WO2015024503A1 - Ingénierie de neurotoxines clostridiales présentant une forte activité catalytique - Google Patents

Ingénierie de neurotoxines clostridiales présentant une forte activité catalytique Download PDF

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WO2015024503A1
WO2015024503A1 PCT/CN2014/084725 CN2014084725W WO2015024503A1 WO 2015024503 A1 WO2015024503 A1 WO 2015024503A1 CN 2014084725 W CN2014084725 W CN 2014084725W WO 2015024503 A1 WO2015024503 A1 WO 2015024503A1
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polypeptide
neurotoxin
vamp2
botulinum neurotoxin
light chain
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PCT/CN2014/084725
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English (en)
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Sheng Chen
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The Hong Kong Polytechnic University
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Priority to CN201480045998.5A priority Critical patent/CN105683212B/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • This invention relates to modification of Clostridia Neurotoxins including Botulinum Neurotoxin and Tetanus Neurotoxin and applications of the derivatives obtained thereof.
  • Clostridia Neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the Botulinum Neurotoxins (BoNT), and spastic paralysis elicited by Tetanus Neurotoxin (TeNT).
  • CNTs are 150 kDa dichain proteins with typical A-B structure-function properties, where the B (binding) domain binds to surface components on the mammalian cell and translocates the A (active) domain to an intracellular location (1).
  • CNTs are organized into three functional domains: an N-terminal catalytic domain (light chain, LC), an internal translocation domain (heavy chain, HCT), and a C-terminal receptor binding domain (heavy chain, HCR) (2).
  • the CNTs are zinc metalloprotease that cleave SNARE (Soluble NSF Attachment REceptor) proteins which interfere with synaptic vesicle fusion to the plasma membrane and ultimately block neurotransmitter release in nerve cells (1, 3).
  • cleave SNARE Soluble NSF Attachment REceptor
  • VAMP2 vesicle associated membrane protein-2
  • SNAP25 Plasma membrane SNARE proteins
  • BoNT serotypes B, D, F and G BoNT serotypes B, D, F and G
  • TeNT cleave VAMP2 BoNT serotypes A and E cleave SNAP25
  • BoNT serotype C cleaves SNAP25 and syntaxin la (3, 5-7).
  • BoNTs are most widely used in protein therapy.
  • BoNT serotype A (BoNT/A) was approved by the United States Food And Drug Administration (FDA) to treat strabismus, blepharospam, hemificial spasm and also for cervical dystonia, cosmetic use, glabellar facial lines and axillary hyperhidrosis as early as 1989.
  • FDA United States Food And Drug Administration
  • BoNT/A was approved by the United States Food And Drug Administration
  • FDA United States Food And Drug Administration
  • the efficacy of BoNT/ A in treating dystonia and other disorders related to involuntary skeletal muscle activity, and the satisfactory safety profile associated with BoNT/A have prompted empirical/off-label use of BoNT/A in a variety of opthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, and painful disorders (8-17).
  • MYOBLOCTM Botulinum Neurotoxin serotype B product
  • BoNT BoNT-like neurotoxin serotype A
  • BoNT/B Botulinum Neurotoxin serotype B
  • BoNTs Since therapeutic properties of BoNTs and development of BoNTs immunoresistance are interrelated, the best way to overcome the immunoresistant problem in BoNTs is to engineer more active BoNTs that could reduce the amounts of toxins required for therapy. Rummel et al have modified a ganglioside binding motif of the heavy chain (HC) domain of BoNT/B that enhances the binding and toxicity to up to three-fold relative to the wild type toxin (43).
  • HC heavy chain
  • BoNT/B ganglioside binding motif of the heavy chain domain of BoNT/B that enhances the binding and toxicity to up to three-fold relative to the wild type toxin (43).
  • engineering of BoNT through modification(s) of its receptor binding sites may affect the selectivity of the binding event.
  • modification of the binding site(s) may not increase the potency significantly enough to prevent the development of immunoresistance. Rather, altering BoNT activity by modification of the light chain (LC) may be a better way to achieve this goal.
  • LC
  • This invention provides methods for modulating activity of Botulinum Neurotoxin and Tetanus Neurotoxin.
  • the present invention provides methods for enhancing substrate recognition of the light chain of Tetanus Neurotoxin (LC/T) and Botulinum Neurotoxin (LC/B) via elevated catalytic activity.
  • LC/T Tetanus Neurotoxin
  • LC/B Botulinum Neurotoxin
  • the present invention provides derivatives of Botulinum Neurotoxin and Tetanus Neurotoxin and their applications thereof.
  • the present invention provides derivatives of the light chain of Tetanus Neurotoxin (LC/T) or derivatives of the entire Tetanus Neurotoxin with altered activity.
  • LC/T Tetanus Neurotoxin
  • the present invention provides derivatives of the light chain of Botulinum Neurotoxin (LC/B) or derivatives of the entire Botulinum Neurotoxin with altered activity.
  • LC/B Botulinum Neurotoxin
  • the LC/B derivatives or Botulinum Neurotoxin derivatives described herein can be used in various therapies, cosmetics or other applications.
  • the LC/B derivatives or Botulinum Neurotoxin derivatives described herein are used in therapy for reducing immunoresistance to Botulinum Neurotoxin.
  • the present invention provides methods to improve current therapy using Botulinum Neurotoxin.
  • the present invention provides method of using derivatives of Botulinum Neurotoxin or light chain of Botulinum Neurotoxin for various therapeutic or cosmetic applications.
  • Figure 1 shows an illustration of the S2', S I ' and SI pockets of the light chain of Tetanus Neurotoxin (LC/T) and light chain of Botulinum Neurotoxin (LC/B), and the P sites of VAMP2. Amino acid residues at the pockets which interacts with different P sites of the VAMP2 are shown.
  • Figure 1A shows the interacting residues of LC/T and the VAMP2, wherein the LC/T is represented as a surface model.
  • Figure IB shows the interacting residues of LC/T and the VAMP2, wherein the LC/T is represented as a cartoon model.
  • Figure 1C show the interacting residues of LC/B and VAMP2, wherein the LC/B is represented as a surface model.
  • Figure ID show the interacting residues of LC/B and VAMP2, wherein the LC/B is represented as a cartoon model.
  • the nomenclatures of the P site of VAMP2 and the S pockets of LC/T are as follow.
  • the two residues that formed the scissile bond of VAMP2 were designated as ⁇ 1- ⁇ from C- to N-terminus.
  • Residues from PI to the C-terminal direction were designated as P2, P3 etc., while residues from ⁇ to the N-terminal direction were designated as P2', P3 ' etc.
  • the pockets in LC/T or LC/B that recognize the specific P sites were designated as the corresponding S pockets.
  • VAMP2 recognizes the PI ' site of VAMP2 (residue F ), is composed of L and I respectively.
  • Figure 2 shows catalytic activities of LC/T, LC B and their derivatives on cleaving substrate VAMP2.
  • Figure 2 A shows the activities of LC/T and derivatives on cleaving VAMP2.
  • Figure 2B shows the activities of LC/B and derivatives on cleaving VAMP2.
  • Error bar represents standard deviation of three independent repeats for each assay.
  • Figure 3 shows orientation of S I ' residue in LC/B, LC/T and the LC/T derivative [K 168 E, L 230 I].
  • Figure 3 A compares the orientation of S I ' pocket residue in LC/B (I 227 ), in LC/T (L 230 )
  • Figure 3B shows a relative flat surface demonstration of S I ' pocket comprising I 227 in LC/B (black), and I 230 in LC/T [K 168 E, L 230 I] (grey).
  • Figure 3C shows the bulky surface of S I ' pocket of LC/T comprising L 230 with a mesh surface demonstration.
  • Figure 3D shows the bulky surface of SI ' pocket of LC/T comprising L 230 with a solid surface demonstration.
  • Figure 4 shows the distances between residues in the SI ' and S2' pockets of LC/T ( Figure 4A), LC/B ( Figure 4B) and LC/T [K 168 E, L 230 I] ( Figure 4C).
  • the distances between the side chains of SI ' residue and S2' residues were measured using the PyMol program.
  • Figure 5 shows the cleavage of endogenous VAMP2 by recombinant LC/B, LC/T and their derivatives.
  • Figure 5A shows the cleavage of endogenous VAMP2 by LC/T and LC/T [K 168 E, L 230 I].
  • Figure 5B shows the cleavage of endogenous VAMP2 by LC/B and LC/B [S 201 P].
  • Upper panel in each figure shows cleavage of VAMP2 analyzed by western blotting using anti- VAMP2 antibody and anti-actin antibody; lower panel shows quantification of the bands obtained from the western blot analysis.
  • BoNTs In order to enhance the therapeutic efficacies of BoNTs and minimize the induction of neutralizing antibody against BoNTs, it is highly desirable to engineer the BoNTs into more potent derivatives such that a lower effective therapeutic dose of BoNT can be used in various treatments and applications.
  • the present invention Through studying the substrate recognition and specificity of the Botulinum Neurotoxin and Tetanus Neurotoxin, the present invention has opened up a new opportunity to engineer Botulinum Neurotoxin and Tetanus Neurotoxin into novel derivatives with enhanced activity and substrate specificity.
  • the present invention provides methods for optimizing substrate recognition of the light chain of Botulinum Neurotoxin B (LC/B) or the light chain of Tetanus Neurotoxin (LC/T), and provides derivatives of these toxins with elevated catalytic activity.
  • LC/B Botulinum Neurotoxin B
  • LC/T Tetanus Neurotoxin
  • the present invention provides methods for modulating activity of Botulinum Neurotoxin and Tetanus Neurotoxin.
  • the present invention provides derivatives of the light chain of Botulinum Neurotoxin B (LC/B) or derivatives of the entire Botulinum Neurotoxin B with elevated activity.
  • the Botulinum Neurotoxin described herein comprises a polypeptide of SEQ ID NO. : 1.
  • derivatives of Botulinum Neurotoxin described herein comprise one or more alteration in the amino acid sequence.
  • derivatives of Botulinum Neurotoxin comprise a change of amino acid at position 201 identified according to SEQ ID NO. : l from serine (S 201 ) to proline (P 201 ).
  • the derivative of Botulinum Neurotoxin [S 201 P] comprises a sequence of SEQ ID NO. : 2.
  • derivatives of Botulinum Neurotoxin comprise a change of amino acid alanine at position 263 identified according to SEQ ID NO. : l .
  • derivatives of Botulinum Neurotoxin comprise a change of amino acid isoleucine at position 264 identified according to SEQ ID NO. : l.
  • the present invention provides derivatives of the light chain of Tetanus Neurotoxin (LC/T) or derivatives of the entire Tetanus Neurotoxin.
  • the Tetanus Neurotoxin described herein comprises a polypeptide of SEQ ID NO. : 3.
  • derivatives of Tetanus Neurotoxin described herein comprise one or more alteration in the amino acid sequence.
  • derivatives of Tetanus Neurotoxin comprise a change of amino acid at position 230 as identified according to SEQ ID NO: 3 from
  • derivatives of Tetanus Neurotoxin comprise a change of amino acid at position 168 as identified according to SEQ ID NO: 3 from lysine (K 168 ) to glutamate (E 168 ).
  • derivative of Tetanus Neurotoxin [L 230 I] comprises a sequence of SEQ ID NO. : 4.
  • derivatives of Tetanus Neurotoxin comprise a change from Leucine-230 (L 230 ) to Isoleucine-230 (I 230 ) and a change from Lysine- 168 (K 168 ) to Glutamate- 168 (E 168 ) identified according to SEQ ID NO: 3.
  • derivative of Tetanus comprises a change from Leucine-230 (L 230 ) to Isoleucine-230 (I 230 ) and a change from Lysine- 168 (K 168 ) to Glutamate- 168 (E 168 ) identified according to SEQ ID NO: 3.
  • Neurotoxin [K 168 E, L 230 I] comprises a sequence of SEQ ID NO: 5.
  • the present invention provides pharmaceutical composition comprising derivatives of Botulinum Neurotoxin or Tetanus Neurotoxin.
  • the Botulinum Neurotoxin, Tetanus Neurotoxin and the derivatives of Botulinum Neurotoxin are examples of Botulinum Neurotoxin and others.
  • Tetanus Neurotoxin described herein are fused or coupled with one or more supplementary polypeptides, wherein the supplementary polypeptide is from Botulinum Neurotoxin, Tetanus
  • the supplementary polypeptides fused or coupled with the present toxins are native or recombinant polypeptides. In another embodiment, the supplementary polypeptide fused or coupled with the present toxins are artificial polypeptides.
  • the DNA of the light chain (LC) and heavy chain (HC) of Botulinum Neurotoxin or Tetanus Neurotoxin or their derivatives described herein are cloned into the same expression vector, thereby expressing a fusion protein comprising the light chain and heavy chain.
  • the light chain and heavy chain are originated from the same serotype of the toxin.
  • the light chain and heavy chain are originated from different serotypes of the toxin.
  • the Botulinum Neurotoxin derivatives described herein are used in a pharmaceutical, clinical, or cosmetic procedure. In one embodiment, the Botulinum Neurotoxin derivatives described herein are used to treat or improve diseases or conditions in various therapies or other applications. In one embodiment, the Botulinum Neurotoxin derivatives described herein can be used in a novel therapy with a lower chance of developing immunoresistance to Botulinum Neurotoxin.
  • the Botulinum Neurotoxin derivatives described herein are used to treat or improve strabismus, blepharospam, hemificial spasm, cervical dystonia, spasticity, glabellar facial lines, axillary hyperhidrosis, lower urinary tract disorders, gastrointestinal tract disorders, spasmodic dysphonia, temporomandibular disorder, sialorrhea, chronic diabetic neuropathy, wound healing, vaginism, musculoskeletal pain, disorders related to involuntary skeletal muscle activity such as involuntary muscle spasm and contractions, or any other applicable diseases or conditions.
  • the present invention provides methods for improving or treating diseases or conditions by administering to the subject an effective amount of the Botulinum Neurotoxin derivatives described herein. In another embodiment, the present invention provides methods for improving current therapy using Botulinum Neurotoxin.
  • the present invention provides methods of using Botulinum Neurotoxin with enhanced therapeutic efficacy or pharmaceutical properties. In another embodiment, the present invention provides methods for reducing immunoresistance to Botulinum Neurotoxin in various therapies and applications. [0041] In one embodiment, the Tetanus Neurotoxin derivatives described herein can be used as a molecular marker or an effective tool to study the mechanisms of exocytosis in central neuron.
  • the present invention provides a modified light chain polypeptide of Botulinum Neurotoxin, wherein the Botulinum Neurotoxin has the amino acid sequence of SEQ ID NO. : l, and the modified light chain comprises one or more mutations at positions as identified according to SEQ ID NO. : 1.
  • the modified light chain polypeptide of Botulinum Neurotoxin described herein comprises an amino acid sequence of SEQ ID NO. : 2.
  • the present invention provides a composition comprising the modified light chain polypeptide of Botulinum Neurotoxin described herein, wherein the polypeptide is fused with or coupled with one or more second polypeptide, and wherein the second polypeptide is a polypeptide from Botulinum Neurotoxin or other organisms.
  • the second polypeptide is native or artificial.
  • the present invention provides a composition comprising the modified light chain polypeptide of Botulinum Neurotoxin described herein and a pharmaceutical acceptable carrier.
  • the present invention provides a method for improving or treating diseases or conditions in a subject, comprising a step of administering to the subject an effective amount of the modified light chain polypeptide of Botulinum Neurotoxin described herein.
  • the diseases or conditions are selected from the group consisting of strabismus, blepharospam, hemificial spasm, cervical dystonia, spasticity, glabellar facial lines, axillary hyperhidrosis, involuntary muscle spasm, lower urinary tract disorders, gastrointestinal tract disorders, spasmodic dysphonia, temporomandibular disorder, sialorrhea, chronic diabetic neuropathy, wound healing, vaginism, musculoskeletal pain and involuntary muscle contractions.
  • the method for improving or treating diseases or conditions described herein, wherein reduced immunoresistance to Botulinum Neurotoxin in a subject is induced in the subject.
  • said reduced immunoresistance to Botulinum Neurotoxin is induced in the subject as compared to treatment with wild-type Botulinum Neurotoxin.
  • the present invention provides a modified light chain polypeptide of Tetanus Neurotoxin, wherein the Tetanus Neurotoxin has the amino acid sequence of SEQ ID NO. : 3, and the modified light chain comprises one or more mutations at positions as identified according to SEQ ID NO. : 3.
  • the modified light chain polypeptide of Tetanus Neurotoxin described herein comprises an amino acid sequence of SEQ ID NO. : 4 or 5.
  • the present invention provides a composition comprising the modified light chain polypeptide of Tetanus Neurotoxin described herein, wherein the polypeptide is fused with or coupled with one or more second polypeptide, and wherein the second polypeptide is a polypeptide from Tetanus Neurotoxin or other organisms.
  • the second polypeptide is native or artificial.
  • composition comprising the modified light chain polypeptide of Tetanus Neurotoxin described herein is used as a molecular marker to study exocytosis in the neuron.
  • the present invention provides a composition comprising the modified light chain polypeptide of Tetanus Neurotoxin described herein and a pharmaceutical acceptable carrier.
  • the present invention provides a method for improving or treating diseases or conditions in a subject, comprising a step of administering to the subject an effective amount of the modified light chain polypeptide of Tetanus Neurotoxin described herein.
  • Plasmids for the expression of LC/T (1-436) (accession no. of the Tetanus Neurotoxin: X06214.1), LC/B (1-440) (accession no. of the Botulinum Neurotoxin: AB084152.1) and VAMP2 (1-97) and subsequent protein expression and purification were performed as previously described (30-32).
  • Site directed mutagenesis of pLC/T, pLC/B and pVAMP2 were performed using QuickChange (Stratagene) protocols as previously described (30, 31). Plasmids were sequenced to confirm the mutation and that additional mutations were not present within the ORFs. Mutated proteins were produced and purified as described (30-33). Linear Velocity And Kinetic Constant Determinations For VAMP2 Cleavage By LC/B And LC/T
  • LC/T derivative [K E, L I] was crystallized by hanging drop vapor diffusion method.
  • LC/T [K 168 E, L 230 I] was stored in a buffer of 10 mM Tris, 20 mM NaCl (pH 7.9) at a concentration of 7.5 mg/ml.
  • Each of the handing drop contained ⁇ ⁇ protein solution and 1 ⁇ mother liquor (250 mM Mg(N0 3 ) 2 and 15% PEG 3350). Crystals were grown at 16°C for 4-5 days until maturation. For data collection, crystals were harvested and cryoprotected in the mother liquor supplemented with 20% glycerol.
  • Neuro-2A cells were cultured in minimum essential medium supplemented with 10% newborn calf serum, 1.4% sodium bicarbonate, and 0.5% penicillin- streptomycin at 37 °C in 5% C0 2 .
  • Confluent cells were harvested and lysed by passing through a 25 gauge needle for 20-30 times on ice. Nuclei and unbroken cells were spun down by centrifuging the lysed product for 5 min at 2500 rpm and the supernatant was collected for assay. After incubating the cell lysate with different amount of LCs at 37°C for 10 min in a reaction volume of 10 ⁇ , the reaction was stopped by adding equal amount of SDS-PAGE sample buffer and boiling the reaction mixture at 100°C for 10 min. Cleavage of VAMP was analyzed by western blotting using anti-VAMP2 and anti-actin antibody.
  • BoNT/B and TeNT were found to cleave substrate VAMP2 at the same scissile bond, while their efficiencies of substrate hydrolysis were different, with LC/B being more active than LC/T by about 20-folds.
  • LC/B and LC/T showed that the active site of the two LCs displayed a similar arrangement for VAMP2 recognition. The major difference between the two systems was found to be at the P2'-S2' substrate recognition site (33, 38).
  • Figure 1 illustrates the active site of LC/B and LC/T for interacting with the substrate VAMP2. Comparison of the structures of LC/B and LC/T revealed that the S2' pocket of the two
  • LCs are similar and comprise an arginine residue (R in LC/B and R in LC/T), while their SI ' pockets were different.
  • the SI ' pocket of LC/B is composed of F 95 , V 200 , S 201 , L 226 and I 227
  • the SI ' pocket of LC/T is composed of F 199 , V 204 , P 205 , L 229 , L 230 and L 231 .
  • Mutation of these SI ' pocket residues to alanine showed no effect on LC/B substrate hydrolysis except for I 227 A, which showed ⁇ 80-fold reduction of k cat , but not K m (38).
  • the different composition of SI ' pocket of LC/B and LC/T may contribute not only to the different recognition of PI ', but also the P2' site of VAMP, which may further affect the different catalytic activity of LC/B and LC/T.
  • SI ' and S2' pockets are in close proximity, the different composition of SI ' pocket of LC/B and LC/T may be correlated to the different properties observed in the S2' pockets of LC/B and LC/T.
  • both S2' pockets of LC/B and LC/T comprise an arginine
  • LC/T [L 230 I], which was engineered for an optimal SI ' pocket, was tested for its activity on cleaving VAMP2 E 78 R. As shown in Table 2, the amount of LC/T required to cleave 50% of VAMP2 was about 120 nM (EC 50 ). LC/T showed no cleavage on VAMP2 E 78 R even at a concentration as high as 36,000 nM.
  • EC 50 refers to the concentration of LC proteins resulting in 50% cleavage of VAMP2 or VAMP2 E'3 ⁇ 4.
  • LC/T (K E, L I) was found to be ⁇ 100-fold more active than Wt-LC/T on cleaving VAMP2, as indicated by the elevated & caf value, but not K m value (Table 1, Figure 2a).
  • One possible explanation for the abolished activity is the combination of three mutations at the active site of LC/T may impair the correct conformation of LC/T (Table 1).
  • Both isoleucine and leucine are hydrophobic residue that are likely to interact with F 77 of VAMP2 through hydrophobic interaction.
  • the isoleucine in the SI ' pocket of LC/B and LC/T was found to be more significant in the interaction. It could be due to different orientation of the isoleucine residue in the pocket that favors interaction with F 77 of VAMP2. Structural analysis of the S I ' pockets of LC/B and LC/T
  • I in LC/B resides at a flatter position than L in LC/T.
  • the bulky leucine in LC/T may push the interacting residue F 77 of VAMP2 outward, thereby restraining the interaction between R 374 and E 78 of VAMP2.
  • the flatter position of isoleucine in LC/B may provide an optimal position for fitting F 77 and E 78 of VAMP2 to the active site of LC/B, hence favors the interactions of I 227 -F 77 and R 370 -E 78 .
  • FIG. 3A illustrates the F 0 -F c electron density of L 230 I mutations in the LC/T (K 168 E, L 230 I) structure.
  • LC/T [K 168 E, L 230 I] can perfectly aligned to Wt LC/T with a Root Mean Square Deviation (RMSD) of 0.150 (370 to 370 atoms with 421 atoms aligned), suggesting that the overall conformation of LC/T [K 168 E, L 230 I] is comparable to that of Wt-LC/T.
  • RMSD Root Mean Square Deviation
  • the present invention found that SI pocket mutation of LC/T from Lysine (K 168 ) to Glutamate 168 (E 168 ) [K 168 E] can increase the rate of cleaving VAMP2 to a level comparable to the rate of LC/B.
  • the side chain of Leucine (L ) of LC/T is more
  • LC/T [K E, L I] attained a higher activity on cleaving native VAMP2 in Neuro2A cell than the wildtype toxin ( Figure 5A).
  • the highly active LC/T derivatives described in the present invention can be used as more effective tools to study the mechanisms of exocytosis in central neuron.
  • LC/B [S 201 P] has a higher activity on cleaving recombinant VAMP2 than the wildtype toxin by more than 10-fold (Table 1) and on cleaving native VAMP2 in Neuro2A cell ( Figure 5B).
  • the LC/B derivative described herein can be used to replace LC/B protein currently used in various therapeutic, cosmetic or other applications, or be developed into novel therapy that may minimize immunoresistance to BoNT.
  • Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond, J Biol Chem 269, 20213-20216.
  • Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexocytosis apparatus, Ann N Y Acad Sci 710, 65-75.

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Abstract

La présente invention concerne des méthodes de modulation de l'activité de la neurotoxine botulique et de la neurotoxine tétanique. Dans un mode de réalisation, la présente invention concerne des dérivés actifs de la chaîne légère de la neurotoxine tétanique (CL/T) et de la neurotoxine botulique (CL/B) ou des dérivés de la neurotoxine tétanique et de la neurotoxine botulique complètes. Dans un autre mode de réalisation, la présente invention concerne des méthodes pour améliorer le traitement actuel au moyen de neurotoxine botulique. Dans un autre mode de réalisation, la présente invention concerne de nouvelles méthodes d'utilisation de la neurotoxine botulique ou de la chaîne légère de la neurotoxine botulique à des fins thérapeutiques ou cosmétiques. Dans un mode de réalisation, la présente invention concerne une méthode de réduction de l'immunorésistance vis-à-vis de la neurotoxine botulique dans diverses applications de la toxine.
PCT/CN2014/084725 2013-08-21 2014-08-19 Ingénierie de neurotoxines clostridiales présentant une forte activité catalytique WO2015024503A1 (fr)

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