acs.jmedchem.5b00495
19
Pharmaceutical Optimization of Peptide 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 (T EM ), and its expression is increased markedly in activated CD4 + and CD8 + T EM /T EMRA 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 T EM cells without affecting other lymphoid subsets 13 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
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Pharmaceutical Optimization of Peptide Toxins for Ion ChannelTargets: Potent, Selective, and Long-Lived Antagonists of Kv1.3Justin 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 SafetySciences, 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 peptidetoxins, naturally occurring disulfide-rich peptides, as ion channelantagonists, more efficient pharmaceutical optimization techno-logies must be developed. Here, we show that the therapeuticproperties of multiple cysteine toxin peptides can be rapidly andsubstantially improved by combining direct chemical strategieswith high-throughput electrophysiology. We applied whole-molecule, brute-force, structure−activity analoging to ShK, apeptide toxin from the sea anemone Stichodactyla helianthusthat inhibits the voltage-gated potassium ion channel Kv1.3, toeffectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequentsite-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolarfunctional 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 fromT cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.
■ INTRODUCTION
Ion channels are attractive targets for the treatment of humandiseases, but the generation of biologic or small molecule drugsthat potently, selectively, and safely modulate ion channelsremains particularly difficult in contemporary drug discovery.1
Toxin peptides represent a class of potential therapeutics fora range of medical indications mediated by ion channel patho-logy, yet clinical applications have been limited primarily tocases where localized administration has been suitable.2 A majorobstacle to more widespread development of toxin peptideshas been effective methods for engineering compounds withdesirable pharmaceutical properties from novel toxin peptideleads.3
Considerable research has focused on venomous animals thathave evolved a diverse repertoire of toxin peptides for predatoryor defensive capabilities. In some cases, the intended biologicalactivity of these toxin peptides overlaps fortuitously with similarmolecular targets that are of human medicinal relevance.4
ShK (1) is a 35 residue three-disulfide peptide originally isolatedfrom the Caribbean sea anemone Stichodactyla helianthus, whosevenom immobilizes prey by targeting ion channels.5,6 ShKinhibits the voltage-gated potassium ion channel Kv1.1,7 whichhas been shown to be critical for neuronal function in mouseand 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 membranepotential and calcium signaling in human effector memory Tcells (TEM), and its expression is increased markedly in activatedCD4+ and CD8+ TEM/TEMRA T cell populations.10 Blockade ofKv1.3 inhibits the activation of T cells and secretion of cytokinesvia the calcineurin pathway by preventing the potassium effluxnecessary for sustained influx of calcium.11,12 As such, Kv1.3represents a target that selectively suppresses activated TEM
cells without affecting other lymphoid subsets13 and a promisinguntapped approach for the treatment of T cell-mediatedautoimmune diseases, such as multiple sclerosis and rheumatoidarthritis, which afflict millions of people.14 Other potentialdisease indications mediated by Kv1.3 have also beenelucidated.15 For toxin peptides to be safe and well-tolerated,undesirable off-target activities and poor pharmacokineticprofiles, particularly rapidly rising and diminishing circulatinglevels, 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, 2015Published: August 19, 2015
Article
pubs.acs.org/jmc
© 2015 American Chemical Society 6784 DOI: 10.1021/acs.jmedchem.5b00495J. Med. Chem. 2015, 58, 6784−6802
toxin peptide concomitantly with polymeric derivatizatizion thatafforded substantial improvement in well-controlled andsustained circulating levels in vivo. Wild-type ShK is composedof 552 atoms. It is often a challenge with molecules of such sizeand complexity to first identify key toxin peptide−ion channelinteractions and, in turn, to discover critical changes that result inimproved properties.16,17 The major obstacles in this context arethe relative ineffectiveness of de novo design approaches giventhe lack of high-resolution structural data for ion channels18 andthe structural intricacy of peptide toxins.19,20 To date, studies onShK have focused on modification of only a couple of sites toachieve moderate selectivity for Kv1.3 over Kv1.1, a challengingendeavor given their 90% amino acid sequence homology in thepore region.7,9b,21 We hypothesized that an effective and generalroute to develop more complete structure−activity relationships(SAR) for peptide toxins, such as ShK, would be the discretechemical preparation of peptide analogues with substitutions ateach site within the molecule with a panel of residues ranging inphysicochemical properties. Building upon the traditional alaninescan that individually replaces each amino acid (excludingcysteines) with one of low aliphatic bulk,16,22 the process wasrepeated throughout the entire molecule with a large aromatic,an acidic, and two different basic amino acid residues. In all, a setof 132 ShK peptide single substitution analogues was chemicallysynthesized. This is an approach we have termed multi attributepositional scan (MAPS) analoging. While greater diversityhas been explored through combinatorial mixtures in short,two-disulfide peptide sequences,23 this work represents, to ourknowledge, the most extensive positional scanning of a long,three-disulfide peptide in discrete format for systematicoptimization of ion channel selectivity. High-throughput screen-ing of this large set of individually prepared Kv1.3 inhibitorypeptides has been facilitated by recent advances in automatedelectrophysiology methods and platforms, i.e., population patchclamp on the IonWorks Quattro (IWQ) system. From the 132analogues prepared and tested, only two peptides displayedpromising selectivity against Kv1.1 with retention of potentactivity at Kv1.3. One of these lead peptide analogues was furthermodified with a poly(ethylene glycol) polymer (PEG), resultingin a remarkable improvement in selectivity, and studiedpharmacologically in a cynomolgus monkey model examiningT cell activation. Weekly administration of this newly identifiedPEGylated ShK peptide analogue suppressed interleukin-17(IL-17) cytokine secretion from T cells in cynomolgus monkeysand was well-tolerated.
■ RESULTS AND DISCUSSIONShK is a 35 amino acid (Xaa) polypeptide acid with six cysteineresidues participating in three disulfide bonds, giving a (Xaa)2-C1-(Xaa)8-C2-(Xaa)4-C3-(Xaa)10-C2-(Xaa)3-C3-(Xaa)2-C1framework (Figure 1).5 The native ShK peptide has picomolar
inhibitory activity at both Kv1.1 and Kv1.3.7 Earlier reportshave focused on modification of the N-terminus and/or position22 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 selectivityover Kv1.1;7 however, such a change concomitantly andimportantly results in a significant lowering of Kv1.3 bindingaffinity and an approximately 103-fold loss in potency forfunctional inhibition of human T cell activation (vide inf ra).Alternatively, N-terminal extension of ShK with phosphotyrosinederivatives can give 100-fold Kv1.3 over Kv1.1 selectivity,21 butsuch molecules nonetheless have short in vivo half-lives with anundesirable pharmacokinetic profile exemplified by a rapid andlarge shift in peak-to-trough circulating levels.24 In this work,we set out to determine if a systematic analoging approach couldbe used to efficiently identify new sites within this constrainedpeptide scaffold that could be modified to significantly improveselectivity for Kv1.3 while retaining potent T cell inhibitoryactivity. A second key goal of this work was to specifically identifya ShK peptide derivative that, in turn, could be modified with ahalf-life-extending group to give a pharmacokinetic profile suitablefor weekly dosing.
Multi Attribute Positional Scan (MAPS) Analoging ofShK. We sought to preferentially disrupt interactions of the ShKpeptide with neuronal Kv1.1 in a novel manner but to maintainthe desired Kv1.3 inhibitory activity. The absence of reliablein silico methods for predicting peptide compounds with suchactivity profiles led us to adopt a brute-force analoging approachvia direct chemical synthesis. Biological display methodologiescould be pursued as an alternative analoging tactic;25 however,such platforms are not currently suited for the identification offunctionally active and, more importantly, selective ion channelinhibitors 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) wereindividually introduced during direct chemical peptide synthesis.The resultant crude linear peptides were then oxidized toestablish the disulfide connectivity and, in turn, purified andtested. An initial set of 132 discrete peptide analogues wassynthesized with modification at all positions except the cysteineframework residues (Figure 2). Aside from conventional alaninepositional substitutions, which primarily tend to indicate whichresidues in a given peptide are critical for overall activity,the effect of increased steric bulk and aromatic hydrophobicityon ion channel interactions was investigated by systematic1-naphthylalanine (1-Nal) substitution. Even though wild-typeShK is already a highly basic peptide, we also decided toexamine the impact on ion channel interactions of both arginineand lysine positional substitutions. While arginine versus lysineexchanges 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 basicplanar δ-guanido group as compared to the sp3-hybridizedprimary ε-amino functionality of lysine. The opposite electro-statically charged substitution, increased positional acidity, wasaccomplished by positional scanning with glutamic acid. Nearlyall of the theoretical number of ShK peptide analogues for thisapproach could be efficiently prepared, but four analogues couldnot be isolated due to technical difficulties with the disulfidebond formation process. Each prepared peptide was individuallytested for its ability to directly inhibit potassium current inChinese hamster ovary (CHO) cells stably expressing the voltage-activated Kv1.3 or Kv1.1 channel using population patch clampon 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) withthree disulfide bonds formed by six cysteines (C3C35, C12C28, andC17C32).
Journal of Medicinal Chemistry Article
DOI: 10.1021/acs.jmedchem.5b00495J. Med. Chem. 2015, 58, 6784−6802
6785
against Kv1.1 current with an IC50 of 20 ± 29 pM in theseassays (n = 31). This is the first time that the activity of nativeShK is reported on the IWQ perforated patch clamp system.While the Kv1.1 IWQ IC50 is similar to that in reports usingother methods, the Kv1.3 IWQ IC50 is about 10-fold higher thanliterature values.7,16b,17,21b The shift in Kv1.3 potency associatedwith this new assay did not prevent the identification of trendsamong the large number of compounds screened in this high-throughput fashion. The activity of important compounds wassubsequently verified using whole-cell patch clamp electro-physiology, which provided better agreement with publisheddata (vide inf ra).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. Thapsigarginchallenge causes unloading of intracellular calcium stores andinitiation of the calcium signaling pathway in T cells, resultingin IL-2 and IFN-γ secretion.6,24,26 In this whole-blood assayformat, the activity of peptides also can be assessed in terms ofthe molecules’ ex vivo metabolic stability over 48 h. The whole-blood assay is a rigorous assessment of sustained Kv1.3inhibition in comparison to electrophysiology (ePhys) becauseePhys assays are generally of short duration (<1−2 h) and useonly physiologically buffered saline with a low concentrationof bovine serum albumin (BSA) in the absence of proteolytic
enzymes. Furthermore, the 48 h time course of the whole-bloodassay may better reflect equilibrium binding kinetics relative toePhys studies. Accordingly, we used a dual screening approachfor the assessment of the peptide analogues: (1) inhibition ofKv1.3 or Kv1.1 by electrophysiology and (2) the inhibition ofIL-2 and IFN-γ secretion in human whole blood (Table 2 andFigure 2). As expected, native ShK was exceptionally potent inthe thapsigargin-induced whole-blood assay, with an IC50 of37 ± 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 inthe Kv1.3 ePhys assay and <1000 pM in the IL-2 and IFN-γwhole-blood assays.The electrophysiological and whole-blood functional testing
of the five families of ShK analogues, Ala, 1-Nal, Arg, Lys, andGlu substitutions, showed that each series provided interestingand unique results and that together a much more completestructure−activity relationship for ShK may be discerned.First, classical alanine scanning replaces the native side chainfunctionality at each position with a small aliphatic group(methyl) that typically weakens the binding interaction for keypositions within the sequence. The alanine ShK analogue seriesindicated that residue positions 11, 22, 23, and 29 were likely tobe 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 analoguefrom the MAPS analoging. Samples were tested against Kv1.3 and Kv1.1 on the IWQ platform. All values are the average ± SD; n ≥ 2. Colorsindicate IC50 values in each assay, with green indicating highly potent, light green meaning moderately potent, yellow indicating weakly potent, andred signifying not potent. Gray indicates no data because the folded peptide analogue was not isolated. Data for wild-type sequence (1 (ShK) IL-2IC50 = 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 indicatedsubstitution 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.5b00495J. Med. Chem. 2015, 58, 6784−6802
6786
Table
1.Kv1.3
andKv1.1
Inhibitory
Activityof
ShKAnaloguesa
positio
nalsubstitution
alanine
1-naphthylalanine
glutam
icacid
arginine
lysine
ShK
residue
residue
positio
nno.
Kv1.3
IC50
(pM)
Kv1.1
IC50
(pM)
no.
Kv1.3
IC50
(pM)
Kv1.1IC
50(pM)
no.
Kv1.3IC
50(pM)
Kv1.1
IC50
(pM)
no.
Kv1.3IC
50(pM)
Kv1.1
IC50
(pM)
no.
Kv1.3IC
50(pM)
Kv1.1
IC50
(pM)
Arg
12
166±
227.7±
2.2
29223±
316.0±
4.0
56151±
8240
±24
WT
WT
110
154±
293.1±
2.4
Ser
23
156±
163.4±
2.2
30257±
456.0±
5.8
5795
±31
1.5±
0.8
8574
±7
1±
1111
146±
448.7±
6.4
Ile4
4179±
372.7±
0.5
31154±
424.2±
1.0
5875
±12
7.6±
3.5
86110±
297±
4112
153±
314.2±
0.2
Asp
5NDb
ND
32>3
333
>3333
59>3
333
1200
±140
87>3
333
3279
±0
113
>3333
458±
296
Thr
65
226±
297.8±
5.1
33153±
3228
±13
60d
171
25.6
88179±
2746
±10
114
129±
172.6±
1.9
Ile7
6201±
129.5±
1.8
34>3
333
305±
134
611308
±316
32±
489
303±
2917.2
±5.1
115
561±
114
>3333
Pro
87
79±
52.8±
0.8
ND
ND
62d
977.3
90447±
187
4.0±
4.0
116d
835.0
Lys
98
119±
1111.0
±6.4
35472±
4611.0±
4.9
63146±
755.9±
4.8
9196
±12
2.7±
1.9
WT
WT
Ser
109
159±
26308±
9136
216±
480.9±
0.3
64133±
84.2±
5.3
9273
±18
3.0±
2.1
117
184±
203.3±
2.9
Arg
1110
431±
142.0±
1.6
37927±
119
27.4±
15.5
65983±
150
29±
9WT
WT
118
133±
132.4±
1.6
Thr
1311
156±
06.8±
2.2
38d
235
15.7
66152±
364.5±
2.1
93152±
3019.5
±6.4
119
154±
314.4±
3.9
Ala
14WTc
WT
3939
±19
48.9±
13.0
67d
110
7.8
9479
±10
1.8±
1.6
120d
735.0
Phe
1512
85±
165.0±
2.5
4073
±20
10.2±
6.5
68267±
2442
±13
9588
±14
15.4
±4.5
121
294±
2952.7
±4.1
Gln
1613
196±
322.2±
1.7
411444
±247
>3333
69197±
3951
±28
96166±
2855
±57
122
352±
302342
±191
Lys
1814
163±
213.5±
0.1
42475±
396.7±
6.5
70120±
144.0±
1.0
97104±
133.8±
1.7
WT
WT
His
1915
240±
295.3±
2.9
43280±
483.3±
1.7
71256±
213
18.1
9895
±22
2.8±
1.8
123
190±
191.7±
0.1
Ser
2016
195±
1718.2
±9.2
44>3
333
>3333
72>3
333
>3333
99>3
333
>3333
124
193±
342.1±
1.7
Met
2117
227±
171.4±
0.3
45853±
149
26.2±
42.9
73>3
333
490±
120
100
259±
4632.4
±8.2
125
798±
2940.0
±15.6
Lys
2218
456±
63760±
285
46>3
333
>3333
74>3
333
>3333
101
>3333
>3333
WT
WT
Tyr
2319
261±
25>3
333
47>3
333
>3333
75>3
333
>3333
102
>3333
>3333
ND
ND
Arg
2420d
625.0
ND
ND
76d
260
14WT
WT
126d
674.0
Leu
2521
115±
31.4±
0.8
48103±
109.1±
3.3
77245±
5420
±15
103
107±
191.8±
1.4
127
100±
92.8±
2.0
Ser
2622
135±
241.1±
0.7
49207±
411018
±148
78320±
4028
±10
104
278±
4770
±7
128
122±
4933.5
±6.3
Phe
2723
275±
173.4±
2.5
50223±
752420
±1300
79>3
333
1800
±300
105
>3333
>3333
129
985±
215
75±
23Arg
2924
70±
61446±
245
51>3
333
>3333
80143±
161.8±
0.5
WT
WT
130
293±
24266±
46Lys
3025
173±
195.0±
3.0
52161±
163.2±
2.5
81278±
515.7±
2.3
106
38±
78.0±
3.2
WT
WT
Thr
3126d
394.0
53126±
376.1±
3.0
82234±
652.3±
1.1
107
>3333
590±
63131d
1417
174
Gly
3327d
755.0
54d
182
6.1
83d
604.9
108
221±
168
3.6±
0132d
122
5.0
Thr
3428
133±
333.7±
1.5
5577
±5
4.4±
0.0
84d
678.3
109
191±
112
2±
2133
153±
395.1±
0.0
aSamples
tested
onIW
Qplatform
(average
±SD
).bND
indicatesnotdeterm
ined
becausefolded
peptideanalogue
was
notisolated.c W
Tindicatessubstitutioncorrespondsto
wild-typesequence;
ShKKv1.3IC
50=132±79
pMandKv1.1IC
50=20
±29
pM.dPercentinhibitio
nas
afunctio
nof
compoundconcentrationwas
measuredas
anaverageof
four
datapointsperconcentration,andthe
resulting
data
setwas
fitto
produceasingleIC
50curve.
Journal of Medicinal Chemistry Article
DOI: 10.1021/acs.jmedchem.5b00495J. Med. Chem. 2015, 58, 6784−6802
6787
Table
2.Inhibition
ofIL-2
andIFN-γ
Secretionin
Hum
anWho
leBlood
byShKAnaloguesa
positio
nalsubstitution
alanine
1-naphthylalanine
glutam
icacid
arginine
lysine
ShK
residue
residue
positio
nno.
WBIL-2
IC50(pM)
WBIFN-γ
IC50(pM)
no.
WBIL-2
IC50
(pM)
WBIFN-γ
IC50(pM)
no.
WBIL-2
IC50
(pM)
WBIFN-γ
IC50
(pM)
no.
WBIL-2
IC50
(pM)
WBIFN-γ
IC50
(pM)
no.
WBIL-2
IC50(pM)
WBIFN-γ
IC50
(pM)
Arg
12
61±
15111±
4429
222±
21248±
9356
214±
26249±
49WT
WT
110
36±
173
±14
Ser
23
42±
1781
±36
30223±
82258±
208
5729
±2
39±
1185
87±
15116±
52111
99±
394
±22
Ile4
423
±16
46±
4531
274±
62318±
128
5831
±26
50±
3686
98±
61123±
62112
23±
1054
±26
Asp
5NDb
ND
3270
400±
54000
>31900
5916
900±
3670
>24600
8740
500±
9530
91700±
22300
113
>100
000
>100
000
Thr
65
56±
1870
±74
33860±
156
962±
303
6074
±6
109±
5288
215±
46304±
178
114
117±
90210±
95Ile
76
748±
270
1980
±932
3411
600±
202
18000±
2690
615000
±3000
19500±
14000
896560
±1730
10100±
1300
115
4670
±4480
13500±
14700
Pro
87
34±
2666
±19
ND
ND
6272
±6
239±
1690
253±
111
1050
±562
116
154±
35975±
532
Lys
98
45±
1381
±29
35368±
186
666±
3363
37±
1356
±10
9176
±48
83±
45WT
WT
Ser
109
822±
1240
1210
±1730
36205±
41255±
117
6423
±0
55±
1792
72±
50129±
74117
52±
682
±16
Arg
1110
881±
469
2150
±921
371140
±123
1650
±150
651320
±600
4540
±2070
WT
WT
118
95±
69252±
244
Thr
1311
391±
228
795±
290
383560
±305
4860
±1430
66785±
175
1100
±94
93398±
242
635±
173
119
257±
39363±
68Ala
14WTc
WT
3989
±5
110±
1567
36±
2132±
2094
205±
81262±
87120
75±
2201±
88Ph
e15
1241
±22
158±
187
40184±
42291±
151
68312±
11575±
438
95143±
52183±
31121
465±
150
991±
74Gln
1613
54±
1090
±27
4128
900±
29700
9550
±2100
69352±
35373±
310
96372±
350
492±
377
122
108±
45151±
110
Lys
1814
32±
1148
±35
42419±
106
375±
197
7047
±35
83±
4897
38±
3056
±38
WT
WT
His
1915
301±
114
556±
318
43153±
7255±
1171
202±
11216±
298
114±
109
282±
208
123
41±
1789
±23
Ser
2016
251±
69809±
375
44>1
00000
>100
000
72>9
2000
>92000
9925
300±
2060
40300±
27000
124
108±
2193±
108
Met
2117
327±
771260
±1030
456590
±2300
8130
±1220
7318
500±
6000
28100±
20900
100
1490
±479
3080
±1960
125
3590
±2360
4820
±628
Lys
2218
1940
±497
4010
±1720
46>1
00000
>100
000
74>1
00000
>100
000
101
>100
000
>100
000
WT
WT
Tyr
2319
3090
±1190
5670
±1600
475970
±698
11200±
8150
75>1
00000
>100
000
102
>100
000
>100
000
ND
ND
Arg
2420
45±
29186±
51ND
ND
761110
±544
4070
±928
WT
WT
126
45±
2108±
36Leu
2521
199±
15641±
444
4861
±69
84±
5477
494±
88678±
27103
137±
57194±
12127
226±
204
366±
4Ser
2622
65±
14120±
4649
357±
141
573±
456
78202±
13400±
173
104
158±
73368±
248
128
273±
30496±
124
Phe
2723
1500
±1550
8010
±9890
501070
±962
2840
±3020
798160
±3880
34000±
8090
105
13000±
2100
59700±
40500
129
3490
±2150
11800±
8830
Arg
2924
2880
±4440
5300
±8150
514720
±2710
8080
±3640
80730±
301960
±1060
WT
WT
130
41±
871
±15
Lys
3025
186±
229
308±
293
5282
±50
148±
5081
20±
2343
±19
106
40±
3058
±39
WT
WT
Thr
3126
55±
15222±
8553
32±
10129±
3382
242±
262
309±
389
107
11100±
2160
39000±
24000
131
5984
±307
27900±
17700
Gly
3327
59±
19122±
6054
439±
551430
±699
8355
±22
176±
54108
89±
36223±
93132
218±
52318±
128
Thr
3428
53±
2483
±36
5518
±14
63±
3784
73±
23124±
114
109
357±
99475±
142
133
67±
6136±
100
aAverage
±SD
.bNDindicatesnotdeterm
ined
becausefolded
peptideanalogue
was
notisolated.cWTindicatessubstitutioncorrespondsto
wild-typesequence;S
hKIL-2
IC50=37
±36
pMandIFN-γ
IC50=48
±43
pM.
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previously published reports on ShK SAR,7,16 and, importantly,these substitutions, along with positions 7, 10, 21, and 27, alsoresulted in considerably reduced activity in the correspondingwhole-blood IL-2 and IFN-γ secretion assays (red). Within thisseries, 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 cytokinesecretion inhibitory activity for these compounds limited theirutility. Under our assay conditions, the substitution of alaninefor lysine at position 18 (14) did not improve selectivity againstKv1.1 as reported in the literature, perhaps due to differencesin electrophysiology platform (IWQ instead of manual patchclamp) and/or cell line (hKv1.1 expressed in HEK293 cellsinstead of mKv1.1 in mouse L929 fibroblasts), and it wasnot tested by manual electrophysiology.20 In short, classic alaninepositional scanning did not result in improvement in eitherpotency or selectivity, necessitating implementation of our MAPSanaloging methodology.The 1-naphthylalanine positional scan of ShK introduces a
large aromatic side chain in place of the wild-type functionalityto examine the effects of increasing hydrophobicity and stericbulk. This series had the largest number of substitutions thatwere disruptive to the Kv1.3 inhibitory activity of ShK. Kv1.3inhibition was adversely affected by 1-Nal incorporation atpositions 5, 7, 9, 11, 16, 18, 20, 21, 22, 23, and 29. Additionally,activity in the whole-blood assay was diminished by substitutionof 1-Nal at positions 13, 27, and 33. This list includes andadds 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 atposition 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 acidswith charged side chains. The glutamic acid substitution serieshad an activity profile similar to that with 1-Nal, with positions5, 7, 11, 13, 20, 21, 22, 23, 24, 27, and 29 not being welltolerated in either the ePhys or whole-blood assays or both,demonstrating the extensive perturbation caused by integrationof an acidic residue into a highly basic peptide sequence.Furthermore, no compound from the glutamic acid substitutionseries appeared to show any selective inhibition for Kv1.3 overKv1.1. One observation unique to the Glu series was thatsubstitution of the native Arg at position 24 caused a loss offunctional activity in the cytokine secretion assays but retainedactivity in the electrophysiology assays, giving some insight intothe 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 leadfor further optimization and study. First, we found that argininesubstitutions at 5, 7, 8, 20, 21, 22, 23, 27, and 31 resulted insignificant loss of Kv1.3 and/or functional inhibitory activity.Lysine substitutions at positions 5, 7, 21, 27, and 31 had similareffects (Figure 3A). Among the different scans, position 31 wasuniquely sensitive to substitution with a basic residue, havingwhole-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 substitutionanalogues at position 7, 115 ([Lys7]ShK), and position 16, 122([Lys16]ShK), were both 6-fold selective, a 40× improvementover native ShK (Figure 3B). However, only 122 retained potent
whole-blood activity with an IL-2 secretion IC50 of 108 ± 45 pMand IFN-γ secretion IC50 of 151 ± 110 pM (n = 67). Bycomparison, 96 ([Arg16]ShK) had no improvement in selec-tivity for Kv1.3 and instead was an approximately 3-fold morepotent inhibitor of Kv1.1 than Kv1.3. The key features of thesequence−activity relationship from the Lys scan are presentedin Figure 3C.To summarize, application of MAPS analoging to ShK led
to the identification of previously unreported sites for potencyand selectivity not found via traditional Ala scanning efforts
Figure 3. (A−C) Functional activity and electrophysiological selectivityof 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 withresidues important for potency in red and bold, residues important forselectivity in blue and underlined, and residues important for both inpurple, bold, and underlined. Note that substitution of lysine at position16 uniquely enhanced Kv1.3 selectivity with retention of potencyagainst cytokine secretion. (D) Consensus findings from MAPSanaloging of ShK with the residues likely to impact potency throughconformational effects in bold and green, residues indicated asimportant for potency by a single series denoted in orange and bold,and the remainder as described in (C).
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(Figure 3D).16 From an overall perspective, only 2 out of the132 initially prepared ShK analogues, 49 and 122, met thefollowing 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 inwhole blood. These two novel lead compounds, not predicteda priori by computational methods,20 were identified only by asystematic approach that scanned the entire molecule multipletimes 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 numberof positions not immediately apparent from the Ala scan aloneand only by balancing the findings from the electrophysio-logical assays with the results of the whole-blood cytokinesecretion assays. Importantly, selectivity against Kv1.1 wasobtained by substitution at positions not identified as critical for
Kv1.3 inhibitory activity. Aside from a better understanding ofwhich surface regions of ShK are important for activity andselectivity, MAPS analoging also provided a more detailed viewof the SAR at each amino acid position.
Structure−Function Relationships of Kv1.3 InhibitoryToxin Peptides. Using racemic crystallography,27 we wereable to solve the X-ray crystal structure of 122 at 1.2 Å resolu-tion (Figure 4). The peptide analogue consists of an extendedconformation at the N-terminus up to residue 8, followed bytwo interlocking turns and then two short helices encompassingresidues 12−19 and 21−24. Substitution of lysine at position 16had no significant effect on the local conformation relative tothe wild-type ShK structure.19a ShK residues Ile7, Arg11, Ser20,Met21, Lys22, Tyr23, and Phe27, each identified as important forbinding to Kv1.3 by an observed >20× loss in functionalactivity 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 andpotency. (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 tothe Kv1.3 channel has been generally described as a “cork ina bottle”, with ShK inserting the side chain of Lys22 into andphysically occluding the channel pore.10,28 To potentially aidour comprehension of the screening data and the selectivity,the 122 structure was docked to a homology model of theKv1.3 channel. Two poses that place Lys16 of 122 near siteswhere 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 inclose contact with the Kv1.3 channel are shown in Figure 5.Despite the availability of this new set of ShK analogues, it isstill unclear which of the proposed binding modes is mostrelevant; however, complementary ion channel site-directedmutagenesis may assist our understanding of the molecularbasis 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 Kv1.3over Kv1.1: Lys7, Lys16, Ala23, 1-Nal26, 1-Nal27, and Ala29, butonly modification of positions 16 and 26 improved Kv1.3selectivity without significantly compromising Kv1.3 potency orfunctional activity (Figure 4E,F). The native residues Gln16 andSer26 are located at the periphery of the putative binding faceand may interact with a portion of the surface of the channelsthat has some structural or sequence difference between Kv1.3
and Kv1.1. However, the unpredictability of the SAR isdemonstrated by substitution of Arg29 with Ala, which islocated more remotely than either position 16 or 26 but led toan increase in selectivity with concomitant loss in potency andunclear effect on overall peptide conformation. Other residueslocated at the border of the binding face, i.e., Thr6, Ser10, Thr13,Arg24, Thr31, and Gly33, have at least one MAPS analogue witha >20× loss in functional activity in the whole-blood assaywithout improvement in Kv1.3 versus Kv1.1 selectivity. Someeffects on activity may be due to conformational disruption ofthe peptide. For example, substitution of a Lys or Arg residue atposition 31 would place the side chains of three basic residues(Lys9 and Lys30) in close proximity and may affect the globalstructure. While our results serve to refine the list of residues inShK with strong Kv1.3 interactions,20 these data also highlightthe importance of residues at the edge of the peptide bindingsurface. While these peripheral residues are typically ignoredby traditional optimization strategies (i.e., alanine scanningand structure-based design), specific changes in charge orhydrophobicity at these sites may serve to elucidate the natureof their contribution to the complex and effect on ion channelselectivity.
[Lys16]ShK Peptide Analogues. Identification of thepotent and moderately selective Kv1.3 inhibitory peptide 122was 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 homologymodel (green ribbons). (A) Side view of pose I. For clarity, two monomers (II and IV) of the homotetrameric channel have been hidden. (B) Topview 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 ofpose 2.
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combination with modifications to reduce oxidative liabilities(Table 3). Shortening the Lys16 side chain by a methyleneunit to orthinine (Orn) resulted in a similar activity profile(134); however, removal of a second methylene unit withdiaminobutyric acid (Dab) led to loss of Kv1.1 selectivity(135). It is known that amidation of the C-terminal acid of ShKprovides a backbone with similar activity and increasedmetabolic 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 proneto epimerization during solid-phase peptide synthesis thancysteine29 and substitution of the oxidizable methionine withnorleucine at position 21 were investigated in combination withthe lysine substitution at position 16 (Table 3). Surprisingly,addition of a C-terminal alanine to [Lys16]ShK resulted inanalogues 137 and 141 with >150-fold selectivity for Kv1.3versus Kv1.1 that retained good activity in blocking T cellcytokine secretion in human whole blood. The improvements inselectivity associated with substitution of lysine at position 16,hydrophobic substitutions at position 21, and extension of theC-terminus have been verified by Pennington and co-workers,including an additive improvement in selectivity through theirapproach of N-terminal modification.30
Electrophysiology of ShK Peptide Analogues. Furtherelectrophysiological 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 patchclamp, due to the large number of compounds that needed tobe tested. A select number of important analogues were testedon 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 ofboth Kv1.3 and Kv1.1 on the PX system. These values were inreasonable 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 tovalues reported in the literature as well as our results in thecytokine secretion assays.7,16b,17,21b Compound 122 was also apotent inhibitor of Kv1.3 on the PX platform and demonstrated>15× selectivity against Kv1.1. The potency and selectivityprofiles for 142 (ShK-Dap22) and 143 (ShK-L5, SupportingInformation Figure S1), which are commercially available,were compared to the results reported previously for theseanalogues.7,21b Molecule 142 showed a significant loss inwhole-blood functional activity, which motivated us to adoptthis assay for the primary screening of our analogues. As dis-cussed earlier, the whole-blood assay is of longer duration andmay better reflect equilibrium binding of the peptide to thetarget. Indeed, while Kalman et al. reported that 142 showedgood potency by electrophysiology (Kv1.3 IC50 = 23 pM)7
similar to our findings, Middleton et al. reported that itsequilibrium binding affinity for Kv1.3 is more than 100 timesweaker than native ShK.31 These latter results are consistentwith our observations in the 48 h whole-blood assay, where 142had IL-2 and IFN-γ IC50 values >3000 pM. In our assays, 143was a potent inhibitor of both Kv1.3 and Kv1.1 as well ascytokine secretion in human whole blood. The disagreementof our selectivity ratio for 143 with published reports may bedue to our use of a different cell line (hKv1.3 in Chinesehamster ovary (CHO) cells rather than mKv1.3 in mouse L929fibroblasts).7,21b
Impact of Conjugation on Potency, Selectivity, andPharmacokinetics of ShK Analogues. The potent wild-typeShK peptide has a very short circulating pharmacokineticprofile in rats (t1/2 ∼ 20 min).32 The short half-life in vivo ofpeptides is typically attributed to rapid metabolic processingand high renal clearance.33 To investigate whether renalclearance was responsible for the short circulating time ofShK, we attempted PEGylation of the molecule as a means toincrease 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. Weexplored a N-terminal reductive amination approach due to thepresence of multiple lysine residues in ShK derivatives and thedifficulty of using cysteine-maleimide chemistry in disulfide-richpeptides. First, a Nα-PEG-ShK conjugate was prepared byreductive alkylation of the N-terminus with a linear 20 kDamonomethoxy PEG aldehyde at pH 4.5 and then purified.Peptide mapping experiments confirmed PEGylation occurredprimarily at the N-terminus of the peptide (data not shown).Testing of 144 (20 kDa-PEG-ShK) revealed that it retainedsubnanomolar potency in inhibiting Kv1.3 and T cell cytokineresponses (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 48122 [Lys16]ShK 352 2342 6.7 108 151134 [Orn16]ShK 140 740 5.3 138 160135 [Dab16]ShK 82 11 0.13 86 223136 [Lys16]ShK-amide 174 600 3.4 223 278137 [Lys16]ShK-Ala 60 9500 158 138 266138 [Nle21]ShK 40 15 0.38 153 303139 [Lys16,Nle21]ShK 130 29 258 225 249 5678140 [Lys16,Nle21]ShK-amide 153 13 220 86 823 1099141 [Lys16,Nle21]ShK-Ala 71 >33 333 >469 305 515
Table 4. Potency and Selectivity of ShK Analogues
cmpd peptide name
Kv1.3PX IC50(pM)
Kv1.1PX IC50(pM)
Kv1.1IC50/Kv1.3IC50
WBIL-2IC50(pM)
WBIFN-γIC50(pM)
1 ShK 39 87 2.2 37 48122 [Lys16]ShK 207 3677 17.8 108 151142 ShK-Dap22 12a 847a 70.6 3763 3112143 ShK-L5 221 214 1.0 31 46
aIndicates 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 havebeen reported to have increased selectivity for Kv1.3, we alsofound 144 to be 5-fold more potent against Kv1.3 than againstKv1.1. Encouraged by the retention of activity, we nextPEGylated our selective [Lys16]ShK analogue at its N-terminuswith linear PEG. The conjugate 145 (20 kDa-PEG-[Lys16]ShK)was found to provide potent blockade of whole-blood IL-2secretion 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 activitywithout impacting Kv1.3, more than 200× the effect thatPEGylation had on native ShK.30 The potency and selectivity of145 are extraordinary when compared to those of the conjugatedwild-type peptide and unconjugated peptide analogues (Figure 6).
The pharmacokinetics and bioactivity of polypeptides canbe significantly altered through the attachment of PEG groupsof differing molecular weight.36 Aside from derivatization with20 kDa-PEG, the [Lys16]ShK peptide was also prepared witheither a 30 kDa linear PEG or a branched PEG consisting oftwo 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 selectivityfor lymphocyte Kv1.3 over neuronal Kv1.1. The linear 30 kDa-PEG-[Lys16]ShK molecule (147) was also a highly potentinhibitor of cytokine secretion in human whole blood. Theseresults suggest that the 122 peptide scaffold is tolerant ofN-terminal derivatization with PEG polymers of differing sizeand architecture. Conjugation of 122 with linear 20 kDa PEGresults in a slightly higher level of Kv1.3 vs Kv1.1 selectivityrelative 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 channelcounterscreens and against other species. Counter screeningrevealed 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 toserve 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 peptideanalogue 145 had no detectable impact on the calcium-activatedK+ channels KCa3.1 (IKCa1) and BKCa.
Cross-Species Activity of 20 kDa-PEG-[Lys16]ShK. Inaddition to its inhibitory activity in human whole blood, 145also inhibited IL-17 and IL-4 secretion from T cells withincynomolgus monkey whole blood with potent IC50 estimatesof 0.09 ± 0.08 nM and 0.17 ± 0.13 nM, respectively. 145 wasalso a potent inhibitor (IC50 = 0.17 nM) of myelin-specificproliferation of the rat T effector memory cell line, PAS.37
Overall, we found that 145 has consistently potent inhibitoryactivity 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 regardsto 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 0.840145 20 kDa-PEG-[Lys16]ShK 0.94 997 1060 0.092 0.160146 20 kDa-brPEG-[Lys16]ShK 2.10 1574 750 0.198 0.399147 30 kDa-PEG-[Lys16]ShK 1.20 1072 890 0.282 0.491
aManual patch clamp electrophysiology.
Figure 6. Graphical comparison of the potency and selectivity of selectnaked and PEGylated peptide analogues relative to ShK. Each pointrepresents one compound with the x-axis value computed as (whole-blood IL-2 IC50)/(ShK whole-blood IL-2 IC50) and the y-axis valuecomputed as ([Kv1.1 IC50/Kv1.3 IC50]/[ShK Kv1.1 IC50/ShK Kv1.3IC50]).
Table 6. Activity of 145 in Counterscreensa
assay IC50 (nM)
human WB IL-2 0.092Kv1.1 997Kv1.2 639Kv1.3 0.94Kv1.4 >10 000Kv1.5 >30 000Kv1.6 466Kv1.7 >10 000IKCa1 >10 000BKCa >10 000hERG (IKr) >10 000Nav1.5 (INa) >30 000Cav1.2 (ICa) >30 000Kv4.3 (Ito) >30 000KvLQT1/minK (IKs) >30 000
an ≥ 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 thatare much shorter (<1 h) than that of our PEG conjugate. Priorto evaluating the pharmacology of the potent and selectiveconjugate 145, its ex vivo plasma stability and pharmacokineticswere determined. The conjugate was found to have high meta-bolic stability in plasma from rat, cynomolgus monkey, andhuman over 2 days at 37 °C (Supporting Information Figure S5).Pharmacokinetic studies in mouse, rat, dog, and cynomolgusmonkey showed good cross-species metabolic stability in vivowith a considerably extended elimination half-life. Moreover,a comparison of 148 (ShK-186), a more advanced derivative of143 containing a C-terminal amide and displaying improvedstability,21c,24 indicates that 145 has a half-life in cynomolgusmonkeys that was 245 times longer than 148 when the same0.5 mg/kg dose was delivered (Table 7). We estimate theexposure of compound 145 over time, as measured by AUC0−∞,was 390 times greater in cynomolgus monkeys than 148, resultingin a clearance rate that was ∼950 times slower in rats andcynomolgus monkeys compared to the 148 peptide. As shown inFigure 7, 145, when dosed subcutaneously in cynomolgusmonkeys at 0.5 mg/kg, achieved a Cmax at 8 h of 254 nM andday 7 serum levels of 28.4 nM. The serum concentration of 145at day 7 after a single dose was approximately 28 and 315 timesgreater 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 monkeysare consistent with a projected weekly dosing profile in humansubjects. It should be noted that despite our PEG conjugateshowing a profoundly longer half-life in vivo than 148 Tarchaet al. report that this peptide analogue shows durablepharmacological effects in monkeys.24 The authors proposethat, although serum levels decline rapidly over the first few hoursafter injection, there could be a slow release from the injectionsite as well as tight binding and slow dissociation from the Kv1.3channel 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 coverageover a narrower dynamic range of serum drug concentrations.Further details on the pharmacokinetics of 145 administeredsubcutaneously are provided in the Supporting Information.Efficacy, Pharmacodynamics, and Safety of 20 kDa-
PEG-[Lys16]ShK. We evaluated the efficacy of 145 in vivousing the adoptive-transfer experimental autoimmune encepha-lomyelitis (AT-EAE) model in rats.38 In this animal model ofmultiple sclerosis, T cells specific for myelin basic protein (MBP)and constitutively expressing Kv1.3 (PAS cells) are activated andinjected into rats, causing inflammation and demyelination of thecentral nervous system (CNS), with symptoms progressing froma distal limp tail to paralysis over the course of a week. Dosing inrats with the Kv1.3 blocker 145 before the onset of EAE caused adelay in the onset of disease. The progression of disease was alsoinhibited with treatment with 145, with an observed dose-dependent effect on reduced disease severity and the preventionof death (Figure 8). In the vehicle-treated animal group,the disease onset occurred on day 4, but, by comparison, inanimals treated with 145, the disease onset was delayed until day4.5 to 5. On day 6, the vehicle-treated rats had developed severedisease (EAE score of 6) and were sacrificed, whereas 145treated animals (at efficacious doses) had only mild disease(EAE score of ∼1) that resolved over time. The molecule 145blocked AT-EAE in a dose-responsive manner with an estimatedED50 of approximately 4 μg/kg on day 7 (Figure 8 and Table
7.Single
DosePharm
acokinetic
(Sub
cutaneou
s)Profile
of145in
CD1Mice,
SpragueDaw
leyRats,BeagleDogs,And
Cynom
olgusMon
keys
Com
paredto
the
Pharm
acokineticsof
148in
SpragueDaw
leyRatsandCynom
olgusMon
keys
24,a
cpmd
species
dose
(mg/kg)
nt 1/2(h)
Tmax(h)
Cmax(ng/mL)
AUC0−
t(ng·h/mL)
AUC0−
∞(ng·h/mL)
Vz/F(m
L/kg)
CL/F(m
L/h/kg)
MRT(h)
145
mouseb
2.0
314.9
4.0
1860
37000
37000
1170
54.1
16.6
145
rat
2.0
3N/A
40±
14531±
9021
900±
2770
21900±
2760
N/A
92±
1336
±2
145
beagle
0.5
342.6±
4.21
18.7±
9.24
1270
±347
95200±
31300
103000±
37300
322±
985.37
±2.14
66.1±
13.5
145
cyno
0.5
364.5±
14.9
8.0
1010
±105
71500±
607
74900±
3260
621±
143
6.68
±0.29
87±
16148c
rat
1.0
30.132
0.083
48NR
11.5
NR
87198
NR
148c
cyno
0.5
20.263
0.083
192
NR
192
NR
6336
NR
aBecause
onlythepeptideportionof
145was
used
incalculatingmg/mLstockconcentrations
andthe[Lys16]ShK
peptideportion(4055Da)
isof
similarMW
to148,equivalent
mg/kg
dosesof
these
twomolecules
generate
similarnm
ol/kgdoses.bSparse
samplingPK
experim
ent.Nostandard
deviations
werecalculated
forPK
parameters.c From
Tarchaet
al.24NR=notreported.
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Supporting Information Figure S6). In a separate study, anequivalent dose (0.01 mg/kg) of PEGylated ShK (144) wasfound to provide greater efficacy in blocking encephalomyelitisthan that of the native ShK peptide (Supporting InformationFigure S7) that has a shorter half-life in animals. Overall, these
data suggest that higher levels of sustained Kv1.3 target coverageappear to result in greater efficacy in this model.The in vivo pharmacodynamics of 145 in cynomolgus
monkeys was also examined. A 12 week pharmacology studywas initiated with three predose baseline measurements duringthe first 2 weeks. This was then followed by four weekly sub-cutaneous doses of 145 at 0.5 mg/kg and an additional 6 weeksof postdosing analysis (Figure 9 and Supporting InformationTables S5−S7). On the basis of earlier pharmacokineticstudies of 145, target coverage with 0.5 mg/kg weekly dosingwas expected to range from 28-times the IL-17 IC95 at theminimum drug concentration in plasma (Cmin) to 249-times IC95at the peak or maximum drug concentration (Cmax). The repeatdosing of the conjugate 145 was well-tolerated. In terms ofgeneral observations, weight gain was normal throughout thestudy, and complete blood counts (CBCs) and blood chemistrywere also found to be normal with respect to predose baselineestimates. Using the cynomolgus monkey whole-blood IL-17pharmacodynamic assay, suppression of T cell inflammation wasachieved during the 4 week dosing period. In terms of repeatdrug exposure, the predicted and observed serum drug troughlevels 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 thirdday (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 anyparameters evaluated. Specifically, there were no PEG-associatedvacuoles observed in renal tubules or tissue macrophages bylight microscopy. On the basis of the absence of adversetoxicity, the no-observed-adverse-effect level (NOAEL) in thisstudy was 2 mg/kg, which correlated with a mean AUC0−168h of584 000 ng·h/mL.
■ CONCLUSIONSThe diverse array of potent biological activities and inherentmetabolic stability of toxin peptides make this class of mole-cules an attractive starting point for drug discovery of ionchannel modulators. We have demonstrated the effectiveness ofthe multi attribute positional scan (MAPS) analoging method-ology to identify potent and subtype-selective analogues ofthe ShK peptide toxin. By scanning the peptide sequence withnot only the traditional Ala residue but also representativebasic, acidic, and hydrophobic residues and screening theresulting >130 analogues via high-throughput electrophysiol-ogy, [Lys16]ShK emerged as a potent antagonist of Kv1.3 withimproved selectivity over Kv1.1.39 Combination with N-terminalconjugation of a 20 kDa poly(ethylene glycol) polymer resultedin an unexpected synergistic increase in Kv1.3 versus Kv1.1selectivity to 1000-fold, with retention of picomolar potency inthe whole-blood T cell assay and prolongation of the half-lifein vivo. A clean selectivity profile against a panel of ion channelsand good plasma stability made 20 kDa-PEG-[Lys16]ShKsuitable for rodent and primate PD studies. Compound 145 wasefficacious in the rat adoptive transfer-experimental autoimmuneencephalitis (AT-EAE) model of multiple sclerosis. The pharma-cokinetic profile of this compound was suitable for weeklydosing in cynomologous monkeys, and it showed suppression ofT cell-mediated inflammation during a 1 month repeat-dosingexperiment without adverse side effects. Through prolongedblockade of Kv1.3 in vivo, 145 or related analogues may allowfurther 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 assayresults: 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 autoimmuneencephalomyelitis in a rat AT-EAE model. The PEGylated ShK or[Lys16]ShK conjugates were delivered subcutaneously (SC) daily fromdays −1 to 7. The rat CD4+ myelin-specific effector memory T cells line,PAS, was delivered by intravenous injection on day 0. The rats weremonitored 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. ClinicalEAE 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 = completehind leg paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribundstate or death. Rats reaching a score of 5.5 were euthanized. Error barsrepresent the standard error of the mean.
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other applications in our laboratory, it appears the MAPSanaloging strategy will be useful to multiple classes of toxinpeptides for ion channel targets as well as small syntheticallyaccessible protein scaffolds for other types of targets.
■ EXPERIMENTAL METHODSPeptide Preparation. Peptide Synthesis. Nα-Fmoc, side chain-
protected amino acids and H-Cys(Trt)-2Cl-Trt resin were purchasedfrom Novabiochem, Bachem, or Sigma-Aldrich. The following sidechain 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 acidsequences were synthesized in a stepwise manner on a CS Bio 336peptide synthesizer by Fmoc-SPPS using DIC/HOBt coupling chemistryat 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 added1.0 mL of 1.0 M N,N′-diisopropylcarbodiimide (DIC) in DMF. Thesolution was agitated with nitrogen bubbling for 15 min to accomplishpreactivation and then added to the resin. The mixture was shakenfor 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 inDMF (5 mL, 2 × 15 min). The first 23 residues were single-coupled
through repetition of the Fmoc-amino acid coupling and Fmoc removalsteps described above. The remaining residues were double-coupledby performing the coupling step twice before proceeding with Fmocremoval.
Following synthesis, the resin was drained, washed sequentially withDCM, DMF, and DCM, and then dried in vacuo. The peptide-resinwas transferred to a 250 mL plastic round-bottomed flask. The peptidewas deprotected and cleaved from the resin by treatment withtriisopropylsilane (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 themixture was stirred for 3 h. The mixture was filtered through a 150 mLsintered glass funnel into a 250 mL plastic round-bottomed flask, and thefiltrate was concentrated in vacuo. The crude peptide was precipitatedby 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 from0.2 mmol), for example, [Lys16]ShK peptide, was dissolved in 16 mLof acetic acid, 64 mL of water, and 40 mL of acetonitrile. The mixturewas stirred rapidly for 15 min to complete dissolution. The peptidesolution was added to a 2 L plastic bottle that contained 1700 mL ofwater and a large stir bar. To the diluted peptide solution was added20 mL of concentrated ammonium hydroxide to increase the pH ofthe solution to 9.5. The pH was adjusted with small amounts of aceticacid or NH4OH as necessary. The solution was stirred at 80 rpmovernight and monitored by LC-MS. Folding was usually judged to becomplete 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 sustainedsuppression of T cell responses, as measured using the ex vivo cynomolgus monkey whole-blood PD assay of inflammation that measured productionof IL-4 (A) and IL-17 (B). Arrows indicate the weekly doses. Each line represents an individual test subject. (C) Predicted versus measured serumconcentrations of 145 in cynomolgus monkeys after weekly subcutaneous (SC) dosing (0.5 mg/kg, n = 6). The measured serum trough levels afterweekly 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 achievedusing linear gradients of buffer B in A (A = 0.1% aqueous TFA; B =90% aq. acetonitrile containing 0.09% TFA), typically 5−65% over90 min at 20 mL/min for preparative separations. Preparative HPLCfractions were characterized by ESMS and photodiode array (PDA)HPLC, combined, and lyophilized. Final analysis (PhenomenexSynergi MAX-RP 2.5 μm, 100 Å, 50 × 2.0 mm column eluted witha 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 absorbanceat 220 nm) was performed for each peptide sample using an Agilent1290 LC-MS. Peptides with >95% purity and correct (m/z) ratiowere screened. (See Supporting Information Table S8 for LC-MScharacterization of ShK and peptide analogues.)PEGylation, Purification, and Analysis. Peptide, for example,
[Lys16]ShK, was selectively PEGylated by reductive alkylation at itsN-terminus using activated linear or branched PEG. Conjugation wasperformed at 2 mg/mL in 50 mM NaH2PO4, pH 4.5, reaction buffercontaining 20 mM sodium cyanoborohydride and a 2 molar excessof 20 kDa monomethoxy-PEG-aldehyde (NOF, Japan). Conjugationreactions were stirred for approximately 5 h at room temperature, andtheir progress was monitored by RP-HPLC. Completed reactions werequenched by 4-fold dilution with 20 mM NaOAc, pH 4, and chilled to4 °C. The PEG-peptides were then purified chromatographically at40 °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 andRP-HPLC and pooling determined by purity >97%. Principlecontaminants observed were di-PEGylated toxin peptide analogue.Selected pools were concentrated to 2−5 mg/mL by centrifugalfiltration against 3 kDa MWCO membranes and dialyzed into 10 mMNaOAc, pH 4, with 5% sorbitol. Dialyzed pools were then sterilefiltered through 0.2 μm filters, and purity was determined to be >97%by SDS-PAGE and RP-HPLC (see Supporting Information Figures S2and S3). Reverse-phase HPLC was performed on an Agilent 1100model 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 monitoringat 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 expressinghuman Kv1.3 or human Kv1.1. Cell lines were from Amgen or BioFocusDPI (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 amplifierfrom Molecular Devices Corp. (Sunnyvale, CA), with 3−5 MΩ pipettespulled from borosilicate glass (World Precision Instruments, Inc.).During data acquisition, capacitive currents were canceled by analoguesubtraction, no series resistance compensation was used, and allcurrents were filtered at 2 kHz. The cells were bathed in an extracellularsolution containing 1.8 mM CaCl2, 5 mM KCl, 135 mM NaCl, 5 mMglucose, 10 mM HEPES, pH 7.4, 290−300 mOsm. The internalsolution contained 90 mM KCl, 40 mM KF, 10 mM NaCl, 1 mMMgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2, 290−300 mOsm. Thecurrents 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 intervalsat a holding potential of −80 mV. To determine IC50, 5−6 peptide orpeptide conjugate concentrations at 1:3 dilutions were made inextracellular solution with 0.1% BSA and delivered locally to cells withRapid Solution Changer RSc-160 (BioLogic Science Instruments).Currents were achieved to steady state for each concentration. Dataanalysis 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 inhibitionat each concentration.
PatchXpress Planar Patch Clamp Electrophysiology. Cells werebathed in an extracellular solution containing 1.8 mM CaCl2, 5 mMKCl, 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. Thepeptide or peptide conjugates were prepared in extracellular solutioncontaining 0.1% BSA. Dendrotoxin-k and Margatoxin were purchasedfrom Alomone Laboratories Ltd. (Jerusalem, Israel); ShK toxin waspurchased from Bachem Bioscience, Inc. (King of Prussia, PA); 4-APwas purchased from Sigma-Aldrich Corp. (St. Louis, MO). Currentswere 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 theSupporting Information Table S1. An extracellular solution with 0.1%BSA was applied first to obtain 100% percent of control (POC), whichwas then followed by 5 different concentrations of 1:3 peptide orpeptide conjugate dilutions for every 400 ms incubation time. At theend, an excess of a specific benchmark ion channel inhibitor was addedto define full or 100% blockage. The residual current present afteraddition of benchmark inhibitor was used in some cases for calculationof zero percent of control. Each individual set of traces or trial werevisually inspected and either accepted or rejected. The general criteriafor 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 firstcompound addition. The POC was calculated from the average peakcurrent of the last 5 sweeps before the next concentration of compoundwas added and exported to Excel for IC50 calculation.
IonWorks Quattro High-Throughput Population Patch ClampElectrophysiology. Electrophysiology was performed on CHO cellsstably expressing hKv1.3 and HEK293 cells stably expressing hKv1.1.The procedure for preparation of the assay plate containing ShKanalogues and conjugates for IWQ electrophysiology was as follows:all analogues were dissolved in extracellular buffer (PBS, with 0.9 mMCa2+ and 0.5 mM Mg2+) with 0.3% BSA and dispensed in row H of96-well polypropylene plates at a concentration of 100 nM fromcolumns 1−10. Columns 11 and 12 were reserved for negative andpositive controls. Serial dilutions at a 1:3 ratio were then made to rowA. IonWorks Quattro (IWQ) electrophysiology and data analysis wereaccomplished as follows: resuspended cells (in extracellular buffer), theassay plate, a population patch clamp (PPC) PatchPlate as well asappropriate 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. Whenthe analogues were added to patch plates, they were further diluted3-fold from the assay plate to achieve a final test concentration rangefrom 33.3 nM to 15 pM with 0.1% BSA. Electrophysiology recordingswere made from the CHO-Kv1.3 and HEK-Kv1.1 cells using anamphotericin-based perforated patch clamp method. Using the voltageclamp circuitry of the IonWorks Quattro, cells were held at a membranepotential of −80 mV, and voltage-activated K+ currents were evoked bystepping the membrane potential to +30 mV for 400 ms. K+ currentswere evoked under control conditions, i.e., in the absence of inhibitorat the beginning of the experiment and after 10 min incubation in thepresence of the analogues and controls. The mean K+ current amplitudewas measured between 430 and 440 ms, and the data were exportedto a Microsoft Excel spreadsheet. The amplitude of the K+ current in thepresence of each concentration of the analogues and controls wasexpressed as a percentage of the K+ current of the precompound currentamplitude in the same well. When these percent of control values wereplotted as a function of concentration, the IC50 value for each compoundcould be calculated using the dose−response fit model 201 in Excel fitprogram.
Measuring Bioactivity in Whole Blood. Ex Vivo Assay toExamine Impact of Toxin Peptide Analogue Kv1.3 Inhibitors onSecretion 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 bloodwas examined using an ex vivo assay that has been described earlier.40
In brief, 50% human whole blood is stimulated with thapsigargin toinduce 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 peptideconjugates were preincubated with the human whole-blood sample for30−60 min prior to addition of the thapsigargin stimulus. After 48 h at37 °C and 5% CO2, conditioned medium was collected and the level ofcytokine 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 robustlyfrom blood isolated from multiple donors. The IL-2 and IFN-γproduced in human whole blood following thapsigargin stimulation wereproduced from T cells, as revealed by intracellular cytokine staining andfluorescence-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 anFc-ShK peptibody conjugate.39 Anti-ShK specific polyclonal antibodieswere affinity-purified from antisera to isolate only those antibodiesspecific for the ShK portion of the conjugate. Following fusion andscreening, hybridomas specific for ShK were selected and isolated.Mouse anti-ShK specific monoclonal antibodies were purified from theconditioned media of the clones. By ELISA analysis, purified anti-ShKpolyclonal and monoclonal antibodies reacted only to the ShK peptidealone and did not cross-react with Fc.Pharmacokinetic (PK) Studies 20 kDa-PEG-ShK Peptide Con-
jugates in Mice, Rats, Dogs, and Monkeys. Single subcutaneousdoses were delivered to animals, and serum was collected at various timepoints after injection. Studies in rats, dogs (beagles), and cynomolgusmonkeys involved two to three animals per dose group, with blood andserum collection occurring at various time points over the course of thestudy. Male Sprague−Dawley (SD) rats (about 0.3 kg), male beagles(about 10 kg), and male cynomolgus monkeys (about 4 kg) were usedin the studies described herein (n = 3 animals per dose group).Approximately 5 male CD-1 mice were used per dose and time point inour mouse pharmacokinetic studies. Serum samples were stored frozenat −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 ShKand 122 peptides alone. Streptavidin microtiter plates were coatedwith 250 ng/mL biotinylated-anti-ShK mouse monoclonal antibody(mAb2.10, Amgen) in I block buffer [per liter: 1000 mL 1× PBSwithout CaCl2, MgCl2, 5 mL of Tween 20 (Thermo Scientific), 2 g ofI block reagent (Tropix)] at 4 °C and incubated overnight withoutshaking. 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 andthen diluted 1:5 (pretreatment) in I block buffer. Pretreated STDs,QCs, and samples were added to the washed plate and incubated atroom temperature for 2 h. (Serial dilutions of STDs and QCs wereprepared in 100% pooled sera. Samples needing dilution were alsoprepared with 100% pooled sera. The pretreatment was done to STDs,QCs, and samples to minimize the matrix effect.) Plates were washedthree times with KPL wash buffer. A HRP-labeled rabbit anti-ShKpolyclonal antibody at 250 ng/mL in I block buffer was added, andplates were incubated at room temperature for 1 h with shaking. Plateswere again washed three times with KPL wash buffer, and the Femto(Thermo Scientific) substrate was added. The plate was read with aLmax II 384 (Molecular Devices) luminometer.Adoptive-Transfer EAE Model of Efficacy. An adoptive transfer
experimental autoimmune encephalomyelitis (AT-EAE) model ofmultiple sclerosis in rats described earlier32 was used to examine theactivity in vivo of our Kv1.3-selective 145 analogue and to compare itsefficacy 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 thesecells 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 antigenstimulation or activation with MBP and irradiated thymocytes (2 days)and propagation with T cell growth factors (5 days). Activation ofPAS T cells (3 × 105/mL) involved incubating the cells for 2 days with10 μg/mL MBP and 15 × 106/mL syngeneic irradiated (3500 rad)thymocytes. On day 2 after in vitro activation, 10−15 × 106 viable PAST cells were IV injected into 6−12 week old female Lewis rats (CharlesRiver Laboratories) by tail. Daily subcutaneous injections of vehicle(2% Lewis rat serum in PBS), 145, 144, or ShK were given from eitherdays −1 to 7 or days −1 to 3, where day −1 represents 1 day prior toinjection of PAS T cells (day 0 in Figure 8). Serum was collected byretro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end ofthe 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 fromthe 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 paresisof each limb and tail. Clinical scoring (EAE score in Figure 8) was asfollows: 0 = no signs, 0.5 = distal limp tail, 1.0 = limp tail, 2.0 = mildparaparesis, ataxia, 3.0 = moderate paraparesis, 3.5 = one hind legparalysis, 4.0 = complete hind leg paralysis, 5.0 = complete hind legparalysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund state ordeath. Rats reaching a score of 5.5 were euthanized.
Pharmacology Study in Cynomolgus Monkey. A repeat-dosepharmacology study was designed and implemented in order toinvestigate the long-term effects of 145 in nonhuman primates. Priorto initiating the study, 6−10 male cynomolgus monkeys were profiledfor a period of 3−10 weeks to allow for assessment of the end points’stability over time and selection of 6 cynomolgus monkeys for thestudy. The end points measured included complete blood counts(CBCs), blood chemistry, FACS analysis of lymphocyte subsets, andthe ex vivo whole-blood PD assay measuring cytokine response andtarget coverage. Subsets analyzed by FACS included lymphocytes,CD4+, CD4+ naive, CD4+ TCM, CD4+ TEM, CD4+CD28−CD95−,CD8+, CD8+ naive, CD8+ TCM, CD8+ TEM, CD8+CD28−CD95−,B cells, NK cells, and NKT cells. Monkeys with the highest level ofCD4+ effector memory T cells were chosen. The design of the 12 weekcynomolgus pharmacology study is illustrated in SupportingInformation Table S5. Male Chinese cynomolgus monkeys that wereused in this study were naive (no earlier exposure to drugs). Care wastaken to avoid undue stress. All injections and blood draws were doneby arm-pull, with the monkeys voluntarily presenting their arm fora grape incentive. The study involved baseline measures for 2 weeks(3 predose samples), 1 month of Kv1.3 block (qw dosing of 145), and6 weeks follow-up analysis.
Ex Vivo Cynomolgus Monkey Whole-Blood Assay To Measurethe Potency of 145 and Its Level of Pharmacodynamic Coverage inVivo. The potency and level of coverage of cynomolgus monkey T cellresponses were determined with an ex vivo whole-blood assaymeasuring thapsigargin-induced IL-4, IL-5, and IL-17. To determinepotency of peptides and peptide conjugates, cynomolgus whole bloodwas obtained from healthy, naive male monkeys in a heparin vacutainer.DMEM complete media was Iscoves DMEM (with L-glutamine and25 mM Hepes buffer) containing 0.1% human albumin (GeminiBioproducts, no. 800-120), 55 μM 2-mercaptoethanol (Gibco), and1× Pen−Strep−Gln (PSG, Gibco, cat. no. 10378-016). Thapsigarginwas obtained from Alomone Laboratories (Jerusalem, Israel). A 10 mMstock solution of thapsigargin in 100% DMSO was diluted with DMEMcomplete media to a 40 μM, 4× solution to provide the 4× thapsigarginstimulus for calcium mobilization. The Kv1.3 inhibitor peptideShK (Stichodacytla helianthus toxin, cat. no. H2358) and the BKCa1inhibitor peptide IbTx (Iberiotoxin, cat. no. H9940) were purchasedfrom Bachem Biosciences, whereas the Kv1.1 inhibitor peptideDTX-k (Dendrotoxin-K) was from Alomone Laboratories (Israel).The calcineurin inhibitor cyclosporin A (CsA) is available commerciallyfrom a variety of vendors. Whereas the BKCa inhibitor IbTx and theKv1.1 inhibitor DTX-k do not inhibit the cytokine response, the Kv1.3inhibitor ShK and the calcineurin inhibitor CsA inhibit the cytokineresponse and are used routinely as standards or positive controls. Ten3-fold serial dilutions of standards, ShK analogues, or ShK conjugateswere prepared in DMEM complete media at 4× final concentration,
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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 themicrotiter plate contained inhibitors (each row with a separate inhibitordilution series), 50 μL of DMEM complete media alone was added tothe 8 wells in column 6 and 100 μL of DMEM complete media alonewas 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, theplate 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 incolumn 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 wholeblood, 100 μL of the supernatant (conditioned media) from each wellof the 96-well plate was transferred to a storage plate. For Meso ScaleDiscovery (MSD) electrochemiluminesence analysis of cytokine pro-duction, the supernatants (conditioned media) were tested using MSDmulti-spot custom coated plates (Meso Scale Discovery, Gaithersburg,MD). The working electrodes on these plates were coated with sevencapture antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFα, hIFNγ, andhIL-17) in advance. After blocking plates with MSD human serumcytokine assay diluent and then washing with PBS containing 0.05%BSA, 25 μL/well of conditioned medium was added to wells of theMSD plate. The plates were covered and placed on a shaking platformfor 1 h. Next, 25 μL of a cocktail of detection antibodies in MSDantibody diluent was added to each well. The cocktail contained sevendetection antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFα, hIFNγ, andhIL-17) at 1 μg/mL each. The plates were covered and placed on ashaking platform overnight (in the dark). The next morning, the plateswere washed three times with PBS buffer. 150 μL of 2× MSD readbuffer T was added to wells of the plate before reading on the MSDsector imager. Since the 8 wells in column 6 of each plate received onlythe thapsigargin stimulus and no inhibitor, the average MSD responsehere was used to calculate the high value for a plate. The calculated lowvalue for the plate was derived from the average MSD response fromthe 8 wells in column 12, which contained no thapsigargin stimulus andno inhibitor. Percent of control (POC) is a measure of the responserelative to the unstimulated versus stimulated controls, where 100 POCis equivalent to the average response of thapsigargin stimulus aloneor the high value. Therefore, 100 POC represents 0% inhibition ofthe response. In contrast, 0 POC represents 100% inhibition of theresponse and would be equivalent to the response where no stimulus isgiven or the low value. To calculate percent of control (POC), thefollowing formula is used: [(MSD response of well) − (low)]/[(high) − (low)] × 100. The potency of the molecules in wholeblood was calculated after curve fitting from the inhibition curve(IC), and IC50 was derived using standard curve fitting software. Theexperimental procedure for adaption of the above method to an ex vivocynomolgus monkey pharmacodynamic (PD) assay to measure thelevel of T cell Kv1.3 coverage in vivo following the dosing of animals isincluded 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 inaccordance with the Guide for the Care and Use of LaboratoryAnimals.41 Animals were individually house in stainless steel cages,except when commingled for environmental enrichment, at an indoorAmerican Association for Accreditation of Laboratory Animal Care(AAALAC) internationally accredited facility in species-specific housing.The research protocol was approved by the Institutional Animal Careand Use Committee. Animals were fed a certified pelleted primate diet(no. 2055C, Harlan Laboratories Inc., Indianapolis, IN) daily in amountsappropriate for the age and size of the animals and had ad libitum accessto water via automatic watering system. Animals were maintained ona 12/12 h light/dark cycle in rooms at 18−26 °C and 30−70% humidityand had access to enrichment opportunities, including cage-enrichmentdevices and commingling.Animals were acclimated for at least 1 week and then randomized
to treatment groups to achieve body weight balance with respect togroups. Fifteen animals were placed into 1 of 5 groups (n = 3/dosegroup) 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) wereadministered via subcutaneous injection. After 2 weeks, all animals wereanesthetized 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 (completeblood count, coagulation, and clinical chemistry), urinalysis, a panel ofurinary 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 equalamounts (by weight) of lyophilized D-[Lys16]ShK and L-[Lys16]ShKin water at 50 mg/mL total concentration (25 mg/mL ofD-[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 withina week. One condition, 0.1 M sodium acetate, pH 4.5, 2−2.5 Mammonium sulfate, produced diffraction-quality crystals. For datacollection, crystals were cryoprotected in mother liquor containing 20%(v/v) glycerol and flash cooled to 100 K. Diffraction data were collectedat the Advanced Light Source, beamline 5.0.2 (Lawrence BerkeleyNational Laboratory, Berkeley, CA), at 100 K. Data were processedand scaled using HKL2000. Attempts to phase the data by molecularreplacement using a NMR structure of ShK (PDB: 1ROO)19a wereunsuccessful, so material labeled with selenomethionine (SeMet) atposition 21 was prepared and used for phasing. Selenomethionine crystalswere grown by mixing native D-[Lys16]ShK with L-[Lys16,SeMet21]ShK.The selenomethionine-labeled crystals grew under identical conditionsas 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 inCCP4.43 Crank used SHELX C/D44 for substructure search, SHELX Efor substructure refinement, and Solomon45 for density modification.This generated a high-quality map, and a polyalanine model was createdusing Coot.46 This model was used for molecular replacement into thenative data for final refinement. Phaser47 was used for molecularreplacement. The space group is R3 with a = 60.78 Å, b = 60.78 Å,c = 43.59 Å, and one molecule in the asymmetric unit. After severalrounds of model building using Coot and refinement with REFMAC5in the CCP4 suite, the R factor and Rfree values converged to 20.4 and22.6%, respectively. Stereochemical analysis of the refined model wasperformed using Coot validation tools and MolProbity.48 Structuralfigures were produced using PyMOL (http://www.pymol.org/).The coordinates and structure factors have been deposited in theBrookhaven Protein Data Bank (accession no. 4Z7P). X-raycrystallography data collection and refinement statistics are includedin Supporting Information Table S9.
Molecular Docking of ShK to Kv1.3 Homology Model. Themolecular modeling program MOE49 was used to construct a homo-logy model of the human Kv1.3 channel using the Kv1.2-Kv2.1 paddlechimera channel structure18b (2R9R.pdb) as a template. The[Lys16]ShK crystal structure was docked to the channel homologymodel using ZDOCK, as implemented in the Discovery Studio 4.1modeling package.50,51 To simplify the docking, the voltage-sensingdomains were excluded from the calculation, and the pore was used asthe receptor (residues 381−491). To limit docked poses to the extra-cellular vestibule, receptor residues 381−415, 437−443, and 459−491were excluded from the peptide−protein interface. An angular step size of6° and distance cutoff of 5 Å were used, resulting in 54 000 potentialposes. The resulting poses were filtered to ensure that residues presumedto be important for binding based on the MAPS analoging ([Lys16]ShKresidues 7, 11, 20, 21, 22, 23, and 27) were within 4 Å of any residue onthe channel. Additionally, poses were retained only if an atom from the[Lys16]ShK toxin was within 4 Å of the key pore-forming Gly residues446 and 448 on the Kv1.3 channel. A total of 777 poses passed thesecriteria and were advanced to further minimization and optimizationusing RDOCK.52 The top poses were visualized with respect to scanningand selectivity data, including the proximity of Lys16 to residuedifferences between the Kv1.1 and Kv1.3 channels.
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■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on the ACSPublications 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 INFORMATIONCorresponding Authors*(J.K.S.) Phone: 1-805-447-3695. E-mail: [email protected].*(L.P.M.) Phone: 1-805-447-9397. E-mail: [email protected] authors declare no competing financial interest.
■ ACKNOWLEDGMENTSWe gratefully thank Ankita Shah, Jason Long, StephanieDiamond, Ryan Holder, and Jingwen Zhang for peptidesynthesis support and Lei Jia and Kaustav Biswas for datavisualization. We wish to thank Dr. Evelyn Beraud (Laboratoired’Immunologie, Faculte de Medecine, Marseille, France) andDr. Christine Beeton (Baylor College of Medicine) forproviding the rat PAS T cell line. The Advanced Light Sourceis supported by the U.S. Department of Energy under contractno. DE-AC02-05CH11231.
■ ABBREVIATIONS USEDAEEA, 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
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