Protoxin-ii variants and methods of use

Patent metadata

Field	Value
Publication number	US-2016287666-A1
Application number	US-201615090328-A
Country	US
Kind code	A1
Filing date	Apr 4, 2016
Priority date	Apr 2, 2015
Publication date	Oct 6, 2016
Grant date	—

Abstract

The present invention relates to Protoxin-II variants, polynucleotides encoding them, and methods of making and using the foregoing.

First claim

We claim:

1 . An isolated Protoxin-II variant that inhibits human Nav1.7 activity, wherein the Protoxin-II variant has at least one amino acid substitution selected from the group consisting of W7Q and W30L; wherein residue numbering is according to SEQ ID NO: 1.

2 . An isolated Protoxin-II variant, wherein the Protoxin-II variant inhibits human Nav1.7 activity with an IC 50 value of about 1×10 −7 M or less, about 1×10 −8 M or less, about 1×10 −9 M or less, about 1×10 −1 ° M or less, about 1×10 −11 M or less, or about 1×10 −12 M or less, wherein the IC 50 value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7, wherein the Protoxin-II variant has a W7Q and/or a W30L substitution, wherein residue numbering is according to SEQ ID NO: 1.

3 . The isolated Protoxin-II variant of claim 1 or 2 , comprising the sequence
X 1 X 2 X 3 CX 4 X 5 WX 6 QX 7 CX 8 X 9 X 10 X 11 X 12 CCX 13 X 14 X 15 X 16 CX 17 LWCX 18 KKLX 19 (SEQ ID NO: 432),
X 1 is G, P, A or deleted;
X 2 is P, A or deleted;
X 3 is S, Q, A, R or Y;
X 4 is Q, R, K, A, S or Y;
X 5 is K, S, Q or R;
X 6 is M or F;
X 7 is T, S, R, K or Q;
X 8 is D, T, or asparagyl-4-aminobutane;
X 9 is 5, A, R, I or V;
X 10 is E, R, N, K, T, Q, Y or glutamyl-4-aminobutane;
X 11 is R or K;
X 12 is K, Q, 5, A or F;
X 13 is E, Q, D, L, N, or glutamyl-4-aminobutane;
X 14 is G, Q or P;
X 15 is M or F;
X 16 is V or S;
X 17 is R, T or N-omega methyl-L-arginine; and
X 18 is K or R; and
X 19 is W or L,
optionally having an N-terminal extension or a C-terminal extension.

4 . The Protoxin-II variant of any of the claims 1 - 3 , wherein the variant has a substitution at one or more residue positions Y1, W7, S11, E12, K14, E17, G18, R22, L29 and W30, when residue numbering is according to SEQ ID NO: 1.

5 . The Protoxin-II variant of any of the claims 1 - 4 comprising the sequence YCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH (SEQ ID NO: 416); wherein residue Y1, S11, E12, K14, E17, G18, L29 and/or W30 is substituted with
a) any other amino acid shown in Table 1; or
b) a non-natural amino acid.

6 . The Protoxin-II variant of any of the claims 1 - 5 comprising the sequence YCQKWMQTCDSERKCCEGMVCRLWCKKKLL-COOH (SEQ ID NO: 422); wherein residue Y1, S11, E12, K14, E17, G18, M19, L29 and/or W30 is substituted with
a) any other amino acid shown in Table 1; or
b) a non-natural amino acid.

7 . The Protoxin-II variant of any of the claims 1 - 6 comprising the amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLL-COOH); wherein the Protoxin-II variant has Q at position 7 and L at position 30, when residue numbering is according to SEQ ID NO: 1

8 . The Protoxin-II variant of any of the claims 1 - 7 comprising the sequence The Protoxin-II variant of any of the claims 1 - 5 comprising the sequence X 1 X 2 X 3 CQKWMQTCDX 4 X 5 RX 6 CCX 7 X 8 X 9 VCRLWCKKKX 10 X 11 (SEQ ID NO: 737);
wherein
X 1 is G, P, A or deleted;
X 2 is P, A or deleted;
X 3 is S, Q, A, R or Y;
X 4 is S, A, R, I or V;
X 5 is E, R, N, K, T, Q, Y or glutamyl-4-aminobutane;
X 6 is K, Q, S, A or F;
X 7 is E, Q, D, L, N or glutamyl-4-aminobutane;
X 8 is G, Q or P;
X 9 is M or F;
X 10 is L, V; and
X 11 is W or L.

9 . The Protoxin-II variant of any of the claims 1 - 8 , wherein the N-terminal extension comprises the amino acid sequence of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385.

10 . The Protoxin-II variant of any of the claims 1 - 9 , wherein the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 or 397.

11 . The Protoxin-II variant of any of the claims 1 - 10 , wherein the N-terminal and/or the C-terminal extension is conjugated to the Protoxin-II variant via a linker.

12 . The Protoxin-II variant of any of the claims 1 - 11 , wherein the linker comprises the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.

13 . The isolated Protoxin-II variant of any of the claims 1 - 12 , that inhibits human Nav1.7 activity with an IC 50 value of about 3×10 −8 M or less, when the IC 50 value is wherein the IC 50 value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.

14 . The isolated Protoxin-II variant of claim 13 that inhibits human Nav1.7 activity with an IC 50 value of between about 3×10 −8 M to about 1×10 −9 M.

15 . The isolated Protoxin-II variant of any of the claims 1 - 14 , wherein the variant inhibits Nav1.7 activity by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when the Nav1.7 activity is measured using QPatch assay according to protocol described in Example 3.

16 . The isolated Protoxin-II variant of any of the claims 1 - 15 , comprising the amino acid sequence
GPQCX 1 X 2 WX 3 QX 4 CX 5 X 6 X 7 X 8 X 9 CCX 10 X 11 FX 12 CX 13 LWCX 14 KKLL (SEQ ID NO: 433), wherein
X 1 is Q, R, K, A or S;
X 2 is K, S, Q or R;
X 3 is M or F;
X 4 is T, S, R, K or Q;
X 5 is D or T;
X 6 is S, A or R;
X 7 is E, R, N, K, T or Q;
X 8 is R or K;
X 9 is K, Q, S or A;
X 10 is E, Q or D;
X 11 is G or Q;
X 12 is V or S;
X 13 is R or T; and
X 14 is K or R.

17 . The isolated Protoxin-II variant of any of the claims 1 - 16 , comprising the amino acid sequence of SEQ ID NOs: 30, 40, 44, 52, 56, 56, 59, 65, 78, 109, 110, 111, 114, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 178, 179, 180, 182, 183, 184, 185, 186, 189, 190, 193, 195, 197, 199, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 224, 226, 227, 231, 232, 243, 244, 245, 247, 249, 252, 255, 258, 261, 263, 264, 265, 266, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 332, 334, 335, 336, 337, 339, 340, 341, 342, 346, 351, 358, 359, 364, 366, 367, 368, 369, 370, 371, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573

Assignees

• Janssen Biotech Inc

Inventors

• Flinspach Mack
• Wickenden Alan
• Fellows Ross
• Xu Qinghao
• Piekarz Andrew

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• A61P25/04

  Centrally acting analgesics, e.g. opioids · CPC title

• A61P19/02

  for joint disorders, e.g. arthritis, arthrosis · CPC title

• A61P21/00

  Drugs for disorders of the muscular or neuromuscular system · CPC title