WO1997032599A1 - Clostridiatoxine chimiquement modifiee a caracteristiques ameliorees - Google Patents
Clostridiatoxine chimiquement modifiee a caracteristiques ameliorees Download PDFInfo
- Publication number
- WO1997032599A1 WO1997032599A1 PCT/US1996/003228 US9603228W WO9732599A1 WO 1997032599 A1 WO1997032599 A1 WO 1997032599A1 US 9603228 W US9603228 W US 9603228W WO 9732599 A1 WO9732599 A1 WO 9732599A1
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- WIPO (PCT)
- Prior art keywords
- neurotoxin
- toxin
- clostridium
- tyrosine residues
- modified
- Prior art date
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- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 108091005990 tyrosine-phosphorylated proteins Proteins 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/08—Clostridium, e.g. Clostridium tetani
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to pharmacologically active compositions of neurotoxins obtained from Clostridium botulinum and Clostridium tetani . Methods for producing the compositions of the invention are also provided.
- BoTx The neurotoxin serotypes produced by Clostridium botulinum (collectively, BoTx) are some of the most potent neuroparalytic agents known to man and are the causative agents for botulism.
- BoTx serotype A (BoTxA) is also considered to be an effective pharmaceutical agent for use in the treatment of certain neuromuscular disorders associated with uncontrolled muscle contractions or spasms such as disorders of the extraocular muscles (e.g., strabismus and nystagmus) as well as dystonias (involuntary contractions of facial, hand and neck muscles) (see, e.g.. The New England Journal of Medicine, 224.: 1186-1194, 1991) .
- BoTxA has been approved for use in temporarily smoothing facial wrinkles.
- BoTxA is believed to produce a reversible, nondegenerative flaccid paralysis of mammalian skeletal muscle, presumably by blocking the exocytosis of acetylcholine at peripheral, presynaptic cholinergic terminals, with limited activity at synapses in the central nervous system (Rabasseda, et al . , Toxicon, 26_:329-326, 1988) .
- BoTx serotypes of BoTx have been identified that have immunologically distinct phenotypes; i.e., serotypes B, Cl, C2, D, E, F and G (Simpson, et al . , Pharmacol . Rev. , 12:155-188, 1981) . All of the BoTx serotypes are believed to be proteins of about 150 kDa molecular weight that are comprised of two polypeptide chains linked by disulphide bridges. The shorter of the two chains (the light chain, LC) is believed to be responsible for the toxicity of the toxin, while the longer of the two chains
- HC the heavy chain
- Botulinum serotypes are believed to be similar in their pharmacological actions (Brin, et al . , "Report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology", Neurology, _0_:1332- 1336, 1990) .
- each of the serotypes cleaves cellular protein substrates which are involved in the release of the acetylcholine neurotransmitter into the synaptic cleft of neurons in the peripheral cholingergic nervous system.
- Protein substrates for BoTx include SNAP-25 (synaptosome-associated protein, cleaved by the A and E serotypes) , syntaxin (cleaved by the C serotype) and synaptobrevin (cleaved by the B, D, F and G serotypes) .
- TeTx Clostridium tetani
- TeTx acts as an anticholinergic, presynaptic neurotoxin.
- Serotypes A and E of the BoTx share a substantial degree of sequence homology with TeTx (DasGupta, et al . , Bioche ie, 21:1193-1200, 1989) .
- TeTx primarily acts on the central (- rather than peripheral) nervous system to produce rigid
- TeTx (rather than flaccid) muscle paralysis, at least one peptide digestion fragment of TeTx (fragment Ibc) has been shown to act peripherally to produce flaccid paralysis in a manner similar to BoTx (Fedinic, et al . , Boll . 1st . Sieroter Milan, £4:35-41, 1985; and, Gawade, et al . , Brain Res . , 3_3_4:139-46, 1985) . TeTx cleaves synaptobrevin.
- TeTx and most BoTx serotypes are available from commercial sources. However, as presently manufactured, up to 90% of the active toxin may be lost during purification to a pharmacologically useful product, resulting in a composition comprised of a combination of active and inactive toxin.
- a pharmacological purified toxin composition is lyophilized for storage, then reconstituted for clinical use with saline or another pharmaceutically acceptable carrier (see, e.g., the manufacturing and lyophilization process described in published European Patent Application No. 0 593 176 A2 [process for limiting the volume of inactive toxin in a purified BoTxA composition] ) .
- the presently available toxin compositions are typically unstable and quickly lose potency at room temperature. Moreover, even when used soon after reconstitution, the relatively low active toxin concentrations present in commercially available toxin compositions limit the activity of each dose, thereby requiring that the toxin be administered repeatedly over a course of time at the same point of entry into tissue.
- BoTx and TeTx compounds which have greater thermal stability and proteolytic activity than do currently available toxin compositions.
- the invention comprises BoTx and TeTx compounds having enhanced proteolytic activity and thermal stability as compared to toxins obtained and purified by conventional techniques . Methods and reagents for use in preparing the toxin compositions of the invention are also provided.
- the invention provides pharmaceutically acceptable compositions of all BoTx serotypes, as well as TeTx compositions.
- tyrosine residues in at least the LC and, preferably, in both the LC and HC of the toxin present are phosphorylated or sulfated to provide a negative charge to each modified tyrosine.
- tyrosine residues in the toxins are substituted with negatively charged amino acids (- e.g., glutamate, aspartate, or negatively charged non- natural amino acids) .
- negatively charged amino acids e.g., glutamate, aspartate, or negatively charged non- natural amino acids
- the substrate On contact with a BoTxA substrate (SNAP-25) , the substrate is cleaved by a 10 nanomolar concentration of phosphorylated toxin in about 5 minutes. In contrast, at even a 50 nanomolar concentration, cleavage of the substrate is not achieved by the unphosphorylated toxin until after 30 minutes or more of contact. Further, at a temperature of 37°C, the unphosphorylated toxin becomes virtually inactivated within about 2 hours, while the phosphorylated toxin retains 50% or more of its proteolytic activity for up to 10 hours at the same temperature. Thus, modification of the toxin to provide it with a negative charge enhances the proteolytic activity and thermal stability of the toxin.
- the invention also provides methods for producing BoTx and TeTx compounds modified according to the invention using either enzymatic or non-enzymatic (chemical) reagents. Pharmaceutical compositions containing such compounds are also provided.
- FIGURE 1 is an autoradiogram depicting phosphorylation of both the heavy chain (HC) and light chain (LC) of BoTxA by a tyrosine kinase (pp60 src ) , measured by incorporation of 32 P into each chain (from [Y- 32 P] ATP) .
- FIGURE 2 is an autoradiogram depicting pp60 src phosphorylation of BoTxA, BoTxE, BoTxB and TeTx, measured by incorporation of 32 P into each chain (from [Y- 32 P] ATP) .
- FIGURE 3 is a graph depicting the rate of phosphorylation of BoTxA by pp60 src (closed circles) and rate of dephosphorylation of pp60 src phosphorylated BoTxA in the presence of protein-tyrosine phosphatase IB (open circles) .
- FIGURE 4 is a graph depicting the rate of cleavage of SNAP-25 by pp60 s c phosphorylated BoTxA during phosphorylation (closed circles) and by the same toxin during protein-tyrosine phosphatase IB dephosphorylation (open circles) .
- FIGURES 5 (a-b) are graphs depicting the rate of cleavage of SNAP-25 by pp60 src phosphorylated BoTxA (closed squares) and unphosphorylated BoTxA (closed circles) .
- Panel A depicts rate of cleavage as a function of toxin concentration
- panel B depicts rate of cleavage as a function of time.
- FIGURES 6 (a-b) are graphs depicting the rate of cleavage of SNAP-25 by pp60 src phosphorylated BoTxE (closed squares) and unphosphorylated BoTxE (closed circles) .
- Panel A depicts rate of cleavage as a function of toxin concentration
- panel B depicts rate of cleavage as a function of time.
- FIGURE 7 is a graph depicting the rate of cleavage of SNAP-25 by pp60 src phosphorylated BoTxA (open squares and circles) and unphosphorylated BoTxA (closed squares and circles) which were preincubated prior to the cleavage array at either 22°C (squares) or 37°C (circles) .
- FIGURES 8 (a-b) are graphs depicting the rate of cleavage of ⁇ NAP-25 by pp60 src phosphorylated BoTxA (closed squares) and unphosphorylated BoTxA (closed circles) , wherein the unphosphorylated starting BoTxA materials were inactivated (to a loss of approximately 90% activity) at 37°C for 6 hours before phosphorylation of one-half of the materials and incubation with SNAP-25.
- FIGURE 9 is a graph depicting the predicted extent of inhibition of [ 3 H] -noradrenaline neurotransmitter from neuronal (PC12) cells on incubation of the cells with pp60 src phosphorylated BoTxE (closed circles) or unphosphorylated BoTxA (open circles) .
- FIGURE 10 depicts the single channel currents of pp60 src phosphorylated BoTxA HC reconstituted in lipid bilayer membranes .
- Unphosphorylated BoTx and TeTx Starting Materials Unphosphorylated TeTx and known serotypes of BoTx for use as starting materials to produce the toxin compositions of the invention may be purified to homogeneity using techniques which will be known to, or can be readily ascertained by, those of ordinary skill in the art .
- descriptions of each purified BoTx serotype are provided in Shone, Clostridium botulinum neurotoxins, their structures and modes of action, "Natural Toxicants in Foods" (Horwood, 1987) at pp 11-57.
- the manufacturing method utilized in producing a commercial BoTxA product supplied by the United Kingdom firm Porton under the tradename DYSPORTTM is set forth at Hambleton, J.
- BoTxA BoTx serotypes A, B and E
- a method useful in producing lyophilized BoTxA is also described in published EPO Application No. 0 593 176 A2.
- the disclosures of these references are each incorporated herein by this reference for the sole purpose of illustrating the state of knowledge in the art regarding the isolation and purification of BoTx and TeTx.
- the products of each isolation and purification technique known in the art are unphosphorylated toxins .
- substantially pure BoTx or TeTx may be obtained through microbial expression, by synthesis, or by purification means known to those skilled in the art, such as affinity chromatography.
- substantially pure as used herein denotes a protein which is substan ⁇ tially free of other compounds with which it may normally be associated in vivo .
- the term refers to homogenous toxin, which homogenicity is determined by reference to purity standards known to those of ordinary skill in the art (e.g., purity sufficient to allow the N-terminal amino acid sequence of the protein to be obtained) .
- BoTx and TeTx peptides can be synthesized by such commonly used methods as t-BOC or Fmoc protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (see.
- Peptides of the invention can also be synthesized by various well known solid phase peptide synthesis methods, such as those described by
- styrene-divinylbenzene containing 0.1-1.0 mMol amines/g polymer.
- the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1/4-1 hours at 0°C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on a "SEPHADEX G-15" or "SEPHAROSE" affinity column.
- Lyophilization of appropri ⁇ ate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
- BoTxA preparations may be obtained from commercial sources. Examples of such sources and their respective products include BoTxA preparations supplied by Allergan, Inc. of Irvine, California under the tradename "BOTOX” and by Porton Products Ltd, of the United Kingdom under the tradename "DYSPORT” .
- BOTOX X-ray photoelectron-activated X
- DYSPORT DYSPORT
- a pentavalent toxoid of all eight known Botulinum serotypes is also available as an investigational drug from the U.S. Center for Disease Control in Atlanta, Georgia. Preparations of individual BoTx serotypes are also available from Wacko of Tokyo, Japan. Of the individual BoTx serotype preparations, BoTxA preparations are currently the most preferred for their known safety and efficacy.
- Tetanus toxins for use as vaccines are also commercially available from, for example, Lederle Laboratories of Wayne, New Jersey under the tradename "TETANUS TOXOID PUROGENATED” as well as from Sigma Chemical of St. Louis, Missouri. and Calbiochem of San Diego, California.
- charge-modified tyrosine residues shall refer to both negatively charged tyrosine residues (prepared according to the invention) and toxins wherein tyrosine residues have been substituted with glutamate, aspartate, or non-naturally occurring amino acids having a negative charge.
- L- bond peptides are susceptible to proteolytic degradation, restricting their application as drugs.
- this obstacle has been successfully bypassed in some cases by synthesizing analogues which contain D-bond amino acids or non-natural amino acids.
- the addition of a single D- amino acid at the C-terminal position is enough to enhance the resistance to proteolytic degradation by exopeptidases, without significantly altering the secondary structure of the peptide [Abiko, supra] .
- Resistance to endopeptidases can be achieved by including individual non-cleavable non-peptidic bonds in points in the peptide sequence that are specially sensitive to enzymatic degradation [Meyer, et al . , J. Med. Chem .
- the invention includes BoTx and TeTx modified to substitute negatively charged, non-natural amino acids for tyrosine residues in the toxins.
- charge-modified toxins of the invention can be synthesized in all-D-conformations. All-D-peptides can be equally active as the original all-L-peptides [Merrifield, et al . , Ciba Founda tion Symposium 186 : 5-20 (1994) ; Wade, et al . , Proc . Na tl . Acad . Sci . USA £2:4761- 4765 (1990)] , capable of successfully resisting enzymatic degradation [Wade, supra ; King, et al . , J. Immunol . 153 :1124-1131 (1994)] and less immunogenic than their all-L-analogues [King, supra] .
- pp60 src is a member of the src family of protein kinases that are known to phosphorylate a focal adhesion kinase and induce morphological transformations (e.g., rounding and detachment) in affected cells.
- pp60 src is also abundant in brain synaptic vesicles. pp60 src is obtainable by isolation and purification from native sources (e.g., from brain tissue homogenates) or may be purchased commercially from sources such as United States Biochemical of Cleveland, Ohio.
- Phosphorylation of tyrosine residues in BoTx and TeTx by pp60 src is selective (i.e., residues other than tyrosine are not phosphorylated) and occurs throughout both the LC and HC (FIGURE 2 and Example 1) .
- residues other than tyrosine are not phosphorylated
- half- maximal phosphorylation of the tyrosine residues appears to occur within about 30 minutes on incubation of 250 nanomolar BoTxA starting material with as little as about 3-6 Units of pp60 src in buffer (FIGURE 3 and Example 1) .
- This ratio of starting material to enzyme (about 250 nanomolar: 3-6 Units) is one in which a minimal amount of enzyme may be used to preserve the enzyme resource and is therefore preferred over effective, but less efficient, protocols in which larger quantities of enzyme are utilized.
- the reaction is reversible (see. FIGURE 3 and Example 1) .
- Similar results are obtainable with another tyrosine kinase recently discovered in neuronal PC12 cells, PYK2 [Lev, et al . , Nature, 376:737-745 (1995)] .
- PYK2 tyrosine kinase
- Tyrosine phosphorylation is a phenomenon which is a key step in signal transduction pathways mediated by membrane proteins.
- other tyrosine kinases such as other members of the src kinase family
- phosphorylating agents in the method of the invention.
- Modification of serine or threonine residues in the toxins does not affect the toxins' activity or stability. Thus, serine phosphorylating agents would not be expected to be useful in the method of the invention.
- phosphorylation may also be produced chemically; i.e., without reliance on a catalytic reaction.
- Reagents known to be useful in phosphorylating tyrosine residues in proteins include di-t-butyl N,N- diethyl-phosphoriamidite (for phosphorylation in a t- butyl phosphate protection reaction; see. e.g.. Perich and Reynolds, Int . J. Pept . Protein Res . , 32:572-575 (1991)
- substitution of tyrosine residues with glutamate or aspartate methods for substituting amino acids in a protein are well-known in the art.
- substitution of tyrosine with aspartate and glutamate may be readily performed by one of ordinary skill in peptide synthesis.
- a desirable mutation may be produced by site-specific mutagenesis using a conventional polymerase chain reaction (PCR) and a primer pair corresponding to the 3 ' and 5 ' regions of the cDNA.
- PCR polymerase chain reaction
- a preferred method of mutation-generating PCR amplification is the overlap extension PCR technique described by Ho, et al .
- this technique accomplishes site- specific mutagenesis of the clone by utilizing a 3 ' primer to add the mismatched mutating bases (primer B in the Ho article, which is used with the 5' primer A in the first PCR cycle described) .
- Amplification using the A and B primers yields an AB fragment .
- a second PCR cycle uses a primer (D) from the 3 ' end of the gene and a 5 ' mutated primer C complementary to primer B.
- the resulting amplification product (fragment CD) will overlap the AB fragment.
- the resulting fusion product will contain the full-length cDNA sequence and the desired mutation.
- Another suitable approach to making single base substitutions or deletions is described by Shaw in U.S. Patent No. 4,904,584 ("Site-Specific Homogenenous Modification of Polypeptides") , the disclosure of which is incorporated herein by this reference for purposes of illustrating knowledge in the art regarding methods for achieving specific mutations in polynucleotides and polypeptides.
- Enhancement of the activity and thermal stability of the toxin products of the inventive process will vary depending on whether the LC, HC or both are phosphorylated.
- both chains will be phosphorylated for maximal enhancement of the toxins' properties, including enhancement of thermal stability (FIGURE 7 and Example 3) .
- phosphorylation of the LC results in enhancement of the toxins' proteolytic properties (see. FIGURES 4 through 6 and Example 2)
- phosphorylation of the HC appears to enhance the toxins' channel gating capabilities by extending the time in which the membrane channels formed by the HC remain open for translocation of the LC into the cytosol (see. FIGURE 10 and Example 6) .
- Selective enhancement of either the channel gating or proteolytic activities of the toxin is achievable by phosphorylating only the HC or LC, respectively.
- Selective phosphorylation of either chain of BoTx or TeTx is performed by cleaving the HC and LC chains for phosphorylation of the tyrosine residues on only the chain to be phosphorylated or by phosphorylating both chains, cleaving the chains and reversing phosphorylation on one chain by applying a dephosphorylating agent such as protein-tyrosine phosphatase IB (FIGURE 4 and Example 2) .
- Compositions containing mixtures of the phosphorylated and unphosphorylated chains are prepared as described below with respect to the fully phosphorylated toxins.
- the scope of the invention encompasses phosphorylation of BoTx and TeTx (and the resulting phosphorylated product) using reagents of varying phosphorylating efficiencies, so long as a demonstrable enhancement of the toxin's proteolytic activity as compared to the unphosphorylated toxin occurs.
- the thermal stability of the phosphorylated toxin is also enhanced as compared to the unphosphorylated toxin.
- BoTx or TeTx having both chains phosphorylated according to the invention can be expected to retain its proteolytic activity in saline at room temperature for at least 5 times longer than the unphosphorylated toxin (FIGURE 7 and Example 4) .
- toxin stability within the meaning of the invention (i.e., as a result of providing a negative charge at the sites of tyrosine residues in the toxin) will be considered to occur when retention of the proteolytic activity of the negatively charged toxin in saline at room temperature (37° C) compared to the unphosphorylated toxin is detectably increased; e.g., by at least about 10%.
- the scope of the invention encompasses negatively charged BoTx and TeTx (and the resulting products) using reagents of varying efficiencies, so long as a demonstrable enhancement of the toxin's stability as compared to the unphosphorylated toxin occurs.
- tyrosine phosphorylation of BoTx and TeTx LC has the unexpected effect of restoring proteolytic activity of unphosphorylated toxins which have become inactivated; e.g., after excessive exposure to moderate heat (see. FIGURE 8 and Example 5) .
- the methods of the invention for preparation of toxins having charge- modified tyrosine residues is useful both in producing pharmaceutically acceptable BoTx and TeTx compositions having enhanced activity as well as restoring activity to conventional unphosphorylated BoTx and TeTx compositions after inactivation.
- Such assays will be useful not only in determining whether modification of the charge of a particular toxin has enhanced its activity or stability within the meaning of the invention, but will also be useful in determining whether a particular modification of the charge adversely affects the secondary structure and catalytic activity of the toxin to aid in selection and design of pharmaceutically useful negatively charged toxins having charge-modified tyrosine residues according to the invention.
- compositions of the invention are prepared by mixing the charge-modified toxin having the desired degree of purity with physiologically acceptable carriers.
- physiologically acceptable carriers will be nontoxic to recipients at the dosages and concentrations employed.
- the preparation of such compositions entails combining the particular protein with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients.
- compositions may be lyophilized for storage and will be reconstituted according to pharmaceutically acceptable means; i.e., suitably prepared and approved for use in the desired application.
- a sodium chloride free buffer is preferred for use as a reconstituting agent .
- BSA or HSA bovine or human serum albumin
- the composition product will be placed into sterile containers (e.g., ampules) for storage and transportation.
- the phosphorylation reaction was conducted in a final volume of 20-40 ⁇ l containing 20 mM MgCl 2 , ImM EGTA, ImM DTT, 20 mM Hepes (pH 7.4) , 3-6 Units of tyrosine kinase pp60 src , 0.1 mM ATP and 4 ⁇ Ci [ 32 P] - ⁇ -ATP (3,000 Ci/mmol; Amersham) .
- the phosphorylation buffer was: 20 mM Hepes, pH7.4, 0.1 mM CaCl 2 , lOmM MgCl 2 , 0.25 mg/ml of L- ⁇ -phosphatidyl-L-serine and 1 mM DTT.
- PKC and PKA phosphorylated samples were subjected to SDS-PAGE on 12% gels. Gels were stained with Coomassie blue R-250, destained, dried and exposed to Kodak X-Omat ARTM x-ray film. For immunoblots, protein bands were electrotransferred onto nitrocellulose membranes. Bands were visualized using the ECL radiographic system (Amersham) . Control samples included samples from which kinases had been omitted. For pp60 src , an additional control sample was preincubated with peptide A.
- BoTxA was strongly phosphorylated by pp60 src but not by the serine kinases PKA or PKC.
- the autoradiograms display incorporation of 32 P into both LC (M r ⁇ 50kD) and HC ( r ⁇ 100kD) (FIGURE 1) .
- pp60 src also phosphorylates the HC and LC of BoTxA, BoTxB, BoTxE and TeTx.
- Autoradiograms display 32 P incorporation into protein (FIGURE 2) .
- the phosphorylation reaction between pp60 src , BoTx and TeTx is reversible.
- dephosphorylation was conducted in vi tro in a final volume of 15 ⁇ l containing 0.1 ⁇ M tyrosine-phosphorylated BoTxA and 50 ng PTP-lB-agarose bead conjugate. Reactions proceed at 37°C for indicated times, and were terminated by centrifugation (14,000 rpm, 1 min) of the PTP-agarose bead conjugate. The extent of BoTxA dephosphorylation was assessed by SDS-PAGE. Controls included omission of PTP or inhibition of PTPwith 200 ⁇ M V0 4 . Phosphorylation of BoTxA was reversed under these conditions.
- Tyrosine phosphorylation of BoTxA is accompanied by augmentation of protease activity.
- Fluorograms display the mobility change of in vi tro translated SNAP-25 due to proteolysis as function of phosphorylation time by Src.
- Last lane shows a control experiment in which BoTxA was omitted, e.
- the concentration of modified and unmodified BoTxA or BoTxE incubated with samples of SNAP- 25 as shown in FIGURE 4 was 10 nM and varied as shown in FIGURES 5 and 6; cleavage reactions were performed at 30°C.
- the unmodified toxin was dephosphorylated as described in Example 1 and the effect of dephosphorylation measured as a function of a decline in proteolytic activity.
- the unmodified toxin was a conventional preparation of BoTxA.
- the unmodified toxin was a conventional preparation of BoTxE.
- the modified BoTxA displayed as much as 50% greater proteolytic activity than the unmodified toxin (closed circles; Panel A displays the results obtained as a function of toxin concentration while Panel B displays the results as a function of time) .
- the enhancement of activity on the part of modified BoTxE (closed squares) as compared to unmodified BoTxE (closed circles) was similar to the enhancement measured in modified BoTxA (Panel A displays the results as a function of toxin concentration while Panel B displays the results as a function of time) .
- FIG. 7 Augmentation of BoTx protease activity by protein tyrosine phosphorylation is accompanied by increased thermal stability (FIGURE 7) .
- This enhancement in thermal stability was determined by measuring the rates of cleavage of in vi tro translated SNAP-25 by tyrosine phosphorylated BoTxA (open squares and circles) or unphosphorylated BoTxA (closed squares and circles) which were preincubated at either 22°C (squares) or 37° (circles) prior to the cleavage assay. The assay was conducted at 30°C at a fixed toxin concentration of 20 nM.
- Example II The modified toxin samples were incubated with SNAP-25 as described in Example II. As shown in FIGURE 8, the modified toxin displayed proteolytic activity similar in magnitude to the modified toxins described in Example III (closed squares) while the unmodified toxin displayed little proteolytic activity (closed circles) .
- Tyrosine-phosphorylated proteins were immunoprecipitated with an antiphosphotoyrosine monoclonal antibody. Immunocomplexes were captured with agarose-con ugated protein G (Pierce) , and analyzed by SDS-PAGE and immunoblotting. Bands were visualized using the ECL system (Amersham) to confirm incorporation of the toxins into the cells. The cells were stimulated with nerve growth factor and the extent of [ 3 H] -labeled noradrenaline neurotransmitter release from the cells measured.
- modified BoTxA were reconstituted into lipid bilayer membranes. As shown in FIGURE 10, the channel formed by the modified toxin exhibits a high probability of being in the open state (top current level) . This indicates augmentation of the ion channel activity of the modified toxin HC in comparison to unmodified toxin.
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Abstract
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PCT/US1996/003228 WO1997032599A1 (fr) | 1996-03-08 | 1996-03-08 | Clostridiatoxine chimiquement modifiee a caracteristiques ameliorees |
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Cited By (7)
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WO2002008268A2 (fr) * | 2000-07-21 | 2002-01-31 | Allergan, Inc. | Motif a base de leucine et neurotoxines clostridiales |
AU2002219850B2 (en) * | 2000-11-17 | 2006-08-10 | Allergan, Inc. | Modified clostridial neurotoxins with altered biological persistence |
US7491799B2 (en) | 2000-07-21 | 2009-02-17 | Allergan, Inc. | Modified botulinum neurotoxins |
US7691983B2 (en) | 2000-07-21 | 2010-04-06 | Allergan, Inc. | Chimera botulinum toxin type E |
US7888469B2 (en) | 2000-11-17 | 2011-02-15 | Allergan, Inc. | Post-translation modification and clostridial neurotoxins |
WO2011023213A1 (fr) * | 2009-08-28 | 2011-03-03 | Merz Pharma Gmbh & Co. Kgaa | Agents de chimiodénervation modifiés |
WO2016110662A1 (fr) * | 2015-01-09 | 2016-07-14 | Ipsen Bioinnovation Limited | Neurotoxines cationiques |
-
1996
- 1996-03-08 WO PCT/US1996/003228 patent/WO1997032599A1/fr active Application Filing
- 1996-03-08 AU AU63268/96A patent/AU6326896A/en not_active Abandoned
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EP1849801A1 (fr) * | 2000-07-21 | 2007-10-31 | Allergan Sales, Inc. | Motif à base de tyrosine et neurotoxines clostridiales |
US7534863B2 (en) * | 2000-07-21 | 2009-05-19 | Allergan, Inc. | Leucine-based motifs and enhanced biological persistence of clostridial neurotoxins |
US8017741B2 (en) | 2000-07-21 | 2011-09-13 | Ester Fernandez-Salas | Chimera botulinum toxin type E |
US7888469B2 (en) | 2000-11-17 | 2011-02-15 | Allergan, Inc. | Post-translation modification and clostridial neurotoxins |
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US11014968B2 (en) | 2015-01-09 | 2021-05-25 | Ipsen Bioinnovation Limited | Cationic neurotoxins |
US12037369B2 (en) | 2015-01-09 | 2024-07-16 | Ipsen Bioinnovation Limited | Cationic neurotoxins |
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