CN1352682A - Cysteine protease and inhibitors for prevention and treatment of neurocysticerocosis - Google Patents
Cysteine protease and inhibitors for prevention and treatment of neurocysticerocosis Download PDFInfo
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- 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)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
- C12N9/641—Cysteine endopeptidases (3.4.22)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
A Taenia cyst wall cysteine proteinase and inhibitors thereof. Also included are pharmaceutical compositions and vaccines for treatment of Taenia infection; methods of inhibiting the activity of a cyst wall cysteine proteinase, and computer-assisted methods for identifying inhibitors of cyst wall cysteine proteinase activity.
Description
The sequence number that the application requires on April 21st, 1999 to apply for is 60/130338 U.S. Provisional Application No., and this paper introduces its full content as a reference.
The statement of government rights
The present invention has obtained the governmental fund support of the U.S./Mexico's foundation (US/Mexico Foundation) and N.I.H.Molecular Parasitology Training Grant For CellBiology of Parasites and Vectors, and fund number is respectively 42-H-94 and AI07322.United States Government enjoys certain right to the present invention.
Background of invention
Neurocysticerocosis is one of modal human central nervous system parasitosis and is the inducement of epileptic seizures in many third world area.At present, the existence at neurocysticerocosis had not both had methods of treatment in full force and effect not have vaccine (Garcia etc., transmissible disease magazine (J.Infec.Dis.), 175:486-489 (1997)) yet.Estimate to have in the world 50,000,000 people to be subjected to the influence of the disease that the packing by taeniasis suis-Taenia solium causes, although the epidemic research result thinks that this estimation numerical value is on the low side.This is that the epileptics sickness rate of developing country in the world is the most probable reason of the twice of developed country.The symptom relevant with neurocysticerocosis comprises epileptic seizures, headache, visual disorder, clouding of consciousness and psychosis.Yet, have the asymptomatic latent period more than 10 years or 10 years, usually near the survival desired value of organizing packing.Result of study shows organizes packing to finish their life cycle by the inflammatory response that suppresses the host, and shows clinical symptom (Evans etc., Emerg.Infect.Dis.3:403-405 (1997)) during packing degeneration and death.
Along with immigrant's increase, cysticercosis becomes a kind of emerging transmissible disease in the U.S..Greater than 1000 examples, as and if this quantity is also rising in the case quantity survey of the annual cysticercosis cellulosae of the U.S..For example, in past three year, what 10% trouble was arranged in the people that Los Angeles County USC MedicalCenter is in hospital because of psychosis is neurocysticerocosis, and its sum reaches about 120 case/years.The infectious rate of certainly, hiding can be higher.The case that this area obtains pellet not, also there has been the data description in San Diego, New York, Chicago and other unexpected area.For example, it is positive that the people of 1.3% in traditional New York Jew community is found to be the neurocysticerocosis test.Although do not reckon with that owing to traditional Jew's fasting pork they have the danger of being infected, they are infected owing to Latin (latino) the immigrant home services personnel's (carrier of tapeworm) that suffered from teniasis pollution according to reports.There is four million peoples to infect to have lived away from home the teniasis of the taeniasis suis in adult stage in the whole world according to reports.Suffer among patients everyone for these, estimate at 10 or more people and infected the taeniasis suis of cystic stage, promptly suffer from cysticercosis.Because worm's ovum can be propagated in environment, so in the popular area of height, almost anyone has the danger of suffering from neurocysticerocosis.
The mankind are unique specific host of armed tapeworm adult, and the armed tapeworm adult sticks on the intestines by its cephalomere and rests in the intestines.In case the people becomes the parasitic accidental intermediate host of packing form, the people has just suffered from neurocysticerocosis, and normally fecal pollution or direct the contact with the tapeworm carrier by the people obtained worm's ovum.Pig is conventional intermediate host.For the people, packing is more prone to reside in central nervous system more, thereby popular neurocysticerocosis.
Because taeniasis suis has only two hosts: people and pig, so The InternationalTask Force for Disease Eradication is defined as neurocysticerocosis need make the target of eradicating effort.Although the high-quality swine rearing of the West Europe and U.S. industry is obtaining some successes aspect the propagation of prevention taeniasis suis, but these methods are domed to failure in developing country, because poor peasant likes selling infected pig in informal stand usually, and is unwilling they to be surrendered and may be punished.In fact some prefer edible pork of suffering from cysticercosis (' measly ') according to reports.
Until today, also do not develop the anti-parasitic vaccine of any mankind that comprises taeniasis suis.Recently the chemical therapeutic method that studies show that large-scale elimination neurocysticerocosis has more prospect and practicality.For example, tentatively use praziquantel (TM) to treat on a large scale, make cysticercosis cellulosae drop to 2.6% from 11.4 in Ecuador.Usually adopt parasiticide compound praziquantel, albendazole (TM) or The combined to come therapeutic activity essence neurocysticerocosis at present.Patient with praziquantel treatment after then with the albendazole treatment, the result shows eliminating having little effect of packing.Yet some patients present the inflammatory reaction of deterioration around the packing, and that the symptom of following comprises is nauseating, vomiting and headache; Some patients develop into cerebral edema.Yet some reports say that these medicines can control neurocysticerocosis patient's epileptic seizures, but do not know that these discoveries are the result of treatment or the result of the deviation of patient selection.What therefore, need especially to pay close attention to is the danger that develops into the acute inflammation relevant with these medicines, cerebral edema and possible chronic hydrocephalus.Although some studies show that albendazole may be more effective than praziquantel, it is also relevant with hematopoietic disorder and hepatic insufficiency and may be teratogenesis/mutagenesis.Carpio and colleagues have carried out maximum once treating with double blind trial at random with albendazole and praziquantel to neurocysticerocosis, as if the conclusion that report recently draws is that any effect is relevant with the natural process that infects, and with any specificity effect of pharmacological agent irrelevant (Arch.Int.Med., 155:1982-1988 (1995)).As if the anti-parasite medicine that only is not used for the neurocysticerocosis treatment.Very clear, need a kind of more effective and specific anti--medicine of neurocysticerocosis, improve existing back work for the treatment of possibly as far as possible.
Summary of the invention
As if found the cyst wall L-Cysteine HCL Anhydrous in the parasite taeniasis suis, this enzyme plays very important biological action in organism.The present invention relates to Taenia cyst wall cysteine protease and a kind of polynucleotide (and complementary strand) thus with nucleotide sequence of encoding cysteine protease.The invention still further relates to one or both the vaccine in a kind of polynucleotide that contain cyst wall L-Cysteine HCL Anhydrous (or its immunogenic polypeptide subunit) or a kind of nucleotide sequence with encoding cysteine protease (or its immunogenic polypeptide subunit).The cyst wall L-Cysteine HCL Anhydrous that is used in the vaccine preferably stems from taeniasis suis or taenia crassiceps.The present invention also comprises the preparation and the using method of this vaccine.
Also found the activity inhibitor of cyst wall L-Cysteine HCL Anhydrous.Therefore the invention still further relates to a kind of pharmaceutical composition, this pharmaceutical composition contains a kind of inhibitor molecules and pharmaceutically acceptable carrier that suppresses the cyst wall cysteine protease activity.Preferably a kind of peptide of described inhibitor molecules or peptide simulated compound, and preferably suppress to derive from the cyst wall L-Cysteine HCL Anhydrous of taeniasis suis and taenia crassiceps.In a preferred embodiment, inhibitor peptide or peptide simulated compound comprise (Xaa)
n-Yaa-Zaa-R; Wherein Xaa and Zaa independently are any amino acid separately; Yaa is a kind of hydrophobic amino acid; R comprises a nucleophilic composition; And n=0-5.More preferably, the N-of inhibitor peptide or peptide simulated compound end contains a protecting group.Described pharmaceutical composition is useful to treatment people's neurocysticerocosis or cysticercosis cellulosae.The present invention also comprises preparation and uses the method that suppresses compound and pharmaceutical composition.For example, the present invention includes the method that suppresses the cyst wall L-Cysteine HCL Anhydrous, this method comprises makes the cyst wall L-Cysteine HCL Anhydrous contact with a kind of inhibitor molecules.The method that suppresses the cyst wall L-Cysteine HCL Anhydrous can be in the environment that does not have cell, in the cell culture, in the organ, in the tissue or carry out in intact animal such as people or the pig.
The present invention also comprises a kind of method of identifying tapeworm cysteine protease activity inhibitor.A kind of candidate inhibitor combines with the tapeworm L-Cysteine HCL Anhydrous and forms a kind of mixture.Then protease substrate Z-Phe-Arg-7-amino-4-trifluoromethyl tonka bean camphor is added in the mixture, measures the cleaved degree of protease substrate.Be construed as that the ingredients of a mixture can add simultaneously or add with the order of any needs.Substrate cracking degree when not having candidate inhibitor to exist is compared, and the reduction of substrate cracking degree represents to suppress the tapeworm cysteine protease activity.
The present invention also comprises a kind of computer-householder method that is used to identify tapeworm cysteine protease activity inhibitor.In one embodiment, provide a kind of computer model of tapeworm cysteine protease activity inhibitor structure, then by calculate or range estimation screening structure storehouse in similar in the candidate compound of described inhibitor.Perhaps, the candidate compound that can project organization be similar to described inhibitor.Randomly, candidate compound can suppress the activity of tapeworm L-Cysteine HCL Anhydrous.In another embodiment of computer-ancillary drug method of design, set up a kind of computer model of tapeworm L-Cysteine HCL Anhydrous by the X-ray crystal structure of tapeworm L-Cysteine HCL Anhydrous, the model of inhibitor molecules is by calculating or range estimation is docked with the protease crystals structure at the binding site of inhibitor.The molecular interaction of inhibitor and proteolytic enzyme is by calculating or estimating and identify, by calculating or estimate the structure candidate compound similar to described inhibitor structure in the screening structural database, perhaps by the calculation Design candidate compound.Then, analyze the molecular interaction of candidate compound and proteolytic enzyme, and randomly analyze the ability that candidate compound suppresses the tapeworm cysteine protease activity by calculating or range estimation.
The accompanying drawing summary
Fig. 1 represents 30 amino acid whose N-terminal sequences (SEQ ID NO:1) of (a) taenia crassiceps L-Cysteine HCL Anhydrous; (b) have other L-Cysteine HCL Anhydrous the catalysis cysteine residues (
*) sequence.
Fig. 2 is a) taenia crassiceps (example I) and b) comparison of the FPLC negatively charged ion-exchange chromatography of the activeconstituents of taeniasis suis (example II) after ACA 54 gels-filtering separation.
Fig. 3 represents by one group of cracking the different peptide substrates assessment purifying that replace a) taenia crassiceps (example I) and the b) substrate specificity of taeniasis suis (example II) L-Cysteine HCL Anhydrous are arranged on P1-P3.
Fig. 4 be pass through a) taenia crassiceps (example I) and b) L-Cysteine HCL Anhydrous of taeniasis suis (example II) represent the western blotting of IgG heavy chain degraded.
Fig. 5 represents to advance splenocyte from being removed by the taenia crassiceps mice infected on one's body by the taeniasis suis enzymatic of purifying.
Fig. 6 represents that multiple inhibitor suppresses its substrate of taeniasis suis protease cracking, Z-Phe-Arg-AFC.
Fig. 7 represents to derive from the scanning electron microscope diagram through the packing surface of the mouse of tapeworm cystatin treatment and control mice.The ratio of enlargement on the two sides in each group is identical.Group A (4000x): SEM represents the strong host immune response reaction at the packing for the treatment of through Z-LLY-FMK.Do not find immunocyte on the packing of not treating mouse deriving from.Group B (25000x): SEM result represents to come from inoblast, hole on the treatment mouse packing skin and the microtriche that comes off.Untreated packing microtriche looks long significantly.Untreated packing group has also been observed anchor and has not been observed anchor in the microtriche of the packing for the treatment of.
Fig. 8 represents the vitro cytotoxicity analysis of Z-LLL-FMK and Z-LLY-FMK inhibitor.
Detailed Description Of The Invention
Found a kind of cysteine proteinase of the host protein that comprises human IgG of can degrading in the cyst wall of people parasite pork tapeworm. Tapeworm cyst wall cysteine proteinase is very crucial for parasitic time to live, thereby important chemotherapy target is provided. The cyst wall cysteine proteinase of pork tapeworm and taenia crassiceps (mouse parasite) has the biochemistry similitude, and has found that inhibitor molecules suppresses the activity of these two kinds of enzymes specifically. The inhibitor of the cysteine proteinase of pork tapeworm and taenia crassiceps is applicable to treat or prevent to comprise people's animal reservoir's infection.
Therefore, the present invention relates to derive from the protease of pork tapeworm, preferably relate to a kind of cyst wall cysteine proteinase of separation, described protease can cracking substrate Z-Phe-Arg-7-amino-4-trifluoromethyl cumarin (Z-Phe-Arg-AFC; Wherein Z is the blocking group CH that also is known as " CBZ "5-CH
2-O-(C=O)-). Z-Phe-Arg-AFC is from Enzyme System Products, Livermore CA, and 94550 buy. Adopt such as this paper, the protein that " stems from " pork tapeworm, such as protease, should be broadly interpreted as and not only comprise the albumen that from pork tapeworm, adopts physical separation method to separate, but also comprise with any other method, have identical activity such as the protein by the preparation of chemical synthesis or recombinant DNA technology or the separation that obtains, and randomly, the protein of same acid sequence. Equally, term " pork tapeworm protein " is appreciated that and comprises protein and the function equivalent synthetic or restructuring thereof that separates from pork tapeworm. The term that this paper is used alternatingly " protease " refers to a kind of key that can crack protein matter, normally connects the enzyme of two amino acid whose peptide bonds.
Preferably, protease of the present invention has one or more following properties:
(1) can be rapidly and the heavy chain of the human IgG of degrading up hill and dale and light chain (for example, by adopting Western blotting to detect, non-cysteine proteinase of limiting the quantity of can be within about 120, the human IgG of the 1 μ g that preferably thoroughly degraded within about 45 minutes);
(2) it has the molecular weight of about 43kD to about 65kD;
(3), approximately has the optimum protein enzymic activity during pH 4.8 at acid pH;
(4) it is sulfydryl-activated; That is, its activity needs the external source mercapto groups, as the L-halfcystine;
(5) must be (preferably, greater than about 10 in the diffusion control limit with Z-Phe-Arg-AFC as the ratio of the Kcat/Km of substrate
8M
-1s
-1);
(6) it has only a substrate binding site;
(7) it meets Michaelis-Menten kinetics; That is, it is non-collaborative;
(8) it is suppressed by cystatin, but can not be suppressed to same degree by the proteinase inhibitor of other types; With
As if the protein decomposing activity of the proteolytic enzyme of new people parasite-taeniasis suis of identifying do not have substantive difference (White etc. with the activity that albuminoid showed of mouse parasite-taenia crassiceps, J.Mol.Biochem.Parasitol., 85:243-253 (1997)).
The invention further relates to a kind of isolating nucleic acid molecule that contains the nucleotide sequence of code book invention proteolytic enzyme.The complement of this nucleic acid molecule is also included among the present invention.
" isolating " biomolecules as polypeptide or polynucleotide, is that a kind of its physical environment of breaking away from adopts recombinant technology preparation, or through the biomolecules of chemistry or enzymic synthesis.Preferably, biomolecules of the present invention is a purifying,, is substantially free of any other biomolecules and relevant cell product or other impurity that is.Term used herein " polypeptide " or " polypeptide subunit " are meant aminoacid polymers and do not mean that the length-specific of aminoacid polymers.Therefore, for example term peptide, oligopeptides, protein and enzyme no matter be to adopt recombinant technology, chemistry or enzymic synthesis or naturally occurring, all are included in the definition of polypeptide.This term also comprises, for example by modification or polypeptides derived such as glycosylation, acetylize, phosphorylations.
The invention further relates to a kind of inhibitor of cyst wall L-Cysteine HCL Anhydrous.This inhibitor molecules suppresses the cyst wall L-Cysteine HCL Anhydrous, and preferably a kind of tapeworm cyst wall L-Cysteine HCL Anhydrous more preferably is the activity of taeniasis suis or taenia crassiceps cyst wall L-Cysteine HCL Anhydrous.Although inhibitor molecules may contain amino acid or deutero-amino acid, but its preferably a kind of peptide (that is a kind of amino acid or amino acid whose molecules of deutero-that contain two or more by the peptide bond connection) or a kind of peptide simulation (peptidomimetic) compound." peptide simulation " compound is the compound of a kind of function and/or a kind of peptide of structural simulation, but it lacks the peptide bond of one or more sign peptides.Therefore the peptide simulated compound is usually as the substrate of proteolytic enzyme and to bring into play the active time in vivo than similar peptide probably long.Except as otherwise noted, the term " peptide " of inhibitor peptide in the context or inhibitor molecules place use comprises peptide and peptide simulated compound.
In a preferred embodiment, inhibitor molecules contains a covalently bound nucleophilicity chemical part at its C-end.The character of nucleophyllic chemical adsorption reagent without limits and can comprise carboxylic acid derivative, amide derivatives, phenyl ring derivative, phenyl derivatives, pyridinyl derivatives etc.Suitable nucleophilic part is including, but not limited to chloromethyl ketone (CMK), methyl fluoride ketone (FMK), α ketone acid (AK), keto-amide (KA), keto ester (KE), vinyl sulfone(Remzaol (VS), pyridyl (Pyr) or its arbitrary combination.Preferred nucleophilic partly is methyl fluoride ketone (FMK).Preferred nucleophilic part can also be hydrophobic, thereby assists inhibitor molecules to pass hemato encephalic barrier.In addition or selectively, the preferred inhibitors peptide in the P2 site (P2 is from second amino acid position of C-end) contain a hydrophobic amino acid, preferred aliphatic series hydrophobic amino acid, more preferably leucine.In a preferred embodiment, although be construed as P1 and the P3 site can be any amino acid, on peptide P1 site, be tyrosine or leucine; And/or on the P3 site, be leucine.Particularly preferred inhibitor peptide comprises available from Enzym SystemProducts, Livermore, the Xaa of CA
n-Leu-Tyr-FMK and Xaa
n-Leu-Leu-FMK, wherein n=0-5; More preferably, n=0-2, for example Z-Leu-Leu-Tyr-methyl fluoride ketone (Z-LLY-FMK) and Z-Leu-Leu-Leu-methyl fluoride ketone (Z-LLL-FMK).Other preferred inhibitors peptide comprises (Ph)
2CHCO-Leu-Phe-CO-NHCH
2-2-pyridyl (Ph2-LF-KP); Z-Leu-Phe-CONH-ethyl (Z-LF-KE); Z-Leu-Phe-CONH-4-Morphophinyl (Z-LF-KM); Z-Leu-Phe-CH
2-CH (OH)-C
6H
4-(4-OPh) (Z-LF-BPh); Boc-Leu-Phe-vinyl sulfone(Remzaol-phenyl (B-LF-VS); With Z-Leu-Leu-Leu-vinyl sulfone(Remzaol-phenyl (Z-LLL-VS).
The N-of inhibitor peptide of the present invention or simulating peptide holds derivatize preferably.For example, it can covalently be connected to blocking group, as " Z " (CH
5-CH
2-O-(C=O)-it is also called " CBZ ") or " Boc " (uncle-butoxy) on.N-end blocking group is known in synthetic organic chemistry and peptide synthetic technical field.
The present invention also provides a kind of treatment and prevention to cause animal infected method by tapeworm.Preferably, described animal is ox, dog, pig or people.More preferably, animal is that infection by Taenia solium can effectively be treated and prevent to pig or people and described method.On the one hand, the medicine composite for curing ground that will contain inhibitor molecules of the present invention is applied to the animal that has infected tapeworm.In the embodiment of a methods of treatment, use inhibitor molecules and can eliminate animal parasite on one's body effectively; In another embodiment, using inhibitor molecules can effectively prevent or delay animal and cysticercosis or neurocysticerocosis occur.On the other hand, the pharmaceutical composition that contains inhibitor molecules of the present invention carries out preventive usage to the animal that is not subjected to cestode infection.In an embodiment of prevention method, parasite is to the infection of animal after using inhibitor molecules and effectively preventing.In another embodiment of prevention method, use inhibitor molecules and can effectively prevent or delay animal and continue and infected parasite and develop into cysticercosis or neurocysticerocosis later on.In another embodiment of prevention method, use inhibitor molecules and can effectively prevent the animal dead that has caused behind the parasite continue having infected.
Because described inhibitor molecules is expected to suppress to derive from the activity of the cyst wall L-Cysteine HCL Anhydrous of the various different tapeworms that comprise taeniasis suis and taenia crassiceps, has also considered the application of human aspect so both considered the application of beasts aspects.The application of beasts aspect comprises the prevention and the treatment of domestic animal, and this also can control to the crowd and propagate infection.Preferably, method of the present invention comprises to having infected tapeworm or having had pig or the people's medication of infecting tapeworm danger.For the mankind, preferably inhibitor is introduced in the blood flow, for example by intravenous injection or oral inhibitor.For pig,, preferably inhibitor is carried out medication as the preventive or the therapeutical agent of anti-cysticercosis cellulosae with the form of feed for the life cycle of tapeworm in the resultant interference pig body.This scheme probably can finally be eliminated human nerve's type cysticercosis.
EXAMPLE V has been described and has been used inhibitor peptide of the present invention to treat the cysticercosis of the mouse model that has infected tapeworm.These studies show that some with the mouse of cysticercosis fight in the highest protection level reported, and inhibitor is very effective at lower concentration.In addition, according to visual observation, do not show tangible toxic action with the mouse of inhibitor for treating.The result who studies these mouse shows that forcefully the inhibitor peptide is specific to existing only in parasite tapeworm cyst wall L-Cysteine HCL Anhydrous on one's body.In other words, inhibiting peptide is considered to disturb significantly the activity of mammalian hosts such as pig or people's proteolytic ferment.Therefore, estimate stronger with inhibitor molecules treatment infected animals of the present invention than the method specificity of the existing treatment neurocysticerocosis that comprises praziquantel and albendazole administration.These two kinds of medicines be find by drug screening method rather than by as be purpose method discovery to suppress specific tapeworm enzymic activity among the present invention.These two kinds of medicines are not very effective and the host are had significant side effects.
Inhibitor molecules of the present invention can be mixed with easily is used for beasts and human pharmaceutical composition.This pharmaceutical composition selectively comprises pharmaceutically useful vehicle or thinner and compatible with inhibitor molecules as carrier.Term " pharmaceutically acceptable carrier " refer to composition in the compatible meaning of other composition on " acceptable " and the carrier harmless to the recipient.Suitable vehicle is, for example, and water, salt solution, glucose, glycerol, ethanol etc. and composition thereof.In addition, if desired, pharmaceutical composition can contain more a spot of auxiliary substance such as wetting agent or emulsifying agent, the assistant agent that pH buffer reagent, salt and/or booster immunization-enhancement component is renderd a service.In preferred embodiments, prepared pharmaceutical composition for the transfer that allows or promote inhibitor molecules to pass through human blood/brain barrier.The present invention also comprises preparation and uses the method for this pharmaceutical composition.
Inhibitor molecules can be formulated into the form of unitary dose and can prepare by known any method in the pharmaceutics field.All methods comprise the step that active compound is combined with the carrier that constitutes one or more ancillary components.In a word, prepare preparation, then, if desired, product is shaped to required preparation by active compound and liquid vehicle, finely divided solid carrier or both are carried out all even the combination closely.
It can be isolating unit such as tablet, lozenge, capsule, lozenge, wafer or cachet that the present invention is suitable for oral preparation, and each all contains powder or the solution in particle inhibitor, the liposome that contains inhibitor or water or the on-aqueous liquid or suspension such as syrup, elixir, emulsion or the Haust (draught) of predetermined amount.This composition and preparation should contain 0.1% active compound at least.The percentage composition of composition and preparation can change and can be suitably given unit dosage form weight about 1% to about 60% between value.
Tablet, lozenge, pill, capsule etc. can also contain one or more following ingredients: tackiness agent such as tragakanta, gum arabic, W-Gum or gelatin; Vehicle such as Lin Suanergai; Disintegrating agent such as W-Gum, yam starch, Lalgine etc.; Lubricant such as Magnesium Stearate; Sweeting agent such as sucrose, fructose, lactose or aspartame; With natural or synthetical seasonings.If unit dosage is a capsule, it can further contain liquid vehicle, as vegetables oil or polyoxyethylene glycol.Other various materials can be the physical conditions that dressing maybe can be modified solid unit dose forms.For example, tablet, pill or capsule can be used dressings such as gelatin, wax, shellac or sugar.Syrup or elixir can contain one or more sweeting agents, sanitas such as methyl p-hydroxybenzoate or propylparaben, prevention sugar crystalline reagent, the reagent that makes any other composition solubilising such as polyhydroxy-alcohol for example glycerine or sorbyl alcohol, stain and seasonings.Be used to prepare material essentially no toxicity in the scope of usage quantity of any unit dosage form.Inhibitor can be incorporated in extended release preparation and the device.
Inhibitor of the present invention can be used as additive, complementary element etc. and directly is incorporated in the feed of animal.Therefore, the present invention further provides a kind of food that contains inhibitor of the present invention.Although more be applicable to this purpose as replenishing or strengthening the processed food of nutrition such as animal-feed, bread, cereal etc., any food all is applicable to this purpose.
The preparation that is suitable for non-enterally administer suitably comprises the sterilized water preparation of inhibitor or contains the sterilized powder dispersion agent of inhibitor, preferably oozes with blood of recipient etc.The grade that comprises in liquid preparation is oozed reagent and is comprised sugar, buffer reagent and sodium-chlor.Used water prepares inhibitor solution, randomly mixes with nontoxic surfactants.The dispersion liquid of used water, ethanol, many alcohol (as glycerine, propylene glycol, liquid macrogol etc.), vegetables oil, glyceryl ester and composition thereof preparation inhibitor.Final dosage form is aseptic, liquid and stable under preparation and storage requirement.For example can by adopt liposome, under the situation of dispersion agent by adopting suitable granularity or by using tensio-active agent can obtain necessary flowability.Can keep any ordinary method of inhibitor activity to carry out the sterilization of liquid preparation by adopting, preferably adopt filtration sterilization.The preferred method of preparation pulvis comprises the vacuum-drying and the lyophilize of sterile solution for injection.By adopting various biocides for example to comprise that antibacterial agent, antiviral agent and the anti-mycotic agent of metagin class, chlorobutanol, phenols, Sorbic Acid, Thiomersalate etc. can prevent microbial contamination afterwards.For example aluminum monostearate and gelatin can make inhibitor discharge for a long time by containing delayed-action activator.
The nose spray agent comprises and contains sanitas and wait the inhibitor pure water solution that oozes reagent.This preparation preferably is adjusted to the pH that fits mutually with nasal mucosa and waits the state that oozes.
The preparation that is used for rectum or vagina medicinal can be the suppository that contains suitable carrier such as Oleum Cocois or hydrogenated fat or hydrogenated fatty acid.
Except with pH and wait be adjusted to with oozing condition optimization match with eyes, can prepare ophthalmic preparation by the method that is similar to nasal spray.
The inhibitor that is dissolved or suspended in the matrix of using in one or more media such as mineral oil, oil, polyhydroxy-alcohol or other topical pharmaceutical formulation is contained in topical preparation.Compound of the present invention is particularly suitable for being admixed in beauty treatment washing lotion, butteriness solution or the sunscreen that skin uses.
The use effective dose of inhibitor molecules of the present invention can be by animal model external activity and activity in vivo relatively come to determine.Calculate that by the active drug dosage of mouse and other animal the method for people's dosage is known in the art; For example, see United States Patent (USP) 4938949, it is hereby incorporated by in full.
Because its high efficiency and tangible specificity estimate that inhibitor molecules of the present invention is better than praziquantel and albendazole for the treatment neurocysticerocosis, and compare with albendazole with praziquantel, can effectively use low drug dose.In some applications, treatment plan comprises the drug combination of praziquantel, albendazole and/or inhibitor molecules of the present invention.
If the avtive spot of the cyst wall L-Cysteine HCL Anhydrous in other parasite is (conserved) that guards, should be appreciated that inhibitor molecules of the present invention can treat other parasitic infection effectively between other important metabolic enzyme.
The present invention also provides a kind of method that is used to identify cyst wall cysteine protease activity inhibitor.A kind of candidate's inhibitor and a kind ofly derive from tapeworm, the cyst wall L-Cysteine HCL Anhydrous of preferred taeniasis suis or taenia crassiceps mixes to form a kind of mixture.With the detected substrate of this proteolytic enzyme, preferably fluorescence molecule Z-Phe-Arg-AFC is added in the mixture then.Take place under the cracked condition being enough to after for some time,, determine whether cracking of substrate according to hereinafter more fully describing.If cracking does not take place, promptly found complete substrate, then identify a kind of cyst wall cysteine protease activity inhibitor likely.
The three-dimensional structure of inhibitor peptide Z-LLY-FMK described herein, Z-LLL-FMK, Ph2-LF-KP or any other inhibitor peptide is applicable to that computer-enhancing shows and manipulation.These structures can be calculated structure by easy-to-use peptide storehouse.By using the technology of general calculation machine in the calculation biology field-auxiliary rational drug design, differentiate the analog of Z-LLY-FMK or Z-LLL-FMK within the scope of the invention, for example have different compositions.The peptide of computer-generation and other small molecule structure storehouse can obtain easily, and can be to carrying out the computer screening with the structural similarity of Z-LLY-FMK or Z-LLL-FMK.Randomly, employing currently known methods in the art can be determined the X-ray crystal structure of tapeworm L-Cysteine HCL Anhydrous, and its coordination can show on computers.Avtive spot can be discerned by computer modeling technique, the potential inhibitor can be identified with combining of avtive spot by inferring.As described below the compound of proof and Z-LLY-FMK or Z-LLL-FMK similar or other are thought that may the bonded compound carrying out in vitro tests by the present invention with described protease activity site determines whether they suppress the activity of cyst wall L-Cysteine HCL Anhydrous then.Except the method for identifying them, the present invention should be understood that the inhibitor molecules that comprises that also the method according to this invention identifies.
The present invention also provides a kind of effective treatment or prevention animal to be formed the infection vaccine of the parasite such as the tapeworm of capsule.This vaccine can be polypeptide vaccine or polynucleotide vaccine, and can contain one or more immunogenic components.Polynucleotide vaccine contains at least a polynucleotide with nucleotide coding district of coding cyst wall L-Cysteine HCL Anhydrous or its immunogenic polypeptide subunit.Similarly, polypeptide vaccine contains at least a cyst wall L-Cysteine HCL Anhydrous or its immunogenic polypeptide subunit.The immunogenic polypeptide subunit of cyst wall L-Cysteine HCL Anhydrous is that a kind of animal host of causing produces antibody-mediated immunne response (promptly, B cell response or humoral immunization), cell-mediated immunne response (that is t cell response) or its bonded immunogenic polypeptide subunit.The immunogenicity of L-Cysteine HCL Anhydrous subunit can be measured by for example adopting the analytical procedure among the embodiment.Randomly, polypeptide vaccine or polynucleotide vaccine contain the immune additional compound of stimulation of host, as following further description.The present invention should be understood that also to comprise the method for preparing and use described polynucleotide and polypeptide vaccine.
Polynucleotide vaccine can contain nucleic acid or its arbitrary combination of DNA, RNA, modification.Preferably, polynucleotide vaccine contains one or more clones or expression vector; More preferably, this vaccine contains one or more expression vectors, and each this expression vector can carry out the autonomous expression in nucleotide coding district so that produce at least a immunogenic polypeptide or cytokine in mammalian cell, as following further description.A kind of expression vector preferably contains the eukaryotic promoter sequence that is connected with one or more coding regions operability ground, more preferably is a kind of nucleotide sequence of eucaryon strong promoter.Promotor is to play the conditioning signal effect and start the dna fragmentation that transcribe coding region, downstream (3 ' direction) in conjunction with RNA polymerase in cell; Transcribe exactly and form the RNA chain according to the genetic information that is contained among the DNA.Promotor is carried out " being connected to operability " or is used to control or regulate transcribing of described nucleotide sequence with nucleotide sequence.The present invention is not limited to use any specific eukaryotic promoter, but the known kind of wide scope; Yet preferably, expression vector contains CMV or RSV promotor.Described promotor can be, but needs not to be, and is allogenic with host cell.The promotor of using is a structure promotor preferably.
The carrier that is used for polynucleotide vaccine of the present invention can be annular or linearity, and is strand or double-stranded and can be a kind of plasmid, clay or episome, but preferably a kind of plasmid.In a preferred embodiment, each nucleotide coding district (no matter being coding immunogenic polypeptide or cytokine) is all on a kind of carrier of separating; Yet, should be understood to one or more coding regions and may reside on the independent carrier, and these coding regions can be by single or multiple promoter regulations.
There is the known plasmid of many those of ordinary skill in the art to be used for the preparation of polynucleotide vaccine.The preferred embodiment of polynucleotide vaccine of the present invention has adopted (the Vical Inc. with plasmid VR1012, San Diego CA), pCMVI.UBF3/2 (S.Johnston, University of Texas) or pcDNA3.1 (InVitrogenCorporation, Carlsbad is CA) as the formation thing of carrier.Plasmid VR1012 and pCMVI.UBF3/2 are particularly preferred.In addition, plasmid constitutes thing can contain the immunostimulatory sequence (ISS) that stimulates animal immune system, as unmethylated dCpG motif.Polynucleotide vaccine constitute thing other may extention comprise the nucleotide sequence of the Codocyte factor, as huge phasmid cell clone stimulating factor (GM-CSF), il-1 2 (IL-12) and jointly-stimulation molecule such as B7-1, B7-2, CD40.Described cytokine can be used for replying of animal immune system that various combinations coordinate well to comprise that antibody and cytotoxic T lymphocyte are replied, and the result produces control or eliminates required special the replying of cestode infection.Polynucleotide vaccine also can be encoded and be contained the fusion product of immunogenic polypeptide and molecule such as CTLA-4, and it can make this fusion product be oriented among the host-antigen presenting cell.Also can transmit plasmid DNA as transfer system, a kind of method that is suitable for the dna vaccination of oral administration with attenuated bacteria.Use the autonomously replicating plasmid transform bacteria, this autonomously replicating plasmid is along with the death of attenuated bacteria in host cell is released in host's the kytoplasm.The another kind of method that polynucleotide are passed to animal comprises the use of virus or bacteria carrier.The example of suitable virus vector comprises adenovirus, poliovirus, and poxvirus such as cowpox, canary pox and fowl pox, simplexvirus comprises catfish virus, carrier relevant with adenovirus and retrovirus.The example of bacteria carrier comprises salmonella, Shigella, catfish tarda, Lu Shi Yersinia and the Listeria monocytogenes of attenuation form.
Polynucleotide vaccine of the present invention can adopt any easy method to give animal-use drug, as intramuscularly, part or the applied dermally skin in animal, or uses particle gun, and the particulate that has wherein wrapped up polynucleotide vaccine is launched into the skin of animal.The amount of the polynucleotide of using to animal is influenced by characteristic, size and disease condition and the medication of animal; For example, the required DNA DNA more required than intramuscularly of particle gun medication lacks usually.And if the Nucleotide of the coding region of the polynucleotide of the co-administered Codocyte factor and coding immunogenic polypeptide, the amount of the polynucleotide of coding immunogenic polypeptide is reduced arbitrarily in the vaccine.
Now, become hundred pieces of publications to report dna vaccination to treating the catching of big small animal model, cancer and autoimmune disease effectively (J.Donnelly etc., Rev.Immunol.15:617 (1997)).The dosage of vaccine for man can easily be inferred according to mouse model by the technician in the inherited immunity field, and the technician who practises medicine now can obtain the lot of documents of relevant Human genome immunity.For example, Wang etc. (Science282:476-480 (1998)) carry out immunity with the proteic plasmid DNA of encoding malaria to the people, and this study group has formulated a preparation and has tested the plan (Hoffman etc., Immunol.Cell.Biol.75:376 (1997)) of polygene plasmodium falciparum liver in the human body-stage D NA vaccine validity.In a word, polynucleotide vaccine of the present invention is to carry out medication with the dosage that contains the essential minimum polynucleotide of effective immunity.Usually give people patient's medication to contain about 20 μ g to the dosage of the plasmid DNA of about 2500 μ g.For indicating 500 μ g or more plasmid DNA in some illustrations.Usually at certain time intervals in, vaccine carries out twice or the multiple injection medication, for example in the timed interval in four weeks.
Polypeptide vaccine of the present invention also can contain the immune assistant agent that stimulates animal except containing cyst wall L-Cysteine HCL Anhydrous or its immunogenic polypeptide subunit.Be applicable to that people or beastly assistant agent are known in the medical field.As polynucleotide vaccine, polypeptide vaccine can adopt any suitable method to give animal-use drug, as the above-mentioned method that is used for the inhibitor molecules medication, and for example intramuscular or peritoneal injection, local application, oral or Nasacort, suction, perfusion etc.Be subjected to characteristic, size and the disease condition of animal and the influence of medication for the polypeptide amount of animal-use drug.The effective dose that is used for people's polypeptide vaccine can easily be determined at the intravital activity in vivo of mouse by measuring it.
Therefore, the present invention further provides method to the vaccine of the anti-cestode infection of animal inoculation pvaccination.Preferably, animal is ox, dog, pig or people.More preferably, animal is pig or people, and infection by Taenia solium can effectively be treated and prevent to described method.On the one hand, the animal of cestode infection is carried out the therapeutic medication of described vaccine.In the embodiment of a methods of treatment, use vaccine and can remove the intravital parasite of animal effectively; In another embodiment, use vaccine and can effectively prevent or delay cysticercosis or neurocysticerocosis in animal performance on one's body.On the other hand, vaccine can not carry out preventive usage to infecting parasitic animal.In the embodiment of a prevention method, use vaccine and can effectively prevent animal and infect parasite in the future.In the embodiment of another prevention method, use vaccine and can effectively prevent or delay animal and in the future developed into cysticercosis or neurocysticerocosis after the parasitic infection.In the embodiment of another prevention method, use that vaccine can prevent to have infected in the future behind the parasite effectively and the animal dead that causes.Because described vaccine can be resisted the various tapeworms that comprise taeniasis suis and taenia crassiceps effectively, has also designed human application so both designed the application of beasts.Beasts are used prevention and the treatment that comprises domestic animal, and also may command is to the propagation of population infection.Preferably, the inventive method comprises that pig or people to having infected tapeworm or PI tapeworm use vaccine.For pig, polynucleotide or polypeptide vaccine preferably carry out the feed medication.
The present invention has further designed in a serial scheme animal has been carried out the medication of different vaccines and/or inhibitor molecules.For example, carry out medication with a kind of dna vaccination based on plasmid as described in this article to animal and come " startup " immunity system, then carry out one or many and (for example use polypeptide vaccine, virus vaccines, contain the coding immune peptide and, the vaccine carrier of the gene of optional cytokine) and/or contain the pharmaceutical composition of the cyst wall cysteine protease activity inhibitor that the present invention identifies.Those skilled in the art can easily determine the medication order of dissimilar compositions by starting efficient immune in animal body, with characteristic with the composition (for example, polypeptide vaccine, plasmid vaccine, vector-viral vaccine, inhibitor molecules) of any given dose medication.
Embodiment
The present invention will be described with the following example.Be to be understood that according to protection scope of the present invention and essence specific embodiment, material, consumption and the method listed are herein carried out the generalized explanation.
The purifying and the sign of the L-Cysteine HCL Anhydrous of mouse parasite taenia crassiceps
The cyst wall L-Cysteine HCL Anhydrous of taenia crassiceps is purified to homogeneity (White etc., Mol.Biochem.Parasitol., 85:243-253 (1997)).This enzyme has a kind of very high conversion capability and the external quick degraded people's of energy IgG to synthetic substrate.This molecule plays critical effect in the tapeworm immunity is hidden, be a kind of important survival strategy that is adopted by parasite.
Because taenia crassiceps and taeniasis suis have tangible biological similarity, the speed of growth (infected 4 week after＞100 capsules) fast, with the simple preservation in BALB/c mouse, so it is often used as the human parasite of research, the model system of taeniasis suis (Hayunga etc., J.Parasitol.75:638-642 (1989); Ambrosio etc., Arch.Med.Res.25:325-330 (1994); White etc., Infect.Agents Dis.1:185-193 (1992)).The natural host of taenia crassiceps comprises rodent (intermediate host) and canine tooth animal (end host).We are purified to homogeneity with a main fertilizer L-Cysteine HCL Anhydrous, this point by it the N-terminal sequence and dye present on the gel single at the silver of SDS-PAGE (SDS-PAGE) and bring confirmation.Adopt acid extraction that described enzyme is dissolved out from complete parasite, and do not find cysteine protease activity in the juice, (Hayunga etc., J.Parasitol.75:638-642 (1989) have also been noticed in this discovery by other people; Ambrosio etc., Arch.Med.Res.25:325-330 (1994)).Owing to compare with the washing composition solubilizing method, acid extraction can be isolated most enzyme, so we suppose that described proteolytic enzyme is positioned on the cyst wall, but unlikely in conformity membrane.These observe the confirmation that had obtained transmission electron microscope afterwards, and cysteine protease activity is positioned in (Khalil etc., J.Paristol.84:513-515 (1998)) on lyase somatocyst and the cell wall.Therefore this L-Cysteine HCL Anhydrous may be positioned on the cyst wall of tapeworm, makes it become chemotherapeutical target.
Gel-filtration (AcA54 gel-filtration column) and fast protein liquid chromatography (F.P.L.C.) anionresin (Mono Q) chromatogram are used to from initial acid extraction thing purifying L-Cysteine HCL Anhydrous 682-doubly.Purifying enzyme with the sharp honeybee of about eluting salt to 1 of 13% activity, is that basis be equivalent to 43kDa and by silver dye acquisition single band with wash-out estimated molecular weight partly by anionresin.(13% salt is effectively, because similarly the taeniasis suis L-Cysteine HCL Anhydrous also carries out wash-out under this percentage composition).Further come to determine purity by the N-terminal sequence.52 amino acid whose results (Fig. 1 (b)) have accurately been obtained.The N-terminal sequence of the material that purifying taenia crassiceps L-Cysteine HCL Anhydrous obtains has confirmed 30 amino acid whose sequences (Fig. 1 a) by the second time.Although this N-terminal sequence 23 contains a halfcystine in the site, described halfcystine is the catalytic halfcystine of papoid Superfamily, and it does not contain apokoinou construction (Hayunga etc., J.Parasitol.75:638-642 (1989) in this Superfamily enzyme.Yet, compare a fertile L-Cysteine HCL Anhydrous the most similar to the enzyme of papoid Superfamily (Fig. 1 b) with the sequence of other L-Cysteine HCL Anhydrous section.Therefore, this enzyme can be represented the L-Cysteine HCL Anhydrous of a new kind, and this L-Cysteine HCL Anhydrous and papoid Superfamily homology are the highest.
Substantially according to White etc., J.Mol.Biochem.Parasitol., described in the 85:243-253 (1997), adopt the synthetic peptide substrates that is connected with the C-end of the amino 4-trifluoromethyl tonka bean camphor of 7-(Z-Phe-Arg-AFC) to analyze the proteolytic activity of the L-Cysteine HCL Anhydrous of purifying.At pH is 4.9 times, the proteolytic enzyme (0.1ug) of purifying is added among the Z-Phe-Arg-AFC (1ug/500uL analysis buffer) of the saturation capacity in the 0.4M Citrate trianion that contains the 10mM halfcystine.With this mixture 37 ℃ of following incubations 18 hours.The L-Cysteine HCL Anhydrous of purifying by tangible sulfydryl dependency (biochemical characteristics of all L-Cysteine HCL Anhydrouss), by cystatin suppress, 5.27 pI and show (referring to the example II) that the significance cracked substrate specificity linearity curve to Z-Phe-Arg-AFC characterizes.The proteolytic enzyme of purifying shows synthetic peptide substrate, and the Kcat/Km that Z-Phe-Arg-AFC is very high (substrate conversion efficiency) equals 2.2 * 10
8M
-1s
-1, near the order of magnitude of enzyme " the diffusion control limit " value, some investigators liken it to the enzyme (second edition, John Wiley and Sons are edited 14:371-400 (1995) for Voet, Biochemistry) of " catalytic perfection ".It is reported and have only few enzyme to have so high catalysis Kcat/Km ratio.Most of enzymes with these characteristics are the enzymes (for example hexokinase and triose-phosphate isomerase) that are easy to tachymetabolism, once running into substrate catalyzed reaction at once.Therefore, the tapeworm L-Cysteine HCL Anhydrous makes the people associate a kind of enzyme (as metabolism) that parasite is had biological significance in the high-level efficiency aspect similar value conversion of substrate.
The taenia crassiceps protease activities is by irreversible cystatin, E64 (L-trans-epoxy succinic acyl-leucyl acid amides-(4-guanidine radicals)-butane) suppress, show that this purifying enzyme is a kind of L-Cysteine HCL Anhydrous.The Km of this enzyme is fully increased, show L-Cysteine HCL Anhydrous to Z-Phe-Arg-AFC substrate substantial loss avidity.This result is consistent with the covalent interaction between E64 and the purifying enzyme, normally the interactional characteristic feature of E64 and L-Cysteine HCL Anhydrous.In order to obtain these kinetic parameters, set up a kind of like this analytical procedure promptly by relative proteolytic enzyme speed (the mol h of concentration of substrate (moles)
-1) the x of Lineweavwer-Burke figure two reciprocal and y intercept calculating K m and Vm respectively.Calculating K cat reciprocal by Vmax.
Example II
The people parasite, the purifying of the L-Cysteine HCL Anhydrous of taeniasis suis and sign
Purifying derives from the people parasite with having characterized, the L-Cysteine HCL Anhydrous of taeniasis suis, and this taeniasis suis is the intermediate host with the pig.Be similar to the L-Cysteine HCL Anhydrous that derives from taenia crassiceps of example I though this proteolytic enzyme is different from, the natural reservoir (of bird flu viruses) of this taenia crassiceps is a rodent, and in BALB/c mouse well-grown.Yet data make the people be sure of that the avtive spot of two kinds of enzymes is height homologous (this is most important prerequisite for special inhibitor).Therefore the proteinase inhibitor of special opposing taeniasis suis L-Cysteine HCL Anhydrous is expected to the L-Cysteine HCL Anhydrous in the special inhibition taenia crassiceps, thereby can test the validity of inhibitor in mouse model.
Obtain the taeniasis suis capsule on one's body from the wild cysticercosis pig in Mexican Guerrero village.As in taenia crassiceps, the cysteine protease activity of most of taeniasis suis capsule can discharge by acid extraction, and does not need to use the sanitising agent solubilising.If adopt the sanitising agent solubilising, the specific activity in the acid extraction thing still exceeds about 5 times.Therefore, as the L-Cysteine HCL Anhydrous of taenia crassiceps, the taeniasis suis L-Cysteine HCL Anhydrous may be positioned on the cyst wall, but is not a kind of conformity membrane albumen.
The purification scheme that is used for taenia crassiceps is used to purifying taeniasis suis L-Cysteine HCL Anhydrous.After the acid extraction, the active part that adopts gel permeation chromatography and wash-out thereby shows that the taeniasis suis L-Cysteine HCL Anhydrous is more bigger than taenia crassiceps L-Cysteine HCL Anhydrous (43 kDa) in the 50-55kDa scope.The optical density readings of FPLC anionresin shows the figure of taeniasis suis and taenia crassiceps enzyme almost overlapping (Fig. 2).From the active part that AcA 54 gel columns obtain, further adopt Mono Q post, separate by the FPLC anion-exchange chromatography.With the 10mM Tris of identical gradient (broken broken line), pH7.6 (solution A) and 10mMTris, pH7.6 add these two kinds of enzymes of 1M NaCl (solution B) wash-out.Collect the 1ml elutriant and analyze (dotted line) with the Z-Phe-Arg-AFC substrate.Measure absorbancy (solid line) at the 280nm place.As observed, two kinds of enzymes wash-out between the salt concn of 12-14% comes out, and shows that these two kinds of enzymes have the similarity and the conservative electric charge conformation of height.The taeniasis suis L-Cysteine HCL Anhydrous is a band on the SDS-PAGE gel that silver dyes.
These data show between taenia crassiceps L-Cysteine HCL Anhydrous and the taeniasis suis L-Cysteine HCL Anhydrous and exist a large amount of similaritys.And fact proved that avtive spot also is a homologous.At first, the optimum pH of taeniasis suis L-Cysteine HCL Anhydrous is identical with the optimum pH (pH4.9) of taenia crassiceps L-Cysteine HCL Anhydrous.And the cracking pattern of substrate is also similar.Be combined into substrate and compare with one, for two kinds of enzymes, Z-Phe-Arg-AFC (Z-FR-AFC) is a most important cleaved substrate (Fig. 3).Can see that Lys-Ala-AFC (KA-AFC) and Arg-AFC (R-AFC) are the better cracking substrates that is only second to Z-FR-AFC of taeniasis suis proteolytic enzyme.The substrate of cracking preferably that these two kinds of substrates also are taenia crassiceps proteolytic enzyme.Any enzyme in two kinds of enzymes for other substrate (for example, LG-AFC, gross activity ZRR-AFC) is all not high enough.These data that show similar cracking pattern between two kinds of enzymes have further confirmed the homology in two kinds of protease activities sites.
As the avtive spot of taenia crassiceps L-Cysteine HCL Anhydrous, the taeniasis suis L-Cysteine HCL Anhydrous is tangible sulfydryl dependency, and is subjected to complete inhibition owing to there not being halfcystine to exist.As shown in table 1, two kinds of L-Cysteine HCL Anhydrouss are subjected to the inhibition of cystatin but are not subjected to the obvious inhibition (＞50%) of the inhibitor of other catalysis kind.For example, the epoxies inhibitor of cystatin (that is: E64 and EP459 (L-trans-epoxy succinic acyl-leucyl acid amides (4-methyl) butane)) can complete inactivation.HgCl
2The mode that relies on concentration suppresses two kinds of enzymes, and this also is the characteristic of L-Cysteine HCL Anhydrous.Methyl chloride inhibitor tosyllysine chloromethylketone (TLCK) and tolylsulfonyl phenylalanyl (phenylanyl) chloromethane ketone (TPCK) have most inhibition, and known its suppresses serine protease and some L-Cysteine HCL Anhydrouss.Comprise that PMSF, 1,10 phenanthroline, ethylenediamine tetraacetic acid (EDTA) (EDTA) or pepstatin do not have obvious suppression at other interior kind inhibitor.In table, only shown a representational experiment, and carried out three experiments in fact at least.Add before the substrate, with inhibitor with enzyme incubation together 30 minutes.Behind the incubation that spends the night, measure fluorescence data.
Table 1. is when with Z-Phe-Arg-AFC during as substrate, (a) taenia crassiceps (example I) and (b) the taeniasis suis L-Cysteine HCL Anhydrous to the susceptibility of the inhibition that causes by one group of inhibitor
A) inhibitor catalytic type concentration % inhibition ± SD
*No halfcystine 100E-64 halfcystine 10
-8M 95 ± 3.0
10
-7M????????98±1.5
10
-6M 99 ± 1.0Ep459 halfcystine 10
-7M 99 ± 1.1Ep475 halfcystine 10
-6M 99 ± 0.9 iodoacetic acid halfcystines 10
-3M 99 ± 0.7HgCl
2Halfcystine 10
-4M 3 ± 2.1
10
-3M????????40±29
4 * 10
3M 77 ± 24PMSF Serine 2 * 10
-3M 47 ± 32APMSF Serine 10
-4 M 0 ± 0TLCK Serine/halfcystine 10
-4M 96 ± 3.1TPCK Serine/halfcystine 10
4M 96 ± 3.51.10 phenanthroline metal-2 * 10
3M 35 ± 38EDTA metal-2 * 10
-3M 27 ± 38Pcpxtatin aspartic acid 10
-4M 34 ± 34
b)
| Inhibitor * | Catalytic type | Concentration | % inhibition ± |
| No halfcystine | |||
| 100 | |||
| E-64 | Halfcystine | ?10 -6M | 100±2.7 |
| ?10 -7M | 100±0.2 | ||
| ?10 -5M | 94±0.9 | ||
| Iodoacetic acid | Halfcystine | ?10 -3M | 100±0.0 |
| HgCl 2 | Halfcystine | ?10 -4M | 9±3.1 |
| ?10 -3M | 41±10.0 | ||
| ?4×10 -3M | 81±1.4 | ||
| PMSF | Serine | ?2×10 -4M | 29+10.0 |
| TLCK | Serine/halfcystine | ?10 -5M | 93+1.0 |
| TPCK | Serine/halfcystine | ?10 -4M | 93±1.0 |
| 1.10 phenanthroline | Metal | ?2×10 -1M | 49±10 |
| EDTA | Metal | ?1×10 -3M | 13±5.0 |
| ?2×10 -3M | 8±2.0 | ||
| Pcpstalin | Aspartic acid | ?1×10 -5 | 34±4.0 |
| ?1×10 -7 | 39±16 | ||
| DCI | 38±11 |
If with human IgG as substrate, the specific cystatin (ability (referring to Fig. 4 b) that for example, Suc-Leu-Tyr-AFC) can suppress the human IgG on taeniasis suis and the taenia crassiceps L-Cysteine HCL Anhydrous crack protein matter trace fully.Therefore, the avtive spot of two kinds of enzymes is by the figure institute mark of height homologous inhibitor.
In addition, taeniasis suis cysteine protease activity site equals 2.84 * 10 at the Kcat/Km ratio of Z-Phe-Arg-AFC
9M
-1s
-1, near the Kcat/Km ratio (2.2 * 10 of taenia crassiceps L-Cysteine HCL Anhydrous
8M
-1s
-1).Therefore the taeniasis suis L-Cysteine HCL Anhydrous is also in the diffusion control ultimate value 10 of the enzyme of " catalytic perfection "
8-10
9The logarithm scope in.Therefore, the taeniasis suis L-Cysteine HCL Anhydrous also demonstrates the remarkable ability of cracking substrate, and we be sure of that thus a kind of so effective enzyme also is important probably to human parasite.
In a word, as the taenia crassiceps enzyme, the feature of taeniasis suis enzyme also is can the degrade heavy chain and the light chain (Fig. 4) of human IgG, and degradation speed is fast.Same a) the taenia crassiceps of human IgG acid extraction thing or b) L-Cysteine HCL Anhydrous (CP) incubation 18 hours together of taeniasis suis purifying.Then under the reductive condition, the incubation test tube is carried out the component classification by SDS-PAGE, trace is to pvdf membrane, and adopt biotinylated resisting-IgG (heavy chain/light chain), vitamin H-peroxidase-conjugated-streptavidin mixture and enhanced chemoluminescence develop the color.In the A group, top and bottom arrow refer to heavy chain and light chain respectively, in the B group, have only shown heavy chain.Do not exist heavy chain and light chain explanation protein to be cracked into peptide.In another experiment, we notice, as taenia crassiceps, and the taeniasis suis enzyme require IgG that just can degrade fully in 30 minutes.This degraded is that sulfydryl is dependent, and by the Gelucystine proteinase inhibitor, E64, suppress, but do not suppressed by the inhibitor of other catalytic type.Under the condition that has IgG to exist, work as specific inhibitor, Z-LLY-FMK (EXAMPLE IV) is during with taeniasis suis L-Cysteine HCL Anhydrous incubation, and the degraded of antibody is suppressed.As if the taeniasis suis L-Cysteine HCL Anhydrous also tend to the degraded of human IgG.For example, when when the concentration that is used for the human IgG Study on degradation is used this substrate, its human albumin of not degrading.
In a word, with regard to avtive spot catalysis, our data show in biological chemistry and biologically taeniasis suis enzyme and taenia crassiceps enzyme be much at one.The specific inhibitor that suppresses the taenia crassiceps L-Cysteine HCL Anhydrous also suppresses taeniasis suis L-Cysteine HCL Anhydrous (EXAMPLE IV).In a word, support the similar evidence of enzyme active sites to comprise: highly similar pH is just when, inhibitor properties (profiles), substrate characteristic, identical anion-exchange column elution mode, similar IgG degraded and the inhibition that is subjected to the inhibitor of high specific.Therefore, the taenia crassiceps capsule can be used as the biological action of research at the proteinase inhibitor of taeniasis suis L-Cysteine HCL Anhydrous fully.
Possible interaction between tapeworm L-Cysteine HCL Anhydrous and host immune system
The cyst wall L-Cysteine HCL Anhydrous of tapeworm plays an important role in the process of tapeworm escape host immune.The tapeworm capsule can be survived for many years in asymptomatic intermediate host.Therefore, the tapeworm capsule must be an immunne response of having escaped the host in some way.And they have necessarily also found the host protein source that can degrade so that obtain the essential amino acid nutrient.We suppose to take place the mechanism of everything, are begun by immune evasion.Yet, be to be understood that scope of the present invention also relates to method and the compound that suppresses the tapeworm cysteine protease activity, but do not mean that and be subjected to any specific mechanism of action or the restriction of pattern.
Shown in the past that IgG transported by hemato encephalic barrier or produces in brain.Found the Fc acceptor on the tapeworm cyst wall, the picked-up that shows IgG may be a kind of active process (Kalinna etc., Parasitol.106:289-296 (1993)).We suppose that IgG is passed on the capsule lysosome in cyst wall contact projection and the cutaneous nerve cell paste.The protein approach that carries out protein degradation behind the lysosome that shuttles back and forth is known (Ambrosio etc., Arch.Med.Res.25:325-330 (1994)) in taenia crassiceps.L-Cysteine HCL Anhydrous also is positioned on the cyst wall.After being sent to lysosome, we suppose that IgG will degrade in lysosome.Hayunga etc. (Hayunga etc., J.Parasitol.75:638-642 (1989)) have observed the IgG unit of degraded in the lysosome vesica of taenia crassiceps.Then IgG is degraded into peptide by special L-Cysteine HCL Anhydrous in lysosome, perhaps even may be degraded to amino acid.Therefore, the IgG of degraded L-Cysteine HCL Anhydrous can assist capsule to hide destructive host IgGs (IgG2 of complement-fixing is relevant with dying back tapeworm).
At last, be easy to become an important metabolic advantage near the peptide that derives from IgG or the amino acid whose benefit of " being arranged in " lysosome.In fact, L-Cysteine HCL Anhydrous can help parasite to utilize host's immunne response, and does not just make it ineffective.As (White etc., J.Mol.Biochem.Parasitol., the 85:243-253 (1997)) that had reported, the IgG because L-Cysteine HCL Anhydrous can be degraded very effectively is so antibody may become the good nutrition source of endoparasite.Total importance of the intravital L-Cysteine HCL Anhydrous of parasite in the growth is illustrated (Khalil etc. by our transmission electron microscope (TEM) research, J.Paristol.84:513-515 (1998)), this studies show that the active high of L-Cysteine HCL Anhydrous in bigger, the older cysticercus of specific activity of the L-Cysteine HCL Anhydrous in less, that metabolism is active and cysticercus growth.These play keying action identical of views at degrade proteins as the IgG process that is used for parasitic nutrition with relevant enzyme than the relation between the cysteine protease activity of the Tibetan household slave's length quickened in the folliculus and increase.
At last, the tapeworm capsule becomes to a kind of balanced responsive, and the IgG of certain level in this host's serum is actually is of value to metabolic, but too much will be harmful to immunity.Perhaps there is a useful baseline physiology IgG level (picked-up of IgG is shown as saturated (Siebert etc., Exp.Parasitol.48:64-174 (1979)) in the taenia crassiceps capsule on the physiological serum level).Therefore, the tapeworm capsule may need the IgGs that is used for immunity of certain level, but the concentration that exceeds this level is deleterious.Perhaps, Here it is, and why high antigenic L-Cysteine HCL Anhydrous (EXAMPLE III) is not positioned at the cyst wall surface, and the reason in cyst wall.Therefore, described capsule may have been evolved and controlled this equilibrated molecular mechanism.For example, excretory tapeworm glycoprotein (Villa etc., Parisotol.112:561-570 (1996)) as if controls the conversion of replying to body fluid t helper cell 2 (Th2) from host's t helper cell 1 (Th1) cell-mediated immune responses, help being increased (this conversion also can assist parasite to avoid destructive Th1 cell response, and they are replied this and do not have a defense) as the output of the antibody of nutrition by parasite.Perhaps, the secretion that reduces these molecules can reduce the conversion to Th2 from Th1, allows parasite otherwise to regulate and control immune environment (therefore, physiology IgG level).
Therefore we have carried out the specific inhibitor of design or discriminating (EXAMPLE IV) tapeworm cyst wall L-Cysteine HCL Anhydrous, and this inhibitor utilizes the dual function pattern performance potential effect of one or two aspect.At first, the special inhibition to L-Cysteine HCL Anhydrous can make host immune sphaeroprotein identification packing so that they are exposed to immunity system.In example VI, we have shown how the packing that stems from the mouse of handling with the special inhibitor of anti-L-Cysteine HCL Anhydrous is wrapped up by immunocyte, and the packing that stems from untreated mice is " clean ".The inhibition that second pattern may be L-Cysteine HCL Anhydrous can weaken packing by making packing lose the amino acid that is produced by IgG, thereby destroys its metabolism." weakened " packing also will be easier to also may be wrecked by immune system recognition or be prevented from further growth and/or propagation at least.Shown in EXAMPLE V, our special cystatin treatment has prevented that mouse suffers from cysticercosis, shows that the growth (by blastogenesis) of packing is suppressed.
Clearly, described L-Cysteine HCL Anhydrous is positioned at these identical zones, it is reported the IgG that has degraded in these zones.Transmission electron microscope (TEM) research has been identified in the tart and lysosomal vacuole on the taenia crassiceps cyst wall, L-Cysteine HCL Anhydrous cracking L-Cysteine HCL Anhydrous substrate, the activity of Z-Phe-Arg-methoxynaphthalene methane amide (MNA) (Khalil etc., J.Paristol.84:513-515 (1998)).Free methoxynaphthalene methane amide and osmium and right-rosaniline coupling, and go up cracking from the Z-Phe-Arg of the carboxyl terminal of the arginine residues of substrate and get off.Compare with the contrast that does not have substrate, around vesica, the intensive settling of electronics occurred, and most vesica is found in the cutaneous nerve cell paste and contact projection that mainly has cyst wall with the cysticercus of substrate incubation.Therefore this research is positioned at L-Cysteine HCL Anhydrous on the cyst wall.If before adding substrate with cysticercus and cystatin, E64, incubation together, electronics conglomerate lack than the electronics conglomerate in the cysticercus that does not have inhibiting and dense degree lower.Therefore, the part that cysteine protease activity is subjected to E64 suppresses, and this has confirmed that further L-Cysteine HCL Anhydrous is present in this zone.Because because the enzyme concn in the vesica is higher and the concentration of this inhibitor in acid vesica may be low excessively than our purified product, so hypothesis adopts E64 that partly inhibition has only taken place.Here our important conclusion is 1) L-Cysteine HCL Anhydrous may be arranged in identical cyst wall zone, observed the IgG and 2 of degraded in this zone) cystatin can original position reduces the activity of L-Cysteine HCL Anhydrous.Back one result has indicated that proteinase inhibitor that a very big possibility is special design can permeate and therefore suppresses cysteine protease activity in the tapeworm cyst wall.
And lysosomal pH (pH4.9) and L-Cysteine HCL Anhydrous are to synthetic substrate, and the active pH of Z-Phe-Arg-AFC is just when identical, and the activity of L-Cysteine HCL Anhydrous all depends on the external source degraded group that also is present in the lysosome.Although Z-Phe-Arg-AFC can be by L-Cysteine HCL Anhydrous and serine stretch protein enzymatic lysis as kallidinogenase, serine protease is unsuitable for playing a role under acid pH and is that sulfydryl relies on.And former research fails to identify the activity of serine protease in tapeworm cysticercus wall with similar peptide.Therefore, the proteolytic activity of measuring with Z-Phe-Arg-AFC under the acid pH in lysosome degraded environment here is likely because the L-Cysteine HCL Anhydrous that we are purified into from the tapeworm cyst wall.The L-Cysteine HCL Anhydrous that we also observe our purifying is the unique a kind of enzyme in the cyst wall.
The tapeworm L-Cysteine HCL Anhydrous is suitable at lysosomal pH and external cracking human IgG in the presence of the degraded group is only arranged.Therefore clearly compare with other proteolytic enzyme in the original acidic extraction thing of purification scheme, this enzyme is the reason of human IgG of degrading fully on the western blotting.Heavy chain and light chain can both be shown that described chain is degraded to peptide or the amino acid of can't see on the SDS-PAGE gel by the complete cracked result of L-Cysteine HCL Anhydrous.The L-Cysteine HCL Anhydrous that dose response experiment shows purifying is with (the enzyme: mole specific energy IgG) fully degraded heavy chain and the light chain of IgG, and degrade with the part that IgG only takes place the mol ratio up to 1: 3000 up to 1: 30.Degraded only need just can be finished and can be by cystatin in 45 minutes fully, and E64 suppresses fully.As mentioned, E64 original position in the tapeworm cyst wall suppresses the activity of L-Cysteine HCL Anhydrous, shows that the cystatin of special design also can suppress the degraded of L-Cysteine HCL Anhydrous to IgG.(in fact, the special inhibition of homologous taeniasis suis L-Cysteine HCL Anhydrous is at Leu-Tyr-AFC-Fig. 4 b).And the substrate specificity general picture of purifying enzyme approaches cathepsin L-sample L-Cysteine HCL Anhydrous most, and this enzyme is a mammiferous lysosomal enzyme and also relevant with the degraded of IgG.Thereby these data declaration taenia crassiceps L-Cysteine HCL Anhydrouss host IgG substrate of having degraded effectively.
Our data show that also the tapeworm L-Cysteine HCL Anhydrous also can be preferably used for the degraded of IgG.As if for example, we have separated a kind of secretion L-Cysteine HCL Anhydrous recently from taenia crassiceps, and its optimal pH is 6.3, and this enzyme can only cause the slight degraded of human IgG heavy chain, and can not cause the degraded of light chain.By relatively, the cyst wall L-Cysteine HCL Anhydrous two kinds of chains of as if degrading.Based on these data, we be sure of that tapeworm cyst wall L-Cysteine HCL Anhydrous plays an important role to the lysosome degraded of the host IgG in the tapeworm, and may be special to this process.
In a word, the very high kinetics cleavage rate of tapeworm cyst wall L-Cysteine HCL Anhydrous, keying action, the entry site (but not too dark in capsule) in cyst wall and its activity possible in parasite nutrition and host immune escape can be made this enzyme become the chemotherapeutical possibility of parasiticide target by the fact that the cystatin original position reduces.
EXAMPLE III
With taeniasis suis L-Cysteine HCL Anhydrous immunoprophylaxis BALB/c mouse
Taeniasis suis L-Cysteine HCL Anhydrous (example II) the immunoprophylaxis BALB/c mouse Freund's complete adjuvant with (the eluting) and partially purified (eluting) of purifying from ACA 54 gel-filtration columns from the MonoQ anion-exchange column.With same preparation mouse is carried out booster immunization after fortnight.Behind 5 first quarter moons of booster immunization, attack mouse, carry out euthanization after effectively infecting for 6 week with the taenia crassiceps capsule.Shown in following table 2, have 72% to avoid cysticercosis with the mouse of the L-Cysteine HCL Anhydrous immunoprophylaxis of purifying, and have only 50% to avoid cysticercosis with the mouse of partially purified L-Cysteine HCL Anhydrous immunoprophylaxis.Therefore as if the taeniasis suis L-Cysteine HCL Anhydrous can be induced the intersection-protection immunne response of BALB/c mouse, and this reply relevant with the purity of L-Cysteine HCL Anhydrous.This phenomenon has confirmed that these enzymes are the similar characteristics of antigen.
After (the following Mono Q behind the ACA54) and partially purified (ACA54) taeniasis suis L-Cysteine HCL Anhydrous immunoprophylaxis of table 2. with purifying, avoid BALB/c mouse to infect the taenia crassiceps cysticercosis
MonoQ 43 38.6 18.6 71.8 1.6 1.3 .6 (purifying) 59 1.8
14 .5ACA54,62 69 5.3 49.8 2 2.1 .12 (part 75 2.3 purifying)
70 2.1 male, 158 137. 18.6 7 7.2 .21
3 (no vaccines) 113 7.5
141??????????????????????????????7.1
We have also obtained splenocyte and have used the taeniasis suis L-Cysteine HCL Anhydrous of purifying or taeniasis suis extract to stimulate them from the taenia crassiceps mice infected.From carry cysticercosis infect the mouse in 6 week and normally, the mouse of the same age of not infection takes out splenocyte on one's body.Normal cell contacts with concanavalin A-A (Con-A).The cell that derives from infecting mouse contacts with initial taeniasis suis extract with the taeniasis suis L-Cysteine HCL Anhydrous of purifying.Insert by the 3H-thymidine and to measure cell proliferation.The proteolytic enzyme of purifying causes to breed in immunocyte replys, and this propagation is replied than the propagation that is caused by extract and replied strongly, is equivalent to the stimulation (Fig. 5) that is caused by mitogen concanavalin A-A.
The bonded of anti-taenia crassiceps of the external evaluation of EXAMPLE IV and taeniasis suis L-Cysteine HCL Anhydrous, efficiently and
Inhibitor optionally
Example II has shown the biological chemistry similarity of the cyst wall proteolytic enzyme of taeniasis suis and taenia crassiceps.Therefore, the proteinase inhibitor of anti-taeniasis suis cyst wall L-Cysteine HCL Anhydrous estimates to suppress the cyst wall L-Cysteine HCL Anhydrous of taenia crassiceps.
Have Langmuir and show that in conjunction with the result of isotherm formula the taeniasis suis L-Cysteine HCL Anhydrous has only a substrate binding site, indicating that it is feasible designing a kind of special inhibitor.Our purpose in this research is to confirm effective, strong and optionally vitro enzyme inhibition, preferably, has at the inhibitor of avtive spot in conjunction with feature.Can also be used to the similarity (Fig. 6) of enzyme analysis avtive spot at the avtive spot binding inhibitors of enzyme.Three kinds of inhibitor of tapeworm cyst wall L-Cysteine HCL Anhydrous have been identified, the protection potentiality of two kinds of inhibitor in these inhibitor of having gone back interior evaluating in treatment mouse cysticercosis process.
Two kinds of proteinase inhibitor: Z-LLL-FMK (Z-LLL-FMK) and Z-LLY-FMK (Z-LLL-FMK) are at the taeniasis suis L-Cysteine HCL Anhydrous that suppresses purifying with the Z-Phe-Arg-AFC of low effective concentration in as the analytical test of substrate.These proteinase inhibitor suppress the activity of taeniasis suis L-Cysteine HCL Anhydrous 100% and 97% respectively with the concentration of nmole.Our dynamics data shows that these inhibitor combine with the avtive spot of L-Cysteine HCL Anhydrous and it is also had very strong deactivation power.The third inhibitor, Ph2-LF-KP also demonstrate and can suppress the target L-Cysteine HCL Anhydrous very effectively, when using with the concentration identical with Z-LLL-FMK and Z-LLY-FMK, can suppress 80% activity of target L-Cysteine HCL Anhydrous.Inhibitor (Z-LLL-FMK and Z-LLY-FMK) also can suppress the taenia crassiceps L-Cysteine HCL Anhydrous similarly.
Z-LLY-FMK and Z-LLL-FMK also suppress the degraded of taeniasis suis L-Cysteine HCL Anhydrous to human IgG.Therefore, we infer that they also may have the potentiality of tapeworm packing degraded human IgG in the blocking-up body.Inhibitor suppresses the strong efficiency of target protease and they with lower concentration the nontoxicity (example VII A) of external immunocyte is shown that these inhibitor can become ideal candidates inhibitor in the mouse model of chemotherapy cysticercosis.
EXAMPLE V adopts specific inhibitor Z-LLY-FMK (Z-LLY-FMK) and Z-LLL-FMK (Z-LLL-FMK) to prevent BALB/c mouse
Infect cysticercosis
May play the prerequisite of keying action based on the cyst wall L-Cysteine HCL Anhydrous to the time to live of tapeworm packing, we have detected effective inhibitors, and Z-LLL-FMK, Z-LLY-FMK and Ph2-LF-KP prevent the ability of mouse infection cysticercosis.In the tentative experiment of BALB/c mouse, Z-LLL-FMK and Z-LLY-FMK are tested respectively as preventive.Injection is undertaken by the methyl-sulphoxide of 150ul.With described inhibitor mouse is carried out two days intraperitoneal preform injection (～1.4 * 10
-2M is dissolved in the volume injected of 150ul 0.15M PBS), follow every mouse and infect with 10 taenia crassiceps packings among the 200ul 0.15M PBS.After this, the time in four weeks, mouse is used same concentration every day.Infect after one month, two groups of euthanize are washed and remove packing with aseptic 0.15M PBS.Minimum two packings that link are be evaluated as how shallow (multilobed) that splits (ML).Through visual inspection, inhibitor does not have toxic action to mouse.
Avoided the infection (table 3) of cysticercosis with mouse 100% ground in the group of Z-LLL-FMK processing.Compare with the control group of handling, have 85%-97% to avoid the infection (table 3 and 4) of cysticercosis with the mouse in the group of Z-LLY-FMK processing.Mouse in the group of handling with Ph2-LF-KP has 40% infection of having avoided cysticercosis.Ensuing research has repeated identical preventive experiment with Z-LLY-FMK, and the result has shown similar protection data (75%-90%).
The vitro inhibition of table 3. taeniasis suis halfcystine and the little %BALB/c minor benefit of the external pork %BALB/c of mutual relationship inhibitor % between the intravital protection are seen with SEM
The packing that the therapeutic of the preventative mouse of tapeworm half Guang ammonia mouse is examined
Lip-deep the exempting from of protection protection of pepsin
Suppressing the intensive Ph2-LF-KP of epidemic disease cell Z-LLL-FMK 100% 100% 60% N/aZ-LLY-FMK 97% 85-97% n/a 80% 40% n/a does not observe " n/a " expression and does not obtain data.
Table 4.Z-leucine-leucine-tyrosine-fluorine ketone is to the result of treatment of the BALB/c mouse that is subjected to the taenia crassiceps packing and attacks
| Not treatment contrast | Packing # | ?Avg | ?SD | The % protection | #ML | ?％ML | ? |
| Mouse | |||||||
| 1 | ?240 | ?197.7 | ?34.3 | ?30 | ?12.5％ | ?14.4 | |
| Mouse | |||||||
| 2 | ?156 | ?16 | ?10.3％ | ||||
| Mouse 3 | ?197 | ?40 | ?20.3％ | ||||
| The treatment contrast | |||||||
| Mouse 4 | ?7 | ?21.3 | ?10.2 | ?96.5％ | ?4 | ?57％ | ?51％ |
| Mouse 5 | ?30 | ?84.8％ | ?13 | ?43 | |||
| Mouse | |||||||
| 6 | ?27 | ?86.4％ | ?14 | ?52％ |
Be that a high proportion of treated still packing of survival demonstrates improper form enjoyably.Many these packings present electrodeless multicell (apoloar multilocularity) (many-the shallow outward appearance of splitting), a kind of improper blastogenesis pattern.Through histology, the how shallow cyst wall of splitting has increased than the normal shallow cyst wall of splitting.
Also carried out the treatment experiment of Z-LLL-FMK.At treatment stage, every day and last week every other day accept 1.48 * 10 in each three week of mouse of treatment
-2Leucine-leucine of ug/ul-tyrosine-fluorine ketone.When mouse infection latter two week of taenia crassiceps packing begin to carry out the treatment of inhibitor, the result shows 60% protection ratio (table 3).Mouse in all groups all survives and does not have significant side effects (for example, film disease, hangover or paresis etc.).
At last, clearly the dose response of vitro inhibition taeniasis suis L-Cysteine HCL Anhydrous is relevant with the endogenous protective of BALB/c mouse.And from the lower inhibitor of validity, the packing that the mouse that Ph2-LF-KP handles is got on one's body neither adheres to immunocyte and does not also damage skin.Example VI. the packing of the mouse of usefulness tapeworm cysteine protease activity inhibitor for treating
Morphological analysis
As if scanning electronic microscope (SEM) detects the result with the minority survival packing of the mouse of Z-LLY-FMK treatment be taken from the EXAMPLE V and shows that the phenomenon that adheres to immunocyte is arranged, the local cyst wall of following this immunocyte to occur just wreck (Fig. 7).We identify the lip-deep scavenger cell of mouse packing, neutrophilic leukocyte, inoblast and the collagen deposition thing that comes from treatment, and do not have these cells and settling on the packing of untreated mice.The atrophy of total surface has also appearred.Compare microtriche (microvillus) or gamut comes off or obviously shorten generally on one's body with the packing of getting from untreated mouse.Found then that for the packing of getting from the mouse of treatment the integrity on packing surface is broken on one's body.Higher magnification ratio shows that near the foreskin the immunocyte has taken place to rot.The packing of getting on one's body from untreated mouse has the long and surperficial uniform feature of packing of no immunocyte, complete sum microtriche.These results are consistent with other result of study, and described other result of study shows the cysticercus alive of pig muscle and do not show host's inflammation on every side from the cysticercus alive that the postmortem human body is got.
Example VII A Z-LLY-FMK and Z-LLL-FMK do not have toxicity to the splenocyte of external BALB/c mouse cell
Taking-up mouse boosting cell and list are handled with inhibitor or with inhibitor and concanavalin A-A (Con-A).Taking out the splenocyte and the list of normal mouse handles with inhibitor or with inhibitor and concanavalin A-A.By
3The H-thymidine is inserted into and measures cell proliferation in the cell.Fig. 8 has drawn a complete inhibitor dose response curve with maximum dose level.Described inhibitor do not induce propagation reply (by
3The H-thymidine inserts mensuration).And described inhibitor does not influence the reactivity of splenocyte to Con-A.And this dosage is three times of the concentration that is used for injecting mouse every day in the prevention experiment (EXAMPLE V).
And even there is inhibitor to exist, trypan blue staining shows still all survivals of mouse cell.When these researchs have confirmed that further our observation is promptly handled host cell in adopting the specific inhibitor body, do not observe host's side reaction yet.Z-LLY-FMK and Z-LLL-FMK thereby may normal BALB/c mouse splenocyte in the body not had toxicity.
Example VII A I
Take the tapeworm cystatin to the people
The inhibitor that is used for medical treatment or animal doctor's purpose is designed to suppress in human body, the pig body or the activity of the tapeworm L-Cysteine HCL Anhydrous among both.In the pig body, the taeniasis suis packing is mainly settled down in muscle.Yet parasite is very neurophilic in human body, so disease is usually expressed as neurocysticerocosis.In neurocysticerocosis, the taeniasis suis packing is settled down in brain, therefore needs design can pass the inhibitor of blood brain barrier.Mouse taenia crassiceps model is fit to measure proscolex (packing) to susceptibility in the body of medicine very much.But also need to measure the ability that potential human medicine passes blood brain barrier.Resembling medicine that albendazole and praziquantel act on the brain packing is like this inferred and can be passed barrier.
Experiment shows that the oil-water partition coefficient of nonelectrolyte absorbs relevant (Levin, J.Med.Chem., 23:682-684 (1980) with their membrane penetrating and the brain in the animal model; Cornford, Exper.Parasitol., 70:25-34 (1990)).Therefore, can determine the partition ratio of inhibitor compound, and if their value prediction be the barrier impermeability, just need design improvement to see through the scheme of possibility.There are many designs by this barrier in known present technique field, comprises that the physics of chemically modified, blood/brain barrier and the blood/brain barrier environment of medicine changes (Abbott etc., Mol.Med.Today, in March, 1996, the summary among the 106-113).For example, Alkermes, Inc.of Cambridge, MA, company of specializing in medicine-transfer system has developed a kind of medicine RMP-7 (Cereport ) that may penetration rate of blood/brain barrier, and this medicine is carrying out the ∏ clinical trial phase.In the another one example, can carry out chemically modified to drug candidate and improve its partition ratio.For example, the FMK substituting group is considered to the film perviousness of medicine is very important and may assists medicine was transported blood/brain barrier.
Whole disclosures of all patents, the patent application that comprises temporary patent application and publication and available electronic material (for example, GenBank amino acid and nucleotide sequence) are quoted as a reference by this paper.Above detailed explanation and embodiment are just in order to be expressly understood that the present invention provides.And should not be interpreted as it is thus to unnecessary restriction of the present invention.Shown in the present invention is not subjected to and the restriction of described detailed content; Many modifications all are conspicuous for those skilled in the art and all will be included in the scope of the present invention that is defined by the claims.
Claims (43)
1. isolating cyst wall L-Cysteine HCL Anhydrous from taeniasis suis.
2. isolating polynucleotide with nucleotide sequence of the described proteolytic enzyme of coding claim 1.
3. isolating is the polynucleotide of the complement of the described nucleic acid molecule of claim 2.
4. as the cyst wall L-Cysteine HCL Anhydrous of vaccine or have the polynucleotide of the nucleotide sequence of coding cyst wall L-Cysteine HCL Anhydrous.
5. cyst wall L-Cysteine HCL Anhydrous or polynucleotide with nucleotide sequence of coding cyst wall L-Cysteine HCL Anhydrous are used to prepare the purposes of vaccine.
6. contain at least a being selected from down and form the vaccine that divides: (a) comprise the polypeptide of cyst wall L-Cysteine HCL Anhydrous or its immune peptide subunit and the polynucleotide that (b) comprise the nucleotide sequence of coded polypeptide, this polypeptide contains cyst wall L-Cysteine HCL Anhydrous or its immune peptide subunit.
7. vaccine as claimed in claim 6, wherein the cyst wall L-Cysteine HCL Anhydrous derives from taeniasis suis or taenia crassiceps.
8. treatment is subjected to the method for the animal of cestode infection, this method comprises with claim 6 or 7 described vaccines carries out medication to infection animal, and wherein the vaccine medication can effectively be eliminated described parasite or prevention on one's body or postpone animal from animal and cysticercosis or neural cysticercosis occur.
9. the method for the opposing cestode infection that watches for animals; this method comprises with claim 6 or 7 described vaccines carries out medication to infection animal not, and wherein the composition medication can effectively be avoided after the animal by described parasitic infection or avoid being suffered from cysticercosis or neural cysticercosis by the animal after the described parasitic infection afterwards.
10. method as claimed in claim 8 or 9, wherein said animal are that pig or people and described cestode infection are infection by Taenia solium.
11. pharmaceutical composition, it contains the inhibitor molecules that suppresses the cyst wall cysteine protease activity, and this inhibitor molecules contains a kind of peptide or peptide simulated compound; And pharmaceutically acceptable carrier.
12. pharmaceutical composition as claimed in claim 11, wherein said peptide or peptide simulated compound contain (Xaa)
n-Yaa-Zaa-R; Wherein Xaa and Zaa independently are any amino acid separately; Yaa is a hydrophobic amino acid; R comprises the nucleophilic part; And n=0-5.
13. pharmaceutical composition as claimed in claim 12, wherein Yaa is a leucine.
14. as claim 12 or 13 described pharmaceutical compositions, wherein R comprises and is selected from following chemical part: carboxylic acid derivative, amide derivatives, benzene derivative, phenyl derivatives, chloromethyl ketone or derivatives thereof, methyl fluoride ketone or derivatives thereof, α ketone acid or derivatives thereof, keto-amide or derivatives thereof, keto ester or derivatives thereof, vinyl sulfone(Remzaol or derivatives thereof and pyridyl or derivatives thereof.
15. the pharmaceutical composition described in claim 12 to 14, wherein R comprises methyl fluoride ketone (FMK).
16. pharmaceutical composition as claimed in claim 15, wherein inhibitor molecules is Z-Leu-Leu-Leu-FMK or Leu-Leu-Tyr-FMK.
17. the pharmaceutical composition described in claim 11 to 16, wherein inhibitor molecules suppresses to derive from the cyst wall L-Cysteine HCL Anhydrous of taeniasis suis or taenia crassiceps.
18. be used for Z-Leu-Leu-Leu-FMK or Z-Leu-Leu-Tyr-FMK as active medicine.
19. Z-Leu-Leu-Leu-FMK or the Z-Leu-Leu-Tyr-FMK that is used for the treatment of human nerve's cysticercosis or cysticercosis cellulosae as claimed in claim 18.
20.Z-Leu-Leu-Leu-FMK or Z-Leu-Leu-Tyr-FMK is used for the purposes of pharmaceutical compositions.
21. the inhibitor molecules that suppresses the tapeworm cysteine protease activity is used for the treatment of the purposes of human nerve's cysticercosis or cysticercosis cellulosae, this inhibitor molecules comprises polypeptide or polypeptide simulated compound.
22. the purposes of inhibitor molecules as claimed in claim 21, wherein said peptide or peptide simulated compound contain (Xaa)
n-Yaa-Zaa-R; Wherein Xaa and Zaa independently are any amino acid separately; Yaa is a hydrophobic amino acid; R comprises the nucleophilic part; And n=0-5.
23. the purposes of inhibitor molecules as claimed in claim 22, wherein Yaa is a leucine.
24. as the purposes of claim 22 or 23 described inhibitor molecules, wherein R comprises and is selected from following chemical part: carboxylic acid derivative, amide derivatives, benzene derivative, phenyl derivatives, chloromethyl ketone or derivatives thereof, methyl fluoride ketone or derivatives thereof, α ketone acid or derivatives thereof, keto-amide or derivatives thereof, keto ester or derivatives thereof, vinyl sulfone(Remzaol or derivatives thereof and pyridyl or derivatives thereof.
25. the purposes of the inhibitor molecules described in claim 22 to 24, wherein R comprises methyl fluoride ketone (FMK).
26. suppress the method for tapeworm cyst wall cysteine protease activity, this method comprises and will comprise (Xaa)
n-Yaa-Zaa-R; Each any naturally amino acid of Xaa and Zaa wherein; Yaa is a hydrophobic amino acid; R comprises the nucleophilic part; With n is that 0 to about 5 inhibitor molecules contacts with tapeworm cyst wall L-Cysteine HCL Anhydrous.
27. method as claimed in claim 26, wherein Yaa is a leucine.
28. as claim 26 or 27 described methods, wherein R comprises and is selected from following chemical part: carboxylic acid derivative, amide derivatives, benzene derivative, phenyl derivatives, chloromethyl ketone or derivatives thereof, methyl fluoride ketone or derivatives thereof, α ketone acid or derivatives thereof, keto-amide or derivatives thereof, keto ester or derivatives thereof, vinyl sulfone(Remzaol or derivatives thereof and pyridyl or derivatives thereof.
29. the method described in claim 26 to 28, wherein R comprises methyl fluoride ketone (FMK).
30. method as claimed in claim 29, wherein inhibitor molecules is Z-Leu-Leu-Leu-FMK or Z-Leu-Leu-Tyr-FMK.
31. the method described in claim 26 to 30, it does not carry out in having the environment of cell.
32. the method described in claim 26 to 30, it carries out in cell culture, in organ or in tissue.
33. the method described in claim 26 to 30, it carries out in complete animal body.
34. as the described method in the claim 26 to 30, wherein the tapeworm L-Cysteine HCL Anhydrous derives from taeniasis suis.
35. identify the method for tapeworm cysteine protease activity inhibitor, this method comprises:
Candidate inhibitor is combined the formation mixture with the tapeworm L-Cysteine HCL Anhydrous;
Protease substrate Z-Phe-Arg-7-amino-4-trifluoromethyl tonka bean camphor is added in the mixture; With
Determine the degree that protease substrate is cleaved; Wherein the substrate cracking degree when not having candidate inhibitor to exist is compared, and the reduction of substrate cracking degree is the index that suppresses the tapeworm cysteine protease activity.
36. method as claimed in claim 35, this method comprise candidate inhibitor is combined the formation mixture with the L-Cysteine HCL Anhydrous that derives from taeniasis suis or taenia crassiceps.
37. identify the computer-householder method of tapeworm cysteine protease activity inhibitor, this method comprises:
The computer organization model of tapeworm cysteine protease activity inhibitor is provided;
From the structure storehouse, screen similar in the candidate compound of described inhibitor or by (ii) calculating or estimate the candidate compound that project organization is similar to described inhibitor by (i) calculating or range estimation; With
Analyze the ability that candidate compound suppresses the tapeworm cysteine protease activity.
38. identify the computer-householder method of tapeworm cysteine protease activity inhibitor, this method comprises:
Set up the computer model of tapeworm L-Cysteine HCL Anhydrous by the X-ray crystal structure of analyzing the tapeworm L-Cysteine HCL Anhydrous;
The computer organization model of tapeworm cysteine protease activity inhibitor is provided;
By calculating or estimating inhibitor structure is docked with the binding site of protease crystals structure at inhibitor;
By calculating or estimate the interaction of identifying between inhibitor and protease molecule;
From the structure storehouse, screen similar in the candidate compound of described inhibitor or by (ii) calculating or estimate the candidate compound that project organization is similar to described inhibitor by (i) calculating or range estimation;
By calculating or estimate the interaction of assessing between candidate compound and described protease molecule;
Analyze the ability that candidate compound suppresses the tapeworm cysteine protease activity.
39. as claim 37 or 38 described methods, wherein inhibitor is Z-Leu-Leu-Leu-FMK or Z-Leu-Leu-Tyr-FMK.
40. treatment is subjected to the method for the animal of cestode infection, this method comprises with the pharmaceutical composition that contains the tapeworm cystatin carries out medication to infection animal, and wherein the composition medication can effectively be eliminated described parasite or prevention on one's body or postpone animal from animal and the sick or neural cysticercosis of bladder worm occur.
The method of opposing cestode infection 41. watch for animals; this method comprises with the pharmaceutical composition that contains the tapeworm cystatin carries out medication to infection animal not, and wherein the composition medication can effectively be avoided after the animal by described parasitic infection or avoid being suffered from cysticercosis or neural cysticercosis by animal after the described parasitic infection afterwards.
42. as claim 40 or 41 described methods, wherein inhibitor is Z-Leu-Leu-Leu-FMK or Z-Leu-Leu-Tyr-FMK.
43. as the described method of claim 40,41 or 42, wherein said animal is that pig or people and described cestode infection are infection by Taenia solium.
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| US13033899P | 1999-04-21 | 1999-04-21 | |
| US60/130,338 | 1999-04-21 |
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| CN00806591A Pending CN1352682A (en) | 1999-04-21 | 2000-04-20 | Cysteine protease and inhibitors for prevention and treatment of neurocysticerocosis |
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| BR (1) | BR0009944A (en) |
| MX (1) | MXPA01010541A (en) |
| WO (1) | WO2000063350A2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002086115A1 (en) | 2001-04-20 | 2002-10-31 | The University Of Georgia Research Foundation, Inc. | Active site inactivators |
| EP2633855A1 (en) | 2012-03-01 | 2013-09-04 | Veterinärmedizinische Universität Wien | Protease inhibitors for treating Trichomonas gallinae infections |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ224597A (en) * | 1988-05-12 | 1992-02-25 | Coopers Animal Health | Taenia ovis antigens and vaccines |
| EP0635058A1 (en) * | 1991-11-12 | 1995-01-25 | The Colorado State University Research Foundation | Protease vaccine against heartworm |
| AU4998993A (en) * | 1992-08-07 | 1994-03-03 | Epimmune, Inc. | Hla binding peptides and their uses |
| ATE352556T1 (en) * | 1997-04-11 | 2007-02-15 | Sangstat Medical Corp | CYTOMODULATING LIPOPHILIC PEPTIDES FOR MODULATING IMMUNE SYSTEM ACTIVITY AND INHIBITING INFLAMMATION |
-
2000
- 2000-04-20 WO PCT/US2000/010672 patent/WO2000063350A2/en active Application Filing
- 2000-04-20 AU AU46505/00A patent/AU4650500A/en not_active Abandoned
- 2000-04-20 MX MXPA01010541A patent/MXPA01010541A/en unknown
- 2000-04-20 CN CN00806591A patent/CN1352682A/en active Pending
- 2000-04-20 BR BR0009944-9A patent/BR0009944A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000063350A3 (en) | 2001-02-08 |
| MXPA01010541A (en) | 2003-08-19 |
| AU4650500A (en) | 2000-11-02 |
| WO2000063350A2 (en) | 2000-10-26 |
| BR0009944A (en) | 2002-01-08 |
| WO2000063350A9 (en) | 2002-06-13 |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |