ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels

Article abstract of DOI:10.1021/acschemneuro.6b00111

The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC₅₀ = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CL_HEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CL_HEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (C_max = 0.20 μM and T_max = 3 h) and favorable CNS distribution (mouse brain/plasma K_p of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.

Full text of DOI:10.1021/acschemneuro.6b00111

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Article
ML418: The first selective, sub-micromolar
pore blocker of Kir7.1 potassium channels
Daniel R. Swale , Haruto Kurata, Sujay V. Kharade , Jonathan H Sheehan,
Rene R. Raphemot , Karl R. Voigtritter , Eric Figueroa , Jens Meiler, Anna
L. Flobaum , Craig W Lindsley, Corey R. Hopkins, and Jerod S. Denton
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00111 • Publication Date (Web): 16 May 2016
Downloaded from http://pubs.acs.org on May 21, 2016
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ML418: The first selective, sub-micromolar pore blocker of Kir7.1 potassium channels
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Daniel R. Swale1€, Haruto Kurata2,3€, Kharade, S. V.1, Jonathan Sheehan4, Rene R. Raphemot1,2, Karl R.
Voigtritter2,3, Eric Figueroa1,2, Jens Meiler4, Anna L. Blobaum2,3, Craig W. Lindsley2,3, Corey R. Hopkins*,2,3
Jerod S. Denton*,1,2
,
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1 Department of Anesthesiology, Vanderbilt University Medical Center
2 Department of Pharmacology, Vanderbilt University Medical Center
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Vanderbilt Center for Neuroscience Drug Discovery and the Vanderbilt Specialized Chemistry Center for
Accelerated Probe Development, Vanderbilt University Medical Center, Nashville, TN 37232
4 Department of Chemistry, Vanderbilt University, Nashville, TN 37232
€Authors contributed equally
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ABSTRACT
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The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator
of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle
contractility during pregnancy. The pharmacological tools available for exploring the physiology and
therapeutic potential of Kir7.1 have been limited to relatively weak and nonꢀselective smallꢀmolecule
inhibitors. Here, we report the discovery in a fluorescenceꢀbased highꢀthroughput screen of a novel
Kir7.1 channel inhibitor, VU714. Siteꢀdirected mutagenesis of poreꢀlining amino acid residues identified
Glutamate 149 and Alanine 150 as essential determinants of VU714 activity. Lead optimization with
medicinal chemistry generated ML418, which exhibits subꢀmicromolar activity (IC50 = 310 nM) and
superior selectivity over other Kir channels (at least 17ꢀfold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3,
Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead
Profiling panel of 64 GPCRs, ionꢀchannels and transporters for offꢀtarget activity of ML418 revealed a
relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes
which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes
regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP)
administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 ꢀM and Tmax = 3 hours)
and favorable CNS distribution (mouse brain:plasma Kp of 10.9 to support in vivo studies for in vivo
studies. ML418, which represents the current stateꢀofꢀtheꢀart in Kir7.1 inhibitors, should be useful for
exploring the physiology of Kir7.1 in vitro and in vivo.
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Keywords: KCNJ13, thallium flux, electrophysiology, comparative modeling, melanocortin signaling,
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myometrium
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INTRODUCTION
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Inward rectifier potassium (Kir) channels play fundamental roles in diverse organ systems, and
could in some cases represent unexploited drug targets for neurological, cardiovascular, endocrine, and
muscle disorders 1, 2. Kir7.1, which is encoded by KCNJ13, one of sixteen genes comprising the Kir
channel family, is expressed in the eye, brain, uterus, kidney, gut, and thyroid gland3ꢀ9. The genetic loss
of Kir7.1 function in retinal pigmented epithelial cells of the eye leads to derangements in subretinal
electrolyte homeostasis and cell degeneration underlying leber congenital amaurosis and snowflake
vitreoretinopathy10ꢀ13. Using a panel of mostly nonꢀspecific inhibitors with differential activities against
Kir channels, GhamariꢀLangroudi and colleagues recently identified Kir7.1ꢀlike currents in neurons of
the paraventricular nucleus (PVN) that are functionally coupled to the melanocortinꢀ4 receptor
(MC4R)14. Agonist binding to MC4R inhibits Kir7.1 activity, depolarizes the membrane potential, and
increases neuronal firing, whereas competitive antagonist binding increases Kir7.1 activity and dampens
neuronal excitability. This model suggests that Kir7.1 coupling to MC4R plays a key role in the central
regulation of food intake and energy homeostasis by the PVN14. Kir7.1 expression in uterine muscles
increases dramatically during pregnancy, thereby hyperpolarizing the membrane potential, inhibiting
calcium signaling, and inducing uterine quiescence during fetal development15. Inhibiting Kir7.1
expression with microRNAs or inhibiting channel function with smallꢀmolecule inhibitors (i.e. VU59016,
MRT0020076917) induces longꢀlasting uterine muscle contractions, lending support to the idea that
Kir7.1 represents a novel drug target for augmentation of labor and treating postꢀpartum hemorrhage17.
The current pharmacological ‘toolkit’ for exploring the physiology and therapeutic potential of
Kir7.1 is inadequate, prompting us to perform a highꢀthroughput screen (HTS) and lead optimization
campaign in order to identify more potent and selective inhibitors. Here, we report the development and
in vitro characterization of ML418, a new stateꢀofꢀtheꢀart inhibitor of Kir7.1.
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RESULTS AND DISCUSSION
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Kir7.1-M125R Assay Development. HTS was performed using a fluorescenceꢀbased assay that
reports the inward movement of the potassium (K+) congener thallium (Tl+) through Kir7.1 channels
expressed in the plasma membrane of TꢀRExꢀHEK293 cells. The higher conductance Kir7.1ꢀM125R
mutant was used as a surrogate to circumvent the weak Tl+ flux observed for wild type (WT) Kir7.1 (see
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ref. ). As shown in Fig. 1, the assay reports robust Kir7.1ꢀM125Rꢀdependent Tl+ flux after induction
Figure 1. Kir7.1 Tl+ flux assay used for HTS. (A) Representative Thallos fluorescence traces recorded from TꢀRExꢀHEKꢀ293ꢀ
Kir7.1ꢀM125R cells cultured overnight with (grey line) or without (black line) tetracycline. Thallium stimulus buffer was added to
each well simultaneously as indicated with the arrow. (B) DMSO tolerance test indicating that DMSO has no effect on Kir7.1ꢀM125Rꢀ
mediated Tl+ flux as concentrations up to 1.3% (v/v). (C) Determination of assay reproducibility. Alternate wells of a 384ꢀwell plate
were treated with DMSO (vehicle) or Kir7.1 inhibitor VU573 (30 ꢁM) before initiating Tl+ flux. Mean fluorescence and 3 S.D. from
the mean for each well population are indicated with a blue dashed line and solid black line, respectively. The mean ± SEM. Z’ for 3
plates assayed on 3 separate days was Z’ = 0.67 ± 0.03.
with tetracycline (Fig. 1A), is DMSO tolerant up to 0.6% v/v (Fig. 1B; screening DMSO concentration
= 0.1% DMSO v/v), and is sufficiently reproducible for HTS (Fig. 1C; mean ± SEM Z’= 0.67 ± 0.03; n
= 3 plates on 3 separate days).
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Discovery and Characterization of VU714. From a pilot screen of 5,230 compounds in the
Vanderbilt Institute of Chemical Biology (VICB) library, 11 putative Kir7.1ꢀM125R inhibitors,
comprising 5 distinct scaffolds, and with differing levels of selectivity over other Kir channels, were
identified (data not shown). VU714 (Fig. 2A) was the most potent and selective inhibitor from the
screen, and was therefore reꢀsynthesized and confirmed from powder to be an authentic Kir7.1ꢀM125R
inhibitor. VU714 inhibited Kir7.1ꢀM125Rꢀmediated Tl+ flux in a doseꢀdependent manner with an IC50
of 5.6 µM (95% Confidence Interval [CI]: 1.9 µM ꢀ 17.5 µM) (Figs 2BꢀC). In “goldꢀstandard” wholeꢀ
cell voltage clamp experiments, the rate of Kir7.1ꢀM125R inhibition by VU714 was concentration
dependent (Fig. 2D), 10 µM VU714 fully inhibited outward and inward Kir7.1ꢀM125Rꢀmediated current
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Figure 2. Discovery and characterization of VU714. (A) Chemical structure of VU714. (B) Doseꢀdependent inhibition of Kir7.1ꢀ
M125Rꢀdependent Tl+ flux by VU714. Cells were preꢀtreated with the indicated concentrations of VU714 for 10 min before adding Tl+
stimulus buffer (arrow). (C) Mean ± SEM % control fluorescence recorded in the indicated concentrations of VU714 (n = 4). (D)
Representative wholeꢀcell patch clamp experiment showing timecourse of VU714ꢀdependent inhibition of Kir7.1 current recorded at ꢀ
120 mV. VU714 concentrations (in ꢁM) are indicated at the top. Experiments were terminated by bath application of 2 mM barium
(Ba). (E) Currentꢀvoltage plot showing inhibition of Kir7.1 by 10 ꢁM VU714 or 2 mM Ba. (F) Mean ± SEM % Kir7.1 inhibition at ꢀ
120 mV. IC50 values were derived by fitting CRC data with a 4ꢀparameter logistical function.
(Fig. 2E), and the IC50 was 1.5 µM (CI: 1.3 µM ꢀ 1.7µM) (Fig. 2F). The 3.7ꢀfold shift in IC50 determined
with patch clamp electrophysiology, as compared with Tl+ flux, is consistent with previous observations
of other Kir channel inhibitors 18ꢀ20. Quantitative Tl+ flux assays were utilized to evaluate the selectivity
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of VU714 for Kir7.1 over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, Kir4.1, and Kir6.2/SUR1, as
reported previously 16, 21, 22. The concentrationꢀresponse curves (CRCs) shown in Fig. 3A revealed that
VU714 is only moderately selective, and inhibits other Kir channels with a rank order potency of Kir7.1
(IC50 = 5.6 µM) > Kir4.1 (IC50 = 13 µM) > Kir1.1 (IC50 = 16 µM) > Kir6.2/SUR1 (IC50 = 30 µM) >
Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2 (IC50 > 30 µM). Kir2.2,Kir2.3, and Kir3.1/3.2 CRCs have been
excluded from Fig. 3A for clarity.
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Figure 3. Analysis of VU714 and ML418 selectivity for Kir7.1 over other Kir channels. (A) VU714 CRCs constructed for Kir7.1ꢀ
M125R over Kir6.2/SUR1 (open diamonds), Kir1.1 (closed circles), Kir2.1 (closed squares), Kir4.1 (open squares) in Tl+ flux assays.
Kir2.2,Kir2.3, Kir3.1/3.2 (IC50s >30 ꢁM) have been excluded for clarity. Data are means ± SEM % control fluorescence (n = 4ꢀ10 per
concentration). (B) ML418 CRCs constructed for the same channels in Tl+ flux assays. (C) Representative wholeꢀcell patch clamp
experiment showing doseꢀdependent inhibition of Kir7.1 current at ꢀ120 mV by the indicated concentration of ML418. The experiment
was terminated by bath application of 2 mM Ba. (D) Comparison of CRCs for VU714 (grey line, data from Fig. 2) and ML418
determined in patch clamp electrophysiology experiments.
VU714 Requires Pore-lining E149 and A150 Residues for Activity. Kir channels are
tetrameric proteins consisting of 8 membraneꢀspanning domains, an ionꢀconduction pore, and
cytoplasmic domain. Most smallꢀmolecule inhibitors studied to date appear to block the conduction
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pathway by interacting with amino acid residues lining the transmembrane pore (reviewed in ). We
therefore performed scanning mutagenesis and voltageꢀclamp electrophysiology to test if VU714
interacts with poreꢀlining amino acids in Kir7.1. Residues that are predicted from homology modeling
(Fig. 4A) to face the pore were mutated to the corresponding residues in Kir2.1 and Kir1.1, which are
only weakly inhibited by (Kir1.1) or insensitive (Kir2.1) to VU714 (Fig. 3A). Mutations were first
introduced into WT Kir7.1, and if they impaired channel activity, were retested in the M125R
background due to its more robust functional expression and ability to rescue the activity of some
mutants. For clarity, only nonꢀfunctional mutants in the M125R background are indicated in Fig. 4B;
however, these mutations were first tested in the WT background and found to be nonꢀfunctional. The
M125R mutation does not alter VU714 sensitivity (Fig. 4C).
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Mutations L146V, I160M, and A161S had no effect on Kir7.1 inhibition by 3 µM VU714,
whereas the T153C/WT mutation significantly (P <0.05) increased channel inhibition. Mutation of E149
to the corresponding Asparagine residue in Kir1.1 (E149N) abolished Kir7.1 activity in both WT and
M125R backgrounds. However, the more conservative mutation, E149Q/M125R, led to a partial, albeit
significant (P<0.05), loss of sensitivity to VU714. The negative charge at position 149 appears to be
important for VU714 activity since mutating E149 to Aspartate (E149D) had no effect on block.
Mutation of the adjacent residue A150 to Serine (A150S) led to a partial loss of inhibition. As shown in
Fig. 4C, the E149Q and A150S mutations significantly (P <0.0001) shifted the IC50 for VU714 from ~2
µM in the WT or M125R mutants to ~18 µM and ~7 µM, respectively. Mutation of both residues
simultaneously led to an additive loss of VU714 sensitivity (Fig. 4B).
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Molecular modeling was used to visualize how VU714 might interact with Kir7.1 to induce
channel block. Interactive ligand placement near E149 and A150, combined with energy minimization,
revealed the best theoretical configuration of VU714 in the Kir7.1 channel pore (Fig. 4DꢀE). The limited
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Figure 4. Identification of pore-lining residues in Kir7.1 required for VU714 activity. (A) Alignment of poreꢀlining M2 helices
from human Kir7.1, Kir2.1, and Kir1.1, with predicted poreꢀfacing residues indicated with arrowheads. (B) Effects of pore mutations on
Kir7.1ꢀWT or Kir7.1ꢀM125R sensitivity to 3 ꢁM VU714. Data are mean ± SEM % inhibition at ꢀ120 mV. * P <0.05 compared to
respective control. N.F., not functional. (C) VU714 CRC for Kir7.1ꢀWT (closed squares; IC50=1.4 ꢁM ), Kir7.1ꢀM125R (open squares;
IC50=1.6 ꢁM), Kir7.1ꢀM125RꢀE148Q (open circles; IC50=18.1 ꢁM), Kir7.1ꢀWTꢀA150S (closed circles; IC50=6.9 ꢁM). (D) Kir7.1
homology model showing lowꢀenergy pose of VU714 near residues E149 and A150. (E) Higherꢀmagnification view (from white box in
D) of VU714 near E149 and A150.
volume of the pore in the putative binding region results in obstruction of the channel by VU714, just
below the selectivity filter. This model suggests a straightforward steric mechanism for the observed
conduction block by VU714. It is notable that this general region of pore is also involved in block of
Kir1.1 by VU59123, Kir2.1 by ML13324, and Kir4.1 by fluoxetine25 (reviewed in ref.1). Pentamidine26
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and chloroquine27 require residues in the cytoplasmic pore for block; however, it is unlike that these
residues participate in VU714 inhibition of Kir7.1 since the double mutation E149Q and A150S
virtually eliminated block of the channel (Fig. 4B).
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VU714 Structure-Activity Relationships. Medicinal chemistry was employed in an effort to
identify VU714 analogs with improved potency and selectivity for Kir7.1. Our first priority was to
reduce the high lipophilicity of VU714 (clogP = 5.83), which may be a liability for offꢀtarget selectivity,
metabolism and toxicity. The initial library to explore SAR (StructureꢀActivity Relationship) in the
rightꢀhand portion of VU714 identified several equipotent moieties, including a simple methyl
compound (1, IC50 = 4.8 ꢀM, clogP = 4.41) without the benzyl moiety (Table 1). Subsequent libraries
with a hydrophilic handle indicated that changing the piperidine ring system to a piperazine was not
productive (amide, sulfonamide, urea and carbamate, 2 – 6), including the direct benzyl analog which
resulted in a 4.5ꢀfold loss of potency compared to VU714 (5, IC50 = 22.0 ꢀM). However, attaching a
hydrophilic handle (ꢀNHR’) at the 4ꢀposition of the piperidine ring system endowed less lipophilic
compounds with equal potency to the HTS hit compound, namely the amide compound (7, IC50 = 8.3
ꢀM, clogP = 3.43) and the carbamate (11, IC50 = 1.7 ꢀM, clogP = 3.65) which were generally more
potent than the corresponding Nꢀalkylated derivatives (12 – 16) (Table 1). Based on these results, we
next evaluated a number of carbamate analogs in order to explore the SAR around this moiety (Table 2).
While the simple methyl carbamate (17, inactive) and ethyl carbamate (18, IC50 = 9.8 ꢀM) analogs were
less active, all of the branched alkyl carbamates (19 – 22) were equipotent with the HTS hit compound.
Among them, the isopropyl (19, IC50 = 1.3 ꢀM, clogP = 3.25) and the tertꢀbutyl carbamate (11, IC50 =
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1.7 ꢀM, clogP = 3.65) were the most active compounds. The benzyl compound (23), ring contracted
compounds (24 and 25) and the spirocyclic compounds (26 and 27) were also tolerated, but less active
than the HTS hit VU714. The next library was focused on rightꢀhand 4ꢀaminopiperidine amide analogs.
The most active amides were aryl analogs in nature (28 – 40, 43) with the 3ꢀchlorophenyl (32, IC50 = 3.1
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ꢀM, clogP = 4.34) being the most potent in this class. Branched alkyl amides (41 – 42) were also
evaluated, but they were less active than the parent compound (Table 2).
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Next, we explored SAR around a leftꢀhand quinoline moiety keeping the 4ꢀmethyl piperidine in
the rightꢀhand portion constant. Unfortunately, structural modifications of this portion of the molecule
were not well tolerated, even minor changes such as changing the chlorine to fluorine and bromine
atoms were less active. The only modification tolerated in our SAR exploration was an introduction of a
methyl group at the 4ꢀposition of the quinoline ring (Fig. S1).
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The SAR data (Fig. S1) is in qualitative agreement with the docking model that illustrates a
plausible binding mode (Fig. 4E). Specifically, position 2 of the quinoline ring faces towards the protein
backbone. There is no space for additional bulk. This is in contrast to positions 3 and 4 of the quinoline
ring where methyl substitution is tolerated. The Nitrogen atom of the quinoline points to a polar pocket
that could provide space for an additional, hydrogenꢀbound water molecule. A Carbon atom could not
engage in these favorable interactions. The chloride and hydroxyl substituents are in tight, polar pockets.
No larger substituents will fit into these pockets. A quantitative analysis of the agreement is beyond the
predictive power of the homology model used for the docking simulation.
Discovery and Characterization of ML418. Having identified several VU714 analogs with
lower lipophilicity and similar potencies in Tl+ flux assays, we next evaluated their activity toward
Kir7.1 using voltage clamp electrophysiology and selectivity among Kir1.1, Kir2.1, Kir2.2, Kir2.3,
Kir3.1/3.2, Kir4.1 and Kir6.2/SUR1 in Tl+ flux assays. The methyl piperidine analog (1) was similar in
activity and selectivity to VU714 (Table 3). However, the carbamate (11 and 19) and amide (32)
analogs exhibited superior inhibitory activity and selectivity. The isopropyl carbamate analog is the most
potent in Kir7.1 activity (19 = ML418) in our library and is at least 23ꢀfold selective against the Kir
channels tested, with the exception of Kir6.2/SUR1 (Fig. 3B; Table 3). ML418 inhibits Kir7.1 doseꢀ
dependently (Fig. 3C) with an IC50 in patch clamp electrophysiology experiments of 310 nM (Fig. 3D;
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Table 3). While the tertꢀbutyl carbamate (11) showed similar potency and selectivity to ML418, the
amide analog (32) exhibited modest activity and greater than 10ꢀfold selectivity over the Kir channels
tested (Table 3). Although compounds 44 and 45 also show selectivity preference over Kir6.2/SUR1, and
should be considered for use as an in vitro tool compound, the totality of the properties favors ML418 as the
first selective Kir7.1 inhibitor.
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In a Lead Profiling Screen (Eurofins) of 64 potential offꢀtargets, ML418 showed a relatively
clean ancillary pharmacology having significant interactions (i.e. >50% radioligand displacement) with
the Lꢀtype calcium channel, voltageꢀgated sodium channel, Dopamine D2S and D4.2 receptors, Sigma
σ1 receptor, and Norepinephrine receptor (Table S1).
DMPK Evaluation for Selected Analogs. We
evaluated selected analogs in our in vitro DMPK panel of
assays, an assessment of intrinsic clearance (CLINT) and
predicted hepatic clearance (CLHEP) in hepatic microsomes
and protein binding in plasma (PPB) in multiple species.
Interestingly, while the lipophilic HTS hit compound
VU714 (cLogP = 5.83) was shown to have high intrinsic
clearance (CLHEP (human) = 16.7 mL/min/kg and CLHEP (rat)
=64.1 mL/min/kg) and low unbound fraction in plasma (%Fu
(mouse) = 0.7, %Fu (rat) = 1.8 and %Fu (human) = 0.5)
across species, more potent analogs than VU714 (32, 11 and
19 = ML418) exhibited lower intrinsic clearance in human
hepatic microsomes (CLHEP (human) = 10.7, 7.9 and 3.6
Figure 5. Time course in vivo PK profile of
ML418. ML418 was dosed at 30 mg/kg in 10%
EtOH, 40% PEG 400, 50% saline vehicle. The
dosing solution was administered by intraperitoneal
mL/min/kg, respectively) and higher unbound fraction in
injection and whole blood collections via the carotid
artery were performed at 0.117, 0.25, 0.5, 1, 2, 4, 7,
and 24 hours post dose.
mouse and rat plasma (%Fu (mouse) = 2.0, 5.6 and 11.4
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and %Fu (rat) = 2.5, 3.6 and 8.8, respectively) as the cLogPs are reduced. While intrinsic clearance in rat
hepatic microsomes of ML418 are slightly reduced (CLHEP (rat) =57.9 mL/min/kg) compared to VU714,
subsequent PK study of ML418 by 30 mg/kg dosage of intraperitoneal administration revealed its
characteristic PK profile (Fig. 5: Cmax = 0.20 ꢀM and Tmax = 3 hours). It may be attributed to
enterohepatic recirculation caused by phenol moiety. In a parallel CNS distribution study in mice,
ML418 demonstrated excellent CNS penetration with a mouse brain:plasma ratio (Kp) of 10.9 (average
of four mice, brain (323.9 ng/g):plasma (29.5 ng/mL)). Thus, ML418 possesses a DMPK profile suitable
for both in vitro and in vivo studies aimed at modulating both peripheral and central Kir7.1 potassium
channels.
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CONCLUSIONS
ML418 is the fifth smallꢀmolecule inhibitor of Kir7.1 reported to date and currently represents
the stateꢀofꢀtheꢀart for the field (Table 5). The first Kir7.1 inhibitor, VU590, was identified in a HTS of
approximately 225,000 compounds for inhibitors of Kir1.1 (IC50 = 0.24 µM) and subsequently found to
have weak offꢀtarget activity toward Kir7.1 (IC50 ~ 8 µM)16. Evaluation of eight VU590 analogs
revealed a flat SAR against Kir7.117. Another compound, VU573 was identified in the same Kir1.1 HTS,
but found to have superior activity toward Kir7.1 (IC50 = 4.9 µM) over Kir1.1 (IC50=19 µM) in Tl+ flux
assays. VU573 also inhibits Kir2.3 and Kir3 channels with singleꢀmicromolar potency18. Lead
optimization efforts failed to generate more potent or selective inhibitors of Kir7.1, but did generate
‘inactive’ analogs that can be useful for determining the specificity of VU573 in pharmacology
experiments targeting Kir7.1 (e.g. see ref.14). ML133 was identified in a HTS of more than 300,000
compounds for modulators of Kir2.1 (IC50 = 0.3 µM at pH 8.0; approximately equal activity toward
Kir2.2, Kir2.3, and Kir2.6) and found to possess weak Kir7.1 activity (IC50=33µM)24. MRT00200769
was discovered in an electrophysiologyꢀbased screen of 7,087 compounds for Kir7.1 inhibitors, but,
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despite being one of the most potent Kir7.1 inhibitors identified, was found to exhibit preferential
activity toward cardiac hERG K+ channels (IC50=0.3 µM) over Kir7.1 (IC50=1.3µM). MRT00200769
also suffers from flat SAR17. In the present study, a HTS of 5,230 compounds from the VICB library led
to the discovery of the novel pore blocker VU714, which was optimized with medicinal chemistry to
generate ML418. The salient features of ML418 include: 1) Kir7.1 IC50=0.3 µM; 2) at least 23ꢀfold
selectivity for Kir7.1 over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir4.1 except for Kir6.2/SUR1; 3) clean
ancillary pharmacology against 58 GPCRs, ion channels, and transporters, including hERG; 4) PK
profile in rat (Cmax = 0.20 ꢀM and Tmax = 3 hours at 30 mg/kg i.p. dosing) and highly CNS penetrant
(mouse brain:plasma Kp of 10.9). Further investigations of ML418 in order to probe the detailed
physiological functions of Kir7.1 will be reported in due course.
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METHODS
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Molecular biology. The open reading frame (ORF) of human Kir7.1 was subꢀcloned into
pcDNA5/TO (Life Technologies) to enable tetracyclineꢀregulated expression (see below). Mutations
were introduced into the Kir7.1 ORF using a QuickChange II mutagenesis kit (Agilent Technologies)
and sequenced to verify incorporation of the intended mutation.
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Tetracycline-inducible stable cell lines. Stably transfected TꢀRExꢀHEKꢀ293 cell lines
expressing the following Kir channels were generated and maintained in culture as described previously:
Kir1.128, Kir2.122, Kir4.121, Kir6.2/SUR122, Kir7.1ꢀM125R18. Kir channel expression was induced by
culturing cells overnight in media containing 1 µg/ml tetracycline.
High-throughput screening (HTS). HTS for Kir7.1ꢀM125R modulators was performed using a
Tl+ꢀflux reporter assay essentially as described previously16, 20, 21. The Kir7.1ꢀM125R mutation increases
the channel unitary conductance and enables robust Tl+ flux measurement that cannot be achieved with
the WT channel18 . TꢀRExꢀHEKꢀ293ꢀKir7.1ꢀM125R cells (20,000/well) were plated in clearꢀbottomed,
blackꢀwalled, 384ꢀwell plates, and cultured overnight in Delbecco’s Modified Eagle’s Medium
(DMEM) containing 10% dialyzed FBS and 1 µg/ml tetracycline to induce Kir7.1ꢀM125R expression.
The following day, the cells were incubated with dyeꢀloading assay buffer (Hank’s Balanced Salt
Solution, 20 mM HEPES, pH 7.3) containing 0.01% (w/v) Pluronic Fꢀ127 (Life Technologies) and 1.2
µM ThallosꢀAM (TEFlabs, Austin, TX) Tl+ reporter dye. The dyeꢀloading solution was replaced after
1hr with 20 µL/well assay buffer. Test compounds from the VICB Library were dispensed into 384ꢀwell
plates using an Echo555 liquid handler (Labcyte, Sunnyvale, CA) and diluted to a 2X concentration.
Cell plates were transferred to a Hamamatsu Functional Drug Screening System 6000 (FDSS6000;
Hamamatsu, Tokyo, Japan) where 20 µL/well test compounds were dispensed into wells to a nominal
concentration of 10 µM. After a 20ꢀmin incubation period, baseline fluorescence (excitation 470 ± 20
nm, emission 540 ± 30 nm) was recorded at 1Hz for 10 sec before addition of thallium stimulus buffer
containing (in mM): 125 NaHCO3, 1.8 CaSO4, 1 MgSO4, 5 glucose, and 1.8 Tl2SO4. Fluorescence data
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were collected for an additional 4 min at 1Hz. Data were analyzed essentially as described previously28,
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using a combination of Microsoft Excel (Microsoft Corporation, Redmond, WA) with XLfit addꢀin
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(IDBS, Guildford, Surrey, UK) and GraphPad Prism(GraphPad Software, San Diego, CA, USA). Raw
data were opened in Microsoft Excel and each data point in a given trace was divided by the first data
point from that trace followed by subtraction of data points from control traces that were generated in
the presence of vehicle controls. The slope of the fluorescence increase beginning 5 sec after Tl+
addition and ending 20 sec after Tl+ addition was calculated. The data were then plotted in Prism
software to generate CRCs. Potencies were calculated from fits to CRC data using a nonꢀlinear
regression analysis.
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Transient transfections and whole-cell patch clamp electrophysiology. Transfections and
wholeꢀcell patch clamp experiments were performed essentially as described previously22. Briefly,
HEKꢀ293T cells were transfected with plasmids encoding WT or mutant Kir7.1 and EGFP (transfection
marker) using Lipofectamine LTX (Life Technologies). The next day, the cells were dissociated with
trypsin, plated on polyꢀLꢀlysineꢀcoated round glass coverslips, and allowed to recover for at least 1hr
before beginning experiments. Coverslips were transferred to a smallꢀvolume recording chamber on the
stage of an inverted fluorescence microscope and superfused with a control bath solution containing (in
mM), 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, 10 HEPES, pH 7.4. Electrodes were pulled with a
FlamingꢀBrown Pꢀ1000 micropipette puller and had resistances between 2ꢀ3 Mꢂ when filled with the
following solution (in mM): 135 mM KCl, 2 MgCl2, 1 EGTA, 10 HEPES, pH 7.3. The cells were
voltage clamped at a holding potential of ꢀ75 mV and then stepped to ꢀ120 mV for 500 msec before
ramping to 120 mV at a rate of 2.4 mv/msec. The cell potential was returned to ꢀ75 msec for 5 sec
before initiating the stepꢀramp protocol again. Doseꢀresponse experiments were performed by
superfusing cells with increasing doses of Kir7.1 inhibitor (e.g. ML418) followed by 4 mM BaCl2 to
fully block Kir7.1. Cells exhibiting less than 90% block by BaCl2 were excluded from analysis. IC50
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values were determined by fitting the Hill equation to CRCs using variableꢀslope, unconstrained,
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nonlinear regression analyses performed with GraphPad Prism (GraphPad Software, San Diego, CA,
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USA). All experiments yielded acceptable Hill slope (>0.8) and r (0.99) values. IC values are
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expressed as mean of n = 5 values. Mean IC values and 95% confidence limits were determined with
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GraphPad InStatTM (GraphPad Software, San Diego, CA, USA). The mean IC
values were
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statistically analyzed using a oneꢀway ANOVA measure with Dunnets multiple comparison test with
significance being represented by P<0.05. Statistical analyses were performed using InStatTM
(GraphPad Software, San Diego, CA, USA).
Comparative homology modeling and in silico docking. A comparative model of Kir7.1 was
generated based on the 3.11 Angstrom resolution crystal structure Kir2.2 (PDB ID 3JYC)30. The
sequence identity between Kir7.1 and Kir2.2 is 33.5% among the 343 residues aligned in each subunit of
the tetramer. MODELLER version 9.9 [REF 2] was used to construct the model. The inhibitor
VU0488714 was docked into the model of Kir7.1 using the Molecular Operating Environment (MOE)
software package 31. Docking was performed by allowing flexibility of the ligand molecule and also the
side chains of the protein model. A representative docking pose was selected from among the 29 lowestꢀ
energy results by choosing the bestꢀscoring pose that features multiple interactions between the inhibitor
and the experimentallyꢀdetermined residues glutamate 149 and alanine 150.
Chemical synthesis. General. All NMR spectra were recorded on a 400 MHz AMX Bruker
1
NMR spectrometer. H and 13C chemical shifts are reported in δ values in ppm downfield with the
deuterated solvent as the internal standard. Data are reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, b = broad, m = multiplet), integration, coupling constant
(Hz). Low resolution mass spectra were obtained on an Agilent 6120 or 6150 with ESI source. Method
A: MS parameters were as follows: fragmentor: 70, capillary voltage: 3000 V, nebulizer pressure: 30
psig, drying gas flow: 13 L/min, drying gas temperature: 350 ºC. Samples were introduced via an
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Agilent 1290 UHPLC comprised of a G4220A binary pump, G4226A ALS, G1316C TCC, and G4212A
DAD with ULD flow cell. UV absorption was generally observed at 215 nm and 254 nm with a 4 nm
bandwidth. Column: Waters Acquity BEH C18, 1.0 x 50 mm, 1.7 um. Gradient conditions: 5% to 95%
CH3CN in H2O (0.1% TFA) over 1.4 min, hold at 95% CH3CN for 0.1 min, 0.5 mL/min, 55 ºC. Method
B: MS parameters were as follows: fragmentor: 100, capillary voltage: 3000 V, nebulizer pressure: 40
psig, drying gas flow: 11 L/min, drying gas temperature: 350 ºC. Samples were introduced via an
Agilent 1200 HPLC comprised of a degasser, G1312A binary pump, G1367B HPꢀALS, G1316A TCC,
G1315D DAD, and a Varian 380 ELSD (if applicable). UV absorption was generally observed at 215
nm and 254 nm with a 4 nm bandwidth. Column: Thermo Accucore C18, 2.1 x 30 mm, 2.6 um. Gradient
conditions: 7% to 95% CH3CN in H2O (0.1% TFA) over 1.6 min, hold at 95% CH3CN for 0.35 min, 1.5
mL/min, 45 ºC. High resolution mass spectra were obtained on an Agilent 6540 UHD QꢀTOF with ESI
source. MS parameters were as follows: fragmentor: 150, capillary voltage: 3500 V, nebulizer pressure:
60 psig, drying gas flow: 13 L/min, drying gas temperature: 275 ºC. Samples were introduced via an
Agilent 1200 UHPLC comprised of a G4220A binary pump, G4226A ALS, G1316C TCC, and G4212A
DAD with ULD flow cell. UV absorption was observed at 215 nm and 254 nm with a 4 nm bandwidth.
Column: Agilent Zorbax Extend C18, 1.8 µm, 2.1 x 50 mm. Gradient conditions: 5% to 95% CH3CN in
H2O (0.1% formic acid) over 1 min, hold at 95% CH3CN for 0.1 min, 0.5 mL/min, 40 ºC. For
compounds that were purified on a Gilson preparative reversedꢀphase HPLC, the system comprised of a
333 aqueous pump with solventꢀselection valve, 334 organic pump, GXꢀ271 or GXꢀ281 liquid hander,
two column switching valves, and a 155 UV detector. UV wavelength for fraction collection was userꢀ
defined, with absorbance at 254 nm always monitored. Method: Phenomenex Axiaꢀpacked Luna C18,
30 x 50 mm, 5 µm column. Mobile phase: CH3CN in H2O (0.1% TFA). Gradient conditions: 0.75 min
equilibration, followed by user defined gradient (starting organic percentage, ending organic percentage,
duration), hold at 95% CH3CN in H2O (0.1% TFA) for 1 min, 50 mL/min, 23 ºC. Solvents for
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extraction, washing and chromatography were HPLC grade. All reagents were purchased from Aldrich
Chemical Co. and were used without purification.
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Synthetic scheme and characterization of ML418. Synthetic scheme of ML418 is shown in
Figure S2 as an example of general synthetic scheme. Experimental procedure for ML418 is described
below. Specific synthetic schemes for each compound are also shown in supplemental information
(Figures S3ꢀ6).
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5-Chloro-8-hydroxyquinoline-7-carbaldehyde (49A): To a solution of 5ꢀchloroquinolinꢀ8ꢀol
48A (8.08 g, 45.0 mmol) in TFA (75 mL) was added hexamethylenetetramine (12.62 g, 90.0 mmol) at
ambient temperature. After a resulting reddish solution was refluxed at 120 ꢀ130oC for 5 hours, 1 mol/L
HClꢀaq (200mL) was added to the reaction mixture at 0oC which was stirred at ambient temperature for
20 min. To the reaction mixture was added ethyl acetate (75 mL) and 5 mol/L NaOHꢀaq (140mL) at 0oC.
The resulting precipitates were collected by filtration and washed with water to give a crude product
(6.59 g) which was triturated with mixed solvent of EtOH (25 mL) and Et2O (20 mL) to yield 5ꢀchloroꢀ
8ꢀhydroxyquinolineꢀ7ꢀcarbaldehyde 49A (3.480 g, 37% yield) as a beige powder.
tert-Butyl (1-((5-chloro-8-hydroxyquinolin-7-yl)methyl)piperidin-4-yl)carbamate (11): To a
suspension of 49A (818 mg, 3.94 mmol) in DCM (25 mL) was added 4ꢀbocꢀaminopiperidine (1.58 g,
7.89 mmol) at ambient temperature. After a resulting greenish solution was stirred at ambient
temperature for 1 hour, sodium triacetoxyborohydride (1.25 g, 5.91 mmol) was added to the reaction
mixture which was stirred at ambient temperature for 18 hours. The reaction mixture was poured into
NaHCO3ꢀaq (150 mL) and it was extracted with DCM (1st: 125 mL, 2nd: 50 mL). Combined organic
extracts were washed with (NaHCO3ꢀaq + water + brine) and dried over MgSO4. The filtrate was
evaporated under reduced pressure to give crude product (2.28 g) which was purified on silica gel
chromatography (DCM/MeOH) after combined with another crude product (1.41 g) by same reaction
conditions from 49A (451 mg, 2.17 mmol) to yield tertꢀbutyl (1ꢀ((5ꢀchloroꢀ8ꢀhydroxyquinolinꢀ7ꢀ
yl)methyl)piperidinꢀ4ꢀyl)carbamate 11 (2.05 g, 86% yield) as a pale yellow powder.
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7-((4-Aminopiperidin-1-yl)methyl)-5-chloroquinolin-8-ol trihydrochloride (51): To
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suspension of 11 (1.034 g, 2.64 mmol) in 1,4ꢀdioxane (10 mL) was added HCl/1,4ꢀdioxane (4 mol/L, 20
mL) at ambient temperature. After a reaction mixture was stirred for 24 hours, the precipitate was
collected by filtration to yield 7ꢀ((4ꢀaminopiperidinꢀ1ꢀyl)methyl)ꢀ5ꢀchloroquinolinꢀ8ꢀol trihydrochloride
51 (1.034 g, 98% yield) as a yellow powder.
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iso-Propyl (1-((5-chloro-8-hydroxyquinolin-7-yl)methyl)piperidin-4-yl)carbamate (19 = ML418):
To a solution of 51 (1.015 g, 2.53 mmol) and DIPEA (1.76 mL, 10.1 mmol) in DCM (25 mL) was added
a solution of isoꢀpropyl chloroformate in toluene (2 mol/L, 1.30 mL, 2.66 mmol) at 0oC. After a
resulting greenish solution was stirred at ambient temperature for 1 hour, it was poured into
ice/NaHCO3ꢀaq, which was extracted with DCM (x 2). Combined organic extracts were dried over
MgSO4 and the filtrate was evaporated under reduced pressure. The residue was purified by Gilson
HPLC separation system using (0.1% TFA in water)/CH3CN as an eluent to give crude product (c.a. 1.1
g) which was recrystallized from Et2O to yield desired product (633 mg 51% yield) as a TFA salt. To a
solution of TFA salt of the desired product (1,236 g, 2.51 mmol) in DCM (37 mL) was added saturated
NaHCO3ꢀaq (12.5 mL) at ambient temperature. Organic phase was separated and aqueous phase was
extracted with DCM (x 2). Combined organic phase was dried over MgSO4. The filtrate was evaporated
under reduced pressure to yield iso-propyl (1ꢀ((5ꢀchloroꢀ8ꢀhydroxyquinolinꢀ7ꢀyl)methyl)piperidinꢀ4ꢀ
yl)carbamate 19 (ML418) (915 mg, 96% yield) as a pale yellow powder. 1H NMR (400.1 MHz, DMSOꢀ
d6): 8.93 (dd, J = 4.2, 1.3 Hz, 1H), 8.47 (dd, J = 8.5, 1.3 Hz, 1H), 7.69 (dd, J = 8.5, 4.2 Hz, 1H), 7.61 (s,
1H), 7.02 (d, J = 7.7 Hz, 1H), 4.73 (sept, J = 6.2 Hz, 1H), 3.51ꢀ3.16 (br, 1H), 2.85ꢀ2.82 (m, 2H), 2.15ꢀ
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2.10 (m, 2H), 1.75ꢀ1.72 (m, 2H), 1.46ꢀ1.39 (m, 2H), 1.16 (d, J = 6.2 Hz, 6H). C NMR (100.6 MHz,
DMSOꢀd6): 155.61, 151.63, 149.47, 139.31, 132.80, 128.72, 125.33, 123.09, 121.32, 118.58, 66.86,
56.62, 52.36, 47.98, 32.29, 22.58. LCMS: RT = 0.773 min, m/z = 378 [M + H]+. HRMS calc’d for:
C19H24ClN3O3 [M+], 377.1506; found 377.1507.
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SUPPORTING INFORMATION
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SAR, chemical synthesis of all analogs, in vitro pharmacology procedures, in vitro PK methods, in vivo
PK methods.
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AUTHOR INFORMATION
Corresponding Authors
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*Jerod S. Denton, Ph.D.
T4208 Medical Center North
1161 21st Avenue South
Nashville, TN 37232
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jerod.s.denton@vanderbilt.edu
T: 615ꢀ343ꢀ7385
*Corey R. Hopkins, Ph.D.
Cool Springs Life Science Center
393 Nichol Mill Lane
Franklin, TN 37067
corey.r.hopkins@vanderbilt.edu
T: 615ꢀ936ꢀ6892
Author Contributions
Conceived and designed the experiments: D.R.S., H.K., K.S.V, J.S., R.R.R, E.F., J.M., A.L.B., C.W.L.,
C.R.H., J.S.D. Performed data analysis: D.R.S., H.K., K.S.V., R.R.R., K.R.V., E.F., J.S., J.M., A.L.B.,
C.W.L., C.R.H., Contributed reagents/materials/analysis tools: D.R.S., H.K., J.S., J.M., C.W.L., Wrote
manuscript: D.R.S., H.K., J.S., J.M., A.L.B., C.W.L., C.R.H., J.S.D.
Funding
This work was funded in part by the Vanderbilt Institute of Clinical and Translational Research pilot
grant (D.R.S, J.S.D.), DK082884 (J.S.D.), and MLPCN (C.R.H, C.W.L.).
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