Protoxin-ii variants and methods of use thereof

Abstract

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

What is claimed is: 
     
         1 . A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises:
 (a) an isolated Protoxin-II variant or a fusion protein thereof to treat the pain, 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; and   (b) a pharmaceutically acceptable excipient.   
     
     
         2 . A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises:
 (a) an isolated Protoxin-II variant or a fusion protein thereof, 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 −10  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 veratridine-induced depolarization inhibition 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; and   (b) a pharmaceutically acceptable excipient.   
     
     
         3 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises the sequence: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 432) 
                 
                     
                   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 , 
                 
             
                
                
                
                
               
            
           
         
         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 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 S, 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, S, 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; 
         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 method of  claim 1 , wherein the isolated Protoxin-II variant has a substitution at one or more residue positions Y 1 , W 7 , S 11 , E 12 , K 14 , E 17 , G 18 , R 22  and L 29 , when residue numbering is according to SEQ ID NO: 1. 
     
     
         5 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises the sequence YCQKWMQTCDSERKCCEGMVCRLWCKKKLW-OH (SEQ ID NO: 424); wherein residue Y 1 , S 11 , E 12 , K 14 , E 17 , G 18 , R 22  and/or L 29  is substituted with
 a) any other amino acid selected from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;   b) a non-natural amino acid; and   c) W30 is substituted by L.   
     
     
         6 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises the sequence YCQKWMQTCDSERKCCEGMVCRLWCKKKLL-OH (SEQ ID NO: 425); wherein residue Y 1 , S 11 , E 12 , K 14 , E 17 , G 18 , M 19  and/or L 29  is substituted with
 a) any other amino acid selected from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or   b) a non-natural amino acid.   
     
     
         7 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLL-COOH); wherein the residue numbering is according to SEQ ID NO: 1. 
     
     
         8 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises 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 method of  claim 1 , wherein the isolated Protoxin-II variant further comprises an N-terminal extension and 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 method of  claim 1 , wherein the isolated Protoxin-II variant further comprises a C-terminal extension and 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 method of  claim 1 , wherein the isolated Protoxin-II variant further comprises an N-terminal extension and/or a C-terminal extension and wherein the N-terminal and/or the C-terminal extension is conjugated to the Protoxin-II variant via a linker. 
     
     
         12 . The method of  claim 11 , wherein the linker comprises the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402. 
     
     
         13 . The method of  claim 1 , wherein the isolated Protoxin-II variant inhibits human Nav1.7 activity with an IC 50  value of about 3×10 −8  M or less, when the IC 50  value is measured using a veratridine-induced depolarization inhibition 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 method of  claim 13 , wherein the isolated Protoxin-II variant inhibits human Nav1.7 activity with an IC 50  value of between about 3×10 −8  M to 1×10 −9  M. 
     
     
         15 . The method of  claim 1 , wherein the isolated Protoxin-II variant inhibits Nav1.7 activity by at least 25%, when the Nav1.7 activity is measured using a patch clamp assay using an extracellular solution containing 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 5 mM glucose, and 10 mM HEPES, pH=7.4, and osmolarity=315 mOsm, an intracellular solution containing 135 mM CsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH=7.3 and osmolarity=290 mOsm and a holding potential of −75 mV. 
     
     
         16 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises the amino acid sequence 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 433) 
                 
                     
                   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, 
                 
             
                
                
                
                
               
            
           
         
         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 method of  claim 1 , wherein the isolated Protoxin-II variant comprises the amino acid sequence of SEQ ID NOs: 30, 40, 44, 52, 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, 478, 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, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735 or 736. 
     
     
         18 . The method of  claim 1 , wherein the isolated Protoxin-II variant comprises a free C-terminal carboxylic acid, amide, methylamide or butylamide group. 
     
     
         19 . The method of  claim 1 , wherein the isolated fusion protein is the Protoxin-II variant conjugated to a half-life extending moiety. 
     
     
         20 . The method of  claim 19 , wherein the half-life extending moiety is human serum albumin (HSA), albumin binding domain (ABD), Fc or polyethylene glycol (PEG). 
     
     
         21 . The method of  claim 1 , wherein the pain is chronic pain, acute pain, neuropathic pain, cancer pain, nociceptive pain, visceral pain, back pain, post-operative pain, thermal pain, phantom limb pain, or pain associated with inflammatory conditions, primary erythemalgia (PE), paroxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis, fibromyalgia, painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN), trigeminal neuralgia (TN), spinal cord injuries or multiple sclerosis. 
     
     
         22 . The method of  claim 21 , wherein the pharmaceutical composition is administered peripherally. 
     
     
         23 . The method of  claim 22 , wherein the pharmaceutical composition is administered locally to a joint, spinal cord, surgical wound, sites of injury or trauma, peripheral nerve fibers, urogenital organs, or inflamed tissues. 
     
     
         24 . The method of  claim 1 , wherein the subject is a human.