Pharmaceutical Optimization of Peptide Toxins for Ion Channel

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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 G. Mcgivern, Colin V Gegg, John K Sullivan, and Les P Miranda J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00495 • Publication Date (Web): 19 Aug 2015 Downloaded from http://pubs.acs.org on August 25, 2015

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Journal of Medicinal Chemistry

Pharmaceutical Optimization of Peptide Toxins for Ion Channel Targets: Potent, Selective, and LongLived Antagonists of Kv1.3 Justin K. Murray,1 Yi-Xin Qian,1 Benxian Liu,2 Robin Elliott,2 Jennifer Aral,1 Cynthia Park,1 Xuxia Zhang,2 Michael Stenkilsson,3 Kevin Salyers,3 Mark Rose,3 Hongyan Li,3 Steven Yu,3 Kristin L. Andrews,5 Anne Colombero,2 Jonathan Werner,4 Kevin Gaida,2 E. Allen Sickmier,1 Peter Miu,1 Andrea Itano,2 Joseph McGivern,1 Colin V. Gegg,1 John K. Sullivan,2,* and Les P. Miranda1,* 1

Therapeutic Discovery, 2Inflammation Research, 3Pharmacokinetics & Drug Metabolism, and

4

Comparative Biology and Safety Sciences, Amgen Inc., One Amgen Center Drive, Thousand

Oaks, CA 91320, USA and 5Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA Kv1.3, ShK, structure-activity, drugs, peptide antagonist, conjugation

ACS Paragon Plus Environment

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ABSTRACT

To realize the medicinal potential of peptide toxins―naturally occurring disulfide-rich peptides―as ion channel antagonists, more efficient pharmaceutical optimization technologies 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 15x selectivity against the closely related neuronal ion channel Kv1.1. Subsequent sitespecific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000x over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological 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.


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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 pathology, yet clinical applications have been limited primarily to cases where localized administration has been suitable.2 A major obstacle to more widespread development of toxin peptides has been effective methods for engineering compounds with desirable pharmaceutical properties from novel toxin peptide leads.3 Considerable research has focused on venomous animals that have evolved a diverse repertoire of toxin peptides for predatory or defensive capabilities. In some cases the intended biological activity of these toxin peptides overlaps fortuitously with similar molecular targets that are of human medicinal relevance.4 ShK (1) is a 35-residue three disulfide peptide originally isolated from the Caribbean sea anemone Stichodactyla helianthus, whose venom immobilizes prey by targeting ion channels.5,6 ShK inhibits the voltage-gated potassium ion channel Kv1.1,7 which has been shown to be critical for neuronal function in mouse and man. In humans, Kv1.1 shares high amino acid sequence homology with another potassium channel family member, Kv1.3, a possible therapeutic target.8,9 Kv1.3 regulates membrane potential and calcium signaling in human effector memory T cells (TEM), and its expression is increased markedly in activated CD4+ and CD8+ TEM/TEMRA T cell populations.10 Blockade of Kv1.3 inhibits the activation of T cells and secretion of cytokines via the calcineurin pathway by preventing the potassium efflux necessary for sustained influx of calcium.11,12 As such, Kv1.3 represents a target that selectively suppresses activated TEM cells without affecting other lymphoid subsets13 and a promising


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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 elimination of unwanted off-target ion channel activity within the ShK toxin peptide concomitantly with polymeric derivatizatizion that afforded substantial improvement in well-controlled and sustained circulating levels in vivo. Wild-type ShK is composed of 552 atoms. It is often a challenge with molecules of such size and complexity to first identify key toxin peptide-ion channel interactions, and in turn, to discover critical changes that result in improved properties.16,17 The major obstacles in this context are the relative ineffectiveness of de novo design approaches given the lack of high-resolution structural data for ion channels18 and the structural intricacy of peptide toxins.19,20 To date, studies on ShK have focused on modification of only a couple of sites to achieve moderate selectivity for Kv1.3 over Kv1.1, a challenging endeavor given their 90% amino acid sequence homology in the pore region.7,9b,21 We hypothesized that an effective and general route to develop more complete structure-activity relationships (SAR) for peptide toxins, such as ShK, would be the discrete chemical preparation of peptide analogs with substitutions at each site within the molecule with a panel of residues ranging in physicochemical properties. Building upon the traditional “alanine scan” that individually replaces each amino acid (excluding the cysteines) with one of low aliphatic bulk,16,22 the process was repeated throughout the entire molecule with a large aromatic, an acidic, and two different basic amino acid residues. In all, a set of 132 ShK peptide single


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substitution analogs were chemically synthesized. This is an approach we have termed Multi Attribute Positional Scan (MAPS) analoging. While greater diversity has been explored through combinatorial mixtures in short, two disulfide peptide sequences,23 this work represents to our knowledge the most extensive positional scanning of a long, three disulfide peptide in discrete format for systematic optimization of ion channel selectivity. High-throughput screening of this large set of individually prepared Kv1.3 inhibitory peptides has been facilitated by recent advances in automated electrophysiology methods and platforms, i.e., population patch clamp on the IonWorks Quattro (IWQ) system. From the 132 analogs prepared and tested, only two peptides displayed promising selectivity against Kv1.1 with retention of potent activity at Kv1.3. One of these lead peptide analogs was further modified with a polyethylene glycol polymer (PEG), resulting in a remarkable improvement in selectivity, and studied pharmacologically in a cynomolgus monkey model examining T cell activation. Weekly administration of this newly identified PEGylated ShK peptide analog suppressed interleukin-17 (IL-17) cytokine secretion from T cells in cynomolgus monkeys and was well-tolerated. RESULTS AND DISCUSSION ShK is a 35-amino acid (Xaa) polypeptide acid with six cysteine residues participating in three disulfide bonds giving a (Xaa)2-C1-(Xaa)8-C2-(Xaa)4-C3-(Xaa)10-C2-(Xaa)3-C3-(Xaa)2-C1 framework (Figure 1).5 The native ShK peptide has picomolar inhibitory activity at both Kv1.1 and Kv1.3.7 Earlier reports have focused on modification of the N-terminus and/or position 22 of ShK for conferring Kv1.3 selectivity.6 In particular, substitution of L-2,3-diaminopropionic acid (Dap) for the native lysine at position 22 can lead to approximately 20-fold selectivity over Kv1.1,7 however such a change concomitantly and importantly results in a significant lowering of Kv1.3 binding affinity and an approximately 103-fold loss in potency for functional inhibition


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of human T cell activation (vide infra). Alternatively, N-terminal extension of ShK with phosphotyrosine derivatives can give 100-fold Kv1.3 over Kv1.1 selectivity,21 but such molecules nonetheless have short in vivo half-lives with an undesirable pharmacokinetic profile exemplified by a rapid and large shift in peak-to-trough circulating levels.24 In this work, we set out to determine if a systematic analoging approach could be used to efficiently identify new sites within this constrained peptide scaffold that could be modified to significantly improve selectivity for Kv1.3 whilst retaining potent T cell inhibitory activity. A second key goal of this work was to specifically identify a ShK peptide derivative that could in turn be modified with a half-life extending group to give a pharmacokinetic profile suitable for weekly dosing. Multi Attribute Positional Scan (MAPS) Analoging of ShK. We sought to preferentially disrupt interactions of the ShK peptide with neuronal Kv1.1 in a novel manner but maintain the desired Kv1.3 inhibitory activity. The absence of reliable in silico methods for predicting peptide compounds with such activity profiles led us to adopt a brute-force analoging approach via direct chemical synthesis. Biological display methodologies could be pursued as an alternative analoging tactic,25 however such platforms are not currently suited to the identification of functionally active and more importantly, selective ion channel inhibitors by electrophysiological screening. To describe the approach, at each position within the ShK peptide, amino acid residues representing different physicochemical attributes (i.e., hydrophobic, basic, and acidic) were individually introduced during direct chemical peptide synthesis. The resultant crude linear peptides were then oxidized to establish the disulfide connectivity, and in turn, purified and tested. An initial set of 132 discrete peptide analogs was synthesized with modification at all positions except the cysteine framework residues (Figure 2). Aside from conventional alanine


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positional substitutions, which primarily tend to indicate which residues in a given peptide are critical for overall activity, the effect of increased steric bulk and aromatic hydrophobicity on ion channel interactions was investigated by systematic 1-naphthylalanine (1-Nal) substitution. Even though wild-type ShK is already a highly basic peptide, we also decided to examine the impact on ion channel interactions of both arginine and lysine positional substitutions. While arginine versus lysine exchanges are sometimes considered to be conservative modifications, these residues are indeed quite different in terms of size, basicity (pKa), and geometry, with arginine having a more basic planar δ-guanido group as compared to the sp3-hybridized primary ε-amino functionality of lysine. The opposite electrostatically charged substitution, increased positional acidity, was accomplished by positional scanning with glutamic acid. Nearly all of the theoretical number of ShK peptide analogs for this approach could be efficiently prepared, but four analogs could not be isolated due to technical difficulties with the disulfide bond formation process. Each prepared peptide was individually tested for its ability to directly inhibit potassium current in Chinese hamster ovary (CHO) cells stably expressing the voltage-activated Kv1.3 or Kv1.1 channel using population patch clamp on the high-throughput IWQ platform (Table 1 and Figure 2). Wild-type ShK blocked Kv1.3 current with an inhibitory concentration (IC50) of 132 ± 79 pM and was similarly effective against Kv1.1 current with an IC50 of 20 ± 29 pM in these assays (n = 31). This is the first time that the activity of native ShK is being reported on the IWQ perforated patch clamp system. While the Kv1.1 IWQ IC50 is similar to reports using other methods, the Kv1.3 IWQ IC50 is about 10-fold higher than literature values.7,16b,17,21b The shift in Kv1.3 potency associated with this new assay did not prevent the identification of trends among the large number of compounds screened in this highthroughput fashion. The activity of important compounds was subsequently verified using whole


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cell patch clamp electrophysiology, which provided better agreement with published data (vide infra). To assess the peptides’ ability to sustain the inhibition of T cell activation in a complex biological matrix, an ex vivo whole blood cytokine secretion assay was employed. Thapsigargin challenge causes unloading of intracellular calcium stores and initiation of the calcium signaling pathway in T cells, resulting in IL-2 and IFN-γ secretion.6,24,26 In this whole blood assay format, the activity of peptides can also be assessed in terms of the molecules’ ex vivo metabolic stability over 48 hours. The whole blood assay is a rigorous assessment of sustained Kv1.3 inhibition in comparison to electrophysiology (ePhys), since ePhys assays are generally of short duration (5x selectivity for Kv1.3 vs. Kv1.1 with IC50 values 3333 >3333 153 ± 32 28 ± 13 >3333 305 ± 134 ND ND 472 ± 46 11.0 ± 4.9 216 ± 48 0.9 ± 0.3 927 ± 119 27.4 ± 15.5 235 15.7 39 ± 19 48.9 ± 13.0 73 ± 20 10.2 ± 6.5 1444 ± 247 >3333 475 ± 39 6.7 ± 6.5 280 ± 48 3.3 ± 1.7 >3333 >3333 853 ± 149 26.2 ± 42.9 >3333 >3333 >3333 >3333 ND ND 103 ± 10 9.1 ± 3.3 207 ± 41 1018 ± 148 223 ± 75 2420 ± 1300 >3333 >3333 161 ± 16 3.2 ± 2.5 126 ± 37 6.1 ± 3.0 182 6.1 77 ± 5 4.4 ± 0.0

# 56 57 58 59 60‡ 61 62‡ 63 64 65 66 67‡ 68 69 70 71 72 73 74 75 76‡ 77 78 79 80 81 82 83‡ 84‡

Positional Substitution Glutamic Acid Kv1.3 IC50 Kv1.1 IC50 (pM) (pM) 151 ± 82 40 ± 24 95 ± 31 1.5 ± 0.8 75 ± 12 7.6 ± 3.5 >3333 1200 ± 140 171 25.6 1308 ± 316 32 ± 4 97 7.3 146 ± 75 5.9 ± 4.8 133 ± 8 4.2 ± 5.3 983 ± 150 29 ± 9 152 ± 36 4.5 ± 2.1 110 7.8 267 ± 24 42 ± 13 197 ± 39 51 ± 28 120 ± 14 4.0 ± 1.0 256 ± 213 18.1 >3333 >3333 >3333 490 ± 120 >3333 >3333 >3333 >3333 260 14 245 ± 54 20 ± 15 320 ± 40 28 ± 10 >3333 1800 ± 300 143 ± 16 1.8 ± 0.5 278 ± 51 5.7 ± 2.3 234 ± 65 2.3 ± 1.1 60 4.9 67 8.3

# 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109

Arginine Kv1.3 IC50 (pM) WT 74 ± 7 110 ± 29 >3333 179 ± 27 303 ± 29 447 ± 187 96 ± 12 73 ± 18 WT 152 ± 30 79 ± 10 88 ± 14 166 ± 28 104 ± 13 95 ± 22 >3333 259 ± 46 >3333 >3333 WT 107 ± 19 278 ± 47 >3333 WT 38 ± 7 >3333 221 ± 168 191 ± 112

Kv1.1 IC50 (pM) WT 1±1 7±4 3279 ± 0 46 ± 10 17.2 ± 5.1 4.0 ± 4.0 2.7 ± 1.9 3.0 ± 2.1 WT 19.5 ± 6.4 1.8 ± 1.6 15.4 ± 4.5 55 ± 57 3.8 ± 1.7 2.8 ± 1.8 >3333 32.4 ± 8.2 >3333 >3333 WT 1.8 ± 1.4 70 ± 7 >3333 WT 8.0 ± 3.2 590 ± 63 3.6 ± 0 2±2

# 110 111 112 113 114 115 116‡ 117 118 119 120‡ 121 122 123 124 125 126‡ 127 128 129 130 131‡ 132‡ 133

Lysine Kv1.3 IC50 Kv1.1 IC50 (pM) (pM) 154 ± 29 3.1 ± 2.4 146 ± 44 8.7 ± 6.4 153 ± 31 4.2 ± 0.2 >3333 458 ± 296 129 ± 17 2.6 ± 1.9 561 ± 114 >3333 83 5.0 WT WT 184 ± 20 3.3 ± 2.9 133 ± 13 2.4 ± 1.6 154 ± 31 4.4 ± 3.9 73 5.0 294 ± 29 52.7 ± 4.1 352 ± 30 2342 ± 191 WT WT 190 ± 19 1.7 ± 0.1 193 ± 34 2.1 ± 1.7 798 ± 29 40.0 ± 15.6 WT WT ND ND 67 4.0 100 ± 9 2.8 ± 2.0 122 ± 49 33.5 ± 6.3 985 ± 215 75 ± 23 293 ± 24 266 ± 46 WT WT 1417 174 122 5.0 153 ± 39 5.1 ± 0.0

* - Samples tested on IWQ platform (Avg. ± SD). # - ND indicates not determined because folded peptide analog was not isolated. † - WT indicates substitution corresponds to wild-type sequence; ShK Kv1.3 IC50 = 132 ± 79 pM and Kv1.1 IC50 = 20 ± 29 pM. ‡ - Percent inhibition as a function of compound concentration was measured as an average of four data points per concentration, and the resulting dataset was fit to produce a single IC50 curve.


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Table 2. Inhibition of IL-2 and IFN-γ Secretion in Human Whole Blood by ShK Analogs* Positional Substitution Alanine

1-Naphthylalanine

Glutamic Acid

Arginine

Lysine

ShK Residue

Residue Position

#

WB IL-2 IC50 (pM)

WB IFN-γ IC50 (pM)

#

WB IL-2 IC50 (pM)

WB IFN-γ IC50 (pM)

#

WB IL-2 IC50 (pM)

WB IFN-γ IC50 (pM)

#

WB IL-2 IC50 (pM)

WB IFN-γ IC50 (pM)

#

WB IL-2 IC50 (pM)

WB IFN-γ IC50 (pM)

Arg

1

2

61 ± 15

111 ± 44

29

222 ± 21

248 ± 93

56

214 ± 26

249 ± 49

-

WT

WT

110

36 ± 1

73 ± 14

Ser

2

3

42 ± 17

81 ± 36

30

223 ± 82

258 ± 208

57

29 ± 2

39 ± 11

85

87 ± 15

116 ± 52

111

99 ± 3

94 ± 22

Ile

4

4

23 ± 16

46 ± 45

31

318 ± 128

58

86

54 ± 26

ND#

ND

32

>31,900

59

>24,600

87

123 ± 62 91,700 ± 22,300

23 ± 10

-

98 ± 61 40,500 ± 9530

112

5

31 ± 26 16,900 ± 3670

50 ± 36

Asp

274 ± 62 70,400 ± 54,000

113

>100,000

>100,000

Thr

6

5

70 ± 74 1980 ± 932

33

860 ± 156

60

109 ± 52 19,500 ± 14,000

88

304 ± 178 10,100 ± 1300

114

210 ± 95 13,500 ± 14,700

Ile

7

6

56 ± 18 748 ± 270

34

11,600 ± 202

962 ± 303 18,000 ± 2690

61

74 ± 6 5000 ± 3000

89

215 ± 46 6560 ± 1730

115

117 ± 90 4670 ± 4480

Pro

8

7

34 ± 26

66 ± 19

-

ND

ND

62

72 ± 6

239 ± 16

90

253 ± 111

1050 ± 562

116

154 ± 35

975 ± 532

Lys

9

8

368 ± 186

666 ± 33

63

37 ± 13

56 ± 10

91

76 ± 48

83 ± 45

-

WT

WT

10

9

36

205 ± 41

129 ± 74

117

52 ± 6

82 ± 16

37

1140 ± 123

65

55 ± 17 4540 ± 2070

72 ± 50

10

23 ± 0 1320 ± 600

92

11

-

WT

WT

118

95 ± 69

252 ± 244

Thr

13

11

38

3560 ± 305

255 ± 117 1650 ± 150 4860 ± 1430

64

Arg

81 ± 29 1210 ± 1730 2150 ± 921 795 ± 290

35

Ser

45 ± 13 822 ± 1240 881 ± 469 391 ± 228

66

785 ± 175

1100 ± 94

93

398 ± 242

635 ± 173

119

257 ± 39

363 ± 68 201 ± 88 991 ± 74

Ala

14

-

WT

Phe

15

12

Gln

16

Lys

†

39

89 ± 5

110 ± 15

67

36 ± 2

132 ± 20

94

205 ± 81

262 ± 87

120

41 ± 22

WT 158 ± 187

40

312 ± 11

575 ± 438

95

143 ± 52

183 ± 31

121

54 ± 10

90 ± 27

41

291 ± 151 9550 ± 2100

68

13

184 ± 42 28,900 ± 29,700

75 ± 2 465 ± 150

69

352 ± 35

373 ± 310

96

372 ± 350

492 ± 377

122

108 ± 45

151 ± 110

18

14

419 ± 106

375 ± 197

70

47 ± 35

83 ± 48

97

38 ± 30

56 ± 38

-

WT

WT

19

15

43

153 ± 7

255 ± 11

71

202 ± 11

216 ± 2

98

89 ± 23

16

251 ± 69

44

>100,000

72

45

6590 ± 2300

73

100

125

4820 ± 628

Lys

22

18

46

>100,000

74

>100,000

>100,000

101

>100,000

>100,000

-

WT

WT

Tyr

23

19

327 ± 77 1940 ± 497 3090 ± 1190

108 ± 2 3590 ± 2360

193 ± 108

17

>92,000 28,100 ± 20,900

124

21

>92,000 18,500 ± 6000

99

Met

>100,000 8130 ± 1220

282 ± 208 40,300 ± 27,000 3080 ± 1960

41 ± 17

20

114 ± 109 25,300 ± 2060 1490 ± 479

123

Ser

48 ± 35 556 ± 318 809 ± 375 1260 ± 1030 4010 ± 1720 5670 ± 1600

42

His

32 ± 11 301 ± 114

47

5970 ± 698

75

>100,000

>100,000

102

>100,000

>100,000

-

ND

ND

>100,000 11200 ± 8150


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


-

ND

ND

76

1110 ± 544

4070 ± 928

-

WT

WT

126

48

61 ± 69

84 ± 54

77

494 ± 88

678 ± 27

103

137 ± 57

194 ± 12

127

49

357 ± 141

78 79

105

158 ± 73 13,000 ± 2100

368 ± 248 59,700 ± 40,500

51

4720 ± 2710

80

730 ± 30

400 ± 173 34,000 ± 8090 1960 ± 1060

128

1070 ± 962

202 ± 13 8160 ± 3880

104

50

573 ± 456 2840 ± 3020 8080 ± 3640

-

WT

52

82 ± 50

148 ± 50

81

20 ± 23

43 ± 19

106

222 ± 85

53

32 ± 10

82

242 ± 262

309 ± 389

59 ± 19

122 ± 60

54

439 ± 55

129 ± 33 1430 ± 699

83

55 ± 22

53 ± 24

83 ± 36

55

18 ± 14

63 ± 37

84

73 ± 23

Arg

24

20

45 ± 29

Leu

25

21

199 ± 15

186 ± 51 641 ± 444

Ser

26

22

Phe

27

23

Arg

29

24

Lys

30

25

65 ± 14 1500 ± 1550 2880 ± 4440 186 ± 229

120 ± 46 8010 ± 9890 5300 ± 8150 308 ± 293

Thr

31

26

55 ± 15

Gly

33

27

Thr

34

28

45 ± 2 226 ± 204

108 ± 36

129

273 ± 30 3490 ± 2150

496 ± 124 11,800 ± 8830

WT

130

41 ± 8

71 ± 15

58 ± 39 39,000 ± 24,000

-

107

40 ± 30 11,100 ± 2160

131

WT 5984 ± 307

WT 27,900 ± 17,700

176 ± 54

108

89 ± 36

223 ± 93

132

218 ± 52

318 ± 128

124 ± 114

109

357 ± 99

475 ± 142

133

67 ± 6

136 ± 100

366 ± 4

* - Avg. ± SD. # - ND indicates not determined because folded peptide analog was not isolated. † - WT indicates substitution corresponds to wild-type sequence; ShK IL-2 IC50 = 37 ± 36 pM and IFN-γ IC50 = 48 ± 43 pM.


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The electrophysiological and whole blood functional testing of the five families of ShK analogs, Ala, 1-Nal, Arg, Lys, and Glu substitutions, showed that each series provided interesting and unique results and that together a much more complete structure-activity relationship for ShK may be discerned. First, classical alanine scanning replaces the native side chain functionality at each position with a small aliphatic group (methyl) that typically weakens the binding interaction for key positions within the sequence. The alanine ShK analog series indicated that residue positions 11, 22, 23, and 29 were likely important for maintaining Kv1.3 or Kv1.1 inhibitory activity (red or yellow in Figure 2). These findings were consistent with previously published reports on ShK SAR7,16 and, importantly, these substitutions, along with positions 7, 10, 21, and 27, also resulted in considerably reduced activity in the corresponding whole blood IL-2 and IFN-γ secretion assays (red). Within this series, only 19 ([Ala23]ShK) and 24 ([Ala29]ShK) showed >5 fold selective inhibition of Kv1.3 over Kv1.1, but unfortunately the concomitant loss in the critical cytokine secretion inhibitory activity for these compounds limited their utility. Under our assay conditions, the substitution of alanine for lysine at position 18 (14) did not improve selectivity against Kv1.1 as reported in the literature, perhaps due to differences in electrophysiology platform (IWQ instead of manual patch clamp) and/or cell line (hKv1.1 expressed in HEK293 cells instead of mKv1.1 in mouse L929 fibroblasts), and it was not tested by manual electrophysiology.20 In short, classic alanine positional scanning did not result in improvement in either potency or selectivity, necessitating implementation of our MAPS analoging methodology. The 1-naphthylalanine positional scan of ShK introduces a large aromatic side chain in place of the wild-type functionality to examine the effects of increasing hydrophobicity and steric bulk. This series had the largest number of substitutions that were disruptive to the Kv1.3 inhibitory


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activity of ShK. Kv1.3 inhibition was adversely affected by 1-Nal incorporation at positions 5, 7, 9, 11, 16, 18, 20, 21, 22, 23, and 29. Additionally, activity in the whole blood assay was diminished by substitution of 1-Nal at positions 13, 27, and 33. This list includes and adds to the key binding residues identified by the Ala scan. Compounds 49 ([1-Nal26]ShK) and 50 ([1Nal27]ShK) had ≥5-fold selectivity over Kv1.1, but only 1-Nal substitution at position 26 retained desirable whole blood activity < 1,000 pM. In addition to varying the hydrophobicity and size at each position of ShK with Ala and 1-Nal, the electrostatic interactions were also probed through incorporation of amino acids with charged side chains. The glutamic acid substitution series had an activity profile similar to 1-Nal with positions 5, 7, 11, 13, 20, 21, 22, 23, 24, 27, and 29 not being well tolerated in either the ePhys or whole blood assays or both, demonstrating the extensive perturbation caused by integration of an acidic residue into a highly basic peptide sequence. Furthermore, no compound from the glutamic acid substitution series appeared to show any selective inhibition for Kv1.3 over Kv1.1. One observation unique to the Glu series was that substitution of the native Arg at position 24 caused a loss of functional activity in the cytokine secretion assays but retained activity in the electrophysiology assays, giving some insight into the SAR for that residue position. The basic arginine and lysine substitution series led to the identification of a selective and potent ShK analog as a lead for further optimization and study. First, we found that arginine substitutions at 5, 7, 8, 20, 21, 22, 23, 27, and 31 resulted in significant loss of Kv1.3 and/or functional inhibitory activity. Lysine substitutions at positions 5, 7, 21, 27 and 31 had similar effects (Figure 3A). Among the different scans, position 31 was uniquely sensitive to substitution with a basic residue having whole blood IC50 values > 5,000 pM for the Arg (107)


13


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and Lys (131) substitution analogs but < 500 pM for the Ala (26), 1-Nal (53), and Glu (82) containing compounds. Interestingly, although no arginine-substituted ShK analogs had any selective inhibition for Kv1.3 over Kv1.1, lysine substitution analogs at position 7, 115 ([Lys7]ShK), and position 16, 122 ([Lys16]ShK), were both 6-fold selective, a 40x improvement over native ShK (Figure 3B). However, only 122 retained potent whole blood activity with an IL-2 secretion IC50 of 108 ± 45 pM and IFN-γ secretion IC50 of 151 ± 110 pM (n = 67). By comparison, 96 ([Arg16]ShK) had no improvement in selectivity for Kv1.3 and instead was an approximately 3-fold more potent inhibitor of Kv1.1 than Kv1.3. The key features of the sequence-activity relationship from the Lys scan are presented in Figure 3C. To summarize, application of MAPS analoging to ShK led to the identification of previously unreported sites for potency and selectivity not found via traditional Ala scanning efforts (Figure 3D).16 From an overall perspective, only 2 out of the 132 initially prepared ShK analogs, 49 and 122, met the following success criteria: 1) 20x loss in functional activity in the whole blood assay without improvement in Kv1.3 versus Kv1.1 selectivity. Some effects on activity may be due to conformational disruption of the peptide. For example, substitution of a Lys or Arg residue at position 31 would place the side chains of three basic residues (Lys9 and Lys30) in close proximity and may affect the global structure. While our results serve to refine the list of residues in ShK with strong Kv1.3 interactions,20 these data also highlight the importance of residues at the edge of the peptide binding surface. While these peripheral residues are typically ignored by traditional optimization strategies (i.e., alanine scanning and structure-based design), specific changes in charge or hydrophobicity at these sites may serve to elucidate the nature of their contribution to the complex and effect on ion channel selectivity.

[Lys16]ShK Peptide Analogs. Identification of the potent and moderately selective Kv1.3 inhibitory peptide 122 was followed up with additional analoging at position 16 and combination with modifications to reduce oxidative liabilities (Table 3). Shortening the Lys16 side chain by a methylene unit to orthinine (Orn) resulted in a similar activity profile (134), however removal of a second methylene unit with diaminobutyric acid (Dab) led to loss of Kv1.1 selectivity (135). It


16

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is known that amidation of the C-terminal acid of ShK provides a backbone with similar activity and increased metabolic stability;21c preparation of the C-terminal amide of [Lys16]ShK yielded a similarly potent and selective derivative (136). Extension of the C-terminus with a residue less prone to epimerization during solid-phase peptide synthesis than cysteine29 and substitution of the oxidizable methionine with norleucine at position 21 were investigated in combination with the lysine substitution at position 16 (Table 3). Surprisingly, addition of a C-terminal alanine to [Lys16]ShK resulted in analogs (137 and 141) with >150-fold selectivity for Kv1.3 versus Kv1.1 that retained good activity in blocking T cell cytokine secretion in human whole blood. The improvements in selectivity associated with substitution of lysine at position 16, hydrophobic substitutions at position 21, and extension of the C-terminus have been verified by Pennington and co-workers, including an additive improvement in selectivity through their approach of Nterminal modification.30 Table 3. Potency and Selectivity of Position 16 ShK Analogs. Cmpd Peptide Name

1 122 134 135 136 137 138 139 140 141

ShK [Lys16]ShK [Orn16]ShK [Dab16]ShK [Lys16]ShK-amide [Lys16]ShK-Ala [Nle21]ShK [Lys16,Nle21]ShK [Lys16,Nle21]ShK-amide [Lys16,Nle21]ShK-Ala

Kv1.3 IWQ IC50 (pM)

Kv1.1 IWQ IC50 (pM)

132 352 140 82 174 60 40 130 153 71

20 2,342 740 11 600 9,500 15 29,258 13,220 >33,333

Kv1.1 IC50 / Kv1.3 IC50 0.15 6.7 5.3 0.13 3.4 158 0.38 225 86 >469

WB IL-2 IC50 (pM)

WB IFN-γ IC50 (pM)

37 108 138 86 223 138 153 249 823 305

48 151 160 223 278 266 303 5,678 1,099 515

Electrophysiology of ShK Peptide Analogs. Further electrophysiological characterization of lead compound 122, the parent ShK peptide, and literature analogs was performed (Table 4).


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Our previous screening experiments utilized a high-throughput 384-well IonWorks Quattro (IWQ) platform, which evaluates receptor inhibition with a population patch clamp, due to the large number of compounds that needed to be tested. A select number of important analogs were tested on a whole cell planar patch clamp platform using the automated PatchXpress (PX) system or manual electrophysiology. As expected, ShK was an exceptionally potent inhibitor of both Kv1.3 and Kv1.1 on the PX system. These values were in reasonable agreement with those obtained by manual whole cell patch clamp electrophysiology where ShK had an IC50 of 16 ± 8 pM for Kv1.3 and 14 ± 3 pM for Kv1.1, similar to values reported in the literature as well as our results in the cytokine secretion assays.7,16b,17,21b Compound 122 was also a potent inhibitor of Kv1.3 on the PX platform and demonstrated >15x selectivity against Kv1.1. The potency and selectivity profiles for 142 (ShK-Dap22) and 143 (ShK-L5, Supporting Information Figure S1), which are commercially available, were compared to the results reported previously for these analogs.7,21b The molecule 142 showed a significant loss in whole blood functional activity, which motivated us to adopt this assay for the primary screening of our analogs. As discussed earlier, the whole blood assay is of longer duration and may better reflect equilibrium binding of the peptide to the target. Indeed, while Kalman et al. reported that 142 showed good potency by electrophysiology (Kv1.3 IC50 = 23 pM)7 similar to our findings, Middleton et al. reported that its equilibrium binding affinity for Kv1.3 is more than 100 times weaker than native ShK.31 These latter results are consistent with our observations in the 48 hour whole blood assay where 142 had IL-2 and IFN-γ IC50 values > 3,000 pM. In our assays, 143 was a potent inhibitor of both Kv1.3 and Kv1.1 as well as cytokine secretion in human whole blood. The disagreement of our selectivity ratio for 143 with published reports may be due to our use of a different cell line


18

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(hKv1.3 in Chinese hamster ovary (CHO) cells rather than mKv1.3 in mouse L929 fibroblasts).7,21b Table 4. Potency and Selectivity of ShK Analogs. Cmpd Peptide Name

Kv1.3 PX IC50 (pM)

Kv1.1 PX IC50 (pM)

ShK 39 87 1 [Lys16]ShK 207 3,677 122 ShK-Dap22 12* 847* 142 ShK-L5 221 214 143 * - Indicates manual whole cell patch clamp data.

Kv1.1 IC50 / Kv1.3 IC50 2.2 17.8 70.6 1.0

WB IL-2 IC50 (pM)

WB IFNγ IC50 (pM)

37 108 3,763 31

48 151 3,112 46

Impact of Conjugation on Potency, Selectivity, and Pharmacokinetics of ShK Analogs. The potent wild-type ShK peptide has a very short circulating pharmacokinetic profile in rats (t1/2 ~ 20 min).32 The short half-life in vivo of peptides is typically attributed to rapid metabolic processing and high renal clearance.33 To investigate whether renal clearance was responsible for the short circulating time of ShK, we attempted PEGylation of the molecule as a means to increase its hydrodynamic radius.34 It was unknown, however, whether attachment of a large polyethylene glycol (PEG) polymer to ShK would significantly impair its activity or not. We explored a N-terminal reductive amination approach due to the presence of multiple lysine residues in ShK derivatives and the difficulty of using cysteine-maleimide chemistry in disulfiderich peptides. First, a Nα-PEG-ShK conjugate was prepared by reductive alkylation of the Nterminus with a linear 20 kDa monomethoxy PEG aldehyde at pH 4.5 and then purified. Peptide mapping experiments confirmed PEGylation occurred primarily at the N-terminus of the peptide (data not shown). Testing of 144 (20kDa-PEG-ShK) revealed that it retained sub-nanomolar potency in inhibiting Kv1.3 and T-cell cytokine responses (Table 5) and exhibited a prolonged half-life in rats (mean residence time of 37 h, Supporting Information Table S2). In agreement


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Page 20 of 62

with other N-terminally derivatized ShK analogs,35 such as 143 (phosphotyrosine-AEEA-ShK), which have been reported to have increased selectivity for Kv1.3, we also found 144 to be 5-fold more potent against Kv1.3 than against Kv1.1. Encouraged by the retention of activity, we next PEGylated our selective [Lys16]ShK analog at its N-terminus with linear PEG. The conjugate 145 (20kDa-PEG-[Lys16]ShK) was found to provide potent blockade of whole blood IL-2 secretion with an IC50 of 92 ± 42 pM (n = 14). More interestingly, selectivity for Kv1.3 over Kv1.1 was not only retained, but showed a synergistic 1000-fold lowering of Kv1.1 activity without impacting Kv1.3, more than 200x the effect that PEGylation had on native ShK.30 The potency and selectivity of 145 is extraordinary when compared to the conjugated wild-type peptide and unconjugated peptide analogs (Figure 6). The pharmacokinetics and bioactivity of polypeptides can be significantly altered through the attachment of PEG groups of differing molecular weight.36 Aside from derivatization with 20kDa-PEG, the [Lys16]ShK peptide was also prepared with either a 30 kDa linear PEG or a branched PEG consisting of two 10 kDa PEG arms (20kDa-brPEG).

The 20kDa-brPEG-

[Lys16]ShK molecule (146) was a potent inhibitor of cytokine secretion in the whole blood assay and had 750-fold selectivity for lymphocyte Kv1.3 over neuronal Kv1.1. The linear 30kDaPEG-[Lys16]ShK molecule (147) was also a highly potent inhibitor of cytokine secretion in human whole blood. These results suggest that the 122 peptide scaffold is tolerant of N-terminal derivatization with PEG polymers of differing size and architecture. Conjugation of 122 with linear 20kDa PEG results in a slightly higher level of Kv1.3 vs. Kv1.1 selectivity relative to the branched or larger PEG chains.


20

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Table 5. Potency and Selectivity of PEGylated ShK Analogs Cmpd Name

Kv1.3 PX IC50 (nM)

20kDa-PEG-ShK 0.299* 144 20kDa-PEG-[Lys16]ShK 0.94 145 2.10 20kDa-brPEG-[Lys16]ShK 146 30kDa-PEG-[Lys16]ShK 1.20 147 * - Manual patch clamp electrophysiology

Kv1.1 PX IC50 (nM)

1.628* 997 1574 1072

Kv1.1 IC50 / Kv1.3 IC50 5 1060 750 890

WB IL-2 IC50 (nM)

WB IFNγ IC50 (nM)

0.380 0.092 0.198 0.282

0.840 0.160 0.399 0.491

In preparation for in vivo studies, the in vitro activity profile of 145 was further characterized in a number of ion channel counterscreens and against other species. Counter-screening revealed that 145 was highly selective over Kv subtypes Kv1.2 (680-fold), Kv1.6 (~500-fold), Kv1.4 (>10,000-fold), Kv1.5 (>10,000-fold), and Kv1.7 (>10,000-fold) (Table 6). Importantly, the conjugate did not impact ion channels that are known to serve a role in human cardiac action potential, exhibiting >10,000-fold selectivity over Nav1.5, Cav1.2, Kv4.3, KvLQT1/minK and hERG. Moreover, the conjugated toxin peptide analog 145 had no detectable impact on the calcium-activated K+ channels KCa3.1 (IKCa1) and BKCa.

Table 6. Activity of 145 in Counterscreens* Assay Human WB IL-2 Kv1.1 Kv1.2 Kv1.3 Kv1.4 Kv1.5 Kv1.6 Kv1.7 IKCa1 BKCa hERG (IKr) Nav1.5 (INa)

IC50 (nM) 0.092 997 639 0.94 >10000 >30000 466 >10000 >10000 >10000 >10000 >30000


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Cav1.2 (ICa) Kv4.3 (Ito) KvLQT1/minK (IKs) * - n ≥ 3 for all.

Page 22 of 62

>30000 >30000 >30000

Cross-Species Activity of 20kDa-PEG-[Lys16]ShK. In addition to its inhibitory activity in human whole blood, 145 also inhibited IL-17 and IL-4 secretion from T cells within cynomolgus monkey whole blood with potent IC50 estimates of 0.09 ± 0.08 nM and 0.17 ± 0.13 nM, respectively. 145 was also a potent inhibitor (IC50 = 0.17 nM) of myelin-specific proliferation of the rat T effector memory cell line, PAS.37 Overall, we found that 145 has consistently potent inhibitory activity towards T cell responses in whole blood assays from rat, monkey, and human (IL-2 IC50 = 0.092 nM). Pharmacokinetics of 20kDa-PEG-[Lys16]ShK. In regards to unconjugated peptides, there are limited pharmacokinetic studies on native ShK32 and the more selective ShK analog, 143,21b indicating these molecules have half-lives in rats that are much shorter (< 1 hour) than our PEG conjugate. Prior to evaluating the pharmacology of the potent and selective conjugate 145, its ex vivo plasma stability and pharmacokinetics were determined. The conjugate was found to have high metabolic stability in plasma from rat, cynomolgus monkey, and human over 2 days at 37°C (Supporting Information Figure S5).

Pharmacokinetic studies in mouse, rat, dog, and

cynomolgus monkey showed good cross-species metabolic stability in vivo with a considerably extended elimination half-life.

Moreover, a comparison of 148 (ShK-186), a more advanced

derivative of 143 containing a C-terminal amide and displaying improved stability,21c,24 indicates 145 has a half-life in cynomolgus monkeys that was 245 times longer than 148 when the same 0.5 mg/kg dose was delivered (Table 7). We estimate the exposure of compound 145 over time as measured by AUC0-inf was 390 times greater in cynomolgus monkeys than 148, resulting in a clearance rate that was ~950 times slower in rats and cynomolgus monkeys compared to the 148


22

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peptide. As shown in Figure 7, 145 when dosed subcutaneously in cynomolgus monkeys at 0.5 mg/kg achieved a Cmax at 8 hr of 254 nM and day 7 serum levels of 28.4 nM. The serum concentration of 145 at day 7 after a single dose was approximately 28 and 315 times greater than the cytokine secretion IC95 (1.0 nM) and IC50 (0.09 nM) estimates in human whole blood, respectively. Therefore, the pharmacokinetics of 145 in cynomolgus monkeys is consistent with a projected weekly dosing profile in human subjects. It should be noted that despite our PEGconjugate showing a profoundly longer half-life in vivo than 148, Tarcha et al. report that this peptide analog shows durable pharmacological effects in monkeys.24 The authors propose that although serum levels decline rapidly over the first few hours after injection, there could be a slow release from the injection site, as well as tight binding and slow dissociation from the Kv1.3 channel on T cells to drive efficacy. Irrespective of these considerations, we show that the conjugate 145 is profoundly longer-lived in vivo, enabling sustained and measurable target coverage over a narrower dynamic range of serum drug concentrations. Further details on the pharmacokinetics of 145 administered subcutaneously are provided in the Supplementary Information. Table 7. Single dose pharmacokinetic (subcutaneous) profile of 145 in CD1 mice, Sprague Dawley rats, beagle dogs, and cynomolgus monkeys compared to the pharmacokinetics of 148 in Sprague Dawley rats and cynomolgus monkeys.24 Because only the peptide portion of 145 was used in calculating mg/mL stock concentrations and the [Lys16]ShK peptide portion (4055 Da) is of similar MW to 148, equivalent mg/kg doses of these two molecules generate similar nmol/kg doses. Cpmd

Species

Dose

n

(mg/kg)

t1/2

Tmax

CL/F

(hr)

Cmax (ng/ml)

Vz/F

(hr)

(ng·hr·ml-1)

AUC0-t

(ng·hr·ml-1)

AUC0-inf

(ml/kg)

(ml·hr-1 ·kg-1)

MRT (hr)

145

Mouse*

2.0

3

14.9

4.0

1860

37,000

37,000

1170

54.1

16.6

145

Rat

2.0

3

N/A

Beagle

0.5

3

145

Cyno

0.5

531 ±90 1270 ±347 1010 ±105

21,900 ±2,770 95,200 ±31,300 71,500 ±607

21,900 ±2,760 103,000 ±37,300 74,900 ±3,260

N/A

145

40 ±14 18.7 ±9.24 8.0

92 ±13 5.37 ±2.14 6.68 ±0.29

36 ±2 66.1 ±13.5 87 ±16

42.6 ±4.21 3 64.5 ±14.9

322 ±98 621 ±143


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Page 24 of 62

148#

Rat

1.0

3 0.132

0.083

48

NR

11.5

NR

87,198

NR

148#

Cyno

0.5

2 0.263

0.083

192

NR

192

NR

6,336

NR

* - Sparse sampling PK experiment. No standard deviations were calculated for PK parameters. #

- from Tarcha, et al., ref. 24. NR = not reported

Efficacy, Pharmacodynamics, and Safety of 20kDa-PEG-[Lys16]ShK. We evaluated the efficacy of 145 in vivo using the adoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE) model in rats.38 In this animal model of multiple sclerosis, T cells specific for myelin basic protein (MBP) and constitutively expressing Kv1.3 (PAS cells) are activated and injected into rats causing inflammation and demyelination of the central nervous system (CNS), with symptoms progressing from a distal limp tail to paralysis over the course of a week. Dosing in rats with the Kv1.3 blocker 145 before the onset of EAE caused a delay in the onset of disease. The progression of disease was also inhibited with treatment with 145, with an observed dosedependent effect on reduced disease severity and the prevention of death (Figure 8). In the vehicle treated animal group, the disease onset occurred on day 4, but by comparison in animals treated with 145 the disease onset was delayed until day 4.5 to 5. On day 6, the vehicle treated rats had developed severe disease (EAE score of 6) and were sacrificed, whereas 145 treated animals (at efficacious doses) had only mild disease (EAE score of ~1) that resolved over time. The molecule 145 blocked AT-EAE in a dose-responsive manner with an estimated ED50 of approximately 4 µg/kg on day 7 (Figure 8 and Supporting Information Figure S6). In a separate study, an equivalent dose (0.01 mg/kg) of PEGylated ShK (144) was found to provide greater efficacy in blocking encephalomyelitis than the native ShK peptide (Supporting Information Figure S7) that has a shorter half-life in animals. Overall, these data suggest that higher levels of sustained Kv1.3 target coverage appear to result in greater efficacy in this model.


24

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The in vivo pharmacodynamics of the molecule 145 in cynomolgus monkeys was also examined. A 12-week pharmacology study was initiated with three pre-dose baseline measurements during the first two weeks. This was then followed by four weekly subcutaneous doses of 145 at 0.5 mg/kg, and an additional six weeks of post dosing analysis (Figure 9 and Supporting Information Tables S5–S7). Based on earlier pharmacokinetic studies of 145, target coverage with 0.5 mg/kg weekly dosing was expected to range from 28-times the IL-17 IC95 at the minimum drug concentration in plasma (Cmin) to 249-times IC95 at the peak or maximum drug concentration (Cmax).

The repeat dosing of the conjugate 145 was well tolerated.

In terms of general

observations, weight gain was normal throughout the study, complete blood counts (CBCs) and blood chemistry were also found to be normal with respect to pre-dose baseline estimates. Using the cynomolgus monkey whole blood IL-17 pharmacodynamic assay, suppression of T cell inflammation was achieved during the four week dosing period.

In terms of repeat drug

exposure, the predicted and observed serum drug trough levels correlated well over the dosing period. The potential toxicity and the toxicokinetics of 145 were evaluated in male cynomolgus monkeys (n = 3 per dose group) after subcutaneous administration of 0.7 mg/kg every third day (4 doses total) or 2 weekly doses at 0.1, 0.5, or 2.0 mg/kg (2 doses total).39 effects on any parameters evaluated.

There were no 145-related

Specifically, there were no PEG-associated vacuoles

observed in renal tubules or tissue macrophages by light microscopy. Based on the absence of adverse toxicity, the no-observed-adverse-effect-level (NOAEL) in this study was 2 mg/kg, which correlated with a mean AUC0-168hr of 584,000 ng·hr/mL. CONCLUSIONS


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Page 26 of 62

The diverse array of potent biological activities and inherent metabolic stability of toxin peptides make this class of molecules an attractive starting point for drug discovery of ion channel modulators. We have demonstrated the effectiveness of the Multi Attribute Positional Scan (MAPS) analoging methodology to identify potent and subtype selective analogs of the ShK peptide toxin. By scanning the peptide sequence with not only the traditional Ala residue but also representative basic, acidic, and hydrophobic residues and screening the resulting >130 analogs via high-throughput electrophysiology, [Lys16]ShK emerged as a potent antagonist of Kv1.3 with improved selectivity over Kv1.1.39 Combination with N-terminal conjugation of a 20 kDa polyethylene glycol polymer resulted in an unexpected synergistic increase in Kv1.3 versus Kv1.1 selectivity to 1000-fold, with retention of picomolar potency in the whole blood T-cell assay and prolongation of the half-life in vivo. A clean selectivity profile against a panel of ion channels and good plasma stability made 20kDa-PEG-[Lys16]ShK suitable for rodent and primate PD studies. Compound 145 was efficacious in the rat adoptive transfer-experimental autoimmune encephalitis (AT-EAE) model of multiple sclerosis. The pharmacokinetic profile of this compound was suitable for weekly dosing in cynomologous monkeys, and it showed suppression of T cell-mediated inflammation during a one month repeat-dosing experiment without adverse side effects. Through prolonged blockade of Kv1.3 in vivo, 145 or related analogs may allow further interrogation of this target for the treatment of autoimmune disease in higher species. In view of these results and other applications in our laboratory, it appears the MAPS analoging strategy will be useful to multiple classes of toxin peptides for ion channel targets, as well as small synthetically accessible protein scaffolds for other types of targets.

EXPERIMENTAL METHODS


26

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Peptide Preparation Peptide Synthesis. Nα-Fmoc, side-chain protected amino acids and H-Cys(Trt)-2Cl-Trt resin were purchased from Novabiochem, Bachem, or Sigma Aldrich. The following side-chain protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Glu(OtBu), His(Trt), Lys(NεBoc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). ShK or other toxin peptide analog amino acid sequences, were synthesized in a stepwise manner on a CS Bio 336 peptide synthesizer by FmocSPPS using DIC/HOBt coupling chemistry at 0.2 mmol scale using H-Cys(Trt)-2Cl-Trt resin (0.2 mmol, 0.32 mmol/g loading). For each coupling cycle, 1 mmol Nα-Fmoc-amino acid was dissolved in 2.5 mL of 0.4 M 1-hydroxybenzotriazole (HOBt) in N,N-dimethylformamide (DMF). To the solution was added 1.0 mL of 1.0 M N,N'-diisopropylcarbodiimide (DIC) in DMF. The solution was agitated with nitrogen bubbling for 15 min to accomplish pre-activation and then added to the resin. The mixture was shaken for 2 h. The resin was filtered and washed three times with DMF, twice with dichloromethane (DCM), and three times with DMF. Fmoc removals were carried out by treatment with 20% piperdine in DMF (5 mL, 2 x 15 min). The first 23 residues were single coupled through repetition of the Fmoc-amino acid coupling and Fmoc removal steps described above.

The remaining residues were double coupled by

performing the coupling step twice before proceeding with Fmoc removal. Following synthesis, the resin was drained, and washed sequentially with DCM, DMF, and DCM, and then dried in vacuo. The peptide-resin was transferred to a 250-mL plastic round bottom flask.

The peptide was deprotected and cleaved from the resin by treatment with

triisopropylsilane (1.5 mL), 3,6-dioxa-1,8-octane-dithiol (DODT, 1.5 mL), water (1.5 mL), trifluoroacetic acid (TFA, 20 mL), and a stir bar, and the mixture was stirred for 3 h. The mixture was filtered through a 150-mL sintered glass funnel into a 250-mL plastic round bottom


27


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flask, and the filtrate was concentrated in vacuo.

Page 28 of 62

The crude peptide was precipitated by

dropwise addition to cold diethyl ether in a 50 mL centrifuge tube, collected by centrifugation, and dried under vacuum. Peptide Folding. The dry crude linear peptide (about 600 mg from 0.2 mmol), for example [Lys16]ShK peptide, was dissolved in 16 mL acetic acid, 64 mL water, and 40 mL acetonitrile. The mixture was stirred rapidly for 15 min to complete dissolution. The peptide solution was added to a 2-L plastic bottle that contained 1700 mL of water and a large stir bar. To the diluted peptide solution was added 20 mL of concentrated ammonium hydroxide to raise the pH of the solution to 9.5. The pH was adjusted with small amounts of acetic acid or NH4OH as necessary. The solution was stirred at 80 rpm overnight and monitored by LC-MS. Folding was usually judged to be complete in 24 to 48 h, and the solution was quenched by the addition 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 highperformance liquid chromatography (RP-HPLC) was performed on a preparative (C18, 10 µm, 2.2 cm × 25 cm) column. Chromatographic separations were achieved using linear gradients of buffer B in A (A = 0.1% aqueous TFA; B = 90% aq. acetonitrile containing 0.09% TFA) typically 5-65% over 90 min at 20 mL/min for preparative separations. Preparative HPLC fractions were characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized. Final analysis (Phenomenex Synergi MAX-RP 2.5 micron, 100 Å, 50 x 2.0 mm column eluted with a 10 to 50% B over 10 min gradient (A: water and B: acetonitrile, 0.1% TFA in each) at a 0.650 mL/min flow rate monitoring UV absorbance at 220 nm) was performed for each peptide sample using an Agilent 1290 LC-MS. Peptides with > 95% purity and correct


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(m/z) ratio were screened. (See Supporting Information Table S8 for LC-MS characterization of ShK and peptide analogs.) PEGylation, Purification and Analysis. Peptide, e.g. [Lys16]ShK, was selectively PEGylated by reductive alkylation at its N-terminus, using activated linear or branched PEG. Conjugation was performed at 2 mg/ml in 50 mM NaH2PO4, pH 4.5 reaction buffer containing 20mM sodium cyanoborohydride and a 2 molar excess of 20 kDa monomethoxy-PEG-aldehyde (NOF, Japan). Conjugation reactions were stirred for approximately 5 hr at room temperature, and their progress was monitored by RP-HPLC. Completed reactions were quenched by 4-fold dilution with 20 mM NaOAc, pH 4 and chilled to 4°C.

The PEG-peptides were then purified

chromatographically at 40°C; using SP Sepharose HP columns (GE Healthcare, Piscataway, NJ) eluted with linear 0-1M NaCl gradients in 20mM NaOAc, pH 4.0. Eluted peak fractions were analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%. Principle contaminants observed were di-PEGylated toxin peptide analog.

Selected pools were

concentrated to 2-5 mg/ml by centrifugal filtration against 3 kDa MWCO membranes and dialyzed into 10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then sterile filtered through 0.2 micron filters and purity determined to be >97% by SDS-PAGE and RP-HPLC (see Supporting Information Figures S2–S3).

Reverse-phase HPLC was performed on an Agilent

1100 model HPLC running a Zorbax® 5µm 300SB-C8 4.6 x 50 mm column (Agilent) in 0.1% TFA/H2O at 1 ml/min and column temperature maintained at 40°C. Samples of PEG-peptide (20 µg) were injected and eluted in a linear 6-60% gradient while monitoring at a wavelength of 215 nm. Electrophysiology


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Cell Lines Expressing Kv1.1 through Kv1.7. CHO K1 cells were stably transfected with human Kv1.3, or, for counterscreens, with hKv1.4, hKv1.6, or hKv1.7; HEK293 cells were stably expressing human Kv1.3 or human Kv1.1. Cell lines were from Amgen or BioFocus DPI (A Galapagos Company). CHO K1 cells stably expressing hKv1.2, for counterscreens, were purchased from Millipore (Cat#.CYL3015). Whole Cell Patch Clamp Electrophysiology. Whole-cell currents were recorded at room temperature using MultiClamp 700B amplifier from Molecular Devices Corp. (Sunnyvale, CA), with 3-5MΩ pipettes pulled from borosilicate glass (World Precision Instruments, Inc). During data acquisition, capacitive currents were canceled by analog subtraction, no series resistance compensation was used, and all currents were filtered at 2 kHz. The cells were bathed in an extracellular solution containing 1.8 mM CaCl2, 5 mM KCl, 135 mM NaCl, 5 mM Glucose, 10 mM HEPES, pH 7.4, 290-300 mOsm. The internal solution containing 90 mM KCl, 40 mM KF, 10 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2, 290-300 mOsm. The currents were evoked by applying depolarizing voltage steps from -80 mV to +30 mV every 30 s ( Kv1.3) or 10 s ( Kv1.1) for 200 ms intervals at holding potential of -80 mV. To determine IC50, 5-6 peptide or peptide conjugate concentrations at 1:3 dilutions were made in extracellular solution with 0.1 % BSA and delivered locally to cells with Rapid Solution Changer RSc-160 (BioLogic Science Instruments). Currents were achieved to steady state for each concentration. Data analysis was performed using pCLAMP (version 9.2) and OriginPro (version 7), and peak currents before and after each test article application were used to calculate the percentage of current inhibition at each concentration. PatchXpress® Planar Patch-Clamp Electrophysiology. Cells were bathed in an extracellular solution containing 1.8 mM CaCl2, 5 mM KCl, 135 mM NaCl, 5 mM Glucose, 10 mM HEPES,


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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 concentrations at 1:3 dilutions were made to determine the IC50s. The peptide or peptide conjugates were prepared in extracellular solution containing 0.1% BSA. Dendrotoxin-k and Margatoxin were purchased from Alomone Labs Ltd. (Jerusalem, Israel); ShK toxin was purchased from Bachem Bioscience, Inc. (King of Prussia, PA); 4-AP was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Currents were recorded at room temperature using a PatchXpress® 7000A electrophysiology system from Molecular Devices Corp. (Sunnyvale, CA). The voltage protocols and recording conditions are shown in the Supporting Information Table S1. An extracellular solution with 0.1% BSA was applied first to obtain 100% percent of control (POC), then followed by 5 different concentrations of 1:3 peptide or peptide conjugate dilutions for every 400 ms incubation time. At the end, excess of a specific benchmark ion channel inhibitor was added to define full or 100% blockage. The residual current present after addition of benchmark inhibitor, was used in some cases for calculation of zero percent of control. Each individual set of traces or trial were visually inspected and either accepted or rejected. The general criteria for acceptance were: 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 a steady-state prior to first compound addition. The POC was calculated from the average peak current of the last 5 sweeps before the next concentration compound addition and exported to Excel for IC50 calculation. IonWorks Quattro High-Throughput Population Patch-Clamp Electrophysiology. Electrophysiology was performed on CHO cells stably expressing hKv1.3 and HEK293 cells stably expressing hKv1.1. The procedure for preparation of the “Assay Plate” containing ShK


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analogs and conjugates for IWQ electrophysiology was as follows: all analogs were dissolved in extracellular buffer (PBS, with 0.9 mM Ca2+ and 0.5 mM Mg2+) with 0.3% BSA and dispensed in the row H of 96-well polypropylene plates at the concentration of 100 nM from column 1 to column 10. Column 11 and 12 were reserved for negative and positive controls, then serial diluted at 1:3 ratio to row A. IonWorks Quattro (IWQ) electrophysiology and data analysis were accomplished as follows: re-suspended cells (in extracellular buffer), the Assay Plate, a Population Patch Clamp (PPC) PatchPlate as well as appropriate intracellular (90 mM potassium gluconate, 20 mM KF, 2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35) and extracellular buffers were positioned on IonWorks Quattro. When the analogs were added to patch plates, they were further diluted 3-fold from the assay plate to achieve a final test concentration range from 33.3 nM to 15 pM with 0.1% BSA. Electrophysiology recordings were made from the CHO-Kv1.3 and HEK-Kv1.1 cells using an amphotericin-based perforated patchclamp method. Using the voltage-clamp circuitry of the IonWorks Quattro, cells were held at a membrane potential of –80 mV and voltage-activated K+ currents were evoked by stepping the membrane potential to +30 mV for 400 ms. K+ currents were evoked under control conditions i.e., in the absence of inhibitor at the beginning of the experiment and after 10-minute incubation in the presence of the analogs and controls. The mean K+ current amplitude was measured between 430 and 440ms and the data were exported to a Microsoft Excel spreadsheet. The amplitude of the K+ current in the presence of each concentration of the analogs and controls was expressed as a percentage of the K+ current of the pre-compound current amplitude in the same well. When these % of control values were plotted as a function of concentration, the IC50 value for each compound could be calculated using the dose-response fit model 201 in Excel fit program.


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Measuring Bioactivity in Whole Blood Ex vivo assay to examine impact of toxin peptide analog Kv1.3 inhibitors on secretion of human IL-2 and IFN-γ. The potency of ShK analogs and conjugates in blocking T cell inflammation in human whole blood was examined using an ex vivo assay that has been described earlier.40 In brief, 50% human whole blood is stimulated with thapsigargin to induce store depletion, calcium mobilization and cytokine secretion. To assess the potency of molecules in blocking T cell cytokine secretion, various concentrations of Kv1.3 blocking peptides and peptide-conjugates were pre-incubated with the human whole blood sample for 30-60 min prior to addition of the thapsigargin stimulus. After 48 hours at 37ºC and 5% CO2, conditioned medium was collected and the level of cytokine secretion was determined using a 4-spot electrochemilluminescent immunoassay from MesoScale Discovery. Using the thapsigargin stimulus, the cytokines IL-2 and IFN-γ were secreted robustly from blood isolated from multiple donors. The IL-2 and IFNγ produced in human whole blood following thapsigargin stimulation were produced from T cells, as revealed by intracellular cytokine staining and fluorescence-activated cell sorting (FACS) analysis. Pharmacokinetic & Pharmacodynamic Studies Detection Antibodies to ShK. Rabbit polyclonal and mouse monoclonal antibodies to ShK were generated by immunization of animals with an Fc-ShK peptibody conjugate.39

Anti-ShK

specific polyclonal antibodies were affinity purified from antisera to isolate only those antibodies specific for the ShK portion of the conjugate. Following fusion and screening, hybridomas specific for ShK were selected and isolated. Mouse anti-ShK specific monoclonal antibodies were purified from the conditioned media of the clones. By ELISA analysis, purified anti-ShK


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polyclonal and monoclonal antibodies reacted only to the ShK peptide alone and did not crossreact with Fc. Pharmacokinetic (PK) studies 20 kDa-PEG-ShK peptide conjugates in mice, rats, dogs and monkeys. Single subcutaneous doses were delivered to animals, and serum was collected at various time points after injection. Studies in rats, dogs (beagles), and cynomolgus monkeys involved two to three animals per dose group, with blood and serum collection occurring at various time points over the course of the study.

Male Sprague-Dawley (SD) rats (about 0.3

kg), male beagles (about 10 kg) and male cynomolgus monkeys (about 4 kg) were used in the studies described herein (n = 3 animals per dose group). Approximately 5 male CD-1 mice were used per dose and time point in our mouse pharmacokinetic studies. Serum samples were stored frozen at -80°C, until analysis in an enzyme-linked immunosorbent assay (ELISA). The following is a brief description of the ELISA protocol for detecting serum levels of conjugates 144 and 145, as well as the ShK and 122 peptides alone. Streptavidin microtiter plates were coated with 250 ng/ml biotinylated-anti-ShK mouse monoclonal antibody (mAb2.10, Amgen) in I block buffer [per liter: 1000 mL 1xPBS without CaCl2, MgCl2, 5 ml Tween 20 (Thermo Scientific), 2 g I block reagent (Tropix)] at 4°C, incubated overnight without shaking. Plates were washed three times with KPL wash buffer (Kirkegaard & Perry Laboratories). Standards (STD), quality controls (QC) and sample dilutions were prepared with 100% pooled sera, then diluted 1/5 (pretreatment) in I block buffer. Pretreated STDs, QCs and samples were added to the washed plate and incubated at room temperature for 2 hours. (Serial dilutions of STDs, QCs were prepared in 100% pooled sera. Samples needing dilution were also prepared with 100% pooled sera. The pretreatment was done to both STDs, QCs and samples to minimize the matrix effect.) Plates were washed three times with KPL wash buffer. A HRP-labeled rabbit


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anti-ShK polyclonal Ab at 250 ng/mL in I block buffer was added and plates were incubated at room temperature for 1 hour with shaking. Plates were again washed three times with KPL wash buffer and the Femto (Thermo Scientific) substrate was added. The plate was read with a Lmax II 384 (Molecular Devices) luminometer. Adoptive-Transfer EAE Model of Efficacy An adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis in rats described earlier32 was used to examine the activity in vivo of our Kv1.3selective 145 analog and compare its efficacy to the less selective molecule 144.

The

encephalomyelogenic CD4+ rat T cell line, PAS, specific for myelin-basic protein (MBP) was kindly provided by Dr. Evelyne Beraud. The maintenance of these cells in vitro and their use in the AT-EAE model has been described.32 PAS T cells were maintained in vitro by alternating rounds of antigen stimulation or activation with MBP and irradiated thymocytes (2 days), and propagation with T cell growth factors (5 days). Activation of PAS T cells (3 × 105/ml) involved incubating the cells for 2 days with 10 µg/ml MBP and 15 × 106/ml syngeneic irradiated (3500 rad) thymocytes. On day 2 after in vitro activation, 10-15 × 106 viable PAS T cells were injected into 6-12 week old female Lewis rats (Charles River Laboratories) by tail IV. Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS), 145 or 144 or ShK were given from either day −1 to day 7 or day -1 today 3, where day −1 represents 1 day prior to injection of PAS T cells (day 0 in Figure 8). Serum was collected by retro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end of the study) for analysis of levels of inhibitor. Rats were weighed on days –1 and days 4 - 8. Animals were scored blinded once a day from the day of cell transfer (day 0) to day 3, and twice a day from day 4 to day 8. Clinical signs were evaluated as the total score of the degree of paresis of each limb and tail. Clinical scoring (“EAE Score” in


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Figure 8): 0 = No signs, 0.5 = distal limp tail, 1.0 = limp tail, 2.0 = mild paraparesis, ataxia, 3.0 = moderate paraparesis, 3.5 = one hind leg paralysis, 4.0 = complete hind leg paralysis, 5.0 = complete hind leg paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund state or death. Rats reaching a score of 5.5 were euthanized. Pharmacology Study in Cynomolgus Monkey A repeat-dose pharmacology study was designed and implemented in order to investigate the long-term effects of the molecule 145 in nonhuman primates. Prior to initiating the study, 6 to 10 male cynomolgus monkeys were profiled for a period of 3-10 weeks to allow for assessment of the end-points’ stability over time and selection of 6 cynomolgus monkeys for the study. The end-points measured included complete blood counts (CBCs), blood chemistry, FACS analysis of lymphocyte subsets, and the ex vivo whole blood PD assay measuring cytokine response and target coverage. Subsets analyzed by FACS included: lymphocytes, CD4+, CD4+ naïve, CD4+ TCM, CD4+ TEM, CD4+CD28-CD95-, CD8+, CD8+ naïve, CD8+ TCM, CD8+ TEM, CD8+CD28CD95-, B cells, NK cells, and NKT cells. Monkeys with the highest level of CD4+ effector memory T cells were chosen.

The design of the 12-week cynomolgus pharmacology study is

illustrated in the Supporting Information Table S5. Male Chinese cynomolgus monkeys that were used in this study were naïve (no earlier exposure to drugs). Care was taken to avoid undue stress. All injections and blood draws were done by arm-pull, with the monkeys voluntarily presenting their arm for a grape incentive. The study involved baseline measures for two weeks (3 predose samples), one month of Kv1.3 block (qw dosing of 145) and 6 weeks follow-up analysis. Ex vivo cynomolgus monkey whole blood assay to measure the potency of 145 and its level of pharmacodynamic coverage in vivo. The potency and level of coverage of cynomolgus monkey


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T cell responses was determined with an ex vivo whole blood assay measuring thapsigargininduced IL-4, IL-5 and IL-17.

To determine potency of peptides and peptide conjugates,

cynomolgus whole blood was obtained from healthy, naïve, male monkeys in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Gemini Bioproducts, #800-120), 55 µM 2mercaptoethanol (Gibco), and 1X Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Jerusalem, Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 µM, 4X solution to provide the 4X thapsigargin stimulus for calcium mobilization.

The Kv1.3 inhibitor peptide ShK

(Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The calcineurin inhibitor cyclosporin A (CsA) is available commercially from a variety of vendors. Whereas the BKCa inhibitor IbTx and the Kv1.1 inhibitor DTX-k do not inhibit the cytokine response, the Kv1.3 inhibitor ShK and the calcineurin inhibitor CsA inhibit the cytokine response and are used routinely as standards or positive controls. Ten 3-fold serial dilutions of standards, ShK analogs or ShK-conjugates were prepared in DMEM complete media at 4X final concentration and 50 µl of each were added to wells of a 96-well Falcon 3075 flat-bottom microtiter plate. Whereas columns 1-5 and 7-11 of the microtiter plate contained inhibitors (each row with a separate inhibitor dilution series), 50 µl of DMEM complete media alone was added to the 8 wells in column 6 and 100 µl of DMEM complete media alone was added to the 8 wells in column 12. To initiate the experiment, 100 µl of whole blood was added to each well of the microtiter plate. The plate was then incubated at 37°C, 5% CO2 for one hour. After one hour, the plate was removed and 50 µL/well of the 4x


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thapsigargin stimulus (40 µM) was added to all wells of the plate, except the 8 wells in column 12. The plates were placed back at 37°C, 5% CO2 for 48 hours. To determine the amount of IL4, IL-5 and IL-17 secreted in whole blood, 100 µL of the supernatant (conditioned media) from each well of the 96-well plate was transferred to a storage plate. For Meso Scale Discovery (MSD) electrochemiluminesence analysis of cytokine production, the supernatants (conditioned media) were tested using MSD Multi-Spot Custom Coated plates (Meso Scale Discovery, Gaithersburg, MD). The working electrodes on these plates were coated with seven Capture Antibodies (hIL-2. hIL-4, hIL-5, hIL-10, hTNFa, hIFNg and hIL-17) in advance. After blocking plates with MSD Human Serum Cytokine Assay Diluent, and then washing with PBS containing 0.05% of BSA, 25 µL/well of conditioned medium was added to wells of the MSD plate. The plates were covered and placed on a shaking platform for 1hr. Next, 25 µL of a cocktail of Detection Antibodies in MSD Antibody Diluent were added to each well. The cocktail contained seven Detection Antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFa, hIFNg and hIL-17) at 1 µg/mL each. The plates were covered and placed on a shaking platform overnight (in the dark). The next morning the plates were washed three times with PBS buffer. 150 µl of 2X MSD Read Buffer T was added to wells of the plate before reading on the MSD Sector Imager. Since the 8 wells in column 6 of each plate received only the thapsigargin stimulus and no inhibitor, the average MSD response here was used to calculate the “High” value for a plate. The calculate “Low” value for the plate was derived from the average MSD response from the 8 wells in column 12 which contained no thapsigargin stimulus and no inhibitor. Percent of control (POC) is a measure of the response relative to the unstimulated versus stimulated controls, where 100 POC is equivalent to the average response of thapsigargin stimulus alone or the “High” value. Therefore, 100 POC represents 0% inhibition of the response. In contrast, 0


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POC represents 100% inhibition of the response and would be equivalent to the response where no stimulus is given or the “Low” value. To calculate percent of control (POC), the following formula is used: [(MSD response of well) – (“Low”)] / [(“High”) – (“Low”)] x 100. The potency of the molecules in whole blood was calculated after curve fitting from the inhibition curve (IC) and IC50 was derived using standard curve fitting software.

The experimental

procedure for adaption of the above method to an ex vivo cynomolgus monkey pharmacodynamic (PD) assay to measure the level of T cell Kv1.3 coverage in vivo following the dosing of animals is included in the Supporting Information. Repeat-dose Toxicology Study Male cynomolgus monkeys (Macaca fascicularis; 2 to 4 years and 2 to 5 kg), were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 7th Edition.41 Animals were individually house in stainless steel cages, except when commingled for environmental enrichment, at an indoor American Association for Accreditation of Laboratory Animal Care (AAALAC), international-accredited facility in species-specific housing. The research protocol was approved by the Institutional Animal Care and Use Committee. Animals were fed a certified pelleted primate diet (#2055C, Harlan Laboratories Inc., Indianapolis, IN) daily in amounts appropriate for the age and size of the animals, and had ad libitum access to water via automatic watering system. Animals were maintained on a 12:12 hr light: dark cycle in rooms at 18°C to 26°C and 30% to 70% humidity and had access to enrichment opportunities, including cageenrichment devices and commingling. Animals were acclimated for at least 1 week, and then randomized to treatment groups to achieve body weight balance with respect to groups. Fifteen animals were placed into 1 of 5 groups (n = 3/dose group) receiving vehicle (10mM NaOAc, 5% Sorbitol, pH 4.0) or 145. Doses of 0.7


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mg/kg every third day for 2 weeks (4 doses total) or 0.1, 0.5, or 2.0 mg/kg weekly for 2 weeks (2 doses total) were administered via subcutaneous injection. After 2 weeks, all animals were anesthetized with sodium pentobarbital, exsanguinated, and necropsied.

The following study

parameters were evaluated: clinical observations, body weights, food consumption, qualitative and quantitative electrocardiograms (ECG), toxicokinetics, routine clinical pathology (complete blood count, coagulation, and clinical chemistry), urinalysis, a panel of urinary biomarkers of renal injury, organ weights, macroscopic observations, and light microscopic observations of a full set of tissues. Crystallization and Structure Determination of [Lys16]ShK Crystallization of racemic [Lys16]ShK was performed by mixing equal amounts (by weight) of lyophilized D-[Lys16]ShK and L-[Lys16]ShK in water at 50 mg/mL total concentration (25 mg/mL of D-[Lys16]ShK and 25 mg/mL of L-[Lys16]ShK). Hanging drop crystallization screens were set up using JCSG core suites 1-4 (Qiagen) at room temperature. Crystals appeared in several conditions within a week. One condition, 0.1 M sodium acetate pH 4.5, 2-2.5 M ammonium sulfate produced diffraction quality crystals. For data collection, crystals were cryoprotected in mother liquor containing 20% (v/v) glycerol and flash cooled to 100 K. Diffraction data were collected at the Advanced Light Source Beamline 5.0.2 (Lawrence Berkeley National Laboratory, Berkeley, CA) at 100 K. Data were processed and scaled using HKL2000. Attempts to phase the data by molecular replacement using a NMR structure of ShK (PDB:1ROO)19a were unsuccessful, so material labeled with selenomethionine (SeMet) at position 21 was prepared and used for phasing. Selenomethionine crystals were grown by mixing native D-[Lys16]ShK with L-[Lys16,SeMet21]ShK. The selenomethionine labeled crystals grew in identical conditions as crystals of the unlabeled peptides. Data sets from


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selenomethionine containing crystals were collected at three wavelengths, peak (0.9792 λ), inflection (0.9794 λ) and remote (0.9824 λ).42 The single selenium atom position was determined with Crank in CCP4.43 Crank used SHELX C/D44 for substructure search, SHELX E for substructure refinement and Solomon45 for density modification. This generated a high quality map, and a poly-alanine model was created using Coot.46 This model was used for molecular replacement into the native data for final refinement. Phaser47 was used for molecular replacement. The space group is R-3 with a = 60.78 Å, b = 60.78 Å, c = 43.59 Å, and one molecule in the asymmetric unit. After several rounds of model building using Coot and refinement with REFMAC5 in the CCP4 suite, the R factor and Rfree values converged to 20.4% and 22.6%, respectively. Stereochemical analysis of the refined model was performed using Coot validation tools and MolProbity.48 Structural figures were produced using PyMol (http://www.pymol.org/). The coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank (accession number: 4Z7P). X-Ray crystallography data collection and refinement statistics are included in the Supporting Information Table S9. Molecular Docking of ShK to Kv1.3 Homology Model The molecular modeling program MOE49 was used to construct a homology model of the human Kv1.3 channel using the Kv1.2-Kv2.1 paddle chimera channel structure18b (2R9R.pdb) as a template. The [Lys16]ShK crystal structure was docked to the channel homology model using ZDOCK as implemented in the Discovery Studio 4.1 modeling package.50,51 To simplify the docking, the voltage-sensing domains were excluded from the calculation, and the pore was used as the receptor (residues 381–491). To limit docked poses to the extracellular vestibule, receptor residues 381–415, 437–443 and 459–491 were excluded from the peptide-protein interface. An angular step size of 6 degrees and distance cutoff of 5 Å were used resulting in 54,000 potential


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poses. The resulting poses were filtered to ensure that residues presumed important for binding based on the MAPS analoging ([Lys16]ShK residues 7, 11, 20, 21, 22, 23, and 27) were within 4 Å of any residue on the channel. Additionally, poses were retained only if an atom from the [Lys16]ShK toxin was within 4 Å of the key pore-forming Gly residues 446 and 448 on the Kv1.3 channel.

A total of 777 poses passed these criteria and were advanced to further

minimization and optimization using RDOCK.52 The top poses were visualized with respect to scanning and selectivity data; including the proximity of Lys16 to residue differences between the Kv1.1 and Kv1.3 channels. Supporting Information. Characterization of ShK peptide analogs, characterization and additional pharmacokinetic and pharmacological data for 145. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Authors. * To whom correspondence should be addressed. John K. Sullivan. Phone: 1-805-447-3695. Email: [email protected]. Les P. Miranda. Phone: 1-805-447-9397. E-mail: [email protected]. Acknowledgement. We gratefully thank Ankita Shah, Jason Long, Stephanie Diamond, Ryan Holder, and Jingwen Zhang for peptide synthesis support and Lei Jia and Kaustav Biswas for data visualization. We wish to thank Dr. Evelyn Beraud (Laboratoire d'Immunologie, Faculte de Medecine, Marseille, France) and Dr. Christine Beeton (Baylor College of Medicine) for providing the rat PAS T cell line. The Advanced Light Source is supported by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.


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ABBREVIATIONS AEEA, 2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid; Boc, tert-butoxycarbonyl; Dap, L-2,3diaminopropionic acid; Fmoc, Nα-9-fluorenylmethoxycarbonyl; 1-Nal, L-1-naphthylalanine; Nle, L-norleucine; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; tBu, tert-butyl; Trt, trityl REFERENCES

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7 Kalman, K.; Pennington, M. W.; Lanigan, M. D.; Nguyen, A.; Rauer, H.; Mahnir, V.; Paschetto, K.; Kem, W. R.; Grissmer, S.; Gutman, G. A.; Christian, E. P.; Cahalan, M. D.; Norton, R. S.; Chandy, K. G. ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide. J. Biol. Chem. 1998, 273, 32697–32707. 8 (a) Chandy, K. G.; DeCoursey, T. E.; Cahalan, M. D.; McLaughlin, C.; Gupta, S. Voltagegated potassium channels are required for human T lymphocyte activation. J. Exp. Med. 1984, 160, 369-385. (b) DeCoursey, T. E.; Chandy, K. G.; Gupta, S.; Cahalan, M. D. Voltagedependent ion channels in T-lymphocytes. J. Neuroimmunol. 1985, 10, 71-95. 9 (a) Wulff, H.; Calabresi, P. A.; Allie, R.; Yun, S.; Pennington, M.; Beeton, C.; Chandy, K. G. The voltage-gated Kv1.3 K+ channel in effector memory T cells as new target for MS. J. Clin. Invest. 2003, 111, 1703–1713. (b) Beeton, C.; Wulff, H.; Standifer, N. E.; Azam, P.; Mullen, K. M.; Pennington, M. W.; Kolski-Andreaco, A.; Wei, E.; Grino, A.; Counts, D. R.; Wang, P. H.; LeeHealey, C. J.; Andrews, B. S.; Sankaranarayanan, A.; Homerick, D.; Roeck, W. W.; Tehranzadeh, J.; Stanhope, K. L.; Zimin, P.; Havel, P. J.; Griffey, S.; Knaus, H. G.; Nepom, G. T.; Gutman, G. A.; Calabresi, P. A.; Chandy, K. G. Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17414–17419. 10 Chandy, K. G.; Wulff, H.; Beeton, C.; Pennington, M.; Gutman, G. A.; Cahalan, M. D.; K+ channels as targets for specific immunomodulation. Trends Pharmacol. Sci. 2004, 25, 280–289. 11 Winslow, M. W.; Neilson, J. R.; Crabtree, G. R. Calcium signaling in lymphocytes. Curr. Op. Immunol. 2003, 15, 299-307. 12 (a) Feske, S.; Prakriya, M.; Rao, A.; Lewis, R. S. A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients. J. Exp. Med. 2005, 202, 651-662.

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blockers as therapeutic agents to interfere with bone resorption of periodontal disease. J. Dent. Res. 2005, 84, 488–499. 16 (a) Pennington, M. W. ; Mahnir, V. M.; Khaytin, I.; Zaydenberg, I.; Byrnes, M. E.; Kem, W. R. An essential binding surface for ShK toxin interaction with rat brain potassium channels. Biochemistry. 1996, 35, 16407–16411. (b) Rauer, H.; Pennington, M.; Cahalan, M.; Chandy, K. G. Structural conservation of the pores of calcium-activated and voltage-gated potassium channels determined by a sea anemone toxin. J. Biol. Chem. 1999, 274, 21885–21892. (c) Pennington, M. W.; Mahnir, V. M.; Krafte, D.S.; Zaydenberg, I; Byrnes, M. E.; Khaytin, I.; Crowley, K.; Kem, W. R. Identification of three separate binding sites on ShK toxin, a potent inhibitor of voltage-dependent potassium channels in human T-lymphocytes and rat brain. Biochem. Biophys. Res. Comm. 1996, 219, 696-701. 17 Lanigan, M. D.; Kalman, K.; Lefievre, Y.; Pennington, M. W.; Chandy, K. G.; Norton, R. S.; Mutating a critical lysine in ShK toxin alters its binding configuration in the pore-vestibule region of the voltage-gated potassium channel, Kv1.3. Biochemistry. 2002, 41, 11963–11971. 18 (a) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998, 280, 69-77.

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MacKinnon, R. Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature. 2007, 450, 376–382. 19 (a) Tudor, J. E.; Pallaghy, P. K.; Pennington, M. W.; Norton, R. S. Solution structure of ShK toxin, a novel potassium channel inhibitor from a sea anemone. Nat. Struct. Biol. 1996, 3, 317–


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320. (b) Pennington, M. W.; Lanigan, M. D.; Kalman, K.; Mahnir, V. M.; Rauer, H.; McVaugh, C. T.; Behm, D.; Donaldson, D.; Chandy, K. G.; Kem, W. R.; Norton, R. S. Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin. Biochemistry. 1999, 38, 14549–14558. 20 (a) Harunur, R. M.; Kuyucak, S. Affinity and selectivity of ShK toxin for the Kv1 potassium channels from free energy simulations. J. Phys. Chem. B. 2012, 116, 4812-4822. (b) Rashid, M. H.; Heinzelmann, G.; Huq, R.; Tajhya, R. B.; Chang, S. C.; Chhabra, S.; Pennington, M. W.; Beeton, C.; Norton, R. S.; Kuyucak, S. A potent and selective peptide blocker of the Kv1.3 channel: prediction from free-energy simulations and experimental confirmation. PloS One. 2013, 8, e78712. doi:10.1371/journal.pone.0078712. 21 (a) Beeton, C.; Wulff, H.; Singh, S.; Botsko, S.; Crossley, G.; Gutman, G. A.; Cahalan, M. D.; Pennington, M.; Chandy, K. G. A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes. J. Biol. Chem. 2003, 278, 9928–9937. (b) Beeton, C.; Pennington, M. W.; Wulff, H.; Singh, S.; Nugent, D.; Crossley, G.; Khaytin, I.; Calabresi, P. A.; Chen, C. Y.; Gutman, G. A.; Chandy, K. G. Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases. Mol. Pharmacol. 2005, 67, 1369–1381. (c) Pennington, M. W.; Beeton, C.; Galea, C. A.; Smith, B. J.; Chi, V.; Monaghan, K. P.; Garcia, A.; Rangaraju, S.; Giuffrida, A.; Plank, D.; Crossley, G.; Nugent, D.; Khaytin, I.; Lefievre, Y.; Peshenko, I.; Dixon, C.; Chauhan, S.; Orzel, A.; Inoue, T.; Hu, X.; Moore, R. V.; Norton, R. S.; Chandy, K. G. Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes. Mol. Pharmacol. 2009, 75, 762–773. 22 Cunningham, B. C.; Wells, J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989, 244, 1081-1085.


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23 Chang, Y.-P.; Banerjee, J.; Dowell, C.; Wu, J.; Gyanda, R.; Houghten, R. A.; Toll, L.; McIntosh, J. M.; Armishaw, C. J. Discovery of a potent and selective α3β4 nicotinic acetylcholine receptor antagonist from an α-conotoxin synthetic combinatorial library. J. Med. Chem. 2014, 57, 3511-3521. 24 Tarcha, E.J.; Chi, V.; Muñoz-Elias, E. J.; Bailey, D.; Londono, L. M.; Upadhyay, S. K.; Norton, K. N.; Olson, A.; Tjong, I.; Nguyen, H. M.; Hu, X.; Rupert, G. W.; Boley, S. E.; Slauter, R.; Sams, J.; Knapp, B.; Kentala, D.; Hansen, Z.; Pennington, M. W.; Beeton, C.; Chandy, K. G.; Iadonato, S. P. Durable pharmacological responses from a single dose of the peptide drug ShK186, a specific Kv1.3 channel inhibitor. J. Pharmacol. Exp. Ther. 2012, 342, 642–653. 25 Moore, S. J.; Cochran, J. R. Engineering knottins as novel binding agents. Meth. Enzymol. 2012, 503, 223-251. 26 Thastrup, O.; Cullen, P. J.; Drøbak, B. K.; Hanley, M. R.; Dawson, A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2466–2470. 27 Dang, B.; Kubota, T.; Mandal, K.; Bezanilla, F.; Kent, S. B. H. Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography. J. Am. Chem. Soc. 2013, 135, 11911-11919. 28 Norton, R. S.; Pennington, M. W.; Wulff, H. Potassium channel blockade by the sea anemone toxin ShK for the treatment of multiple sclerosis and other autoimmune diseases. Curr. Med. Chem. 2004, 11, 3041–3052. 29 Pennington, M. W.; Rashid, M. H.; Tajhya, R. B.; Beeton, C.; Kuyucak, S.; Norton, R. S. A C-terminally amidated analogue of ShK is a potent and selective blocker of the voltage-gated potassium channel Kv1.3. FEBS Lett. 2012, 586, 3996-4001.


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30 Pennington, M. W.; Chang, S. C.; Chauhan, S.; Huq, R.; Tajhya, R. B.; Chhabra, S.; Norton, R. S.; Beeton, C. Development of highly selective Kv1.3-blocking peptide based on the sea anemone peptide ShK. Mar. Drugs 2015, 13, 529-542. 31 Middleton, R. E.; Sanchez, M.; Linde, A. R.; Bugianesi, R. M.; Dai, G.; Felix, J. P.; Koprak, S. L.; Staruch, M. J.; Bruguera, M.; Cox, R.; Ghosh, A.; Hwang, J.; Jones, S.; Kohler, M.; Slaughter, R. S.; McManus, O. B.; Kaczorowski, G. J.; Garcia, M. L.; Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile. Biochemistry 2003, 42, 13698–13707. 32 Beeton, C.; Wulff, H.; Barbaria, J.; Clot-Faybesse, O.; Pennington, M.; Bernard, D.; Cahalan, M. D.; Chandy, K. G.; Béraud, E. Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13942–13947. 33 Beeton, C.; Smith, B. J.; Sabo, J. K.; Crossley, G.; Nugent, D.; Khaytin, I.; Chi, V.; Chandy, K. G.; Pennington, M. W.; Norton, R. S. The D-diastereomer of ShK toxin selectively blocks voltage-gated K+ channels and inhibits T lymphocyte proliferation. J. Biol. Chem. 2008, 283, 988–997. 34 Edwards, W.; Fung-Leung, W.-P.; Huang, C.; Chi, E.; Wu, N.; Liu, Y.; Maher, M. P.; Bonesteel, R.; Connor, J.; Fellows, R.; Garcia, E.; Lee, J.; Lu, L.; Ngo, K.; Scott, B.; Zhou, H.; Swanson, R. V.; Wickendon, A. D. Targeting the ion channel Kv1.3 with scorpion venom peptides engineered for potency, selectivity, and half-life. J. Biol. Chem. 2014, 289, 2270422714. 35 Chang, S. C.; Huq, R.; Chhabra, S.; Beeton, C.; Pennington, M. W.; Smith, B. J.; Norton, R. S. N-terminally extended analogues of the K+ channel toxin from Stichodactyla helianthus as


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potent and selective blockers of the voltage-gated potassium channel Kv1.3. FEBS J. 2015, DOI: 10.1111/febs.13294. 36 Harris, J. M.; Chess, R. B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discovery 2003, 2, 214–221. 37 Conjugates were tested in vitro for their activity in inhibiting antigen (myelin)-mediated proliferation (3H-thymidine incorporation) of the rat T effector memory cell line, PAS. The methods employed here were similar to those described in ref. 32. 38 Beeton, C.; Barbaria, J.; Giraud, P.; Devaux, J.; Benoliel, A.-M.; Gola, M.; Sabatier, J. M.; Bernard, D.; Crest, M.; Beraud, E. Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation. J. Immunol. 2001, 166, 936-944. 39 Sullivan, J. K.; Miranda, L. P.; Gegg, C. V.; Hu, S.-F. S.; Belouski, E. J.; Murray, J. K.; Nguyen, H.; Walker, K. W.; Arora, T.; Jacobsen, F. W.; Li, Y.-S.; Boone, T. C. Selective and potent peptide inhibitors of Kv1.3. PCT Int. Appl. WO 2010108154 A2 20100923, 2010. 40 Sullivan, J. K.; McGivern, J. G.; Miranda, L. P.; Nguyen, H. Q.; Walker, K. W.; Hu, S.-F. S.; Gegg, C. V.; Arora Khare, T.; Adler, B. S.; Martin, F. H.Toxin peptide therapeutic agents. PCT Int. Appl. WO 2008088422 A2 20080724, 2008. 41 Guide for the Care and Use of Laboratory Animals, 7th ed.; National Academy Press: Washington, D.C., 1996. 42 Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307–326. 43 Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.;


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Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. Overview of the CCP4 suite and current developments. Acta Cryst. 2011, 67, 235–242. 44 Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Cryst. 2010, D66, 479–485. 45 Abrahams, J. P.; Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Cryst. 1996, D52, 30-42. 46 Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004, 60, 2126–2132. 47 McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 2005, 61, 458–464. 48 Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G.J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 12–21. 49 Molecular Operating Environment (MOE), 2007.09; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2007. 50 Chen, R.; Li, L.; Weng, Z. ZDOCK: an initial-stage protein docking algorithm. Proteins. 2003, 52, 80-87. 51 Accelrys Software Inc., Discovery Studio Modeling Environment, Release 2.1, San Diego: Accelrys Software Inc., 2008. 52 Chen, R.; Li, L.; Weng, Z. RDOCK: refinement of rigid-body protein docking predictions. Proteins. 2003, 53, 693–707.


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Figure 1. Amino acid sequence of the ShK toxin peptide (1) with three disulfide bonds formed by the six cysteines (C3—C35, C12—C28, and C17—C32).

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 analog from the MAPS analoging. Samples were tested against Kv1.3 and Kv1.1 on the IWQ platform. All values are avg. ± SD, n ≥ 2. Colors indicate IC50 values in each assay, with green indicating highly potent, light green meaning moderately potent, yellow indicating weakly potent, and red signifying not potent. Gray indicates no data because the folded peptide analog was not isolated. Data for wild-type sequence (1 (ShK) IL-2 IC50 = 37 ± 36 pM, IFN-γ IC50 = 48 ± 43 pM, Kv1.3 IC50 = 132 ± 79 pM, Kv1.1 IC50 = 20 ± 29 pM) has been included wherever the indicated substitution is the same as the native residue (Ala14, Arg1, Arg11, Arg24, Arg29, Lys9, Lys18, Lys22, and Lys30) and marked with a black rectangle.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 3. A–C) Functional activity and electrophysiological selectivity of lysine scan ShK analogs. A) Inhibition of IL-2 and IFN-γ secretion in whole blood assay. (Top concentration tested was 100 nM.) B) Kv1.1/Kv1.3 selectivity ratio. C) ShK peptide sequence with residues important for potency in red and bold, residues important for selectivity in blue and underlined, and residue important for both in purple, bold, and underlined. Note that substitution of lysine at position 16 uniquely enhanced Kv1.3 selectivity with retention of potency against cytokine secretion. D) Consensus findings from MAPS analoging of ShK with the residues likely to impact potency through conformational effects in bold and green, residues indicated as important for potency by a single series denoted in orange and bold, and the remainder as described in C).


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Figure 4. Crystal structure of [Lys16]ShK. A–B) 122 with side chains rendered as sticks and backbone secondary structure indicated with ribbons. C–F) Surface rendering of 122 with residues colored according to putative interaction with Kv1.3 during binding. Blue indicates direct contact; yellow and orange residues make peripheral contact with yellow substitutions affecting selectivity and orange substitutions impacting selectivity and potency. A, C, and E) View of the putative binding interface of the peptide. B, D, and F) Side view with Lys22 facing downward.


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Figure 5. Molecular docking of 122 (grey ribbon with residues important to binding in blue and Lys16 and Ser26 in yellow) to Kv1.3 homology model (green ribbons). A) Side view of pose I. For clarity, two monomers (II and IV) of the homotetrameric channel have been hidden. B) Top view of pose 1. C) Side view of pose 2. For clarity, two monomers (I and III) of the homotetrameric channel have been hidden. D) Top view of pose 2.


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Figure 6. Graphical comparison of the potency and selectivity of select naked and PEGylated peptide analogs relative to ShK. Each point represents one compound with the x-axis value computed as (whole blood IL-2 IC50) / (ShK whole blood IL-2 IC50) and y-axis value computed as ([Kv1.1 IC50 / Kv1.3 IC50] / [ShK Kv1.1 IC50 / ShK Kv1.3 IC50]).


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Figure 8. Pharmacokinetic profiles of a single subcutaneous dose (mouse and rat, dose = 2 mg/kg; beagle and cyno, dose = 0.5 mg/kg) of 145 (with target coverage estimates based on whole blood assay results: cynomolgus monkey IL-17 IC50 = 0.09 and human IL-2 IC50 = 0.092 nM).


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Figure 8. Comparison of the in vivo efficacy of 20kDa-PEG-ShK (144) and the Kv1.3 selective inhibitor 145 in blocking autoimmune encephalomyelitis in a rat AT-EAE model. The PEGylated ShK or [Lys16]ShK conjugates were delivered subcutaneously (SC) daily from day 1 through day 7. The rat CD4+ myelin-specific effector memory T cells line, PAS, was delivered by intravenous injection on day 0. The rats were monitored for signs of EAE once or twice per day in a blinded fashion, and 5 or 6 female Lewis rats were used per treatment group. Clinical EAE scores were: 0 = no signs, 0.5 = distal limp tail, 1.0 = limp tail, 2.0 = mild paraparesis, ataxia, 3.0 = moderate paraparesis, 3.5 = one hind leg paralysis, 4.0 = complete hind leg paralysis, 5.0 = complete hind leg paralysis and incontinence, 5.5 = tetraplegia, 6.0 = moribund state or death. Rats reaching a score of 5.5 were euthanized. Error bars represent the standard error of the mean.


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Figure 9. 12-week pharmacology study in cynomolgus monkeys. Weekly dosing of cynomolgus monkeys with 145 provided sustained suppression of T cell responses, as measured using the ex vivo cynomolgus monkey whole blood PD assay of inflammation that measured production of IL-4 (Figure 9A) and IL-17 (Figure 9B). Arrows indicate the weekly doses. Each line represents an individual test subject. Figure 9C shows predicted versus measured serum concentrations of 145 in cynomolgus monkeys after weekly subcutaneous (SC) dosing (0.5 mg/kg, n = 6). The measured serum trough levels after weekly dosing (open squares), matched closely those predicted based on repeat-dose modeling of the single-dose pharmacokinetic data (solid line). Figure 9D shows weight gain was normal for each animal during the 12-week cynomolgus monkey pharmacology study; arrows on x-axis indicate SC dosing with 145.


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Table of Contents Graphic


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Pharmaceutical Optimization of Peptide Toxins for Ion Channel

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