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United States Patent (19) 11 Patent Number: 4,929,718 Possani Et Al

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United States Patent (19) 11 Patent Number: 4,929,718 Possani Et Al United States Patent (19) 11 Patent Number: 4,929,718 Possani et al. 45 Date of Patent: May 29, 1990 54 SYNTHETIC NOXIUSTOXIN RELATED Fletcher B. Taylor, Jr., et al., Thrombosis Research, PEPTDES “Clots Lysis: Effects of Protein C''', 37: 639-649, 1985. Hector H. Valdiva, et al., Elsevier Science Publishers 75 Inventors: Lourival D. P. Possani; Georgina B. B.V. (Biomedical Divison) “FEBS Letters', 226(2); Gurrola, both of Cuernavaca; Marco 280-284, 1988. A. A. C. Bayon; Maria B. Sitges, both E. Carbone, et al., European Journal of Physiology, of Mexico, all of Mexico "Blocking of the Squid Axon L-- Channel by Noxius 73 Assignee: Universidad Nacional Autonoma De toxin', 408; 423-431, 1987. Mexico, Dependence, Mexico Maria Sitges, et al., The Journal of Neuroscience, 21 Appl. No.: 132,169 “NTX-Induced Transmitter Release and K-- Permea bility’, 6(6): 1570-1574, 1986. 22 Filed: Dec. 14, 1987 51) Int. Cl. ........................... C07K 7/06; C07K7/08 Primary Examiner-Lester L. Lee 52 U.S. Cl. .................................... 530/326; 530/328; Attorney, Agent, or Firm-Fleit, Jacobson, Cohn, Price, 530/324; 530/858 Holman & Stern 58 Field of Search ............... 530/324, 858, 328, 326; 57 ABSTRACT 514/12 The invention relates to a novel immunogenic synthetic 56) References Cited peptide that is capable of specifically blocking potas U.S. PATENT DOCUMENTS sium channels of excitable membranes and inducing immunity against an effective lethal dose of Noxius 4,499,080 2/1985 Duflot et al. ........................ 530/324 toxin. The invention also relates to the discovery that OTHER PUBLICATIONS the immunogenic and blocking properties of the peptide L. D. Possani et al., Carlsburg Res. Commun., "Primary are severable. Peptides possessing either the immuno Structure of Noxiustoxin', 47: 285–289, 1982. genic or the blocking property are disclosed. Anthony T. Tu, Marcel Dekker, Inc. New York & Basel, “Handbook of Natural Toxins',m 2: 513–543. 3 Claims, 3 Drawing Sheets U.S. Patent May 29, 1990 Sheet 1 of 3 4929,718 O. E SOO5 CN C OO O 5 O Time (min) F. G. A O. 6O s OO5 3On CN amp C g OO O Time (min) F. G. B U.S. Patent May 29, 1990 Sheet 2 of 3 4,929.718 Cud U C) H ol U d >C m 2 S2 H L C H 2 > CO (%) asdale wave-H U.S. Patent May 29, 1990 Sheet 3 of 3 4,929,718 - - - - - ( : 2 \ s 2 4 21 4. 3. 3 FIG. 3 4,929,718 1. 2 nesses such as cardiac arrhythmias or others which SYNTHETIC NOXUSTOXN RELATED cause alterations at the level of ionic channels. PEPTDES It is therefore an objective of this invention to pro vide polypeptides with immunogenic properties, syn BACKGROUND TO THE INVENTION 5 thesized according to the amino acid sequences of Noxi The use of toxins as tools in neurophysiological stud ustoxin with molecular weights in the range of 1,000 to ies began with the discovery of Tetrodotoxin. Thereaf 4,200. ter the application of neurotoxins isolated from scorpion Another objective of this invention is to obtain poly venoms has been widely investigated. peptides which have the property of blocking potassium More than 110 different polypeptides have been iso 10 ionic channels. lated and characterized from the venom of 21 different A further objective is to provide polypeptides which species of scorpions which exist in different geographi are of use in the preparation of drugs and vaccines. cal regions of the world. An additional objective of this invention is to provide The chemical structure of many toxins from scorpion products which can be used in the treatment of cardiac venom has been studied and has been found to consist of 15 arrhythmias and/or illnesses which cause alterations to compact molecules, stabilized 3 to 4 disulphide bridges the level of the ionic channels. with two-and-half turns of alpha-helix and three-strand These objectives, among others, can be appreciated stretch of antiparallel beta sheet which runs parallel to in greater detail in the following section. the alpha-helix. These data were obtained by X-ray diffraction and circular dichroism studies. The use of 20 BRIEF DESCRIPTION OF THE FIGURES optical rotatory dispersion measurements has shown FIG. 1a represents the separation by HPLC of syn that some toxins from the venom of Centruroides noxius thetic peptide NTX 1-9 through a molecular weight and Tityus serrulatus are highly thermostable: they can sieving column (Whatman I-125) in triethylamine-phos be boiled for five minutes and still fold back to their phoric acid buffer, pH 2.25, in the presence of acetoni original three-dimensional structure, preserving their 25 trile (ratio 2:1 by volume), at a flow rate of 1 ml/min. toxicity. Two families of toxins have been isolated: one FIG. 1b represents separation by HPLC of synthetic with a short chain (approximately 39 amino acid resi peptide NTX 1-9 through a C18 reverse phase column. dues) and another with a long chain (61 to 70 amino acid A gradient from 100% A (0.12% trifluoroacetic acid residues). In order to obtain information on the active site of 30 (TFA) in water) to 60% B (0.12% TFA in acetonitrile) these peptides, several chemical modifications have was run for 70 minutes with a flow rate of 1 mi./min. 50 been carried out on some known toxins and it has been ug of peptide was applied to the column. The peak on found that a reduction of the disulphide bridges, a modi the elution profile labeled “P” is pure NTX 1-9 and that fication of the carboxylic groups and acetylation of labeled 'A' is an artifact of the solvent wash. us amino groups can change their toxicity. This suggests 35 FIG. 2 depicts the effect of native Noxiustoxin and the importance of some amino acids in the toxin's activ the synthetic peptides on GABA release from Brain ity. Synaptosomes. The applicant has carried out comparative studies - FIG. 3 shows the result of immunodiffusion on 0.8% into the primary structure of scorpion toxins using the agarose gel in phosphate-saline buffer. method of metric analysis with which it has established 40 DETALED DESCRIPTION OF THE the existence of at least five different groups of toxins NVENTION with similary homologous amino acid sequences in which the preservation of cysteines in all the toxins This invention is related to polypeptides of 9 to 39 studied seems to be very important for the stabilization amino acids with a similar sequence to that of Noxius of the molecules, and hence for their function. 45 toxin characterized by the applicant with the objective Similarly, the applicant has synthesized polypeptides of producing it synthetically by some appropriate whose structure is related to the functioning of neuro method. toxins and which are the basis for the preparation of The basic sequence synthesized is the following: synthetic vaccines and drugs. Threonine-Isoleucine-Isoleucine-Asparagine-Valine This invention is based on synthetically obtaining 50 Lysine-Cysteine-Threonine-Serine-Proline-Lysine polypeptides of the Noxiustoxin type from the scorpion Glutamine-Cysteine-Serine-Lysine-Proline-Cysteine Centruroides noxius. This toxin represents only 1% of Lysine-Glutamic acid-Leucine-Tyrosine-Glycine the scorpion's soluble venom and its effective lethal Serine-Serine-Alanine-Glycine-Alanine-Lysine-Cys dose is in the order of 100 g. per 20 gr. of body weight teine-Methionine-Asparagine-Glycine-Lysine-Cys of albino mice. These compounds belong to the short 55 teine-Lysine-Cysteine-Tyrosine-Asparagine-Asparag chain peptide family (39 amino acid residues) which are 18. stabilized by three disulphide bridges and block potas Several polypeptides containing a partial amino acid sium channels from excitable membranes. sequence of the basic sequence described above were synthesized. SUMMARY OF THE INVENTION The results obtained were outstanding due to the fact This invention refers to polypeptides with an amino that polypeptides corresponding to the C-terminal re acid sequence similar to that of Noxiustoxin: they con gion of the Noxiustoxin sequence present immunogenic sist of a number of amino acids which varys from 9 to properties and have no toxic effect. For this reason they 39; offer good immunogenic responses and for this rea are of use in the preparation of vaccines and/or im son are useful in the preparation of an antitoxin scorpion 65 provement of the serum used against scorpion stings. vaccine and some of them, which have the property of The polypeptides corresponding to the N-terminal blocking potassium channels, can be used in the prepa region of Noxiustoxin present a toxic effect to mice and ration of new drugs particularly for treatment of ill block the potassium channels of excitable membranes 4,929,718 3 4. such as brain synaptosomes, dorsal root ganglion cells another three times with methylene chloride. The BOC and cardiac tissue. These characteristics allow the pep Asparagine derivative is dissolved in 10 ml. of DMF tides to be used as tools to study ionic channels or as plus 20 ml. of methylene chloride and the mixture is new drugs for the treatment of illnesses like cardiac added to the amino methyl resin. It is left in agitation for arrhythmias involving the potassium channels. 10 minutes after which 1.3 millimoles of dicyclohexyl Some examples of synthesized polypeptides are pres carbodiimide (DCC) are added and the product is left ented below in order to illustrate more fully the objec under agitation at room temperature for 36 hours. tives of this invention. (E) Acethylation of the free amino groups I-Synthesis of Peptides with PAM-Resins 10 In order to block undesirable free amino groups 2 ml.
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