HK1226746A1

HK1226746A1 - Protoxin-ii variants and methods of use

Info

Publication number: HK1226746A1
Application number: HK17100274.8A
Authority: HK
Inventors: Flinspach Mack; Wickenden Alan; Fellows Ross; Neff Robert; Liu Yi; Hagan Rebecca; Xu Qinghao
Original assignee: Janssen Biotech, Inc.
Priority date: 2013-10-03
Filing date: 2014-10-03
Publication date: 2017-10-06

Description

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Voltage-gated sodium channels (VGSC) are present in all excitable cells including cardiac and skeletal muscle cells and central and peripheral neurons. In neuronal cells, sodium channels are responsible for amplifying sub-threshold depolarizations and generating the rapid upstroke of the action potential. As such, sodium channels are essential to the initiation and propagation of electrical signals in the nervous system. Aberrant sodium channel function is thought to underlie a variety of medical disorders (Hübner and Jentsch, Hum Mol Genet 11:2435-45, 2002), including epilepsy (Yogeeswari et al., Curr Drug Targets 5:589-602, 2004), arrhythmia (Tfelt-Hansen et al., J Cardiovasc Electrophysiol 21:107-15, 2010), myotonia (Cannon and Bean, J Clin Invest 120:80-3, 2010), and pain (Cregg et al., J Physiol 588:1897-904, 2010). Sodium channels are typically a complex of various subunits, the principle one being the pore-forming alpha-subunit, which is alone sufficient for function.

Nine known members of the family of voltage-gated sodium channel alpha subunits exist in humans, Nav1.1 - Nav1.9. The Nav1.x subfamily can be pharmacologically subdivided into two groups, the tetrodotoxin (TTX) -sensitive and TTX-resistant. Nav1.7, (a.k.a. PN1 or hNE) is encoded by the SCN9A gene, is TTX-sensitive and is primarily expressed in peripheral sympathetic and sensory neurons. Nav1.7 accumulates at nerve fiber endings and amplifies small sub-threshold depolarizations and acts as a threshold channel that regulates excitability.

Nav1.7 function is implicated in various pain states, including acute, inflammatory and/or neuropathic pain. In man, gain of function mutations of Nav1.7 have been linked to primary erythermalgia (PE), a disease characterized by burning pain and inflammation of the extremities (Yang et al., J Med Genet 41:171-4, 2004), and paroxysmal extreme pain disorder (PEPD)(Fertleman et al., Neuron 52:767-74, 2006). Consistent with this observation, non-selective sodium channel blockers lidocaine, mexiletine and carbamazepine can provide symptomatic relief in these painful disorders (Legroux-Crespel et al., Ann Dermatol Venereol 130:429-33, 2003; Fertleman et al., Neuron 52:767-74, 2006).

Loss-of-function mutations of Nav1.7 in humans cause congenital indifference to pain (CIP), a rare autosomal recessive disorder characterized by a complete indifference or insensitivity to painful stimuli (Cox et al., Nature 444:894-8, 2006; Goldberg et al, Clin Genet 71:311-9, 2007; Ahmad et al., Hum Mol Genet 16:2114-21, 2007).

Single nucleotide polymorphisms in the coding region of SCN9A have been associated with increased nociceptor excitability and pain sensitivity. For example, a polymorphism rs6746030 resulting in R1150W substitution in human Nav1.7 has been associated with osteoarthritis pain, lumbar discectomy pain, phantom pain, and pancreatitis pain (Reimann et al., Proc Natl Acad Sci USA 107:5148-53, 2010). DRG neurons expressing the R1150W mutant Nav1.7 display increased firing frequency in response to depolarization (Estacion et al., Ann Neurol 66:862-6, 2009). A disabling form of fibromyalgia has been associated with SCN9A sodium channel polymorphism rs6754031, indicating that some patients with severe fibromyalgia may have a dorsal root ganglia sodium channelopathy (Vargas-Alarcon et al., BMC Musculoskelet Disord 13:23, 2012).

In mice, deletion of the SCN9A gene in nociceptive neurons leads to reduction in mechanical and thermal pain thresholds and reduction or abolition of inflammatory pain responses (Nassar et al., Proc Natl Acad Sci USA 101:12706-11, 2004). Ablating SCN9A in all sensory neurons abolished mechanical pain, inflammatory pain and reflex withdrawal responses to heat. Deleting SCN9A in both sensory and sympathetic neurons abolished mechanical, thermal and neuropathic pain, and recapitulated the pain-free phenotype seen in humans with Nav1.7 loss-of-function mutations (Minett et al., Nat Commun 3:791, 2012). Nav1.7 inhibitors or blockers may therefore be useful in the treatment of a wide range of pain associated with various disorders.

Spider venoms are known to contain a large number of sodium channel blocking peptides, including Huwentoxin-IV (HwTx-IV) (Peng et al., J Biol Chem 277:47564-71, 2002), Protoxin-I, Protoxin-II (Middleton et al., Biochemistry 41:14734-47, 2002) and Phrixotoxin-III (Bosmans et al., Mol Pharmacol 69:419-29, 2006). WO 2012/004664 and Yang et. al. (Yang et. al., Proc Natl Acad Sci USA, 110(43), 17534-17539, 2013) relate to peptides that inhibit the Nav1.7 sodium channel. There is a need for identification of additional Nav1.7 blockers for treatment of a wide range of pain indications. In particular, there is a need for new Nav1.7 blockers with selectivity for Nav1.7 over other voltage gated sodium channel isoforms.

SUMMARY OF THE INVENTION

The invention provides an isolated Protoxin-II variant comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 40, 44, 56, 59, 65, 78, 109, 110, 111, 114, 115, 116, 117, 118, 119, 121, 122, 123, 129, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 158, 159, 172, 173, 175, 177, 178, 183, 184, 185, 186, 189, 190, 193, 196, 197, 199, 205, 207, 210, 211, 216, 217, 224, 230, 269, 270, 271, 272, 273, 274, 275, 277, 278, 279, 280, 281, 282, 283, 284, 287, 288, 289, 290, 291, 292, 297, 298, 299, 301, 302, 304, 305, 307, 308, 309, 310, 312, 314, 315, 318, 319,320, 321, 322, 323, 324, 325, and 326, wherein the variant inhibits human Nav1.7 activity with an IC₅₀ value of 30x10^-9 M or less, wherein the IC₅₀ value is measured using a FLIPR Tetra membrane depolarisation assay, the assay comprising fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7 and using bis-1,3(diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390- 420 nm and measuring FRET at 515-575 nm.

The invention also provides the protein conjugate comprising the Protoxin-II variant as defined by the claims conjugated to a half-life extending moiety; wherein the conjugate is a fusion protein comprising the Protoxin-II variant and a human serum albumin (HSA), albumin binding domain (ABD), or a Fc polypeptide; or the conjugate comprises polyethylene glycol (PEG) chemically coupled to the Protoxin-II variant.

The invention also provides an isolated polynucleotide encoding the Protoxin-II variant as defined by the claims.

The invention also provides a vector comprising the isolated polynucleotide as defined by the claims.

The invention also provides a host cell comprising the vector as defined by the claims.

The invention also provides a method of producing an isolated Protoxin-II variant, comprising culturing the host cell as defined by the claims and recovering the Protoxin-II variant produced by the host cell.

The invention also provides a pharmaceutical composition comprising the isolated Protoxin-II variant as defined by the claims and a pharmaceutically acceptable excipient.

The invention also provides the Protoxin-II variant as defined by the claims for use in treating Nav1.7-mediated pain in a subject in need thereof.

The disclosure provides an isolated Protoxin-II variant comprising the sequence X₁X₂X₃CX₄X₅WX₆QX₇CX₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄FX₁₅CX₁₆LWCX₁₇KKLW (SEQ ID NO: 403), wherein

X1: is G, P, A or deleted;
X2: is P, A or deleted;
X3: is S, Q, A, R or Y;
X4: is Q, R, K, A or S;
X5: is K, S, Q or R;
X6: is M or F;
X7: is T, S, R, K or Q;
X8: is D or T;
X9: is S, A or R;
X10: is E, R, N, K, T or Q;
X11: is R or K;
X12: is K, Q, S or A;
X13: is E, Q or D;
X14: is G or Q;
X15: is V or S;
X16: is R or T; and
X17: is K or R;

optionally having an N-terminal extension or a C-terminal extension, wherein the polypeptide inhibits human Nav1.7 activity with an IC₅₀ value of about 1x10^-7 M or less, wherein the IC₅₀ value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7. The disclosure also provides an isolated Protoxin-II variant 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: 78 (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH), wherein the amino acid sequence has Q at position 1, Q at position 7 and F at position 19, when residue numbering is according to SEQ ID NO: 1; the polypeptide inhibits human Nav1.7 activity with an IC₅₀ value of about 30x10^-9 M or less, wherein the IC₅₀ value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and the polypeptide selectively inhibits Nav1.7.

Another embodiment of the invention is a polynucleotide encoding the Protoxin-II variant of the invention.

Another embodiment of the invention is a vector comprising the isolated polynucleotide of the invention.

Another embodiment of the invention is a host cell comprising the vector of the invention.

Another embodiment of the invention is a method for producing the Protoxin-II variant of the invention, comprising culturing the host cell of the invention and recovering the Protoxin-II variant from the cell.

Another embodiment of the invention is a pharmaceutical composition comprising the isolated Protoxin-II variant of the invention and a pharmaceutically acceptable excipient.

Another embodiment of the invention is a Protoxin-II variant as defined by the claims for use in treating Nav1.7-mediated pain in a subject, comprising administering to a subject an effective amount of the Protoxin-II variant of the invention to treat the Nav1.7-mediated pain.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the genus amino acid sequence of Protoxin-II variants that inhibit Nav1.7 with an IC50 value of 30 nM or less in a FLIPR Tetra assay. Residue numbering is according to wild-type Protoxin-II of SEQ ID NO: 1. Genus SEQ ID NO: 403.
Figure 2 shows the IC50 values for Nav1.7 and Nav1.6 inhibition in a QPatch assay, and selectivity of each variant calculated by ratio of IC50(Nav1.6)/IC50(Nav1.7) obtained in QPatch assay. SE: standard error.
Figure 3 shows the sequences and the genus sequence of Protoxin-II variants that inhibit Nav1.7 with an IC50 value of 30 nM or less in a FLIPR Tetra assay, and are over 30-fold selective over Nav1.6. Selectivity of each variant was calculated by ratio of IC50(Nav1.6)/IC509av1.7) obtained in QPatch assay. Residue numbering is according to wild-type Protoxin-II of SEQ ID NO: 1.
Figure 4A shows efficacy of NV1D3034 (NV1D3034-OH) (SEQ ID NO: 78) against CFA-induced thermal hyperalgesia assessed by measurement of paw withdrawal latency in the Hargreaves test before (pre-CFA) and after CFA injection (0) and 1-day after peptide administration (1). ***P<0.001 vs. PBS, two-way ANOVA followed by Bonferroni's multiple comparison.
Figure 4B shows efficacy of NV1D3034 (NV1D3034-OH) (SEQ ID NO: 78) in CFA-induced thermal hyperalgesia expressed as percent MPE (maximum possible effect) (MPE%) at each dose on day1 following peptide administration. *P<0.05 vs PBS, one-way ANOVA followed by Bonferroni's multiple comparison.
Figure 5A shows efficacy of NV1D3368 (NV1D3368-OH) (SEQ ID NO: 198) against CFA-induced thermal hyperalgesia assessed by measurement of paw withdrawal latency in the Hargreaves test before (pre-CFA) and after CFA injection (0) and 1-day after peptide administration (1). **P<0.01 and ****P<0.0001 vs.
PBS, two-way ANOVA followed by Bonferroni's multiple comparison
Figure 5B shows efficacy of NV1D3368 (NV1D3368-OH) (SEQ ID NO: 198) in CFA-induced thermal hyperalgesia expressed as percent MPE (MPE%) at each dose on day1 following peptide administration. *P<0.05 and **P<0.01 vs PBS, one-way ANOVA followed by Bonferroni's multiple comparison.
Figure 6A shows efficacy of NV1D2775-OH (SEQ ID NO: 56) against CFA-induced thermal hyperalgesia assessed by measurement of paw withdrawal latency in the Hargreaves test before (pre-CFA) and after CFA injection (0) and 1-day after peptide administration (1). ****P<0.0001 vs. PBS, two-way ANOVA followed by Bonferroni's multiple comparison.
Figure 6B shows efficacy of NV1D2775-OH (SEQ ID NO: 56) in CFA-induced thermal hyperalgesia expressed as percent MPE (MPE%) at each dose on day1 following peptide administration.. ***P<0.001 and ****P<0.0001 vs PBS, one-way ANOVA followed by Bonferroni's multiple comparison.
Figure 6C shows efficacy of NV1D2775-OH (SEQ ID NO: 56) against CFA-induced tactile allodynia. Tactile thresholds of hind paw before (pre-CFA) and after CFA (0) and 1-day after peptide administration (1). ****P<0.0001 vs. PBS, two-way ANOVA followed by Bonferroni's multiple comparison.
Figure 6D shows efficacy of NV1D2775-OH (SEQ ID NO: 56) against CFA-induced tactile allodynia expressed as percent MPE (MPE%) on day1 following peptide. ***P<0.001 vs PBS, one-way ANOVA followed by Bonferroni's multiple comparison.
Figure 7A shows time course of NV1D2775-OH mediated reversal of thermal hyperalgesia in the CFA model as assessed by measurement of paw withdrawal latency in the Hargreaves test before and after CFA and at various time points post-peptide administration. **P<0.01 vs. PBS, two-way ANOVA followed by Bonferroni's multiple comparison. Shaded areas indicate compound delivery period (0-24hr).
Figure 7B shows time course of NV1D2775-OH mediated reversal of tactile allodynia in the CFA model as assessed by measurement of tactile threshold before and after CFA and at various time points post-peptide administration. **P<0.01 vs. PBS, two-way ANOVA followed by Bonferroni's multiple comparison. Shaded areas indicate compound delivery period (0-24hr).
Figure 8 shows that NV1D2775-OH produced significant analgesia in the hotplate test. Thermal withdrawal latency was evaluated at 50 and 55°C pre- and post-pump implantation. Pump implantation had no impact on the latency in the control PBS group. One day after pump, NV1D2775-OH treated-mice exhibited prolonged latency compared to the PBS group. *P<0.05 and ****P<0.0001 vs. PBS, one-way ANOVA followed by Bonferroni's multiple comparison.
Figure 9 shows that NV1D2775-OH pretreatment protected animals from carrageenan-induced thermal hyperalgesia. Paw withdrawal latencies were measured pre- and on day1 post-pump before intraplantar carrageenan injection. Latencies were measured again at 2, 3 and 4hr following carrageenan.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and in the claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which an invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, exemplary compositions and methods are described herein.

The term "polypeptide" means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides". Polypeptides may also be referred as "proteins".

The term "polynucleotide" means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNAs and RNAs are typical examples of polynucleotides.

The term "complementary sequence" means a second isolated polynucleotide sequence that is antiparallel to a first isolated polynucleotide sequence and that comprises nucleotides complementary to the nucleotides in the first polynucleotide sequence.

The term "vector" means a non-natural polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain a cDNA encoding a protein of interest and additional elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.

The term "expression vector" means a vector that can be utilized in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

The term "variant" as used herein refers to a polypeptide or a polynucleotide that differs from wild type Protoxin-II polypeptide of SEQ ID NO: 1 or the polynucleotide encoding the wild type Protoxin-II having the sequence of SEQ ID NO: 107 by one or more modifications for example, substitutions, insertions or deletions of nucleotides or amino acids.

Throughout the specification, residues that are substituted in the Protoxin-II variants are numbered corresponding to their position in the wild-type Protoxin-II of SEQ ID NO: 1. For example, "Y1A" in the specification refers to the substitution of tyrosine at residue position that corresponds to the position 1 in the wild type Protoxin-II of SEQ ID NO:1 with alanine.

"Complementary DNA" or "cDNA" refers to a well-known synthetic polynucleotide that shares the arrangement of sequence elements found in native mature mRNA species with contiguous exons, with the intervening introns present in genomic DNA are removed. The codons encoding the initiator methionine may or may not be present in cDNA. cDNA may be synthesized for example by reverse transcription or synthetic gene assembly.

"Synthetic" or "non-natural" as used herein refers to a polynucleotide or a polypeptide molecule not present in nature.

"Nav1.7" (also referred to as hNE or PN1) or "hNav1.7" as used herein refers to the well-known human sodium channel protein type 9 subunit alpha having a sequence shown in GenBank accession number NP_002968.1 and in SEQ ID NO: 79.

The term "wild type Protoxin-II" or "wild type ProTx-II" as used herein refers to the tarantula Thrixopelma pruriens (Peruvian green velvet tarantula) toxin peptide having the amino acid sequence YCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH (SEQ ID NO: 1) as described in Middleton et al., Biochemistry 41(50):14734-47, 2002.

The term "recombinant Protoxin-II" or recombinant ProTx-II" as used herein refers to the recombinant Protoxin-II obtained from expression and subsequent cleavage of a Protoxin-II fusion protein having the sequence of GPYCQKWMWTCDSERKCCEGMVCRLWCKKKLW-OH as shown in SEQ ID NO: 2. Recombinant Protoxin-II incorporates a two amino acid N-terminal extension (residues G and P) when compared to the wild type Protoxin-II.

"Blocks human Nav1.7 activity" or "inhibits human Nav1.7 activity" as used herein refers to the ability of the Protoxin-II variant of the invention to reduce membrane depolarization induced by veratridine (3-veratroylveracevine) with an IC₅₀ value of about 1x10^-7 M or less in a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET), where veratridine-induced depolarization is measured as a reduction in FRET signal using DISBAC2(3) ([bis-(1,3-diethylthiobarbituric acid) trimethine oxonol]) as an acceptor and PTS18 (trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate) as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm in a cell line stably expressing human Nav1.7.

"FLIPR® Tetra membrane depolarization assay" as used herein is the assay described in Example 3.

The term "substantially identical" as used herein means that the two Protoxin-II variant amino acid sequences being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of 1, 2, 3, 4, 5, 6, or 7 amino acids in the Protoxin-II variant amino acid sequence that do not adversely affect peptide properties. Amino acid sequences substantially identical to the Protoxin-II variants disclosed herein are within the scope of the application. In some instances, the sequence identity can be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity can be determined for example by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carslbad, CA). The protein sequences of the present invention may be used as a query sequence to perform a search against public or patent databases, for example, to identify related sequences. Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov), or the GenomeQuest™ (GenomeQuest, Westborough, MA) suite using the default settings.

Conventional one and three-letter amino acid codes are used herein as shown in Table 1.

Table 1.

Amino acid	Three letter code	One letter code
Alanine	Ala	A
Arginine	Arg	R
Asparagine	Asn	N
Aspartate	Asp	D
Cysteine	Cys	C
Glutamate	Glu	E
Glutamine	Gln	Q
Glycine	Gly	G
Histidine	His	H
Isoleucine	Ile	I
Leucine	Leu	L
Lysine	Lys	K
Methionine	Met	M
Phenylalanine	Phe	F
Proline	Pro	P
Serine	Ser	S
Threonine	Thr	T
Tryptophan	Trp	W
Tyrosine	Tyr	Y
Valine	Val	V

The present invention provides isolated Protoxin-II (ProTx-II) variant polypeptides as defined by the claims that inhibit human Nav1.7 activity, polynucleotides encoding them, vectors, host cells, and methods of using the polynucleotides and polypeptides of the invention. The polypeptides of the invention inhibit depolarization resulting from Nav1.7 activation, and therefore may be useful in the treatment of various conditions associated with pain and conditions associated with sensory or sympathetic neuron dysfunction.

The variants of the invention are potent inhibitors of Nav1.7. The current invention is based, at least in part, on the finding that certain residue substitutions in Protoxin-II enhance selectivity, synthetic yield and/or homogeneity without adversely affecting the potency of the generated Protoxin-II variants, specifically W7 and M19, and additionally residues Y1 and S11, and further additionally residues E12, R22 and (residue numbering according to SEQ ID NO: 1).

One embodiment of the invention is an isolated Protoxin-II variant, wherein the Protoxin-II variant inhibits human Nav1.7 activity with an IC₅₀ value of about 1x10^-9 M or less, about 1x10^-10 M or less, about 1x10^-11 M or less, or about 1x10^-12 M or less, wherein the IC₅₀ value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7 and using bis-1,3(diethylthiobarbituric acid) trimethine oxonol as an electron acceptor and trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390- 420 nm and measuring FRET at 515-575 nm.

optionally having an N-terminal extension or a C-terminal extension, wherein the polypeptide inhibits human Nav1.7 activity with an IC₅₀ value of about 1x10^-7 M or less, wherein the IC₅₀ value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.

The N-terminal extension can comprise the amino acid sequences of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385.

The C-terminal extension can comprise the amino acid sequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 or 397.

The N-terminal and/or the C-terminal extension may be conjugated to the Protoxin-II variant via a linker.

The linker can comprise the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.

The N-terminal extension can consist of the amino acid sequences of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385.

The C-terminal extension can consist of the amino acid sequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 or 397.

The linker can consist of the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.

The Protoxin-II variants of the invention are potent Nav1.7 inhibitors. Recombinant Protoxin-II (SEQ ID NO: 2) has an IC₅₀ value of about 4x10^-9 M for human Nav1.7 in a veratridine-induced depolarization inhibition assay measuring decline in FRET (fluorescence resonance energy transfer) in cells stably expressing Nav1.7 using FLIPR® Tetra instrument (Molecular Devices) using experimental details described in Example 3. A Protoxin-II variant is "a potent" Nav1.7 inhibitor when the IC₅₀ value in the assay described above and in Experiment 3 is about 30x10^-9 M or less i.e. within 10 fold of recombinant Protoxin-II. For clarity, an IC₅₀ of 30x10^-9 M is identical to IC₅₀ of 3.0x10^-8 M.

The Protoxin-II variant polypeptides of the invention may be produced by chemical synthesis, such as solid phase peptide synthesis, on an automated peptide synthesizer. Alternatively, the polypeptides of the invention may be obtained from polynucleotides encoding the polypeptides by the use of cell-free expression systems such as reticulocyte lysate based expression systems, or by recombinant expression systems. Those skilled in the art will recognize other techniques for obtaining the polypeptides of the invention. In an exemplary method, the Protoxin-II variants of the invention are generated by expressing them as human serum albumin (HSA) fusion proteins utilizing a glycine-rich linker such as (GGGGS)₄ (SEQ ID NO:80) or (GGGGS)₆ (SEQ ID NO: 81) coupled to a protease cleavable linker such as a recognition sequence for HRV3C protease (Recombinant type 14 3C protease from human rhinovirus) LEVLFQGP (HRV3C linker) (SEQ ID NO: 82), and cleaving the expressed fusion proteins with the HRV3C protease to release the recombinant Protoxin-II variant peptides. Hexahistidine (SEQ ID NO: 108) or other tags may be used to facilitate purification using well known methods.

Protoxin-II variants of the invention may be purified using methods described herein. In an exemplary method, Protoxin-II variants of the invention expressed as HSA fusion proteins and cleaved with HRV3C protease may be purified using sold phase extraction (SPE) as described herein.

Generation of the Protoxin-II variants optionally having N-terminal and/or C-terminal extensions, and Protoxin-II variant fusion proteins is typically achieved at the nucleic acid level. The polynucleotides may be synthesized using chemical gene synthesis according to methods described in U.S. Pat. No. 6,521,427 and 6,670,127 , utilizing degenerate oligonucleotides to generate the desired variants, or by standard PCR cloning and mutagenesis. Libraries of variants may be generated by standard cloning techniques to clone the polynucleotides encoding the Protoxin-II variants into the vector for expression.

The Protoxin-II variants may incorporate additional N- and/or C-terminal amino acids when compared to the wild type Protoxin-II of SEQ ID NO: 1, for example resulting from cloning and/or expression schemes. For example, cleavage from HSA after expression of the variant as HSA-linker-HRV3C cleavable peptide-Protoxin-II variant fusion protein may result in the incorporation of additional two residues to the N-terminus of each Protoxin-II variant, such as G and P.

The Protoxin-II variants of the invention are tested for their ability to inhibit human Nav1.7 using methods described herein. An exemplary assay is a veratridine-induced depolarization inhibition assay measuring decline in FRET (fluorescence resonance energy transfer) in cells stably expressing Nav1.7. Another exemplary assay employs electrophysiological recordings to measure changes in Nav1.7-mediated currents using well known patch clamp techniques and as described herein.

Examples of isolated Protoxin-II variants are those comprising the amino acid sequence of SEQ ID NOs: 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 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, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 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, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 35, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368 367, 370 or 371.

The Protoxin-II variants of the invention may inhibit human Nav1.7 with an IC₅₀ value of about 1x10^-9 or less, about 1x10^-10 M or less, about 1x10^-11 M or less, or about 1x10^-12 M or less. Exemplary variants demonstrating the range of IC₅₀ values are variants having amino acid sequences shown in SEQ ID NOs: 30, 40, 44, 52, 56, 56, 59, 65, 78, 109, 110, 111, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 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, or 368.

Table 2 and Table 3 show the sequences of select Protoxin-II variants.

Table 2.

Protein name	Protoxin-II variant peptide name	SEQ ID NO:	Protein amino acid sequence
	wild type	1	YCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D12	2	GPYCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D748	3	GPACQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D751	4	GPQCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D2292	5	GPRCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D750	6	GPSCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D1328	7	GPYCQKWFWTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D774	8	GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D786	9	GPYCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH
	NV1D2300	10	GPYCQKWMWTCDRERKCCEGMVCRLWCKKKLW-COOH
	NV1D791	11	GPYCQKWMWTCDSKRKCCEGMVCRLWCKKKLW-COOH
	NV1D1332	12	GPYCQKWMWTCDSNRKCCEGMVCRLWCKKKLW-COOH
	NV1D2512	13	GPYCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH
	NV1D1336	14	GPYCQKWMWTCDSERKCCEGLVCRLWCKKKLW-COOH
	NV1D1337	15	GPYCQKWMWTCDSERKCCEGMVCTLWCKKKLW-COOH
	NV1D2308	16	GPYCQKWMWTCDSERKCCEGMVCRLWCRKKLW-COOH
NV1G953	NV1D2670	17	GPACQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH
NV1G951	NV1D2674	18	GPACQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G909	NV1D2664	19	GPACQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH
NV1G963	NV1D2671	20	GPQCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH
NV1G949	NV1D2675	21	GPQCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G977	NV1D2665	22	GPQCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH
NV1G957	NV1D2668	23	GPRCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH
NV1G965	NV1D2672	24	GPRCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G973	NV1D2662	25	GPRCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH
NV1G975	NV1D2669	26	GPSCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH
NV1G971	NV1D2673	27	GPSCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G995	NV1D2663	28	GPSCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH
NV1G961	NV1D2676	29	GPYCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G911	NV1D2666	30	GPYCQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH
	NV1D2816	31	GPACQKWFQTCDSERKCCEGMVCRLWCKKKLW-COOH
NV1G905	NV1D2735	32	GPACQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH
NV1G919	NV1D2739	33	GPACQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH
NV1G979	NV1D2731	34	GPACQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH
	NV1D2810	35	GPQCQKWFQTCDSERKCCEGMVCRLWCKKKLW-COOH
NV1G1099	NV1D2732	36	GPQCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G1011	NV1D2740	37	GPQCQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2819	38	GPRCQKWFWTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G1105	NV1D2729	39	GPRCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G1013	NV1D2733	40	GPRCQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH
	NV1D2814	41	GPSCQKWFQTCDSERKCCEGMVCRLWCKKKLW-COOH
	NV1D2820	42	GPSCQKWFWTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G983	NV1D2730	43	GPSCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G1003	NV1D2734	44	GPSCQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH
NV1G1009	NV1D2738	45	GPSCQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2851	46	GPYCQKWFKTCDAERKCCEGMVCRLWCKKKLW-COOH
	NV1D2850	47	GPYCQKWFQTCDAERKCCEGMVCRLWCKKKLW-COOH
NV1G987	NV1D2667	48	GPYCQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2867	49	GPACQKWFQTCDAERKCCEGMVCRLWCKKKLW-COOH
	NV1D2881	50	GPACQKWFQTCDSERKCCEGFVCRLWCKKKLW-COOH
	NV1D2882	51	GPACQKWFQTCDSERKCCEGLVCRLWCKKKLW-COOH
NV1G899	NV1D2774	52	GPACQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH
NV1G1077	NV1D2902	53	GPACQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2861	54	GPQCQKWFQTCDAERKCCEGMVCRLWCKKKLW-COOH
	NV1D2870	55	GPQCQKWFQTCDSERKCCEGLVCRLWCKKKLW-COOH
NV1G1007	NV1D2775	56	GPQCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH
NV1G1067	NV1D2893	57	GPQCQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2887	58	GPRCQKWFWTCDAERKCCEGFVCRLWCKKKLW-COOH
NV1G1005	NV1D2772	59	GPRCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH
NV1G1061	NV1D2896	60	GPRCQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2877	61	GPSCQKWFQTCDSERKCCEGFVCRLWCKKKLW-COOH
	NV1D2878	62	GPSCQKWFQTCDSERKCCEGLVCRLWCKKKLW-COOH
	NV1D2889	63	GPSCQKWFWTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2889	64	GPSCQKWFWTCDAERKCCEGFVCRLWCKKKLW-COOH
NV1G1001	NV1D2773	65	GPSCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2890	66	GPSCQKWFWTCDAERKCCEGLVCRLWCKKKLW-COOH
NV1G1109	NV1D2899	67	GPSCQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2905	68	GPYCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2906	69	GPYCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2921	70	GPACQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2922	71	GPACQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2909	72	GPQCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2910	73	GPQCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2913	74	GPRCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2914	75	GPRCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH
	NV1D2917	76	GPSCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH
	NV1D2918	77	GPSCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH
NV1G1153	NV1D3034	78	GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH

Table 3.

Protein name	Protoxin-II variant peptide name	SEQ ID NO:	Protein amino acid sequence
(-GP) NV1G1001	(-GP) NV1D2773	109
(-GP) NV1G1001-NH-Me	(-GP) NV1D2773-NH2	110
NV1G1007-NH2	NV1D2775-NH2	111
NV1G1107-NH2	NV1D2890-NH2	112
NV1G1137	NV1D2974	113
(-GP) N-Ac-NV1G1137-NH2	(-GP) N-Ac-NV1D2974-NH2	114
(-GP) N-Ac-NV1G1137-	(-GP) N-Ac-NV1D2974	115
NV1G1153	NV1D3034	116
NV1G1153-NH2	NV1D3034-NH2	117
NV1G1153-NH-butyl	NV1D3034-NH-butyl	118
NV1G1153-NH-methyl	NV1D3034-NH-methyl	119
(-GP) N-Ac-NV1G1153	(-GP) N-Ac-NV1D3034	120
(-GP) N-Ac-NV1G1153-NH2	(-GP) N-Ac-NV1D3034-NH2	121
NV1G1818	NV1D3368	122
NV1G1818-NH2	NV1D3368-NH2	123
NV1G1147	NV1D2969	124
NV1G1145	NV1D2970	125
NV1G1143	NV1D2971	126
NV1G1141	NV1D2972	127
NV1G1139	NV1D2973	128
NV1G1137	NV1D2974	129
NV1G1137-NH2	NV1D2974-NH2	130
NV1G1517	NV1D3004	131
NV1G1515	NV1D3005	132
NV1G1519	NV1D3006	133
NV1G1513	NV1D3007	134
NV1G1523	NV1D3012	135
NV1G1525	NV1D3013	136
NV1G1255	NV1D3014	137
NV1G1187	NV1D3015	138
NV1G1257	NV1D3016	139
NV1G1221	NV1D3017	140
NV1G1521	NV1D3018	141
NV1G1531	NV1D3019	142
NV1G1239	NV1D3020	143
NV1G1583	NV1D3030	144
NV1G1527	NV1D3031	145
NV1G1511	NV1D3032	146
NV1G1509	NV1D3033	147
NV1G1231	NV1D3035	148
NV1G1211	NV1D3036	149
NV1G1267	NV1D3044	150
NV1G1269	NV1D3045	151
NV1G1215	NV1D3048	152
NV1G1593	NV1D3050	153
NV1G1263	NV1D3051	154
NV1G1585	NV1D3052	155
NV1G1623	NV1D3056	156
NV1G1613	NV1D3057	157
NV1G1259	NV1D3058	158
NV1G1265	NV1D3062	159
NV1G1273	NV1D3109	160
NV1G1225	NV1D3121	161
NV1G1886	NV1D3249	162
NV1G1633	NV1D3251	163
NV1G1631	NV1D3252	164
NV1G1885	NV1D3254	165
NV1G1884	NV1D3256	166
NV1G1881	NV1D3257	167
NV1G1879	NV1D3259	168
NV1G1883	NV1D3260	169
NV1G1880	NV1D3261	170
NV1G1882	NV1D3262	171
NV1G1776	NV1D3339	172
NV1G1775	NV1D3340	173
NV1G1768	NV1D3341	174
NV1G1777	NV1D3342	175
NV1G1770	NV1D3344	176
NV1G1767	NV1D3345	177
NV1G1769	NV1D3346	178
NV1G1774	NV1D3347	179
NV1G1771	NV1D3348	180
NV1G1778	NV1D3349	181
NV1G1773	NV1D3350	182
NV1G1779	NV1D3351	183
NV1G1772	NV1D3352	184
NV1G1868	NV1D3353	185
NV1G1824	NV1D3354	186
NV1G1863	NV1D3356	187
NV1G1826	NV1D3357	188
NV1G1810	NV1D3358	189
NV1G1836	NV1D3359	190
NV1G1834	NV1D3360	191
NV1G1829	NV1D3361	192
NV1G1820	NV1D3362	193
NV1G1828	NV1D3363	194
NV1G1827	NV1D3365	195
NV1G1857	NV1D3366	196
NV1G1823	NV1D3367	197
NV1G1818	NV1D3368	198
NV1G1811	NV1D3369	199
NV1G1853	NV1D3370	200
NV1G1817	NV1D3371	201
NV1G1814	NV1D3372	202
NV1G1831	NV1D3374	203
NV1G1819	NV1D3375	204
NV1G1859	NV1D3376	205
NV1G1825	NV1D3377	206
NV1G1821	NV1D3378	207
NV1G1835	NV1D3379	208
NV1G1815	NV1D3380	209
NV1G1833	NV1D3381	210
NV1G1812	NV1D3382	211
NV1G1782	NV1D3383	212
NV1G1783	NV1D3384	213
NV1G1785	NV1D3385	214
NV1G1784	NV1D3386	215
NV1G1780	NV1D3387	216
NV1G1781	NV1D3388	217
NV1G1786	NV1D3389	218
NV1G1851	NV1D3390	219
NV1G1852	NV1D3391	220
NV1G1854	NV1D3392	221
NV1G1860	NV1D3393	222
NV1G1789	NV1D3394	223
NV1G1787	NV1D3396	224
NV1G1856	NV1D3397	225
NV1G1855	NV1D3398	226
NV1G1788	NV1D3399	227
NV1G1849	NV1D3400	228
NV1G1795	NV1D3401	229
NV1G1803	NV1D3403	230
NV1G1807	NV1D3408	231
NV1G1806	NV1D3409	232
NV1G1805	NV1D3410	233
NV1G1809	NV1D3413	234
NV1G1850	NV1D3414	235
NV1G1793	NV1D3419	236
NV1G1822	NV1D3423	237
NV1G1813	NV1D3424	238
NV1G1840	NV1D3425	239
NV1G1848	NV1D3426	240
NV1G1841	NV1D3427	241
NV1G1844	NV1D3428	242
NV1G1842	NV1D3430	243
NV1G1846	NV1D3431	244
NV1G1843	NV1D3432	245
NV1G1892	NV1D3439	246
NV1G1916	NV1D3465	247
NV1G1922	NV1D3466	248
NV1G1915	NV1D3467	249
NV1G1924	NV1D3470	250
NV1G1709	NV1D3510	251
NV1G1681	NV1D3511	252
NV1G1693	NV1D3512	253
NV1G1705	NV1D3513	254
NV1G1689	NV1D3514	255
NV1G1711	NV1D3515	256
NV1G1685	NV1D3516	257
NV1G1697	NV1D3517	258
NV1G1695	NV1D3518	259
NV1G1701	NV1D3519	260
NV1G1691	NV1D3520	261
NV1G1679	NV1D3521	262
NV1G1683	NV1D3523	263
NV1G1707	NV1D3524	264
NV1G1713	NV1D3525	265
NV1G1687	NV1D3526	266
NV1G1699	NV1D3527	267
NV1G1675	NV1D3528	268
NV1G1754	NV1D3529	269
NV1G1748	NV1D3530	270
NV1G1747	NV1D3531	271
NV1G1752	NV1D3532	272
NV1G1722	NV1D3533	273
NV1G1744	NV1D3534	274
NV1G1742	NV1D3535	275
NV1G1723	NV1D3536	276
NV1G1745	NV1D3537	277
NV1G1757	NV1D3538	278
NV1G1762	NV1D3539	279
NV1G1763	NV1D3540	280
NV1G1728	NV1D3541	281
NV1G1730	NV1D3542	282
NV1G1760	NV1D3543	283
NV1G1727	NV1D3544	284
NV1G1729	NV1D3545	285
NV1G1867	NV1D3546	286
NV1G1759	NV1D3547	287
NV1G1758	NV1D3548	288
NV1G1766	NV1D3549	289
NV1G1761	NV1D3550	290
NV1G1726	NV1D3551	291
NV1G1721	NV1D3552	292
NV1G1765	NV1D3553	293
NV1G1764	NV1D3554	294
NV1G1732	NV1D3555	295
NV1G1862	NV1D3556	296
NV1G1751	NV1D3558	297
NV1G1866	NV1D3559	298
NV1G1865	NV1D3560	299
NV1G1716	NV1D3561	300
NV1G1724	NV1D3562	301
NV1G1717	NV1D3563	302
NV1G1743	NV1D3564	303
NV1G1720	NV1D3565	304
NV1G1735	NV1D3566	305
NV1G1734	NV1D3568	306
NV1G1741	NV1D3569	307
NV1G1719	NV1D3570	308
NV1G1718	NV1D3571	309
NV1G1725	NV1D3572	310
NV1G1869	NV1D3573	311
NV1G1755	NV1D3574	312
NV1G1756	NV1D3575	313
NV1G1746	NV1D3576	314
NV1G1733	NV1D3577	315
NV1G1738	NV1D3578	316
NV1G1737	NV1D3579	317
NV1G1740	NV1D3580	318
NV1G1864	NV1D3581	319
NV1G1739	NV1D3582	320
NV1G1870	NV1D3583	321
NV1G1715	NV1D3584	322
NV1G1753	NV1D3585	323
NV1G1750	NV1D3586	324
NV1G1750-NH2	NV1D3586-NH2	325
NV1G1749	NV1D3587	326
NV1G1871	NV1D3772	327
NV1G1839	NV1D3774	328
NV1G1877	NV1D3775	329
NV1G1872	NV1D3777	330
NV1G1941	NV1D3782	331
NV1G1990	NV1D3788	332
NV1G1991	NV1D3789	333
NV1G1989	NV1D3791	334
NV1G1993	NV1D3792	335
NV1G1967	NV1D3793	336
NV1G1969	NV1D3795	337
NV1G1974	NV1D3796	338
NV1G1950	NV1D3797	339
NV1G1948	NV1D3798	340
NV1G2057	NV1D3799	341
NV1G1954	NV1D3800	342
NV1G1956	NV1D3801	343
NV1G1961	NV1D3802	344
NV1G1960	NV1D3803	345
NV1G1977	NV1D3804	346
NV1G1982	NV1D3805	347
NV1G1984	NV1D3806	348
NV1G1985	NV1D3808	349
NV1G1983	NV1D3809	350
NV1G1973	NV1D3810	351
NV1G1976	NV1D3811	352
NV1G1980	NV1D3812	353
NV1G1952	NV1D3813	354
NV1G1957	NV1D3814	355
NV1G1981	NV1D3815	356
NV1G1959	NV1D3818	357
NV1G1986	NV1D3819	358
NV1G1968	NV1D3822	359
NV1G1945	NV1D3823	360
NV1G1972	NV1D3824	361
NV1G1946	NV1D3825	362
NV1G1970	NV1D3826	363
NV1G1949	NV1D3828	364
NV1G1951	NV1D3829	365
NV1G1971	NV1D3830	366
NV1G1975	NV1D3832	367
NV1G1978	NV1D3833	368
NV1G1979	NV1D3834	369
NV1G2043	NV1D3835	370
NV1G1955	NV1D3838	371

Some isolated Protoxin-II variants inhibit human Nav1.7 activity with an IC₅₀ value of about 3x10^-8 M or less.

Some isolated Protoxin-II variants inhibit human Nav1.7 activity with an IC₅₀ value of between about 3x10^-8 M to about 1x10^-9 M.

The disclosure also provides an isolated Protoxin-II variant comprising the amino acid sequence GPQCX₁X₂WX₃QX₄CX₅X₆X₇X₈X₉CCX₁₀X₁₁FX₁₂CX₁₃LWCX₁₄KKLW (SEQ ID NO: 404), wherein

X1: is Q, R, K, A or S;
X2: is K, S, Q or R;
X3: is M or F;
X4: is T, S, R, K or Q;
X5: is D or T;
X6: is S, A or R;
X7: is E, R, N, K, T or Q;
X8: is R or K;
X9: is K, Q, S or A;
X10: is E, Q or D;
X11: is G or Q;
X12: is V or S;
X13: is R or T; and
X14: is K or R.

Exemplary Protoxin-II variants that inhibit human Nav1.7 activity with an IC₅₀ value of about 30x10^-9 M or less are variants comprising the amino acid sequences of SEQ ID NOs: 56, 78, 111, 114, 117, 118, 119, 122, 123, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 145,146, 147, 149 ,150, 151, 152, 153, 154, 156, 158, 159, 165, 172, 173, 175, 177, 178, 183, 184, 185, 186, 189, 190, 193, 197, 199, 207, 210, 211, 216, 217, 224, 266, 273, 282 or 335.

In some embodiments, the isolated Protoxin-II variant selectively inhibits human Nav1.7. The Protoxin-II variants of the invention may be more selective towards Nav1.7 when compared to the recombinant Protoxin-II (SEQ ID NO: 2). In the QPatch electrophysiology assay, recombinant Protoxin-II has an IC₅₀ of about 2.2x10^-9 M for Nav1.7 and an IC₅₀ of about 62x10^-9 M for Nav1.6, and therefore the ratio of IC₅₀ for Nav1.6 to IC₅₀ for Nav1.7 about 28 fold. "Selectivity" or "selective" or "more selective" or "selectively blocks" or "selectively inhibits" when used herein refers to a Protoxin-II variant that has a ratio of IC₅₀ for Nav1.6 to IC₅₀ for Nav1.7 (IC₅₀(Nav1.6)/ IC₅₀(Nav1.7)) equal or over about 30. IC₅₀ for Nav1.6 may be assayed in a QPatch electrophysiology assay using cell lines stably expressing Nav1.6 using similar methods to those described for Nav1.7.

Residue positions in Protoxin-II that can be mutagenized to improve selectivity include residues 7 and 19, and optionally residues 1 and 11, and further optionally 12, 20, 22 and 26 (residue numbering according to SEQ ID NO: 1). Exemplary substitutions to improve selectivity are Y1Q, W7Q, S11R, S11A, E12T, M19F, V20S,, R22T, and K26R. Exemplary Protoxin-II variants with improved selectivity are variants of SEQ ID NOs: 56, 59, 65, 78, 111, 114, 117, 118, 119, 121, 122, 123, 129, 130, 133, 150, 190, 217, 281, 324, 325 or 326.

An isolated Protoxin-II variant may comprise the sequence GPX₁CQKWMQX₂CDX₃X₄RKCCX₅GFX₆CX₇LWCX₈KKLW (SEQ ID NO: 405); wherein

X1: is Y, Q, A, S or R;
X2: is T or S;
X3: is S, R or A;
X4: is E, T or N;
X5: is E or Q;
X6: is V or S;
X7: is R or T; and
X8: is K or R;

wherein the Protoxin-II variant inhibits human Nav1.7 activity with an IC₅₀ value of about 3x10^-8 M or less, and selectively inhibits human Nav1.7.

An isolated Protoxin-II variant may comprise the sequence GPQCQKWMQX₁CDX₂X₃RKCCX₄GFX₅CX₆LWCX₈KKLW (SEQ ID NO: 406); wherein

X1: is T or S;
X2: is S, R or A;
X3: is E, T or N;
X4: is E or Q;
X5: is V or S;
X6: is R or T; and
X7: is K or R.

An isolated Protoxin-II variant may comprise 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: 78 (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH); wherein the amino acid sequence has Q at position 1, Q at position 7 and F at position 19, when residue numbering is according to SEQ ID NO: 1; the polypeptide inhibits human Nav1.7 activity with an IC₅₀ value of about 30x10^-9 M or less, wherein the IC₅₀ value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7; and the polypeptide selectively inhibits Nav1.7.

In some embodiments, the isolated Protoxin-II variant has a free C-terminal carboxylic acid, amide, methylamide or butylamide group, which are generated via routine synthetic methods.

The disclosure also provides an isolated fusion protein comprising the Protoxin-II variant of SEQ ID NOs: 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 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, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 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, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 35, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368 367, 370 or 371. Such second polypeptides may be well known leader or secretory signal sequences, or synthetic sequences resulting for example from cloning steps, or tags such as hexahistidine tag (SEQ ID NO: 108). Such second polypeptide may be a half-life extending moiety. In one embodiment, the isolated fusion protein comprises the Protoxin-II variant of the invention conjugated to a half-life extending moiety.

Exemplary half-life extending moieties that can be used include well known human serum albumin, transthyretin (TTR), a thyroxine-binding globulin (TGB), albumin-binding domains, or an Fc or fragments thereof. Biologically suitable polymers or copolymers may also be used, for example ethylene glycol or polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20000, dextran, polylysine, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, octane, or carbohydrates (dextran, cellulose, oligo- or polysaccharides). These moieties may be direct fusions with the Protoxin-II variant polypeptides and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced Protoxin-II variants of the invention.

In another embodiment, the half-life extending moiety of the fusion protein of the invention is human serum albumin, albumin binding domain (ABD), or polyethylene glycol (PEG).

In another embodiment, the half-life extending moiety of is conjugated to the Protoxin-II variant via a linker. Suitable linkers are well known and include linkers having the sequence shown in SEQ ID NOs: 80 or 81.

Exemplary fusion proteins incorporating Protoxin-II variants of the invention are those having the polypeptide sequence of SEQ ID NOs: 83, 85, 87 or 103.

Protoxin-II variants of the invention incorporating additional moieties may be compared for functionality by several well-known assays. For example, pharmacokinetic properties of Protoxin-II variants coupled to PEG may be evaluated in well known in vivo models.

Additional substitutions to the Protoxin-II variants can be made as long as the resulting variant or the fusion protein retains similar characteristics when compared to the parent peptide. Exemplary modifications are for example conservative substitutions that will result in Protoxin-II variants with similar characteristics to those of the parent molecules. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981). Non-conservative substitutions can be made to the Protoxin-II variants that involve substitutions of amino acid residues between different classes of amino acids to improve properties of the Protoxin-II variants and Protoxin-II variant fusion proteins. Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a fashion similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement takes place can readily be tested in the same manner.

Another embodiment of the invention is an isolated synthetic polynucleotide comprising a polynucleotide encoding the Protoxin-II variant of the invention.

Certain exemplary synthetic polynucleotides are disclosed herein, however, other synthetic polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the Protoxin-II variants and Protoxin-II variant fusion proteins of the invention are also within the scope of the invention. Exemplary synthetic polynucleotides are for example polynucleotide sequences shown in SEQ ID NOs: 84, 86, 88 and 104, which encode the Protoxin-II variant fusion proteins of the invention. Those skilled in the art can readily identify the polynucleotide segments in the fusion proteins that encode the Protoxin-II variant itself. The synthetic polynucleotide sequences encoding the Protoxin-II variants or fusion proteins of the invention can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended host cell. The synthetic polynucleotide may be a cDNA.

The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer. Alternatively, the polynucleotides of the invention may be produced by other techniques such as PCR based duplication, vector based duplication, or restriction enzyme based DNA manipulation techniques. Techniques for producing or obtaining polynucleotides of known sequences are well known.

The polynucleotides of the invention may also comprise at least one non-coding sequence, such as transcribed but not translated sequences, termination signals, ribosome binding sites, mRNA stabilizing sequences, introns and polyadenylation signals. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids. These additional polynucleotide sequences may, for example, encode a marker or well-known tag sequences such as a hexa-histidine (SEQ ID NO: 108) or a HA tag which facilitate the purification of fused polypeptides.

Another embodiment of the invention is a vector comprising the polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotide of the invention into a given organism or genetic background by any means. For example, polynucleotides encoding the Protoxin-II variants or the Protoxin-II variant fusion proteins of the invention are inserted into an expression vector and may be operably linked to control sequences in the expression vector to ensure efficient expression, such as signal sequences, promoters (e.g. naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the Protoxin-II variant or the Protoxin-II variant fusion protein of the invention. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the proteins encoded by the incorporated polynucleotides.

Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 PGK1, ENO or PYK1 promoter and the like, or a regulatable promoter such as a GAL1 or GAL10 promoter. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

An exemplary vector for expression of the Protoxin-II variants or Protoxin-II variant fusion proteins is a vector having ampicillin-resistance selection marker, CMV promoter, CMV intron, signal peptide, neomycin resistance, f1 origin of replication, SV40 polyadenylation signal, and cDNA encoding the Protoxin-II variant or the Protoxin-II variant fusion protein of the invention.

Another embodiment of the invention is a host cell comprising the vector of the invention. The term "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells.

Escherichia coli, bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species, are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61) or DG44.

Introduction of a polynucleotide, such as a vector, into a host cell can be effected by methods well known to those skilled in the art. Exemplary methods are calcium phosphate transfection, DEAE-Dextran mediated transfection, microinjection, cationic lipid-mediated transfection and electroporation.

Another embodiment of the invention is a method for producing the Protoxin-II variant of the invention comprising the steps of providing a host cell of the invention; culturing the host cell under conditions sufficient for the expression of at least one Protoxin-II variant of the invention; and recovering the Protoxin-II variant produced by the host cell.

Host cells can be cultured under any conditions suitable for maintaining or propagating a given type of host cell and sufficient for expressing a polypeptide. Culture conditions, media, and related methods sufficient for the expression of polypeptides are well known in the art. For example, many mammalian cell types can be aerobically cultured at 37°C using appropriately buffered DMEM media while bacterial, yeast and other cell types may be cultured at 37°C under appropriate atmospheric conditions in LB media.

In the methods of the invention, the expression of the Protoxin-II variant can be confirmed using a variety of well-known methods. For example, expression of a polypeptide can be confirmed using detection reagents, such as using SDS-PAGE or HPLC.

Another aspect of the disclosure is a method of modulating the activity of Nav1.7 in a biological tissue, the method comprising contacting the biological tissue expressing Nav1.7 with a Nav1.7-modulating amount of the Protoxin-II variant of the invention.

USES OF PROTOXIN-II VARIANTS

Protoxin-II variants of the invention may be utilized in any therapy where it is desired to treat, reduce or alleviate symptoms of pain or other disorders of sensory or sympathetic neuron dysfunction.

Pain treated with the Protoxin-II variants of the invention may be any type of pain, such as chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, pain associated with inflammatory conditions, post-operative pain, thermal pain or pain associated with disease and degeneration.

Pain treated with the Protoxin-II variants of the invention may be Nav1.7-mediated pain.

Nav1.7-mediated pain as used herein refers to pain resulting at least partially from increased Nav1.7 channel activity.

The Protoxin-II variants of the invention may be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.

The pain and/or Nav1.7-mediated pain may result from one or more causes, such as peripheral neuropathy, central neuropathy, nerve compression or entrapment syndromes such as carpal tunnel syndrome, tarsus tunnel syndrome, ulnar nerve entrapment, compression radiculopathy, lumbar spinal stenosis, sciatic nerve compression, spinal root compression, intercostal neuralgia, compression radiculopathy and radicular lower back pain, spinal root lesions, neuritis, autoimmune diseases, general inflammation, chronic inflammatory conditions, arthritis, rheumatic diseases, lupus, osteoarthritis, general gastrointestinal disorders, colitis, gastric ulceration, duodenal ulcers, inflammatory bowel disorders, irritable bowel syndrome, pain associated with diarrhea, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, sunburn, carditis, dermatitis, myositis, neuritis, collagen vascular diseases, inflammatory pain and associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, multiple sclerosis, demyelinating diseases, diabetes, diabetic neuropathy pain, causalgia, pain resulting from amputation or abscess, phantom limb pain, fracture pain, bone injury, direct trauma, HIV infection, acquired immune deficiency syndrome ("AIDS"), small pox infection, herpes infection, exposure to toxins or other foreign particles or molecules, invasive cancer, cancer, chemotherapy, radiotherapy, hormonal therapy, burns, congenital defect, dental pain, gout pain, fibromyalgias, encephalitis, chronic alcoholism, hypothyroidism, uremia and vitamin deficiencies, trigeminal neuralgia, stroke, thalamic pain syndrome, general headache, migraine, cluster headache, tension headache, mixed- vascular and non-vascular syndromes, sympathetically maintained pain, deafferentation syndromes, asthma, epithelial tissue damage or dysfunction, disturbances of visceral motility at respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns, allergic skin reactions, pruritis, vasomotor or allergic rhinitis, or bronchial disorders, dysmenorrhoea, pain during labor and delivery, dyspepsia, gastroesophageal reflux, pancreatitis, and visceralgia.

Other disorders of sensory or sympathetic neuron dysfunction that may be alleviated by the Protoxin-II variants of the invention include itch, cough and asthma. In mice, global deletion of the SCN9A gene leads to complete insensitivity to histamine-induced itch (Gingras et al., American Pain Society Meeting Abstract 2013 and U.S. Pat. Publ. No. 2012/0185956 ). This finding suggests that peptide Nav1.7 blockers may have utility in the treatment of itch, which may arise from various sources, such as dermatological or inflammatory disorders; or inflammatory disorders such as renal or hepatobiliary disorders, immunological disorders, medication reactions and unknown/idiopathic conditions, including dermatitis, psoriasis, eczema, insect sting or bite. Nav1.7 is also expressed in sensory nerves innervating the airways (Muroi et al., J Physiol. 2011 Dec 1;589(Pt 23):5663-76; Muroi et al., Am J Physiol Regul Integr Comp Physiol. 2013 Apr 10), suggesting that peptide Nav1.7 blockers may be beneficial in the treatment of cough e.g., acute or chronic cough, or cough caused by irritation from gastroesophageal reflux disease, and inflammatory diseases of the airways such as asthma and allergy-related immune responses, bronchospasm, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and hiccups (hiccoughs, singultus). Silencing Nav1.7 in vivo in nodose ganglia of guinea pigs using shRNA nearly abolished the cough reflex induced by mechanical probing (Muroi et al., Am J Physiol Regul Integr Comp Physiol. 2013 Apr 10).

The Protoxin-II variants of the invention can be used in a method of alleviating or treating itch, cough or asthma in a subject by administering a therapeutically effective amount of the Protoxin-II variant of the invention to a subject in need thereof for a time sufficient to alleviate the itch, cough or asthma.

The Protoxin-II variants of the invention can be used in a method of alleviating or treating Nav1.7-mediated itch, Nav1.7-mediated cough or Nav1.7-mediated asthma in a subject by administering a therapeutically effective amount of the Protoxin-II variant of the invention to a subject in need thereof for a time sufficient to alleviate the itch, cough or asthma.

Nav1.7-mediated itch as used herein refers to itch resulting at least partially from increased Nav1.7 channel activity.

Nav1.7-mediated cough as used herein refers to cough resulting at least partially from increased Nav1.7 channel activity.

Nav1.7-mediated asthma as used herein refers to asthma resulting at least partially from increased Nav1.7 channel activity.

Protoxin-II variants of the invention may be tested for their effect in reducing or alleviating pain and/or Nav1.7-mediated pain using animal models described herein, and models such as the rat spinal nerve ligation (SNL) model of neuropathic pain, carageenan induced allodynia model, the Freund's complete adjuvant (CFA)-induced allodynia model, the thermal injury model, the formalin model and the Bennett Model, and other models as described in U.S. Pat. Appl. No. 2011/0124711 and U.S. Pat. No. 7,998,980 . Carageenan induced allodynia and CFA-induced allodynia are models of inflammatory pain. The Bennett model provides an animal model for chronic pain including post-operative pain, complex regional pain syndrome, and reflex sympathetic dystrophy.

Any of the foregoing animal models may be used to evaluate the efficacy of Protoxin-II variants of the invention inhibitor in treating pain and/or NAv1.7-mediated pain. The efficacy of the Protoxin-II variants of the invention may be compared to a no treatment or placebo control. Additionally or alternatively, efficacy may be evaluated in comparison to one or more known pain-relieving medicaments.

The present invention provides Protoxin-II variants for use in treating Nav1.7-mediated pain using the Protoxin-II variants of the invention. It has been discovered in the pending application by the inventors ( U.S. Patent Application Number 61/781,276 ) that administration of Nav1.7 blocking peptides are efficacious in treating and/or alleviating pain in various animal models of pain, contrary to what was disclosed and suggested in the literature. While peptide inhibitors of Nav1.7 have been shown to be potent and/or selective towards Nav1.7 in in vitro cell culture models using overexpressed Nav1.7 or on isolated neurons in which the blood-nerve barrier is subverted through desheathing or hypertonic saline injection, they have so far proven non-efficacious in in vivo animal models of pain, where the lack of efficacy has been reported to result from the inability of the peptides to pass the blood-nerve barrier. Several publications describe lack of efficacy of Nav1.7 blocking peptides in animal models of pain or in isolated nerves. For example Hackel et al., Proc Natl Acad Sci 109:E2018-27, 2012, describes the inability of ProTx-II to inhibit action potential firing in isolated nerves unless the perineural barrier, which provides a diffusion barrier in this model, is compromised. ProTx-II was found non-efficacious in rodent models of acute and inflammatory pain; a likely explanation stated the inability of ProTx-II to cross the blood-nerve barrier (Schmalhofer et al., Mol Pharmacol 74:1476-1484, 2008). It has been proposed that Nav1.7 peptide toxin blockers have poor oral bioavailability and they are difficult to deliver to nerve endings, implying that their use as therapeutic agents remain limited (Dib-Hajj et al., Nature Rev Neuroscience 14, 49-62, 2013).

Nav1.7 is expressed in the peripheral nervous system e.g., in nociceptive dorsal root ganglions (DRG), most notably in nociceptive small-diameter DRG neurons, in particular in peripheral terminals in the skin, with little representation in the brain. Nav1.7 distribution (e.g. sensory ending) and physiology predispose it to a major role in transmitting painful stimuli.

One embodiment of the invention is a Protoxin-II variant for use in treating Nav1.7-mediated pain by administering a therapeutically effective amount of the Protoxin-II variant of the invention to a subject in need thereof for a time sufficient to treat the Nav1.7-mediated pain.

The Protoxin-II variants of the invention Nav1.7 may be utilized in any therapy where it is desired to treat Nav1.7-mediated pain or other disorders of sensory or sympathetic neuron dysfunction. "Treat" or "treatment" of pain is meant to include partially or completely to prevent, stop, inhibit, reduce, or delay the perception of pain.

In some embodiments, the Nav1.7-mediated pain is chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, post-operative pain, thermal pain, phantom limb pain, or pain associated with inflammatory conditions, primary erythemalgia (PE), paraoxysmal 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, or pain associated with disease and degeneration.

Neuropathic pain includes for example painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN). Other causes of neuropathic pain include spinal cord injuries, multiple sclerosis, phantom limb pain, post-stroke pain and HIV-associated pain. Conditions such as chronic back pain, osteoarthritis and cancer may also result in the generation of neuropathic-related pain and thus are potentially suitable for treatment with the Protoxin-II variants of the invention.

In another embodiment, the Nav1.7-mediated pain is associated with primary erythemalgia (PE), paraoxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis or fibromyalgia.

The Protoxin-II variants of the invention may be conjugated to a second polypeptide to form a fusion protein. Such fusion proteins are for example the well-known Fc fusions or fusions to human serum albumin to extend half-life of the peptide inhibitors. The conjugation may be a direct conjugation via a linker, such as a glycine-serine rich linker. Such linkers are well known in the art. The Protoxin-II variants of the invention incorporating additional moieties may be compared for their Nav1.7 blocking ability and efficacy in treatment or reducing pain using well known methods and those described herein.

Other disorders of sensory or sympathetic neuron dysfunction that can be treated with the Protoxin-II variants of the invention, including asthma, cough, heart-burn, itch, dermatitis, bladder instability, and Reynaud's disease.

PHARMACEUTICAL COMPOSITIONS

The Protoxin-II variants of the invention may be formulated in a pharmaceutically acceptable vehicle or carrier. One embodiment of the invention is a pharmaceutical composition comprising the isolated Protoxin-II variant of the invention and a pharmaceutically acceptable excipient.

A suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. These solutions are sterile and generally free of particulate matter, and may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable excipients as required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. Suitable vehicles and their formulation and packaging are described, for example, in Remington: The Science and Practice of Pharmacy (21st ed., Troy, D. ed., Lippincott Williams & Wilkins, Baltimore, MD (2005) Chapters 40 and 41).

The Protoxin-II variants of the invention may be administered by peripheral administration. "Peripheral administration" or "administered peripherally" means introducing an agent into a subject outside of the central nervous system. Peripheral administration encompasses any route of administration other than direct administration to the spine or brain.

Peripheral administration can be local or systemic. Local administration may be used to concentrate the therapeutic to the site of action, such as local administration to joints, spinal cord, surgical wounds, sites of injury/trauma, peripheral nerve fibers, various organs (GI, urogenital, etc) or inflamed tissues. Systemic administration results in delivery of a pharmaceutical composition to essentially the entire peripheral nervous system of the subject and may also result in delivery to the central nervous system depending on the properties of the composition.

Routes of peripheral administration encompass, without limitation, topical administration, intravenous or other injection, and implanted mini-pumps or other extended release devices or formulations.

Pharmaceutical compositions of the invention include formulations involving the Protoxin-II variants of the invention in sustained- or controlled-delivery formulations. These formulations may be achieved through use of for example injectable microspheres, bio-erodible particles, microemulsions, nanoparticles, nanocapsules, macroemulsions, polymeric compounds (such as polyesters, polyamino acids, hydrogels, poly(lactic acid), polyglycolic acid or ethylene vinylacetate copolymers), beads or liposomes, hyaluronic acid or implantable drug delivery devices.

The Protoxin-II variants of the invention may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous), intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices, or any other administration, particularly in the form of liquid solutions or suspensions; for buccal or sublingual administration such as in the form of tablets or capsules; or intranasally such as in form of powders, nasal drops or aerosols or certain agents; transdermally in a form of a gel, ointment, lotion, cream or dusting powder, suspension or patch delivery system with chemical enhancers to either modify the skin structure or to increase the drug concentration in the transdermal patch, or with agents that enable the application of formulations containing proteins and peptides onto the skin (Int. Pat. Publ. No. WO98/53847 ), or applications of electric fields to create transient transport pathways such as electroporation, or to increase the mobility of charged drugs through the skin such as iontophoresis, or application of ultrasound such as sonophoresis ( U.S. Pat. Nos. 4,309,989 and 4,767,402 ). The composition also may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.

In certain embodiments, where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

The concentration of the Protoxin-II variants of the invention or other peptide inhibitors of Nav1.7 in such pharmaceutical formulation can vary widely, for example from about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or between 2% to 5%, up to as much as 15%, 20%, 30%, 40%, 50%, 60% or 70% by weight and will be selected primarily based on fluid volumes, viscosities and other factors, according to the particular mode of administration selected. The Protoxin-II variants of the invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective with conventional protein preparations. Lyophilization and reconstitution techniques are well known in the art.

An exemplary pharmaceutical compositions of the present invention may comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol, sucrose, Tween-20 and/or a suitable substitute thereof.

The appropriate therapeutically effective dose may be determined readily by those skilled in the art. An effective dose refers to an amount or dosage sufficient to produce a desired result, i.e. to partially or completely prevent, stop, inhibit, reduce, or delay the perception of pain associated with any painful medical condition. The effective amount may vary depending on the specific vehicle and the Protoxin-II variants of the invention selected, and is also dependent on a variety of factors and conditions related to the subject to be treated and the severity of the pain. For example, factors such as age, weight and health of the subject to be administered with the pharmaceutical compositions of the invention as well as dose response curves and toxicity data obtained in preclinical animal work could be among those considered. A determined dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician or other person skilled in the relevant art (e.g. nurse, veterinarian, or veterinary technician) during the treatment period. The determination of an effective amount or a therapeutically effective amount for a given agent is well within the ability of those skilled in the art.

Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg, about 50 ng to about 30 mg or about 5 mg to about 25 mg of a Protoxin-II variant of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg or about 5 mg to about 25 mg of the Protoxin-II variants of the invention. Actual methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, "Remington's Pharmaceutical Science", 15th ed., Mack Publishing Company, Easton, PA.

The present invention will now be described with reference to the following specific, non-limiting examples.

Example 1: Design and generation of Protoxin-II variants

Protoxin-II single position limited amino acid scanning library substitution was designed to assess to what degree selectivity, peptide yield, and homogeneity can be improved.

Protoxin-II variants were designed as HRV3C protease cleavable HSA fusion proteins in the following format from N-to C- terminus: 6xHis-HSA-linker-HRV3C cleavable peptide-Protoxin-II variant ("6xHis" disclosed as SEQ ID NO: 108); linker being (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 80, HSA having the sequence of SEQ ID NO: 106, HRV3C cleavable peptide having the sequence of SEQ ID NO: 82). Each Protoxin-II variant, after cleavage from HSA had a residual N-terminal GP from the cleavage site.

The variants were characterized in membrane depolarization assays using FLIPR® Tetra as described in Example 3 FLIPR® Tetra membrane depolarization assay, and in whole cell patch clamp experiments using the QPatch assay as described in Example 3.

Combinatorial libraries were designed to test for additive effects of select single position hits in an attempt to generate Nav1.7 antagonists with further improved potency and selectivity profile compared to the native peptide.

Construction of the Expression Vectors

The designed Protoxin-II variant genes were generated using synthetic gene assembly technology as described in U.S. Pat. No. US6,521,427 . The amino acid sequences of the designed peptide variants were back-translated to DNA sequences using human high-frequency codons. The DNA sequence of each variant gene, together with a portion of vector DNA including the DNA cloning sites, was synthesized as multiple oligonucleotides, some of which contained degenerate codons, and assembled into full-length DNA fragments. The assembled DNA fragments were amplified by PCR and PCR products were subsequently cloned as a pool. Pooled PCR products were digested with the appropriate restriction enzymes and cloned into the designed expression vector in such a manner as to fuse each toxin variant gene to the signal peptide and the fusion partner (6xHis-HSA-linker-HRV3C cleavable peptide ("6xHis" disclosed as SEQ ID NO: 108) contained in the vector. Standard molecular biology techniques were used to identify a positive clone for each designed variant. The plasmid DNA from these positive clones was purified and sequence confirmed before expressing the Protoxin-II peptide variant fusion proteins using standard methods.

Protein Expression

HEK 293-F cells were maintained in 293 Freestyle™ media (Invitrogen Cat # 12338) and split when the cell concentration was between 1.5 and 2.0 x 10⁶ cells per ml. The cells were grown in suspension, shaking at 125 RPM in a humidified incubator set at 37°C and 8% CO₂. HEK 293F cells were transiently transfected using a DNA/lipid complex after they were diluted to 1.0 x 10⁶ cells per ml. To generate the complex, 1.25 µg DNA per ml of transfection was diluted in 1.0 ml of OptiPro media (Invitrogen Cat # 12309) and 1.25 ml of Freestyle™ Max transfection reagent (Invitrogen Cat # 16447) was diluted in 1.0 ml of OptiPro media. The DNA and Max transfection reagent were mixed together and incubated for 10 minutes at room temperature before adding to the cells. Transfected cells were placed in a humidified incubator set at 37°C and 8% CO₂ for 4 days shaking at 125 RPM. The supernatant was separated from the cells by centrifugation at 5,000 x g for 10 minutes and filtered through a 0.2µm filter (Corning; Cat #431153), then concentrated 10 and 50 fold using an Amicon Ultra Concentrator 10K (Cat #UFC901096), and centrifuging for approximately 10 minutes at 3,750 x g.

Example 2: Purification of Protoxin-II variants

Protoxin-II variants were expressed as HSA fusion proteins as indicated in Example 1 and the Protoxin-II variant peptides were cleaved with HRV3C protease prior to purification. Two methodologies were tested for efficient purification of the Protoxin-II variants.

Protein Purification Purification of Protoxin-II Variants by RP-HPLC

The secreted proteins were purified from the expression supernatants via IMAC using 1 ml HisTrap HP columns (GE Healthcare Cat# 17-5247-01). The chromatography method was run using an AKTA Xpress and protein was eluted from the column using a step gradient of Imidazole. Peak fractions were pooled and digested overnight with HRV 3C protease (1µg protease / 150µg fusion).

Cleaved peptide-fusion pools were further purified using a Dionex HPLC system with a reverse phase Phenomenex Luna 5 µm C18(2) column (Cat# 00B-4252-P0-AX). Samples were eluted from the column with a 0-68% Acetonitrile (0.05% TFA) linear gradient. Elution fractions were pooled, lyophilized overnight and reconstituted in HEPES buffered saline, pH 7.4 (10 mM HEPES, 137mM NaCl, 5.4 mM KCl, 5 mM glucose, 2mM CaCl₂, 1mM MgCl₂).

Table 4 shows yields of Protoxin-II variants purified by RP-HPLC. The average mg yield/L was 0.01615.

Purification of Protoxin-II Variants by Solid Phase Extraction (SPE)

The secreted proteins were purified from the expression supernatants via IMAC using 1 ml HisTrap HP columns (GE Healthcare Cat# 17-5247-01). The chromatography method was run using an AKTA Xpress and protein was eluted from the column using a step gradient of Imidazole. Peak fractions were pooled and digested overnight with HRV3C protease (1µg protease / 150µg fusion). The cleaved sample was loaded into a 50 kDa molecular weight cut off centrifugal filter unit (Millipore UFC805096) and cleaved peptide collected in the filtrate fraction.

Peptide pools were loaded onto a 96-well solid phase extraction block (Agilent Bond Elut Plexa A3969030) for further purification, desalting, and concentration. Blocks were used in conjunction with a vacuum manifold (Whatman). Peptide samples were loaded and washed in 0.05% TFA in water and eluted with a step gradient of acetonitrile with 0.05% TFA in water. Elution fractions were then lyophilized overnight and reconstituted in HEPES buffered saline, pH 7.4 (10 mM HEPES, 137mM NaCl, 5.4 mM KCl, 5 mM glucose, 2mM CaCl₂, 1mM MgCl₂).

Peptides were reconstituted in supplemented HEPES buffered saline, pH 7.4 (10 mM HEPES, 137mM NaCl, 5.4 mM KCl, 5 mM glucose, 2mM CaCl₂, 1mM MgCl₂) and absorbance was measured at 280 nm. Concentration values were then calculated using each sample's extinction coefficient. 2 µg of each peptide were loaded onto an Invitrogen NuPAGE® Novex® Bis-Tris Gel 15 well gel and run in MES buffer non-reduced.

Samples were analyzed on Agilent 1100 HPLC using 4-80% acetonitrile in 0.05% TFA linear gradient with a Phenomenex Luna C18(2) analytical column (Cat#00A-4041-B0). Concentrations of all peptides were normalized and 10 µl of each were injected for a total of 1.3 µg per sample. Absorbance at 220 nm was monitored and chromatograms analyzed were using Chromeleon software.

Table 5 shows yields (mg) of Protoxin-II variants purified by SPE. The average mg yield/L was 0.05353.

The benefits of the SPE purification process are ease and throughput of purification since samples are processed in parallel in a 96-well block rather than serially on RP-HPLC, and improvement in yield. There was, on average, more than 3-fold higher yield (mg/L) for variants purified by SPE versus RP-HPLC.

Table 5.

Protoxin-II Variant Peptide ID	yield (mg)	Protoxin-II Variant Peptide ID	yield (mg)
NV1D12	0.0054	NV1D2734	0.0602
NV1D2659	0.0234	NV1D2772	0.2050
NV1D2664	0.0060	NV1D2775	0.2225
NV1D2666	0.0225	NV1D2738	0.0512
NV1D2708	0.0721	NV1D2740	0.0373
NV1D2725	0.0144	NV1D2733	0.1913
NV1D2739	0.0053	NV1D788	0.0000
NV1D2765	0.0097	NV1D757	0.0021
NV1D2748	0.0995	NV1D791	0.0007
NV1D2771	0.0103	NV1D2310	0.0011
NV1D2770	0.0121	NV1D2308	0.0014
NV1D2778	0.0644	NV1D778	0.0019
NV1D2782	0.0202	NV1D2294	0.0000
NV1D2756	0.0466	NV1D856	0.0047
NV1D2759	0.0218	NV1D2309	0.0023
NV1D2712	0.0558	NV1D846	0.0020
NV1D12	0.0127	NV1D2896	0.0504
NV1D2673	0.0625	NV1D2913	0.0203
NV1D2662	0.0433	NV1D2910	0.0253
NV1D2669	0.2661	NV1D2893	0.0569
NV1D2665	0.0389	NV1D2909	0.0195
NV1D2731	0.2547	NV1D2917	0.0339
NV1D2767	0.0238	NV1D2914	0.0201
NV1D2730	0.2566	NV1D2922	0.0554
NV1D2766	0.0198	NV1D2902	0.0061
NV1D2667	0.0050	NV1D2889	0.0022
NV1D2769	0.0142	NV1D2887	0.0025
NV1D2719	0.0675	NV1D2878	0.0272
NV1D2776	0.0633	NV1D2877	0.0129
NV1D2663	0.0344	NV1D2851	0.0029
NV1D2709	0.1841	NV1D2850	0.0026
NV1D2720	0.0538	NV1D2820	0.0020
NV1D12	0.0095	NV1D2819	0.0015
NV1D2773	0.1921	NV1D2814	0.0163
NV1D2810	0.0086	NV1D2918	0.0256
NV1D2732	0.0262	NV1D2921	0.0533
NV1D757	0.0026	NV1D2905	0.0126
NV1D791	0.0206	NV1D2906	0.0189
NV1D2310	0.0085	NV1D2881	0.0207
NV1D2308	0.0179	NV1D2882	0.0223
NV1D778	0.0094	NV1D2869	0.0038
NV1D856	0.0247	NV1D2870	0.0187
NV1D2309	0.0035	NV1D2867	0.0147
NV1D846	0.0043	NV1D2888	0.0045
NV1D2889	0.0107	NV1D2816	0.0133
NV1D2887	0.0061	NV1D2885	0.0025
NV1D2861	0.0469	NV1D2974	0.0418
NV1D2729	0.1101	NV1D2972	0.1089
NV1D2890	0.0088	NV1D2971	0.0407
NV1D2899	0.0402	NV1D2970	0.0557
NV1D2804	0.0044	NV1D2969	0.0799

Example 3: Characterization of Protoxin-II variants

Select Protoxin-II variants were characterized in membrane depolarization and whole cell patch clamp assays to assess their potency and selectivity towards Nav1.7.

FLIPR® Tetra membrane depolarization assay

The ability of the generated peptides to inhibit membrane depolarization induced by Nav1.7 agonist veratridine (3-Veratroylveracevine; Biomol, Catalog# NA125) was measured with a FRET (fluorescence resonance energy transfer) assay on FLIPR® Tetra using DISBAC2(3) (Invitrogen, K1018) as an electron acceptor and PTS18 (Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate) (Sigma) as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm.

HEK293 cells stably expressing human Nav1.7 were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 400 pg/mL geneticin and 100 µM NEAAs (all reagents from Invitrogen). 50 µL of harvested cells were plated at 25,000 cells/well into poly-lysine coated 384-well black clear bottom plates. The plates were incubated at room temperature (RT) for 15 min followed by an overnight incubation at 37°C. All incubations were done in the dark unless otherwise stated. The next day, the wells were washed 4 times with assay buffer (137 mM NaCl, 4mM KCl, 2mM MgCl₂, 2mM CaCl₂, 5 mM Glucose, 10 mM HEPES, pH 7.4), and resuspended in 25 µL of assay buffer. 2x stock (6 µM) of the PTS18 dye was prepared by suspending the dye in 10% pluronic F127 in DMSO at 1:1 (v/v ratio). 25 µL of the 2x PTS18 stock was added into the wells and the cells were stained for 30 min at RT, after which the dye was washed off with the assay buffer. Peptides were suspended at 3x their final concentration in the assay buffer containing 10 µM DISBAC2(3) and 400 µM VABSC-1 to suppress background fluorescence (Sigma, cat# 201987). 25 pL/well of the suspended peptides were added into each well, and incubated for 60 minutes at RT. Depolarization was induced by 25 µM final concentration of veratridine (by adding 25 µL/well of 75µM (3x) stock solution), and the reduction in the mean intensity of FRET dye fluorescence was measured 30-100 seconds after adding the agonist. A 1.3X dilution of each measured peptide occurred after adding veratridine by convention, the concentration at the beginning of the FLIPR® Tetra assay is reported.

Concentration-response curves of synthetic Protoxin-II (Peptide International) were constructed in each experimental series and were used as controls. Fluorescence counts for each well were converted to % response by normalizing the signal to the difference between negative control (response to agonist veratridine alone) and positive control (response to veratridine in the presence of 10 µM tetracaine) values. For measurements, "spatial uniformity correction" (all fluorescence traces are normalized to the average initial starting intensity) and "subtract bias value" (subtract the initial starting intensity from each trace) were turned on in FLIPR® Tetra. Each data point represented the response in an individual well. All individual data points were used in a non-linear least-squares fitting procedure to find the best fit to a Hill function using Origin (Microcal). IC₅₀ values were extracted from the resultant fitted curve. The mean responses of the positive (P) and negative (N) controls were used to calculate the % response in a well as follows: % response = 100^∗(N-R)/(N-P).

Assay plates were accepted if the potency of control antagonists for that day were within ±0.5 log units of their historical mean.

QPatch Assay

HEK293 cells stably expressing human Nav1.5 (SEQ ID NO: 105), Nav1.7 (SEQ ID NO: 79) or Nav1.6 (SEQ ID NO: 407) were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, 400 µg/mL Geneticin and 100 µM NEAAs (all reagents from Invitrogen). Cells were maintained at 37°C and in 5% CO2 and assayed upon reaching ∼50-90% confluency. CHO cells stably expressing human Nav1.6 in a tetracycline-inducible manner(SEQ ID NO: 407) were cultured in HAMs F12, supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, 10 pg/mL Blasticidin and 400 µg/mL Zeocin. Cells were maintained at 37°C and in 5% CO2, and assayed upon reaching ∼50-90% confluency. Nav1.6 expression was induced with 1 µg/ml of tetracycline, 24-48h prior to an experiment.

Before testing in QPatch HT (Sophion), cells were first dissociated using 0.05% trypsin (5 min at 37°C), resuspended in CHO-S-SFM media (Life Technologies) and gently triturated to break up cell clumps. Cell density was adjusted to 1-2×10⁶/mL with the same media and cells were the transferred to a cell "hotel" in QPatch HT and used in experiments for several hours. For giga-ohm seal formation and whole-cell patch clamp recording, the extracellular solution contained 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, and 10 mM HEPES, pH = 7.4 and osmolarity = 315 mOsm. The intracellular solution contained 135 mM CsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH = 7.3 and osmolarity = 290 mOsm. The voltage protocol used in the assay was as follows. From a holding potential of -75 mV (Nav1.7), -60 mV (Nav1.6), or -105 mV (Nav1.5) cells were first hyperpolarized to -120 mV for 2 sec and then depolarized to 0 mV for 5 ms before returning to the holding potential. This protocol was repeated once every 60 sec during liquid applications (see below). Cells were otherwise held at the holding potential when the above voltage protocol was not executed. Upon establishment of the whole-cell recording configuration, a total of five applications of the extracellular solution (all containing 0.1% bovine serum albumin (BSA) with or without test compound, except for the last application, which contained 1 µM TTX or 10 mM lidocaine as a positive control) were made on to cells being recorded. The first liquid application contained only the control buffer (5 µl). The voltage protocol was executed 10 times (for a total duration of 10 min) five sec after the application. The next three liquid applications (5 µl each) contained a test compound (same compound at the same concentration for all three applications) or control buffer (for control cells only). Five seconds after each of these applications, the voltage protocol was again executed 10 times (also once per min). The last application contained positive (composed of three 10µl sub-applications, each separated by 2 sec), five seconds after which the same voltage protocol was executed twice to obtain the baseline current. Currents were sampled at 25 kHz and filtered at 5 kHz with an 8-pole Bessle filter. The series resistance compensation level was set at 80%. For each cell, the peak current amplitude at 0 mV for each current trace in the first four liquid applications was first subtracted from that of the last trace in the presence of positive control and then normalized to that of the last trace in the first (control buffer) application as % inhibition. To control for current rundown, this (% inhibition) value for each cell in the presence of a test compound was further normalized to the average % inhibition value for control (typically 5-6) cells in the same experiment. The mean of the last two such values in the last compound application (i.e., the corrected % inhibition value for each concentration of a test compound) were taken as the % inhibition value for each cell at the particular compound concentration tested. The % inhibition values for all cells tested at each compound concentration were averaged and used in concentration response calculations. All experiments were performed at room temperature (∼22 °C). Data are expressed as mean±se. Wild type Protoxin-II was included in each experiment as a positive control. Data were accepted only if the potency of Protoxin-II was within ±0.5 log units of its historical mean.

IC₅₀ values for Nav1.7 for select Protoxin-II variants obtained using the FLIPR® Tetra are shown in Table 6.

Table 6.

Protein ID	Protoxin-II Variant Peptide ID	Protoxin-II variant Peptide SEQ ID NO:	hNav1.7
			TETRA

NV1D12_5	NV1D12	2	4.1±3.6
NV1G1045	NV1D791	11	4.8±0.4
NV1D1332_1	NV1D1332	12	6.7±0.5
NV1D1336_1	NV1D1336	14	10.5±1.2
NV1D1337_1	NV1D1337	15	10.3±1.0
NV1G1049	NV1D2308	16	4.5±0.4
NV1 G953	NV1D2670	17	22.2±3.3
NV1 G951	NV1D2674	18	4.0±0.2
NV1 G963	NV1D2671	20	31.5±6.4
NV1G949	NV1D2675	21	4.3±0.3
NV1 G977	NV1D2665	22	4.9±0.4
NV1G957	NV1D2668	23	17.5±2.6
NV1 G965	NV1D2672	24	4.5±0.3
NV1 G973	NV1D2662	25	4.0±0.4
NV1 G975	NV1D2669	26	18.4±5.7
NV1 G971	NV1D2673	27	4.3±0.5
NV1 G995	NV1D2663	28	4.2±0.4
NV1 G961	NV1D2676	29	26.5±2.9
NV1G911	NV1D2666	30	66.5±36.7
NV1G1133	NV1D2816	31	667±93.6
NV1 G905	NV1D2735	32	60.0±16.2
NV1 G979	NV1D2731	34	20.7±7.2
NV1G1097	NV1 D281 0	35	339±5750
NV1G1099	NV1D2732	36	126±26.9
NV1G1011	NV1D2740	37	3.6±9.9
NV1G1105	NV1D2729	39	8.0±0.9
NV1G1013	NV1D2733	40	7.5±2.9
NV1G1095	NV1D2814	41	754±51.3
NV1 G983	NV1D2730	43	25.5±4.3
NV1G1003	NV1D2734	44	13.4±0.8
NV1G1009	NV1D2738	45	2.6±0.2
NV1G1129	NV1D2867	49	>1000
NV1G1121	NV1D2881	50	488±72.2
NV1G1123	NV1D2882	51	857±65.7
NV1 G899	NV1D2774	52	50.5±15.2
NV1G1103	NV1D2861	54	>1000
NV1G1127	NV1D2870	55	784±84.8
NV1G1007	NV1D2775	56	25.4±2.0
NV1G1067	NV1D2893	57	75.5±10.5
NV1G1005	NV1D2772	59	15.6±1.8
NV1G1061	NV1D2896	60	80.3±7.1
NV1G1085	NV1D2877	61	441±73.3
NV1G1083	NV1D2878	62	680±40.7
NV1G1079	NV1D2889	64	12.1±1.5
NV1G1001	NV1D2773	65	18.8±1.5
NV1G1107	NV1D2890	66	25.8±4.2
NV1G1109	NV1D2899	67	33.3±6.7
NV1G1117	NV1D2905	68	713±87.3
NV1G1119	NV1D2906	69	940±86.7
NV1G1115	NV1D2921	70	586±71.7
NV1G1075	NV1D2922	71	204±45.7
NV1G1069	NV1D2909	72	97.1±10.1
NV1G1065	NV1D2910	73	441±41.7
NV1G1063	NV1D2913	74	79.7±9.3
NV1G1073	NV1D2914	75	135±7.8
NV1G1071	NV1D2917	76	197±48.3
NV1G1113	NV1D2918	77	983±98.7
NV1G1153	NV1D3034	78	10.3±2.1

Select Protoxin-II variants were tested for selectivity against human Nav1.5 using QPatch. IC₅₀ values for both Nav1.7 and Nav1.5 for select peptides obtained using QPatch are shown in Table 7.

Table 7.

Protein ID	Protoxin-II Variant Peptide ID	Protoxin-II variant Peptide SEQ ID NO:	hNav1.7	hNav1.5
			QPatch

NV1D12_5	NV1D12	2	2.2±1.3	>1000
NV1G899	NV1D2774	52	18.7±13.6	>3000
NV1G1007	NV1D2775	56	4.0±8.9	>3000
NV1G1005	NV1D2772	59	6.2±3.2	>3000
NV1G1001	NV1D2773	65	4.3±3.3	>3000
NV1G1153	NV1D3034	78	4.3±4.3	>1000

Example 4. Generation and characterization of combinatorial Protoxin-II variants

Combinatorial libraries were designed to test for additive effects of select single position hits in an attempt to generate Nav1.7 antagonists with further improved potency and selectivity profile compared to the native peptide using several approaches.

A limited amino acid scan was conducted at all non-cysteine Protoxin-II positions using A, D, Q, R, K and S for diversification. In these experiments, Protoxin-II was expressed and tested as monovalent Fc fusion protein as described in Example 1. From this scan, substitutions Y1Q, W7Q, S11A, were identified that improved potency and/or selectivity of the resulting variants.

A full amino acid scan (excluding cys and trp) at positions M6 and M19 was also conducted. M19F substitution was identified from this scan that improved potency and/or selectivity of the resulting variants.

Protoxin-II/Huwentoxin-IV single position chimeras were designed bidirectionally. The purpose of this library was to obtain Protoxin-II variants that retained potency and selectivity profile of the wild type Protoxin-II and would achieve beneficial refolding properties associated with Huwentoxin-IV. Substitutions R22T and E12N were identified from this scan.

Peptide NV1G1153 was further engineered by diversifying position Y1 by a limited amino acid scan using R, K, T, A, D, E, Q and S, and by charge cluster engineering, where all sets of charged residues in the three-dimensional structure of the peptide (D10/E12, K4/E17, D10/E12/R13) were mutated.

N- and C-terminal extensions were introduced to select peptides, including NV1G1153 with the purpose of improving peptide distribution to the site of action and of improving half-life of the peptides without significantly increasing the molecular weight of the resulting peptide. The N- and C-terminal extensions that were used are shown in Table 8 and 9, respectively, and are described in Oi et. al., Neuroscience Letters 434, 266-272, 2008; Whitney et. al., Nature Biotechnology 2011 29:4, 352-356; Sockolosky et. al., (2012) 109:40, 16095-16100. Cell penetrating peptides HIV Tat and polyarginine were also used. Various linkers were used to couple the Protoxin-II variant to the N- and/or C-terminal extensions. The linkers used are shown in Table 10.

Protoxin-II variants from each campaign were tested for their potency and selectivity for Nav1.7 using methods described in Example 3. The amino acid sequences of the variants that inhibited Nav1.7 with an IC₅₀ value of 200 nM or less are shown in Table 3. Table 11 shows the amino acid substitutions in select variant when compared to the wild type Protoxin-II, and the IC₅₀ values for Nav1.7 inhibition in the FLIPR Tetra assay.

Table 8.

N-terminal extension
Amino acid sequence	SEQ ID NO:
GPAAAAA	372
GPAPAPA	373
GGGGGG	374
GPCCNCSSKWCRDHSRCC	375
GPSPGARAF	376
GPDGPWRKM	377
GPFGQKASS	378
GPCRTIGPSVC	379
GPSHSNTQTLAKAPEHTG	380
GPQRFVTGHFGGLYPANG	381
GPGWCGDPGATCGKLRLYCCSGFCDSYTKTCKDKSSA	382
APAPAPAPAP	383
GPYGRKKRRQRRR	384
GPRRRRRRRRRRR	385

Table 9.

C-terminal extensions
Amino acid sequence	SEQ ID NO:
CRTIGPSVC	386
YGRKKRRQRRR	387
GGGGG	374
DGPWRKM	388
CCNCSSKWCRDHSRCC	389
RRRRRRRRRRR	390
SHSNTQTLAKAPEHTG	391
APAPA	392
AAAAA	393
FGQKASS	394
QRFVTGHFGGLYPANG	395
SPGARAF	396
GPGWCGDPGATCGKLRLYCCSGFCDAYTKTCKDKSSA	397

Table 10.

Linkers
Amino acid sequence	SEQ ID NO:
GSAPAPAPAPAPGS	398
GSAPAPAPAPAPAPAPAPAPAPGS	399
	400
APAPA	392
	401
APAPAPAPAP	383
APAPAPAPAPAPAPAPAPAP	402

Table 11.

Protein name	Protoxin-II variant peptide name	Protein SEQ ID NO:	Substitutions		SE
NV1G1728	NV1D3541	281	Y1A,W7Q, S11R, E12N, M19F, R22T,K26R	9.4	1.2
NV1G1870	NV1D3583	321	Y1A,W7Q,S11A,E12R,M19F ,V20S	13.1	1.57
NV1G1752	NV1D3532	272	Y1A,W7Q,S11A,E12K,M19F ,R22T,K26R	17.3	2
NV1G1749	NV1D3587	326	Y1A,W7Q,S11A, E12N, M19F, V20S	18.3	2.6
NV1G1725	NV1D3572	310	Y1A,W7Q,S11A,E12R,M19F ,R22T	19.8	2.2
NV1G1745	NV1D3537	277	Y1A,W7Q,S11A,E12K,M19F ,V20S,R22T,K26R	21.4	4.1
NV1G1720	NV1D3565	304	Y1A,W7Q,S11A,E12R,M19F ,V20S,R22T	23	2.8
NV1G1761	NV1D3550	290	Y1A,W7Q,S11R,M19F,R22T, K26R	25.8	2.7
NV1G1746	NV1D3576	314	Y1A,W7Q,S11A,E12N,M19F ,R22T	26.7	5.2
NV1G979	NV1D2731	34	Y1A,W7Q,S11A	20.7	7.2
NV1G953	NV1D2670	17	Y1A,W7Q	22.2	3.3
NV1G1519	NV1D3006	133	Y1Q,W7Q,S11A,E12R, M19F	4.03	1.05
NV1G1007-NH2	NV1D2775-NH2	111	Y1Q,W7Q,S11A,M19F	5.06	0.473
NV1G1517	NV1D3004	131	Y1Q,W7Q,S11R,M19F	6.23	1.56
(-GP) N-Ac-NV1G1137-NH2	(-GP) N-Ac-NV1D2974-NH2	114	Y1Q,W7Q,S11A,M19F,V20S ,R22T	6.43	1.06
NV1G1776	NV1D3339	172	Y1Q, Q3R,W7Q,S11R,M19F,R22T ,K26R	6.57	0.675
NV1G1153-NH-methyl	NV1D3034-NH-methyl	119	Y1Q,W7Q,S11R,M19F,R22T ,K26R	7.1	0.9
(-GP) N-Ac-NV1G1153-NH2	(-GP) N-Ac-NV1D3034-NH2	121	Y1Q,W7Q,S11R,M19F,R22T ,K26R	7.63	1.04
NV1G1523	NV1D3012	135	Y1Q,W7Q,S11R,E12N,M19F	7.74	0.904
NV1G1515	NV1D3005	132	Y1Q,W7Q,S11A,E12N,M19F	7.83	1.38
NV1G1187	NV1D3015	138	Y1Q,W7Q,S11R,M19F,K26R	8.86	2.28
NV1G1521	NV1D3018	141	Y1Q,W7Q,S11A,E12N,M19F ,K26R	9.79	2.91
NV1G1267	NV1D3044	150	Y1Q,W7Q,S11R,E12N,M19F ,R22T,K26R	9.8	0.849
NV1G1153	NV1D3034	78	Y1Q,W7Q,S11R,M19F,R22T ,K26R	10.3	2.14
NV1G1836	NV1D3359	190	YI0,W7Q,T8S, S11R,M19F, R22T,K26R	10.5	0.739
NV1G1593	NV1D3050	153	Y1Q,W7Q,S11R,E12K,M19F	10.8	1.3
NV1G1215	NV1D3048	152	Y1Q,W7Q,S11A,E12K,M19F	11.1	1.05
NV1G1868	NV1D3353	185	Y10,W7Q,T8R,S11R,M19F, R22T,K26R	11.2	1.25
NV1G1525	NV1D3013	136	Y1Q,W7Q,S11R,E12R,M19F	11.3	1.83
NV1G1775	NV1D3340	173	Y1Q,Q3K,W7Q,S11R,M19F, R22T,K26R	11.5	0.798
NV1G1833	NV1D3381	210	Y1Q,W7Q,S11RK14Q, M19F, R22T,K26R	12.2	1.56
NV1G1153-NH2	NV1D3034-NH2	117	Y1Q,W7Q,S11R,M19F,R22T ,K26R	12.2	1
NV1G1777	NV1D3342	175	Y1Q,Q3A,W7Q,S11R,M19F,	12.8	2.67
	R22T,K26R
NV1G1259	NV1D3058	158	Y1Q,W7Q,S11A,E12K,M19F ,R22T,K26R	12.9	1.29
NV1G1511	NV1D3032	146	Y1Q,W7Q,S11R,E12N,M19F ,K26R	13	203
NV1G1527	NV1D3031	145	Y1Q,W7Q,S11R,E12R,M19F ,R22T	13	1.36
NV1G1265	NV1D3062	159	Y1Q,W7Q,S11R,E12K,M19F ,R22T,K26R	13.2	1.43
NV1G1781	NV1D3388	217	Y1Q,W7Q,S11RE17Q,M19F, R22T,K26R	13.5	1.14
NV1G1824	NV1D3354	186	Y1Q,W7Q,T8K,S11R,M19F, R22T,K26R	13.9	1.12
NV1G1772	NV1D3352	184	Y1Q,K4S,W7Q,S11R,M19F, R22T,K26R	14.2	2.01
NV1G1509	NV1D3033	147	Y1Q,W7Q,S11R,E12R,M19F ,K26R	14.5	2.18
NV1G1779	NV1D3351	183	Y1Q,K4Q,W7Q,S11R,M19F, R22T,K26R	15.3	2.39
NV1G1687	NV1D3526	266	Y1Q,W7Q,S11R,M19F,R22T ,K26R	15.4
NV1G1269	NV1D3045	151	Y1Q,W7Q,S11R,E12R,M19F ,R22T,K26R	15.6	1.39
NV1G1623	NV1D3056	156	Y1Q,W7Q,S11R,E12K,M19F ,R22T	16.2	2.99
NV1G1859	NV1D3376	205	Y1Q,W7Q,S11R,K14R,M19F ,R22T,K26R	16.3	2.53
NV1G1153-NH-butyl	NV1D3034-NH-butyl	118	Y1Q,W7Q,S11R,M19F,R22T ,K26R	16.6	1.4
NV1G1211	NV1D3036	149	Y1Q,W7Q,S11A,E12R,M19F ,R22T,K26R	17.2	1.55
NV1G1885	NV1D3254	165	Y1Q,W7Q,S11A,M19F	17.5	2.45
NV1G1730	NV1D3542	282	Y1Q,W7Q,S11R,E12N,M19F ,V20S,R22T,K26R	17.7	2.5
NV1G1263	NV1D3051	154	Y1Q,W7Q,S11A,E12K,M19F ,R22T	17.9	1.78
NV1G1818	NV1D3368	122	Y1Q,W7Q,S11R,E12T, M19F, R22T,K26R	17.9	1.89
NV1G1153 (synthetic)	NV1D3034	116	Y1Q,W7Q,S11R,M19F,R22T ,K26R	18	2.5
NV1G1823	NV1D3367	197	Y1Q,W7Q,S11R,E12Q,M19F ,R22T,K26R	18.6	2.17
NV1G1820	NV1D3362	193	Y1Q,W7Q,D10T,S11R,M19F ,R22T,K26R	20.1	2.32
NV1G1811	NV1D3369	199	Y1Q,W7Q,S11R,R13K,M19F ,R22T,K26R	20.4	2.44
NV1G1810	NV1D3358	189	Y1Q,W7Q,T8Q,S11R,M19F, R22T,K26R	20.5	2.11
NV1G1818-NH2	NV1D3368-NH2	123	Y1Q,W7Q,S11R,E12T,M19F ,R22T,K26R	20.5	2.8
NV1G1137 (synthetic)	NV1D2974	129	Y1Q,W7Q,S11A,M19F,V20S ,R22T	21.6	1.34
NV1G1221	NV1D3017	140	Y1Q,W7Q,S11A,E12R,M19F ,R22T	21.9	2.48
NV1G1722	NV1D3533	273	Y1Q,W7Q,S11A,E12K,M19F ,V20S,R22T,K26R	22.4	3.5
NV1G1767	NV1D3345	177	Y1Q,Q3S,W7Q,S11R,M19F, R22T,K26R	22.4	2.52
NV1G1769	NV1D3346	178	Y1Q,K4R,W7Q,S11R,M19F, R22T,K26R	23.2	3.39
NV1G1780	NV1D3387	216	Y1Q,W7Q,S11R,E17D,M19F ,R22T,K26R	23.7	2.85
NV1G1886	NV1D3249	162	Y1Q,W7Q,S11A,M19F	24.1	11.5
NV1G1812	NV1D3382	211	Y1Q,W7Q,S11R,K14S,M19F ,R22T,K26R	24.3	2.14
NV1G1857	NV1D3366	196	Y1Q,W7Q,D10S,S11R,M19F ,R22T,K26R	24.6	3.8
NV1G1821	NV1D3378	207	Y1Q,W7Q,S11R,K14A,M19F ,R22T,K26R	24.8	2.66
NV1G1993	NV1D3792	335	Y1Q,W7Q,S11R,M19F,R22T ,K26R	25.3	2.8
NV1G1007	NV1D2775	56	Y1Q,W7Q,S11A,M19F	25.4	2
NV1G1787	NV1D3396	224	Y1Q,W7Q,S11R,G18Q,M19 F,R22T,K26R	26.4	3.17
NV1G1257	NV1D3016	139	Y1Q,W7Q,S11A,E12N,M19F ,R22T	26.6	3.1
NV1G1153 (synthetic)	NV1D3034	116	Y1Q,W7Q,S11R,M19F,R22T ,K26R	27.3	2.02
NV1G1803	NV1D3403	230	Y1Q,W7Q,S11R,M19F,R22T ,K26R,K27A	28.3	1.97
(-GP) N-Ac-NV1G1137	N-Ac-NV1D2974	115	Y1Q,W7Q,S11A,M19F,V20S ,R22T	28.6	2.23
NV1G1531	NV1D3019	142	Y1Q,W7Q,S11A,E12R,M19F ,K26R	28.7	4.78
NV1G1513	NV1D3007	134	Y1Q,W7Q,S11A,M19F,K26R	29.6	9.17
NV1G1991	NV1D3789	333	Y1Q,W7Q,S11R,M19F,R22T ,K26R	29.9	5.19
NV1G1013	NV1D2733	40	Y1R,W7Q,M19F	7.54	2.9
NV1G1740	NV1D3580	318	Y1R,W7Q,S11A,E12R,M19F, V20S	8.4	1.5
NV1G1757	NV1D3538	278	Y1R,W7Q,S11R,E12N,M19F ,R22T,K26R	11.6	1.4
NV1G1741	NV1D3569	307	Y1R,W7Q,S11A,E12R,M19F, R22T	11.9	0.8
NV1G1715	NV1D3584	322	Y1R,W7Q,S11A,E12N,M19F ,V20S	13.9	1.4
NV1G1754	NV1D3529	269	Y1R,W7Q,S11A,E12K,M19F, R22T,K26R	14.6	1.7
NV1G1005	NV1D2772	59	Y1R,W7Q,S11A,M19F	15.6	1.8
NV1G1733	NV1D3577	315	Y1R,W7Q,S11A,M19F,V20S	18.8	2.2
NV1G1744	NV1D3534	274	Y1R,W7Q,S11A,E12K,M19F, V20S,R22T,K26R	20.6	2.2
NV1G1724	NV1D3562	301	Y1R,W7Q,S11A,E12R,M19F, V20S,R22T	23.6	2.7
NV1G1735	NV1D3566	305	Y1R,W7Q,S11A,M19F,R22T	23.7	2.5
NV1G1760	NV1D3543	283	Y1R,W7Q,S11R,E12N,M19F ,V20S,R22T,K26R	23.8	1.9
NV1G1759	NV1D3547	287	Y1R,W7Q,S11R,M19F,R22T, K26R	26.5	2.1
NV1G1751	NV1D3558	297	Y1R,W7Q,S11A,E12N,M19F ,V20S,R22T	26.7	3.4
NV1G1726	NV1D3551	291	Y1R,W7Q,S11R,M19F,V20S, R22T,K26R	29.3	3.8
NV1G1105	NV1D2729	39	Y1R,W7Q,S11A	8	8.85E-01
NV1G957	NV1D2668	23	Y1R,W7Q	17.5	2.6
(-GP) NV1G1001	(-GP) NV1D2773	109	Y1S,W7Q,S11A,M19F	9.47	1.28
(-GP) NV1G1001-NH-methyl	(-GP) NV1D2773-NH-methyl	110	Y1S,W7Q,S11A,M19F	11.5	0.61
NV1G1003	NV1D2734	44	Y1S,W7Q,M19F	13.4	0.8
NV1G1864	NV1D3581	319	Y1S,W7Q,S11A,E12R,M19F, V20S	14.6	1.7
NV1G1748	NV1D3530	270	Y1S,W7Q,S11A,E12K,M19F, R22T,K26R	15.6	2.2
NV1G1758	NV1D3548	288	Y1S,W7Q,S11R,M19F,R22T, K26R	17.6	1.9
NV1G1727	NV1D3544	284	Y1S,W7Q,S11R,E12N,M19F, V20S,R22T,K26R	17.8	2.2
NV1G1719	NV1D3570	308	Y1S,W7Q,S11A,E12R,M19F, R22T	18.1	1.5
NV1G1742	NV1D3535	275	Y1S,W7Q,S11A,E12K,M19F, V20S,R22T,K26R	18.7	2.8
NV1G1001	NV1D2773	65	Y1S,W7Q,S11A,M19F	18.8	1.5
NV1G1753	NV1D3585	323	Y1S,W7Q,S11A,E12N,M19F ,V20S	19.4	2.1
NV1G1762	NV1D3539	279	Y1S,W7Q,S11R,E12N,M19F, R22T,K26R	19.4	1.8
NV1G1755	NV1D3574	312	Y1S,W7Q,S11A,E12N,M19F ,R22T	22.3	2.7
NV1G1717	NV1D3563	302	Y1S,W7Q,S11A,E12R,M19F, V20S,R22T	22.4	2.4
NV1G1866	NV1D3559	298	Y1S,W7Q,S11A,E12N,M19F ,V20S,R22T	26.5	5.02
NV1G1721	NV1D3552	292	Y1S,W7Q,S11R,M19F,V20S, R22T,K26R	28.1	3.7
NV1G975	NV1D2669	26	Y1S,W7Q	18.4	5.7
NV1G983	NV1D2730	43	Y1S,W7Q,S11A	25.5	4.3
NV1G1750-NH2	NV1D3586-NH2	325	W7Q,S11A,E12N,M19F,V20 S	4.23	0.33
NV1G1747	NV1D3531	271	W7Q,S11A,E12K,M19F,R22 T,K26R	13	2.1
NV1G1763	NV1D3540	280	W7Q,S11R,E12N,M19F,R22 T,K26R	16	1.5
NV1G1739	NV1D3582	320	W7Q,S11A,E12R,M19F,V20 S	17.8	2.2
NV1G1750	NV1D3586	324	W7Q, S11A, E12N, M19F, V20S	20.5	2.2
NV1G1718	NV1D3571	309	W7Q,S11A,E12R,M19F,R22 T	21	2.3
NV1G1865	NV1D3560	299	W7Q,S11A,E12N,M19F,V20 S,R22T	27.2	3.42
NV1G1766	NV1D3549	289	W7Q,S11R,M19F,R22T,K26 R	27.5	3.2
NV1G961	NV1D2676	29	W7Q,S11A	26.5	2.9
NV1G951	NV1D2674	18	Y1A,S11A	4.03	0.2
NV1G1011	NV1D2740	37	Y1Q,S11A,M19F	3.62	9.9
NV1G977	NV1D2665	22	Y1Q,M19F	4.9	0.4
NV1G949	NV1D2675	21	Y1Q,S11A	4.33	0.3
NV1G973	NV1D2662	25	Y1R,M19F	4.03	0.4
NV1G965	NV1D2672	24	Y1R,S11A	4.5	0.3
NV1G1009	NV1D2738	45	Y1S,S11A,M19F	2.57	0.2
NV1G995	NV1D2663	28	Y1S,M19F	4.19	0.4
NV1G1107-NH2	NV1D2890-NH2	112	Y1S,M6F,S11A,M19L	9.12	1.17
NV1G971	NV1D2673	27	Y1S,S11A	4.31	0.5
NV1G1782	NV1D3383	212	Y1Q,W7Q,S11R,E17R,M19F ,R22T,K26R,	30.3	4.06
NV1G1990	NV1D3788	332	Y1Q,W7Q,S11R,M19F,R22T ,K26R,	30.3	4.78
(-GP) N-Ac-NV1G1153-	(-GP) N-Ac-NV1D3034	120	Y1Q,W7Q,S11R,M19F,R22T ,K26R	30.4	2.96
NV1G1786	NV1D3389	218	Y1Q,W7Q,S11R,E17S,M19F ,R22T,K26R,	30.8	4.48
NV1G1147	NV1D2969	124	Y1S, W7Q, S11A, M19F, V20S	31	6.15
NV1G1764	NV1D3554	294	Y1A,W7Q,S11R,M19F,V20S ,R22T,K26R	31.4	3.3
NV1G963	NV1D2671	20	Y1Q,W7Q	31.5	6.4
NV1G1835	NV1D3379	208	Y1Q,K4D,W7Q,S11R,M19F, R22T,K26R	31.6	2.88
NV1G1231	NV1D3035	148	Y1Q,W7Q,S11A,E12N,M19 F,R22T,K26R	32	4.9
NV1G1743	NV1D3564	303	W7Q,S11A,E12R,M19F,V2 0S,R22T	32.3	3.1
NV1G1960	NV1D3803	345	Y1Q,W7Q,S11R,M19F,R22 T,K26R	32.3	5.33
NV1G1924	NV1D3470	250	Y1Q,W7Q,S11R,M19L,R22 T,K26R	32.5	403
NV1G1756	NV1D3575	313	W7Q,S11A,E12N,M19F,R22 T	33.2	3.9
NV1G1109	NV1D2899	67	Y1S,W7Q,S11A,M19L	33.3	6.7
NV1G1818	NV1D3368	122	Y1Q,W7Q,S11R,E12T,M19 F,R22T,K26R	33.5	10.7
NV1G1784	NV1D3386	215	Y1Q,W7Q,S11R,E17A,M19 F,R22T,K26R	33.6	4.71
NV1G1141	NV1D2972	127	Y1Q,W7Q,S11A,M19F,V20 S	34.1	6.2
NV1G1774	NV1D3347	179	Y1Q,K4T,W7Q,S11R,M19F, R22T,K26R	34.2	5.99
NV1G1881	NV1D3257	167	Y1Q,W7Q,S11A,M19F	34.2	2.81
NV1G1915	NV1D3467	249	Y1Q,W7Q,S11R,E17G,M19 F,R22T,K26R	34.5	4
NV1G1984	NV1D3806	348	Y1Q,W7Q,S11R,M19F,R22 T,K26R	35.1	4.56
NV1G1716	NV1D3561	300	Y1A,W7Q,S11A,E12N,M19F ,V20S,R22T,	35.6	5
NV1G1255	NV1D3014	137	Y1Q,W7Q,S11R,M19F,R22T	36.1	5.37
NV1G1959	NV1D3818	357	Y1Q,W7Q,S11R,M19F,R22 T,K26R	36.3	204
NV1G1825	NV1D3377	206	Y1Q,W7Q,S11R,K14T,M19 F,R22T,K26R	36.4	4.83
NV1G1723	NV1D3536	276	W7Q,S11A,E12K,M19F,V20 S,R22T,K26R	37	5.4
NV1G1732	NV1D3555	295	Y1R,W7Q,S11A,M19F, V20S, R22T,	37.4	4.3
NV1G1983	NV1D3809	350	Y1Q,W7Q,S11R,M19F,R22 T,K26R	38.9	4.81
NV1G1982	NV1D3805	347	Y1Q,W7Q,S11R,M19F,R22 T,K26R	41.2	5.44
NV1G1785	NV1D3385	214	Y1Q,W7Q,S11R,E17T,M19 F,R22T,K26R	41.5	6.5
NV1G1583	NV1D3030	144	Y1Q,W7Q,S11R,E12N,M19 F,R22T	41.9	5.15
NV1G1729	NV1D3545	285	W7Q,S11R,E12N,M19F,V20 S,R22T,K26R	42.8	4.6
NV1G1007	NV1D2775	56	Y1Q,W7Q,S11A,M19F	42.9	6.7
NV1G1734	NV1D3568	306	Q1A,W7Q,S11A,M19F,R22T	44	8.3
NV1G1683	NV1D3523	263	Y1Q,W7Q,S11R,M19F,R22 T,K26R	44.7
NV1G1834	NV1D3360	191	Y1Q,W7Q,D10R,S11R,M19 F,R22T,K26R	45.2	3.79
NV1G1795	NV1D3401	229	Y1Q,W7Q,S11R,M19F,R22 T,K26R,K27R	45.5	6.58
NV1G1689	NV1D3514	255	Y1Q,W7Q,S11R,M19F,R22 T,K26R	46.4
NV1G2043	NV1D3835	370	Y1Q,W7Q,S11R,M19F,R22 T,K26R	46.4	4.09
NV1G1783	NV1D3384	213	Y1Q,W7Q,S11R,E17K,M19 F,R22T,K26R	46.8	7.39
NV1G1239	NV1D3020	143	Y1Q,W7Q,S11A,M19F,R22 T,K26R	47.2	7.84
NV1G1788	NV1D3399	227	Y1Q,W7Q,S11R,M19F,V20 T,R22T,K26R	47.3	6.36
NV1G899	NV1D2774	52	Y1A,W7Q,S11A,M19F	50.5	15.2
NV1G2057	NV1D3799	341	Y1Q,W7Q,S11R,M19F,R22 T,K26R	50.6	6.33
NV1G1738	NV1D3578	316	W7Q,S11A,M19F,V20S,	50.7	5.7
NV1G1713	NV1D3525	265	Y1Q,W7Q,S11R,M19F,R22 T,K26R	52.3
NV1G1765	NV1D3553	293	W7Q,S11R,M19F,V20S,R22 T,K26R	52.4	10
NV1G1916	NV1D3465	247	Y1Q,W5F,W7Q,S11R,M19F ,R22T,K26R	52.8	10.3
NV1G1977	NV1D3804	346	Y1Q,W7Q,S11R,M19F,R22 T,K26R	53.6	6.27
NV1G1879	NV1D3259	168	Y1Q,W7Q,S11A,M19F	54.9	7.62
NV1G1884	NV1D3256	166	Y1Q,W7Q,S11A,M19F	55.7	10.5
NV1G1986	NV1D3819	358	Y1Q,W7Q,S11R,M19F,R22 T,K26R	56	6.57
NV1G1633	NV1D3251	163	Y1Q,W7Q,S11A,M19F	56.1	13.9
NV1G1880	NV1D3261	170	Y1Q,W7Q,S11A,M19F	57	6.25
NV1G1985	NV1D3808	349	Y1Q,W7Q,S11R,M19F,R22 T,K26R	57	6.74
NV1G1849	NV1D3400	228	Y1Q,W7Q,S11R,M19F,V20 Q,R22T,K26R	57.3	9.52
NV1G1883	NV1D3260	169	Y1Q,W7Q,S11A,M19F	57.6	6.91
NV1G1145	NV1D2970	125	Y1S,W7Q,S11A,M19F,R22T	58	18.8
NV1G1697	NV1D3517	258	Y1Q,W7Q,S11R,M19F,R22 T,K26R	58.5
NV1G1737	NV1D3579	317	Y1A,W7Q,S11A,M19F,V20S	59.9	9.6
NV1G1978	NV1D3833	368	Y1Q,W7Q,S11R,M19F,R22 T,K26R	60.3	9.57
NV1G1954	NV1D3800	342	Y1Q,W7Q,S11R,M19F,R22 T,K26R	60.9	6.43
NV1G1989	NV1D3791	334	Y1Q,W7Q,S11R,M19F,R22 T,K26R	61.8	8.66
NV1G1815	NV1D3380	209	Y1Q,K4E,W7Q,S11R,M19F, R22T,K26R	64	10.5
NV1G1967	NV1D3793	336	Y1Q,W7Q,S11R,M19F,R22 T,K26R	64.6	8.19
NV1G1869	NV1D3573	311	Y1R,W7Q,S11A,E12N,M19 F,R22T	64.7	50.7
NV1G1872	NV1D3777	330	Y1Q,W7Q,S11R,M19F,R22 T,K26R	64.9	15.3
NV1G1979	NV1D3834	369	Y1Q,W7Q,S11R,M19F,R22 T,K26R	65.5	7.59
NV1G1827	NV1D3365	195	Y1Q,W7Q,D10Q,S11R,M19 F,R22T,K26R	66.1	10.1
NV1G1768	NV1D3341	174	Y1Q,Q3T,W7Q,S11R,M19F, R22T,K26R	66.2	9.32
NV1G911	NV1D2666	30	W7Q,M19F	66.5	36.7
NV1G1856	NV1D3397	225	Y1Q,W7Q,S11R,G18S,M19 F,R22T,K26R	66.7	7.31
NV1G1973	NV1D3810	351	Y1Q,W7Q,S11R,M19F,R22 T,K26R	66.9	7.04
NV1G1855	NV1D3398	226	Y1Q,W7Q,S11R,M19F,V20 S,R22T,K26R	67.3	11
NV1G1961	NV1D3802	344	Y1Q,W7Q,S11R,M19F,R22 T,K26R	68	8.23
NV1G1846	NV1D3431	244	Y1Q,K4E,W7Q,S11R,E17K, M19F,R22T,K26R	68.6	13.9
NV1G1771	NV1D3348	180	Y1Q,K4A,W7Q,S11R,M19F, R22T,K26R	70.6	15.9
NV1G1691	NV1D3520	261	Y1Q,W7Q,S11R,M19F,R22	71.4
	T,K26R
NV1G1681	NV1D3511	252	Y1Q,W7Q,S11R,M19F,R22 T,K26R	71.5
NV1G1968	NV1D3822	359	Y1Q,W7Q,S11R,M19F,R22 T,K26R	74.2	11.1
NV1G1813	NV1D3424	238	Y1Q,W7Q,D10K,S11R,E12K ,M19F,R22T,K26R	75.2	12.2
NV1G1067	NV1D2893	57	Y1Q,W7Q,S11A,M19L	75.5	10.5
NV1G1867	NV1D3546	286	Y1A,W7Q,S11R,E12N,M19 F,V20S,R22T,K26R	76	17.6
NV1G1143	NV1D2971	126	Y1S,W7Q,S11A,M19F,V20S ,R22T	77.5	22.1
NV1G1806	NV1D3409	232	Y1Q,W7Q,S11R,M19F,R22 T,K26R,K28T	79.1	11.3
NV1G1061	NV1D2896	60	Y1R,W7Q,S11A,M19L	80.3	7.13
NV1G1793	NV1D3419	236	Y1Q,W7Q,S11R,M19F,R22 T,K26R,W30D	80.9	11.9
NV1G1613	NV1D3057	157	Y1Q,W7Q,S11R,E12K,M19 F,K26R	83.4	16.6
NV1G1585	NV1D3052	155	Y1Q,W7Q,S11A, E12K,M19F,K26R	84.8	28.8
NV1G1707	NV1D3524	264	Y1Q,W7Q,S11R,M19F,R22 T,K26R	84.9
NV1G1773	NV1D3350	182	Y1Q,K4E,W7Q,S11R,M19F, R22T,K26R	85.6	14.4
NV1G1949	NV1D3828	364	Y1Q,W7Q,S11R,M19F,R22 T,K26R	87.5	11
NV1G1976	NV1D3811	352	Y1Q,W7Q,S11R,M19F,R22 T,K26R	87.7	15.7
NV1G1956	NV1D3801	343	Y1Q,W7Q,S11R,M19F,R22 T,K26R	88.1	11.4
NV1G1975	NV1D3832	367	Y1Q,W7Q,S11R,M19F,R22 T,K26R	88.4	12.3
NV1G1839	NV1D3774	328	Y1Q,W7Q,S11R,M19F,R22 T,K26R	88.6	19.6
NV1G1971	NV1D3830	366	Y1Q,W7Q,S11R,M19F,R22 T,K26R	88.6	9.88
NV1G1882	NV1D3262	171	Y1Q,W7Q,S11A,M19F	89.2	8.32
NV1G1950	NV1D3797	339	Y1Q,W7Q,S11R,M19F,R22 T,K26R	91.1	13.5
NV1G1828	NV1D3363	194	Y1Q,W7Q,D10A,S11R,M19 F,R22T,K26R	93.1	15.3
NV1G1139	NV1D2973	128	Y1Q,W7Q,S11A,M19F,R22 T	93.9	19.5
NV1G1842	NV1D3430	243	Y1Q,K4D,W7Q,S11R,E17K, M19F,R22T,K26R	93.9	14.1
NV1G1948	NV1D3798	340	Y1Q,W7Q,S11R,M19F,R22 T,K26R	94.5	17.8
NV1G1807	NV1D3408	231	Y1Q,W7Q,S11R,M19F,R22 T,K26R,K28R	94.8	17.8
NV1G1137	NV1D2974	129	Y1Q,W7Q,S11A,M19F,V20 S,R22T	95.7	16.2
NV1G1843	NV1D3432	245	Y1Q,K4E,W7Q,S11R,E17R, M19F,R22T,K26R	95.9	10.4
NV1G1822	NV1D3423	237	Y1Q,W7Q,D10R,S11R,E12R ,M19F,R22T,K26R	99.5	9.45
NV1G1862	NV1D3556	296	W7Q,S11A,M19F,V20S,R22 T	100	18.5
NV1G1969	NV1D3795	337	Y1Q,W7Q,S11R,M19F,R22 T,K26R	100	14.5
NV1G1980	NV1D3812	353	Y1Q,W7Q,S11R,M19F,R22 T,K26R	101	23.6
NV1G1850	NV1D3414	235	Y1Q,W7Q,S11R,M19F,R22 T,K26R,K28S	102	19.4
NV1G1981	NV1D3815	356	Y1Q,W7Q,S11R,M19F,R22 T,K26R	102	13.5
NV1G1851	NV1D3390	219	Y1Q,W7Q,S11R,G18R,M19 F,R22T,K26R	108	15.5
NV1G1922	NV1D3466	248	Y1Q,W7Q,S11E,M19F,R22 T,K26R	108	922
NV1G1778	NV1D3349	181	Y1Q,K4D,W7Q,S11R,M19F, R22T,K26R	109	16
NV1G1972	NV1D3824	361	Y1Q,W7Q,S11R,M19F,R22 T,K26R	110	16.1
NV1G1974	NV1D3796	338	Y1Q,W7Q,S11R,M19F,R22 T,K26R	110	19.6
NV1G1826	NV1D3357	188	Y1Q,W7Q,T8E,S11R,M19F, R22T,K26R	111	15.1
NV1G1892	NV1D3439	246	Y1Q,W7Q,S11R,M19F,R22 T,K26R,W30G	112	13.2
NV1G1819	NV1D3375	204	Y1Q,W7Q,S11R,R13S,M19 F,R22T,K26R	113	1270
NV1G1805	NV1D3410	233	Y1Q,W7Q,S11R,M19F,R22 T,K26R,K28A	114	21.5
NV1G1831	NV1D3374	203	Y1Q,W7Q,S11R,R13Q,M19 F,R22T,K26R	114	1600
NV1G1693	NV1D3512	253	Y1Q,W7Q,S11R,M19F,R22 T,K26R	115.6
NV1G1854	NV1D3392	221	Y1Q,W7Q,S11R,G18T,M19 F,R22T,K26R	117	21.8
NV1G1951	NV1D3829	365	Y1Q,W7Q,S11R,M19F,R22 T,K26R	122	13.3
NV1G1860	NV1D3393	222	Y1Q,W7Q,S11R,G18A,M19 F,R22T,K26R	125	24.8
NV1G1099	NV1D2732	36	Y1Q,W7Q,S11A	126	26.9
NV1G1705	NV1D3513	254	Y1Q,W7Q,S11R,M19F,R22 T,K26R	131.2
NV1G1848	NV1D3426	240	Y1Q,W7Q,D10K,S11R,E12K ,R13D,M19F,R22T,K26R	135	39.9
NV1G1952	NV1D3813	354	Y1Q,W7Q,S11R,M19F,R22 T,K26R	139	30.1
NV1G1631	NV1D3252	164	Y1Q,W7Q,S11A,M19F	145	53
NV1G1817	NV1D3371	201	Y1Q,W7Q,S11R,R13A,M19 F,R22T,K26R	151	33.7
NV1G1789	NV1D3394	223	Y1Q,W7Q,S11R,G18D,M19 F,R22T,K26R	155	41.4
NV1G1852	NV1D3391	220	Y1Q,W7Q,S11R,G18K,M19 F,R22T,K26R	157	23.1
NV1G1709	NV1D3510	251	Y1Q,W7Q,S11R,M19F,R22 T,K26R	159
NV1G1840	NV1D3425	239	Y1Q,W7Q,D10R,S11R, E12R, R13D,M19F,R22T,K26R	161	27.9
NV1G1809	NV1D3413	234	Y1Q,W7Q,S11R,M19F,R22 T,K26R,K28Q	164	43.7
NV1G1863	NV1D3356	187	Y1Q,W7Q,T8D,S11R,M19F, R22T,K26R	167	32.2
NV1G1699	NV1D3527	267	Y1Q,W7Q,S11R,M19F,R22 T,K26R	169.1
NV1G1844	NV1D3428	242	Y1Q,W7Q,D10K,S11R,E12 K,R13E,M19F,R22T,K26R	180	52.4
NV1G1853	NV1D3370	200	Y1Q,W7Q,S11R,R13T,M19 F,R22T,K26R	181	25.1
NV1G1946	NV1D3825	362	Y1Q,W7Q,S11R,M19F,R22 T,K26R	194	28.4

The wild-type Protoxin-II inhibits Navl.7 with an IC₅₀ value of about 4 nM in FLIPR assay as described in Example 3. Variants retaining significant Navl.7 potency were characterized further. Figure 1 shows the sequence genus of generated Protoxin-II variants that inhibit Navl.7 with an IC₅₀ value of 30 nM or less.

Select Protoxin-II variants were tested for their inhibition of Navl.7 and for their selectivity against human Navl.6 using QPatch. IC₅₀ values for both Navl.7 and Navl.6 for select peptides obtained using QPatch are shown in Figure 2. These peptides inhibited Navl.7 with an IC₅₀ of 30 nM or less, and were at least 30-fold selective over Navl.7 when compared to Navl.6.

The amino acid sequences of the peptides shown in Figure 2 are shown in Figure 3. All these peptides had W7Q and M19F substitutions when compared to the wild type Protoxin-II.

The protoxin-II variants were expressed and purified as described in Example 1, or synthesized by standard solid phase synthesis methods. The yields of the recombinant or synthetic peptides were compared to the yields of the wild-type protoxin. Table 12 shows that the yields of the select protoxin-II variants were significantly higher than that of protoxin-II, indicating improved folding properties of the variants. The scale of the solid-phase synthesis was 0.5 mmol.

Table 12.

Peptide	Solid phase synthesis			Recombinant expression
Peptide	Total yield	Yield from Crude	Yield From Linear	% active isomer
Protoxin-II	52 mg	2.7%	7.3%	54.0%
NV1D2775	84 mg	4.5%	18.7%	89.1%
NV1D3034	149 mg	8.0%	21.0%	85.2%
NV1D3368	83 mg	4.0%	24.0%	93.8%

Example 5. Protoxin-II variants are efficient in in vivo models of pain Materials and methods

Animals Male C57Bl/6 mice (24-26g), ordered from Charles River and housed individually, were used for this study.

Behavioral Tests

Von Frey Test: Mechanical (tactile) threshold was assessed by Von Frey Hairs following the Up-Down method (Dixon, 1980, Chaplan et al., 1994). 7 graded stimuli (von Frey filaments: 0.03, 0.07, 0.16, 0.4, 0.6, 1, 2 g; Stoelting, Wood Dale, IL) were used. Von Frey hairs were presented perpendicularly against the center plantar area (between toris) on a hindpaw. Sufficient force was applied to bend the filament slightly and held for 3 seconds. Per the Chaplan paper, a positive response can be either 1) a sharp withdrawal or 2) immediate flinching upon removal of the filament. See Chaplan et al for more details. Mice were acclimated to the wire mesh in the testing chamber for 30-60 minutes prior to testing.

Hargreaves Test: A modified Hargreaves box was used to measure thermal paw withdrawal latency (PWL) (Hargreaves et al., 1988, Pain, 32:77-88; Dirig et al.,1997, J Neurosci. Methods, 76:183-191). This box consists of a chamber with a raised glass floor maintained at a constant temperature (27°C). The thermal nociceptive stimulus originates from a projection bulb light beam below the glass surface. The light beam is aimed at the area between toris (center plantar). The "start" button will turn on the light and start the timer. Movements (such as a sudden withdrawal) of the stimulated paw will trigger the switch to turn off the light and stop the timer. The latency in seconds is displayed. If no movement occurs, the bulb will be turned off after 20 seconds (cutoff) to prevent tissue injury. The animals were allowed to habituate on the glass surface for 30-60 minutes before PWL measurement. Constant amperage was used throughout the study, which resulted in Pre-test paw withdrawal latencies between 8-12 seconds when averaged over 3 to 6 read-outs taken at least 5 minutes apart.

MPE% Calculation: Percent maximum possible effect (MPE%) = (T₁ - T₀) / (Tc - T₀) x 100%. T₀: threshold on day0 (post-CFA, pre-pump); T₁: threshold on dayl post pump implantation; Tc: cut-off of the test (20s for the Hargreaves test and 2g for the Von Frey test) Hotplate Test: Animals were placed on a 10" x 10" metal plate surrounded by 4 Plexiglas walls (15 inches high). The plate was maintained at a temperature of either 50 or 55°C. The response latency (time when the animal first flinches or licks its hind paw, jumps, or vocalizes) was measured and the animal removed from the plate. Animals showing no response were removed from the plate after 40s (50°C) or 20s (55°C) to prevent any possible tissue damage. This trial was repeated 2-5 times every 15-60 minutes in a day.

Inflammatory Pain Models

CFA Model: Animals were anesthetized with isoflurane (4% induction and 2% maintenance) and 20 µL of 100% Complete Freund's Adjuvant (CFA; Sigma-Aldrich; Saint Louis, MO) was injected into the center plantar area on one hind paw using a 27gauge needle attached to a 50µL Hamilton syringe. Carrageenan model: Animals were anesthetized with isoflurane (4% induction and 2% maintenance) and 25 µL of 2% λ-carrageenan (Sigma-Aldrich; Saint Louis, MO) dissolved in normal saline was injected into the center plantar area on hind paws using an insulin syringe (BD; Franklin Lakes, New Jersey).

Implantation of Mini Pumps

Alzet micro-osmotic mini pumps (Durect Corporation Model 1003D and 2001D) were filled and primed per manufacturer's guide. Mice were anesthetized with isoflurane (5% induction; 2% maintenance). Their backs were shaved, wiped down with isopropyl alcohol and povidone iodine, and a small incision was made between the scapulae. Using a pair of forceps or hemostat, a small pocket was formed by spreading the subcutaneous connective tissues apart. The pump was inserted into the pocket with the flow moderator pointing away from the incision. The skin incision was then closed using 7mm staples and the animals were allowed to recover in their home cages.

Data Analysis

Data are represented as mean ± s.e.m. Prism (Graphpad Software Inc., La Jolla, CA) was used for graphing and statistical analysis. For comparison of threshold values over time, a two-way ANOVA followed by Bonferroni's multiple comparison test was used with a significance level of p<0.05. Hotplate and data were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test.

Results

Efficacy of variants NV1D3034-OH (NV1D3034-COOH), NV1D3368-OH (NV1D3368-COOH) and NV1D2775-OH (NV1D2775-COOH) was studied in the CFA model, a commonly used model of inflammatory pain. The injection of CFA in the hindpaw induced paw edema (not shown) and hypersensitivity to thermal stimuli (thermal hyperalgesia), as indicated by the lowered thermal latency in the injected paw on day0 (Figure 6A ). Thermal hyperalgesia was completely reversed by NV1D3034-OH at 684 and 1824µg/day, when administered by a subcutaneous osmotic mini-pump (Figure 4A and 4 B).

NV1D3368-OH fully reversed CFA-induced thermal hyperalgesia at 684 and 1824µg/day (Figure 5A and 5 B). NV1D2775-OH demonstrated strong efficacy in the CFA model. Thermal latencies reached values close to the cut-off following NV1D2775 administration (Figure 6A and 6 B, 1824µg/day), suggesting a strong analgesia effect on top of the anti-hyperalgesia effect. In addition, NV1D2775-OH reversed CFA-induced tactile allodynia (Figure 6C and 6 D, 1824µg/day). The anti-hyperalgesic effect of NV1D2775-OH was seen as early as 4hr post-pump implantation (Figure 7A ). The effect reached the maximum at 8hr in both the thermal and tactile tests (Figure 7A and 7 B), which was maintained at 24hr. Thermal latency and tactile threshold returned the control level by 48h post pump implantation (approximately 24h after the pumps were predicted to be empty) (Figure 7A and 7 B).

CFA-induced thermal hyperalgesia was readily reversed by two additional peptides, NV1D3368-amide (NV1D3368-NH₂) and NV1D3034-N-methylamide (NV1D3034-NHMe). Thermal from the experiments is summarized in Table 13.

Table 13.

Peptide	Dose (µg/day/mouse)
Peptide	Vehicle (PBS)	228	684	1824
NV1D3034-OH	20±7 (11)	22±6 (6)	48±10* (8)	50±6* (8)
NV1D3368-OH	13±7 (8)	23±8 (7)	42±9* (7)	47±6** (8)
NV1D2775-OH	15±4 (20)	35±8 (8)	57±12*** (8)	85±6**** (12)
	15±13 (6)	27±4 (4)	46±9 (4)	55±15 (6)
NV1D3034-NHMe	5±25 (3)			49±17 (6)
P<0.05, P<0.01, P<0.001 and **P<0.0001 vs. PBS, one-way ANOVA followed by Bonferroni's multiple comparison.

NV1D2775-OH also exhibited strong, dose-dependent efficacy in the hotplate test (Figure 8 ). Latencies at 50 and 55°C reached values near cut-off following the administration of 1824µg/day. At 228µg/day, NV1D2775-OH produced a modest yet significant increase in the thermal latency, compared to the PBS control.

The efficacy of NV1D2775-OH was evaluated in another model of inflammatory pain, the carrageenan model. Animals were implanted with NV1D2775-OH or PBS pumps. Thermal withdrawal latencies were measured pre-and on dayl post-pump. λ-carrageenan was injected into the hindpaws and thermal latencies were measured again on 2, 3 and 4hr following carrageenan. NV1D2775-OH at 1824µg/day produced significant analgesia (Figure 9 ). Injection of λ-carrageenan in the hindpaws induced inflammation (not shown) and lowered thermal paw withdrawal latency in the Hargreaves test over the 4hr test-period (Figure 9 , PBS group). Animals pretreated with NV1D2775-OH at 1824µg/day were fully protected from carrageenan-induced hyperalgesia.

SEQUENCE LISTING

<110> Janssen Biotech, Inc.
<120> PROTOXIN-II VARIANTS AND METHODS OF USE
<130> P068172EP
<140> EP14790422.1 <141> 2013-10-03
<150> PCT/US2014/058972 <151> 2013-10-03
<150> 61/886,100 <151> 2013-10-03
<160> 407
<170> PatentIn version 3.5
<210> 1 <211> 30 <212> PRT <213> Thrixopelma pruriens
<220> <221> MOD_RES <222> (30)..(30) <223> -COOH
<400> 1
<210> 2 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D12 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 2
<210> 3 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D748 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 3
<210> 4 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D751 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 4
<210> 5 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2292 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 5
<210> 6 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D750 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 6
<210> 7 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D1328 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 7
<210> 8 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D774 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 8
<210> 9 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D786 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 9
<210> 10 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2300 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 10
<210> 11 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D791 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 11
<210> 12 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D1332 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 12
<210> 13 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2512 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 13
<210> 14 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D1336 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 14
<210> 15 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D1337 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 15
<210> 16 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2308 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 16
<210> 17 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2670 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 17
<210> 18 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2674 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 18
<210> 19 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2664 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 19
<210> 20 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2671 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 20
<210> 21 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2675 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 21
<210> 22 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2665 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 22
<210> 23 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2668 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 23
<210> 24 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2672 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 24
<210> 25 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2662 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 25
<210> 26 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2669 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 26
<210> 27 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2673 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 27
<210> 28 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2663 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 28
<210> 29 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2676 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 29
<210> 30 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2666 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 30
<210> 31 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2816 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 31
<210> 32 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2735 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 32
<210> 33 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2739 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 33
<210> 34 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2731 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 34
<210> 35 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2810 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 35
<210> 36 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2732 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 36
<210> 37 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2740 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 37
<210> 38 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2819 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 38
<210> 39 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2729 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 39
<210> 40 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2733 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 40
<210> 41 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2814 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 41
<210> 42 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2820 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 42
<210> 43 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2730 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 43
<210> 44 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2734 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 44
<210> 45 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2738 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 45
<210> 46 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2851 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 46
<210> 47 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2850 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 47
<210> 48 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2667 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 48
<210> 49 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2867 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 49
<210> 50 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2881 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 50
<210> 51 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2882 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 51
<210> 52 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2774 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 52
<210> 53 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2902 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 53
<210> 54 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2861 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 54
<210> 55 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2870 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 55
<210> 56 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2775 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 56
<210> 57 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2893 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 57
<210> 58 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2887 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 58
<210> 59 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2772 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 59
<210> 60 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2896 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 60
<210> 61 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2877 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 61
<210> 62 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2878 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 62
<210> 63 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2889 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 63
<210> 64 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2889 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 64
<210> 65 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2773 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 65
<210> 66 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2890 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 66
<210> 67 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2899 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 67
<210> 68 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2905 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 68
<210> 69 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2906 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 69
<210> 70 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2921 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 70
<210> 71 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2922 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 71
<210> 72 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2909 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 72
<210> 73 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2910 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 73
<210> 74 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2913 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 74
<210> 75 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2914 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 75
<210> 76 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2917 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 76
<210> 77 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D2918 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 77
<210> 78 <211> 32 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1D3034 polypeptide
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 78
<210> 79 <211> 1977 <212> PRT <213> Homo Sapiens
<400> 79
<210> 80 <211> 20 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic Linker peptide
<400> 80
<210> 81 <211> 30 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic Linker polypeptide
<400> 81
<210> 82 <211> 8 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic HRV3C cleavage site peptide
<400> 82
<210> 83 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1007 (NV1D2775) Y1Q, W7Q, S11A, M19F polypeptide
<400> 83
<210> 84 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1007 (NV1D2775) Y1Q, W7Q, S11A, M19F polynucleotide
<400> 84
<210> 85 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1005 (NV1D2772) Y1R, W7Q, S11A, M19F polypeptide
<400> 85
<210> 86 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1005 (NV1D2772) Y1R, W7Q, S11A, M19F polynucleotide
<400> 86
<210> 87 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1001 (NV1D2773) Y1S, W7Q, S11A, M19F polypeptide
<400> 87
<210> 88 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1001 (NV1D2773) Y1S, W7Q, S11A, M19F polynucleotide
<400> 88
<210> 89 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1079 (NV1D2889) Y1S,M6F,S11A,M19F polypeptide
<400> 89
<210> 90 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1079 (NV1D2889) Y1S,M6F,S11A,M19F polynucleotide
<400> 90
<210> 91 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1107 (NV1D2890) Y1S,M6F,S11A,M19L polypeptide
<400> 91
<210> 92 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1107 (NV1D2890) Y1S,M6F,S11A,M19L polynucleotide
<400> 92
<210> 93 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1063 (NV1D2913) Y1R,M6F,W7Q,S11A,M19F polypeptide
<400> 93
<210> 94 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1063 (NV1D2913) Y1R,M6F,W7Q,S11A,M19F polynucleotide
<400> 94
<210> 95 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1097 (NV1D2810), Y1Q,M6F,W7Q polypeptide
<400> 95
<210> 96 <211> 1959 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1097 (NV1D2810), Y1Q,M6F,W7Q polynucleotide
<400> 96
<210> 97 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1123 (NV1D2882), Y1A,M6F,W7Q,M19L polypeptide
<400> 97
<210> 98 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1123 (NV1D2882), Y1A,M6F,W7Q,M19L polynucleotide
<400> 98
<210> 99 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1127 (NV1D2870), Y1Q,M6F,W7Q,M19L polypeptide
<400> 99
<210> 100 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1127 (NV1D2870), Y1Q,M6F,W7Q,M19L polynucleotide
<400> 100
<210> 101 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1083 (NV1D2878), Y1S,M6F,W7Q,M19L polypeptide
<400> 101
<210> 102 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1083 (NV1D2878), Y1S,M6F,W7Q,M19L polynucleotide
<400> 102
<210> 103 <211> 672 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1153 (NV1D3034) fusion protein
<400> 103
<210> 104 <211> 2019 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic NV1G1153 (NV1D3034) fusion polynucleotide
<400> 104
<210> 105 <211> 2016 <212> PRT <213> Homo sapiens
<400> 105
<210> 106 <211> 612 <212> PRT <213> Homo sapiens
<400> 106
<210> 107 <211> 93 <212> DNA <213> Thrixopelma pruriens
<400> 107
<210> 108 <211> 6 <212> PRT <213> Artificial Sequence
<220> <223> Description of Artificial Sequence: Synthetic 6xHis tag
<400> 108
<210> 109 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> variant (-GP) NV1D2773
<220> <221> MOD_RES <222> (30)..(30) <223> -COOH
<400> 109
<210> 110 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> Variant (-GP) NV1D2773-NH2
<220> <221> MOD_RES <222> (30)..(30) <223> -NH2
<400> 110
<210> 111 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2775-NH2
<220> <221> MOD_RES <222> (32)..(32) <223> -NH2
<400> 111
<210> 112 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2890-NH2
<220> <221> MOD_RES <222> (30)..(30) <223> -NH2
<400> 112
<210> 113 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2974
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 113
<210> 114 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> Variant (-GP) N-Ac-NV1D2974-NH2
<220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION
<220> <221> MOD_RES <222> (30)..(30) <223> -NH2
<400> 114
<210> 115 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> Variant (-GP) N-Ac-NV1D2974
<220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION
<220> <221> MOD_RES <222> (30)..(30) <223> -NH2
<400> 115
<210> 116 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3034
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 116
<210> 117 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3034-NH2
<220> <221> MOD_RES <222> (32)..(32) <223> -NH2
<400> 117
<210> 118 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> NV1D3034-NH-butyl
<220> <221> MOD_RES <222> (32)..(32) <223> NH-CH2-CH2-CH2-CH3
<400> 118
<210> 119 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3034-NH-methyl
<220> <221> MOD_RES <222> (32)..(32) <223> -NH-CH3
<400> 119
<210> 120 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> Variant (-GP) N-Ac-NV1D3034
<220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION
<220> <221> MOD_RES <222> (30)..(30) <223> -COOH
<400> 120
<210> 121 <211> 30 <212> PRT <213> Artificial sequence
<220> <223> Variant (-GP) N-Ac-NV1D3034-NH2
<220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION
<220> <221> MOD_RES <222> (30)..(30) <223> -NH2
<400> 121
<210> 122 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3368
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 122
<210> 123 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3368-NH2
<220> <221> MOD_RES <222> (32)..(32) <223> -NH2
<400> 123
<210> 124 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2969
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 124
<210> 125 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2970
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 125
<210> 126 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2971
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 126
<210> 127 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2972
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 127
<210> 128 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2973
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 128
<210> 129 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2974
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 129
<210> 130 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D2974-NH2
<220> <221> MOD_RES <222> (32)..(32) <223> -NH2
<400> 130
<210> 131 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3004
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 131
<210> 132 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3005
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 132
<210> 133 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3006
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 133
<210> 134 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3007
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 134
<210> 135 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3012
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 135
<210> 136 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3013
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 136
<210> 137 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3014
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 137
<210> 138 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3015
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 138
<210> 139 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3016
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 139
<210> 140 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3017
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 140
<210> 141 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3018
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 141
<210> 142 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3019
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 142
<210> 143 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3020
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 143
<210> 144 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3030
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 144
<210> 145 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3031
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 145
<210> 146 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3032
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 146
<210> 147 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3033
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 147
<210> 148 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3035
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 148
<210> 149 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3036
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 149
<210> 150 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3044
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 150
<210> 151 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3045
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 151
<210> 152 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3048
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 152
<210> 153 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3050
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 153
<210> 154 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3051
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 154
<210> 155 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3052
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 155
<210> 156 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3056
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 156
<210> 157 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3057
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 157
<210> 158 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3058
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 158
<210> 159 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3062
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 159
<210> 160 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3109
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 160
<210> 161 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3121
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 161
<210> 162 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3249
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 162
<210> 163 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3251
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 163
<210> 164 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3252
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 164
<210> 165 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3254
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 165
<210> 166 <211> 64 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3256
<220> <221> MOD_RES <222> (64)..(64) <223> -COOH
<400> 166
<210> 167 <211> 62 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3257
<220> <221> MOD_RES <222> (62)..(62) <223> -COOH
<400> 167
<210> 168 <211> 74 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3259
<220> <221> MOD_RES <222> (74)..(74) <223> -COOH
<400> 168
<210> 169 <211> 74 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3260
<220> <221> MOD_RES <222> (74)..(74) <223> -COOH
<400> 169
<210> 170 <211> 72 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3261
<220> <221> MOD_RES <222> (72)..(72) <223> -COOH
<400> 170
<210> 171 <211> 72 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3262
<220> <221> MOD_RES <222> (72)..(72) <223> -COOH
<400> 171
<210> 172 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3339
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 172
<210> 173 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3340
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 173
<210> 174 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3341
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 174
<210> 175 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3342
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 175
<210> 176 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3344
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 176
<210> 177 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3345
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 177
<210> 178 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3346
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 178
<210> 179 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3347
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 179
<210> 180 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3348
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 180
<210> 181 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3349
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 181
<211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3350
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 182
<210> 183 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3351
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 183
<210> 184 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3352
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 184
<210> 185 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3353
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 185
<210> 186 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3354
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 186
<210> 187 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3356
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 187
<210> 188 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3357
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 188
<210> 189 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3358
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 189
<210> 190 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3359
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 190
<210> 191 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3360
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 191
<210> 192 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3361
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 192
<210> 193 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3362
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 193
<210> 194 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3363
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 194
<210> 195 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3365
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 195
<210> 196 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3366
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 196
<210> 197 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3367
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 197
<210> 198 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3368
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 198
<210> 199 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3369
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 199
<210> 200 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3370
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 200
<210> 201 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3371
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 201
<210> 202 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3372
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 202
<211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3374
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 203
<210> 204 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3375
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 204
<210> 205 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3376
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 205
<210> 206 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3377
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 206
<210> 207 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3378
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 207
<210> 208 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3379
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 208
<210> 209 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3380
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 209
<210> 210 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3381
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 210
<210> 211 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3382
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 211
<210> 212 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3383
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 212
<210> 213 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3384
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 213
<210> 214 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3385
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 214
<210> 215 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3386
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 215
<210> 216 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3387
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 216
<210> 217 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3388
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 217
<210> 218 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3389
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 218
<210> 219 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3390
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 219
<210> 220 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3391
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 220
<210> 221 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3392
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 221
<210> 222 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3393
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 222
<210> 223 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3394
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 223
<210> 224 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3396
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 224
<210> 225 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3397
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 225
<210> 226 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3398
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 226
<210> 227 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3399
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 227
<210> 228 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3400
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 228
<210> 229 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3401
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 229
<210> 230 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3403
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 230
<210> 231 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3408
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 231
<210> 232 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3409
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 232
<210> 233 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3410
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 233
<210> 234 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3413
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 234
<210> 235 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3414
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 235
<210> 236 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3419
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 236
<210> 237 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3423
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 237
<210> 238 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3424
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 238
<210> 239 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3425
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 239
<210> 240 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3426
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 240
<210> 241 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3427
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 241
<210> 242 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3428
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 242
<210> 243 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3430
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 243
<210> 244 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3431
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 244
<210> 245 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3432
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 245
<210> 246 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3439
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 246
<210> 247 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3465
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 247
<210> 248 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3466
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 248
<210> 249 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3467
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 249
<210> 250 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3470
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 250
<210> 251 <211> 44 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3510
<220> <221> MOD_RES <222> (44)..(44) <223> -COOH
<400> 251
<210> 252 <211> 39 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3511
<220> <221> MOD_RES <222> (39)..(39) <223> -COOH
<400> 252
<210> 253 <211> 49 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3512
<220> <221> MOD_RES <222> (49).. (49) <223> -COOH
<400> 253
<210> 254 <211> 44 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3513
<220> <221> MOD_RES <222> (44)..(44) <223> -COOH
<400> 254
<210> 255 <211> 39 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3514
<220> <221> MOD_RES <222> (39)..(39) <223> -COOH
<400> 255
<210> 256 <211> 49 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3515
<220> <221> MOD_RES <222> (49).. (49) <223> -COOH
<400> 256
<210> 257 <211> 44 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3516
<220> <221> MOD_RES <222> (44)..(44) <223> -COOH
<400> 257
<210> 258 <211> 39 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3517
<220> <221> MOD_RES <222> (39)..(39) <223> -COOH
<400> 258
<210> 259 <211> 58 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3518
<220> <221> MOD_RES <222> (58)..(58) <223> -COOH
<400> 259
<210> 260 <211> 53 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3519
<220> <221> MOD_RES <222> (53)..(53) <223> -COOH
<400> 260
<210> 261 <211> 48 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3520
<220> <221> MOD_RES <222> (48)..(48) <223> -COOH
<400> 261
<210> 262 <211> 53 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3521
<220> <221> MOD_RES <222> (53)..(53) <223> -COOH
<400> 262
<210> 263 <211> 43 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3523
<220> <221> MOD_RES <222> (43)..(43) <223> -COOH
<400> 263
<210> 264 <211> 53 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3524
<220> <221> MOD_RES <222> (53)..(53) <223> -COOH
<400> 264
<210> 265 <211> 48 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3525
<220> <221> MOD_RES <222> (48)..(48) <223> -COOH
<400> 265
<210> 266 <211> 43 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3526
<220> <221> MOD_RES <222> (43)..(43) <223> -COOH
<400> 266
<210> 267 <211> 42 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3527
<220> <221> MOD_RES <222> (42)..(42) <223> -COOH
<400> 267
<210> 268 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3528
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 268
<210> 269 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3529
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 269
<210> 270 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3530
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 270
<210> 271 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3531
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 271
<210> 272 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3532
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 272
<210> 273 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3533
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 273
<210> 274 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3534
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 274
<210> 275 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3535
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 275
<210> 276 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3536
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 276
<210> 277 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3537
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 277
<210> 278 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3538
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 278
<210> 279 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3539
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 279
<210> 280 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3540
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 280
<210> 281 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3541
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 281
<210> 282 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3542
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 282
<210> 283 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3543
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 283
<210> 284 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3544
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 284
<210> 285 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3545
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 285
<210> 286 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3546
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 286
<210> 287 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3547
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 287
<210> 288 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3548
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 288
<210> 289 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3549
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 289
<210> 290 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3550
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 290
<210> 291 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3551
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 291
<210> 292 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3552
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 292
<210> 293 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3553
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 293
<210> 294 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3554
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 294
<210> 295 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3555
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 295
<210> 296 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3556
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 296
<210> 297 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3558
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 297
<210> 298 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3559
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 298
<210> 299 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3560
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 299
<210> 300 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3561
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 300
<210> 301 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> variant NV1D3562
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 301
<210> 302 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3563
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 302
<210> 303 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3564
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 303
<210> 304 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3565
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 304
<210> 305 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3566
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 305
<210> 306 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3568
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 306
<210> 307 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3569
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 307
<210> 308 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3570
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 308
<210> 309 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3571
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 309
<210> 310 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> variant NV1D3572
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 310
<210> 311 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3573
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 311
<210> 312 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3574
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 312
<210> 313 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3575
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 313
<210> 314 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3576
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 314
<210> 315 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3577
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 315
<210> 316 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3578
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 316
<210> 317 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3579
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 317
<210> 318 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3580
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 318
<210> 319 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3581
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 319
<210> 320 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3582
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 320
<210> 321 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3583
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 321
<210> 322 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3584
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 322
<210> 323 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3585
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 323
<210> 324 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3586
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 324
<210> 325 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3586-NH2
<220> <221> MOD_RES <222> (32)..(32) <223> -NH2
<400> 325
<210> 326 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3587
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 326
<210> 327 <211> 48 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3772
<220> <221> MOD_RES <222> (48)..(48) <223> -COOH
<400> 327
<210> 328 <211> 68 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3774
<220> <221> MOD_RES <222> (68)..(68) <223> -COOH
<400> 328
<210> 329 <211> 58 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3775
<220> <221> MOD_RES <222> (58)..(58) <223> -COOH
<400> 329
<210> 330 <211> 48 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3777
<220> <221> MOD_RES <222> (48)..(48) <223> -COOH
<400> 330
<210> 331 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3782
<220> <221> MOD_RES <222> (32)..(32) <223> -COOH
<400> 331
<210> 332 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3788
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 332
<210> 333 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3789
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 333
<210> 334 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3791
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 334
<210> 335 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3792
<220> <221> MOD_RES <222> (37)..(37) <223> -COOH
<400> 335
<210> 336 <211> 74 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3793
<220> <221> MOD_RES <222> (74)..(74) <223> -COOH
<400> 336
<210> 337 <211> 72 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3795
<220> <221> MOD_RES <222> (72)..(72) <223> -COOH
<400> 337
<210> 338 <211> 62 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3796
<220> <221> MOD_RES <222> (62)..(62) <223> -COOH
<400> 338
<210> 339 <211> 72 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3797
<220> <221> MOD_RES <222> (72)..(72) <223> -COOH
<400> 339
<210> 340 <211> 74 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3798
<220> <221> MOD_RES <222> (74)..(74) <223> -COOH
<400> 340
<210> 341 <211> 62 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3799
<220> <221> MOD_RES <222> (62)..(62) <223> -COOH
<400> 341
<210> 342 <211> 64 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3800
<220> <221> MOD_RES <222> (64)..(64) <223> -COOH
<400> 342
<210> 343 <211> 49 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3801
<220> <221> MOD_RES <222> (49).. (49) <223> -COOH
<400> 343
<210> 344 <211> 44 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3802
<220> <221> MOD_RES <222> (44)..(44) <223> -COOH
<400> 344
<210> 345 <211> 39 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3803
<220> <221> MOD_RES <222> (39)..(39) <223> -COOH
<400> 345
<210> 346 <211> 49 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3804
<220> <221> MOD_RES <222> (49).. (49) <223> -COOH
<400> 346
<210> 347 <211> 44 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3805
<220> <221> MOD_RES <222> (44)..(44) <223> -COOH
<400> 347
<210> 348 <211> 39 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3806
<220> <221> MOD_RES <222> (39)..(39) <223> -COOH
<400> 348
<210> 349 <211> 44 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3808
<220> <221> MOD_RES <222> (44)..(44) <223> -COOH
<400> 349
<210> 350 <211> 39 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3809
<220> <221> MOD_RES <222> (39)..(39) <223> -COOH
<400> 350
<210> 351 <211> 58 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3810
<220> <221> MOD_RES <222> (58)..(58) <223> -COOH
<400> 351
<210> 352 <211> 53 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3811
<220> <221> MOD_RES <222> (53)..(53) <223> -COOH
<400> 352
<210> 353 <211> 48 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3812
<220> <221> MOD_RES <222> (48)..(48) <223> -COOH
<400> 353
<210> 354 <211> 53 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3813
<220> <221> MOD_RES <222> (53)..(53) <223> -COOH
<400> 354
<210> 355 <211> 48 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3814
<220> <221> MOD_RES <222> (48)..(48) <223> -COOH
<400> 355
<210> 356 <211> 43 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3815
<220> <221> MOD_RES <222> (43)..(43) <223> -COOH
<400> 356
<210> 357 <211> 43 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3818
<220> <221> MOD_RES <222> (43)..(43) <223> -COOH
<400> 357
<210> 358 <211> 42 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3819
<220> <221> MOD_RES <222> (42)..(42) <223> -COOH
<400> 358
<210> 359 <211> 107 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3822
<220> <221> MOD_RES <222> (107)..(107) <223> -COOH
<400> 359
<210> 360 <211> 107 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3823
<220> <221> MOD_RES <222> (107)..(107) <223> -COOH
<400> 360
<210> 361 <211> 107 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3824
<220> <221> MOD_RES <222> (107)..(107) <223> -COOH
<400> 361
<210> 362 <211> 107 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3825
<220> <221> MOD_RES <222> (107)..(107) <223> -COOH
<400> 362
<210> 363 <211> 107 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3826
<220> <221> MOD_RES <222> (107)..(107) <223> -COOH
<400> 363
<210> 364 <211> 84 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3828
<220> <221> MOD_RES <222> (84)..(84) <223> -COOH
<400> 364
<210> 365 <211> 82 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3829
<220> <221> MOD_RES <222> (82)..(82) <223> -COOH
<400> 365
<210> 366 <211> 84 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3830
<220> <221> MOD_RES <222> (84)..(84) <223> -COOH
<400> 366
<210> 367 <211> 61 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3832
<220> <221> MOD_RES <222> (61)..(61) <223> -COOH
<400> 367
<210> 368 <211> 51 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3833
<220> <221> MOD_RES <222> (51)..(51) <223> -COOH
<400> 368
<210> 369 <211> 46 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3834
<220> <221> MOD_RES <222> (46).. (46) <223> -COOH
<400> 369
<210> 370 <211> 41 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3835
<220> <221> MOD_RES <222> (41)..(41) <223> -COOH
<400> 370
<210> 371 <211> 46 <212> PRT <213> Artificial sequence
<220> <223> Variant NV1D3838
<220> <221> MOD_RES <222> (46).. (46) <223> -COOH
<400> 371
<210> 372 <211> 7 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 372
<210> 373 <211> 7 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 373
<210> 374 <211> 6 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 374
<210> 375 <211> 18 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 375
<210> 376 <211> 9 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 376
<210> 377 <211> 9 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 377
<210> 378 <211> 9 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 378
<210> 379 <211> 11 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 379
<210> 380 <211> 18 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 380
<210> 381 <211> 18 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 381
<210> 382 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 382
<210> 383 <211> 10 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 383
<210> 384 <211> 13 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 384
<210> 385 <211> 13 <212> PRT <213> Artificial sequence
<220> <223> N-terminal extension
<400> 385
<210> 386 <211> 9 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 386
<210> 387 <211> 11 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 387
<210> 388 <211> 7 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 388
<210> 389 <211> 16 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 389
<210> 390 <211> 11 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 390
<210> 391 <211> 16 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 391
<210> 392 <211> 5 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 392
<210> 393 <211> 5 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 393
<210> 394 <211> 7 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 394
<210> 395 <211> 16 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 395
<210> 396 <211> 7 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 396
<210> 397 <211> 37 <212> PRT <213> Artificial sequence
<220> <223> C-terminal extension
<400> 397
<210> 398 <211> 14 <212> PRT <213> Artificial sequence
<220> <223> Linker
<400> 398
<210> 399 <211> 24 <212> PRT <213> Artificial sequence
<220> <223> Linker
<400> 399
<210> 400 <211> 40 <212> PRT <213> Artificial sequence
<220> <223> Linker
<400> 400
<210> 401 <211> 34 <212> PRT <213> Artificial sequence
<220> <223> Linker
<400> 401
<210> 402 <211> 20 <212> PRT <213> Artificial sequence
<220> <223> Linker
<400> 402
<210> 403 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Genus protoxin-II sequence
<220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is Gly, Pro, Ala or deleted
<220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is Pro, Ala or deleted
<220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is Ser, Gln, Ala, Arg or Tyr
<220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is Gln, Arg, Lys, Ala or Ser
<220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is Lys, Ser, Gln or Arg
<220> <221> MISC_FEATURE <222> (8)..(8) <223> Xaa is Met or Phe
<220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa is Thr, Ser, Arg, Lys or Gln
<220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is Asp or Thr
<220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is Ser, Ala or Arg
<220> <221> MISC_FEATURE <222> (14)..(14) <223> Xaa is Glu, Arg, Asn, Lys, Thr or Gln
<220> <221> MISC_FEATURE <222> (15)..(15) <223> Xaa is Arg or Lys
<220> <221> MISC_FEATURE <222> (16)..(16) <223> Xaa is Lys, Gln, Ser or Ala
<220> <221> MISC_FEATURE <222> (19)..(19) <223> Xaa is Glu, Gln or Asp
<220> <221> MISC_FEATURE <222> (20)..(20) <223> Xaa is Glu or Gln
<220> <221> MISC_FEATURE <222> (22)..(22) <223> Xaa is Val or Ser
<220> <221> MISC_FEATURE <222> (24)..(24) <223> Xaa is Arg or Thr
<220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is Lys or Arg
<400> 403
<210> 404 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Protoxin-II genus 2
<220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is Gln, Arg, Lys, Ala or Ser
<220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is Lys, Ser, Gln or Arg
<220> <221> MISC_FEATURE <222> (8)..(8) <223> Xaa is Met or Phe
<220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa is Thr, Ser, Arg, Lys or Gln
<220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is Asp or Thr
<220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is Ser, Ala or Arg
<220> <221> MISC_FEATURE <222> (14)..(14) <223> Xaa is Glu, Arg, Asn, Lys, Thr or Gln
<220> <221> MISC_FEATURE <222> (15)..(15) <223> Xaa is Arg or Lys
<220> <221> MISC_FEATURE <222> (16)..(16) <223> Xaa is Lys, Gln, Ser or Ala
<220> <221> MISC_FEATURE <222> (19)..(19) <223> Xaa is Glu, Gln or Asp
<220> <221> MISC_FEATURE <222> (20)..(20) <223> Xaa is is Gly or Gln
<220> <221> MISC_FEATURE <222> (22)..(22) <223> Xaa is Val or Ser
<220> <221> MISC_FEATURE <222> (24)..(24) <223> Xaa is Arg or Thr
<220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is Lys or Arg
<400> 404
<210> 405 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Protoxin-II genus 3
<220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is Tyr, Gln, Ala, Ser or Arg
<220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa is Thr or Ser
<220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is Ser, Arg or Ala
<220> <221> MISC_FEATURE <222> (14)..(14) <223> Xaa is Glu, Thr or Asn
<220> <221> MISC_FEATURE <222> (19)..(19) <223> Xaa is Glu or Gln
<220> <221> MISC_FEATURE <222> (22)..(22) <223> Xaa is Val or Ser
<220> <221> MISC_FEATURE <222> (24)..(24) <223> Xaa is Arg or Thr
<220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is Lys or Arg
<400> 405
<210> 406 <211> 32 <212> PRT <213> Artificial sequence
<220> <223> Protoxin-II genus 4
<220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa is Thr or Ser
<220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa is Ser, Arg or Ala
<220> <221> MISC_FEATURE <222> (14)..(14) <223> Xaa is Glu, Thr or Asn
<220> <221> MISC_FEATURE <222> (19)..(19) <223> Xaa is Glu or Gln
<220> <221> MISC_FEATURE <222> (22)..(22) <223> Xaa is Val or Ser
<220> <221> MISC_FEATURE <222> (24)..(24) <223> Xaa is Arg or Thr
<220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is Lys or Arg
<400> 406
<210> 407 <211> 1980 <212> PRT <213> Homo sapiens
<400> 407

Claims

An isolated Protoxin-II variant comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 40, 44, 56, 59, 65, 78, 109, 110, 111, 114, 115, 116, 117, 118, 119, 121, 122, 123, 129, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 158, 159, 172, 173, 175, 177, 178, 183, 184, 185, 186, 189, 190, 193, 196, 197, 199, 205, 207, 210, 211, 216, 217, 224, 230, 269, 270, 271, 272, 273, 274, 275, 277, 278, 279, 280, 281, 282, 283, 284, 287, 288, 289, 290, 291, 292, 297, 298, 299, 301, 302, 304, 305, 307, 308, 309, 310, 312, 314, 315, 318, 319, 320, 321, 322, 323, 324, 325, and 326, wherein the variant inhibits human Nav1.7 activity with an IC₅₀ value of 30x10^-9 M or less, wherein the IC₅₀ value is measured using a FLIPR Tetra membrane depolarisation assay, the assay comprising fluorescence resonance energy transfer (FRET) in the presence of 25x10^-6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7 and using bis-1,3(diethylthiobarbituric acid)trimethine oxonol as an electron acceptor and trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate as a donor by exciting the donor at 390- 420 nm and measuring FRET at 515-575 nm.
The isolated Protoxin-II variant of claim 1, wherein the variant selectively inhibits human Navl.7.
The isolated Protoxin-II variant of claim 2, comprising an amino acid sequence selected from the group consisting of the sequence of SEQ ID NOs: 56, 78, 114, 129, 133, and 146.
The isolated Protoxin-II variant of any of the claims 1, 2 or 3, having a free C-terminal carboxylic acid, amide, methylamide or butylamide group.
A conjugate comprising the Protoxin-II variant of any of the claims 1-3 conjugated to a half-life extending moiety; wherein the conjugate is a fusion protein comprising the Protoxin-II variant and a human serum albumin (HSA), albumin binding domain (ABD), or a Fc polypeptide; or the conjugate comprises polyethylene glycol (PEG) chemically coupled to the Protoxin-II variant.
An isolated polynucleotide encoding the Protoxin-II variant of any one of claims 1-3.
A vector comprising the isolated polynucleotide of claim 6.
A host cell comprising the vector of claim 7.
A method of producing an isolated Protoxin-II variant, comprising culturing the host cell of claim 8 and recovering the Protoxin-II variant produced by the host cell.
A pharmaceutical composition comprising the isolated Protoxin-II variant of any one of claims 1-4 or the conjugate of claim 5 and a pharmaceutically acceptable excipient.
The Protoxin-II variant of any one of claims 1-4 or the pharmaceutical composition of claim 10 for use in treating Navl.7-mediated pain in a subject in need thereof; wherein optionally
(a) pain is chronic pain, acute pain, neuropathic 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; or

(b) the Protoxin-II variant is administered peripherally; or

(c) the Protoxin-II variant is administered locally to a joint, spinal cord, surgical wound, sites of injury or trauma, peripheral nerve fibers, urogenital organs, or inflamed tissues; or

(d) the subject is a human.