Investigation of the structure activity relationship of flufenamic acid derivatives at the human TRESK channel K2P18.1

Article abstract of DOI:10.1016/j.bmcl.2016.09.020

TRESK (Twik RElated Spinal cord K⁺channel) is a member of the Twin Pore Domain potassium channel (K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal (TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward K⁺current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in the opening action of its ligands are still poorly understood, making the development of new potent and selective openers challenging. In this work we present a structure activity relationship (SAR) of the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects of chemical modifications to build a TRESK homology model to support the biological results. A plausible binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding site.

Full text of DOI:10.1016/j.bmcl.2016.09.020

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigation of the structure activity relationship of flufenamic acid
derivatives at the human TRESK channel K2P18.1
Aymeric Monteillier a, Alexandre Loucif a, Kiyoyuki Omoto b, Edward B. Stevens a, Sergio L. Vicente a,
Pierre-Philippe Saintot a, Lishuang Cao a, David C. Pryde b,
⇑
a Pfizer Neuroscience and Pain Research Unit, Portway Building, Granta Park, Great Abington, Cambridge CB21 6GS, UK
b Worldwide Medicinal Chemistry, Pfizer Neuroscience and Pain Research Unit, Portway Building, Granta Park, Great Abington, Cambridge CB21 6GS, UK
a r t i c l e i n f o
a b s t r a c t
TRESK (Twik RElated Spinal cord K+ channel) is a member of the Twin Pore Domain potassium channel
(K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal
(TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward
K+ current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for
pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in
the opening action of its ligands are still poorly understood, making the development of new potent
and selective openers challenging. In this work we present a structure activity relationship (SAR) of
the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects
of chemical modifications to build a TRESK homology model to support the biological results. A plausible
binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding
site.
Article history:
Received 12 July 2016
Revised 5 September 2016
Accepted 6 September 2016
Available online xxxx
Keywords:
TRESK
Channel
Opener
Fenamate
Ó 2016 Elsevier Ltd. All rights reserved.
Ion channels are transmembrane proteins forming a water-
filled pore allowing an often highly selective passive flow of ions
across the membrane of cells. This transport is crucial to the estab-
lishment and control of membrane potential (resting potential),
where changes in voltage can induce a wide range of cellular
responses such as depolarization or hyperpolarisation of sensory
neurons. Their implication in many different clinical states includ-
ing hypersensitivity, insensitivity to pain and migraine has made
ion channels very compelling new targets for drug discovery.1
Ion channels are classified according to their ion selectivity,
leading to five major classes: sodium, calcium, chloride, potassium
and nonspecific cation channels.2 Among the potassium channel
family are the Twin Pore Domain (K2P) potassium channel subfam-
ily, containing 15 members expressed in neurons, muscle cells and
endocrine cells.3
The work in this Letter is focused on TRESK (K2P18.1), a K2P
channel expressed mainly in dorsal root ganglia (DRG) neurons
and trigeminal neurons, peripheral sensory neurons having a key
role in pain transmission and migraine respectively. The KCNK18
gene was first cloned in 2003 from human spinal cord, with the
K
2P18.1 channel identified shortly thereafter.4
Of all K2P’s expressed in DRG neurons, TRESK has been shown
to be the most abundant5 where knockout leads to an enhance-
ment of DRG neuron excitability in rat.6 Activation of TRESK using
pharmacological openers gives rise to outward currents responsi-
ble for
a hyperpolarisation of the neuronal cells, inhibiting
excitability and thus pain transmission. Moreover, a specific
N
N
N
NH
O
OH
H
N
F
H
N
H
N
F
N
F
O
3
Abbreviations: K2P, twin pore potassium channel; TRESK, Twik RElated Spinal
cord K+ channel; DRG, dorsal root ganglion; TG, trigeminal; SAR, structure activity
relationship; FFA, flufenamic acid; hTRESK, human-TRESK; HB, hydrogen bond;
HBD, hydrogen bond donor; TM domain, transmembrane domain.
1
2
Flufenamic acid
TRESK opener
EC50 >100
BL-1249
TRESK opener
EC50 ~6µM, Emax ~75%
Lidocaine
TRESK blocker
µ
IC50 >100 M
⇑
Corresponding author at present address: Curadev Pharma Ltd, Innovation
µ
M
House, Discovery Park, Sandwich, Kent CT14 9ND, UK. Tel.: +44 (0) 1304 806842.
E-mail address: david@curadev.co.uk (D.C. Pryde).
Figure 1. Structures of some reported TRESK ligands.
http://dx.doi.org/10.1016/j.bmcl.2016.09.020
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
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2
A. Monteillier et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
aura.7 Interestingly, an indirect long term inhibition of this K2P
channel by cyclosporin A or tacrolimus, two immunosuppressant
drugs administrated after organ transplant, have been shown to
induce new migraine-like headache syndromes.8 As most neuro-
logical pain conditions still represent an unmet medical need, find-
ing new potent and selective TRESK openers could provide an
interesting alternative to current pain treatments.
The physiological activation of TRESK is described in the litera-
ture as an indirect mechanism, mainly mediated by the intracellu-
lar calcium concentration, as its elevation activates calcineurin
responsible for the dephosphorylation of two intracellular serine
residues that downregulate channel activity when phosphory-
lated.9 However, this channel can also be modulated via direct
interaction with either openers, such as flufenamic acid10 (FFA,
1), the structurally related BL-124911 (2) or blockers such as lido-
caine12 (3) shown in Figure 1. As the crystal structure of TRESK
O
OH
H
N
O
OH
F
F
O
F
F
F
F
5
4
O
OH
O
OH
O
OH
H
N
H
N
H
N
6
8
7
Figure 2. Initial set of designed FFA analogues.
dominant-negative mutation of KCNK18, the gene coding for
TRESK, has been related to a congenital form of migraine with
A
Molecule 4
Molecule 1
300
300
200
100
0
200
100
0
0.05
0.05
0.05
0.10
0.15
0.20
0.20
0.20
0.25
0.25
0.25
0.05
0.10
0.15
0.20
0.25
Time (s)
-100
-200
Time (s)
-100
-200
Molecule 5
Molecule 6
300
200
100
0
300
200
100
0
0.05
0.10
0.15
0.20
0.25
0.10
0.15
Time (s)
Time (s)
-100
-200
-100
-200
Molecule 7
Molecule 8
300
200
100
0
300
200
100
0
0.05
0.10
0.15
0.20
0.25
0.10
0.15
Time (s)
Time (s)
-100
-200
-100
-200
B
100
1
4
5
6
7
8
50
0
-50
-100
FFA and analogs (100 uM)
Figure 3. Activity results for the first set of analogues. Panel A: Baseline traces are depicted in black, and the red traces were taken in the presence of compound. Panel B:
Summary of current enhancement data in the presence of compounds, taken from the Panel A data.
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A. Monteillier et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
O
O
OH
OH
O
OH
has not been elucidated yet, little is known about the binding site
of these ligands, which makes the discovery of new and more
potent molecules difficult.13
O
O
F
H
N
F
F
F
N
N
F
F
Recently, the structure of a related K2P channel TREK-214 has
been described which represents a breakthrough in further eluci-
dating the structure–function of this overall family of proteins.
Both FFA and BL-1249 are also known to have activity at TREK
channels.15
11
9
10
O
OH
O
OH
H
N
H
N
H
N
O
A docking study was also published recently in which TRESK
inhibitors were docked into a mouse TRESK homology model based
on a MthK channel template structure.16 These studies indicated
several ligands, including 3, to bind in the vicinity of a panel of
Phe residues in the second and fourth transmembrane (TM)
regions, with alanine mutations of the human equivalent of
Phe145 and Phe352 showing diminished blockade by 3. This study
12
14
13
Figure 4. Second round of target structures, designed to probe the importance of
the amino group and the m-substituted phenyl ring.
A
Molecule 10
Molecule 9
400
200
0
400
200
0
0.05
0.10
Time (s)
0.15
0.20
0.25
0.05
0.10
Time (s)
0.15
0.20
0.25
-200
-200
Molecule 11
Molecule 12
1500
1000
500
0
1500
1000
500
0
0.05
0.10
0.15
0.20
0.25
0.05
0.10
Time (s)
0.15
0.20
0.25
Time (s)
Molecule 13
Molecule 14
1000
500
0
1000
500
0
0.05
0.10
0.15
0.20
0.25
0.05
0.10
0.15
0.20
0.25
Time (s)
Time (s)
B
300
200
100
0
9
10
11
12
13
14
-100
FFA analogs (100uM)
Figure 5. Activity results for the second tranche of analogues. Panel A: Baseline traces are depicted in black, and the red traces were taken in the presence of compound. Panel
B: Summary of current enhancement data in the presence of compounds, taken from the Panel A data.
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4
A. Monteillier et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
N
N
N
NH
was intriguing to us, in that it suggested a small molecule binding
site in the fenestration region that could support binding of other
known TRESK ligands, possibly including channel openers from
the fenamate chemotype such as 1 and 2.
O
NH2
F
H
N
F
H
N
CN
H
N
F
F
F
F
In terms of other ligands for the TRESK protein, a recent screen
of the LOPAC library has been disclosed, in which several inhibitors
and activators of the channel were discovered using a thallium flux
assay.17 For example, the histamine antagonist loratadine was
17
15
16
N
NH
O
OH
O
OH
N
H
N
H
N
F
H
N
found to be a sub-1 lM inhibitor of the TRESK channel, through
F
NC
MeO
F
binding in the vicinity of the same Phe145 in TM2 and Phe352 in
the TM4 domain targeted in the previous work, two residues which
are uniquely located in human TRESK within the K2P family.17
In this Letter, we describe studies in which a set of diverse
structural analogues of 1 and 2 were designed and tested against
18
20
19
Figure 6. Third round of targets, designed to probe the acid group and the m-
substituted phenyl ring.
A
Molecule 15
Molecule 13
2000
1000
1500
1000
500
0
500
0
0.05
0.10
Time (s)
0.15
0.20
0.25
0.05
0.10
Time (s)
0.15
0.20
0.20
0.20
0.25
0.25
0.25
-500
Molecule 16
Molecule 17
600
400
200
0
600
400
200
0
0.05
0.10
Time (s)
0.15
0.20
0.25
0.05
0.10
Time (s)
0.15
-200
-200
Molecule 18
Molecule 19
600
400
200
0
600
400
200
0
0.05
0.10
Time (s)
0.15
0.20
0.25
0.05
16
0.10
Time (s)
0.15
-200
-200
B
13
15
17
18
19
20
300
200
100
0
Molecule 20
400
300
200
100
0
0.05
0.10
0.15
0.20
0.25
Time (s)
-100
FFA analogs (100 uM)
-100
Figure 7. Activity results for the third tranche of analogues. Panel A: Baseline traces are depicted in black, and the red traces were taken in the presence of compound. Panel
B: Summary of current enhancement data in the presence of compounds, taken from the Panel A data.
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A. Monteillier et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 1
group in 7 or removing it completely in 6 all lost activity. These
data suggested the existence of a quite specific lipophilic cavity
crucial to opener activity. Swapping the amine linker with an oxy-
gen atom in 5 again removed opener activity, suggesting that the
amine functional group was also crucial to activity. Adding further
methyl groups to 7 to give the tri-methyl analogue 8 produced sig-
nificant channel block.
Comparative opening efficacy of 1, 13 and 15 at human TRESK
Compound
cLogP (LogD)
EC50 (lM)
1
13
15
5.5 (2.2)
6.2 (3.2)
4.9 (2.7)
474
45
18.8
A second set of analogues was designed (Fig. 4) with structures
aimed at refining our initial observations. 9 and 10 probed the
importance of the amino group as either a hydrogen bond donor
(HBD) or supporting an intramolecular HB that could constrain
the conformation of the carboxylic acid moiety. 11–14 were
designed to explore the hypothesis of a cavity being filled by a lipo-
philic meta substituent. Test results (Fig. 5) showed that 9 and 10
had no activity at all, suggesting that both the NH of flufenamic
acid and the acid group were important for TRESK activity. Inter-
estingly, 11 and 12 showed blocking activity, suggesting the size
of any putative opener cavity to be limited.
13 and 14 both showed an opener effect, either equivalent to 1
in the case of 14, or greater for 13, indicating that growing the tri-
fluoromethyl group could offer improved potency provided this
growth in size was limited.
A further set of analogues were designed to explore the acid
group and the phenyl substitution pattern more fully (Fig. 6) and
its role in channel opening (Fig. 7).
N
N
N
NH
N
N
N
NH
N
N
N
NH
H
N
H
N
F
F
H
N
F
22
15
2
TRESK blocker
TRESK opener
EC50 ~6 M, Emax ~75%
TRESK opener
EC50 18.8µM, Emax 102%
µ
O
OH
O
OH
O
OH
H
N
F
H
N
H
N
F
F
13
1
21
TRESK opener
EC50 45µM, Emax 354%
TRESK opener
EC50 >100µM
TRESK blocker
Figure 8. Comparative efficacy of 1 and analogues.
Replacing the acid in 13 with a tetrazole isostere 15 resulted in
a modest increase in opening efficacy compared to the acid equiv-
alent. Replacement of the tetrazole with the nitrile group in 16, or
with the non-acidic HBD carboxamide 17 both resulted in signifi-
cant channel block. Replacement with the non-acidic triazole 19
resulted in a complete loss of activity.
These results served to underline the importance of the acidic
functionality in 1 and 2 for channel opening efficacy. Finally, the
phenyl group was furnished with small substituents, either a
nitrile 18 or a methoxy group 20, both of which were inactive,
and emphasized the importance of a larger lipophilic group in this
region of the chemotype.
The TRESK opening functional efficacy of 13 and 15 was directly
compared to that of 1 as shown in Table 1 and confirmed 13 to
have an EC50 of 45 lM with 15 moderately more potent at 18.8 lM.
It was interesting to compare directly the opening efficacy of
the trifluoromethyl group contained in 1 with either a tert-Bu
group or a fused cyclohexyl group (Fig. 8). Compounds 21 and 22
were synthesized to complete the set of analogues, and were both
found to be channel blockers. Direct replacement of the trifluo-
hTRESK expressing cells for their ability to open or block the chan-
nel. The data from these studies was used to build a docking model
of hTRESK and thereby develop hypotheses of the molecular basis
of the observed structure–activity and structure–function
relationships.
1 is a weak opener of TRESK, with 2 being a significantly more
potent activator in our hands. Activity was assessed by manual
patch clamp electrophysiology assaying of HEK293 cells overex-
pressing K2P18.1. We wished to determine if we could further build
on the promising activity of 2 through iterative structure–activity
based enhancements within a fenamic acid structural template.
Our first step was to design analogues of 1 with simple modifi-
cations of the functional groups likely to be involved in engaging
the channel. A first set of molecules was designed to explore the
nature of the central heteroatom linker between the two aromatic
rings and the aryl group substitution (Fig. 2) and tested for their
hTRESK activity (Fig. 3).
Initial results showed that minor structural modifications led to
drastic changes in activity. Indeed, none of the initial set of ana-
logues were openers, with some compounds showing significant
TRESK block. For instance, simply moving the trifluoromethyl
group of FFA to the para position in 4, converting it into a methyl
romethyl group with
a tert-Bu group (1 ? 13) moderately
increased potency, but a fused cyclohexyl group converted 1 into
a blocker (1 ? 21). Converting the acid of 1 into a tetrazole con-
Figure 9. Docked structures of 1 and 2 bound in a homology model of human TRESK.
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A. Monteillier et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
verted 1 into a blocker (1 ? 22), whilst the fused cyclohexyl equiv-
alent 2 was the most potent compound in the study, and of similar
potency to the tert-Bu congener 15. It is also notable that the
potency of 2 was greater than that of either 13 or 15, but both
these latter compounds showed greater opening efficacy, indicat-
ing a mismatch between binding affinity and functional efficacy.
To support these biological results, 1 and its derivatives were
docked into a TRESK homology model built using the published
structure of TRAAK and TWIK-1 in order to provide a better under-
standing of the binding site shape and the residues likely to be
involved in the interaction with openers (Fig. 9).
In summary, a plausible model has been developed to provide
good support of the experimental voltage clamp results through
proposing a fenamate binding site on hTRESK.
Note
The authors declare no competing financial interest.
Acknowledgment
We thank the Pfizer Neusentis electrophysiology team for their
technical support of these studies.
Figure 9 shows docked structures of 1 and 2. The suggested
binding site was a space that extended from the pore to the fenes-
tration site, being similar to the ligand binding site highlighted in
the TREK-2 crystal structure.14 Phe145 and Phe352 (highlighted
by circles), which Kim et al.16 suggested to be key residues for
interactions with small molecules, were in close proximity with
the docked ligands. The amine linkers of both ligands formed
hydrogen bonds with the backbone oxygen of Leu321 in the
pore-loop (depicted in green), suggesting their significant contri-
bution to the activity of the ligands. Interestingly, hydrogen bond
interactions with the pore-loop have been reported for other
potassium channels, sodium channels, and calcium channels.18
This was consistent with the SAR that swapping the amine with
an oxygen or methylation of the amine resulted in complete loss
of activity. A further hydrogen bond was formed between the
tetrazole of 2 and the side chain of Thr323, however, this interac-
tion is not observed for the docked carboxylic acid of 1, which is
oriented quite differently to 2. The models were unable to rational-
ize the SAR for the acid replacements 17 and 19. The hydrophobic
aryl groups were accommodated in the fenestration site, and were
surrounded by hydrophobic residues (Ile286, Ile289, Val290, and
Ile294). The observation that position and size of the hydrophobic
substituents were key for opener activity was consistent with the
docking models. Aryl groupings with a para substituent were less
able to fit this hydrophobic region effectively. The importance of
the size of these substituents also reflected the space constraints
within this hydrophobic region as the bulkier hydrophobic groups
as in 11 and 12 were less well tolerated and began to show block-
ing activity. This latter observation suggested the fenestration site
changes shape in transitioning between open and closed states and
bulkier groups fit better to the latter. Further validation of this
hypothesis is the subject of ongoing investigation.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.09.
020.
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The combination of biological results and molecular modeling
allowed a possible fenamate opener binding site to be proposed,
showing good predictivity for ligands with improved efficacy com-
pared to FFA. Although the opening mechanism is still poorly
understood for TRESK, we can hypothesize that FFA and its opener
derivatives act through a stabilization of an open state where the
transmembrane domains TM2 (in blue), TM3 (in magenta) and
TM4 (in red) of each subunit remain distant to each other. We also
showed SAR suggesting that the opener binding site cavity is of
limited size, potentially restricting the possibilities of further opti-
mization at this region of the binding site.
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The blocking effect observed in this study is more complicated
to explain. The broad structural similarity of the blockers described
herein suggests they share the same binding site. Our modeling
work indicates binding to be through a p–p interaction with three
phenylalanine residues Phe-130, Phe-487 and Phe-490, resulting in
the bringing together of the TM4 and TM2 subunits, stabilizing a
closed state.
Please cite this article in press as: Monteillier, A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.09.020