CN102241744A - Virus infection blocker, and its drug composition and application - Google Patents
Virus infection blocker, and its drug composition and application Download PDFInfo
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- CN102241744A CN102241744A CN201010174788XA CN201010174788A CN102241744A CN 102241744 A CN102241744 A CN 102241744A CN 201010174788X A CN201010174788X A CN 201010174788XA CN 201010174788 A CN201010174788 A CN 201010174788A CN 102241744 A CN102241744 A CN 102241744A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
The invention relates to a blocker for preventing and treating a virus infection. Through a change of modifications of a tail end N and a tail end C, a stability of the blocker and an effectiveness of virus infection blocking can be influenced obviously. The invention also relates to an application of the blocker in the prevention and the treatment of a virus infection.
Description
Technical field
The application relates to a kind of virus infection blocker and application thereof, and particularly, the application relates to the polypeptide that HBV capable of blocking that N-terminal and C-terminal all modify infects.
Background technology
1.HBV epidemiology
It is one of the most serious public health problem in the whole world that HBV infects, and is the 3rd common disease that is only second to venereal disease and varicella.The whole world has 2,000,000,000 people to infect HBV approximately, and 75% world population is lived in high incidence of hepatitis b, and the chronic HBV infection person surpasses 3.5 hundred million.The annual death toll relevant with the HBV infection is up to 1,000,000, and annual newly-increased the infection estimates it is that HIV increases 2.5～4 times that infect newly.75% HBV chronic infection person concentrates on Asia (being about 2.87 hundred million) in the world, China is the high popular district of hepatitis B, the end of the seventies, the beginning of the nineties and the nearest sick investigation data of learning of three national hepatitis epidemics show, China infected HBV person 6.9 hundred million, infection rate is 57.6%, HBV person 0.87 hundred million is carried in the whole nation for a long time, surplus the existing chronic hepatitis B patient 2,000 ten thousand.
2.HBV prevention of infecting and treatment present situation
The importance of HBV obtains worldwide already extensively generally acknowledging that its prevention and treatment also are placed in precedence.But still lack the medicine that can remove HBV in the body so far, the definite relatively antiviral of curative effect mainly is interferon-alpha and nucleoside analog (lamivudine, Adefovir, Entecavir and Te Bifuding etc.).Interferon-alpha is mainly by immunomodulatory with induce in the target cell antiviral protein and bring into play antivirus action; Nucleoside analog then acts on the HBV reversed transcriptive enzyme, suppresses the synthetic of viral DNA chain, thereby suppresses duplicating of HBV DNA.Standard course of therapy through 1 year, about 33% interferon-alpha treatment patient and Yue 16% lamivudine therapy patient can obtain thoroughly or the part serology is replied (HBsAg turns out cloudy, HBeAg all turns out cloudy, occur or do not occur resisting-HBe antibody), virusology is replied (HBV DNA turns out cloudy) and biochemical responses (alanine aminotransferase interval continuous 2 detections in 1 month are all normal, and liver function is normal again) completely.
After Krugman in 1970 obtained the earliest hepatitis B vaccine, various countries utilized asymptomatic HBsAg carrier's blood plasma to extract haematogenous HBsAg to prepare the HBV vaccine in succession.Haematogenous HBV vaccine is proved to be safe and effective through life-time service, but because its source is limited, preparation cost is high, is replaced by gene recombination HBV vaccine.Main recombiant vaccine comprises the reconstituted hepatitis B vaccine of yeast and Chinese hamster ovary cell expression at present.These vaccine immunogenicities are strong, and the omnidistance immunity of 3 pins back anti-HBsAg antibody male rotary rate is not less than 85%.But it is slower that its main drawback is an antibody response, and HBV infects newborn infant's initial immunity of puerpera if surpass more than 7 days, then loses the HBV vaccine is blocked vertical transmission to the newborn infant effect.And 10%～15% the inoculator do not produce and reply or low replying, this crowd still can be infected by HBV.
Hepatitis B immune globulin (HBIG) is to use behind the healthy people of hepatitis b vaccination, the tire immunoglobulin preparation of blood plasma or the preparation of serum separation and Extraction of the height of collection, and its antibody titer is more than 100IU/ml.Be applicable to that burst inadvertent contamination crowd, immunologic hypofunction person and HBV infect the neonatal passive immunization prevention of puerpera.The chronic HBV infection person of China has 30%-50% approximately by mother-to-baby transmission; be blocking-up HBV mother and baby perinatal transmission; generally with HBIG and Hepatitis B virus vaccine combined utilization; though protection ratio can reach more than 80%; but the blocking-up that HBIG provides contribution is also not obvious; combined utilization is only than the blocking-up rate of Hepatitis B virus vaccine list with raising 5%～10%, and the still more healthy puerpera newborn infant of HBV infection puerpera newborn infant's HBV infection rate is high 4 times after the combined utilization.In recent years find that intrauterine infection may be the important channel of mother-to-baby transmission, the HBV infection sign recall rate that HBV infects pregnant woman's induced labor foetus liver or blood can reach 40%, the positive mother's of HBsAg baby's intrauterine infection rate about 16%.Therefore domestic some hospital infects the HBIG of pregnant woman's shot in every month greater than 200IU in last 3～4 middle of the month of gestation to HBV, in the hope of prevention fetus HBV intrauterine infection.But this method Intrauterine Transmission barrier effect is unsatisfactory, and may cause the appearance and the immunoreactive generation of immunocomplex pathology of HBV S district variant.
HBIG also is widely used in the prevention of the relevant after liver transplantation HBV infection and recurrence of hepatitis B.Do not having under the situation of preventive measures, in the relevant after liver transplantation of hepatitis B 6 months HBV again infection rate up to about 40%, 2 year in again infection rate up to about 60%.Majority cases of infection is again gone through acute, chronic hepatitis and is developed into liver cirrhosis, liver failure, and long-term prognosis is not good, needs liver retransplantation.Adopt separately lamivudine prevention recurrent HBV after liver transplantation, its Long-term HBV-DNA, HBeAg and HBsAg negative conversion rate are about 60～70%, and the HBV transgenation easily takes place, and YMDD resistance aberration rate is up to 21%.How long the phase was used antiviral before the patient transplanted in addition, and antiviral opposing phenomenon appears in majority, has increased the difficulty of transplanting back virus infection recurrence rate and prevention.Therefore state adds with heavy dose of HBIG with the relevant after liver transplantation hepatitis B recurrence of prevention hepatitis B.After liver transplantation is not accepted to occur the HBsAb of different titers in the early stage body of postoperative among the receptor of HBIG treatment, and serum HBsAb fades away subsequently, and uses the higher titre of patients serum HBsAb energy long term maintenance of HBIG.High dosage unrestriction HBIG single therapy can stop 65%～80% patient recurrence, but HBIG costs an arm and a leg, and expense every year is up to about 1,300,000 yuans.Though it is good that domestic employing lamivudine and low dose of HBIG combined utilization stop the curative effect of transplanting back HBV infection and recurrence to show, confirms without the large sample clinical trial.And the immune pressure that prolonged application HBIG brings can cause HBV genovariation and the immunologic escape phenomenon occur, makes HBIG be used for the prevention that the relevant after liver transplantation HBV of hepatitis B infects again and is very limited.
In sum, be used to prevent and treat the active passive immunity that means that HBV infects mainly are limited to nucleotide analog inhibition, antiviral cell factor and neutrality antibody at present, lack that other infect the inhibition means of links to HBV virus.Polypeptide involved in the present invention can combine with liver cell, and directly blocking virus is to hepatocellular infection, and treatment and the prevention infected for HBV provide new means.
3. the polypeptide that infects about HBV capable of blocking disclosed by the invention
The HBV peplos contain greatly (L), in (M), little (S) three kinds of surface antigen proteins, these albumen are by the single open reading frame coding of the S district gene with 3 different translation initiation sites, i.e. L (Pre-S1+PreS2+S), M (Pre S2+S) and S (S).The L albumen Pre-S1 district of HBV exists and liver cell specific receptors bonded key amino acid sequence, and the present invention synthesizes crucial sequences polypeptide, and transformation is modified at its two ends, has improved polypeptide to the usefulness that HBV infects blocking-up, has strengthened the stability of polypeptide.The chronically infected medicine of treatment HBV all acts on HBV time multiplexed cell system link at present, and the polypeptide that the present invention relates to can efficiently directly suppress the infection of HBV pair cell, and very stable, and the control of infecting for HBV provides good medicinal compound.
Summary of the invention
The present invention relates to the polypeptide that a kind of HBV capable of blocking infects, this polypeptide is held to the C end from N has aminoacid sequence SEQ ID NO:1, and its N end is modified by hydrophobic group, and its C holds stabilized modification.In one embodiment, the N of described polypeptide end is modified by myristoylation modification, stearic acid modification, palmitinic acid modification, cholesterol, and its C end is refined modification by amidation (Amidation, amination) modification or isoamyl two.Wherein the hydrophobic grouping of N end is modified and can be strengthened the usefulness that described polypeptide blocking-up HBV infects, and the stability of C end is modified the stability that can strengthen described polypeptide.The invention still further relates to a kind of pharmaceutical composition, it contains polypeptide disclosed by the invention and pharmaceutically acceptable carrier.The invention still further relates to described polypeptide and prevent and/or treat purposes in the medicament of hepatitis B in preparation.
Particularly, the application provides a peptide species, and this polypeptide is selected from:
(1) aminoacid sequence shown in the SEQ ID NO:1; Or
(2) the active aminoacid sequence that on the aminoacid sequence basis shown in the SEQ ID NO:1, has 1-3 aminoacid replacement, disappearance or insertion and keep the blocking-up HBV of aminoacid sequence shown in the SEQ ID NO:1 to infect;
Wherein, this polypeptide N end has that hydrophobic grouping is modified and C holds stabilized modification.
In one embodiment, described N end is hydrophobically modified modifies or stearic acid is modified or palmitinic acid is modified or the cholesterol modification for myristoylation.
In one embodiment, described C end stabilization is modified to amidation modification or isoamyl two alcoholization modifications.
In one embodiment, described N end is hydrophobically modified to be modified for myristoylation, and described C end stabilization is modified to amidation and modifies.
In one embodiment, described amino acid sequence of polypeptide is shown in SEQ ID NO:2,3 or 4.
In one embodiment, amino acid sequence of polypeptide is shown in SEQ ID NO:2,3 or 4, and the N of described polypeptide end is for myristoylation is modified, the C end is modified for amidation.
In one embodiment, described amino acid sequence of polypeptide is the aminoacid sequence shown in the SEQ ID NO:1, and its N end is for myristoylation is modified, the C end is modified for amidation.
The application provides a kind of pharmaceutical composition, and this pharmaceutical composition contains described polypeptide of the application and pharmaceutically acceptable carrier.
In one embodiment, described amino acid sequence of polypeptide is as described in arbitrary sequence among the SEQ ID NO:1-4.
In one embodiment, described pharmaceutical composition contains the aminoacid sequence shown at least one SEQ ID NO:1-4.
In one embodiment, the polypeptide in the described pharmaceutical composition is the aminoacid sequence shown in the SEQ ID NO:1, and its N end is for myristoylation is modified, the C end is modified for amidation.
In one embodiment, described amino acid sequence of polypeptide is the aminoacid sequence shown in the SEQ ID NO:1, and its N end is for myristoylation is modified, the C end is modified for amidation.
In one embodiment, the concentration of polypeptide is more than or equal to 20ng/ml in the pharmaceutical composition, is preferably more than to equal 100ng/ml.
The application also relates to the purposes of described polypeptide in the medicament of preparation treatment HBV infection usefulness.
In one embodiment, described amino acid sequence of polypeptide is the aminoacid sequence shown in the SEQ ID NO:1, and its N end is for myristoylation is modified, the C end is modified for amidation.
In one embodiment, described object is for infecting the people of HBV or PI HBV.
Description of drawings
Fig. 1 shows to have SEQ ID NO:1 aminoacid sequence, and the amidation of C end is modified, the blocking-up that N end hydrophobic grouping modified polypeptides infects HBV.
Fig. 2 shows to have SEQ ID NO:1 aminoacid sequence, and N end myristoylation is modified, the stability of C end stabilization modified polypeptides.
Fig. 3 shows to have SEQ ID NO:1 aminoacid sequence, and N end myristoylation is modified, the blocking-up that C end stabilization modified polypeptides infects HBV.
Influence and stability that the sudden change of Fig. 4 show amino acid is infected SEQ ID NO:1-4 aminoacid sequence polypeptide blocking-up HBV.
Embodiment
As mentioned above, the invention provides the polypeptide that a kind of HBV of inhibition infects.This polypeptide is held to C end from N has aminoacid sequence shown in the SEQ ID NO:1, and its N end has hydrophobic grouping to be modified, and its C holds stabilized modification.Above-described N end hydrophobic grouping is modified, and these hydrophobic groupings are myristic acid, palmitinic acid, stearic acid, oleic acid, linolic acid, cholesterol, arachidonic acid and similar group thereof preferably.Further preferably being myristic acid, palmitinic acid, stearic acid or cholesterol, more preferably is myristic acid.Above-described C end stabilization is modified, and these stabilizations are modified and comprised that (being that amination is modified) modified in amidation, isoamyl two alcoholization are modified and other peptide C end stabilizations are modified.Be preferably amidation and modify (being that amination is modified).
In a preferred version of the present invention; sequence is that the C-terminal of the polypeptide of SEQ ID NO:1 is modified through amidation; and its N-terminal has carried out myristoylation, stearic acid, palmitinic acid and cholesterol modification respectively, and wherein preferably N end myristoylation modified polypeptides has higher virus infection inhibition activity.In the another one preferred version; sequence is that the N-terminal of the polypeptide of SEQ ID NO:1 is modified through myristoylation; and its C-terminal has carried out the amidation modification respectively and isoamyl two alcoholization are modified, and wherein preferably C end amidation modified polypeptides has higher virus infection inhibition activity and advantages of excellent stability.Therefore in the present invention further optimization scheme, sequence is that the C-terminal of the polypeptide of SEQ ID NO:1 is modified through amidation, and N-terminal has carried out the myristoylation modification.
Any homologue of the related polypeptide of the application is the application's a part, comprise through one or more amino acid mutations, as comprise the peptide sequence that 1-10 amino acid, a preferable 1-8 amino acid, a better 1-5 amino acid, a better 1-3 amino acid whose deletion, conservative/non-conserved amino acid are replaced or inserted and obtain.Here " homologue " comprises that also relating to polypeptide with the application has polypeptide greater than 30% (as 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or bigger) homology.In a preferred version of the present invention, be that the polypeptide of SEQ ID NO:1 carries out the peptide sequence (SEQ ID NO:2-4) that 1-3 amino acid whose sudden change obtains to sequence.
Particularly, amino acid is generally divided into four classes: (1) acidity---aspartic acid and L-glutamic acid; (2) alkalescence---Methionin, arginine, Histidine; (3) nonpolar---L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane; (4) uncharged polarity---glycine, l-asparagine, glutamine, halfcystine, Serine, Threonine, tyrosine.Sometimes phenylalanine, tryptophane and tyrosine are classified as aromatic amino acid.For example, have reason to predict: replace leucine, replace aspartic acid, replace Threonine with Isoleucine or Xie Ansuan separately with Serine with L-glutamic acid, perhaps with the similarly conservative amino acid of aminoacid replacement relevant on the structure, such substituting will can not have material impact to biological activity.
The application comprises that also the polypeptide that will provide is as blocking or prevent medicine that HBV infects and the pharmaceutical preparation of forming with suitable pharmaceutically acceptable carrier.The application also comprises the pharmaceutical composition that contains described polypeptide.
Pharmaceutical preparation of the present invention or pharmaceutical composition contain the polypeptide of the present invention of significant quantity.As used herein, described " significant quantity " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.For example, for liquid preparation or composition, the concentration of described polypeptide can be for more than the 20ng/ml, and for example 50ng/ml is above, 80ng/ml is above, 100ng/ml is above or higher.
Described " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.This term refers to some medicament carriers like this: they itself are not necessary activeconstituents, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.Acceptable carrier can contain liquid on combination of traditional Chinese medicine is learned, as water, salt solution, damping fluid.In addition, also may there be complementary material in these carriers, as weighting agent, lubricant, glidant, wetting agent or emulsifying agent, pH buffer substance etc.
Can adopt the suitable method of the various routines in this area to give object pharmaceutical composition of the present invention or pharmaceutical preparation, described mode includes but not limited to: oral, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, slowly-releasing give etc.
The measure of infecting as treatment or prevention HBV with the polypeptide that provided also is provided the application, and relates to the application's polypeptide in the needed any prevention of interior patient, treatment measure.The application comprises especially and suppresses prevention and the treatment measure that HBV infects in the application's polypeptide body, comprising stoping HBV to propagate in the cell of organism uninfection.The preventive measures here are meant the possibility that reduces or avoid patient infection HBV, and the treatment measure is meant all measures of improvement or stable status of patient.The patient of indication is any people who is infected, may be infected by HBV and may soon be infected by HBV by HBV.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the described condition of laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment one: have SEQ ID NO:1 aminoacid sequence, the amidation of C end is modified, the blocking-up that N end hydrophobic grouping modified polypeptides infects HBV
1, the preparation of polypeptide and modification
By standard Fmoc scheme, initial with the 0.25mM resin with AB 431A type Peptide synthesizer, the residue extension is synthetic one by one to aminoterminal from carboxyl terminal according to SEQ ID NO:1 sequence, adds hydrophobic group at last and modifies.Behind the peptide end of synthesis, through the cutting of cutting liquid, G6 glass sand hourglass filtering resin, the filtrate vacuum is drained, the further amidation of the C-terminal of polypeptide.Deionized water dissolving polypeptide product,
Explorer 100 type medium pressure liguid chromatograph C18 column purification, substep is collected main peak.Target peak is collected sample and is identified with the anti-phase high pressure liquid chromatography Symmetry C18 of Delta 600 types analytical column purity, API 2000LC/MS/MS type mass spectrograph molecular weight identification.The collection liquid freeze-drying of medium pressure liquid chromatography purifying gained is dissolved in PBS and forms the polypeptide storage liquid, 0.20 μ M filtration sterilization, and-80 ℃ are frozen.
2, the cultivation of tree shrew primary hepatocyte
Tree shrew anesthesia is after portal catheterization, two step of the classics perfusion that provides according to BD company hepatocyte culture medium service manual digests liver organization, separate primary hepatocyte, after washing, be plated on liver cell culture plate (the product B D354408 of BD company), adopt hepatocyte culture medium to cultivate (the product B D355056 of BD company), changed substratum in per 3 days, 37 degree, 5% carbon dioxide environment are cultivated.
3, HBV virus is to hepatocellular infection.
The serum virus 2ml of the blended HBV infected patient of the clinical collection of China is added the tree shrew primary hepatocyte of cultivating 3 days, hatch for 37 ℃ and infected 12 hours.Remove infection serum after infection is finished, washed cell 3 times replenished the fresh culture cultured continuously 12 days.
4, HBV infects the detection of back liver cell culture supernatant HBsAg
HBsAg in the culture supernatant detects by sandwich sandwich assay (in-house sandwich) enzyme immunoassay (ELISA).37 ℃ of bags of 1ug/ml anti-hbs monoclonal antibodies were by 96 orifice plates 2 hours.Behind the thorough washing (0.1%Tween 20PBS washing 3 times, PBS washing 2 times), 37 ℃ of sealings of 10% foetal calf serum 1 hour.Remove confining liquid, the liver cell culture supernatant 100 μ l that add the HBV infection were hatched 12 hours for 4 ℃.Remove culture supernatant, thorough washing adds peroxidase link coupled anti-HBsAg antibody and hatched 1 hour for 37 ℃.Remove unnecessary antibody, add phenylenediamine-H
2O
2Reaction substrate room temperature reaction 15 minutes, 2N H
2SO
4Stopped reaction, the OD492 of assaying reaction product calculates and infects HBsAg content in the supernatant.
The blocking-up of 5, have SEQ ID NO:1 aminoacid sequence, C holding amidation modification, N end hydrophobic grouping modified polypeptides that HBV is infected
The tree shrew primary hepatocyte was cultivated 3 days in 24 well culture plates, in the HBV virus infection, added terminal modified each 20ng/ml of polypeptide of different N in infecting supernatant, hatched jointly 12 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 12 days.Shown in Figure 1A, not to be subjected to polypeptide to handle hepatocellular HBV infection is contrast, have N and hold different hydrophobic grouping modified polypeptides that the HBV infected liver cell is had in various degree blocking effect, wherein N-terminal be that myristic acid is modified, stearic acid is modified, palmitinic acid is modified, cholesterol is modified and the literalness polypeptide of N-terminal can suppress respectively HBV to liver cell infect 57.4%, 49.2%, 46.8%43.2% and 9.7%.As seen, N-terminal infects retarding effect for the myristic acid modified polypeptides has higher H BV, and the fluorescently-labeled N of FITC, C-terminal are that the SEQ ID NO:1 polypeptide that myristic acid and amidation are modified can combine (Figure 1B) with primary hepatocyte.
Embodiment two: have SEQ ID NO:1 aminoacid sequence, N end myristoylation is modified, the stability of C end stabilization modified polypeptides
1, the preparation of polypeptide and modification
By standard Fmoc scheme, initial with the 0.25mM resin with AB 431A type Peptide synthesizer, the residue extension is synthetic one by one to aminoterminal from carboxyl terminal according to SEQ ID NO:1 sequence, and last myristic acid is modified.Behind the peptide end of synthesis, through the cutting of cutting liquid, G6 glass sand hourglass filtering resin, the filtrate vacuum is drained, and further amidation of the C-terminal of polypeptide or isoamyl two alcoholization are modified or the C end is not modified.Deionized water dissolving polypeptide product,
Explorer 100 type medium pressure liguid chromatograph C18 column purification, substep is collected main peak.Target peak is collected sample and is identified with the anti-phase high pressure liquid chromatography Symmetry C18 of Delta 600 types analytical column purity, API 2000LC/MS/MS type mass spectrograph molecular weight identification.The collection liquid freeze-drying of medium pressure liquid chromatography purifying gained is dissolved in PBS and forms the polypeptide storage liquid, 0.20 μ M filtration sterilization, and-80 ℃ are frozen.
2, the stability of polypeptide
Polypeptide is dissolved in 0.02M PBS, forms 0.25mg/ml concentration solution, places 37 degree to place 3 days.Adopt Luna C18,150 * 4.6mm, 5 μ, the 100A chromatographic column adopts the anti-phase high pressure liquid chromatograph of Delta 600 types to carry out the HPLC purity check respectively.As shown in Figure 2, N holds all and modifies through myristoylation, and purity is respectively 98.2%, 98.7% and 98.3% (Fig. 2 A) before the polypeptide 37 degree placements of C-terminal amidation modification respectively, isoamyl two alcoholization modifications and C-terminal unmodified; 37 degree place that purity is respectively 95.4%, 95.6% and 95.5% (Fig. 2 B) after 12 hours; 37 degree place that purity is reduced to 83.9%, 87.7% and 43.9% (Fig. 2 C) respectively after 3 days.As seen the polypeptide deficient in stability of C end unmodified obviously increases and polypeptide is stable after C end amidation modification and isoamyl two alcoholization modifications.
Embodiment three: have SEQ ID NO:1 aminoacid sequence, N end myristoylation is modified, the blocking-up that C end stabilization modified polypeptides infects HBV
1, the preparation of polypeptide and modification (with example two).
2, the infection of the cultivation of tree shrew primary hepatocyte and HBV virus (with embodiment one)
3, HBV infects the detection (with embodiment one) of back liver cell culture supernatant HBsAg
4, have SEQ ID NO:1 aminoacid sequence, N end myristoylation is modified, the blocking-up that C end stabilization modified polypeptides infects HBV
1) blocking effect that under the polypeptide lower concentration HBV is infected: the tree shrew primary hepatocyte was cultivated 3 days in 24 well culture plates, in the HBV virus infection, added terminal modified each 20ng/ml of polypeptide of different C in infecting supernatant, hatched jointly 12 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 12 days.As shown in Figure 3A; not to be subjected to polypeptide to handle hepatocellular HBV infection is contrast; N-terminal is all modified by myristoylation and C end has that different stabilizations are modified and the polypeptide of C end unmodified has in various degree blocking effect to the HBV infected liver cell, and wherein C-terminal is that amidation modification, isoamyl two alcoholization are modified and the polypeptide of C end unmodified can suppress HBV respectively to 55.9%, 42.4% and 57.3% of liver cell infection.As seen, C-terminal acid amides modified polypeptide and C end unmodified polypeptide is suitable to the blocking effect that HBV infects under low consistency conditions, and two alcoholization of C-terminal isoamyl are modified and then influenced the blocking effect of polypeptide to virus infection.
2) blocking effect that under the polypeptide high density HBV is infected: the tree shrew primary hepatocyte was cultivated 3 days in 24 well culture plates, in the HBV virus infection, added terminal modified each 100ng/ml of polypeptide of different C in infecting supernatant, hatched jointly 12 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 12 days.Shown in Fig. 3 B; not to be subjected to polypeptide to handle hepatocellular HBV infection is contrast; N-terminal is all modified by myristoylation and C end is modified for amidation modification, isoamyl two alcoholization and the polypeptide of C end unmodified has in various degree blocking effect to the HBV infected liver cell, wherein C-terminal be amidation modification, isoamyl two alcoholization modify and the polypeptide of C end unmodified can suppress respectively HBV to liver cell infect 95.1%, 60.9% and 81.5%.As seen, blocking-up HBV effect is still held unmodified not as good as C polypeptide is modified in two alcoholization of C-terminal isoamyl.And unexpectedly, C-terminal amidation modified polypeptides polypeptide blocking-up HBV effect than C end unmodified when high density obviously improves.May there be two aspect reasons in this reinforcing effect: 1) polypeptide is after amidation is modified, and the transformation period prolongs, and the polypeptide effective concentration in test blocking-up time (12 hours) is increased.But Study on Stability shows among the embodiment two, and last C end unmodified was compared the degraded indifference with C end amidation modified polypeptides in 12 hours.Therefore can not explain that C end amidation modification is to the enhancement of polypeptide virus blocking ability under the high density condition with the transformation period advantage.2) some biological character of having strengthened polypeptide (as the polymer under spatial configuration of molecules, hydrophilic and hydrophobic, the high density condition etc.) is modified in the amidation of C end, is more suitable for the competition with HBV, and then has strengthened the effect that the polypeptide blocking-up is infected.But, can't expect and explain therefore that C end amidation modification is to the enhancement of polypeptide virus blocking ability under the high density condition because the biological character of this polypeptide is not still understood at present.
Embodiment four: the influence that amino acid mutation infects SEQ ID NO:1 aminoacid sequence polypeptide blocking-up HBV
1, the preparation of polypeptide and modification
Press standard Fmoc scheme with AB 431A type Peptide synthesizer, initial with the 0.25mM resin, (have 1 sudden change F13L respectively according to SEQ ID NO:2-4 sequence, 2 sudden change F13L, H39Q and 3 suddenly change F13L, H39Q, N44D) the residue extension is synthetic one by one to aminoterminal from carboxyl terminal, and last myristic acid is modified.Behind the peptide end of synthesis, through the cutting of cutting liquid, G6 glass sand hourglass filtering resin, the filtrate vacuum is drained, and the further amidation of the C-terminal of polypeptide is modified.Deionized water dissolving polypeptide product,
Explorer 100 type medium pressure liguid chromatograph C18 column purification, substep is collected main peak.Target peak is collected sample and is identified with the anti-phase high pressure liquid chromatography Symmetry C18 of Delta 600 types analytical column purity, API 2000LC/MS/MS type mass spectrograph molecular weight identification.The collection liquid freeze-drying of medium pressure liquid chromatography purifying gained is dissolved in PBS and forms the polypeptide storage liquid, 0.20 μ M filtration sterilization, and-80 ℃ are frozen.
2, the infection of the cultivation of tree shrew primary hepatocyte and HBV virus (with embodiment one).
3, HBV infects the detection (with embodiment one) of back liver cell culture supernatant HBsAg.
4, have SEQ ID NO:1-4 aminoacid sequence, the amination of N end myristoyl is modified, the blocking-up that C end amidation modified polypeptides infects HBV
1) the tree shrew primary hepatocyte was cultivated 3 days in 24 well culture plates, in the HBV virus infection, added each 20ng/ml of SEQ ID NO:1-4 polypeptide of sequence in infecting supernatant, hatched jointly 12 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 12 days.Shown in Fig. 4 A, be contrast not to be subjected to polypeptide to handle hepatocellular HBV infection, SEQID NO:1-4 polypeptide of sequence can suppress HBV respectively to 56.7%, 55.9%, 59.3% and 52.6% of liver cell infection.As seen, the sudden change of 1-3 aminoacid sequence does not influence the effect that SEQ ID NO:1 polypeptide of sequence blocking-up HBV infects.
2) the tree shrew primary hepatocyte was cultivated 3 days in 24 well culture plates, in the HBV virus infection, added each 100ng/ml of SEQ ID NO:1-4 polypeptide of sequence in infecting supernatant, hatched jointly 12 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 12 days.Shown in Fig. 4 B, be contrast not to be subjected to polypeptide to handle hepatocellular HBV infection, SEQID NO:1-4 polypeptide of sequence can suppress HBV respectively to 94.7%, 96.2%, 95.7% and 93.9% of liver cell infection.As seen, the sudden change of 1-3 aminoacid sequence does not influence the effect that SEQ ID NO:1 polypeptide of sequence blocking-up HBV infects.
5, the stability of mutant polypeptide
Polypeptide is dissolved in 0.02M PBS, forms 0.25mg/ml concentration solution, places 37 degree to place 3 days.Adopt Luna C18,150 * 4.6mm, 5 μ, the 100A chromatographic column adopts the anti-phase high pressure liquid chromatograph of Delta 600 types to carry out the HPLC purity check respectively.Purity was respectively 98.2%, 98.9%, 98.6% and 98.8% before SEQ ID NO:1-4 polypeptide 37 degree was placed, and shown in Fig. 4 C, 37 degree place that purity is reduced to 83.9%, 84.1%, 82.7% and 83.2% respectively after 3 days.As seen, the sudden change of 1-3 aminoacid sequence does not influence SEQ ID NO:1 polypeptide of sequence stability.
Above-mentioned specific embodiment only is illustrative, and nonrestrictive.The application's protection domain will be limited by claim.It will be understood by those skilled in the art that under situation without departing from the spirit and scope of the present invention, can make various modifications and changes to technical scheme of the present invention, these modifications and change still comprise within the scope of the present invention.
Sequence table
＜110〉Liu Hongli
＜120〉a kind of virus infection blocker, its pharmaceutical composition and application thereof
<130>101685
<160>4
<170>PatentIn?version?3.3
<210>1
<211>47
<212>PRT
＜213〉HBV surface L protein
<400>1
Gly?Thr?Asn?Leu?Ser?Val?Pro?Asn?Pro?Leu?Gly?Phe?Phe?Pro?Asp?His
1 5 10 15
Gln?Leu?Asp?Pro?Ala?Phe?Gly?Ala?Asn?Ser?Asn?Asn?Pro?Asp?Trp?Asp
20 25 30
Phe?Asn?Pro?Asn?Lys?Asp?His?Trp?Pro?Glu?Ala?Asn?Gln?Val?Gly
35 40 45
<210>2
<211>47
<212>PRT
＜213〉HBV surface L protein derived peptide
<400>2
Gly?Thr?Asn?Leu?Ser?Val?Pro?Asn?Pro?Leu?Gly?Phe?Leu?Pro?Asp?His
1 5 10 15
Gln?Leu?Asp?Pro?Ala?Phe?Gly?Ala?Asn?Ser?Asn?Asn?Pro?Asp?Trp?Asp
20 25 30
Phe?Asn?Pro?Asn?Lys?Asp?His?Trp?Pro?Glu?Ala?Asn?Gln?Val?Gly
35 40 45
<210>3
<211>47
<212>PRT
＜213〉HBV surface L protein derived peptide
<400>3
Gly?Thr?Asn?Leu?Ser?Val?Pro?Asn?Pro?Leu?Gly?Phe?Leu?Pro?Asp?His
1 5 10 15
Gln?Leu?Asp?Pro?Ala?Phe?Gly?Ala?Asn?Ser?Asn?Asn?Pro?Asp?Trp?Asp
20 25 30
Phe?Asn?Pro?Asn?Lys?Asp?Gln?Trp?Pro?Glu?Ala?Asn?Gln?Val?Gly
35 40 45
<210>4
<211>47
<212>PRT
＜213〉HBV surface L protein derived peptide
<400>4
Gly?Thr?Asn?Leu?Ser?Val?Pro?Asn?Pro?Leu?Gly?Phe?Leu?Pro?Asp?His
1 5 10 15
Gln?Leu?Asp?Pro?Ala?Phe?Gly?Ala?Asn?Ser?Asn?Asn?Pro?Asp?Trp?Asp
20 25 30
Phe?Asn?Pro?Asn?Lys?Asp?Gln?Trp?Pro?Glu?Ala?Asp?Gln?Val?Gly
35 40 45
Claims (10)
1. a peptide species is selected from:
(1) aminoacid sequence shown in the SEQ ID NO:1; Or
(2) the active aminoacid sequence that on the aminoacid sequence basis shown in the SEQ ID NO:1, has 1-3 aminoacid replacement, disappearance or insertion and keep the blocking-up HBV of aminoacid sequence shown in the SEQ ID NO:1 to infect;
Wherein, this polypeptide N end has that hydrophobic grouping is modified and C holds stabilized modification.
2. polypeptide as claimed in claim 1 is characterized in that, it is that myristoylation is modified or stearic acid is modified or palmitinic acid is modified or cholesterol is modified that described N holds hydrophobically modified.
3. polypeptide as claimed in claim 1 is characterized in that, described C end stabilization is modified to the amidation modification or isoamyl two alcoholization are modified.
4. the described polypeptide of claim 1 is characterized in that, described N end is hydrophobically modified for myristoylation is modified, described C end stabilization is modified to amidation and modifies.
5. the described polypeptide of claim 1 is characterized in that, described amino acid sequence of polypeptide is shown in SEQ IDNO:2,3 or 4.
6. polypeptide as claimed in claim 5 is characterized in that, the N end of described polypeptide is for myristoylation is modified, the C end is modified for amidation.
7. polypeptide as claimed in claim 1 is characterized in that, described amino acid sequence of polypeptide is the aminoacid sequence shown in the SEQID NO:1, and its N end is for myristoylation is modified, the C end is modified for amidation.
8. a pharmaceutical composition is characterized in that, it contains each described polypeptide and pharmaceutically acceptable carrier among the claim 1-7.
9. each described polypeptide infects purposes in the medicament of usefulness at preparation treatment HBV among the claim 1-7.
10. purposes as claimed in claim 9 is characterized in that, described amino acid sequence of polypeptide is the aminoacid sequence shown in the SEQID NO:1, and its N end is for myristoylation is modified, the C end is modified for amidation.
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| CN201010174788.XA CN102241744B (en) | 2010-05-14 | 2010-05-14 | Virus infection blocker, and its drug composition and application |
| PCT/CN2011/073963 WO2011140984A1 (en) | 2010-05-14 | 2011-05-12 | Anti-hbv polypeptide, pharmaceutical composition and use thereof |
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| CN201010174788.XA CN102241744B (en) | 2010-05-14 | 2010-05-14 | Virus infection blocker, and its drug composition and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015000371A1 (en) * | 2013-07-01 | 2015-01-08 | 上海贺普药业股份有限公司 | He pula peptide formulation |
| CN109310737A (en) * | 2016-05-30 | 2019-02-05 | 上海贺普药业股份有限公司 | The composition and method for treating metabolic disease |
| WO2019080919A1 (en) * | 2017-10-27 | 2019-05-02 | 上海贺普药业股份有限公司 | Drug and method for treating liver diseases related to hepatitis b viruses in full-dose condition |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733798A (en) * | 2005-08-12 | 2006-02-15 | 上海汐群生物科技有限公司 | Hepatitis B virus surface L protein related peptide |
| WO2009092612A1 (en) * | 2008-01-25 | 2009-07-30 | Universitätsklinikum Heidelberg | Hydrophobic modified pres-derived peptides of hepatitis b virus (hbv) and their use as vehicles for the specific delivery of compounds to the liver |
-
2010
- 2010-05-14 CN CN201010174788.XA patent/CN102241744B/en active Active
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1733798A (en) * | 2005-08-12 | 2006-02-15 | 上海汐群生物科技有限公司 | Hepatitis B virus surface L protein related peptide |
| WO2009092612A1 (en) * | 2008-01-25 | 2009-07-30 | Universitätsklinikum Heidelberg | Hydrophobic modified pres-derived peptides of hepatitis b virus (hbv) and their use as vehicles for the specific delivery of compounds to the liver |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015000371A1 (en) * | 2013-07-01 | 2015-01-08 | 上海贺普药业股份有限公司 | He pula peptide formulation |
| CN104274827A (en) * | 2013-07-01 | 2015-01-14 | 上海贺普药业股份有限公司 | He Pula peptide preparation |
| JP2016523267A (en) * | 2013-07-01 | 2016-08-08 | 上海賀普薬業股▲分▼有限公司Ｓｈａｎｇｈａｉ Ｈｅｐ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｃｏ．， Ｌｔｄ． | Heprapeptide formulation |
| EP3025723A4 (en) * | 2013-07-01 | 2016-12-21 | Shanghai Hep Pharmaceutical Co Ltd | He pula peptide formulation |
| JP2019070016A (en) * | 2013-07-01 | 2019-05-09 | 上海賀普薬業股▲分▼有限公司Ｓｈａｎｇｈａｉ Ｈｅｐ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｃｏ．， Ｌｔｄ． | Formulation of he pula peptide |
| US10603352B2 (en) | 2013-07-01 | 2020-03-31 | Shanghai Hep Pharmaceutical Co., Ltd. | Formulations of hepalatide |
| CN104274827B (en) * | 2013-07-01 | 2020-07-14 | 上海贺普药业股份有限公司 | Formulations of He Pula peptide |
| CN109310737A (en) * | 2016-05-30 | 2019-02-05 | 上海贺普药业股份有限公司 | The composition and method for treating metabolic disease |
| CN109310737B (en) * | 2016-05-30 | 2023-05-05 | 上海贺普药业股份有限公司 | Compositions and methods for treating metabolic disorders |
| WO2019080919A1 (en) * | 2017-10-27 | 2019-05-02 | 上海贺普药业股份有限公司 | Drug and method for treating liver diseases related to hepatitis b viruses in full-dose condition |
| CN111417402A (en) * | 2017-10-27 | 2020-07-14 | 上海贺普药业股份有限公司 | Medicine and method for treating hepatitis B virus related liver disease under full dosage condition |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011140984A1 (en) | 2011-11-17 |
| CN102241744B (en) | 2015-03-04 |
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