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![previously published reports on ShK SAR,7,16
and, importantly,
these substitutions, along with positions 7, 10, 21, and 27, also
resulted in considerably reduced activity in the corresponding
whole-blood IL-2 and IFN-γ secretion assays (red). Within this
series, only 19 ([Ala23]ShK) and 24 ([Ala29]ShK) showed
>5-fold selective inhibition of Kv1.3 over Kv1.1, but,
unfortunately, the concomitant loss in the critical cytokine
secretion inhibitory activity for these compounds limited their
utility. Under our assay conditions, the substitution of alanine
for lysine at position 18 (14) did not improve selectivity against
Kv1.1 as reported in the literature, perhaps due to differences
in electrophysiology platform (IWQ instead of manual patch
clamp) and/or cell line (hKv1.1 expressed in HEK293 cells
instead of mKv1.1 in mouse L929 fibroblasts), and it was
not tested by manual electrophysiology.20
In short, classic alanine
positional scanning did not result in improvement in either
potency or selectivity, necessitating implementation of our MAPS
analoging methodology.
The 1-naphthylalanine positional scan of ShK introduces a
large aromatic side chain in place of the wild-type functionality
to examine the effects of increasing hydrophobicity and steric
bulk. This series had the largest number of substitutions that
were disruptive to the Kv1.3 inhibitory activity of ShK. Kv1.3
inhibition was adversely affected by 1-Nal incorporation at
positions 5, 7, 9, 11, 16, 18, 20, 21, 22, 23, and 29. Additionally,
activity in the whole-blood assay was diminished by substitution
of 1-Nal at positions 13, 27, and 33. This list includes and
adds to the key binding residues identified by the Ala scan.
Compounds 49 ([1-Nal26]ShK) and 50 ([1-Nal27]ShK) had
≥5-fold selectivity over Kv1.1, but only 1-Nal substitution at
position 26 retained desirable whole-blood activity <1000 pM.
In addition to varying the hydrophobicity and size at each
position of ShK with Ala and 1-Nal, the electrostatic inter-
actions were also probed through incorporation of amino acids
with charged side chains. The glutamic acid substitution series
had an activity profile similar to that with 1-Nal, with positions
5, 7, 11, 13, 20, 21, 22, 23, 24, 27, and 29 not being well
tolerated in either the ePhys or whole-blood assays or both,
demonstrating the extensive perturbation caused by integration
of an acidic residue into a highly basic peptide sequence.
Furthermore, no compound from the glutamic acid substitution
series appeared to show any selective inhibition for Kv1.3 over
Kv1.1. One observation unique to the Glu series was that
substitution of the native Arg at position 24 caused a loss of
functional activity in the cytokine secretion assays but retained
activity in the electrophysiology assays, giving some insight into
the SAR for that residue position.
The basic arginine and lysine substitution series led to the
identification of a selective and potent ShK analogue as a lead
for further optimization and study. First, we found that arginine
substitutions at 5, 7, 8, 20, 21, 22, 23, 27, and 31 resulted in
significant loss of Kv1.3 and/or functional inhibitory activity.
Lysine substitutions at positions 5, 7, 21, 27, and 31 had similar
effects (Figure 3A). Among the different scans, position 31 was
uniquely sensitive to substitution with a basic residue, having
whole-blood IC50 values >5000 pM for the Arg (107) and Lys
(131) substitution analogues but <500 pM for the Ala (26),
1-Nal (53), and Glu (82) containing compounds. Interestingly,
although no arginine-substituted ShK analogues had any se-
lective inhibition for Kv1.3 over Kv1.1, lysine substitution
analogues at position 7, 115 ([Lys7]ShK), and position 16, 122
([Lys16]ShK), were both 6-fold selective, a 40× improvement
over native ShK (Figure 3B). However, only 122 retained potent
whole-blood activity with an IL-2 secretion IC50 of 108 ± 45 pM
and IFN-γ secretion IC50 of 151 ± 110 pM (n = 67). By
comparison, 96 ([Arg16]ShK) had no improvement in selec-
tivity for Kv1.3 and instead was an approximately 3-fold more
potent inhibitor of Kv1.1 than Kv1.3. The key features of the
sequence−activity relationship from the Lys scan are presented
in Figure 3C.
To summarize, application of MAPS analoging to ShK led
to the identification of previously unreported sites for potency
and selectivity not found via traditional Ala scanning efforts
Figure 3. (A−C) Functional activity and electrophysiological selectivity
of lysine scan ShK analogues. (A) Inhibition of IL-2 and IFN-γ secre-
tion in whole-blood assay. (Top concentration tested was 100 nM.)
(B) Kv1.1/Kv1.3 selectivity ratio. (C) ShK peptide sequence with
residues important for potency in red and bold, residues important for
selectivity in blue and underlined, and residues important for both in
purple, bold, and underlined. Note that substitution of lysine at position
16 uniquely enhanced Kv1.3 selectivity with retention of potency
against cytokine secretion. (D) Consensus findings from MAPS
analoging of ShK with the residues likely to impact potency through
conformational effects in bold and green, residues indicated as
important for potency by a single series denoted in orange and bold,
and the remainder as described in (C).
Journal of Medicinal Chemistry Article
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![(Figure 3D).16
From an overall perspective, only 2 out of the
132 initially prepared ShK analogues, 49 and 122, met the
following success criteria: (1) <500 pM potent Kv1.3 inhibitor,
(2) 5-fold or greater selectivity for Kv1.3 over Kv1.1, and (3)
<1000 pM inhibitor of IL-2 and IFN-γ cytokine secretion in
whole blood. These two novel lead compounds, not predicted
a priori by computational methods,20
were identified only by a
systematic approach that scanned the entire molecule multiple
times with amino acids with different physicochemical pro-
perties, not just alanine. Furthermore, a consensus list of Kv1.3-
interacting residues within ShK was identified with a number
of positions not immediately apparent from the Ala scan alone
and only by balancing the findings from the electrophysio-
logical assays with the results of the whole-blood cytokine
secretion assays. Importantly, selectivity against Kv1.1 was
obtained by substitution at positions not identified as critical for
Kv1.3 inhibitory activity. Aside from a better understanding of
which surface regions of ShK are important for activity and
selectivity, MAPS analoging also provided a more detailed view
of the SAR at each amino acid position.
Structure−Function Relationships of Kv1.3 Inhibitory
Toxin Peptides. Using racemic crystallography,27
we were
able to solve the X-ray crystal structure of 122 at 1.2 Å resolu-
tion (Figure 4). The peptide analogue consists of an extended
conformation at the N-terminus up to residue 8, followed by
two interlocking turns and then two short helices encompassing
residues 12−19 and 21−24. Substitution of lysine at position 16
had no significant effect on the local conformation relative to
the wild-type ShK structure.19a
ShK residues Ile7
, Arg11
, Ser20
,
Met21
, Lys22
, Tyr23
, and Phe27
, each identified as important for
binding to Kv1.3 by an observed >20× loss in functional
activity for at least three MAPS analogues, cluster in the tertiary
Figure 4. Crystal structure of [Lys16]ShK. (A, B) 122 with side chains rendered as sticks and backbone secondary structure indicated with ribbons.
(C−F) Surface rendering of 122 with residues colored according to putative interaction with Kv1.3 during binding. Blue indicates direct contact;
yellow and orange residues make peripheral contact, with yellow substitutions affecting selectivity and orange substitutions impacting selectivity and
potency. (A, C, E) View of the putative binding interface of the peptide. (B, D, F) Side view with Lys22
facing downward.
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![structure of the peptide (Figure 4C,D). The binding of ShK to
the Kv1.3 channel has been generally described as a “cork in
a bottle”, with ShK inserting the side chain of Lys22
into and
physically occluding the channel pore.10,28
To potentially aid
our comprehension of the screening data and the selectivity,
the 122 structure was docked to a homology model of the
Kv1.3 channel. Two poses that place Lys16
of 122 near sites
where Kv1.3 differs from Kv1.1 in amino acid sequence (His451
versus Tyr379
and Gly427
versus His355
for Kv1.3 and Kv1.1,
respectively) while maintaining key ShK binding residues in
close contact with the Kv1.3 channel are shown in Figure 5.
Despite the availability of this new set of ShK analogues, it is
still unclear which of the proposed binding modes is most
relevant; however, complementary ion channel site-directed
mutagenesis may assist our understanding of the molecular
basis for the selectivity of 122.
There were a total of six ShK substitution analogues from the
MAPS analoging that resulted in 5-fold selectivity for Kv1.3
over Kv1.1: Lys7
, Lys16
, Ala23
, 1-Nal26
, 1-Nal27
, and Ala29
, but
only modification of positions 16 and 26 improved Kv1.3
selectivity without significantly compromising Kv1.3 potency or
functional activity (Figure 4E,F). The native residues Gln16
and
Ser26
are located at the periphery of the putative binding face
and may interact with a portion of the surface of the channels
that has some structural or sequence difference between Kv1.3
and Kv1.1. However, the unpredictability of the SAR is
demonstrated by substitution of Arg29
with Ala, which is
located more remotely than either position 16 or 26 but led to
an increase in selectivity with concomitant loss in potency and
unclear effect on overall peptide conformation. Other residues
located at the border of the binding face, i.e., Thr6
, Ser10
, Thr13
,
Arg24
, Thr31
, and Gly33
, have at least one MAPS analogue with
a >20× loss in functional activity in the whole-blood assay
without improvement in Kv1.3 versus Kv1.1 selectivity. Some
effects on activity may be due to conformational disruption of
the peptide. For example, substitution of a Lys or Arg residue at
position 31 would place the side chains of three basic residues
(Lys9
and Lys30
) in close proximity and may affect the global
structure. While our results serve to refine the list of residues in
ShK with strong Kv1.3 interactions,20
these data also highlight
the importance of residues at the edge of the peptide binding
surface. While these peripheral residues are typically ignored
by traditional optimization strategies (i.e., alanine scanning
and structure-based design), specific changes in charge or
hydrophobicity at these sites may serve to elucidate the nature
of their contribution to the complex and effect on ion channel
selectivity.
[Lys16]ShK Peptide Analogues. Identification of the
potent and moderately selective Kv1.3 inhibitory peptide 122
was followed up with additional analoging at position 16 and
Figure 5. Molecular docking of 122 (gray ribbon with residues important to binding in blue and Lys16
and Ser26
in yellow) to Kv1.3 homology
model (green ribbons). (A) Side view of pose I. For clarity, two monomers (II and IV) of the homotetrameric channel have been hidden. (B) Top
view of pose 1. (C) Side view of pose 2. For clarity, two monomers (I and III) of the homotetrameric channel have been hidden. (D) Top view of
pose 2.
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DOI: 10.1021/acs.jmedchem.5b00495
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![combination with modifications to reduce oxidative liabilities
(Table 3). Shortening the Lys16
side chain by a methylene
unit to orthinine (Orn) resulted in a similar activity profile
(134); however, removal of a second methylene unit with
diaminobutyric acid (Dab) led to loss of Kv1.1 selectivity
(135). It is known that amidation of the C-terminal acid of ShK
provides a backbone with similar activity and increased
metabolic stability;21c
preparation of the C-terminal amide of
[Lys16]ShK yielded a similarly potent and selective derivative
(136). Extension of the C-terminus with a residue less prone
to epimerization during solid-phase peptide synthesis than
cysteine29
and substitution of the oxidizable methionine with
norleucine at position 21 were investigated in combination with
the lysine substitution at position 16 (Table 3). Surprisingly,
addition of a C-terminal alanine to [Lys16]ShK resulted in
analogues 137 and 141 with >150-fold selectivity for Kv1.3
versus Kv1.1 that retained good activity in blocking T cell
cytokine secretion in human whole blood. The improvements in
selectivity associated with substitution of lysine at position 16,
hydrophobic substitutions at position 21, and extension of the
C-terminus have been verified by Pennington and co-workers,
including an additive improvement in selectivity through their
approach of N-terminal modification.30
Electrophysiology of ShK Peptide Analogues. Further
electrophysiological characterization of lead compound 122,
the parent ShK peptide, and literature analogues was performed
(Table 4). Our previous screening experiments utilized a
high-throughput 384-well IonWorks Quattro (IWQ) platform,
which evaluates receptor inhibition with a population patch
clamp, due to the large number of compounds that needed to
be tested. A select number of important analogues were tested
on a whole-cell planar patch clamp platform using the auto-
mated PatchXpress (PX) system or manual electrophysiology.
As expected, ShK was an exceptionally potent inhibitor of
both Kv1.3 and Kv1.1 on the PX system. These values were in
reasonable agreement with those obtained by manual whole-
cell patch clamp electrophysiology where ShK had an IC50 of
16 ± 8 pM for Kv1.3 and 14 ± 3 pM for Kv1.1, similar to
values reported in the literature as well as our results in the
cytokine secretion assays.7,16b,17,21b
Compound 122 was also a
potent inhibitor of Kv1.3 on the PX platform and demonstrated
>15× selectivity against Kv1.1. The potency and selectivity
profiles for 142 (ShK-Dap22) and 143 (ShK-L5, Supporting
Information Figure S1), which are commercially available,
were compared to the results reported previously for these
analogues.7,21b
Molecule 142 showed a significant loss in
whole-blood functional activity, which motivated us to adopt
this assay for the primary screening of our analogues. As dis-
cussed earlier, the whole-blood assay is of longer duration and
may better reflect equilibrium binding of the peptide to the
target. Indeed, while Kalman et al. reported that 142 showed
good potency by electrophysiology (Kv1.3 IC50 = 23 pM)7
similar to our findings, Middleton et al. reported that its
equilibrium binding affinity for Kv1.3 is more than 100 times
weaker than native ShK.31
These latter results are consistent
with our observations in the 48 h whole-blood assay, where 142
had IL-2 and IFN-γ IC50 values >3000 pM. In our assays, 143
was a potent inhibitor of both Kv1.3 and Kv1.1 as well as
cytokine secretion in human whole blood. The disagreement
of our selectivity ratio for 143 with published reports may be
due to our use of a different cell line (hKv1.3 in Chinese
hamster ovary (CHO) cells rather than mKv1.3 in mouse L929
fibroblasts).7,21b
Impact of Conjugation on Potency, Selectivity, and
Pharmacokinetics of ShK Analogues. The potent wild-type
ShK peptide has a very short circulating pharmacokinetic
profile in rats (t1/2 ∼ 20 min).32
The short half-life in vivo of
peptides is typically attributed to rapid metabolic processing
and high renal clearance.33
To investigate whether renal
clearance was responsible for the short circulating time of
ShK, we attempted PEGylation of the molecule as a means to
increase its hydrodynamic radius.34
It was unknown, however,
whether attachment of a large poly(ethylene glycol) (PEG)
polymer to ShK would significantly impair its activity. We
explored a N-terminal reductive amination approach due to the
presence of multiple lysine residues in ShK derivatives and the
difficulty of using cysteine-maleimide chemistry in disulfide-rich
peptides. First, a Nα
-PEG-ShK conjugate was prepared by
reductive alkylation of the N-terminus with a linear 20 kDa
monomethoxy PEG aldehyde at pH 4.5 and then purified.
Peptide mapping experiments confirmed PEGylation occurred
primarily at the N-terminus of the peptide (data not shown).
Testing of 144 (20 kDa-PEG-ShK) revealed that it retained
subnanomolar potency in inhibiting Kv1.3 and T cell cytokine
responses (Table 5) and exhibited a prolonged half-life in rats
(mean residence time of 37 h, Supporting Information Table S2).
Table 3. Potency and Selectivity of Position 16 ShK Analogues
cmpd peptide name Kv1.3 IWQ IC50 (pM) Kv1.1 IWQ IC50 (pM) Kv1.1 IC50/Kv1.3 IC50 WB IL-2 IC50 (pM) WB IFN-γ IC50 (pM)
1 ShK 132 20 0.15 37 48
122 [Lys16]ShK 352 2342 6.7 108 151
134 [Orn16]ShK 140 740 5.3 138 160
135 [Dab16]ShK 82 11 0.13 86 223
136 [Lys16]ShK-amide 174 600 3.4 223 278
137 [Lys16]ShK-Ala 60 9500 158 138 266
138 [Nle21]ShK 40 15 0.38 153 303
139 [Lys16,Nle21]ShK 130 29 258 225 249 5678
140 [Lys16,Nle21]ShK-amide 153 13 220 86 823 1099
141 [Lys16,Nle21]ShK-Ala 71 >33 333 >469 305 515
Table 4. Potency and Selectivity of ShK Analogues
cmpd peptide name
Kv1.3
PX IC50
(pM)
Kv1.1
PX IC50
(pM)
Kv1.1
IC50/
Kv1.3
IC50
WB
IL-2
IC50
(pM)
WB
IFN-γ
IC50
(pM)
1 ShK 39 87 2.2 37 48
122 [Lys16]ShK 207 3677 17.8 108 151
142 ShK-Dap22 12a
847a
70.6 3763 3112
143 ShK-L5 221 214 1.0 31 46
a
Indicates manual whole-cell patch clamp data.
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![In agreement with other N-terminally derivatized ShK ana-
logues,35
such as 143 (phosphotyrosine-AEEA-ShK), which have
been reported to have increased selectivity for Kv1.3, we also
found 144 to be 5-fold more potent against Kv1.3 than against
Kv1.1. Encouraged by the retention of activity, we next
PEGylated our selective [Lys16]ShK analogue at its N-terminus
with linear PEG. The conjugate 145 (20 kDa-PEG-[Lys16]ShK)
was found to provide potent blockade of whole-blood IL-2
secretion with an IC50 of 92 ± 42 pM (n = 14). More interes-
tingly, selectivity for Kv1.3 over Kv1.1 was not only retained,
but it showed a synergistic 1000-fold lowering of Kv1.1 activity
without impacting Kv1.3, more than 200× the effect that
PEGylation had on native ShK.30
The potency and selectivity of
145 are extraordinary when compared to those of the conjugated
wild-type peptide and unconjugated peptide analogues (Figure 6).
The pharmacokinetics and bioactivity of polypeptides can
be significantly altered through the attachment of PEG groups
of differing molecular weight.36
Aside from derivatization with
20 kDa-PEG, the [Lys16]ShK peptide was also prepared with
either a 30 kDa linear PEG or a branched PEG consisting of
two 10 kDa PEG arms (20 kDa-brPEG). The 20 kDa-brPEG-
[Lys16]ShK molecule (146) was a potent inhibitor of cytokine
secretion in the whole-blood assay and had 750-fold selectivity
for lymphocyte Kv1.3 over neuronal Kv1.1. The linear 30 kDa-
PEG-[Lys16]ShK molecule (147) was also a highly potent
inhibitor of cytokine secretion in human whole blood. These
results suggest that the 122 peptide scaffold is tolerant of
N-terminal derivatization with PEG polymers of differing size
and architecture. Conjugation of 122 with linear 20 kDa PEG
results in a slightly higher level of Kv1.3 vs Kv1.1 selectivity
relative to the branched or larger PEG chains.
In preparation for in vivo studies, the in vitro activity profile
of 145 was further characterized in a number of ion channel
counterscreens and against other species. Counter screening
revealed that 145 was highly selective over Kv subtypes Kv1.2
(680-fold), Kv1.6 (∼500-fold), Kv1.4 (>10 000-fold), Kv1.5
(>10 000-fold), and Kv1.7 (>10 000-fold) (Table 6). Importantly,
the conjugate did not impact ion channels that are known to
serve a role in human cardiac action potential, exhibiting
>10 000-fold selectivity over Nav1.5, Cav1.2, Kv4.3, KvLQT1/
minK, and hERG. Moreover, the conjugated toxin peptide
analogue 145 had no detectable impact on the calcium-activated
K+
channels KCa3.1 (IKCa1) and BKCa.
Cross-Species Activity of 20 kDa-PEG-[Lys16]ShK. In
addition to its inhibitory activity in human whole blood, 145
also inhibited IL-17 and IL-4 secretion from T cells within
cynomolgus monkey whole blood with potent IC50 estimates
of 0.09 ± 0.08 nM and 0.17 ± 0.13 nM, respectively. 145 was
also a potent inhibitor (IC50 = 0.17 nM) of myelin-specific
proliferation of the rat T effector memory cell line, PAS.37
Overall, we found that 145 has consistently potent inhibitory
activity toward T cell responses in whole-blood assays from rat,
monkey, and human (IL-2 IC50 = 0.092 nM).
Pharmacokinetics of 20 kDa-PEG-[Lys16]ShK. In regards
to unconjugated peptides, there are limited pharmacokinetic
Table 5. Potency and Selectivity of PEGylated ShK Analogues
cmpd name Kv1.3 PX IC50 (nM) Kv1.1 PX IC50 (nM) Kv1.1 IC50/Kv1.3 IC50 WB IL-2 IC50 (nM) WB IFN-γ IC50 (nM)
144 20 kDa-PEG-ShK 0.299a
1.628a
5 0.380 0.840
145 20 kDa-PEG-[Lys16]ShK 0.94 997 1060 0.092 0.160
146 20 kDa-brPEG-[Lys16]ShK 2.10 1574 750 0.198 0.399
147 30 kDa-PEG-[Lys16]ShK 1.20 1072 890 0.282 0.491
a
Manual patch clamp electrophysiology.
Figure 6. Graphical comparison of the potency and selectivity of select
naked and PEGylated peptide analogues relative to ShK. Each point
represents one compound with the x-axis value computed as (whole-
blood IL-2 IC50)/(ShK whole-blood IL-2 IC50) and the y-axis value
computed as ([Kv1.1 IC50/Kv1.3 IC50]/[ShK Kv1.1 IC50/ShK Kv1.3
IC50]).
Table 6. Activity of 145 in Counterscreensa
assay IC50 (nM)
human WB IL-2 0.092
Kv1.1 997
Kv1.2 639
Kv1.3 0.94
Kv1.4 >10 000
Kv1.5 >30 000
Kv1.6 466
Kv1.7 >10 000
IKCa1 >10 000
BKCa >10 000
hERG (IKr) >10 000
Nav1.5 (INa) >30 000
Cav1.2 (ICa) >30 000
Kv4.3 (Ito) >30 000
KvLQT1/minK (IKs) >30 000
a
n ≥ 3 for all.
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![studies on native ShK32
and the more selective ShK analogue,
143,21b
indicating that these molecules have half-lives in rats that
are much shorter (<1 h) than that of our PEG conjugate. Prior
to evaluating the pharmacology of the potent and selective
conjugate 145, its ex vivo plasma stability and pharmacokinetics
were determined. The conjugate was found to have high meta-
bolic stability in plasma from rat, cynomolgus monkey, and
human over 2 days at 37 °C (Supporting Information Figure S5).
Pharmacokinetic studies in mouse, rat, dog, and cynomolgus
monkey showed good cross-species metabolic stability in vivo
with a considerably extended elimination half-life. Moreover,
a comparison of 148 (ShK-186), a more advanced derivative of
143 containing a C-terminal amide and displaying improved
stability,21c,24
indicates that 145 has a half-life in cynomolgus
monkeys that was 245 times longer than 148 when the same
0.5 mg/kg dose was delivered (Table 7). We estimate the
exposure of compound 145 over time, as measured by AUC0−∞,
was 390 times greater in cynomolgus monkeys than 148, resulting
in a clearance rate that was ∼950 times slower in rats and
cynomolgus monkeys compared to the 148 peptide. As shown in
Figure 7, 145, when dosed subcutaneously in cynomolgus
monkeys at 0.5 mg/kg, achieved a Cmax at 8 h of 254 nM and
day 7 serum levels of 28.4 nM. The serum concentration of 145
at day 7 after a single dose was approximately 28 and 315 times
greater than the cytokine secretion IC95 (1.0 nM) and IC50
(0.09 nM) estimates in human whole blood, respectively.
Therefore, the pharmacokinetics of 145 in cynomolgus monkeys
are consistent with a projected weekly dosing profile in human
subjects. It should be noted that despite our PEG conjugate
showing a profoundly longer half-life in vivo than 148 Tarcha
et al. report that this peptide analogue shows durable
pharmacological effects in monkeys.24
The authors propose
that, although serum levels decline rapidly over the first few hours
after injection, there could be a slow release from the injection
site as well as tight binding and slow dissociation from the Kv1.3
channel on T cells to drive efficacy. Irrespective of these con-
siderations, we show that the conjugate 145 is profoundly longer-
lived in vivo, enabling sustained and measurable target coverage
over a narrower dynamic range of serum drug concentrations.
Further details on the pharmacokinetics of 145 administered
subcutaneously are provided in the Supporting Information.
Efficacy, Pharmacodynamics, and Safety of 20 kDa-
PEG-[Lys16]ShK. We evaluated the efficacy of 145 in vivo
using the adoptive-transfer experimental autoimmune encepha-
lomyelitis (AT-EAE) model in rats.38
In this animal model of
multiple sclerosis, T cells specific for myelin basic protein (MBP)
and constitutively expressing Kv1.3 (PAS cells) are activated and
injected into rats, causing inflammation and demyelination of the
central nervous system (CNS), with symptoms progressing from
a distal limp tail to paralysis over the course of a week. Dosing in
rats with the Kv1.3 blocker 145 before the onset of EAE caused a
delay in the onset of disease. The progression of disease was also
inhibited with treatment with 145, with an observed dose-
dependent effect on reduced disease severity and the prevention
of death (Figure 8). In the vehicle-treated animal group,
the disease onset occurred on day 4, but, by comparison, in
animals treated with 145, the disease onset was delayed until day
4.5 to 5. On day 6, the vehicle-treated rats had developed severe
disease (EAE score of 6) and were sacrificed, whereas 145
treated animals (at efficacious doses) had only mild disease
(EAE score of ∼1) that resolved over time. The molecule 145
blocked AT-EAE in a dose-responsive manner with an estimated
ED50 of approximately 4 μg/kg on day 7 (Figure 8 and
Table7.SingleDosePharmacokinetic(Subcutaneous)Profileof145inCD1Mice,SpragueDawleyRats,BeagleDogs,AndCynomolgusMonkeysComparedtothe
Pharmacokineticsof148inSpragueDawleyRatsandCynomolgusMonkeys24,a
cpmdspeciesdose(mg/kg)nt1/2(h)Tmax(h)Cmax(ng/mL)AUC0−t(ng·h/mL)AUC0−∞(ng·h/mL)Vz/F(mL/kg)CL/F(mL/h/kg)MRT(h)
145mouseb
2.0314.94.018603700037000117054.116.6
145rat2.03N/A40±14531±9021900±277021900±2760N/A92±1336±2
145beagle0.5342.6±4.2118.7±9.241270±34795200±31300103000±37300322±985.37±2.1466.1±13.5
145cyno0.5364.5±14.98.01010±10571500±60774900±3260621±1436.68±0.2987±16
148c
rat1.030.1320.08348NR11.5NR87198NR
148c
cyno0.520.2630.083192NR192NR6336NR
a
Becauseonlythepeptideportionof145wasusedincalculatingmg/mLstockconcentrationsandthe[Lys16]ShKpeptideportion(4055Da)isofsimilarMWto148,equivalentmg/kgdosesofthese
twomoleculesgeneratesimilarnmol/kgdoses.b
SparsesamplingPKexperiment.NostandarddeviationswerecalculatedforPKparameters.c
FromTarchaetal.24
NR=notreported.
Journal of Medicinal Chemistry Article
DOI: 10.1021/acs.jmedchem.5b00495
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![Supporting Information Figure S6). In a separate study, an
equivalent dose (0.01 mg/kg) of PEGylated ShK (144) was
found to provide greater efficacy in blocking encephalomyelitis
than that of the native ShK peptide (Supporting Information
Figure S7) that has a shorter half-life in animals. Overall, these
data suggest that higher levels of sustained Kv1.3 target coverage
appear to result in greater efficacy in this model.
The in vivo pharmacodynamics of 145 in cynomolgus
monkeys was also examined. A 12 week pharmacology study
was initiated with three predose baseline measurements during
the first 2 weeks. This was then followed by four weekly sub-
cutaneous doses of 145 at 0.5 mg/kg and an additional 6 weeks
of postdosing analysis (Figure 9 and Supporting Information
Tables S5−S7). On the basis of earlier pharmacokinetic
studies of 145, target coverage with 0.5 mg/kg weekly dosing
was expected to range from 28-times the IL-17 IC95 at the
minimum drug concentration in plasma (Cmin) to 249-times IC95
at the peak or maximum drug concentration (Cmax). The repeat
dosing of the conjugate 145 was well-tolerated. In terms of
general observations, weight gain was normal throughout the
study, and complete blood counts (CBCs) and blood chemistry
were also found to be normal with respect to predose baseline
estimates. Using the cynomolgus monkey whole-blood IL-17
pharmacodynamic assay, suppression of T cell inflammation was
achieved during the 4 week dosing period. In terms of repeat
drug exposure, the predicted and observed serum drug trough
levels correlated well over the dosing period.
The potential toxicity and the toxicokinetics of 145 were
evaluated in male cynomolgus monkeys (n = 3 per dose group)
after subcutaneous administration of 0.7 mg/kg every third
day (4 doses total) or 2 weekly doses at 0.1, 0.5, or 2.0 mg/kg
(2 doses total).39
There were no 145-related effects on any
parameters evaluated. Specifically, there were no PEG-associated
vacuoles observed in renal tubules or tissue macrophages by
light microscopy. On the basis of the absence of adverse
toxicity, the no-observed-adverse-effect level (NOAEL) in this
study was 2 mg/kg, which correlated with a mean AUC0−168h of
584 000 ng·h/mL.
■ CONCLUSIONS
The diverse array of potent biological activities and inherent
metabolic stability of toxin peptides make this class of mole-
cules an attractive starting point for drug discovery of ion
channel modulators. We have demonstrated the effectiveness of
the multi attribute positional scan (MAPS) analoging method-
ology to identify potent and subtype-selective analogues of
the ShK peptide toxin. By scanning the peptide sequence with
not only the traditional Ala residue but also representative
basic, acidic, and hydrophobic residues and screening the
resulting >130 analogues via high-throughput electrophysiol-
ogy, [Lys16]ShK emerged as a potent antagonist of Kv1.3 with
improved selectivity over Kv1.1.39
Combination with N-terminal
conjugation of a 20 kDa poly(ethylene glycol) polymer resulted
in an unexpected synergistic increase in Kv1.3 versus Kv1.1
selectivity to 1000-fold, with retention of picomolar potency in
the whole-blood T cell assay and prolongation of the half-life
in vivo. A clean selectivity profile against a panel of ion channels
and good plasma stability made 20 kDa-PEG-[Lys16]ShK
suitable for rodent and primate PD studies. Compound 145 was
efficacious in the rat adoptive transfer-experimental autoimmune
encephalitis (AT-EAE) model of multiple sclerosis. The pharma-
cokinetic profile of this compound was suitable for weekly
dosing in cynomologous monkeys, and it showed suppression of
T cell-mediated inflammation during a 1 month repeat-dosing
experiment without adverse side effects. Through prolonged
blockade of Kv1.3 in vivo, 145 or related analogues may allow
further interrogation of this target for the treatment of auto-
immune disease in higher species. In view of these results and
Figure 7. Pharmacokinetic profiles of a single subcutaneous dose
(mouse and rat, dose = 2 mg/kg; beagle and cyno, dose = 0.5 mg/kg)
of 145 (with target coverage estimates based on whole-blood assay
results: cynomolgus monkey IL-17 IC50 = 0.09 and human IL-2 IC50 =
0.092 nM).
Figure 8. Comparison of the in vivo efficacy of 20 kDa-PEG-ShK (144)
and the Kv1.3-selective inhibitor 145 in blocking autoimmune
encephalomyelitis in a rat AT-EAE model. The PEGylated ShK or
[Lys16]ShK conjugates were delivered subcutaneously (SC) daily from
days −1 to 7. The rat CD4+
myelin-specific effector memory T cells line,
PAS, was delivered by intravenous injection on day 0. The rats were
monitored for signs of EAE once or twice per day in a blinded fashion,
and 5 or 6 female Lewis rats were used per treatment group. Clinical
EAE scores were as follows: 0 = no signs, 0.5 = distal limp tail, 1.0 =
limp tail, 2.0 = mild paraparesis, ataxia, 3.0 = moderate paraparesis, 3.5 =
one hind leg paralysis, 4.0 = complete hind leg paralysis, 5.0 = complete
hind leg paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund
state or death. Rats reaching a score of 5.5 were euthanized. Error bars
represent the standard error of the mean.
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![other applications in our laboratory, it appears the MAPS
analoging strategy will be useful to multiple classes of toxin
peptides for ion channel targets as well as small synthetically
accessible protein scaffolds for other types of targets.
■ EXPERIMENTAL METHODS
Peptide Preparation. Peptide Synthesis. Nα
-Fmoc, side chain-
protected amino acids and H-Cys(Trt)-2Cl-Trt resin were purchased
from Novabiochem, Bachem, or Sigma-Aldrich. The following side
chain protection strategy was employed: Asp(OtBu), Arg(Pbf),
Cys(Trt), Glu(OtBu), His(Trt), Lys(Nε
-Boc), Ser(OtBu), Thr(OtBu),
and Tyr(OtBu). ShK or other toxin peptide analogue amino acid
sequences were synthesized in a stepwise manner on a CS Bio 336
peptide synthesizer by Fmoc-SPPS using DIC/HOBt coupling chemistry
at 0.2 mmol scale using H-Cys(Trt)-2Cl-Trt resin (0.2 mmol,
0.32 mmol/g loading). For each coupling cycle, 1 mmol Nα
-Fmoc-
amino acid was dissolved in 2.5 mL of 0.4 M 1-hydroxybenzotriazole
(HOBt) in N,N-dimethylformamide (DMF). To the solution was added
1.0 mL of 1.0 M N,N′-diisopropylcarbodiimide (DIC) in DMF. The
solution was agitated with nitrogen bubbling for 15 min to accomplish
preactivation and then added to the resin. The mixture was shaken
for 2 h. The resin was filtered and washed three times with DMF,
twice with dichloromethane (DCM), and three times with DMF.
Fmoc removals were carried out by treatment with 20% piperdine in
DMF (5 mL, 2 × 15 min). The first 23 residues were single-coupled
through repetition of the Fmoc-amino acid coupling and Fmoc removal
steps described above. The remaining residues were double-coupled
by performing the coupling step twice before proceeding with Fmoc
removal.
Following synthesis, the resin was drained, washed sequentially with
DCM, DMF, and DCM, and then dried in vacuo. The peptide-resin
was transferred to a 250 mL plastic round-bottomed flask. The peptide
was deprotected and cleaved from the resin by treatment with
triisopropylsilane (1.5 mL), 3,6-dioxa-1,8-octane-dithiol (DODT, 1.5 mL),
water (1.5 mL), trifluoroacetic acid (TFA, 20 mL), and a stir bar, and the
mixture was stirred for 3 h. The mixture was filtered through a 150 mL
sintered glass funnel into a 250 mL plastic round-bottomed flask, and the
filtrate was concentrated in vacuo. The crude peptide was precipitated
by dropwise addition to cold diethyl ether in a 50 mL centrifuge tube,
collected by centrifugation, and dried under vacuum.
Peptide Folding. The dry crude linear peptide (about 600 mg from
0.2 mmol), for example, [Lys16]ShK peptide, was dissolved in 16 mL
of acetic acid, 64 mL of water, and 40 mL of acetonitrile. The mixture
was stirred rapidly for 15 min to complete dissolution. The peptide
solution was added to a 2 L plastic bottle that contained 1700 mL of
water and a large stir bar. To the diluted peptide solution was added
20 mL of concentrated ammonium hydroxide to increase the pH of
the solution to 9.5. The pH was adjusted with small amounts of acetic
acid or NH4OH as necessary. The solution was stirred at 80 rpm
overnight and monitored by LC-MS. Folding was usually judged to be
complete in 24 to 48 h, and the solution was quenched by the addition
Figure 9. Twelve week pharmacology study in cynomolgus monkeys. Weekly dosing of cynomolgus monkeys with 145 provided sustained
suppression of T cell responses, as measured using the ex vivo cynomolgus monkey whole-blood PD assay of inflammation that measured production
of IL-4 (A) and IL-17 (B). Arrows indicate the weekly doses. Each line represents an individual test subject. (C) Predicted versus measured serum
concentrations of 145 in cynomolgus monkeys after weekly subcutaneous (SC) dosing (0.5 mg/kg, n = 6). The measured serum trough levels after
weekly dosing (open squares), matched closely those predicted based on repeat-dose modeling of the single-dose pharmacokinetic data (solid line).
(D) Weight gain for each animal during the 12 week cynomolgus monkey pharmacology study; arrows on x-axis indicate SC dosing with 145.
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![of acetic acid and TFA (pH 2.5). The aqueous solution was filtered
(0.45 μm cellulose membrane).
Reversed-Phase HPLC Purification and Analysis and Mass
Spectrometry. Reversed-phase high-performance liquid chromatog-
raphy (RP-HPLC) was performed on a preparative (C18, 10 μm,
2.2 cm × 25 cm) column. Chromatographic separations were achieved
using linear gradients of buffer B in A (A = 0.1% aqueous TFA; B =
90% aq. acetonitrile containing 0.09% TFA), typically 5−65% over
90 min at 20 mL/min for preparative separations. Preparative HPLC
fractions were characterized by ESMS and photodiode array (PDA)
HPLC, combined, and lyophilized. Final analysis (Phenomenex
Synergi MAX-RP 2.5 μm, 100 Å, 50 × 2.0 mm column eluted with
a 10 to 50% B over 10 min gradient [A: water and B: acetonitrile, 0.1%
TFA in each] at a 0.650 mL/min flow rate monitoring UV absorbance
at 220 nm) was performed for each peptide sample using an Agilent
1290 LC-MS. Peptides with >95% purity and correct (m/z) ratio
were screened. (See Supporting Information Table S8 for LC-MS
characterization of ShK and peptide analogues.)
PEGylation, Purification, and Analysis. Peptide, for example,
[Lys16]ShK, was selectively PEGylated by reductive alkylation at its
N-terminus using activated linear or branched PEG. Conjugation was
performed at 2 mg/mL in 50 mM NaH2PO4, pH 4.5, reaction buffer
containing 20 mM sodium cyanoborohydride and a 2 molar excess
of 20 kDa monomethoxy-PEG-aldehyde (NOF, Japan). Conjugation
reactions were stirred for approximately 5 h at room temperature, and
their progress was monitored by RP-HPLC. Completed reactions were
quenched by 4-fold dilution with 20 mM NaOAc, pH 4, and chilled to
4 °C. The PEG-peptides were then purified chromatographically at
40 °C using SP Sepharose HP columns (GE Healthcare, Piscataway,
NJ) and eluted with linear 0−1 M NaCl gradients in 20 mM NaOAc,
pH 4.0. Eluted peak fractions were analyzed by SDS-PAGE and
RP-HPLC and pooling determined by purity >97%. Principle
contaminants observed were di-PEGylated toxin peptide analogue.
Selected pools were concentrated to 2−5 mg/mL by centrifugal
filtration against 3 kDa MWCO membranes and dialyzed into 10 mM
NaOAc, pH 4, with 5% sorbitol. Dialyzed pools were then sterile
filtered through 0.2 μm filters, and purity was determined to be >97%
by SDS-PAGE and RP-HPLC (see Supporting Information Figures S2
and S3). Reverse-phase HPLC was performed on an Agilent 1100
model HPLC running a Zorbax 5 μm 300SB-C8 4.6 × 50 mm column
(Agilent) in 0.1% TFA/H2O at 1 mL/min, and the column tem-
perature was maintained at 40 °C. Samples of PEG-peptide (20 μg)
were injected and eluted in a linear 6−60% gradient while monitoring
at a wavelength of 215 nm.
Electrophysiology. Cell Lines Expressing Kv1.1−Kv1.7. CHO K1
cells were stably transfected with human Kv1.3 or, for counterscreens,
with hKv1.4, hKv1.6, or hKv1.7; HEK293 cells were stably expressing
human Kv1.3 or human Kv1.1. Cell lines were from Amgen or BioFocus
DPI (A Galapagos Company). CHO K1 cells stably expressing hKv1.2,
for counterscreens, were purchased from Millipore (cat. no. CYL3015).
Whole-Cell Patch Clamp Electrophysiology. Whole-cell currents
were recorded at room temperature using MultiClamp 700B amplifier
from Molecular Devices Corp. (Sunnyvale, CA), with 3−5 MΩ pipettes
pulled from borosilicate glass (World Precision Instruments, Inc.).
During data acquisition, capacitive currents were canceled by analogue
subtraction, no series resistance compensation was used, and all
currents were filtered at 2 kHz. The cells were bathed in an extracellular
solution containing 1.8 mM CaCl2, 5 mM KCl, 135 mM NaCl, 5 mM
glucose, 10 mM HEPES, pH 7.4, 290−300 mOsm. The internal
solution contained 90 mM KCl, 40 mM KF, 10 mM NaCl, 1 mM
MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2, 290−300 mOsm. The
currents were evoked by applying depolarizing voltage steps from
−80 to +30 mV every 30 s (Kv1.3) or 10 s (Kv1.1) for 200 ms intervals
at a holding potential of −80 mV. To determine IC50, 5−6 peptide or
peptide conjugate concentrations at 1:3 dilutions were made in
extracellular solution with 0.1% BSA and delivered locally to cells with
Rapid Solution Changer RSc-160 (BioLogic Science Instruments).
Currents were achieved to steady state for each concentration. Data
analysis was performed using pCLAMP (version 9.2) and OriginPro
(version 7), and peak currents before and after each test article
application were used to calculate the percentage of current inhibition
at each concentration.
PatchXpress Planar Patch Clamp Electrophysiology. Cells were
bathed in an extracellular solution containing 1.8 mM CaCl2, 5 mM
KCl, 135 mM NaCl, 5 mM glucose, 10 mM HEPES, pH 7.4, 290−
300 mOsm. The internal solution contained 90 mM KCl, 40 mM KF,
10 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2,
290−300 mOsm. Usually, 5 peptide or peptide conjugate concen-
trations at 1:3 dilutions were made to determine the IC50s. The
peptide or peptide conjugates were prepared in extracellular solution
containing 0.1% BSA. Dendrotoxin-k and Margatoxin were purchased
from Alomone Laboratories Ltd. (Jerusalem, Israel); ShK toxin was
purchased from Bachem Bioscience, Inc. (King of Prussia, PA); 4-AP
was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Currents
were recorded at room temperature using a PatchXpress 7000A electro-
physiology system from Molecular Devices Corp. (Sunnyvale, CA).
The voltage protocols and recording conditions are shown in the
Supporting Information Table S1. An extracellular solution with 0.1%
BSA was applied first to obtain 100% percent of control (POC), which
was then followed by 5 different concentrations of 1:3 peptide or
peptide conjugate dilutions for every 400 ms incubation time. At the
end, an excess of a specific benchmark ion channel inhibitor was added
to define full or 100% blockage. The residual current present after
addition of benchmark inhibitor was used in some cases for calculation
of zero percent of control. Each individual set of traces or trial were
visually inspected and either accepted or rejected. The general criteria
for acceptance were as follows: (1) baseline current must be stable, (2)
initial peak current must be >300 pA, (3) intitial Rm and final Rm must
>300 Ohm, and (4) peak current must achieve steady state prior to first
compound addition. The POC was calculated from the average peak
current of the last 5 sweeps before the next concentration of compound
was added and exported to Excel for IC50 calculation.
IonWorks Quattro High-Throughput Population Patch Clamp
Electrophysiology. Electrophysiology was performed on CHO cells
stably expressing hKv1.3 and HEK293 cells stably expressing hKv1.1.
The procedure for preparation of the assay plate containing ShK
analogues and conjugates for IWQ electrophysiology was as follows:
all analogues were dissolved in extracellular buffer (PBS, with 0.9 mM
Ca2+
and 0.5 mM Mg2+
) with 0.3% BSA and dispensed in row H of
96-well polypropylene plates at a concentration of 100 nM from
columns 1−10. Columns 11 and 12 were reserved for negative and
positive controls. Serial dilutions at a 1:3 ratio were then made to row
A. IonWorks Quattro (IWQ) electrophysiology and data analysis were
accomplished as follows: resuspended cells (in extracellular buffer), the
assay plate, a population patch clamp (PPC) PatchPlate as well as
appropriate intracellular (90 mM potassium gluconate, 20 mM KF,
2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35)
and extracellular buffers were positioned on IonWorks Quattro. When
the analogues were added to patch plates, they were further diluted
3-fold from the assay plate to achieve a final test concentration range
from 33.3 nM to 15 pM with 0.1% BSA. Electrophysiology recordings
were made from the CHO-Kv1.3 and HEK-Kv1.1 cells using an
amphotericin-based perforated patch clamp method. Using the voltage
clamp circuitry of the IonWorks Quattro, cells were held at a membrane
potential of −80 mV, and voltage-activated K+
currents were evoked by
stepping the membrane potential to +30 mV for 400 ms. K+
currents
were evoked under control conditions, i.e., in the absence of inhibitor
at the beginning of the experiment and after 10 min incubation in the
presence of the analogues and controls. The mean K+
current amplitude
was measured between 430 and 440 ms, and the data were exported
to a Microsoft Excel spreadsheet. The amplitude of the K+
current in the
presence of each concentration of the analogues and controls was
expressed as a percentage of the K+
current of the precompound current
amplitude in the same well. When these percent of control values were
plotted as a function of concentration, the IC50 value for each compound
could be calculated using the dose−response fit model 201 in Excel fit
program.
Measuring Bioactivity in Whole Blood. Ex Vivo Assay to
Examine Impact of Toxin Peptide Analogue Kv1.3 Inhibitors on
Secretion of Human IL-2 and IFN-γ. The potency of ShK analogues
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![and conjugates in blocking T cell inflammation in human whole blood
was examined using an ex vivo assay that has been described earlier.40
In brief, 50% human whole blood is stimulated with thapsigargin to
induce store depletion, calcium mobilization, and cytokine secretion.
To assess the potency of molecules in blocking T cell cytokine secretion,
various concentrations of Kv1.3 blocking peptides and peptide
conjugates were preincubated with the human whole-blood sample for
30−60 min prior to addition of the thapsigargin stimulus. After 48 h at
37 °C and 5% CO2, conditioned medium was collected and the level of
cytokine secretion was determined using a four-spot electrochemillumi-
nescent immunoassay from MesoScale Discovery. Using the thapsi-
gargin stimulus, the cytokines IL-2 and IFN-γ were secreted robustly
from blood isolated from multiple donors. The IL-2 and IFN-γ
produced in human whole blood following thapsigargin stimulation were
produced from T cells, as revealed by intracellular cytokine staining and
fluorescence-activated cell sorting (FACS) analysis.
Pharmacokinetic and Pharmacodynamic Studies. Detection
Antibodies to ShK. Rabbit polyclonal and mouse monoclonal anti-
bodies to ShK were generated by immunization of animals with an
Fc-ShK peptibody conjugate.39
Anti-ShK specific polyclonal antibodies
were affinity-purified from antisera to isolate only those antibodies
specific for the ShK portion of the conjugate. Following fusion and
screening, hybridomas specific for ShK were selected and isolated.
Mouse anti-ShK specific monoclonal antibodies were purified from the
conditioned media of the clones. By ELISA analysis, purified anti-ShK
polyclonal and monoclonal antibodies reacted only to the ShK peptide
alone and did not cross-react with Fc.
Pharmacokinetic (PK) Studies 20 kDa-PEG-ShK Peptide Con-
jugates in Mice, Rats, Dogs, and Monkeys. Single subcutaneous
doses were delivered to animals, and serum was collected at various time
points after injection. Studies in rats, dogs (beagles), and cynomolgus
monkeys involved two to three animals per dose group, with blood and
serum collection occurring at various time points over the course of the
study. Male Sprague−Dawley (SD) rats (about 0.3 kg), male beagles
(about 10 kg), and male cynomolgus monkeys (about 4 kg) were used
in the studies described herein (n = 3 animals per dose group).
Approximately 5 male CD-1 mice were used per dose and time point in
our mouse pharmacokinetic studies. Serum samples were stored frozen
at −80 °C until analysis in an enzyme-linked immunosorbent assay
(ELISA).
The following is a brief description of the ELISA protocol for
detecting serum levels of conjugates 144 and 145 as well as the ShK
and 122 peptides alone. Streptavidin microtiter plates were coated
with 250 ng/mL biotinylated-anti-ShK mouse monoclonal antibody
(mAb2.10, Amgen) in I block buffer [per liter: 1000 mL 1× PBS
without CaCl2, MgCl2, 5 mL of Tween 20 (Thermo Scientific), 2 g of
I block reagent (Tropix)] at 4 °C and incubated overnight without
shaking. Plates were washed three times with KPL wash buffer
(Kirkegaard & Perry Laboratories). Standards (STD), quality controls
(QC), and sample dilutions were prepared with 100% pooled sera and
then diluted 1:5 (pretreatment) in I block buffer. Pretreated STDs,
QCs, and samples were added to the washed plate and incubated at
room temperature for 2 h. (Serial dilutions of STDs and QCs were
prepared in 100% pooled sera. Samples needing dilution were also
prepared with 100% pooled sera. The pretreatment was done to STDs,
QCs, and samples to minimize the matrix effect.) Plates were washed
three times with KPL wash buffer. A HRP-labeled rabbit anti-ShK
polyclonal antibody at 250 ng/mL in I block buffer was added, and
plates were incubated at room temperature for 1 h with shaking. Plates
were again washed three times with KPL wash buffer, and the Femto
(Thermo Scientific) substrate was added. The plate was read with a
Lmax II 384 (Molecular Devices) luminometer.
Adoptive-Transfer EAE Model of Efficacy. An adoptive transfer
experimental autoimmune encephalomyelitis (AT-EAE) model of
multiple sclerosis in rats described earlier32
was used to examine the
activity in vivo of our Kv1.3-selective 145 analogue and to compare its
efficacy to that of the less selective molecule 144. The encephalomyelo-
genic CD4+
rat T cell line, PAS, specific for myelin-basic protein (MBP),
was kindly provided by Dr. Evelyne Beraud. The maintenance of these
cells in vitro and their use in the AT-EAE model has been described.32
PAS T cells were maintained in vitro by alternating rounds of antigen
stimulation or activation with MBP and irradiated thymocytes (2 days)
and propagation with T cell growth factors (5 days). Activation of
PAS T cells (3 × 105
/mL) involved incubating the cells for 2 days with
10 μg/mL MBP and 15 × 106
/mL syngeneic irradiated (3500 rad)
thymocytes. On day 2 after in vitro activation, 10−15 × 106
viable PAS
T cells were IV injected into 6−12 week old female Lewis rats (Charles
River Laboratories) by tail. Daily subcutaneous injections of vehicle
(2% Lewis rat serum in PBS), 145, 144, or ShK were given from either
days −1 to 7 or days −1 to 3, where day −1 represents 1 day prior to
injection of PAS T cells (day 0 in Figure 8). Serum was collected by
retro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end of
the study) for analysis of levels of inhibitor. Rats were weighed on days
−1 and 4−8. Animals were scored in a blinded fashion once a day from
the day of cell transfer (day 0) to day 3 and twice a day from days 4 to 8.
Clinical signs were evaluated as the total score of the degree of paresis
of each limb and tail. Clinical scoring (EAE score in Figure 8) was as
follows: 0 = no signs, 0.5 = distal limp tail, 1.0 = limp tail, 2.0 = mild
paraparesis, ataxia, 3.0 = moderate paraparesis, 3.5 = one hind leg
paralysis, 4.0 = complete hind leg paralysis, 5.0 = complete hind leg
paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund state or
death. Rats reaching a score of 5.5 were euthanized.
Pharmacology Study in Cynomolgus Monkey. A repeat-dose
pharmacology study was designed and implemented in order to
investigate the long-term effects of 145 in nonhuman primates. Prior
to initiating the study, 6−10 male cynomolgus monkeys were profiled
for a period of 3−10 weeks to allow for assessment of the end points’
stability over time and selection of 6 cynomolgus monkeys for the
study. The end points measured included complete blood counts
(CBCs), blood chemistry, FACS analysis of lymphocyte subsets, and
the ex vivo whole-blood PD assay measuring cytokine response and
target coverage. Subsets analyzed by FACS included lymphocytes,
CD4+
, CD4+
naïve, CD4+
TCM, CD4+
TEM, CD4+
CD28−
CD95−
,
CD8+
, CD8+
naïve, CD8+
TCM, CD8+
TEM, CD8+
CD28−
CD95−
,
B cells, NK cells, and NKT cells. Monkeys with the highest level of
CD4+
effector memory T cells were chosen. The design of the 12 week
cynomolgus pharmacology study is illustrated in Supporting
Information Table S5. Male Chinese cynomolgus monkeys that were
used in this study were naïve (no earlier exposure to drugs). Care was
taken to avoid undue stress. All injections and blood draws were done
by arm-pull, with the monkeys voluntarily presenting their arm for
a grape incentive. The study involved baseline measures for 2 weeks
(3 predose samples), 1 month of Kv1.3 block (qw dosing of 145), and
6 weeks follow-up analysis.
Ex Vivo Cynomolgus Monkey Whole-Blood Assay To Measure
the Potency of 145 and Its Level of Pharmacodynamic Coverage in
Vivo. The potency and level of coverage of cynomolgus monkey T cell
responses were determined with an ex vivo whole-blood assay
measuring thapsigargin-induced IL-4, IL-5, and IL-17. To determine
potency of peptides and peptide conjugates, cynomolgus whole blood
was obtained from healthy, naïve male monkeys in a heparin vacutainer.
DMEM complete media was Iscoves DMEM (with L-glutamine and
25 mM Hepes buffer) containing 0.1% human albumin (Gemini
Bioproducts, no. 800-120), 55 μM 2-mercaptoethanol (Gibco), and
1× Pen−Strep−Gln (PSG, Gibco, cat. no. 10378-016). Thapsigargin
was obtained from Alomone Laboratories (Jerusalem, Israel). A 10 mM
stock solution of thapsigargin in 100% DMSO was diluted with DMEM
complete media to a 40 μM, 4× solution to provide the 4× thapsigargin
stimulus for calcium mobilization. The Kv1.3 inhibitor peptide
ShK (Stichodacytla helianthus toxin, cat. no. H2358) and the BKCa1
inhibitor peptide IbTx (Iberiotoxin, cat. no. H9940) were purchased
from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide
DTX-k (Dendrotoxin-K) was from Alomone Laboratories (Israel).
The calcineurin inhibitor cyclosporin A (CsA) is available commercially
from a variety of vendors. Whereas the BKCa inhibitor IbTx and the
Kv1.1 inhibitor DTX-k do not inhibit the cytokine response, the Kv1.3
inhibitor ShK and the calcineurin inhibitor CsA inhibit the cytokine
response and are used routinely as standards or positive controls. Ten
3-fold serial dilutions of standards, ShK analogues, or ShK conjugates
were prepared in DMEM complete media at 4× final concentration,
Journal of Medicinal Chemistry Article
DOI: 10.1021/acs.jmedchem.5b00495
J. Med. Chem. 2015, 58, 6784−6802
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![and 50 μL of each was added to wells of a 96-well Falcon 3075 flat-
bottomed microtiter plate. Whereas columns 1−5 and 7−11 of the
microtiter plate contained inhibitors (each row with a separate inhibitor
dilution series), 50 μL of DMEM complete media alone was added to
the 8 wells in column 6 and 100 μL of DMEM complete media alone
was added to the 8 wells in column 12. To initiate the experiment,
100 μL of whole blood was added to each well of the microtiter plate.
The plate was then incubated at 37 °C, 5% CO2 for 1 h. After 1 h, the
plate was removed, and 50 μL/well of the 4× thapsigargin stimulus
(40 μM) was added to all wells of the plate, except the 8 wells in
column 12. The plates were placed back at 37 °C, 5% CO2 for 48 h.
To determine the amount of IL-4, IL-5, and IL-17 secreted in whole
blood, 100 μL of the supernatant (conditioned media) from each well
of the 96-well plate was transferred to a storage plate. For Meso Scale
Discovery (MSD) electrochemiluminesence analysis of cytokine pro-
duction, the supernatants (conditioned media) were tested using MSD
multi-spot custom coated plates (Meso Scale Discovery, Gaithersburg,
MD). The working electrodes on these plates were coated with seven
capture antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFα, hIFNγ, and
hIL-17) in advance. After blocking plates with MSD human serum
cytokine assay diluent and then washing with PBS containing 0.05%
BSA, 25 μL/well of conditioned medium was added to wells of the
MSD plate. The plates were covered and placed on a shaking platform
for 1 h. Next, 25 μL of a cocktail of detection antibodies in MSD
antibody diluent was added to each well. The cocktail contained seven
detection antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFα, hIFNγ, and
hIL-17) at 1 μg/mL each. The plates were covered and placed on a
shaking platform overnight (in the dark). The next morning, the plates
were washed three times with PBS buffer. 150 μL of 2× MSD read
buffer T was added to wells of the plate before reading on the MSD
sector imager. Since the 8 wells in column 6 of each plate received only
the thapsigargin stimulus and no inhibitor, the average MSD response
here was used to calculate the high value for a plate. The calculated low
value for the plate was derived from the average MSD response from
the 8 wells in column 12, which contained no thapsigargin stimulus and
no inhibitor. Percent of control (POC) is a measure of the response
relative to the unstimulated versus stimulated controls, where 100 POC
is equivalent to the average response of thapsigargin stimulus alone
or the high value. Therefore, 100 POC represents 0% inhibition of
the response. In contrast, 0 POC represents 100% inhibition of the
response and would be equivalent to the response where no stimulus is
given or the low value. To calculate percent of control (POC), the
following formula is used: [(MSD response of well) − (low)]/
[(high) − (low)] × 100. The potency of the molecules in whole
blood was calculated after curve fitting from the inhibition curve
(IC), and IC50 was derived using standard curve fitting software. The
experimental procedure for adaption of the above method to an ex vivo
cynomolgus monkey pharmacodynamic (PD) assay to measure the
level of T cell Kv1.3 coverage in vivo following the dosing of animals is
included in the Supporting Information.
Repeat-Dose Toxicology Study. Male cynomolgus monkeys
(Macaca fascicularis; 2 to 4 years and 2 to 5 kg) were cared for in
accordance with the Guide for the Care and Use of Laboratory
Animals.41
Animals were individually house in stainless steel cages,
except when commingled for environmental enrichment, at an indoor
American Association for Accreditation of Laboratory Animal Care
(AAALAC) internationally accredited facility in species-specific housing.
The research protocol was approved by the Institutional Animal Care
and Use Committee. Animals were fed a certified pelleted primate diet
(no. 2055C, Harlan Laboratories Inc., Indianapolis, IN) daily in amounts
appropriate for the age and size of the animals and had ad libitum access
to water via automatic watering system. Animals were maintained on
a 12/12 h light/dark cycle in rooms at 18−26 °C and 30−70% humidity
and had access to enrichment opportunities, including cage-enrichment
devices and commingling.
Animals were acclimated for at least 1 week and then randomized
to treatment groups to achieve body weight balance with respect to
groups. Fifteen animals were placed into 1 of 5 groups (n = 3/dose
group) receiving vehicle (10 mM NaOAc, 5% sorbitol, pH 4.0) or 145.
Doses of 0.7 mg/kg every third day for 2 weeks (4 doses total) or
0.1, 0.5, or 2.0 mg/kg weekly for 2 weeks (2 doses total) were
administered via subcutaneous injection. After 2 weeks, all animals were
anesthetized with sodium pentobarbital, exsanguinated, and necropsied.
The following study parameters were evaluated: clinical observations,
body weights, food consumption, qualitative and quantitative electro-
cardiograms (ECG), toxicokinetics, routine clinical pathology (complete
blood count, coagulation, and clinical chemistry), urinalysis, a panel of
urinary biomarkers of renal injury, organ weights, macroscopic observa-
tions, and light microscopic observations of a full set of tissues.
Crystallization and Structure Determination of [Lys16]ShK.
Crystallization of racemic [Lys16]ShK was performed by mixing equal
amounts (by weight) of lyophilized D-[Lys16]ShK and L-[Lys16]ShK
in water at 50 mg/mL total concentration (25 mg/mL of
D-[Lys16]ShK and 25 mg/mL of L-[Lys16]ShK). Hanging drop cry-
stallization screens were set up using JCSG core suites 1−4 (Qiagen)
at room temperature. Crystals appeared in several conditions within
a week. One condition, 0.1 M sodium acetate, pH 4.5, 2−2.5 M
ammonium sulfate, produced diffraction-quality crystals. For data
collection, crystals were cryoprotected in mother liquor containing 20%
(v/v) glycerol and flash cooled to 100 K. Diffraction data were collected
at the Advanced Light Source, beamline 5.0.2 (Lawrence Berkeley
National Laboratory, Berkeley, CA), at 100 K. Data were processed
and scaled using HKL2000. Attempts to phase the data by molecular
replacement using a NMR structure of ShK (PDB: 1ROO)19a
were
unsuccessful, so material labeled with selenomethionine (SeMet) at
position 21 was prepared and used for phasing. Selenomethionine crystals
were grown by mixing native D-[Lys16]ShK with L-[Lys16,SeMet21]ShK.
The selenomethionine-labeled crystals grew under identical conditions
as those for crystals of the unlabeled peptides. Data sets from seleno-
methionine-containing crystals were collected at three wavelengths:
peak (0.9792 λ), inflection (0.9794 λ), and remote (0.9824 λ).42
The single selenium atom position was determined with Crank in
CCP4.43
Crank used SHELX C/D44
for substructure search, SHELX E
for substructure refinement, and Solomon45
for density modification.
This generated a high-quality map, and a polyalanine model was created
using Coot.46
This model was used for molecular replacement into the
native data for final refinement. Phaser47
was used for molecular
replacement. The space group is R3̅ with a = 60.78 Å, b = 60.78 Å,
c = 43.59 Å, and one molecule in the asymmetric unit. After several
rounds of model building using Coot and refinement with REFMAC5
in the CCP4 suite, the R factor and Rfree values converged to 20.4 and
22.6%, respectively. Stereochemical analysis of the refined model was
performed using Coot validation tools and MolProbity.48
Structural
figures were produced using PyMOL (http://www.pymol.org/).
The coordinates and structure factors have been deposited in the
Brookhaven Protein Data Bank (accession no. 4Z7P). X-ray
crystallography data collection and refinement statistics are included
in Supporting Information Table S9.
Molecular Docking of ShK to Kv1.3 Homology Model. The
molecular modeling program MOE49
was used to construct a homo-
logy model of the human Kv1.3 channel using the Kv1.2-Kv2.1 paddle
chimera channel structure18b
(2R9R.pdb) as a template. The
[Lys16]ShK crystal structure was docked to the channel homology
model using ZDOCK, as implemented in the Discovery Studio 4.1
modeling package.50,51
To simplify the docking, the voltage-sensing
domains were excluded from the calculation, and the pore was used as
the receptor (residues 381−491). To limit docked poses to the extra-
cellular vestibule, receptor residues 381−415, 437−443, and 459−491
were excluded from the peptide−protein interface. An angular step size of
6° and distance cutoff of 5 Å were used, resulting in 54 000 potential
poses. The resulting poses were filtered to ensure that residues presumed
to be important for binding based on the MAPS analoging ([Lys16]ShK
residues 7, 11, 20, 21, 22, 23, and 27) were within 4 Å of any residue on
the channel. Additionally, poses were retained only if an atom from the
[Lys16]ShK toxin was within 4 Å of the key pore-forming Gly residues
446 and 448 on the Kv1.3 channel. A total of 777 poses passed these
criteria and were advanced to further minimization and optimization
using RDOCK.52
The top poses were visualized with respect to scanning
and selectivity data, including the proximity of Lys16 to residue
differences between the Kv1.1 and Kv1.3 channels.
Journal of Medicinal Chemistry Article
DOI: 10.1021/acs.jmedchem.5b00495
J. Med. Chem. 2015, 58, 6784−6802
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![■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00495.
Characterization of ShK peptide analogues; character-
ization and additional pharmacokinetic and pharmaco-
logical data for 145 (PDF).
■ AUTHOR INFORMATION
Corresponding Authors
*(J.K.S.) Phone: 1-805-447-3695. E-mail: jsulliva@amgen.com.
*(L.P.M.) Phone: 1-805-447-9397. E-mail: lesm@amgen.com.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We gratefully thank Ankita Shah, Jason Long, Stephanie
Diamond, Ryan Holder, and Jingwen Zhang for peptide
synthesis support and Lei Jia and Kaustav Biswas for data
visualization. We wish to thank Dr. Evelyn Beraud (Laboratoire
d’Immunologie, Faculte de Medecine, Marseille, France) and
Dr. Christine Beeton (Baylor College of Medicine) for
providing the rat PAS T cell line. The Advanced Light Source
is supported by the U.S. Department of Energy under contract
no. DE-AC02-05CH11231.
■ ABBREVIATIONS USED
AEEA, 2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid; Boc,
tert-butoxycarbonyl; Dap, L-2,3-diaminopropionic acid; Fmoc,
Nα
-9-fluorenylmethoxycarbonyl; 1-Nal, L-1-naphthylalanine;
Nle, L-norleucine; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-
5-sulfonyl; tBu, tert-butyl; Trt, trityl
■ REFERENCES
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Journal of Medicinal Chemistry Article
DOI: 10.1021/acs.jmedchem.5b00495
J. Med. Chem. 2015, 58, 6784−6802
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![CHROMB17026[1]](https://cdn.slidesharecdn.com/ss_thumbnails/b0d6b51c-4b0a-4d84-b2c7-32fb051a4561-160507012648-thumbnail.jpg?width=640&height=640&fit=bounds)
acs.jmedchem.5b00495
- 1. Pharmaceutical Optimization ofPeptide Toxins for Ion Channel Targets: Potent, Selective, and Long-Lived Antagonists of Kv1.3 Justin K. Murray,† Yi-Xin Qian,† Benxian Liu,‡ Robin Elliott,‡ Jennifer Aral,† Cynthia Park,† Xuxia Zhang,‡ Michael Stenkilsson,§ Kevin Salyers,§ Mark Rose,§ Hongyan Li,§ Steven Yu,§ Kristin L. Andrews,⊥ Anne Colombero,‡ Jonathan Werner,∥ Kevin Gaida,‡ E. Allen Sickmier,† Peter Miu,† Andrea Itano,‡ Joseph McGivern,† Colin V. Gegg,† John K. Sullivan,*,‡ and Les P. Miranda*,† † Therapeutic Discovery, ‡ Inflammation Research, § Pharmacokinetics & Drug Metabolism, and ∥ Comparative Biology and Safety Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States ⊥ Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States *S Supporting Information ABSTRACT: To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization techno- logies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole- molecule, brute-force, structure−activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmaco- logical potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys. ■ INTRODUCTION Ion channels are attractive targets for the treatment of human diseases, but the generation of biologic or small molecule drugs that potently, selectively, and safely modulate ion channels remains particularly difficult in contemporary drug discovery.1 Toxin peptides represent a class of potential therapeutics for a range of medical indications mediated by ion channel patho- logy, yet clinical applications have been limited primarily to cases where localized administration has been suitable.2 A major obstacle to more widespread development of toxin peptides has been effective methods for engineering compounds with desirable pharmaceutical properties from novel toxin peptide leads.3 Considerable research has focused on venomous animals that have evolved a diverse repertoire of toxin peptides for predatory or defensive capabilities. In some cases, the intended biological activity of these toxin peptides overlaps fortuitously with similar molecular targets that are of human medicinal relevance.4 ShK (1) is a 35 residue three-disulfide peptide originally isolated from the Caribbean sea anemone Stichodactyla helianthus, whose venom immobilizes prey by targeting ion channels.5,6 ShK inhibits the voltage-gated potassium ion channel Kv1.1,7 which has been shown to be critical for neuronal function in mouse and man. In humans, Kv1.1 shares high amino acid sequence homology with another potassium channel family member, Kv1.3, a possible therapeutic target.8,9 Kv1.3 regulates membrane potential and calcium signaling in human effector memory T cells (TEM), and its expression is increased markedly in activated CD4+ and CD8+ TEM/TEMRA T cell populations.10 Blockade of Kv1.3 inhibits the activation of T cells and secretion of cytokines via the calcineurin pathway by preventing the potassium efflux necessary for sustained influx of calcium.11,12 As such, Kv1.3 represents a target that selectively suppresses activated TEM cells without affecting other lymphoid subsets13 and a promising untapped approach for the treatment of T cell-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, which afflict millions of people.14 Other potential disease indications mediated by Kv1.3 have also been elucidated.15 For toxin peptides to be safe and well-tolerated, undesirable off-target activities and poor pharmacokinetic profiles, particularly rapidly rising and diminishing circulating levels, must be addressed. Herein, we present an effective direct chemical strategy, coupled with high-throughput electrophysiology, for the elimina- tion of unwanted off-target ion channel activity within the ShK Received: March 26, 2015 Published: August 19, 2015 Article pubs.acs.org/jmc © 2015 American Chemical Society 6784 DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802
- 2. toxin peptide concomitantlywith polymeric derivatizatizion that afforded substantial improvement in well-controlled and sustained circulating levels in vivo. Wild-type ShK is composed of 552 atoms. It is often a challenge with molecules of such size and complexity to first identify key toxin peptide−ion channel interactions and, in turn, to discover critical changes that result in improved properties.16,17 The major obstacles in this context are the relative ineffectiveness of de novo design approaches given the lack of high-resolution structural data for ion channels18 and the structural intricacy of peptide toxins.19,20 To date, studies on ShK have focused on modification of only a couple of sites to achieve moderate selectivity for Kv1.3 over Kv1.1, a challenging endeavor given their 90% amino acid sequence homology in the pore region.7,9b,21 We hypothesized that an effective and general route to develop more complete structure−activity relationships (SAR) for peptide toxins, such as ShK, would be the discrete chemical preparation of peptide analogues with substitutions at each site within the molecule with a panel of residues ranging in physicochemical properties. Building upon the traditional alanine scan that individually replaces each amino acid (excluding cysteines) with one of low aliphatic bulk,16,22 the process was repeated throughout the entire molecule with a large aromatic, an acidic, and two different basic amino acid residues. In all, a set of 132 ShK peptide single substitution analogues was chemically synthesized. This is an approach we have termed multi attribute positional scan (MAPS) analoging. While greater diversity has been explored through combinatorial mixtures in short, two-disulfide peptide sequences,23 this work represents, to our knowledge, the most extensive positional scanning of a long, three-disulfide peptide in discrete format for systematic optimization of ion channel selectivity. High-throughput screen- ing of this large set of individually prepared Kv1.3 inhibitory peptides has been facilitated by recent advances in automated electrophysiology methods and platforms, i.e., population patch clamp on the IonWorks Quattro (IWQ) system. From the 132 analogues prepared and tested, only two peptides displayed promising selectivity against Kv1.1 with retention of potent activity at Kv1.3. One of these lead peptide analogues was further modified with a poly(ethylene glycol) polymer (PEG), resulting in a remarkable improvement in selectivity, and studied pharmacologically in a cynomolgus monkey model examining T cell activation. Weekly administration of this newly identified PEGylated ShK peptide analogue suppressed interleukin-17 (IL-17) cytokine secretion from T cells in cynomolgus monkeys and was well-tolerated. ■ RESULTS AND DISCUSSION ShK is a 35 amino acid (Xaa) polypeptide acid with six cysteine residues participating in three disulfide bonds, giving a (Xaa)2- C1-(Xaa)8-C2-(Xaa)4-C3-(Xaa)10-C2-(Xaa)3-C3-(Xaa)2-C1 framework (Figure 1).5 The native ShK peptide has picomolar inhibitory activity at both Kv1.1 and Kv1.3.7 Earlier reports have focused on modification of the N-terminus and/or position 22 of ShK for conferring Kv1.3 selectivity.6 In particular, substitution of L-2,3-diaminopropionic acid (Dap) for the native lysine at position 22 can lead to approximately 20-fold selectivity over Kv1.1;7 however, such a change concomitantly and importantly results in a significant lowering of Kv1.3 binding affinity and an approximately 103 -fold loss in potency for functional inhibition of human T cell activation (vide infra). Alternatively, N-terminal extension of ShK with phosphotyrosine derivatives can give 100-fold Kv1.3 over Kv1.1 selectivity,21 but such molecules nonetheless have short in vivo half-lives with an undesirable pharmacokinetic profile exemplified by a rapid and large shift in peak-to-trough circulating levels.24 In this work, we set out to determine if a systematic analoging approach could be used to efficiently identify new sites within this constrained peptide scaffold that could be modified to significantly improve selectivity for Kv1.3 while retaining potent T cell inhibitory activity. A second key goal of this work was to specifically identify a ShK peptide derivative that, in turn, could be modified with a half-life-extending group to give a pharmacokinetic profile suitable for weekly dosing. Multi Attribute Positional Scan (MAPS) Analoging of ShK. We sought to preferentially disrupt interactions of the ShK peptide with neuronal Kv1.1 in a novel manner but to maintain the desired Kv1.3 inhibitory activity. The absence of reliable in silico methods for predicting peptide compounds with such activity profiles led us to adopt a brute-force analoging approach via direct chemical synthesis. Biological display methodologies could be pursued as an alternative analoging tactic;25 however, such platforms are not currently suited for the identification of functionally active and, more importantly, selective ion channel inhibitors by electrophysiological screening. To describe the approach, at each position within the ShK peptide, amino acid residues representing different physico- chemical attributes (i.e., hydrophobic, basic, and acidic) were individually introduced during direct chemical peptide synthesis. The resultant crude linear peptides were then oxidized to establish the disulfide connectivity and, in turn, purified and tested. An initial set of 132 discrete peptide analogues was synthesized with modification at all positions except the cysteine framework residues (Figure 2). Aside from conventional alanine positional substitutions, which primarily tend to indicate which residues in a given peptide are critical for overall activity, the effect of increased steric bulk and aromatic hydrophobicity on ion channel interactions was investigated by systematic 1-naphthylalanine (1-Nal) substitution. Even though wild-type ShK is already a highly basic peptide, we also decided to examine the impact on ion channel interactions of both arginine and lysine positional substitutions. While arginine versus lysine exchanges are sometimes considered to be conservative modifi- cations, these residues are indeed quite different in terms of size, basicity (pKa), and geometry, with arginine having a more basic planar δ-guanido group as compared to the sp3 -hybridized primary ε-amino functionality of lysine. The opposite electro- statically charged substitution, increased positional acidity, was accomplished by positional scanning with glutamic acid. Nearly all of the theoretical number of ShK peptide analogues for this approach could be efficiently prepared, but four analogues could not be isolated due to technical difficulties with the disulfide bond formation process. Each prepared peptide was individually tested for its ability to directly inhibit potassium current in Chinese hamster ovary (CHO) cells stably expressing the voltage- activated Kv1.3 or Kv1.1 channel using population patch clamp on the high-throughput IWQ platform (Table 1 and Figure 2). Wild-type ShK blocked Kv1.3 current with an inhibitory con- centration (IC50) of 132 ± 79 pM and was similarly effective Figure 1. Amino acid sequence of the ShK toxin peptide (1) with three disulfide bonds formed by six cysteines (C3 C35 , C12 C28 , and C17 C32 ). Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6785
- 3. against Kv1.1 currentwith an IC50 of 20 ± 29 pM in these assays (n = 31). This is the first time that the activity of native ShK is reported on the IWQ perforated patch clamp system. While the Kv1.1 IWQ IC50 is similar to that in reports using other methods, the Kv1.3 IWQ IC50 is about 10-fold higher than literature values.7,16b,17,21b The shift in Kv1.3 potency associated with this new assay did not prevent the identification of trends among the large number of compounds screened in this high- throughput fashion. The activity of important compounds was subsequently verified using whole-cell patch clamp electro- physiology, which provided better agreement with published data (vide infra). To assess the peptides’ ability to sustain the inhibition of T cell activation in a complex biological matrix, an ex vivo whole- blood cytokine secretion assay was employed. Thapsigargin challenge causes unloading of intracellular calcium stores and initiation of the calcium signaling pathway in T cells, resulting in IL-2 and IFN-γ secretion.6,24,26 In this whole-blood assay format, the activity of peptides also can be assessed in terms of the molecules’ ex vivo metabolic stability over 48 h. The whole- blood assay is a rigorous assessment of sustained Kv1.3 inhibition in comparison to electrophysiology (ePhys) because ePhys assays are generally of short duration (<1−2 h) and use only physiologically buffered saline with a low concentration of bovine serum albumin (BSA) in the absence of proteolytic enzymes. Furthermore, the 48 h time course of the whole-blood assay may better reflect equilibrium binding kinetics relative to ePhys studies. Accordingly, we used a dual screening approach for the assessment of the peptide analogues: (1) inhibition of Kv1.3 or Kv1.1 by electrophysiology and (2) the inhibition of IL-2 and IFN-γ secretion in human whole blood (Table 2 and Figure 2). As expected, native ShK was exceptionally potent in the thapsigargin-induced whole-blood assay, with an IC50 of 37 ± 36 pM against IL-2 and 48 ± 43 pM for IFN-γ secretion (n = 42). Our initial desire was to identify compound(s) with >5× selectivity for Kv1.3 vs Kv1.1 with IC50 values <500 pM in the Kv1.3 ePhys assay and <1000 pM in the IL-2 and IFN-γ whole-blood assays. The electrophysiological and whole-blood functional testing of the five families of ShK analogues, Ala, 1-Nal, Arg, Lys, and Glu substitutions, showed that each series provided interesting and unique results and that together a much more complete structure−activity relationship for ShK may be discerned. First, classical alanine scanning replaces the native side chain functionality at each position with a small aliphatic group (methyl) that typically weakens the binding interaction for key positions within the sequence. The alanine ShK analogue series indicated that residue positions 11, 22, 23, and 29 were likely to be important for maintaining Kv1.3 or Kv1.1 inhibitory activity (red or yellow in Figure 2). These findings were consistent with Figure 2. Heat map showing inhibition of Kv1.3 and Kv1.1 and inhibition of IL-2 and IFN-γ secretion in human whole blood for each ShK analogue from the MAPS analoging. Samples were tested against Kv1.3 and Kv1.1 on the IWQ platform. All values are the average ± SD; n ≥ 2. Colors indicate IC50 values in each assay, with green indicating highly potent, light green meaning moderately potent, yellow indicating weakly potent, and red signifying not potent. Gray indicates no data because the folded peptide analogue was not isolated. Data for wild-type sequence (1 (ShK) IL-2 IC50 = 37 ± 36 pM, IFN-γ IC50 = 48 ± 43 pM, Kv1.3 IC50 = 132 ± 79 pM, Kv1.1 IC50 = 20 ± 29 pM) has been included wherever the indicated substitution is the same as the native residue (Ala14 , Arg1 , Arg11 , Arg24 , Arg29 , Lys9 , Lys18 , Lys22 , and Lys30 ) and marked with a black rectangle. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6786
- 4.
- 5.
- 6. previously published reportson ShK SAR,7,16 and, importantly, these substitutions, along with positions 7, 10, 21, and 27, also resulted in considerably reduced activity in the corresponding whole-blood IL-2 and IFN-γ secretion assays (red). Within this series, only 19 ([Ala23]ShK) and 24 ([Ala29]ShK) showed >5-fold selective inhibition of Kv1.3 over Kv1.1, but, unfortunately, the concomitant loss in the critical cytokine secretion inhibitory activity for these compounds limited their utility. Under our assay conditions, the substitution of alanine for lysine at position 18 (14) did not improve selectivity against Kv1.1 as reported in the literature, perhaps due to differences in electrophysiology platform (IWQ instead of manual patch clamp) and/or cell line (hKv1.1 expressed in HEK293 cells instead of mKv1.1 in mouse L929 fibroblasts), and it was not tested by manual electrophysiology.20 In short, classic alanine positional scanning did not result in improvement in either potency or selectivity, necessitating implementation of our MAPS analoging methodology. The 1-naphthylalanine positional scan of ShK introduces a large aromatic side chain in place of the wild-type functionality to examine the effects of increasing hydrophobicity and steric bulk. This series had the largest number of substitutions that were disruptive to the Kv1.3 inhibitory activity of ShK. Kv1.3 inhibition was adversely affected by 1-Nal incorporation at positions 5, 7, 9, 11, 16, 18, 20, 21, 22, 23, and 29. Additionally, activity in the whole-blood assay was diminished by substitution of 1-Nal at positions 13, 27, and 33. This list includes and adds to the key binding residues identified by the Ala scan. Compounds 49 ([1-Nal26]ShK) and 50 ([1-Nal27]ShK) had ≥5-fold selectivity over Kv1.1, but only 1-Nal substitution at position 26 retained desirable whole-blood activity <1000 pM. In addition to varying the hydrophobicity and size at each position of ShK with Ala and 1-Nal, the electrostatic inter- actions were also probed through incorporation of amino acids with charged side chains. The glutamic acid substitution series had an activity profile similar to that with 1-Nal, with positions 5, 7, 11, 13, 20, 21, 22, 23, 24, 27, and 29 not being well tolerated in either the ePhys or whole-blood assays or both, demonstrating the extensive perturbation caused by integration of an acidic residue into a highly basic peptide sequence. Furthermore, no compound from the glutamic acid substitution series appeared to show any selective inhibition for Kv1.3 over Kv1.1. One observation unique to the Glu series was that substitution of the native Arg at position 24 caused a loss of functional activity in the cytokine secretion assays but retained activity in the electrophysiology assays, giving some insight into the SAR for that residue position. The basic arginine and lysine substitution series led to the identification of a selective and potent ShK analogue as a lead for further optimization and study. First, we found that arginine substitutions at 5, 7, 8, 20, 21, 22, 23, 27, and 31 resulted in significant loss of Kv1.3 and/or functional inhibitory activity. Lysine substitutions at positions 5, 7, 21, 27, and 31 had similar effects (Figure 3A). Among the different scans, position 31 was uniquely sensitive to substitution with a basic residue, having whole-blood IC50 values >5000 pM for the Arg (107) and Lys (131) substitution analogues but <500 pM for the Ala (26), 1-Nal (53), and Glu (82) containing compounds. Interestingly, although no arginine-substituted ShK analogues had any se- lective inhibition for Kv1.3 over Kv1.1, lysine substitution analogues at position 7, 115 ([Lys7]ShK), and position 16, 122 ([Lys16]ShK), were both 6-fold selective, a 40× improvement over native ShK (Figure 3B). However, only 122 retained potent whole-blood activity with an IL-2 secretion IC50 of 108 ± 45 pM and IFN-γ secretion IC50 of 151 ± 110 pM (n = 67). By comparison, 96 ([Arg16]ShK) had no improvement in selec- tivity for Kv1.3 and instead was an approximately 3-fold more potent inhibitor of Kv1.1 than Kv1.3. The key features of the sequence−activity relationship from the Lys scan are presented in Figure 3C. To summarize, application of MAPS analoging to ShK led to the identification of previously unreported sites for potency and selectivity not found via traditional Ala scanning efforts Figure 3. (A−C) Functional activity and electrophysiological selectivity of lysine scan ShK analogues. (A) Inhibition of IL-2 and IFN-γ secre- tion in whole-blood assay. (Top concentration tested was 100 nM.) (B) Kv1.1/Kv1.3 selectivity ratio. (C) ShK peptide sequence with residues important for potency in red and bold, residues important for selectivity in blue and underlined, and residues important for both in purple, bold, and underlined. Note that substitution of lysine at position 16 uniquely enhanced Kv1.3 selectivity with retention of potency against cytokine secretion. (D) Consensus findings from MAPS analoging of ShK with the residues likely to impact potency through conformational effects in bold and green, residues indicated as important for potency by a single series denoted in orange and bold, and the remainder as described in (C). Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6789
- 7. (Figure 3D).16 From anoverall perspective, only 2 out of the 132 initially prepared ShK analogues, 49 and 122, met the following success criteria: (1) <500 pM potent Kv1.3 inhibitor, (2) 5-fold or greater selectivity for Kv1.3 over Kv1.1, and (3) <1000 pM inhibitor of IL-2 and IFN-γ cytokine secretion in whole blood. These two novel lead compounds, not predicted a priori by computational methods,20 were identified only by a systematic approach that scanned the entire molecule multiple times with amino acids with different physicochemical pro- perties, not just alanine. Furthermore, a consensus list of Kv1.3- interacting residues within ShK was identified with a number of positions not immediately apparent from the Ala scan alone and only by balancing the findings from the electrophysio- logical assays with the results of the whole-blood cytokine secretion assays. Importantly, selectivity against Kv1.1 was obtained by substitution at positions not identified as critical for Kv1.3 inhibitory activity. Aside from a better understanding of which surface regions of ShK are important for activity and selectivity, MAPS analoging also provided a more detailed view of the SAR at each amino acid position. Structure−Function Relationships of Kv1.3 Inhibitory Toxin Peptides. Using racemic crystallography,27 we were able to solve the X-ray crystal structure of 122 at 1.2 Å resolu- tion (Figure 4). The peptide analogue consists of an extended conformation at the N-terminus up to residue 8, followed by two interlocking turns and then two short helices encompassing residues 12−19 and 21−24. Substitution of lysine at position 16 had no significant effect on the local conformation relative to the wild-type ShK structure.19a ShK residues Ile7 , Arg11 , Ser20 , Met21 , Lys22 , Tyr23 , and Phe27 , each identified as important for binding to Kv1.3 by an observed >20× loss in functional activity for at least three MAPS analogues, cluster in the tertiary Figure 4. Crystal structure of [Lys16]ShK. (A, B) 122 with side chains rendered as sticks and backbone secondary structure indicated with ribbons. (C−F) Surface rendering of 122 with residues colored according to putative interaction with Kv1.3 during binding. Blue indicates direct contact; yellow and orange residues make peripheral contact, with yellow substitutions affecting selectivity and orange substitutions impacting selectivity and potency. (A, C, E) View of the putative binding interface of the peptide. (B, D, F) Side view with Lys22 facing downward. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6790
- 8. structure of thepeptide (Figure 4C,D). The binding of ShK to the Kv1.3 channel has been generally described as a “cork in a bottle”, with ShK inserting the side chain of Lys22 into and physically occluding the channel pore.10,28 To potentially aid our comprehension of the screening data and the selectivity, the 122 structure was docked to a homology model of the Kv1.3 channel. Two poses that place Lys16 of 122 near sites where Kv1.3 differs from Kv1.1 in amino acid sequence (His451 versus Tyr379 and Gly427 versus His355 for Kv1.3 and Kv1.1, respectively) while maintaining key ShK binding residues in close contact with the Kv1.3 channel are shown in Figure 5. Despite the availability of this new set of ShK analogues, it is still unclear which of the proposed binding modes is most relevant; however, complementary ion channel site-directed mutagenesis may assist our understanding of the molecular basis for the selectivity of 122. There were a total of six ShK substitution analogues from the MAPS analoging that resulted in 5-fold selectivity for Kv1.3 over Kv1.1: Lys7 , Lys16 , Ala23 , 1-Nal26 , 1-Nal27 , and Ala29 , but only modification of positions 16 and 26 improved Kv1.3 selectivity without significantly compromising Kv1.3 potency or functional activity (Figure 4E,F). The native residues Gln16 and Ser26 are located at the periphery of the putative binding face and may interact with a portion of the surface of the channels that has some structural or sequence difference between Kv1.3 and Kv1.1. However, the unpredictability of the SAR is demonstrated by substitution of Arg29 with Ala, which is located more remotely than either position 16 or 26 but led to an increase in selectivity with concomitant loss in potency and unclear effect on overall peptide conformation. Other residues located at the border of the binding face, i.e., Thr6 , Ser10 , Thr13 , Arg24 , Thr31 , and Gly33 , have at least one MAPS analogue with a >20× loss in functional activity in the whole-blood assay without improvement in Kv1.3 versus Kv1.1 selectivity. Some effects on activity may be due to conformational disruption of the peptide. For example, substitution of a Lys or Arg residue at position 31 would place the side chains of three basic residues (Lys9 and Lys30 ) in close proximity and may affect the global structure. While our results serve to refine the list of residues in ShK with strong Kv1.3 interactions,20 these data also highlight the importance of residues at the edge of the peptide binding surface. While these peripheral residues are typically ignored by traditional optimization strategies (i.e., alanine scanning and structure-based design), specific changes in charge or hydrophobicity at these sites may serve to elucidate the nature of their contribution to the complex and effect on ion channel selectivity. [Lys16]ShK Peptide Analogues. Identification of the potent and moderately selective Kv1.3 inhibitory peptide 122 was followed up with additional analoging at position 16 and Figure 5. Molecular docking of 122 (gray ribbon with residues important to binding in blue and Lys16 and Ser26 in yellow) to Kv1.3 homology model (green ribbons). (A) Side view of pose I. For clarity, two monomers (II and IV) of the homotetrameric channel have been hidden. (B) Top view of pose 1. (C) Side view of pose 2. For clarity, two monomers (I and III) of the homotetrameric channel have been hidden. (D) Top view of pose 2. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6791
- 9. combination with modificationsto reduce oxidative liabilities (Table 3). Shortening the Lys16 side chain by a methylene unit to orthinine (Orn) resulted in a similar activity profile (134); however, removal of a second methylene unit with diaminobutyric acid (Dab) led to loss of Kv1.1 selectivity (135). It is known that amidation of the C-terminal acid of ShK provides a backbone with similar activity and increased metabolic stability;21c preparation of the C-terminal amide of [Lys16]ShK yielded a similarly potent and selective derivative (136). Extension of the C-terminus with a residue less prone to epimerization during solid-phase peptide synthesis than cysteine29 and substitution of the oxidizable methionine with norleucine at position 21 were investigated in combination with the lysine substitution at position 16 (Table 3). Surprisingly, addition of a C-terminal alanine to [Lys16]ShK resulted in analogues 137 and 141 with >150-fold selectivity for Kv1.3 versus Kv1.1 that retained good activity in blocking T cell cytokine secretion in human whole blood. The improvements in selectivity associated with substitution of lysine at position 16, hydrophobic substitutions at position 21, and extension of the C-terminus have been verified by Pennington and co-workers, including an additive improvement in selectivity through their approach of N-terminal modification.30 Electrophysiology of ShK Peptide Analogues. Further electrophysiological characterization of lead compound 122, the parent ShK peptide, and literature analogues was performed (Table 4). Our previous screening experiments utilized a high-throughput 384-well IonWorks Quattro (IWQ) platform, which evaluates receptor inhibition with a population patch clamp, due to the large number of compounds that needed to be tested. A select number of important analogues were tested on a whole-cell planar patch clamp platform using the auto- mated PatchXpress (PX) system or manual electrophysiology. As expected, ShK was an exceptionally potent inhibitor of both Kv1.3 and Kv1.1 on the PX system. These values were in reasonable agreement with those obtained by manual whole- cell patch clamp electrophysiology where ShK had an IC50 of 16 ± 8 pM for Kv1.3 and 14 ± 3 pM for Kv1.1, similar to values reported in the literature as well as our results in the cytokine secretion assays.7,16b,17,21b Compound 122 was also a potent inhibitor of Kv1.3 on the PX platform and demonstrated >15× selectivity against Kv1.1. The potency and selectivity profiles for 142 (ShK-Dap22) and 143 (ShK-L5, Supporting Information Figure S1), which are commercially available, were compared to the results reported previously for these analogues.7,21b Molecule 142 showed a significant loss in whole-blood functional activity, which motivated us to adopt this assay for the primary screening of our analogues. As dis- cussed earlier, the whole-blood assay is of longer duration and may better reflect equilibrium binding of the peptide to the target. Indeed, while Kalman et al. reported that 142 showed good potency by electrophysiology (Kv1.3 IC50 = 23 pM)7 similar to our findings, Middleton et al. reported that its equilibrium binding affinity for Kv1.3 is more than 100 times weaker than native ShK.31 These latter results are consistent with our observations in the 48 h whole-blood assay, where 142 had IL-2 and IFN-γ IC50 values >3000 pM. In our assays, 143 was a potent inhibitor of both Kv1.3 and Kv1.1 as well as cytokine secretion in human whole blood. The disagreement of our selectivity ratio for 143 with published reports may be due to our use of a different cell line (hKv1.3 in Chinese hamster ovary (CHO) cells rather than mKv1.3 in mouse L929 fibroblasts).7,21b Impact of Conjugation on Potency, Selectivity, and Pharmacokinetics of ShK Analogues. The potent wild-type ShK peptide has a very short circulating pharmacokinetic profile in rats (t1/2 ∼ 20 min).32 The short half-life in vivo of peptides is typically attributed to rapid metabolic processing and high renal clearance.33 To investigate whether renal clearance was responsible for the short circulating time of ShK, we attempted PEGylation of the molecule as a means to increase its hydrodynamic radius.34 It was unknown, however, whether attachment of a large poly(ethylene glycol) (PEG) polymer to ShK would significantly impair its activity. We explored a N-terminal reductive amination approach due to the presence of multiple lysine residues in ShK derivatives and the difficulty of using cysteine-maleimide chemistry in disulfide-rich peptides. First, a Nα -PEG-ShK conjugate was prepared by reductive alkylation of the N-terminus with a linear 20 kDa monomethoxy PEG aldehyde at pH 4.5 and then purified. Peptide mapping experiments confirmed PEGylation occurred primarily at the N-terminus of the peptide (data not shown). Testing of 144 (20 kDa-PEG-ShK) revealed that it retained subnanomolar potency in inhibiting Kv1.3 and T cell cytokine responses (Table 5) and exhibited a prolonged half-life in rats (mean residence time of 37 h, Supporting Information Table S2). Table 3. Potency and Selectivity of Position 16 ShK Analogues cmpd peptide name Kv1.3 IWQ IC50 (pM) Kv1.1 IWQ IC50 (pM) Kv1.1 IC50/Kv1.3 IC50 WB IL-2 IC50 (pM) WB IFN-γ IC50 (pM) 1 ShK 132 20 0.15 37 48 122 [Lys16]ShK 352 2342 6.7 108 151 134 [Orn16]ShK 140 740 5.3 138 160 135 [Dab16]ShK 82 11 0.13 86 223 136 [Lys16]ShK-amide 174 600 3.4 223 278 137 [Lys16]ShK-Ala 60 9500 158 138 266 138 [Nle21]ShK 40 15 0.38 153 303 139 [Lys16,Nle21]ShK 130 29 258 225 249 5678 140 [Lys16,Nle21]ShK-amide 153 13 220 86 823 1099 141 [Lys16,Nle21]ShK-Ala 71 >33 333 >469 305 515 Table 4. Potency and Selectivity of ShK Analogues cmpd peptide name Kv1.3 PX IC50 (pM) Kv1.1 PX IC50 (pM) Kv1.1 IC50/ Kv1.3 IC50 WB IL-2 IC50 (pM) WB IFN-γ IC50 (pM) 1 ShK 39 87 2.2 37 48 122 [Lys16]ShK 207 3677 17.8 108 151 142 ShK-Dap22 12a 847a 70.6 3763 3112 143 ShK-L5 221 214 1.0 31 46 a Indicates manual whole-cell patch clamp data. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6792
- 10. In agreement withother N-terminally derivatized ShK ana- logues,35 such as 143 (phosphotyrosine-AEEA-ShK), which have been reported to have increased selectivity for Kv1.3, we also found 144 to be 5-fold more potent against Kv1.3 than against Kv1.1. Encouraged by the retention of activity, we next PEGylated our selective [Lys16]ShK analogue at its N-terminus with linear PEG. The conjugate 145 (20 kDa-PEG-[Lys16]ShK) was found to provide potent blockade of whole-blood IL-2 secretion with an IC50 of 92 ± 42 pM (n = 14). More interes- tingly, selectivity for Kv1.3 over Kv1.1 was not only retained, but it showed a synergistic 1000-fold lowering of Kv1.1 activity without impacting Kv1.3, more than 200× the effect that PEGylation had on native ShK.30 The potency and selectivity of 145 are extraordinary when compared to those of the conjugated wild-type peptide and unconjugated peptide analogues (Figure 6). The pharmacokinetics and bioactivity of polypeptides can be significantly altered through the attachment of PEG groups of differing molecular weight.36 Aside from derivatization with 20 kDa-PEG, the [Lys16]ShK peptide was also prepared with either a 30 kDa linear PEG or a branched PEG consisting of two 10 kDa PEG arms (20 kDa-brPEG). The 20 kDa-brPEG- [Lys16]ShK molecule (146) was a potent inhibitor of cytokine secretion in the whole-blood assay and had 750-fold selectivity for lymphocyte Kv1.3 over neuronal Kv1.1. The linear 30 kDa- PEG-[Lys16]ShK molecule (147) was also a highly potent inhibitor of cytokine secretion in human whole blood. These results suggest that the 122 peptide scaffold is tolerant of N-terminal derivatization with PEG polymers of differing size and architecture. Conjugation of 122 with linear 20 kDa PEG results in a slightly higher level of Kv1.3 vs Kv1.1 selectivity relative to the branched or larger PEG chains. In preparation for in vivo studies, the in vitro activity profile of 145 was further characterized in a number of ion channel counterscreens and against other species. Counter screening revealed that 145 was highly selective over Kv subtypes Kv1.2 (680-fold), Kv1.6 (∼500-fold), Kv1.4 (>10 000-fold), Kv1.5 (>10 000-fold), and Kv1.7 (>10 000-fold) (Table 6). Importantly, the conjugate did not impact ion channels that are known to serve a role in human cardiac action potential, exhibiting >10 000-fold selectivity over Nav1.5, Cav1.2, Kv4.3, KvLQT1/ minK, and hERG. Moreover, the conjugated toxin peptide analogue 145 had no detectable impact on the calcium-activated K+ channels KCa3.1 (IKCa1) and BKCa. Cross-Species Activity of 20 kDa-PEG-[Lys16]ShK. In addition to its inhibitory activity in human whole blood, 145 also inhibited IL-17 and IL-4 secretion from T cells within cynomolgus monkey whole blood with potent IC50 estimates of 0.09 ± 0.08 nM and 0.17 ± 0.13 nM, respectively. 145 was also a potent inhibitor (IC50 = 0.17 nM) of myelin-specific proliferation of the rat T effector memory cell line, PAS.37 Overall, we found that 145 has consistently potent inhibitory activity toward T cell responses in whole-blood assays from rat, monkey, and human (IL-2 IC50 = 0.092 nM). Pharmacokinetics of 20 kDa-PEG-[Lys16]ShK. In regards to unconjugated peptides, there are limited pharmacokinetic Table 5. Potency and Selectivity of PEGylated ShK Analogues cmpd name Kv1.3 PX IC50 (nM) Kv1.1 PX IC50 (nM) Kv1.1 IC50/Kv1.3 IC50 WB IL-2 IC50 (nM) WB IFN-γ IC50 (nM) 144 20 kDa-PEG-ShK 0.299a 1.628a 5 0.380 0.840 145 20 kDa-PEG-[Lys16]ShK 0.94 997 1060 0.092 0.160 146 20 kDa-brPEG-[Lys16]ShK 2.10 1574 750 0.198 0.399 147 30 kDa-PEG-[Lys16]ShK 1.20 1072 890 0.282 0.491 a Manual patch clamp electrophysiology. Figure 6. Graphical comparison of the potency and selectivity of select naked and PEGylated peptide analogues relative to ShK. Each point represents one compound with the x-axis value computed as (whole- blood IL-2 IC50)/(ShK whole-blood IL-2 IC50) and the y-axis value computed as ([Kv1.1 IC50/Kv1.3 IC50]/[ShK Kv1.1 IC50/ShK Kv1.3 IC50]). Table 6. Activity of 145 in Counterscreensa assay IC50 (nM) human WB IL-2 0.092 Kv1.1 997 Kv1.2 639 Kv1.3 0.94 Kv1.4 >10 000 Kv1.5 >30 000 Kv1.6 466 Kv1.7 >10 000 IKCa1 >10 000 BKCa >10 000 hERG (IKr) >10 000 Nav1.5 (INa) >30 000 Cav1.2 (ICa) >30 000 Kv4.3 (Ito) >30 000 KvLQT1/minK (IKs) >30 000 a n ≥ 3 for all. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6793
- 11. studies on nativeShK32 and the more selective ShK analogue, 143,21b indicating that these molecules have half-lives in rats that are much shorter (<1 h) than that of our PEG conjugate. Prior to evaluating the pharmacology of the potent and selective conjugate 145, its ex vivo plasma stability and pharmacokinetics were determined. The conjugate was found to have high meta- bolic stability in plasma from rat, cynomolgus monkey, and human over 2 days at 37 °C (Supporting Information Figure S5). Pharmacokinetic studies in mouse, rat, dog, and cynomolgus monkey showed good cross-species metabolic stability in vivo with a considerably extended elimination half-life. Moreover, a comparison of 148 (ShK-186), a more advanced derivative of 143 containing a C-terminal amide and displaying improved stability,21c,24 indicates that 145 has a half-life in cynomolgus monkeys that was 245 times longer than 148 when the same 0.5 mg/kg dose was delivered (Table 7). We estimate the exposure of compound 145 over time, as measured by AUC0−∞, was 390 times greater in cynomolgus monkeys than 148, resulting in a clearance rate that was ∼950 times slower in rats and cynomolgus monkeys compared to the 148 peptide. As shown in Figure 7, 145, when dosed subcutaneously in cynomolgus monkeys at 0.5 mg/kg, achieved a Cmax at 8 h of 254 nM and day 7 serum levels of 28.4 nM. The serum concentration of 145 at day 7 after a single dose was approximately 28 and 315 times greater than the cytokine secretion IC95 (1.0 nM) and IC50 (0.09 nM) estimates in human whole blood, respectively. Therefore, the pharmacokinetics of 145 in cynomolgus monkeys are consistent with a projected weekly dosing profile in human subjects. It should be noted that despite our PEG conjugate showing a profoundly longer half-life in vivo than 148 Tarcha et al. report that this peptide analogue shows durable pharmacological effects in monkeys.24 The authors propose that, although serum levels decline rapidly over the first few hours after injection, there could be a slow release from the injection site as well as tight binding and slow dissociation from the Kv1.3 channel on T cells to drive efficacy. Irrespective of these con- siderations, we show that the conjugate 145 is profoundly longer- lived in vivo, enabling sustained and measurable target coverage over a narrower dynamic range of serum drug concentrations. Further details on the pharmacokinetics of 145 administered subcutaneously are provided in the Supporting Information. Efficacy, Pharmacodynamics, and Safety of 20 kDa- PEG-[Lys16]ShK. We evaluated the efficacy of 145 in vivo using the adoptive-transfer experimental autoimmune encepha- lomyelitis (AT-EAE) model in rats.38 In this animal model of multiple sclerosis, T cells specific for myelin basic protein (MBP) and constitutively expressing Kv1.3 (PAS cells) are activated and injected into rats, causing inflammation and demyelination of the central nervous system (CNS), with symptoms progressing from a distal limp tail to paralysis over the course of a week. Dosing in rats with the Kv1.3 blocker 145 before the onset of EAE caused a delay in the onset of disease. The progression of disease was also inhibited with treatment with 145, with an observed dose- dependent effect on reduced disease severity and the prevention of death (Figure 8). In the vehicle-treated animal group, the disease onset occurred on day 4, but, by comparison, in animals treated with 145, the disease onset was delayed until day 4.5 to 5. On day 6, the vehicle-treated rats had developed severe disease (EAE score of 6) and were sacrificed, whereas 145 treated animals (at efficacious doses) had only mild disease (EAE score of ∼1) that resolved over time. The molecule 145 blocked AT-EAE in a dose-responsive manner with an estimated ED50 of approximately 4 μg/kg on day 7 (Figure 8 and Table7.SingleDosePharmacokinetic(Subcutaneous)Profileof145inCD1Mice,SpragueDawleyRats,BeagleDogs,AndCynomolgusMonkeysComparedtothe Pharmacokineticsof148inSpragueDawleyRatsandCynomolgusMonkeys24,a cpmdspeciesdose(mg/kg)nt1/2(h)Tmax(h)Cmax(ng/mL)AUC0−t(ng·h/mL)AUC0−∞(ng·h/mL)Vz/F(mL/kg)CL/F(mL/h/kg)MRT(h) 145mouseb 2.0314.94.018603700037000117054.116.6 145rat2.03N/A40±14531±9021900±277021900±2760N/A92±1336±2 145beagle0.5342.6±4.2118.7±9.241270±34795200±31300103000±37300322±985.37±2.1466.1±13.5 145cyno0.5364.5±14.98.01010±10571500±60774900±3260621±1436.68±0.2987±16 148c rat1.030.1320.08348NR11.5NR87198NR 148c cyno0.520.2630.083192NR192NR6336NR a Becauseonlythepeptideportionof145wasusedincalculatingmg/mLstockconcentrationsandthe[Lys16]ShKpeptideportion(4055Da)isofsimilarMWto148,equivalentmg/kgdosesofthese twomoleculesgeneratesimilarnmol/kgdoses.b SparsesamplingPKexperiment.NostandarddeviationswerecalculatedforPKparameters.c FromTarchaetal.24 NR=notreported. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6794
- 12. Supporting Information FigureS6). In a separate study, an equivalent dose (0.01 mg/kg) of PEGylated ShK (144) was found to provide greater efficacy in blocking encephalomyelitis than that of the native ShK peptide (Supporting Information Figure S7) that has a shorter half-life in animals. Overall, these data suggest that higher levels of sustained Kv1.3 target coverage appear to result in greater efficacy in this model. The in vivo pharmacodynamics of 145 in cynomolgus monkeys was also examined. A 12 week pharmacology study was initiated with three predose baseline measurements during the first 2 weeks. This was then followed by four weekly sub- cutaneous doses of 145 at 0.5 mg/kg and an additional 6 weeks of postdosing analysis (Figure 9 and Supporting Information Tables S5−S7). On the basis of earlier pharmacokinetic studies of 145, target coverage with 0.5 mg/kg weekly dosing was expected to range from 28-times the IL-17 IC95 at the minimum drug concentration in plasma (Cmin) to 249-times IC95 at the peak or maximum drug concentration (Cmax). The repeat dosing of the conjugate 145 was well-tolerated. In terms of general observations, weight gain was normal throughout the study, and complete blood counts (CBCs) and blood chemistry were also found to be normal with respect to predose baseline estimates. Using the cynomolgus monkey whole-blood IL-17 pharmacodynamic assay, suppression of T cell inflammation was achieved during the 4 week dosing period. In terms of repeat drug exposure, the predicted and observed serum drug trough levels correlated well over the dosing period. The potential toxicity and the toxicokinetics of 145 were evaluated in male cynomolgus monkeys (n = 3 per dose group) after subcutaneous administration of 0.7 mg/kg every third day (4 doses total) or 2 weekly doses at 0.1, 0.5, or 2.0 mg/kg (2 doses total).39 There were no 145-related effects on any parameters evaluated. Specifically, there were no PEG-associated vacuoles observed in renal tubules or tissue macrophages by light microscopy. On the basis of the absence of adverse toxicity, the no-observed-adverse-effect level (NOAEL) in this study was 2 mg/kg, which correlated with a mean AUC0−168h of 584 000 ng·h/mL. ■ CONCLUSIONS The diverse array of potent biological activities and inherent metabolic stability of toxin peptides make this class of mole- cules an attractive starting point for drug discovery of ion channel modulators. We have demonstrated the effectiveness of the multi attribute positional scan (MAPS) analoging method- ology to identify potent and subtype-selective analogues of the ShK peptide toxin. By scanning the peptide sequence with not only the traditional Ala residue but also representative basic, acidic, and hydrophobic residues and screening the resulting >130 analogues via high-throughput electrophysiol- ogy, [Lys16]ShK emerged as a potent antagonist of Kv1.3 with improved selectivity over Kv1.1.39 Combination with N-terminal conjugation of a 20 kDa poly(ethylene glycol) polymer resulted in an unexpected synergistic increase in Kv1.3 versus Kv1.1 selectivity to 1000-fold, with retention of picomolar potency in the whole-blood T cell assay and prolongation of the half-life in vivo. A clean selectivity profile against a panel of ion channels and good plasma stability made 20 kDa-PEG-[Lys16]ShK suitable for rodent and primate PD studies. Compound 145 was efficacious in the rat adoptive transfer-experimental autoimmune encephalitis (AT-EAE) model of multiple sclerosis. The pharma- cokinetic profile of this compound was suitable for weekly dosing in cynomologous monkeys, and it showed suppression of T cell-mediated inflammation during a 1 month repeat-dosing experiment without adverse side effects. Through prolonged blockade of Kv1.3 in vivo, 145 or related analogues may allow further interrogation of this target for the treatment of auto- immune disease in higher species. In view of these results and Figure 7. Pharmacokinetic profiles of a single subcutaneous dose (mouse and rat, dose = 2 mg/kg; beagle and cyno, dose = 0.5 mg/kg) of 145 (with target coverage estimates based on whole-blood assay results: cynomolgus monkey IL-17 IC50 = 0.09 and human IL-2 IC50 = 0.092 nM). Figure 8. Comparison of the in vivo efficacy of 20 kDa-PEG-ShK (144) and the Kv1.3-selective inhibitor 145 in blocking autoimmune encephalomyelitis in a rat AT-EAE model. The PEGylated ShK or [Lys16]ShK conjugates were delivered subcutaneously (SC) daily from days −1 to 7. The rat CD4+ myelin-specific effector memory T cells line, PAS, was delivered by intravenous injection on day 0. The rats were monitored for signs of EAE once or twice per day in a blinded fashion, and 5 or 6 female Lewis rats were used per treatment group. Clinical EAE scores were as follows: 0 = no signs, 0.5 = distal limp tail, 1.0 = limp tail, 2.0 = mild paraparesis, ataxia, 3.0 = moderate paraparesis, 3.5 = one hind leg paralysis, 4.0 = complete hind leg paralysis, 5.0 = complete hind leg paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund state or death. Rats reaching a score of 5.5 were euthanized. Error bars represent the standard error of the mean. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6795
- 13. other applications inour laboratory, it appears the MAPS analoging strategy will be useful to multiple classes of toxin peptides for ion channel targets as well as small synthetically accessible protein scaffolds for other types of targets. ■ EXPERIMENTAL METHODS Peptide Preparation. Peptide Synthesis. Nα -Fmoc, side chain- protected amino acids and H-Cys(Trt)-2Cl-Trt resin were purchased from Novabiochem, Bachem, or Sigma-Aldrich. The following side chain protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Glu(OtBu), His(Trt), Lys(Nε -Boc), Ser(OtBu), Thr(OtBu), and Tyr(OtBu). ShK or other toxin peptide analogue amino acid sequences were synthesized in a stepwise manner on a CS Bio 336 peptide synthesizer by Fmoc-SPPS using DIC/HOBt coupling chemistry at 0.2 mmol scale using H-Cys(Trt)-2Cl-Trt resin (0.2 mmol, 0.32 mmol/g loading). For each coupling cycle, 1 mmol Nα -Fmoc- amino acid was dissolved in 2.5 mL of 0.4 M 1-hydroxybenzotriazole (HOBt) in N,N-dimethylformamide (DMF). To the solution was added 1.0 mL of 1.0 M N,N′-diisopropylcarbodiimide (DIC) in DMF. The solution was agitated with nitrogen bubbling for 15 min to accomplish preactivation and then added to the resin. The mixture was shaken for 2 h. The resin was filtered and washed three times with DMF, twice with dichloromethane (DCM), and three times with DMF. Fmoc removals were carried out by treatment with 20% piperdine in DMF (5 mL, 2 × 15 min). The first 23 residues were single-coupled through repetition of the Fmoc-amino acid coupling and Fmoc removal steps described above. The remaining residues were double-coupled by performing the coupling step twice before proceeding with Fmoc removal. Following synthesis, the resin was drained, washed sequentially with DCM, DMF, and DCM, and then dried in vacuo. The peptide-resin was transferred to a 250 mL plastic round-bottomed flask. The peptide was deprotected and cleaved from the resin by treatment with triisopropylsilane (1.5 mL), 3,6-dioxa-1,8-octane-dithiol (DODT, 1.5 mL), water (1.5 mL), trifluoroacetic acid (TFA, 20 mL), and a stir bar, and the mixture was stirred for 3 h. The mixture was filtered through a 150 mL sintered glass funnel into a 250 mL plastic round-bottomed flask, and the filtrate was concentrated in vacuo. The crude peptide was precipitated by dropwise addition to cold diethyl ether in a 50 mL centrifuge tube, collected by centrifugation, and dried under vacuum. Peptide Folding. The dry crude linear peptide (about 600 mg from 0.2 mmol), for example, [Lys16]ShK peptide, was dissolved in 16 mL of acetic acid, 64 mL of water, and 40 mL of acetonitrile. The mixture was stirred rapidly for 15 min to complete dissolution. The peptide solution was added to a 2 L plastic bottle that contained 1700 mL of water and a large stir bar. To the diluted peptide solution was added 20 mL of concentrated ammonium hydroxide to increase the pH of the solution to 9.5. The pH was adjusted with small amounts of acetic acid or NH4OH as necessary. The solution was stirred at 80 rpm overnight and monitored by LC-MS. Folding was usually judged to be complete in 24 to 48 h, and the solution was quenched by the addition Figure 9. Twelve week pharmacology study in cynomolgus monkeys. Weekly dosing of cynomolgus monkeys with 145 provided sustained suppression of T cell responses, as measured using the ex vivo cynomolgus monkey whole-blood PD assay of inflammation that measured production of IL-4 (A) and IL-17 (B). Arrows indicate the weekly doses. Each line represents an individual test subject. (C) Predicted versus measured serum concentrations of 145 in cynomolgus monkeys after weekly subcutaneous (SC) dosing (0.5 mg/kg, n = 6). The measured serum trough levels after weekly dosing (open squares), matched closely those predicted based on repeat-dose modeling of the single-dose pharmacokinetic data (solid line). (D) Weight gain for each animal during the 12 week cynomolgus monkey pharmacology study; arrows on x-axis indicate SC dosing with 145. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6796
- 14. of acetic acidand TFA (pH 2.5). The aqueous solution was filtered (0.45 μm cellulose membrane). Reversed-Phase HPLC Purification and Analysis and Mass Spectrometry. Reversed-phase high-performance liquid chromatog- raphy (RP-HPLC) was performed on a preparative (C18, 10 μm, 2.2 cm × 25 cm) column. Chromatographic separations were achieved using linear gradients of buffer B in A (A = 0.1% aqueous TFA; B = 90% aq. acetonitrile containing 0.09% TFA), typically 5−65% over 90 min at 20 mL/min for preparative separations. Preparative HPLC fractions were characterized by ESMS and photodiode array (PDA) HPLC, combined, and lyophilized. Final analysis (Phenomenex Synergi MAX-RP 2.5 μm, 100 Å, 50 × 2.0 mm column eluted with a 10 to 50% B over 10 min gradient [A: water and B: acetonitrile, 0.1% TFA in each] at a 0.650 mL/min flow rate monitoring UV absorbance at 220 nm) was performed for each peptide sample using an Agilent 1290 LC-MS. Peptides with >95% purity and correct (m/z) ratio were screened. (See Supporting Information Table S8 for LC-MS characterization of ShK and peptide analogues.) PEGylation, Purification, and Analysis. Peptide, for example, [Lys16]ShK, was selectively PEGylated by reductive alkylation at its N-terminus using activated linear or branched PEG. Conjugation was performed at 2 mg/mL in 50 mM NaH2PO4, pH 4.5, reaction buffer containing 20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDa monomethoxy-PEG-aldehyde (NOF, Japan). Conjugation reactions were stirred for approximately 5 h at room temperature, and their progress was monitored by RP-HPLC. Completed reactions were quenched by 4-fold dilution with 20 mM NaOAc, pH 4, and chilled to 4 °C. The PEG-peptides were then purified chromatographically at 40 °C using SP Sepharose HP columns (GE Healthcare, Piscataway, NJ) and eluted with linear 0−1 M NaCl gradients in 20 mM NaOAc, pH 4.0. Eluted peak fractions were analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%. Principle contaminants observed were di-PEGylated toxin peptide analogue. Selected pools were concentrated to 2−5 mg/mL by centrifugal filtration against 3 kDa MWCO membranes and dialyzed into 10 mM NaOAc, pH 4, with 5% sorbitol. Dialyzed pools were then sterile filtered through 0.2 μm filters, and purity was determined to be >97% by SDS-PAGE and RP-HPLC (see Supporting Information Figures S2 and S3). Reverse-phase HPLC was performed on an Agilent 1100 model HPLC running a Zorbax 5 μm 300SB-C8 4.6 × 50 mm column (Agilent) in 0.1% TFA/H2O at 1 mL/min, and the column tem- perature was maintained at 40 °C. Samples of PEG-peptide (20 μg) were injected and eluted in a linear 6−60% gradient while monitoring at a wavelength of 215 nm. Electrophysiology. Cell Lines Expressing Kv1.1−Kv1.7. CHO K1 cells were stably transfected with human Kv1.3 or, for counterscreens, with hKv1.4, hKv1.6, or hKv1.7; HEK293 cells were stably expressing human Kv1.3 or human Kv1.1. Cell lines were from Amgen or BioFocus DPI (A Galapagos Company). CHO K1 cells stably expressing hKv1.2, for counterscreens, were purchased from Millipore (cat. no. CYL3015). Whole-Cell Patch Clamp Electrophysiology. Whole-cell currents were recorded at room temperature using MultiClamp 700B amplifier from Molecular Devices Corp. (Sunnyvale, CA), with 3−5 MΩ pipettes pulled from borosilicate glass (World Precision Instruments, Inc.). During data acquisition, capacitive currents were canceled by analogue subtraction, no series resistance compensation was used, and all currents were filtered at 2 kHz. The cells were bathed in an extracellular solution containing 1.8 mM CaCl2, 5 mM KCl, 135 mM NaCl, 5 mM glucose, 10 mM HEPES, pH 7.4, 290−300 mOsm. The internal solution contained 90 mM KCl, 40 mM KF, 10 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2, 290−300 mOsm. The currents were evoked by applying depolarizing voltage steps from −80 to +30 mV every 30 s (Kv1.3) or 10 s (Kv1.1) for 200 ms intervals at a holding potential of −80 mV. To determine IC50, 5−6 peptide or peptide conjugate concentrations at 1:3 dilutions were made in extracellular solution with 0.1% BSA and delivered locally to cells with Rapid Solution Changer RSc-160 (BioLogic Science Instruments). Currents were achieved to steady state for each concentration. Data analysis was performed using pCLAMP (version 9.2) and OriginPro (version 7), and peak currents before and after each test article application were used to calculate the percentage of current inhibition at each concentration. PatchXpress Planar Patch Clamp Electrophysiology. Cells were bathed in an extracellular solution containing 1.8 mM CaCl2, 5 mM KCl, 135 mM NaCl, 5 mM glucose, 10 mM HEPES, pH 7.4, 290− 300 mOsm. The internal solution contained 90 mM KCl, 40 mM KF, 10 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2, 290−300 mOsm. Usually, 5 peptide or peptide conjugate concen- trations at 1:3 dilutions were made to determine the IC50s. The peptide or peptide conjugates were prepared in extracellular solution containing 0.1% BSA. Dendrotoxin-k and Margatoxin were purchased from Alomone Laboratories Ltd. (Jerusalem, Israel); ShK toxin was purchased from Bachem Bioscience, Inc. (King of Prussia, PA); 4-AP was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Currents were recorded at room temperature using a PatchXpress 7000A electro- physiology system from Molecular Devices Corp. (Sunnyvale, CA). The voltage protocols and recording conditions are shown in the Supporting Information Table S1. An extracellular solution with 0.1% BSA was applied first to obtain 100% percent of control (POC), which was then followed by 5 different concentrations of 1:3 peptide or peptide conjugate dilutions for every 400 ms incubation time. At the end, an excess of a specific benchmark ion channel inhibitor was added to define full or 100% blockage. The residual current present after addition of benchmark inhibitor was used in some cases for calculation of zero percent of control. Each individual set of traces or trial were visually inspected and either accepted or rejected. The general criteria for acceptance were as follows: (1) baseline current must be stable, (2) initial peak current must be >300 pA, (3) intitial Rm and final Rm must >300 Ohm, and (4) peak current must achieve steady state prior to first compound addition. The POC was calculated from the average peak current of the last 5 sweeps before the next concentration of compound was added and exported to Excel for IC50 calculation. IonWorks Quattro High-Throughput Population Patch Clamp Electrophysiology. Electrophysiology was performed on CHO cells stably expressing hKv1.3 and HEK293 cells stably expressing hKv1.1. The procedure for preparation of the assay plate containing ShK analogues and conjugates for IWQ electrophysiology was as follows: all analogues were dissolved in extracellular buffer (PBS, with 0.9 mM Ca2+ and 0.5 mM Mg2+ ) with 0.3% BSA and dispensed in row H of 96-well polypropylene plates at a concentration of 100 nM from columns 1−10. Columns 11 and 12 were reserved for negative and positive controls. Serial dilutions at a 1:3 ratio were then made to row A. IonWorks Quattro (IWQ) electrophysiology and data analysis were accomplished as follows: resuspended cells (in extracellular buffer), the assay plate, a population patch clamp (PPC) PatchPlate as well as appropriate intracellular (90 mM potassium gluconate, 20 mM KF, 2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35) and extracellular buffers were positioned on IonWorks Quattro. When the analogues were added to patch plates, they were further diluted 3-fold from the assay plate to achieve a final test concentration range from 33.3 nM to 15 pM with 0.1% BSA. Electrophysiology recordings were made from the CHO-Kv1.3 and HEK-Kv1.1 cells using an amphotericin-based perforated patch clamp method. Using the voltage clamp circuitry of the IonWorks Quattro, cells were held at a membrane potential of −80 mV, and voltage-activated K+ currents were evoked by stepping the membrane potential to +30 mV for 400 ms. K+ currents were evoked under control conditions, i.e., in the absence of inhibitor at the beginning of the experiment and after 10 min incubation in the presence of the analogues and controls. The mean K+ current amplitude was measured between 430 and 440 ms, and the data were exported to a Microsoft Excel spreadsheet. The amplitude of the K+ current in the presence of each concentration of the analogues and controls was expressed as a percentage of the K+ current of the precompound current amplitude in the same well. When these percent of control values were plotted as a function of concentration, the IC50 value for each compound could be calculated using the dose−response fit model 201 in Excel fit program. Measuring Bioactivity in Whole Blood. Ex Vivo Assay to Examine Impact of Toxin Peptide Analogue Kv1.3 Inhibitors on Secretion of Human IL-2 and IFN-γ. The potency of ShK analogues Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6797
- 15. and conjugates inblocking T cell inflammation in human whole blood was examined using an ex vivo assay that has been described earlier.40 In brief, 50% human whole blood is stimulated with thapsigargin to induce store depletion, calcium mobilization, and cytokine secretion. To assess the potency of molecules in blocking T cell cytokine secretion, various concentrations of Kv1.3 blocking peptides and peptide conjugates were preincubated with the human whole-blood sample for 30−60 min prior to addition of the thapsigargin stimulus. After 48 h at 37 °C and 5% CO2, conditioned medium was collected and the level of cytokine secretion was determined using a four-spot electrochemillumi- nescent immunoassay from MesoScale Discovery. Using the thapsi- gargin stimulus, the cytokines IL-2 and IFN-γ were secreted robustly from blood isolated from multiple donors. The IL-2 and IFN-γ produced in human whole blood following thapsigargin stimulation were produced from T cells, as revealed by intracellular cytokine staining and fluorescence-activated cell sorting (FACS) analysis. Pharmacokinetic and Pharmacodynamic Studies. Detection Antibodies to ShK. Rabbit polyclonal and mouse monoclonal anti- bodies to ShK were generated by immunization of animals with an Fc-ShK peptibody conjugate.39 Anti-ShK specific polyclonal antibodies were affinity-purified from antisera to isolate only those antibodies specific for the ShK portion of the conjugate. Following fusion and screening, hybridomas specific for ShK were selected and isolated. Mouse anti-ShK specific monoclonal antibodies were purified from the conditioned media of the clones. By ELISA analysis, purified anti-ShK polyclonal and monoclonal antibodies reacted only to the ShK peptide alone and did not cross-react with Fc. Pharmacokinetic (PK) Studies 20 kDa-PEG-ShK Peptide Con- jugates in Mice, Rats, Dogs, and Monkeys. Single subcutaneous doses were delivered to animals, and serum was collected at various time points after injection. Studies in rats, dogs (beagles), and cynomolgus monkeys involved two to three animals per dose group, with blood and serum collection occurring at various time points over the course of the study. Male Sprague−Dawley (SD) rats (about 0.3 kg), male beagles (about 10 kg), and male cynomolgus monkeys (about 4 kg) were used in the studies described herein (n = 3 animals per dose group). Approximately 5 male CD-1 mice were used per dose and time point in our mouse pharmacokinetic studies. Serum samples were stored frozen at −80 °C until analysis in an enzyme-linked immunosorbent assay (ELISA). The following is a brief description of the ELISA protocol for detecting serum levels of conjugates 144 and 145 as well as the ShK and 122 peptides alone. Streptavidin microtiter plates were coated with 250 ng/mL biotinylated-anti-ShK mouse monoclonal antibody (mAb2.10, Amgen) in I block buffer [per liter: 1000 mL 1× PBS without CaCl2, MgCl2, 5 mL of Tween 20 (Thermo Scientific), 2 g of I block reagent (Tropix)] at 4 °C and incubated overnight without shaking. Plates were washed three times with KPL wash buffer (Kirkegaard & Perry Laboratories). Standards (STD), quality controls (QC), and sample dilutions were prepared with 100% pooled sera and then diluted 1:5 (pretreatment) in I block buffer. Pretreated STDs, QCs, and samples were added to the washed plate and incubated at room temperature for 2 h. (Serial dilutions of STDs and QCs were prepared in 100% pooled sera. Samples needing dilution were also prepared with 100% pooled sera. The pretreatment was done to STDs, QCs, and samples to minimize the matrix effect.) Plates were washed three times with KPL wash buffer. A HRP-labeled rabbit anti-ShK polyclonal antibody at 250 ng/mL in I block buffer was added, and plates were incubated at room temperature for 1 h with shaking. Plates were again washed three times with KPL wash buffer, and the Femto (Thermo Scientific) substrate was added. The plate was read with a Lmax II 384 (Molecular Devices) luminometer. Adoptive-Transfer EAE Model of Efficacy. An adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis in rats described earlier32 was used to examine the activity in vivo of our Kv1.3-selective 145 analogue and to compare its efficacy to that of the less selective molecule 144. The encephalomyelo- genic CD4+ rat T cell line, PAS, specific for myelin-basic protein (MBP), was kindly provided by Dr. Evelyne Beraud. The maintenance of these cells in vitro and their use in the AT-EAE model has been described.32 PAS T cells were maintained in vitro by alternating rounds of antigen stimulation or activation with MBP and irradiated thymocytes (2 days) and propagation with T cell growth factors (5 days). Activation of PAS T cells (3 × 105 /mL) involved incubating the cells for 2 days with 10 μg/mL MBP and 15 × 106 /mL syngeneic irradiated (3500 rad) thymocytes. On day 2 after in vitro activation, 10−15 × 106 viable PAS T cells were IV injected into 6−12 week old female Lewis rats (Charles River Laboratories) by tail. Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS), 145, 144, or ShK were given from either days −1 to 7 or days −1 to 3, where day −1 represents 1 day prior to injection of PAS T cells (day 0 in Figure 8). Serum was collected by retro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end of the study) for analysis of levels of inhibitor. Rats were weighed on days −1 and 4−8. Animals were scored in a blinded fashion once a day from the day of cell transfer (day 0) to day 3 and twice a day from days 4 to 8. Clinical signs were evaluated as the total score of the degree of paresis of each limb and tail. Clinical scoring (EAE score in Figure 8) was as follows: 0 = no signs, 0.5 = distal limp tail, 1.0 = limp tail, 2.0 = mild paraparesis, ataxia, 3.0 = moderate paraparesis, 3.5 = one hind leg paralysis, 4.0 = complete hind leg paralysis, 5.0 = complete hind leg paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund state or death. Rats reaching a score of 5.5 were euthanized. Pharmacology Study in Cynomolgus Monkey. A repeat-dose pharmacology study was designed and implemented in order to investigate the long-term effects of 145 in nonhuman primates. Prior to initiating the study, 6−10 male cynomolgus monkeys were profiled for a period of 3−10 weeks to allow for assessment of the end points’ stability over time and selection of 6 cynomolgus monkeys for the study. The end points measured included complete blood counts (CBCs), blood chemistry, FACS analysis of lymphocyte subsets, and the ex vivo whole-blood PD assay measuring cytokine response and target coverage. Subsets analyzed by FACS included lymphocytes, CD4+ , CD4+ naïve, CD4+ TCM, CD4+ TEM, CD4+ CD28− CD95− , CD8+ , CD8+ naïve, CD8+ TCM, CD8+ TEM, CD8+ CD28− CD95− , B cells, NK cells, and NKT cells. Monkeys with the highest level of CD4+ effector memory T cells were chosen. The design of the 12 week cynomolgus pharmacology study is illustrated in Supporting Information Table S5. Male Chinese cynomolgus monkeys that were used in this study were naïve (no earlier exposure to drugs). Care was taken to avoid undue stress. All injections and blood draws were done by arm-pull, with the monkeys voluntarily presenting their arm for a grape incentive. The study involved baseline measures for 2 weeks (3 predose samples), 1 month of Kv1.3 block (qw dosing of 145), and 6 weeks follow-up analysis. Ex Vivo Cynomolgus Monkey Whole-Blood Assay To Measure the Potency of 145 and Its Level of Pharmacodynamic Coverage in Vivo. The potency and level of coverage of cynomolgus monkey T cell responses were determined with an ex vivo whole-blood assay measuring thapsigargin-induced IL-4, IL-5, and IL-17. To determine potency of peptides and peptide conjugates, cynomolgus whole blood was obtained from healthy, naïve male monkeys in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Gemini Bioproducts, no. 800-120), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen−Strep−Gln (PSG, Gibco, cat. no. 10378-016). Thapsigargin was obtained from Alomone Laboratories (Jerusalem, Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, cat. no. H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, cat. no. H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Laboratories (Israel). The calcineurin inhibitor cyclosporin A (CsA) is available commercially from a variety of vendors. Whereas the BKCa inhibitor IbTx and the Kv1.1 inhibitor DTX-k do not inhibit the cytokine response, the Kv1.3 inhibitor ShK and the calcineurin inhibitor CsA inhibit the cytokine response and are used routinely as standards or positive controls. Ten 3-fold serial dilutions of standards, ShK analogues, or ShK conjugates were prepared in DMEM complete media at 4× final concentration, Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6798
- 16. and 50 μLof each was added to wells of a 96-well Falcon 3075 flat- bottomed microtiter plate. Whereas columns 1−5 and 7−11 of the microtiter plate contained inhibitors (each row with a separate inhibitor dilution series), 50 μL of DMEM complete media alone was added to the 8 wells in column 6 and 100 μL of DMEM complete media alone was added to the 8 wells in column 12. To initiate the experiment, 100 μL of whole blood was added to each well of the microtiter plate. The plate was then incubated at 37 °C, 5% CO2 for 1 h. After 1 h, the plate was removed, and 50 μL/well of the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate, except the 8 wells in column 12. The plates were placed back at 37 °C, 5% CO2 for 48 h. To determine the amount of IL-4, IL-5, and IL-17 secreted in whole blood, 100 μL of the supernatant (conditioned media) from each well of the 96-well plate was transferred to a storage plate. For Meso Scale Discovery (MSD) electrochemiluminesence analysis of cytokine pro- duction, the supernatants (conditioned media) were tested using MSD multi-spot custom coated plates (Meso Scale Discovery, Gaithersburg, MD). The working electrodes on these plates were coated with seven capture antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFα, hIFNγ, and hIL-17) in advance. After blocking plates with MSD human serum cytokine assay diluent and then washing with PBS containing 0.05% BSA, 25 μL/well of conditioned medium was added to wells of the MSD plate. The plates were covered and placed on a shaking platform for 1 h. Next, 25 μL of a cocktail of detection antibodies in MSD antibody diluent was added to each well. The cocktail contained seven detection antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFα, hIFNγ, and hIL-17) at 1 μg/mL each. The plates were covered and placed on a shaking platform overnight (in the dark). The next morning, the plates were washed three times with PBS buffer. 150 μL of 2× MSD read buffer T was added to wells of the plate before reading on the MSD sector imager. Since the 8 wells in column 6 of each plate received only the thapsigargin stimulus and no inhibitor, the average MSD response here was used to calculate the high value for a plate. The calculated low value for the plate was derived from the average MSD response from the 8 wells in column 12, which contained no thapsigargin stimulus and no inhibitor. Percent of control (POC) is a measure of the response relative to the unstimulated versus stimulated controls, where 100 POC is equivalent to the average response of thapsigargin stimulus alone or the high value. Therefore, 100 POC represents 0% inhibition of the response. In contrast, 0 POC represents 100% inhibition of the response and would be equivalent to the response where no stimulus is given or the low value. To calculate percent of control (POC), the following formula is used: [(MSD response of well) − (low)]/ [(high) − (low)] × 100. The potency of the molecules in whole blood was calculated after curve fitting from the inhibition curve (IC), and IC50 was derived using standard curve fitting software. The experimental procedure for adaption of the above method to an ex vivo cynomolgus monkey pharmacodynamic (PD) assay to measure the level of T cell Kv1.3 coverage in vivo following the dosing of animals is included in the Supporting Information. Repeat-Dose Toxicology Study. Male cynomolgus monkeys (Macaca fascicularis; 2 to 4 years and 2 to 5 kg) were cared for in accordance with the Guide for the Care and Use of Laboratory Animals.41 Animals were individually house in stainless steel cages, except when commingled for environmental enrichment, at an indoor American Association for Accreditation of Laboratory Animal Care (AAALAC) internationally accredited facility in species-specific housing. The research protocol was approved by the Institutional Animal Care and Use Committee. Animals were fed a certified pelleted primate diet (no. 2055C, Harlan Laboratories Inc., Indianapolis, IN) daily in amounts appropriate for the age and size of the animals and had ad libitum access to water via automatic watering system. Animals were maintained on a 12/12 h light/dark cycle in rooms at 18−26 °C and 30−70% humidity and had access to enrichment opportunities, including cage-enrichment devices and commingling. Animals were acclimated for at least 1 week and then randomized to treatment groups to achieve body weight balance with respect to groups. Fifteen animals were placed into 1 of 5 groups (n = 3/dose group) receiving vehicle (10 mM NaOAc, 5% sorbitol, pH 4.0) or 145. Doses of 0.7 mg/kg every third day for 2 weeks (4 doses total) or 0.1, 0.5, or 2.0 mg/kg weekly for 2 weeks (2 doses total) were administered via subcutaneous injection. After 2 weeks, all animals were anesthetized with sodium pentobarbital, exsanguinated, and necropsied. The following study parameters were evaluated: clinical observations, body weights, food consumption, qualitative and quantitative electro- cardiograms (ECG), toxicokinetics, routine clinical pathology (complete blood count, coagulation, and clinical chemistry), urinalysis, a panel of urinary biomarkers of renal injury, organ weights, macroscopic observa- tions, and light microscopic observations of a full set of tissues. Crystallization and Structure Determination of [Lys16]ShK. Crystallization of racemic [Lys16]ShK was performed by mixing equal amounts (by weight) of lyophilized D-[Lys16]ShK and L-[Lys16]ShK in water at 50 mg/mL total concentration (25 mg/mL of D-[Lys16]ShK and 25 mg/mL of L-[Lys16]ShK). Hanging drop cry- stallization screens were set up using JCSG core suites 1−4 (Qiagen) at room temperature. Crystals appeared in several conditions within a week. One condition, 0.1 M sodium acetate, pH 4.5, 2−2.5 M ammonium sulfate, produced diffraction-quality crystals. For data collection, crystals were cryoprotected in mother liquor containing 20% (v/v) glycerol and flash cooled to 100 K. Diffraction data were collected at the Advanced Light Source, beamline 5.0.2 (Lawrence Berkeley National Laboratory, Berkeley, CA), at 100 K. Data were processed and scaled using HKL2000. Attempts to phase the data by molecular replacement using a NMR structure of ShK (PDB: 1ROO)19a were unsuccessful, so material labeled with selenomethionine (SeMet) at position 21 was prepared and used for phasing. Selenomethionine crystals were grown by mixing native D-[Lys16]ShK with L-[Lys16,SeMet21]ShK. The selenomethionine-labeled crystals grew under identical conditions as those for crystals of the unlabeled peptides. Data sets from seleno- methionine-containing crystals were collected at three wavelengths: peak (0.9792 λ), inflection (0.9794 λ), and remote (0.9824 λ).42 The single selenium atom position was determined with Crank in CCP4.43 Crank used SHELX C/D44 for substructure search, SHELX E for substructure refinement, and Solomon45 for density modification. This generated a high-quality map, and a polyalanine model was created using Coot.46 This model was used for molecular replacement into the native data for final refinement. Phaser47 was used for molecular replacement. The space group is R3̅ with a = 60.78 Å, b = 60.78 Å, c = 43.59 Å, and one molecule in the asymmetric unit. After several rounds of model building using Coot and refinement with REFMAC5 in the CCP4 suite, the R factor and Rfree values converged to 20.4 and 22.6%, respectively. Stereochemical analysis of the refined model was performed using Coot validation tools and MolProbity.48 Structural figures were produced using PyMOL (http://www.pymol.org/). The coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank (accession no. 4Z7P). X-ray crystallography data collection and refinement statistics are included in Supporting Information Table S9. Molecular Docking of ShK to Kv1.3 Homology Model. The molecular modeling program MOE49 was used to construct a homo- logy model of the human Kv1.3 channel using the Kv1.2-Kv2.1 paddle chimera channel structure18b (2R9R.pdb) as a template. The [Lys16]ShK crystal structure was docked to the channel homology model using ZDOCK, as implemented in the Discovery Studio 4.1 modeling package.50,51 To simplify the docking, the voltage-sensing domains were excluded from the calculation, and the pore was used as the receptor (residues 381−491). To limit docked poses to the extra- cellular vestibule, receptor residues 381−415, 437−443, and 459−491 were excluded from the peptide−protein interface. An angular step size of 6° and distance cutoff of 5 Å were used, resulting in 54 000 potential poses. The resulting poses were filtered to ensure that residues presumed to be important for binding based on the MAPS analoging ([Lys16]ShK residues 7, 11, 20, 21, 22, 23, and 27) were within 4 Å of any residue on the channel. Additionally, poses were retained only if an atom from the [Lys16]ShK toxin was within 4 Å of the key pore-forming Gly residues 446 and 448 on the Kv1.3 channel. A total of 777 poses passed these criteria and were advanced to further minimization and optimization using RDOCK.52 The top poses were visualized with respect to scanning and selectivity data, including the proximity of Lys16 to residue differences between the Kv1.1 and Kv1.3 channels. Journal of Medicinal Chemistry Article DOI: 10.1021/acs.jmedchem.5b00495 J. Med. Chem. 2015, 58, 6784−6802 6799
- 17. ■ ASSOCIATED CONTENT *SSupporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00495. Characterization of ShK peptide analogues; character- ization and additional pharmacokinetic and pharmaco- logical data for 145 (PDF). ■ AUTHOR INFORMATION Corresponding Authors *(J.K.S.) Phone: 1-805-447-3695. E-mail: jsulliva@amgen.com. *(L.P.M.) Phone: 1-805-447-9397. E-mail: lesm@amgen.com. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS We gratefully thank Ankita Shah, Jason Long, Stephanie Diamond, Ryan Holder, and Jingwen Zhang for peptide synthesis support and Lei Jia and Kaustav Biswas for data visualization. We wish to thank Dr. Evelyn Beraud (Laboratoire d’Immunologie, Faculte de Medecine, Marseille, France) and Dr. Christine Beeton (Baylor College of Medicine) for providing the rat PAS T cell line. The Advanced Light Source is supported by the U.S. Department of Energy under contract no. DE-AC02-05CH11231. ■ ABBREVIATIONS USED AEEA, 2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid; Boc, tert-butoxycarbonyl; Dap, L-2,3-diaminopropionic acid; Fmoc, Nα -9-fluorenylmethoxycarbonyl; 1-Nal, L-1-naphthylalanine; Nle, L-norleucine; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran- 5-sulfonyl; tBu, tert-butyl; Trt, trityl ■ REFERENCES (1) Clare, J. J. Targeting ion channels for drug discovery. Disc. Med. 2010, 9, 253−260. (2) Lee, S.; Hwang, S. W. Peptide neurotoxins targeting voltage-gated ion channels and their therapeutic implications. Front. Clin. Drug Res.: Cent. Nerv. Syst. 2013, 1, 100−165. (3) Kuyucak, S.; Norton, R. S. Computational approaches for designing potent and selective analogs of peptide toxins as novel therapeutics. Future Med. Chem. 2014, 6, 1645−1658. (4) King, G. F. Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opin. Biol. 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