CN103819554A - Synthetic SP series polypeptides and application thereof - Google Patents
Synthetic SP series polypeptides and application thereof Download PDFInfo
- Publication number
- CN103819554A CN103819554A CN201410088581.9A CN201410088581A CN103819554A CN 103819554 A CN103819554 A CN 103819554A CN 201410088581 A CN201410088581 A CN 201410088581A CN 103819554 A CN103819554 A CN 103819554A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- cell
- cancer
- leukemia
- pp2a
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses synthetic SP series polypeptides and application thereof in treating cancer. The specific amino acid sequences of the synthetic SP series polypeptides are as shown in SEQ ID No. 1-11. The SP series polypeptides can be used for treating chronic myelogenous leukemia. The SP series polypeptides performs the function of treating leukemia by activating the activity of intracellular PP2A and conducting dephosphorylation on BCR/ABL, and also has a treatment effect on imatinib-resistant leukemia. The SP series polypeptides perform the function through SET/PP2A path, and have wide application in treating cancer.
Description
Technical field
The invention belongs to the polypeptide drugs technical field in biological chemistry, be specifically related to the purposes of polypeptide, SP series polypeptide has the purposes for the treatment of cancer.
Background technology
Chronic granulocytic leukemia (CML), also claims chronic myelocytic leukemia (CGL), is that a class originates in the cell generation canceration that forms blood in marrow, and infiltrates the cancer of blood.Suffer from after CML, leukemia cell assembles in vivo need to be for a long time, many patients in several years even without any symptom.Meanwhile, other histoorgans of cancer cell infiltration body, comprise spleen.CML also can be transformed into the mushroom acute leukemia of cancer cells, and its cancer cells can infiltrate each organ of body fast.Any hematopoietic cell or lymphoidocyte can be transformed into leukemia cell.Once produce such variation, these leukemia cells just can not carry out normal ripe program.Some leukemia cells may accelerate by rate of propagation, but in most cases, but can be not dead at specified time.They continue survive and gather together, and often squeeze normal medullary cell, cause erythrocyte quantity to die-off.As time goes on, leukemiacell infiltration periphery cell is also transferred to its hetero-organization, thereby affects body correlation function.
The case of suffering from CML accounts for 10% left and right of total leukemia case, and the male sex suffers from the possibility of this disease a little more than women, and the ill possibility of white people is higher than non-descendants American.Suffer from the patient age of CML greatly about 64 years old, almost patient ages more than half 65 years old and more than.Most of CML betide between adult, and only a few betides between children, generally speaking, similar with adult to children's treatment.American Cancer Society in 2014 announces approximately has 5980 patients to be diagnosed as CML (3130 male patients and 2850 female patients), wherein approximately 810 people's death (550 male patients and 260 female patients).
The symptom of CML is conventionally fuzzyyer, and often caused by other reasons, comprising: weakness, fatigue, night sweat, lose weight, fever, ostalgia, spleen expand (sensation left side thorax has foreign matter), belly has pain or swelling sense and eat a light meal and just feel there is satiety.But these have more than is the symptom that CML there will be, and they also there will be in other cancer symptoms, or in the disease of non-cancer.
In the time being made a definite diagnosis, there is not any symptom in many CML patients.Leukemia normally patient finds doing in other uncorrelated disease examinations, even and existing partial symptoms, they are often fuzzy and nonspecific.Conventionally can do FBE, in the common body of CML patient, quantity of leucocyte increases, and contains many immature cells.Sometimes in patient body, red corpuscle and hematoblastic quantity can reduce, although these symptoms are shown to be leukemia, patient generally still will carry out other blood and bone marrow examination to make a definite diagnosis.
The treatment of CML is mainly comprised to the following aspects: targeted therapy, immunotherapy, chemotherapy, radiation therapy, operation, stem cell transplantation, wherein targeted therapy is Main Means.
Conventional chemotherapy is often referred to any medicine that can treat cancer, mainly refers to conventional cell toxicity medicament here, mainly enters blood circulation of human body by mode oral or injection, kills those proliferation and differentiation cell rapidly.But tumor growth rapidly cell not only have cancer cells, in normal cell, also have, as marrow inner cell, oral cavity and intestines inner cell and hair follicle inner cell etc., they also can be attacked by chemotherapeutics conventionally, thereby cause the side effects such as alopecia, stomatocace, nausea and vomiting, easily stasis of blood purple is hemorrhage, tired.Be generally used for now auxiliary target to pharmacological agent, or use after targeted drug loses medicinal effect.
The oncogene that has BCR-ABL by name in CML cell, is caused by 9,22 chromosome rearrangements, in normal cell, does not exist.This genetic transcription and translation becomes BCR-ABL protein, can cause the breeding of CML Growth of Cells to exceed control.BCR-ABL is a class tyrosine-kinase zymoprotein, and nowadays, the tyrosine kinase inhibitor of target BCR-ABL (TKIs) medicine is the choice drug for the treatment of CML.These medicines are very micro-to normal impact cell, so the medicine that their side effect is used for the treatment of CML than other is much smaller, and for example chemicals or Interferon, rabbit.Conventional targeted drug is as follows:
Imatinib (imatinib mesylate) is the medicine of first target BCR-ABL, becomes very soon the choice drug for the treatment of CML, becomes famous with first-generation tyrosine kinase inhibitor.Nearly all CML patient replys treatment with imatinib, and Most patients can be lived many years.But this medicine can not be cured leukemia completely, thus need patient to take every day, until lose result for the treatment of.For some patient, imatinib finally loses treatment function, so-called imatinib resistance that Here it is.Imatinib resistance is mainly that in CML cell, some transgenation causes, and some resistance can solve by strengthening dosage, but some needs another kind of medicine, and this just introduces TKIs s-generation product---Dasatinib/nilotinib.They are generally used for imatinib resistance or the insupportable situation of imatinib side effect patient, but still have very large side effect, and dosage will be higher than imatinib.Pu Na is the TKIs product of up-to-date target BCR-ABL for Buddhist nun.In the time that every other TKIs product is ineffective, just use Pu Na to replace Buddhist nun to carry out CML treatment.Or in the time there is T315I sudden change in CML cell, also adopt Pu Na to replace Buddhist nun to treat.T315I sudden change conventionally betides other TKIs products treatments of life-time service CML and produces after resistance, so far, has and only have Pu Na to replace the Buddhist nun can this resistance behavior of continual cure.But Pu Na is quite serious to the side effect of patient body for Buddhist nun, except common weakness, headache, stomachache, measles or other tetter, also can produce in vivo clot, easily causes heart trouble, apoplexy and block artery of extremity vein etc.More seriously this medicine can make cardiac muscle atrophy, will cause pancreatitis, liver failure and congestive heart failure etc.
Therefore, need in a hurry now a kind of medicine of new mechanism of action, can safe and effective treatment CML.
Summary of the invention
COG133 is the polypeptide of first anti-inflammatory and neuroprotective material, derives from lipophorin (apoE) receptors bind region 133-149 amino acids sequence in human body.COG112 is that the N end of COG133 has merged the fusogenic peptide that comes from tactile keratic protein delivery structural domain (PTD) the sequence formation of Drosophila, and biological activity improves greatly compared with COG133.ApoE series polypeptide has the polypeptide of apoE albumen 133-149 amino acids sequence, research before this finds that it has treatment disorder disease, comprises central nervous system (CNS) inflammation, traumatic brain injury, inflammatory bowel, cerebral ischemia disease, atherosclerosis, septicemia, sacroiliitis, alzheimer's disease and other cerebral nerve disorders.
The present invention is by COG133 is carried out to polypeptide transformation, synthetic a series of SP series polypeptide, as follows:
COG133?Ac-LRVRLASHLRKLRKRLL-NH
2?(SEQ?ID?NO:1)
SP2?Ac-LRVRLASVLRKLVKRLL-NH
2?(SEQ?ID?NO:2)
SP2?Ac-LRVRLASHLRkLRKRLL-NH
2?(SEQ?ID?NO:3)
SP4?Ac-LRVRLASHLRKLRkRLL-NH
2?(SEQ?ID?NO:4)
SP5?Ac-LRVRLASHLRkLRkRLL-NH
2?(SEQ?ID?NO:5)
SP6?Ac-LRVRLASHLrKLRKRLL-NH
2?(SEQ?ID?NO:6)
SP7?Ac-LRVRLASHLRKLrKRLL-NH
2?(SEQ?ID?NO:7)
SP8?Ac-LRVRLASHLrKLrKRLL-NH
2?(SEQ?ID?NO:8)
SP9?Ac-ASHLRkLRKRLL-NH
2?(SEQ?ID?NO:9)
SP10?Ac-ASHLRKLRkRLL-NH
2?(SEQ?ID?NO:10)
CU063?Ac-LRVRLASVLRKLVkRLL-NH
2?(SEQ?ID?NO:11)
Wherein small letter k represents D type Methionin, and small letter r represents D type arginine, and Ac represents acetylize carboxyl terminal.
For CU063, the present invention expects, by introducing D type amino acid, can delay polypeptide degraded, obtains better pharmacologically active long half-lift of obtaining in vivo more.
The invention provides and be used for the treatment of leukemic method, comprise when at least one SP series polypeptide is not existed with described peptide, can occur compared with reduce disease symptoms amount use.In one embodiment, described leukemia is chronic granulocytic leukemia (CML).SET, is also called as I
2pP2A is a PP2A inhibitor on effective physiological level.SET is overexpression and inhibition PP2A activity in CML cell, maintains oncogene BCR/ABL kinases in sustained activation state.Add after SP series polypeptide, can activate CML cell interior PP2A activity, the instrumentality of dephosphorylation relevant cell propagation and survival, thus suppress BCR/ABL kinase activity, reach and reduce the object that leukemia produces.This mechanism is different with tyrosine kinase inhibitor (imatinib, Dasatinib) mechanism of action, and the latter directly acts on BCR/ABL kinases, can be subject to this kinase mutant and affects pharmaceutical activity.Therefore, SP series polypeptide has the ability of the CML for the treatment of Bcr/Abl transgenation.
In one embodiment of the invention, SP series polypeptide increases the activity of PP2A in the cell being subject to processing.PP2A is an important silk Threonine Phosphatases, it is a heterogeneous triplet, comprise two common components, a catalytic subunit (PP2Ac) and a support subunit (PP2A A) composition catalytic center dimer are connecting an adjusting B subunit by four kinds of different family gene codings simultaneously.PP2A can regulate most biological processes in cell, comprises cell proliferation, apoptosis, growth and migration.The forfeiture of PP2A activity or suppressed meeting cause PP2A seriously to lose the function that suppresses tumour.The inventor has reason to believe that SP series polypeptide can reduce one or more symptoms relevant to cancer, includes but are not limited to tumour formation, tumor growth, cancer cells number, cancer cells to the diffusion of health tissues and the survival of minimizing.SP series polypeptide can be used for treating multiple leukemia (acute lymphoblastic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia etc.), mammary cancer, cervical cancer, ovarian cancer, prostate cancer, colorectal carcinoma, lung cancer, carcinoma of the pancreas, the cancer of the brain, skin carcinoma (melanoma and non-black element knurl), bladder cancer, carcinoma of endometrium, renal cell carcinoma, cancer of the stomach, esophagus cancer, liver cancer, lymphoma and sarcoma etc.
Polypeptide of the present invention can be by standard technique generation as known in the art.Peptide of the present invention can add multiple mark part if radio-labeling, fluorescent mark are for detecting and spike.Fluorescent mark includes but are not limited to fluorescein, eosin, rhodamine, tonka bean camphor and NBD fluorophore etc.
Can be easy to be reached by those skilled in the art to strengthen the functionally active relevant to these peptides to the modification of peptide disclosed herein.For example, for the peptide of the inventive method can be through chemically modified or coupling in other molecules to strengthen as the parameter such as solvability, serum stability, and reservation function activity.Particularly; peptide of the present invention can be held acetylize and/or hold amidation at C-at N-; or coupling, compound or be blended in and strengthen the molecule of its serum stability, include but are not limited to albumin, immunoglobulin (Ig) and fragment thereof, Transferrins,iron complexes, lipoprotein, liposome, PEG and dextran etc.
Peptide of the present invention not only comprises the aminoacid sequence of all disclosed SP series polypeptide, also comprise that those pass through for example homologous recombination, fixed point or PCR mutagenesis simultaneously, and the corresponding peptides of other animal species, wherein said animal species includes but are not limited to rabbit, rat, pig, ox, sheep, horse and non-human primates species, and comprise that peptide wherein uses that part except naturally occurring amino acid is substituted, chemistry, enzyme or other appropriate means and the derivative of covalent modification.
SP series polypeptide disclosed by the invention, includes but are not limited to SP series polypeptide and derivative thereof, also can be free form or salt form, and wherein said salt is pharmaceutically acceptable.This comprises the inorganic salt of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese etc.The also available multiple organic salt that includes but are not limited to the described peptides of preparation such as acetic acid, propionic acid, pyruvic acid, toxilic acid, succsinic acid, tartrate, citric acid, phenylformic acid, styracin, Whitfield's ointment.
SP disclosed by the invention series polypeptide can with pharmaceutically acceptable carrier combinations.Described carrier can be liquid, thereby makes said composition be applicable to parenteral administration, or can be solid, and preparation is for oral tablet or pill.In addition, described carrier can be sprayable liquid or solid form, thereby makes described composition be applicable to suck.Peptide of the present invention also can be formulated as for topical application, the form of for example emulsifiable paste or gel, and topical formulations is particularly useful for treatment skin carcinoma.
SP disclosed by the invention series polypeptide can be by different approaches to human body administration, and that concrete route of administration comprises is oral, parenteral administration, oral cavity suction, rectal administration, intranasal administration, sublingual administration and by the administration of the property implanted container.That parenteral administration is included in is again subcutaneous, in intravenously, intramuscular, intraarticular, synovial membrane chamber, in breastbone, in sheath, in liver, in damage location and intracranial injection or infiltration.
SP series polypeptide disclosed by the invention is when for human body diseases prevention and treatment, dosage used is subject to the impact of many factors, these factors comprise age, body weight, healthy state, sex, race, food habits, administration time, micturition frequency and whether use other drug, etc.
Peptide of the present invention can be used for the treatment of separately cancer or use with other therapeutic combinations that are generally used for treating cancer, for example chemotherapeutics of described therapeutical agent (Chlorambucil, endoxan), reflunomide (prednisone, prednisolone), fludarabine, pentostatin, CldAdo, imatinib, Dasatinib, hormonotherapy etc.
Accompanying drawing explanation
The cytotoxicity of Fig. 1 COG133, SP2, CU063 polypeptide effect K562, BaF3p210WT, BaF3p210T315I cell strain 72h.
Fig. 2 COG133, SP2, CU063 polypeptide are in 24h, 48h, the 72h cytotoxicity to K562, BaF3p210WT, BaF3p210T315I cell strain respectively.
Fig. 3 COG133, SP2 polypeptide to K562, BaF3p210WT, the effect of BaF3p210T315I cell strain after, SET in cell, PP2A-C, BCR-ABL, the expression of the albumen such as p-BCR-ABL.
Fig. 4 COG133, SP2 polypeptide and respectively with okadaic acid (OA) coupling, act on after K562, BaF3p210WT, BaF3p210T315I cell strain the activity of PP2A in cell.
Embodiment
List following embodiment for the present invention is described, but should not be considered as limitation of the present invention.
Embodiment 1:
BCR-ABL fusion can produce the protein (p is the mass unit of metering intracellular protein, is equivalent to KDa) of weigh p210 or a p180.Select imatinib persister---the mouse source BaF3p210T315I cell with T315I sudden change, carry out cytotoxicity screening.
Detect the half-inhibition concentration (ICs of polypeptide to cytosis 24h, the 48h such as K562, BaF3p210WT, BaF3p210T315I, 72h such as COG133, SP2-SP10, CU063
50).
Experimental procedure:
1 inoculates K562, BaF3p210WT, BaF3p210T315I cell (2.5-4 × 10, every hole in 96 orifice plates
3individual cell), after 12-18h, every hole adds the substratum that 100 μ l contain polypeptide, and the peak concentration of polypeptide is 64 μ M, and 2 times of concentration are successively decreased.
2 after polypeptide effect 24h, 48h, 72h, plate is taken out from incubator, under room temperature, place after 30min, add CellTiter Glo Reagent(reagent balance under room temperature, the 96 every holes of orifice plate add 25 μ l), plate is placed in to the 5min(300 – 500rpm that vibrates on vibrator), under room temperature, place the concrete time of 10min-1h(and determine according to cell category).
3. after reading, ask IC
50(Perkin Elemer2103Multipliable Reader).
In K562, BaF3p210WT cell strain, detect COG133, the half inhibiting rate of SP2-SP10 polypeptide to cell.
The screening active ingredients of polypeptide in table 1:K562/BaF3p210WT cell strain
| IC 50(μM) | K562 | BaF3p210WT |
| COG133 | 24.7±1.6 | 21.3±2.0 |
| SP2 | 2.2±0 | 2.43±0.2 |
| SP3 | 18.4±0.8 | 18.0±1.1 |
| SP4 | 24.5±3.8 | 20.8±0.4 |
| SP5 | 24.1±1.0 | 20.0±1.0 |
| SP6 | 22.8±0.9 | 19.2±0.8 |
| SP7 | 24.6±1.1 | 22.0±2.0 |
| SP8 | 24.5±0.9 | 23.1±2.2 |
| SP9 | 19.0±1.0 | 24.4±1.0 |
| S10 | 17.3±0.6 | 17.3±2.9 |
Experimental result finds that SP2 polypeptide has good cytotoxicity, and its activity improves nearly 10 times than COG133.All the other polypeptide still can keep original COG133 cytoactive.
Introduce the amino acid whose CU063 of D type, active do not have considerable change (Fig. 1) than SP2.Illustrate at cell levels D type amino acid and do not play a role.
Can observe simultaneously, As time goes on (24h, 48h, 72h), polypeptide is continual and steady (Fig. 2) to the lethal effect of cell.
Embodiment 2:
Investigate SET, PP2A-C, BCR-ABL, the changing conditions of the albumen such as p-BCR-ABL under COG133 and the effect of SP2 polypeptide.
Experimental procedure:
1 inoculates K562, BaF3p210WT, BaF3p210T315I cell (2-3 × 10, every hole in six orifice plates
5individual cell, substratum 2ml), after 12-18h, add the polypeptide mother liquor of certain volume, make the final concentration of polypeptide be respectively COG133:20,10,5 μ M; SP2:2,1,0.5 μ M, after polypeptide effect 24h, cell is moved in 1.5ml EP pipe, centrifugation, abandon supernatant, clean cell precipitation with PBS and once discard afterwards PBS, add 120-150 μ l to add the cell pyrolysis liquid of proteinase inhibitor, high speed thermal agitation 5 seconds, allow cell precipitation suspend completely and to scatter, after ice bath 15-30min, in 4 ℃ of high speed centrifugation 5min, draw immediately supernatant to the EP pipe of precooling.
2 adopt BCA method to measure the protein concentration of cell pyrolysis liquid.
3SDS-PAGE electrophoresis (the every hole of total protein applied sample amount 20-40 μ g, resolving gel concentration is 12%), the about 20min of 90V electrophoresis, switches to the about 1h of 150V electrophoresis.
4 adopt wet type transferring film method, the sandwich of wet type transferring film is arranged as: sponge/paper/glue/film/paper/sponge, all between close-packed arrays, particularly glue and film, can not leave bubble, the direction that sandwich is laid confirms that correct negative pole side is the albumen in electronegative glue, (film) electromigration to positive pole side.Pvdf membrane need to soak 1-2 minute in methyl alcohol, then hatches in ice-cold electricity and turn in damping fluid 5 minutes, glue also need be in ice-cold electricity turns damping fluid balance 3-5 minute, otherwise can cause band distortion when transferring film.Transferring film condition: 300-350mA, constant current electrophoresis 50-55min.
5 use 5%BSA to soak, and 4 ℃ of sealings one hour, hatch primary antibodie (self-control sealing bag is hatched, the TBST preparation containing 1-5%BSA for primary antibodie), and 4 ℃ are cleaned film 3 times, each 10min with TBST after spending the night.Hatch two and resist, room temperature 1-2h.Again clean film 3 times with TBST, each 10min.
6 exposure colour developings, are used chemoluminescence method: HRP chemical luminous substrate (ECL method).
Found that polypeptide does not affect SET, PP2A, BCR/ABL protein expression, only make p-BCR/ABL dephosphorylation (Fig. 3), the positive polypeptide that echoes bibliographical information only affects the interaction between SET and PP2A, make PP2A dissociate out from mixture, thereby a series of downstreams of the dephosphorylation factor, reaches the effect that suppresses tumour.
Embodiment 3:
Investigate the polypeptide (COG133, SP2) of different concns, okadaic acid (okadaic acid, OA) is separately and after the cell certain hour such as combined action K562, the changing conditions of PP2A activity in cell pyrolysis liquid.The PP2A immunoprecipitation determination of activity test kit that this experiment adopts Merck & Co., Inc. to produce is measured.
Experimental procedure:
1. in six orifice plates, inoculate K562, BaF3p210WT, BaF3p210T315I cell (2-3 × 10, every hole
5individual cell, substratum 3ml), after 12-18h, add the polypeptide mother liquor (first add OA in compound sample, add again corresponding polypeptide after 12h) of certain volume, make the final concentration of polypeptide as shown in table 1 (COG133:10,20 μ M; SP2:1,2 μ M)
Table 1:Peptide-X, OA(okadaic acid) concentration arranges table
2. drug effect moves into cell in the EP pipe of 1.5mL after complete, 4 ℃, the centrifugal 5min of 1200g, supernatant discarded, with TBS cleaning cell 2 times (each 1.0ml), the centrifugal 5min of 1200g again, after supernatant discarded, add cell pyrolysis liquid (the 20mM Tris-HCl of 120 μ l, 150mM NaCl, 1mM EDTA, 1mM MgCl
2, 0.5%NP-40, proteinase inhibitor cocktail, pH7.5) and in lysing cell 30min on ice.4 ℃, centrifugal 8min under 14500rpm.Supernatant is proceeded in the EP pipe of an other 0.5mL, adopt BCA method to carry out the mensuration of protein concentration.
3. in the lysis supernatant of total protein 100 μ g, respectively add the PP2A-c antibody of 2 μ g, and add the albumin A agarose slurries of 25 μ l, then add the TBS of certain volume, make its cumulative volume be 500 μ l(protein concentrations be 0.2 μ g/ μ l).At 4 ℃
Under, on shaking table, hatch 2h.
4. hatched rear centrifugally, first cleaned agarose pearl 3 times, each 800 μ l with TBS.Measure buffer solution for cleaning once with 400 μ l pNPPSer/Thr subsequently, carry out immediately phosphate radical detection.If do not polluted by phosphate radical, add the phosphorylated peptide (end reaction concentration is 642 μ M) after 45 μ l dilutions, then add the pNPP Ser/Thr of 25 μ l to measure damping fluid.At 30 ℃, on shaking table, hatch 15min.
5. hatched rear recentrifuge, got 25 μ l supernatant liquors in 96 hole microwell plates, every hole adds the Victoria Green WPB of 100 μ l to measure damping fluid.Under room temperature, place 15min, use microplate reader to measure absorbancy under 650nm, and obtain corresponding phosphate concentration according to typical curve.
6. the mensuration of typical curve: the distilled water of getting 125 μ l solution C and 1125 μ l is mixed and made into the working fluid that phosphate concn is 0.1mM.Press table 2 drawing standard curve.
Table 2: typical curve arranges table
Standard curve determination step: (1) respectively gets various phosphorus concentration hydrochlorate reference liquid 25 μ l in the 96 each holes of orifice plate by the requirement of table 2.(2) each hole adds the Victoria Green WPB inspection measurements determination damping fluid (by specification preparation) of 100 μ l again, adds fashionable attention and prevent the generation of bubble.(3) under room temperature, react 15min.(4) under 650nm, measure absorbancy.(5) with absorbancy to phosphate concentration (pmoles) drawing standard curve.
Discharge the PP2A activity of measuring increases (Fig. 4) by phosphoric acid under SP series polypeptide exists, and shows that SP series polypeptide alleviated the baseline of PP2A activity and suppressed.As anticipation, in the time of okadaic acid Individual existence, PP2A is active to be reduced.But SP series polypeptide increases PP2A activity in the time that okadaic acid exists, show in cell, to have the balance between active PP2A and non-activity PP2A, and removable this of SP series polypeptide weighs to regulate the amount of active PP2A enzyme.Therefore, in cell, the activity of PP2A can be by SP series polypeptides for modulating.
Claims (9)
1. come from the synthetic polypeptide of artificial design of lipophorin, be called SP series polypeptide, its aminoacid sequence is SEQ ID NO:1-11, has the purposes for the treatment of cancer.
2. according to the polypeptide of claim 1, it is characterized in that, the N end of described polypeptide is modified through amidation through acetylation modification, C end.
3. cancer according to claim 1, refers to leukemia.
4. leukemia according to claim 3, refers to chronic granulocytic leukemia (CML).
5. polypeptide according to claim 1, also comprises that those pass through for example homologous recombination, fixed point or PCR mutagenesis, and the corresponding peptides of other animal species.
6. polypeptide according to claim 1, also comprises the polypeptide existing with free form or salt form.
7. polypeptide according to claim 1, also comprise can with the polypeptide of pharmaceutically acceptable carrier combinations.
8. polypeptide according to claim 1, can be used for the treatment of separately cancer or use with other therapeutic combinations that are generally used for treating cancer.
9. polypeptide according to claim 1, can be by different approaches to human body administration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410088581.9A CN103819554A (en) | 2014-03-11 | 2014-03-11 | Synthetic SP series polypeptides and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410088581.9A CN103819554A (en) | 2014-03-11 | 2014-03-11 | Synthetic SP series polypeptides and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103819554A true CN103819554A (en) | 2014-05-28 |
Family
ID=50754878
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410088581.9A Pending CN103819554A (en) | 2014-03-11 | 2014-03-11 | Synthetic SP series polypeptides and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103819554A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834252A (en) * | 2017-02-21 | 2017-06-13 | 中国人民解放军军事医学科学院毒物药物研究所 | A kind of high stable type MazF mutant and its application |
| CN109134616A (en) * | 2018-07-06 | 2019-01-04 | 泰安市启航生物科技有限公司 | A kind of synthetic peptide sp4 and its application |
| CN109232718A (en) * | 2018-11-09 | 2019-01-18 | 泰安市启航生物科技有限公司 | A kind of synthetic peptide sp2 and its application |
| CN115925881A (en) * | 2022-08-26 | 2023-04-07 | 中国药科大学 | Polypeptide for inhibiting SET protein nucleoplasm shuttle and application thereof |
-
2014
- 2014-03-11 CN CN201410088581.9A patent/CN103819554A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834252A (en) * | 2017-02-21 | 2017-06-13 | 中国人民解放军军事医学科学院毒物药物研究所 | A kind of high stable type MazF mutant and its application |
| CN106834252B (en) * | 2017-02-21 | 2019-10-11 | 中国人民解放军军事医学科学院毒物药物研究所 | A kind of high stable type MazF mutant and its application |
| CN109134616A (en) * | 2018-07-06 | 2019-01-04 | 泰安市启航生物科技有限公司 | A kind of synthetic peptide sp4 and its application |
| CN109134616B (en) * | 2018-07-06 | 2020-04-24 | 泰安市启航生物科技有限公司 | Synthetic peptide sp4 and application thereof |
| EP3696187A4 (en) * | 2018-07-06 | 2021-12-08 | Taian City Qihang Biotechnology Co. | Synthetic peptide sp4 and use thereof |
| CN109232718A (en) * | 2018-11-09 | 2019-01-18 | 泰安市启航生物科技有限公司 | A kind of synthetic peptide sp2 and its application |
| CN109232718B (en) * | 2018-11-09 | 2020-04-14 | 泰安市启航生物科技有限公司 | Synthetic peptide sp2 and application thereof |
| WO2020093427A1 (en) * | 2018-11-09 | 2020-05-14 | 泰安市启航生物科技有限公司 | Synthetic peptide sp2 and use thereof |
| JP2021527664A (en) * | 2018-11-09 | 2021-10-14 | 泰安市啓航生物科技有限公司 | Synthetic peptide sp2 and its uses |
| JP7102026B2 (en) | 2018-11-09 | 2022-07-19 | 泰安市啓航生物科技有限公司 | Synthetic peptide sp2 |
| CN115925881A (en) * | 2022-08-26 | 2023-04-07 | 中国药科大学 | Polypeptide for inhibiting SET protein nucleoplasm shuttle and application thereof |
| CN115925881B (en) * | 2022-08-26 | 2023-07-11 | 中国药科大学 | Polypeptide for inhibiting SET protein nucleoplasm shuttle and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zimmers et al. | STAT3 in the systemic inflammation of cancer cachexia | |
| Zhang et al. | WNT signaling underlies the pathogenesis of neuropathic pain in rodents | |
| Jackman et al. | The molecular basis of skeletal muscle atrophy | |
| Lin et al. | VEGF and its receptor-2 involved in neuropathic pain transmission mediated by P2X2/3 receptor of primary sensory neurons | |
| Kawarazaki et al. | Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiency | |
| Mariotti et al. | N-cadherin as a therapeutic target in cancer | |
| Petreaca et al. | Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability | |
| Breitbart et al. | Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting | |
| Shen et al. | Effects of palmatine on rats with comorbidity of diabetic neuropathic pain and depression | |
| Ji et al. | Lithium alleviates blood-brain barrier breakdown after cerebral ischemia and reperfusion by upregulating endothelial Wnt/β-catenin signaling in mice | |
| Pang et al. | Effect of apelin on the cardiac hemodynamics in hypertensive rats with heart failure | |
| Zhang et al. | The role of P2X4 receptors in chronic pain: A potential pharmacological target | |
| Omori et al. | Enhanced phosphorylation of PTEN in rat brain after transient middle cerebral artery occlusion | |
| Zhang et al. | Gastrodin alleviates cerebral ischaemia/reperfusion injury by inhibiting pyroptosis by regulating the lncRNA NEAT1/miR-22-3p axis | |
| Zhao et al. | The sensitization of peripheral C-fibers to lysophosphatidic acid in bone cancer pain | |
| Liu et al. | Role of glial cell line-derived neurotrophic factor in perineural invasion of pancreatic cancer | |
| CN103819554A (en) | Synthetic SP series polypeptides and application thereof | |
| Yin et al. | Netrin‑1 induces the proliferation of gastric cancer cells via the ERK/MAPK signaling pathway and FAK activation Corrigendum in/10.3892/or. 2018.6863 | |
| Dembiński et al. | Dual, time-dependent deleterious and protective effect of anandamide on the course of cerulein-induced acute pancreatitis. Role of sensory nerves | |
| Hu et al. | G protein-coupled receptor 91 signaling in diabetic retinopathy and hypoxic retinal diseases | |
| Xu et al. | Ghrelin inhibits ovarian epithelial carcinoma cell proliferation in vitro | |
| Song et al. | Hemokinins modulate endothelium function and promote angiogenesis through neurokinin-1 receptor | |
| Olona et al. | Cardiac glycosides cause cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis | |
| Zhao et al. | PTEN inhibition protects against experimental intracerebral hemorrhage-induced brain injury through PTEN/E2F1/β-catenin pathway | |
| Tan et al. | Chemokine-like factor 1-derived C-terminal peptides induce the proliferation of dermal microvascular endothelial cells in psoriasis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140528 |