughly 10-fold compared with SKA-31 but preserved selectivity for KCa3.1 over KCa2.3 to ∼100-fold (SKA-111).
selectivity, whereas introduction of bromide (SKA-87) resulted However, replacement of the CH3 group with other, larger

350
Coleman et al.
carbon containing electron-donating groups such as 2OCH3
(SKA-117) or electron withdrawing groups such as –CN (SKA-
107) again reduced potency and selectivity. Attaching two of
the obviously favorable CH3 groups in positions 6 and 8 of the
ring system (SKA-109) instead of the 5-position preserved
selectivity over KCa2.3 but reduced potency on KCa3.1 by
10-fold. Installation of an ethylene bridge connecting the 5-
and 6- position reduced both potency and selectivity and
resulted in a compound (SKA-73) that activated both KCa2.3
and KCa3.1 equipotently with an EC50 of 1 mM. We further
tried replacing the terminal ring of SKA-31 with a thiophene
(SKA-110) or an aliphatic cyclopentyl (SKA-81) but again only
saw a reduction in potency.
To better understand the full extent of the pharmacophore
and potentially obtain patentable compounds, we explored
alternative scaffolds (Fig. 2, red compounds). Moving away
from the 2-aminothiazole system by replacing the 2-position
NH2 group with a CH3 group (SKA-74), as well as isosterically
replacing the S atom with an O (SKA-103 and SKA-104) or
geminal CH3 groups (SKA-92), completely abolished activity.
However, if the 2-position NH2 group was retained and only
the S isosterically replaced with an O as in the SKA-102,
which basically constitutes an oxazole analogous SKA-31, ac-
tivity was regained, and the resulting compound activated
K
Ca3.1 with an EC50 of 2.7 mM. Introduction of a CH3 group in
Fig. 3. X-ray crystal structures of SKA-120 (top) and SKA-121 (bottom).
5-position, which previously was found to increase selectivity
of the napthothiazole SKA-111 for KCa3.1 to 100-fold, had
a similar effect on the 2-aminonaphthooxazole system. The
two regioisomers, SKA-120 and SKA-121, which resulted from SKA-111 and the naphthooxazole SKA-121, we determined
the synthesis and had to be separated by flash chromatogra- seven-point concentration-response curves on KCa2.1, KCa2.2,
phy, exhibited EC50 values of 180 and 109 nM for KCa3.1 and
K
Ca2.3 and KCa3.1 with 250 nM free Ca21 in the internal
EC50 values of 9.2 and 4.4 mM for KCa2.3, corresponding to solution (Fig. 4). SKA-111 and SKA-121 displayed nearly
a ∼50- or 40-fold selectivity. The correct structural assign- identical EC50 values on KCa3.1 (111 6 27 nM and 109 6 14 nM).
ment of the two regioisomers was confirmed by the different Similar to the template SKA-31 (Sankaranarayanan et al.,
chemical shift of proton 9-H in the 800 MHz 1H-NMR since 2009), these effects plateaued at a roughly 30-fold maximal
this proton is shielded differently depending on whether it is current increase with this intracellular Ca21 concentration.
in proximity to either N or O in the adjacent oxazole ring. We Both compounds exhibited 40- to 120-fold selectivity over the
further grew crystals of SKA-120 and SKA-121 and had them three KCa2 channels (Fig. 4; Table 1). The Hill coefficient nH
subjected to X-ray analysis, which allowed us to “see” the varied between 1.6 and 3.1 in most cases, which was again
exact position of the N and O in the two compounds (Fig. 3). similar to what had been previously reported for the template
The crystal structures show two very different hydrogen bond- SKA-31.
ing networks. Although SKA-120 exists as a dimer, SKA-121
has a hydrogen bonding motif that leads to a tetramer forming 121 over more distantly related channels. At the highest
ribbon structures (Supplemental Fig. 1). reasonable and well-dissolvable test concentrations, 25 mM
We next determined the selectivity of SKA-111 and SKA-
In summary, this SAR study demonstrated that it is for SKA-111 and 50 mM for SKA-121, both compounds blocked
possible to generate KCa3.1-selective activators using the representative members of the major KV channel families
naphthothiazole and the isosteric naphthooxazole scaffolds. (KV1.3, KV2.1, KV3.1, and KV11.1) by 10% to 46% (Table 1).
In both cases, the presence of the 2-amino group was Similarly, neuronal (NaV1.2, NaV1.7), skeletal muscle (NaV1.4),
absolutely required for activity on both KCa2.3 and KCa3.1 and cardiac (NaV1.5) sodium channels, as well as L-type Ca21
channels. It was further necessary for the annulated three- channels (CaV1.2), were blocked by 20% to 50% by 25 mM of
ring system to be fully aromatic. Replacement of the terminal SKA-111 or 50 mM SKA-121. SKA-111 and SKA-121 thus
or the internal ring system with aliphatic rings reduced displayed at least 200- to 400-fold selectivity for KCa3.1 over
activity on both channels. The key position able to confer both these physiologically relevant channels. Interestingly, while
high potency and selectivity for KCa3.1 seems to be the 5-position, performing this selectivity screen, we found that N1E-115
which proved to have a very “tight” SAR. Whereas the large, neuroblastoma cells, a cell line established in the 1970s from
lipophilic, and relatively “soft” chloride endowed the compounds a spontaneous mouse neuroblastoma tumor, are highly suit-
with potency on both KCa2.3 and KCa3.1 (SKA-72), only CH3 able for automated electrophysiology. The cell line produced
in this position produced selectivity for KCa3.1 on both the large NaV1.2 currents comparable to NaV1.5 currents in a
naphthothiazole (SKA-111) and the isosteric naphthooxazole stable HEK-293 cell line (Supplemental Fig. 2).
(SKA-121) system.
SKA-121 is a Positive Gating Modulator of KCa3.1.
SKA-111 and SKA-121 Are Selective KCa3.1 Activa- Classic KCa activators like EBIO and NS309 have been shown
tors. To fully evaluate the selectivity of the naphthothiazole to increase the apparent Ca21-sensitivity of KCa channels by

Positive Gating Modulation of KCa3.1
351
Fig. 4. SKA-111 and SKA-121 are potent and selective KCa3.1 activators. (A) Example traces of KCa3.1 and KCa2.3 activation by SKA-111 and
concentration-response curves for KCa3.1 (EC50 111 6 27 nM, nH 1.5), KCa2.3 (EC50 13.7 6 6.9 mM, nH 1.9), KCa2.1 (EC50 8.1 6 0.4 mM, nH 4.5), and KCa2.2
(EC50 7.7 6 1.9 mM, nH 2.3). (B) Example traces of KCa3.1 and KCa2.3 activation by SKA-121 and concentration-response curves for KCa3.1 (EC50 109 6 14 nM,
nH 3.0), KCa2.3 (EC50 4.4 6 2.3 mM, nH 1.6), KCa2.1 (EC50 8.7 6 1.6 mM, nH 4.1), and KCa2.2 (EC50 6.8 6 2.2 mM, nH 1.7). All data points are mean 6 S.D.
(C) Representative currents from inside-out patches in the presence of 0.3 mM (top) and 1 mM (bottom) Ca2+ before and after application of 1 mM
SKA-121. (D) KCa3.1 current at 275 mV in an inside-out patch exposed to varying Ca2+ concentrations as a function of time. (Note: SKA-121 applied with
500 nM Ca2+ was washed out with 1 mM Ca2+). (E) Ca2+ concentration-response curve for KCa3.1 activation measured from inside-out patches in the
absence or presence of 1 mM SKA-121. Currents from individual patches were normalized to the effect of 10 mM Ca2+ in the absence of SKA-121. Data are
mean 6 S.D. (n = 3–5/data point; *P , 0.05, unpaired Student t test). An extra sum-of-squares F test (GraphPad Prism5) to compare the two curves
rendered a P , 0.0001 for comparison of the EC50 values in the presence and absence of SKA-121. (F) Blockade of KCa3.1 and KCa2.3 currents activated
by SKA-121 by TRAM-34 and UCL1684.
stabilizing the interaction between CaM and KCa channels SKA-121 at every Ca21 concentration. The EC50 of the
(Pedarzani et al., 2001; Li et al., 2009) Since this phenomenon Ca21-concentration response curve (Fig. 4E) shifted from
manifests in a leftward shift of the Ca21 concentration-response 650 6 50 nM to 380 6 95 nM in the presence of 1 mM of
curve, we performed inside-out experiments in which we varied SKA-121, whereas the Hill coefficient was not changed by
the intracellular [Ca21]i concentration and investigated the SKA-121 (nH ∼3 in both cases). Interestingly, SKA-121 not
ability of 1 mM of SKA-121 to further activate KCa3.1 currents only shifted the curve to the left, but it also substantially
at different [Ca21]i concentrations. As shown in Fig. 4C, increased the maximal achievable current at 1 and 10 mM,
inside-out patches pulled from hKCa3.1-expressing HEK-293 suggesting that the compound might be able to further in-
cells exhibited Ca21-dependent K1 currents, reversing at crease the open probability of KCa3.1 at these Ca21 concen-
0 mV in symmetrical K1, which could be increased further by trations. As expected, KCa3.1 currents activated by 1 mM

352
Coleman et al.
TABLE 1
Selectivity of SKA-111 and SKA-121 over selected ion channels
The number in parentheses indicates the number of cells used to determining the EC50 values or the % of current
inhibition.
Channel
KCa1.1
SKA-111 EC50
SKA-121 EC50
120% of current at 25 mM (3)
8.1 6 0.4 (10)
115% of current at 50 mM (3)
8.7 6 1.6 (8)
6.8 6 1.7 (12)
4.4 6 2.6 (18)
0.019 6 0.014 (21)
SKA-121% current inhibition at 50 mM
27.5 6 7.2% (3)
K
K
K
K
Ca2.1
Ca2.2
Ca2.3
Ca3.1
7.7 6 1.9 (10)
13.7 6 6.9 (15)
0.111 6 0.027 (24)
SKA-111% current inhibition at 25 mM
28.5 6 2.3% (4)
Channel
KV1.3
KV2.1
KV3.1
KV11.1 (hERG)
NaV1.2
NaV1.4
NaV1.5
NaV1.7
CaV1.2
37.3 6 9.0% (4)
43.2 6 12.5% (3)
46.3 6 16.5% (4)
16.7 6 9.7% (5)
15.2 6 12.7% (5)
25.7 6 1.3% (5)
32.1 6 11.1% (5)
28.5 6 1.8% (5)
48.9 6 2.7% (5)
32.9 6 1.3% (3)
10.1 6 7.7% (5)
19.5 6 6.9% (5)
33.4 6 10.5% (5)
39.9 6 11.1% (5)
27.5 6 0.9% (3)
49.5 6 17.0% (5)
SKA-121 could be completely inhibited by 1 mM of the KCa3.1 drop in mean arterial blood pressure (MAP) by ∼25 mmHg
pore blocker TRAM-34 (Wulff et al., 2000), whereas the KCa starting 20–30 minutes after injection (Fig. 6A, left). This
2
channel pore blocker UCL1684 (Rosa et al., 1998) had a decrease in MAP was significant compared with vehicle (peanut
similar effect on KCa2.3 currents activated by 20 mM SKA-121 oil)-treated mice. The blood pressure drop was accompanied
(Fig. 4F).
by a severe reduction in HR by ∼400 bpm (Fig. 6A, left). To
SKA-111 and SKA-121 Increase Bradykinin-Induced avoid fatal hypothermia or circulatory collapse, we handled
Vasodilation. In addition to modulating the contractile these severely bradycardic mice for ∼2 hours and increased
state of the underlying vascular smooth muscle by releasing RT to 34°C. As shown in Fig. 6A, these maneuvers increased
nitric oxide and prostacyclin, the vascular endothelium can MAP transiently, presumably because of a sympathetic input
induce an endothelium-derived hyperpolarization (EDH) on total peripheral resistance, but not HR, and the low HR
in response to stimulation with acetylcholine or bradykinin persisted over another 10 hours before the mice slowly re-
(BK). These agonists increase [Ca21]i in the endothelium, ac- covered (Fig. 6A, left). SKA-121 at 100 mg/kg also lowered
tivate KCa3.1 and KCa2.3, and induce KCa, channel-mediated blood pressure by ∼20 mmHg (Fig. 6A, right). However, this
hyperpolarization and arterial relaxation (Ng et al., 2008; drop was more transient and lasted ∼3 hours. HR was only
Grgic et al., 2009; Dalsgaard et al., 2010; Edwards et al., 2010; moderately reduced (Fig. 6A, right). A lower dose of 30 mg/kg
Köhler et al., 2010; Wulff and Köhler, 2013). To demonstrate of both compounds did not produce significant alterations in
that SKA-111 and SKA-121 efficiently augment native KCa3.1 MAP (Supplemental Fig. 3), whereas SKA-111 decreased HR
in PCAs and thereby potentiate BK-induced relaxation, we to a minor extent (∼250 bpm for ∼2 hours after injection
performed isometric myography on PCA precontracted with (Supplemental Fig. 3). The vehicles, peanut oil (for SKA-111) or
0.2 mM of the vasospasmic thromboxane mimetic, U46619. peanut oil/DMSO (9:1 v/v, for SKA-121), did not cause
SKA-111, as well as SKA-121, both at 1 mM, potentiated BK significant alterations in MAP or HR (Fig. 6A).
(1 mM)-induced relaxation to ꢀ200% and ꢀ300%, respectively
We next tested whether SKA-111 and SKA-121 are also
(Fig. 5). The KCa3.1 blocker TRAM-34 (1 mM) prevented this efficient in lowering the higher MAP caused by systemic
potentiation and the combination of TRAM-34 and the KCa2.3 inhibition of nitric oxide production by L-NAME administered
blocker UCL-1684 (1 mM) inhibited this potentiation slightly in the drinking water (Fig. 6B). However, for these experi-
more effectively than TRAM-34 alone (Fig. 5).
ments, we used a lower dose (60 mg/kg) of SKA-111 to avoid
These data from ex vivo vessel experimentation demonstrate causing such a severe drop of HR as observed with 100 mg/kg.
that the KCa3.1-selective activators SKA-111 and SKA-121 are The 60 mg/kg dose produced only a minor drop in MAP and
capable of positively modulating a physiologic response, (e.g., HR. In contrast, SKA-121 at 100 mg/kg produced a significant
EDH-type vasorelaxation, in which KCa3.1/KCa2.3 functions drop in MAP by ∼25 mmHg over 6 hours. This drop was ac-
have been implicated before) (Edwards et al., 2010; Wulff and companied by a minor decrease in HR (Fig. 6B, right).
Köhler, 2013).
We next evaluated the KCa3.1 selectivity by using KCa3.12/2
Systemic Cardiovascular Effects of SKA-111 and mice and found that SKA-111 at 100 mg/kg also produced
SKA-121. Since we had mice implanted with telemetry a significant drop in MAP by ∼15 mmHg lasting ∼16 hours in
leads available, we performed telemetric blood pressure mea- these animals (Fig. 6C, left). Similar to wild-type mice, HR
surements on wild-type and KCa3.12/2 mice to evaluate the decreased substantially by ∼400 beats/minute over ∼22 hours
cardiovascular activity and selectivity of SKA-111 and SKA- (Fig. 6C, left). Similar to the results in wild-type mice, the
121 before performing pharmacokinetic studies. However, lower dose of 30 mg/kg also significantly reduced HR,
since we did not know the half-life when these experiments although less dramatically and not for as long as the higher
were done, we chose to start with the relatively high dose of dose. MAP did not change with the 30 mg/kg dose of SKA-111
100 mg/kg for both compounds, reasoning that we could (Fig. 6C, left). In contrast to SKA-111, SKA-121 at 100 mg/kg
lower the dose in subsequent experiments. In wild-type mice, had no significant effects on MAP and HR in the KCa3.12/2
i.p. injection of 100 mg/kg SKA-111 produced a substantial mice (Fig. 6C, right).

Positive Gating Modulation of KCa3.1
353
Fig. 5. Isometric myography. SKA-111 and SKA-121 (both at 1 mM) increased BK-induced EDH-type relaxation of U46619-precontracted PCA rings in
the presence of blockers of NO synthesis (L-NNA) and cyclooxygenases (indomethacin). TRAM-34 (1 mM) alone and the combination of TRAM-34 and
UCL-1684 (1 mM) prevented the increase of relaxation. Data are mean 6 S.E.M., n = 5–22 PCA. *P , 0.05, unpaired Student’s t test.
Taken together, these telemetry experiments showed detectable in both plasma and brain. SKA-111 proved highly
that both compounds exhibited cardiovascular activity in brain penetrant with a brain-to-plasma ratio of 9.3 6 5.3.
vivo as they substantially reduce basal blood pressure and SKA-121 was less brain penetrant but still very effectively
the higher blood pressure caused by NO deficiency. Moreover, partitioned into the brain with a brain/plasma ratio of 3.3 6 2.9
SKA-121 produced this blood pressure–lowering action in a (Fig. 7C).
K
Ca3.1-dependent manner as suggested by lack of MAP-
Taken together, these results explain why SKA-111 had
lowering effects in KCa3.12/2 mice. In contrast, SKA-111 lowered a much more prolonged blood pressure–lowering effect in
MAP and induced a strong HR reduction independently of the telemetry experiments in Fig. 6 than did SKA-121. The
KCa3.1.
fact that SKA-111 is highly brain penetrant and probably
Pharmacokinetics of SKA-111 and SKA-121. To help achieved total brain concentrations in the range of 10–30 mM
us better interpret the results of the telemetry experi- for hours after i.p. administration at 100 mg/kg also pro-
ments, we established ultraperformance liquid chromatogra- vides an explanation for why this KCa3.1 selective compound
phy (UPLC)/mass spectrometry (MS) assays for SKA-111 and “lost” its selectivity in vivo and reduced blood pressure and
SKA-121 based on an high-performance LC/MS assay we had heart rate, side effects that are presumably mediated by KCa2
previously published for SKA-31 (Sankaranarayanan et al., channel activation in the central nervous system as well
2009) and performed some basic pharmacokinetic studies as KCa2 channel activation in resistance arteries and the
with both compounds in mice. After i.v. injection at 10 mg/kg heart (Radtke et al., 2013) and also occurred in KCa3.12/2
into the tail vein, total SKA-111 plasma concentrations fell mice (Fig. 6C, left). In keeping with its shorter half-life of
biexponentially, reflecting a two-compartment model with only 20 minutes and its lower brain penetration, SKA-121
very rapid distribution from blood into tissue (∼2 minutes), induced a significant but less prolonged drop in MAP in the
followed by elimination with a half-life of 4.7 60.6 hours (Fig. 7A). telemetry experiments when administered i.p. at 100 mg/kg
SKA-121, in contrast, had a much shorter half-life (∼20 minutes), (Fig. 6A right) and exhibited KCa3.1 selectivity in vivo as
and plasma decay was extremely rapid (21.3 6 2.4 mM at suggested by the lack of MAP-lowering effects in KCa3.12/2
5 minutes; 483 6 231 nM at 1 hour and 53 6 44 nM at mice and its insignificant effect on heart rate in wild-type
4 hours). Since SKA-121 is relatively well soluble (logP 5 1.79) mice.
and could potentially be added to drinking water in animal
experiments, we also administered it orally and found that it
had an oral availability of roughly 25% (Fig. 7B). But again,
Discussion
plasma levels dropped rapidly from 1.1 6 0.1 mM at 1 hour after
oral administration to 27 6 5 nM at 8 hours. Plasma protein
binding was 59% 6 2% (n 5 3) for SKA-111 and 81% 6 4%
(n 5 2) for SKA-121. Since we had also removed brains from
the mice when obtaining blood samples by cardiac puncture
(which had been done at every third blood collection), we
further determined total brain concentrations at various
time points and obtained averaged brain/plasma ratios for
both compounds from times when the compounds were
We here used our previously described mixed KCa2/3 channel
activator SKA-31 (Sankaranarayanan et al., 2009) as a tem-
plate for the design of two selective KCa3.1 activators. Both
molecules, SKA-111 and SKA-121, activate KCa3.1 with EC50
s
of ∼110 nM and display 40- to 120-fold selectivity for KCa3.1
over the three KCa2 channels (KCa2.1, KCa2.2, and KCa2.3).
These compounds constitute the first pharmacological or chem-
ical biology tools that can be used to selectively activate KCa3.1
channels in tissue preparations or in vivo without performing

354
Coleman et al.
Fig. 6. Blood pressure telemetry. (A) Injections (i.p.) of SKA-111 (panels on left) and SKA-121 (panels on right) reduced MAP in wild-type mice. SKA-111
(on left), but not SKA-121 (on right), severely reduced HR. As indicated by the dashed line (right panel), SKA-111–treated animals were initially handled
and warmed to avoid fatal hypothermia during strong bradycardia. Ve, vehicle control. Black and white marked intervals of x-axis indicate dark and
light periods. (B) SKA-121 (on right) but, to a lesser extent, SKA-111 (on left), reduced blood pressure in L-NAME–treated moderately hypertensive mice.
HR remained virtually stable. (C) Cardiovascular actions of SKA-111 and SKA-121 in KCa3.12/2 mice. At 100 mg/kg, SKA-111 but not SKA-121 reduced
MAP and HR. SKA-111 also reduced HR at 30 mg/kg. Data points are means 6 S.E.M.; n = 3 or 4 experiments/strain and compound. Lines indicate time
periods when pressures or HRs were significantly different from Ve. *P , 0.05, unpaired Student’s t test.
additional manipulations such as genetically knocking out or stretch of 16 amino acids that connects S6 to the CaMBD and
pharmacologically blocking KCa2 channels.
was not visible in the EBIO/CaM/CaMBD crystal becomes
According to the definition of positive-gating modulation, com- visible, suggesting that it undergoes a transition to a well-
pounds like EBIO and NS309 act by shifting the Ca21-activation defined structure. Other manipulations of this S6-CaMBD
curve of KCa2/3 channels to the left, meaning that the deter- linker region, such as cross-linking a residue in the region to
mined EC50 values for Ca21-dependent channel activation a residue in the CaMBD, also increase channel activity and
calculated from Ca21-concentration response curves decrease apparent Ca21 sensitivity, demonstrating that this linker re-
in the presence of the modulator molecule. For NS309, studies gion plays a crucial role in coupling Ca21 binding to CaM to
using CaM mutants making unstable association with the the mechanical opening of KCa channels (Zhang et al., 2013).
CaMB of KCa2.2 have shown that NS309 increases the ap- By changing the confirmation of this linker region, NS309 is
parent Ca21-sensitivity of KCa channels by stabilizing the thus “truly” a gating modulator and we assume that SKA-111
interaction between CaM and the CaMBD of the KCa channels and SKA-121 are exerting their effects in a similar manner.
(Pedarzani et al., 2001; Li et al., 2009). More recently, Zhang We have not yet mapped their binding sites, but we have also
et al. (2012) crystallized CaM bound to the CaMBD of KCa2.2 observed that mutations of the analogous residues in the
and afterward soaked EBIO into the crystal. In a subsequent CaMBD of KCa2.3, which had been reported to increase or
study, the same group obtained a cocrystal of the CaM/CaMBD decrease the potency of EBIO for activating KCa2.2 (Zhang
with NS309 (Zhang et al., 2013). Both molecules reside in et al., 2012), also significantly altered the potency of SKA-31
a pocket formed at the interface between CaM/CaMBD. (Brown et al., 2014) suggesting that, similar to EBIO and
Interestingly, on NS309 binding, an intrinsically disordered NS309, benzothiazole-type KCa2/3 activators bind at the

Positive Gating Modulation of KCa3.1
355
interface between CaM/CaMBD. This interface pocket is
relatively “tight,” and the CaMBD shows a number of se-
quence differences between the four KCa channels, making it
appear plausible that a “minor” structural change such as
adding a -CH3 in 5-position of SKA-31 can increase selectivity
for KCa3.1 from 10-fold to 100-fold in SKA-111. This hy-
pothesis that SKA-31, SKA-111, and SKA-121 are binding to
the same site in the CaM/CaMBD interface as NS309 agrees
well with the relatively steep structure-activity-relationship
we observed in our study. For the napthobenzothiazole and
the isosteric naphthooxazole system, potency and selectivity
for KCa3.1 over KCa2 channels were very sensitive to the exact
position and electronic nature of substituents (e.g., SKA-106
and SKA-109 in Fig. 2). Another interesting observation in
this context is that SKA-121 not only shifted the concentration-
response curve for Ca21-dependent KCa3.1 activation to the
left but also increased the maximal achievable current at
1 and 10 mM in inside-out patches (Fig. 4). Since none of the
benzothiazole-type KCa2/3 activators, including SKA-31,
SKA-111, SKA-121, and their many derivatives, had ever
increased KCa3.1 or KCa2 currents in our hands at Ca21
concentrations lower than 100 nM or in the absence of Ca21
with KF-based pipette solutions, we do not ascribe this effect
to a direct channel opening component in their mechanism of
action, like that reported for GW542573X and (-)-CM-TMPF
for KCa2.1 (Hougaard et al., 2009, 2012). Unlike SKA-121, which
we think is binding at the CaM/CaMBD interface, (-)CM-TMPF
has been found to interact with positions deep within the inner
pore vestibule (Hougaard et al., 2012) close to the selectivity
filter, where the gate of KCa2/3 channels seems to be located
(Bruening-Wright et al., 2002, 2007; Garneau et al., 2009;
Klein et al., 2007). It therefore seems reasonable to attribute
the Ca21-independent KCa2.1 channel activation by (-)-CM-TMPF
to a directly opening effect on the gate and use this ex-
planation to account for the fact that (-)-CM-TMPF increases
K
Ca2.1 currents to roughly 40% of their maximal activity at
Ca21 concentrations between 10 and 100 nM and then levels
off in its opening/activating activity at higher Ca21 concentra-
tions (Hougaard et al., 2012). Since this is clearly not the case
for SKA-121, which in contrast further increases maximal
channel activity at 1 and 10 mM of Ca21, we believe that SKA-
121 is a “classic” positive gating modulator, which requires
the presence of Ca21 to enhance KCa channel activity but by
stabilizing the interaction between CaM and the CaMBD of
K
Ca3.1 is also able to further increase the Ca21-dependent
open channel probability Po(max) value of KCa3.1. Unlike KCa
2
channels, which are assumed to be fully open at saturating
[Ca21]i concentrations, KCa3.1 channels have been reported
to have a relatively low Ca21-dependent Po(max), which can
be increased significantly by the addition of 1.6 mM MgATP
(Gerlach et al., 2001; Jones et al., 2007) or by mutations of
residues in S5 (Garneau et al., 2014). We here left out ATP from
the internal solutions for inside-out and whole-cell recordings
on purpose to not confuse the analysis by having too many
variables.
Since KCa3.1 is involved in EDH-mediated vasodilator re-
sponses and has been accordingly suggested as a potential new
antihypertensive pharmacological target (Grgic et al., 2009;
Dalsgaard et al., 2010; Edwards et al., 2010; Köhler et al.,
2010), we tested the effect of both of our new KCa3.1 selective
activators, SKA-111 and SKA-121, on BK-induced EDH re-
sponses in vitro on porcine coronary arteries and on blood
Fig. 7. Pharmacokinetics of SKA-111 and SKA-121. (A) Total SKA-111
plasma concentration (mean 6 S.D.) after i.v. administration of 10 mg/kg
to mice (n = 2 to 3 per time point; t1/2 Distribution ∼2 minutes; t1/2
elimination = 4.7 6 0.6 hours). Inset: The first 10 hours on a log scale. (B)
Total SKA-121 plasma concentrations (mean 6 S.D.) after i.v. (black) and
oral (red) application of 10 mg/kg to mice (n = 3/time point; t1/2 ∼20
minutes; oral availability ∼25%). Inset: The first 10 hours after i.v.
administration on a log scale. (C) Brain-to-plasma concentration ratios for
SKA-111 and SKA-121 determined from multiple paired brain and plasma
samples obtained during the experiments shown in (A) and (B) (n = 8 for
SKA-111 and n = 5 for SKA-121).

356
Coleman et al.
Blank T, Nijholt I, Kye MJ, Radulovic J, and Spiess J (2003) Small-conductance,
Ca21-activated K1 channel SK3 generates age-related memory and LTP deficits.
Nat Neurosci 6:911–912.
Brähler S, Kaistha A, Schmidt VJ, Wölfle SE, Busch C, Kaistha BP, Kacik M,
Hasenau AL, Grgic I, and Si H et al. (2009) Genetic deficit of SK3 and IK1 channels
disrupts the endothelium-derived hyperpolarizing factor vasodilator pathway and
causes hypertension. Circulation 119:2323–2332.
Brown BM, Coleman N, Oliván-Viguera A, Köhler R, Wulff H (2014) Positive KCa
channel gating modulators with selectivity for KCa3.1. FASEB J 28:1057.6.
Bruening-Wright A, Lee WS, Adelman JP, and Maylie J (2007) Evidence for a deep
pore activation gate in small conductance Ca21-activated K1 channels. J Gen
Physiol 130:601–610.
Bruening-Wright A, Schumacher MA, Adelman JP, and Maylie J (2002) Localization
of the activation gate for small conductance Ca21-activated K1 channels. J Neurosci
22:6499–6506.
Dalsgaard T, Kroigaard C, and Simonsen U (2010) Calcium-activated potassium
channels - a therapeutic target for modulating nitric oxide in cardiovascular disease?
Expert Opin Ther Targets 14:825–837.
Damkjaer M, Nielsen G, Bodendiek S, Staehr M, Gramsbergen JB, de Wit C, Jensen
BL, Simonsen U, Bie P, and Wulff H et al. (2012) Pharmacological activation of
KCa3.1/KCa2.3 channels produces endothelial hyperpolarization and lowers blood
pressure in conscious dogs. Br J Pharmacol 165:223–234.
Debono MW, Le Guern J, Canton T, Doble A, and Pradier L (1993) Inhibition by
riluzole of electrophysiological responses mediated by rat kainate and NMDA
receptors expressed in Xenopus oocytes. Eur J Pharmacol 235:283–289.
Devor DC, Singh AK, Frizzell RA, and Bridges RJ (1996) Modulation of Cl- secretion
by benzimidazolones. I. Direct activation of a Ca(21)-dependent K1 channel. Am J
Physiol 271:L775–L784.
Duprat F, Lesage F, Patel AJ, Fink M, Romey G, and Lazdunski M (2000) The
neuroprotective agent riluzole activates the two P domain K(1) channels TREK-1
and TRAAK. Mol Pharmacol 57:906–912.
pressure in mice. Both compounds potentiated BK effects in
vitro and robustly lower blood pressure in mice in vivo. How-
ever, as these experiments and subsequently performed
pharmacokinetic studies showed, both compounds do not have
ideal properties for development into a potential antihyper-
tensive drug candidate. SKA-111 is so highly brain penetrant
that it achieves roughly ∼10-fold higher concentrations in the
central nervous system and may thus cause complex neurologic
side effects by activating neuronal KCa2 channels (Adelman
et al., 2012). Moreover, SKA-111 induces the same severe
bradycardia that had also been a problem when the unselective
SKA-31 was dosed at 100 mg/kg in connexin 40–deficient mice
(Radtke et al., 2013). This bradycardia is especially impressive
in that it occurs also in KCa3.12/2 mice (Fig. 6) and is probably
due to direct effects on KCa2 channels in cardiac pacemaker
tissue (Radtke et al., 2013), as well as to a possible a central
decrease in sympathetic drive through activation of neuronal
KCa2 channels. SKA-121 is less brain penetrant and largely
maintains its KCa3.1 selectivity in vivo. It lowers blood pressure
in both normotensive and hypertensive mice without significantly
reducing heart rate or affecting blood pressure in KCa3.12/2 mice.
However, a problem with SKA-121 is the extremely short
20-minute half-life in mice, which would necessitate continuous
infusion for blood pressure studies, a depot, or very frequently
repeated drug applications. In this respect, it should, of course,
be explored if SKA-121 possibly has a longer half-life in larger
animals such as dogs, pigs, or primates.
In summary, with SKA-111 and SKA-121, we have iden-
tified two KCa3.1-selective positive gating modulators that
constitute novel pharmacological tools for further dissecting
the role of KCa3.1 in EDH and systemic blood pressure and
that could help determine whether KCa3.1 activators could
eventually be developed into a new class of endothelial
targeted antihypertensive agents. Other potential indications
for KCa3.1 activators could be intrasurgical hypertension,
acute vasospasm, or preservation of endothelial function in
large vascular organs such as the heart or kidneys or in vessel
grafts during storage and transplantation. KCa3.1 activators
have also long been suggested for enhancing fluid secretion in
cystic fibrosis (Singh et al., 2001). Although SKA-111 and
SKA-121 are not ideal candidate molecules, they could serve
as templates for the design of derivatives with pharmacoki-
netic properties more suitable for further development and
innovation.
Edwards G, Félétou M, and Weston AH (2010) Endothelium-derived hyper-
polarising factors and associated pathways: a synopsis. Pflugers Arch 459:
863–879.
Fanger CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K,
Chandy KG, Cahalan MD, and Aiyar J (1999) Calmodulin mediates calcium-
dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol
Chem 274:5746–5754.
Garneau L, Klein H, Banderali U, Longpré-Lauzon A, Parent L, and Sauvé R (2009)
Hydrophobic interactions as key determinants to the KCa3.1 channel closed con-
figuration: an analysis of KCa3.1 mutants constitutively active in zero Ca21. J Biol
Chem 284:389–403.
Garneau L, Klein H, Lavoie MF, Brochiero E, Parent L, and Sauvé
R (2014)
Aromatic-aromatic interactions between residues in KCa3.1 pore helix and S5
transmembrane segment control the channel gating process. J Gen Physiol 143:
289–307.
Gerlach AC, Syme CA, Giltinan L, Adelman JP, and Devor DC (2001) ATP-dependent
activation of the intermediate conductance, Ca21-activated K1 channel, hIK1, is
conferred by a C-terminal domain. J Biol Chem 276:10963–10970.
Göblyös A, Santiago SN, Pietra D, Mulder-Krieger T, von Frijtag Drabbe Künzel J,
Brussee J, and Ijzerman AP (2005) Synthesis and biological evaluation of
2-aminothiazoles and their amide derivatives on human adenosine receptors.
Lack of effect of 2-aminothiazoles as allosteric enhancers. Bioorg Med Chem 13:
2079–2087.
Grgic I, Kaistha BP, Hoyer J, and Köhler R (2009) Endothelial Ca1-activated K1
channels in normal and impaired EDHF-dilator responses: relevance to cardiovascular
pathologies and drug discovery. Br J Pharmacol 157:509–526.
Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz
MJ, Auperin DD, and Chandy KG (1994) Pharmacological characterization of five
cloned voltage-gated K1 channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably
expressed in mammalian cell lines. Mol Pharmacol 45:1227–1234.
Grunnet M, Jespersen T, Angelo K, Frøkjaer-Jensen C, Klaerke DA, Olesen SP, and Jensen
BS (2001) Pharmacological modulation of SK3 channels. Neuropharmacology 40:
879–887.
Hougaard C, Eriksen BL, Jørgensen S, Johansen TH, Dyhring T, Madsen LS,
Strøbaek D, and Christophersen P (2007) Selective positive modulation of the SK3
and SK2 subtypes of small conductance Ca21-activated K1 channels. Br J Pharmacol
151:655–665.
Hougaard C, Hammami S, Eriksen BL, Sørensen US, Jensen ML, Strøbæk D,
and Christophersen P (2012) Evidence for a common pharmacological interaction
site on K(Ca)2 channels providing both selective activation and selective inhibition
of the human K(Ca)2.1 subtype. Mol Pharmacol 81:210–219.
Acknowledgments
The authors thank Dr. Eduardo Romanos-Alfonos and Susana
Murillo-Pola of the Unit of Functional Evaluations of the IACS for
excellent technical support (telemetry).
Hougaard C, Jensen ML, Dale TJ, Miller DD, Davies DJ, Eriksen BL, Strøbaek D, Trezise
DJ, and Christophersen P (2009) Selective activation of the SK1 subtype of human small-
conductance Ca21-activated K1 channels by 4-(2-methoxyphenylcarbamoyloxymethyl)-
piperidine-1-carboxylic acid tert-butyl ester (GW542573X) is dependent on serine 293
in the S5 segment. Mol Pharmacol 76:569–578.
Jenkins DP, Yu W, Brown BM, Løjkner LD, and Wulff H (2013) Development of
a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and
activators. Assay Drug Dev Technol 11:551–560.
Joiner WJ, Wang LY, Tang MD, and Kaczmarek LK (1997) hSK4, a member of
a novel subfamily of calcium-activated potassium channels. Proc Natl Acad Sci
USA 94:11013–11018.
Jones HM, Bailey MA, Baty CJ, Macgregor GG, Syme CA, Hamilton KL, and Devor
DC (2007) An NH2-terminal multi-basic RKR motif is required for the ATP-
dependent regulation of hIK1. Channels (Austin) 1:80–91.
Authorship Contributions
Participated in research design: Coleman, Brown, Valero, Oliván-
Viguera, Köhler, Wulff.
Conducted experiments: Coleman, Brown, Singh, Valero, Oliván-
Viguera, Olmstead.
Performed data analysis: Coleman, Brown, Heike Wulff.
Wrote or contributed to the writing of the manuscript: Coleman,
Brown, Singh, Oliván-Viguera, Köhler, Wulff.
References
Adelman JP, Maylie J, and Sah P (2012) Small-conductance Ca21-activated K1
channels: form and function. Annu Rev Physiol 74:245–269.
Jordan AD, Luo C, and Reitz AB (2003) Efficient conversion of substituted aryl
thioureas to 2-aminobenzothiazoles using benzyltrimethylammonium tribromide.
J Org Chem 68:8693–8696.
Kasumu AW, Hougaard C, Rode F, Jacobsen TA, Sabatier JM, Eriksen BL, Strøbæk
D, Liang X, Egorova P, and Vorontsova D et al. (2012) Selective positive modulator
Balut CM, Hamilton KL, and Devor DC (2012) Trafficking of intermediate (KCa3.1)
and small (KCa2.x) conductance, Ca(21)-activated K(1) channels: a novel target for
medicinal chemistry efforts? ChemMedChem 7:1741–1755.

Positive Gating Modulation of KCa3.1
357
of calcium-activated potassium channels exerts beneficial effects in a mouse model
of spinocerebellar ataxia type 2. Chem Biol 19:1340–1353.
Schuart J and Müller HK (1973) [2-Aminooxazoles and 2-iminooxazolines. 1: reaction of
racemic alpha-methylaminopropiophenone using cyanobromide]. Pharmazie 28:438–439.
Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A 64:112–122.
Singh S, Syme CA, Singh AK, Devor DC, and Bridges RJ (2001) Benzimidazolone
activators of chloride secretion: potential therapeutics for cystic fibrosis and
chronic obstructive pulmonary disease. J Pharmacol Exp Ther 296:600–611.
Strøbaek D, Teuber L, Jørgensen TD, Ahring PK, Kjaer K, Hansen RS, Olesen SP,
Christophersen P, and Skaaning-Jensen B (2004) Activation of human IK and SK
Ca21 -activated K1 channels by NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime).
Biochim Biophys Acta 1665:1–5.
Klein H, Garneau L, Banderali U, Simoes M, Parent L, and Sauvé R (2007) Structural
determinants of the closed KCa3.1 channel pore in relation to channel gating: results
from a substituted cysteine accessibility analysis. J Gen Physiol 129:299–315.
Köhler M, Hirschberg B, Bond CT, Kinzie JM, Marrion NV, Maylie J, and Adelman
JP (1996) Small-conductance, calcium-activated potassium channels from mammalian
brain. Science 273:1709–1714.
Köhler R (2012) Cardiovascular alterations in KCa3.1/KCa2.3-deficient mice and
after acute treatment with KCa3.1/KCa2.3 activators. EDHF 2012: 10th Anniversary
Meeting June 27th - 30th, 2012; Feletou M, Vanhoutte PM (eds) vol. 49, pp 1–54
Vaux-de-Cernay, France.
Köhler R, Kaistha BP, and Wulff H (2010) Vascular KCa-channels as therapeutic targets
in hypertension and restenosis disease. Expert Opin Ther Targets 14:143–155.
Li W, Halling DB, Hall AW, and Aldrich RW (2009) EF hands at the N-lobe of
calmodulin are required for both SK channel gating and stable SK-calmodulin
interaction. J Gen Physiol 134:281–293.
Ng KF, Leung SW, Man RY, and Vanhoutte PM (2008) Endothelium-derived
hyperpolarizing factor mediated relaxations in pig coronary arteries do not involve
Gi/o proteins. Acta Pharmacol Sin 29:1419–1424.
Pedarzani P, Mosbacher J, Rivard A, Cingolani LA, Oliver D, Stocker M, Adelman JP,
and Fakler B (2001) Control of electrical activity in central neurons by modulating the
gating of small conductance Ca21-activated K1 channels. J Biol Chem 276:9762–9769.
Radtke J, Schmidt K, Wulff H, Köhler R, and de Wit C (2013) Activation of KCa3.1 by
SKA-31 induces arteriolar dilatation and lowers blood pressure in normo- and
hypertensive connexin40-deficient mice. Br J Pharmacol 170:293–303.
Rosa JC, Galanakis D, Ganellin CR, Dunn PM, and Jenkinson DH (1998) Bis-quinolinium
cyclophanes: 6,10-diaza-3(1,3),8(1,4)-dibenzena-1,5(1,4)- diquinolinacyclodecaphane
(UCL 1684), the first nanomolar, non-peptidic blocker of the apamin-sensitive Ca(21)-
activated K1 channel. J Med Chem 41:2–5.
Sankaranarayanan A, Raman G, Busch C, Schultz T, Zimin PI, Hoyer J, Köhler R,
and Wulff H (2009) Naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a new activator of
KCa2 and KCa3.1 potassium channels, potentiates the endothelium-derived hyper-
polarizing factor response and lowers blood pressure. Mol Pharmacol 75:281–295.
Schmitz A, Sankaranarayanan A, Azam P, Schmidt-Lassen K, Homerick D, Hänsel
W, and Wulff H (2005) Design of PAP-1, a selective small molecule Kv1.3 blocker,
Wei AD, Gutman GA, Aldrich R, Chandy KG, Grissmer S, and Wulff H (2005) In-
ternational Union of Pharmacology. LII. Nomenclature and molecular relation-
ships of calcium-activated potassium channels. Pharmacol Rev 57:463–472.
Wulff
H and Köhler R (2013) Endothelial small-conductance and intermediate-
conductance KCa channels: an update on their pharmacology and usefulness as
cardiovascular targets. J Cardiovasc Pharmacol 61:102–112.
Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, and Chandy KG (2000)
Design of a potent and selective inhibitor of the intermediate-conductance Ca21
-activated K1 channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci
USA 97:8151–8156.
Wulff H and Zhorov BS (2008) K1 channel modulators for the treatment of neuro-
logical disorders and autoimmune diseases. Chem Rev 108:1744–1773.
Xia XM, Fakler B, Rivard A, Wayman G, Johnson-Pais T, Keen JE, Ishii T,
Hirschberg B, Bond CT, and Lutsenko S et al. (1998) Mechanism of calcium gating
in small-conductance calcium-activated potassium channels. Nature 395:503–507.
Zhang M, Pascal JM, Schumann M, Armen RS, and Zhang JF (2012) Identification
of the functional binding pocket for compounds targeting small-conductance
Ca²⁺-activated potassium channels. Nat Commun 3:1021.
Zhang M, Pascal JM, and Zhang JF (2013) Unstructured to structured transition of
an intrinsically disordered protein peptide in coupling Ca²⁺-sensing and SK chan-
nel activation. Proc Natl Acad Sci USA 110:4828–4833.
Address correspondence to: Heike Wulff, Department of Pharmacology,
Genome and Biomedical Sciences Facility, Room 3502, 451 Health Sciences
Drive, University of California, Davis, Davis, CA 95616. E-mail: hwulff@
ucdavis.edu
for the suppression of effector memory
T cells in autoimmune diseases. Mol
Pharmacol 68:1254–1270.