NOVEL MUTANT OF a-CONOTOXIN PEPTIDE TxID, PHARMACEUTICAL COMPOSITION AND USE THEREOF

Abstract

The invention belongs to the field of biochemistry and molecular biology. The present invention provides a mutant of -conotoxin peptide TxID, a pharmaceutical composition and use thereof. The TxID mutant is capable of specifically blocking acetylcholine receptors (nAChRs), in particular, 34, or 6/34 subtype, and has application prospects in the manufacture of a medicament for smoking cessation and detoxification and analgesia, a medicament for treating psychiatric diseases and cancers, regulating appetite, and a tool medicine for neuroscience.

Claims

1. An isolated polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 in which the serine at position 9 is substituted by a different L-form or D-form amino acid.

2. An isolated polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 2-3, 7, 9, 11-33, respectively.

3. The polypeptide according to claim 1, wherein counted from the N-terminus of the polypeptide: the first cysteine forms a disulfide bond with the third cysteine, and the second cysteine forms a disulfide bond with the fourth cysteine; or the first cysteine forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine; or the first cysteine forms a disulfide bond with the second cysteine, and the third cysteine forms a disulfide bond with the forth cysteine.

4. An isolated fusion protein comprising at least one polypeptide according to claim 1.

5. An isolated polynucleotide encoding the polypeptide according to claim 1.

6. A nucleic acid construct comprising the polynucleotide according to claim 5.

7. A transformed cell comprising the polynucleotide according to claim 5.

8. A pharmaceutical composition comprising at least one polypeptide according to claim 1.

9.-10. (canceled)

11. A method of blocking an acetylcholine receptor or modulating a acetylcholine level in vivo or in vitro, comprising a step of administering to a subject or administering to a cell an effective amount of the polypeptide according to claim 1, wherein the acetylcholine receptor is an 34 acetylcholine receptor, or an 64* acetylcholine receptor.

12.-13. (canceled)

14. A method for treating and/or preventing a nervous system disease or cancer, or a method for killing pests, analgesia, smoking cessation, detoxification or promoting appetite, comprising a step of administering to a subject in need thereof an effective amount of the polypeptide according to claim 1.

15. The polypeptide according to claim 1, wherein the serine at position 9 in the sequence as shown in SEQ ID NO: 1 is substituted with alanine, 2-aminobutyric acid, histidine, arginine, tyrosine, threonine, lysine, leucine, phenylalanine, D-arginine, D-serine, glutamic acid or aspartic acid.

16. The polypeptide according to claim 2, wherein counted from the N-terminus of the polypeptide: the first cysteine forms a disulfide bond with the third cysteine, and the second cysteine forms a disulfide bond with the fourth cysteine; or the first cysteine forms a disulfide bond with the fourth cysteine, and the second cysteine forms a disulfide bond with the third cysteine; or the first cysteine forms a disulfide bond with the second cysteine, and the third cysteine forms a disulfide bond with the forth cysteine.

17. The polypeptide according to claim 1, wherein the carboxy terminus of the polypeptide is amidated.

18. The polypeptide according to claim 2, wherein the carboxy terminus of the polypeptide is amidated.

19. The pharmaceutical composition according to claim 8, which further comprising a pharmaceutically acceptable excipient.

20. The method according to claim 11, wherein the 64* acetylcholine receptor is an 6/34 acetylcholine receptor.

21. The method according to claim 14, wherein the nervous system disease is at least one of addiction, neuralgia, Parkinson's disease, dementia, schizophrenia and depression.

22. The method according to claim 21, wherein the addiction is caused by at least one psychoactive substance.

23. The method according to claim 22, wherein the psychoactive substance is nicotine, opium, heroin, methamphetamine (ice), morphine, marijuana, cocaine or alcohol.

24. The method according to claim 21, wherein the neuralgia is selected from at least one of the following: sciatica, trigeminal neuralgia, lymphatic neuralgia, multi-point motor neuralgia, acute strenuous spontaneous neuralgia, crush neuralgia, and compound neuralgia.

25. The method according to claim 21, wherein the neuralgia is caused by at least one of the following factors: cancer, cancer chemotherapy, alcoholism, diabetes, sclerosis, herpes zoster, mechanical injury, surgical injury, AIDS, head neuralgia, drug poisoning, Industrial pollution poisoning, myeloma, chronic congenital sensory neuropathy, angiitis, vasculitis, ischemia, uremia, childhood bile liver disease, chronic respiratory disorder, multiple organ failure, sepsis/pyaemia, hepatitis, Porphyria, vitamin deficiency, chronic liver disease, native biliary sclerosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis or allergies.

26. The method according to claim 14, wherein the cancer is a lung cancer, ovarian cancer, leukemia, neuroblastoma or breast cancer.

27. The method according to claim 26, wherein the lung cancer is small cell lung cancer.

Description

DRAWINGS

[0073] FIG. 1: HPLC chromatogram and mass spectrum of TxID and TxID[S9A]. FIG. 1A, HPLC chromatogram of TxID with a peak time of 23.31 min. FIG. 1B, ESI-MS mass spectrum of TxID, actually measured molecular weight of 1488.56 Da, consistent with the theoretical value. FIG. 1C, HPLC chromatogram of TxID[S9A] with a peak time of 25.08 min. FIG. 1D, ESI-MS spectrum of TxID[S9A], actually measured molecular weight of 1472.56 Da, consistent with the theoretical value.

[0074] FIG. 2: FIGS. 2A-2D show the strong blocking activities of 1 M of TxID, TxID[S9H], TxID [S9L] or TxID [S9Y] to rat 34 nAChRs current, respectively.

[0075] Rat 34 nAChRs were expressed in Xenopus oocytes, and the cell membrane was clamped at 70 mV, giving an Ach pulse of 1 s every minute. In each figure, a representative current trace of one polypeptide on one oocyte is shown. After obtaining the control current, 1 M of toxin peptide was added, and the first Ach pulse current after 5 min incubation was the current trace of the peptide affecting the receptor, indicated by arrows in the figure. The polypeptide is then eluted, and the magnitude of the current generated by the Ach pulse during the elution and its trace are also measured simultaneously. C in the figure indicates the control current generated by ACh excitation. The identification in the following current trace diagram is the same as this description.

[0076] FIG. 3: FIGS. 3A-3D show the blocking effects of 1 M of TxID[14D], TxID[14H], TxID[I14L] or TxID[S9(D-Arg)] on rat 34 nAChRs current, respectively.

[0077] FIG. 4: FIGS. 4A-4D show the blocking effects of 1 M of TxID[S9(D-Ser)], TxID[S9A], TxID[S9Abu] or TxID[S9F] on rat 34 nAChRs current, respectively.

[0078] FIG. 5: FIGS. 5A-5D show the blocking effects of 1 M of TxID[S9K], TxID[S9T], TxID[S12Y] or TxID[S9R] on rat 34 nAChRs current, respectively.

[0079] FIG. 6: FIGS. 6A-6E show that 10 M of TxID[10R], TxID[H5W,S9A], TxID[H5W], TxID[M11H] or TxID[10R] have no blocking effects on the current of rat 34 nAChRs.

[0080] FIG. 7: FIGS. 7A-7D show the blocking effects of 1 M of TxID[S9Abu], TxID[S9F], TxID or TxID[I14L] on rat 6/34 nAChRs current, respectively.

[0081] FIG. 8: FIGS. 8A-8D show the blocking effects of 1 M of TxID[S9(D-Arg)], TxID[S9A], TxID[S7H] or TxID[S9L] on rat 6/34 nAChRs currents, respectively.

[0082] FIG. 9: FIGS. 9A-9D show the blocking effects of 1 M of TxID[S9R)], TxID[S9T], TxID[S9Y] or TxID[S12Y] on rat 6/34 nAChRs current, respectively.

[0083] FIG. 10: FIGS. 10A-10D show that 10 M of TxID[9R], TxID[10R], TxID[H5W,S9A], or TxID[H5W] have no blocking effect on the current of rat 6/34 nAChRs.

[0084] FIG. 11: FIGS. 11A-11C show that 10 M of TxID[S9D], TxID[S9E] or TxID[S9K] have no blocking effect on the current of rat 6/34 nAChRs.

[0085] FIG. 12: Effects of TxID or TxID[S9A] at 100 nM or 10 nM on the current of rat 34 and 6/34 nAChRs both shows the different degrees of discrimination. Rat 34 and 6/34 nAChRs were expressed in Xenopus oocytes, and the cell membrane was clamped at 70 mV, giving an Ach pulse of 1 s every minute. A representative current trace of one polypeptide on one oocyte is shown in the figure. After obtaining the control current, 100 nM or 10 nM of toxin peptide was added, and the first Ach pulse current after 5 min incubation was the current trace of the polypeptide affecting the receptor, indicated by the arrows in the figure. The polypeptide is then eluted, and the magnitude of the current generated by the Ach pulse during the elution and its trace were also measured simultaneously. C in the figure indicates the control current generated by ACh excitation.

[0086] FIG. 12A shows the effect of 100 nM TxID on the current of rat 34 nAChRs.

[0087] FIG. 12B shows the effect of 100 nM TxID on the current of rat 6/34 nAChRs.

[0088] FIG. 12C shows the effect of 100 nM TxID[S9A] on the current of rat 34 nAChRs.

[0089] FIG. 12D shows the effect of 100 nM TxID[S9A] on the current of rat 6/34 nAChRs.

[0090] FIG. 12E shows the effect of 10 nM TxID on the current of rat 34 nAChRs.

[0091] FIG. 12F shows the effect of 10 nM TxID on the current of rat 6/34 nAChRs.

[0092] FIG. 12G shows the effect of 10 nM TxID[S9A] on the current of rat 34 nAChRs.

[0093] FIG. 12H shows the effect of 10 nM TxID[S9A] on the current of rat 6/34 nAChRs.

[0094] FIG. 13 shows the concentration response curves of TxID or TxID[S9A] to rat 34 and 6/34 nAChRs. In the figure, the abscissa is the logarithm of the molar concentration (M) of the polypeptide used; the ordinate is the percentage of dose response (% Response), which is the ratio percentage of the acetylcholine receptor current to the control current at the corresponding concentration of toxin. The corresponding half-blocking dose (IC.sub.50) is shown in Table 2. The individual values in the figure are the average values of currents taken from 5-21 Xenopus oocytes. FIG. 13A, Concentration dose response curves of TxID to two subtypes of 34 nAChRs or 6/34 nAChRs. FIG. 13B, Concentration dose response curves of TxID[S9A] to two subtypes of 34 nAChRs and 6/34 nAChRs.

[0095] FIG. 14: FIG. 14A shows the effect of 1 M TxID[S9K] on the current of rat 34 nAChRs. FIG. 14B shows the effect of 10 M TxID[S9K] on the current of rat 6/34 nAChRs. FIGS. 14A and 14B show that TxID[S9K] has a very high degree of discrimination between the two subtypes of 34 and 6/34 nAChRs which are extremely similar.

[0096] FIG. 15 shows the comparison of secondary chemical shift (ordinate) analysis of TxID[S9A] (isomer 1) and TxID (isomer 1).

[0097] The amino acid sequences of TxID[S9A] and TxID are shown in the figure. The amino acid S in parentheses indicates that the serine (Ser) at position 9 of TxID is substituted with alanine (Ala, A) and then mutated into mutant TxID[S9A]. The black bar in the figure represents TxID[S9A], and the gray bar represents TxID.

[0098] FIG. 16: FIG. 16A, Molecular binding model of TxID to 34 acetylcholine receptor.

[0099] FIG. 16B, Molecular binding model of TxID to 649 acetylcholine receptor.

[0100] In FIGS. 16A and 16B, the 3 subunit is shown in pink, the 6 subunit is shown in blue, and the 4 subunit is shown in green. Hydrogen bonds are formed between Ser-9 and Lys-81, indicated by dashed lines. The amino acid numbers on the 4 subunit are numbered according to the full length of the rat 4 subunit precursor sequence (UniProt identifier P12392).

[0101] FIG. 16C shows the results of molecular dynamics (MD) simulations of TxID in 50 ns. The distance between the Ser-9 side chain hydroxyl group of the TxID and the 4 Lys-81 side chain nitrogen atom is shown as a function of time.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0102] Embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, when specific techniques or conditions are not indicated, they are carried out in accordance with the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. When the reagents or instruments used are not indicated by the manufacturer, they are conventional products that can be obtained commercially.

Example 1: Sequence Design and Artificial Synthesis of a Novel Mutant of -Conotoxin TxID

[0103] Based on the -conotoxin TxID mature peptide (the amino acid sequence thereof is shown in SEQ ID NO: 1 in Table 1 below, and its C-terminal was amidated), the inventors creatively designed a series of new polypeptide mutants. The amino acid sequences thereof are shown in SEQ ID NO: 2-37 in Table 1 below.

TABLE-US-00001 TABLE1 Sequencesof-conotoxinTxIDanditsmutants SEQID NO: Name Sequence.sup.a 1 TxID GCCSHPVCSAMSPIC* 2 TxID[G1A] ACCSHPVCSAMSPIC* 3 TxID[S4A] GCCAHPVCSAMSPIC* 4 TxID[H5A] GCCSAPVCSAMSPIC* 5 TxID[P6A] GCCSHAVCSAMSPIC* 6 TxID[V7A] GCCSHPACSAMSPIC* 7 TxID[S9A] GCCSHPVCAAMSPIC* 8 TxID[M11A] GCCSHPVCSAASPIC* 9 TxID[S12A] GCCSHPVCSAMAPIC* 10 TxID[P13A] GCCSHPVCSAMSAIC* 11 TxID[I14A] GCCSHPVCSAMSPAC* 12 TxID[M11I] GCCSHPVCSAISPIC* 13 TxID[S9A,M11L] GCCSHPVCAALSPIC* 14 TxID[I14R] GCCSHPVCSAMSPRC* 15 TxID[I14Y] GCCSHPVCSAMSPYC* 16 TxID[I14D] GCCSHPVCSAMSPDC* 17 TxID-[S9Abu] GCCSHPVCBAMSPIC* 18 Tx1D-[S9H] GCCSHPVCHAMSPIC* 19 Tx1D-[S9R] GCCSHPVCRAMSPIC* 20 TxID-[S9Y] GCCSHPVCYAMSPIC* 21 TxID-[S9T] GCCSHPVCTAMSPIC* 22 TxID-[S12Y] GCCSHPVCSAMYPIC* 23 TxID-[I14L] GCCSHPVCSAMSPLC* 24 TxID-[S9K] GCCSHPVCKAMSPIC* 25 TxID-[S9L] GCCSHPVCLAMSPIC* 26 TxID-[S9F] GCCSHPVCFAMSPIC* 27 TxID-[14H] GCCSHPVCSAMSPHIC* 28 TxID-[S9(D-Arg)] GCCSHPVC(D-Arg)AMSPIC* 29 TxID-[14D] GCCSHPVCSAMSPDIC* 30 TxID-[S9(D-Ser)] GCCSHPVC(D-Ser)AMSPIC* 31 TxID-[S9E] GCCSHPVCEAMSPIC* 32 TxID-[S9D] GCCSHPVCDAMSPIC* 33 TxID-[M11H] GCCSHPVCSAHSPIC* 34 TxID-[H5W,S9A] GCCSWPVCAAMSPIC* 35 TxID-[H5W] GCCSWPVCSAMSPIC* 36 TxID-[9R] GCCSHPVCRSAMSPIC* 37 TxID-[10R] GCCSHPVCSRAMSPIC* .sup.a Mutation sites for each mutant are underlined. B in the sequence represents 2-aminobutyric acid (Abu). *indicates C-terminal amidation.

[0104] Linear peptides of the polypeptides listed in Table 1 were artificially synthesized by the Fmoc method. The specific method is as follows:

[0105] The resin peptides were artificially synthesized by Fmoc chemical method, and the resin peptides could be synthesized by a peptide synthesizer or a manual synthesis method. In addition to cysteine, the remaining amino acids were protected with standard side chain protecting groups. The SH groups of the first and third cysteines (Cys) of each polypeptide were protected by Trt (S-trityl), and the SH groups of the second and fourth cysteines were protected by Acm (S-acetamidomethyl), and the disulfide linkages after oxidative folding were Cys1-Cys3 and Cys2-Cys4, i.e., Cys (1-3, 2-4). The synthesis procedure was as follows: a linear peptide was synthesized on the ABI Prism 433a polypeptide synthesizer by Fmoc and FastMoc methods in solid phase synthesis. The side chain protecting groups of Fmoc amino acids were: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys). Using Fmoc HOBT DCC method, the resin and Fmoc amino acids were amidated by Rink, and the synthesis steps were carried out with reference to the instrument synthesis manual. In order to complete the reaction, the piperidine deprotection time and the coupling time were appropriately extended, and the refractory amino acids were double-coupled to obtain a resin peptide. The linear peptide was cleaved from the resin with reagent K (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole; 90:5:2.5:7.5:5, v/v/v/v/v) and precipitated and washed with ice diethyl ether to recycle a crude linear peptide.

[0106] Preparative reverse HPLC C18 column (Vydac) was used for purification, in which the elution gradient was 10-40% B90 in 0-20 min. The solvent B90 was composed of 90% ACN (acetonitrile), 0.5% TFA (trifluoroacetic acid), and balance of pure water; the solvent A was an aqueous solution of 0.65% TFA. UV absorption analysis was performed at a wavelength of 214 nm. The purified linear peptide was subjected to purity detection using an analytical HPLC C18 column (Vydac), and the elution gradient was the same as above. It had a purity of over 95% and was used for oxidative folding.

[0107] According to references (Dowell, C.; Olivera, B. M.; Garrett, J. E.; Staheli, S. T.; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. M.; McIntosh, J. M., Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience. 2003, 23 (24), 8445-52), a two-step oxidative folding reaction of the above linear peptide was described as follows:

[0108] The first pair of disulfide bonds were first formed between the two cysteines of the Trt protecting groups by potassium ferricyanide oxidation (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min). The monocyclic peptide was purified by reverse-phase HPLC C18 column (Vydac) and then oxidized with iodine (10 mM iodine in H2O:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min), the Acm on other two cysteines were removed, and the second pair of disulfide bonds were formed between these two cysteines. The bicyclic peptide was purified by reverse phase HPLC C18 column (Vydac), and the linear gradient of elution was still 10-40% B90 within 0-20 min. Ultraviolet absorption analysis was carried out at a wavelength of 214 nm. Thus, an -conotoxin with disulfide bonds formed in oriented manner between the corresponding cysteines in order from the N-terminus to the C-terminus was obtained.

[0109] The purified peptides with 2 pairs of disulfide bonds were tested for purity and molecular weight by HPLC and mass spectrometry, and the results were all correct. The HPLC chromatographic conditions were as follows: Vydac C18 HPLC reverse phase analytical column, gradient elution in 20 minutes with B solution from 10% to 40% and solution A from 90% to 60%, in which the solution A was 0.65% trifluoroacetic acid (TFA), the B solution was an aqueous solution of 0.5% TFA and 90% acetonitrile. The UV analysis wavelength was 214 nm, and the peak time of TxIC, i.e., the retention time, was 23.366 minutes.

[0110] For example, FIGS. 1A-1D show HPLC chromatograms and mass spectra of TxID and TxID[S9A]. The peak time of TxID was 23.31 min (FIG. 1A) and was identified as correct by mass spectrometry (ESI-MS) (FIG. 1B). The monoisotopic mass of TxID after oxidative folding was consistent with the measured molecular weight: the theoretical molecular weight of TxID was 1488.559 Da, and the molecular weight of TxID was 1488.56 Da. The peak time of TxID[S9A] was 25.08 min (FIG. 1C) and was also confirmed by ESI-MS mass spectrometry (FIG. 1D). The monoisotopic mass of the oxidatively folded TxID[S9A] was also consistent with the measured molecular weight: the theoretical molecular weight of TxID[S9A] was 1472.564 Da, and the measured molecular weight of TxID[S9A] was 1472.56 Da.

[0111] Through the above steps, all the 37 polypeptides listed in Table 1 were correctly synthesized, and oxidatively folded peptides with disulfide linkages of Cys (1-3, 2-4) were formed, which could be used for subsequent activity research and structural analysis.

[0112] The concentrations of the polypeptide were determined by colorimetry at a wavelength of 280 nm, and the concentrations and masses of the polypeptides were calculated according to the Beer-Lambert equation, and used in the experiments in the following examples.

Example 2: Experiment of -Conotoxin TxID New Mutant Specifically Blocking 34 nAChR Subtype

[0113] Referring to the methods in the literature (Luo S, Zhangsun D, Zhu X, Wu Y, Hu Y, Christensen S, Harvey P J, Akcan M, Craik D J, McIntosh J M. Characterization of a novel -conotoxin TxID from Conus textile that potently blocks rate 34 nicotinic acetylcholine receptors. Journal of Medicinal Chemistry. 2013, 56: 9655-9663. Azam L, Yoshikami D, McIntosh J M. Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A]. J Biol Chem. 2008; 283(17):11625-32.), as well as the specification of in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, Tex.)), various rat neural nAChRs subtypes (34, 6/34, 910, 42, 44, 34, 22, 24, 7), human 34 and 6/34, and mouse muscular nAChRs (11) cRNA were prepared, and their concentrations were measured and calculated by OD values at UV 260 nm. Xenopus laveis were dissected and oocytes (frog eggs) were collected, cRNA was injected into frog eggs with 5-10 ng cRNA per subunit. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after the collection of frog eggs, and voltage clamp for nAChRs was recorded within 1-4 days after injection.

[0114] One frog egg injected with cRNA was placed in a 30 L Sylgard recording groove (depth 4 mmdiameter 2 mm), gravity infusion was performed with a ND96 perfusate (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl.sub.2, 1.0 mM MgCl.sub.2, 5 mM HEPES, pH 7.1-7.5) containing 0.1 mg/ml BSA (bovine serum albumin), or a ND96 (ND96A) containing 1 mM atropine, in a flow rate of 1 ml/min. All conotoxin solutions also contained 0.1 mg/ml BSA to reduce non-specific adsorption of toxins, a switch valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, Calif.) was used to switch between infusion of toxins and acetylcholine (ACh), and a series of three-way solenoid valves (model 161TO31, Neptune Research, Northboro, Ma.) were used to freely switch between perfusion ND96 and ACh. Online recordation was performed when the Ach-gated current was set at slow clamp by a two-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, Conn.) and the clamp gain at the maximum (2000) position. A glass electrode was drawn from glass capillary (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, Fla.) with 1 mm outer diameter0.75 inner diameter mm, and filled with 3M KCl as a voltage and current electrode. The membrane voltage was clamped at 70 mV. The entire system was controlled and recorded by a computer. The ACh pulse was automatic perfusion with ACh for 1 s every 5 min. The concentrations of ACh were 10 M for the muscular nAChRs and the neuronal 910 nAChRs, 200 M for the 7 of expression neuronal nAChRs, and 100 M for other subtypes. The current responses and the current traces of at least 5 eggs expressing a certain subtype to different toxin concentrations were recorded.

[0115] The current data of the test were statistically analyzed by GraphPad Prism software (San Diego, Calif.), and a dose-response curve was drawn to calculate various parameters of conotoxin, such as half-blocking concentration IC.sub.50 and the like, about toxin blocking nAChRs.

[0116] The experimental results are shown in Table 2 below. The ratios of the blocking activities of these mutants to 34 nAChR to that of TxID are also summarized in Table 2.

TABLE-US-00002 TABLE 2 Blocking activities of -conotoxin TxID and its mutants on rat 34 and 6/34 acetylcholine receptor subtypes (half-blocking dose, IC.sub.50). IC.sub.50 ratio.sup.f of SEQ ID Polypeptide 34 34 6/34 6/34 6/34 NO: name.sup.a IC.sub.50, nM.sup.c IC.sub.50 ratio.sup.d IC.sub.50, nM.sup.c IC.sub.50 ratio.sup.e vs. 34 1 TxID 3.64 (1.8-7.3) 1.0 33.9 (23.6-48.7) 1.0 9.3 2 TxID[G1A] 61.9 (32.4-118.0) 17.0 278 (154-503) 8.2 4.5 3 TxID[S4A] 10.8 (8.6-13.4) 3.0 64.1 (41.6-98.7) 1.9 5.9 4 TxID[H5A] >10000.sup.b >10000.sup.b 5 TxID[P6A] >10000.sup.b >10000.sup.b 6 TxID[V7A] >10000.sup.b >10000.sup.b 7 TxID[S9A] 3.89 (2.5-5.9) 1.1 178.1 (137.0-231.5) 5.2 45.8 8 TxID[M11A] >10000.sup.b >10000.sup.b 9 TxID[S12A] 17.4 (8.6-3.5) 4.8 39.3 (25.6-59.8) 1.2 2.3 10 TxID[P13A] >10000.sup.b >10000.sup.b 11 TxID[I14A] 16.1 (9.1-28.5) 4.4 45.8 (33.7-62.2) 1.3 2.8 12 TxID[M11I] 74.9 (55.0-102.1) 20.6 50.4 (31.3-81.2) 1.5 0.7 13 TxID[S9A, M11I] 30.7 (14.1-66.8) 8.4 101.0 (55.8-183.2) 3.0 0.3 14 TxID[I14R] 9.2 (5.0-16.9) 2.5 67.4 (42.8-106) 2.0 7.3 15 TxID[I14Y] 10.0 (6.4-15.6) 2.7 38.7 (23.0-65.0) 1.1 3.9 16 TxID[I14D] 9.8 (6.6-14.4) 2.7 40.8 (32.9-50.5) 1.2 4.2 17 TxID[S9Abu] 1.87 (1.48-2.37) 0.5 4.91 (3.65-6.6) 0.1 2.6 18 TxID[S9H] 2.61 (2.27-2.99) 0.7 14.69 (10.57-20.41) 0.4 5.6 19 TxID[S9R] 5.26 (3.87-7.13) 1.5 264.1 (177.1-393.88) 7.8 50.2 20 TxID[S9Y] 7.66 (6.06-9.67) 2.1 17.76 (12.42-25.37) 0.5 2.3 21 TxID[S9T] 7.8 (6.16-9.87) 2.2 39.06 (31.95-47.75) 1.2 5 22 TxID[S12Y] 9.09 (7.04-11.73) 2.5 34.23 (28.18-41.57) 1.0 3.8 23 TxID[I14L] 9.98 (8.33-11.97) 2.8 28.98 (22.11-38) 0.9 2.9 24 TxID[S9K] 10.13 (8.07-12.72) 2.8 >10000.sup.b 25 TxID[S9L] 13.11 (10.25-16.78) 3.6 16.18 (11.34-23.08) 0.5 1.2 26 TxID[S9F] 13.85 (10.48-18.3) 3.8 7.11 (6.02-8.39) 0.2 0.5 27 TxID[14H] 32.97 (24.61-44.18) 9.2 312.02 (198.26-491.01) 9.2 9.5 28 TxID[S9(D-Arg)] 47.93 (34.65-66.31) 13.3 338.93 (250.26-458.88) 10.0 7.1 29 TxID[14D] 50.58 (37.49-68.25) 14.1 2258.5 (1342.4-3798.94) 66.6 44.7 30 TxID[S9(D-Ser)] 115.88 (84.85-158.14) 32.2 395.84 (284.2-551.4) 11.7 3.4 31 TxID[S9E] 307.61 (231.28-409.17) 85.4 >10000.sup.b 32 TxID[S9D] 379.5 (230.7-568.3) 105.4 >10000.sup.b 303 33 TxID[M11H] >10000.sup.b 3492.82 (1601.66-7611.49) 103 34 TxID[H5W, S9A] >10000.sup.b >10000.sup.b 35 TxID[H5W| >10000.sup.b >10000.sup.b 36 TxID[9R] >10000.sup.b >10000.sup.b 37 TxID[10R] >10000.sup.b >10000.sup.b .sup.aCysteine mode is C.sub.1C.sub.2-C.sub.3-C.sub.4, and disulfide bond mode is 1-3, 2-4. .sup.bBlocking current is less than 50% at a high concentration of 10 M. .sup.cNumber in parentheses indicates the range of half-blocking dose (IC.sub.50) for 95% confidence interval. .sup.dRatio of the IC.sub.50 of TxID mutant to 34 nAChR subtype to the IC.sub.50 of wild-type TxID to 34 nAChR subtype. .sup.eRatio of the IC.sub.50 of TxID mutant to 6/34 nAChR subtype to the IC.sub.50 of wild-type TxID to 6/34 nAChR subtype. .sup.fRatio of the IC.sub.50 of each peptide to 6/34 nAChR subtype to its IC.sub.50 to 34 nAChR subtype.

[0117] The results show:

[0118] The 26 mutants shown in SEQ ID NO: 2-3, 7, 9, 11-32 (prepared in Example 1) all had nanomolar blocking activity to rat 34 nAChR (see Table 2, and FIGS. 2A-2D, FIGS. 3A-3D, FIGS. 4A-4D, and FIGS. 5A-5D), and their half-blocking doses (IC.sub.50) varied from 1.87 nM to 379.5 nM (Table 2). Among them, most of the mutants (23 polypeptides in total) maintained strong blocking activity against 34 nAChR, and their IC.sub.50 values were all below 100 nM; and 19 polypeptides had stronger blocking activity against 34 nAChR, and their IC.sub.50 were all below 36 nM.

[0119] Most of the mutants had a strong blocking activity against 34 nAChR, their IC.sub.50 values were close to that of TxID and all below 10 nM, and some of them were even stronger than the blocking activity of TxID (IC.sub.50, 3.6 nM); for example, the IC.sub.50 values of SEQ ID NO: 17 TxID[S9Abu] and SEQ ID NO: 18 TxID[S9H] were only 1.87 nM and 2.61 nM, respectively (Table 2), and they were the specific blockers to 34 nAChR with the strongest blocking activity discovered so far.

[0120] The elution rates of TxID and its new mutants after blocking the 34 nAChR current were different (FIGS. 2A-2D, FIGS. 3A-3D, FIGS. 4A-4D, FIGS. 5A-5D). As shown in FIGS. 2A-2D, after 1 M of the new mutants TxID[S9H], TxID[S9L] and TxID[S9Y] blocked the 34 nAChR current, the elution was very slow, and the current after 2 min of elution was almost 0 nA. However, after blocking with 1 M wild-type TxID, its current returned to the level of control current C within 2 min of elution. Also, after TxID[S9Abu] and TxID[S9F](4A-4D) blocked the 34 nAChR current, the elution was also very slow. The elution rates of the remaining polypeptides were relatively fast, but the respective elution rates were different (FIGS. 3A-3D, FIGS. 4A-4D, FIGS. 5A-5D). The elution rate reflected an important feature of the binding between polypeptide and receptor, namely the rate at which the reaction binding dissociated.

[0121] Only the three mutant polypeptides, SEQ ID NO: 30 TxID[S9(D-Ser)], SEQ ID NO: 31 TxID[S9E], SEQ ID NO: 32 TxID[S9D], had an IC.sub.50 between 115 and 380 nM. There were also 10 polypeptides, SEQ ID NOs: 4, 5, 6, 8, 10, and 33-37, which lost blocking activity on 34 nAChR (Table 2, FIGS. 6A-6E), and at a high concentration of 10 M, their blocking activities to 34 nAChR current were less than 50% and their IC.sub.50 were greater than 10000 nM (see Table 2, and FIGS. 6A-6E).

[0122] In addition, the present inventors also determined the blocking activities of TxID and its new mutants (such as Table 1) on human 34 nAChR, and their activities were similar to those to rat 34 nAChR, and there was no significant difference between the two species.

Example 3: Experiment of -Conotoxin TxID New Mutants Specifically Blocking 6/34 nAChR Subtypes

[0123] Referring to the electrophysiological method in Example 2, the blocking activities of TxID and all polypeptides of its new mutant in Table 1 against 6/34 (equivalent to 64*, * representing the remaining possible subunits) nAChR were determined, and the results were shown in Table 2, as well as FIGS. 7A-7D, FIGS. 8A-8D, FIGS. 9A-9D, FIGS. 10A-10D, and FIGS. 11A-11C. The ratios of the blocking activities of these mutants to 6/34 nAChR compared to TxID were also summarized in Table 2.

[0124] The results show:

[0125] Total of 24 new peptides, SEQ ID NOs: 2-3, 7, 9, 11-23, 25-30 and 33 (prepared in Example 1), had different degrees of blocking effect on rat 6/34 nAChR (please See Table 2, and FIGS. 7A-7D, FIGS. 8A-8D and FIGS. 9A-9D), and half-blocking doses (IC.sub.50) ranging from 4.91 nM to 3492.82 nM (Table 2).

[0126] Among them, most of the mutants in total of 22 polypeptides maintained blocking activities on 6/34 nAChR, and their IC.sub.50 were all below 1000 nM, in which there were 7 polypeptides, namely SEQ ID NO: 2, 7, 13, 19, 27, 28 and 30, presenting IC.sub.50 to 6/34 nAChR in a range of 100-1000 nM. There were 15 mutants that had stronger blocking activity on 6/34 nAChR, and their IC.sub.50 values were close to that of TxID and all below than 100 nM, and some of them were stronger than the blocking activity of TxID on 6/34 nAChR (IC.sub.50, 33.9 nM); for example, the IC.sub.50 values of SEQ ID NOS: 20, 23, 25 and 26 were 17.76 nM, 28.98 nM, 16.18 nM and 7.11 nM, respectively (Table 2). There were two mutant polypeptides, SEQ ID NO: 29 and SEQ ID NO: 33, which showed IC.sub.50 in a range between 1 M and 10 M.

[0127] In addition, there were 12 polypeptides, SEQ ID NOs: 4-6, 8, 10, 24, 31-32, 34-37, which almost lost blocking activity against 6/34 nAChR (Table 2, FIGS. 10A-10D, and FIGS. 11A-11C), and their IC50 values were greater than 10000 nM (i.e., 10 M).

[0128] The elution rates of TxID and its new mutants after blocking 6/34 nAChR current were different (FIGS. 7A-7D, FIGS. 8A-8D, FIGS. 9A-9D). As shown in FIGS. 7A-7D, the elution rate of TxID[S9F] was significantly slower than those of other polypeptides. In general, they are similar to the elution rate and peak shape of wild-type TxID after blocking 6/34 nAChR current (FIGS. 7A-7D, FIGS. 8A-8D, FIGS. 9A-9D), and could generally return to the level of the control current C after elution for 2 min.

[0129] In addition, the inventors also determined the blocking activities of TxID and its new mutants (as shown in Table 1) on human 6/34 nAChR, indicating that their activities were similar to those on rat 6/34 nAChR, and there were no significant difference between the two species.

Example 4: Comparison of Activities of -Conotoxin TxID New Mutants on Two Subtypes of 34 and 6/34 nAChR

[0130] The present inventors compared the activities of the -conotoxin TxID new mutants on the two subtypes of 34 and 6/34 nAChR according to the experimental data in Table 2 above, in which the comparison results were expressed by the degrees of discrimination, the degree of discrimination=(IC.sub.50 of mutant on rat 6/34)/IC.sub.50 of mutant on rat 34), and the unit was nM. The results are shown in Table 3 below. In addition, the inventors also list the currently known conotoxins acting on 34 nAChRs and their discriminations between two very similar subtypes 34 and 6/34 nAChR in Table 3 by literature investigation.

TABLE-US-00003 TABLE 3 Alpha-conotoxins acting on 34 and 6/34 acetylcholine receptor subtypes and their discrimination for the two subtypes SEQ ID 34 6/34 Discrim- Selectivity to NO: Name Sequence .sup.a Source IC.sub.50[nM] IC.sub.50[nM] ination .sup.b nAChR subtype Reference .sup.d 1 TxID GCCSHPVCSAMSPIC* C. textile 3.64 33.9 9.3 34 > 6/34 Luo S. et al. 2013b 7 TxID[S9A] GCCSHPVCAAMSPIC* synthetic 3.89 178.1 45.8 34 > 6/34 19 TxID[S9R] GCCSHPVCRAMSPIC* Artificially 5.26 264.1 50.2 34 > 6/34 synthesized 24 TxID[S9K] GCCSHPVCKAMSPIC* Artificially 10.13 >10000.sup.c 34 > 6/34 synthesized 29 TxID[14D] GCCSHPVCSAMSPDIC* Artificially 50.58 2258.5 44.7 34 > 6/34 synthesized 17 TxID[S9Abu] GCCSHPVCBAMSPIC* Artificially 1.87 4.91 2.6 34 >= 6/34 synthesized 18 TxID[S9H] GCCSHPVCHAMSPIC* Artificially 2.61 14.69 5.6 34 > 6/34 synthesized 31 TxID[S9E] GCCSHPVCEAMSPIC* Artificially 307.61 >10000.sup.c 34 synthesized 38 TP-2212-59 GCCSHPBCFBZYC* Artificially 2.3 N.D. 34 Chang et al. synthesized 2014 39 AuIB GCCSYPPCFATNPDC* C. aulicus 750 N.D. 34 Luo S. et al. 1998 40 RegIIA GCCSHPACNVNNPHIC* C. regius 47.3 147 3.1 32 > 34 Franco et al. 62 > 7 > 64 2012 41 RegIIA[N11, GCCSHPACNVAAPHIC* Artificially 370 5100 13.8 34 > 6/34* Kompella 12 A] synthesized et al. 2015 42 PeIA GCCSHPACSVNHPELC* C. pergrandis 480 1500 3.1 910 > 32 > McIntosh 62 > 34 > 7 et al. 2005 43 PIA RDPCCSNPVCTVHNPQIC* C. 518 30 17.3 62* >> 64 Dowell et al. purpurascens 32 > 34 2003 44 BuIA GCCSTPPCAVLYC* C. bullatus 27.7 2.1 13.2 63* > 32 > Azam et al. 34 > 44 > 2005) 2* .sup.a B represents 2-aminobutyric acid (Abu). Z represents pentane. .sup.b Ratio between the IC.sub.50 of each polypeptide to 6/34 nAChR subtype and the IC.sub.50 to 34 nAChR subtype. .sup.c Blocking less than 50% of current at a high concentration of 10 M. *indicates C-terminal amidation. N.D. indicates not determined, no reference value. .sup.d References: Luo S. et al. 2013b: Luo S, Zhangsun D, Zhu X, Wu Y, Hu Y, Christensen S, Harvey P J, Akcan M, Craik D J, McIntosh J M. 2013a. Characterization of a novel -conotoxin TxID from Conus textile that potently blocks rat 34 nicotinic acetylcholine receptors. Journal of Medicinal Chemistry 56: 9655-9663. Chang et al. 2014: Discovery of a potent and selective 34 nicotinic acetylcholine receptor antagonist from an -conotoxin synthetic combinatorial library. Chang Y P, Banerjee J, Dowell C, Wu J, Gyanda R, Houghten R A, Toll L, McIntosh J M, Armishaw C J. J Med Chem. 2014 Apr. 24; 57(8): 3511-21. Luo S. et al. 1998: Luo S, Kulak J M, Cartier G E, Jacobsen R B, Yoshikami D, Olivera B M, McIntosh J M. 1998. alpha-conotoxin AulB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. J Neurosci 18: 8571-8579. Franco et al. 2012: Franco A, Kompella S N, Akondi K B, Melaun C, Daly N L, Luetje C W, Alewood P F, Craik D J, Adams D J, Mari F. 2012. RegIIA: an alpha4/7-conotoxin from the venom of Conus regius that potently blocks alpha3beta4 nAChRs. Biochem Pharmacol 83: 419-426. Kompella et al. 2015: Kompella S N, Hung A, Clark R J, Mari F, Adams D J. 2015. Alanine scan of alpha-conotoxin RegIIA reveals a selective alpha3beta4 nicotinic acetylcholine receptor antagonist. J Biol Chem 290: 1039-1048. McIntosh et al. 2005: McIntosh J M, Plazas P V, Watkins M, Gomez-Casati M E, Olivera B M, Elgoyhen A B. 2005. A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors. J Biol Chem 280: 30107-30112. Dowell et al. 2003: Dowell C, Olivera B M, Garrett J E, Staheli S T, Watkins M, Kuryatov A, Yoshikami D, Lindstrom J M, McIntosh J M. 2003. Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. J Neurosci 23: 8445-8452. Azam et al. 2005: Azam L, Dowell C, Watkins M, Stitzel J A, Olivera B M, McIntosh J M. 2005. Alpha-conotoxin BulA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors. J Biol Chem 280: 80-87.

[0131] It can be seen from Table 2 and Table 3:

[0132] Most of the new mutants of -conotoxin TxID retained the blocking activity against two similar subtypes of 34 and 64* nAChRs (Table 2), and the discrimination degrees between the two subtypes were mostly within 10 times. The discrimination of wild-type TxID between the two similar subtypes of 34 and 64* nAChRs was only 9.3 times (Table 3).

[0133] However, a good discrimination between the two subtypes 34 and 64* nAChRs was observed in five new TxID mutants, including SEQ ID NO: 7 that was TxID[S9A], SEQ ID NO: 19 that was TxID[S9R], SEQ ID NO: 24 that was TxID[S9K], SEQ ID NO: 29 that was TxID[14D], and SEQ ID NO: 31 that was TxID[S9E] as shown in Table 2-3. Among them, the polypeptides of SEQ ID NO: 7, 19 and 29 had a high degree of discrimination between the two similar subtypes of 34 and 64* nAChRs, the discrimination degrees of the three polypeptides were relatively close, and the discrimination degrees were in range of 45-50 times.

[0134] It is particularly noteworthy that the novel mutant SEQ ID NO: 24, TxID[S9K], was found to have the best discrimination between the two subtypes, and was a ligand substance so far found with the highest discrimination degree between the two similarly subtypes of 34 and 64* nAChRs. TxID[S9K] was highly active against 34 nAChR with a half-blocking dose of only 10.13 nM, while the blocking activity on 64* nAChR was lost, its blocking current was less than 50% at high concentration of 10 M, and its half-blocking dose was >10000 nM (Table 2, Table 3, FIG. 11C). SEQ ID NO: 31, that is, TxID [S9E], had relatively weak activity against 34 nAChR, and its half-blocking dose was 307.61 nM, but it lost blocking activity to 64* nAChR, and it had no blocking activity on 64* nAChR at high concentration of 10 M (FIG. 11B, Table 2, Table 3), this mutant peptide showed well discrimination between the two similar subtypes of 34 and 64* nAChRs.

[0135] However, all of the conotoxins found in the prior art have a discrimination degree of less than 20 times for the two subtypes (Table 3); for example, RegIIA, RegIIA [N11, 12A], PeIA, PIA, BuIA show a discrimination degree of 3.1-17.3 times for the two subtypes.

[0136] In addition, the present inventors also analyzed in detail the current blocking effect of wild-type TxID, mutant TxID[S9A] and mutant TxID[S9K] at different concentrations on the two subtypes of 34 and 64* nAChRs, respectively. The results are shown in FIGS. 12-14. The specific experimental steps refer to the Examples 2-3 above.

[0137] FIG. 12 and FIG. 13 show the comparison of current blocking effect between wild-type TxID and mutant TxID[S9A] on the two subtypes of 34 and 64* nAChRs, respectively (FIGS. 12A-12H), and the concentration-dose response curves (FIGS. 13A-13B).

[0138] TxID at 100 nM or 10 nM showed similar current blocking strength on the two subtypes (FIGS. 12A, 12B, 12E, 12F). TxID at 100 nM blocked the two subtypes of 34 and 64* nAChRs in current by 97% and 85%, respectively (FIGS. 12A-12B). TxID at 10 nM blocked the two subtypes of 34 and 64* nAChRs in current by 67% and 46%, respectively (FIGS. 12E-12F).

[0139] TxID[S9A] at 100 nM or 10 nM showed very different effects on the current blocking for the two receptor subtypes and could distinguish them (FIGS. 12C, 12D, 12G, 12H). TxID[S9A] at 100 nM blocked the two subtypes 34 and 64* nAChRs in current by 98% and 26%, respectively (FIGS. 12C-12D), showing significant difference. TxID[S9A] at 10 nM blocked the current of the 34 nAChRs subtype by 75% (FIG. 12G), and TxID[S9A] at 10 nM completely lost the blocking activity to the 64* nAChRs subtype (FIG. 12H), and its current was the same of the control current.

[0140] The concentration dose response curves of FIGS. 13A-13B also reflect that TxID[S9A] had a good discrimination for the two subtypes of 34 and 64* nAChRs.

[0141] TxID[S9K] at 1 M completely blocked the current of 34 nAChR (FIGS. 14A-14B), while TxID[S9K] with a high concentration of 10 M showed extremely weak blocking activity on 6/34 nAChR, i.e., less than of the control current (FIGS. 14A-14B).

Example 5: Spatial Structure Analysis of TxID and TxID[S9A] by Nuclear Magnetic Resonance (NMR)

[0142] The inventors further analyzed the spatial structure of TxID and TxID[S9A] by nuclear magnetic resonance (NMR), and the specific method referred to Luo S, Zhangsun D, Zhu X, Wu Y, Hu Y, Christensen S, Harvey P J, Akcan M, Craik D J, McIntosh J M. 2013a. Characterization of a novel -conotoxin TxID from Conus textile that potently blocks rat 34 nicotinic acetylcholine receptors. Journal of Medicinal Chemistry 56: 9655-9663. FIG. 15 showed the analysis results of the secondary chemical shift (ordinate) of TxID[S9A] (isomer 1) and TxID (isomer 1).

[0143] NMR structural analysis showed that TxID[S9A] had two conformers in aqueous solution, called isomer 1 (trans-trans isomer, trans isomer) and isomer 2 (cis-trans isomer). At 308 K, the ratio of the two spatial isomers was 70:30. These spatial isomers were formed by the cis-trans isomerization of the peptide bonds preceding the prolines (Pro, P) at the 6th and 13th positions. If the secondary H chemical shift is 0.1 ppm greater than the random curl value, it indicates that the polypeptide has typical structural features, i.e., the positive shift represents (3-type folding structure, and the negative shift represents the -helical structure (Wishart D S, Bigam C G). Holm A, Hodges R S, Sykes B D. 1995. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. 1. Investigations of nearest-neighbor effects. Journal of Biomolecular NMR 5: 67-81). FIG. 15 shows that the trans isomer of TxID[S9A] (isomer 1) has no significant difference in secondary structure characteristics compared to the trans isomer of TxID. Mutation of serine (Ser-9) at position 9 of TxID into alanine (Ala-9) enhanced the -helical structure of the middle portion of the polypeptide. For cis and trans isomers, both TxID and TxID[S9A] tended to adopt a random coil structure (data were not shown).

[0144] Referring to the method of Yu et al. 2012 (Yu, R., Kaas, Q., and Craik, D J (2012). Delineation of the unbinding pathway of alpha-conotoxin ImI from the alpha7 nicotinic acetylcholine receptor. The Journal of Physical Chemistry B 116, 6097-6105), a molecular docking analysis of the interaction of -conotoxin TxID with the ligand binding domain of rat 34 or 64 nAChRs was performed. The original homology model was constructed using Modeller9v13 (Sali and Blundell 1993) software and two molecular templates. These two molecular templates were the crystal structures of acetylcholine binding protein (AChBP) and -conotoxin TxIA variant (PDB identifier 2uz6), respectively (Dutertre, S., Ulens, C., Buttner, R., Fish, A., van Elk, R., Kendel, Y., Hopping, G., Alewood, P F, Schroeder, C., Nicke, A., et al. (2007). AChBP-targeted alpha-conotoxin correlates distinct binding orientations With nAChR subtype selectivity. The EMBO Journal 26, 3858-3867), and the crystal structure of the isolated human 9 subunit (PDB identifier 4d01), (Zouridakis et al. 2014). It was assumed that 34 and 64 nAChRs were pentamers composed of two -subunits and three -subunits, and the binding site of conotoxin to the receptor was the interface formed between the (right side) and (complementary side) subunits. By using molecular dynamics (MD) simulation software and method with amber ff99SB-ILDN (Lindorff-Larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., and Shaw, D. E. (2010). Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 78, 1950-1958) molecular force field and Gromacs 5.1 MD engine (Pronk, S., Pall, S., Sc Hulz, R., Larsson, P., Bjelkmar, P., Apostolov, R., Shirts, M. R., Smith, J. C., Kasson, P. M., van der Spoel, D., et al. (2013). GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845-854), the molecular docking binding model for the interaction of TxID with rat 34 or 64 nAChRs was refined respectively, and three molecular dynamics (MD) simulation results were obtained (FIG. 16).

[0145] The molecular docking binding models of TxID to 34 and 64* nAChRs (FIGS. 16A-16B) show that although the overall difference between the two was very small, there were still differences, mainly due to the different shape of the binding site, and caused by difference in the amino acid species near the binding site on the 3 and 6 subunits. Molecular dynamics (MD) simulations performed within 50 ns showed that at the binding site to 64* nAChRs, a weak hydrogen bond was formed between Ser-9 of TxID and 4 Lys-81 (FIG. 16B), while this hydrogen bond was absent at the binding site to 34 nAChRs (FIG. 16A).

[0146] The time-dependent distance between the Ser-9 side chain hydroxyl group of TxID and the 4 Lys-81 side chain nitrogen atom was shown in FIG. 16C. Substitution of Ser-9 with alanine disrupted this hydrogen bonding interaction. The results showed that the blocking activity of TxID[S9A] on 64* nAChRs was reduced by 5 times compared with that of TxID, which might be attributed to the fact that at 300 K, this point mutation caused an energy decrease of about 1 kcal/mol, which was consistent with the energy of losing a hydrogen bond (Bowie J U. 2011. Membrane protein folding: how important are hydrogen bonds? Curr Opin Struct Biol 21: 42-49). In contrast, at the binding site to 34 nAChRs, there was no interaction between Ser-9 of TxID and the receptor, and thus the substitution of S9A had no effect on the blocking activity of 34 nAChRs, which was consistent with the experimental results (Table 2-3, FIG. 16A).

[0147] When the glycine (Gly-1) at position 1 of TxID was substituted with alanine (G1A), the blocking activities against 34 and 6/34 nAChRs were decreased by 17 times and 8 times, respectively. According to the molecular docking binding model of FIG. 16, there might be a charge interaction between the negatively charged Asp-192 on the 4 subunit and the glycine at position 1 of TxID. This interaction could be attenuated by changes in the bone conformation of TxID [G1A] compared to the wild-type -conotoxin TxID. The methionine (Met-11) at position 11 of TxID was replaced by isoleucine (Ile) (M11I), resulting in a 20-fold decrease in the blocking activity against 34 nAChRs, but there was not a decrease in the blocking activity against 6/34 nAChRs. According to the molecular docking model, Met-11 was in contact with Cys-218 on the C-loop of 3 subunit, and the substitution of M11A might cause a change in binding mode, because a larger side chain would cause a steric hindrance. The M11A mutations resulted in a total loss of blocking activity to both 34 and 6/34 nAChRs, and their half-blocking doses were greater than 10000 nM (Table 2). In contrast, in the binding model to 64, Met-11 could be substituted by Ile without causing steric hindrance to the binding site on the 6 subunit, thus not affecting the blocking activity of wild-type TxID and point mutation TxID[M11I] against 6/34 nAChRs (FIGS. 16A-16B, Table 2).

Example 6: Re-Assay of the Blocking Activities of Some New Mutants of -Conotoxin TxID on Two Subtypes of 34 and 6/34 nAChRs

[0148] The blocking activities of some new mutants of TxID against rat 34 and 6/34 nAChRs was measured again by the electrophysiological method in Example 2 using the newly purchased Xenopus oocytes. The results are shown in Table 4. The results showed that the blocking activities of these mutants were basically the same as those of Examples 2 and 3, except that their half-blocking doses (IC.sub.50) had slight changes, which were within the range of normal error variation. These results further confirmed the blocking activities of the above mutants on 34 and 6/34 nAChRs.

TABLE-US-00004 TABLE 4 Blocking activities of some new mutants of -conotoxin TxID on 34 and 6/34 nAChRs (half-blocking dose, IC.sub.50) 34 6/34 SEQ ID NO: Polypeptide name.sup.a IC.sub.50, nM.sup.c IC.sub.50, nM.sup.c 17 TxID[S9Abu] 1.94 (1.53-2.46) 6.24 (4.64-8.39) 18 TxID[S9H] 1.96 (1.45-2.28) 18.66 (13.43-25.94) 19 TxID[S9R] 5.38 (3.98-7.28) 350 (232.6-526.6) 20 TxID[S9Y] 7.93 (6.29-10) 22.86 (16.24-32.19) 21 TxID[S9T] 7.36 (5.91-9.16) 49.64 (40.61-60.68) 24 TxID[S9K] 6.86 (5.27-8.94) >10,000.sup.b 25 TxID[S9L] 9.89 (8.07-12.13) 20.56 (14.41-29.33) 26 TxID[S9F] 10.7 (8.41-13.62) 10.22 (8.53-12.24) 28 TxID[S9(D-Arg)] 52.48 (38.89-70.82) 430.7 (318.1-583.2) 30 TxID[S9(D-Ser)] 124.9 (90.05-173.16) 502.98 (361.19-700.77) 31 TxID[S9E] 319.1 (239.9-424.5) >10,000.sup.b 32 TxID[S9D] 375.6 (239.4-589.5) >10,000.sup.b .sup.aCysteine mode is C.sub.1C.sub.2-C.sub.3-C.sub.4, and disulfide bond linkage mode is 1-3, 2-4. .sup.bBlocking current is less than 50% at a high concentration of 10 M. .sup.cThe number in parentheses indicates the range of half-blocking dose (IC.sub.50) for 95% confidence interval.

[0149] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.