CN102180951B - Cholesterol modified anti-human immunodeficiency virus (HIV) polypeptide medicament and application thereof - Google Patents

Abstract

The invention discloses a cholesterol modified anti-human immunodeficiency virus (HIV) polypeptide medicament and application thereof. The cholesterol modified anti-HVI polypeptide is named CHFI, and the amino acid sequence of the polypeptide is shown as the sequence 2 in a sequence table, wherein the cysteine residue at the 33rd position of the sequence 2 is connected with cholesterol through a thioether bond. Bromoacetic acid cholesterol ester is grafted to the side chain of polypeptide chain cysteine by thioether formation reaction with extremely high chemical selectivity to obtain the CHFI. The CHFI has the important advantages of strong anti-virus activity, targeting property, long half-life period, good water solubility and the like, and is easily synthesized.

Description

Anti-HIV polypeptide drugs that SUV is modified and uses thereof

Technical field

The present invention relates to the polypeptide drug of a kind of efficient HIV inhibiting (HIV) and the purposes of method of design and treatment AIDS thereof.

Background technology

Present global HIV number of the infected surpasses 6,000 ten thousand, wherein about 2,000 ten thousand death.New number of the infected and death toll increase year by year.Estimate just have every day 1.6 ten thousand New Developments to infect now according to WHO; Infer that nearly 100,000,000 people infect after 10 years, this will be a melt-down of human society undoubtedly.Vaccine is the best means that prevent AIDS, but two during the last ten years, the clinical protocol of going back none HIV vaccine is proved to be effective.In one quite long from now on period, effectively the HIV vaccine is feared difficulty has important breakthrough.Therefore, the medicine of studying the HIV-1 infection mechanism energetically and designing the different duplicate stage of blocking virus in view of the above is one of emphasis strategy of present prevention and control AIDS.Up to now, the medicine of treating the HIV infection clinically can be divided into RTI, proteinase inhibitor, integrase inhibitor and HIV entry inhibitor, and they are directed against the different links of virus replication respectively.Most drug belongs to RTI and proteinase inhibitor; These two types of medicines are induced the sudden change of HIV resistance clinically easily; And human body had bigger toxic side effect, and cause a lot of patients can't continue to receive treatment, greatly limited AIDS patient's clinical treatment.Therefore, research and develop new AIDS-treating medicine, especially the suppressor factor to viral route of entry is current key content.

HIV-1 gp41 structure and functional study

HIV-1 gets into the process of target cell mainly by encapsulating gp gp160 mediation.This albumen by surperficial subunit gp120 with stride film subunit gp41 and be connected through non covalent bond.Under native state, gp160 is a tripolymer, and wherein termolecular gp120 forms a globular complex, and three molecule gp41 then resemble three pin, inserts in the peplos ^[1]In HIV target cell infection process, the gp120 major function is to combine with acceptor CD4 molecule and accessory receptor (chemokine receptor CCR 5 or CXCR4 etc.), and the gp41 molecule mainly mediates the film fusion of virus and cell.I type transmembrane protein as other is the same, and HIV-1 gp41 molecule is striden film district and intracellular region and formed by the film outskirt.Discover that gp41 film outskirt comprises several important function district: the hydrophobicity fusogenic peptide (fusion peptide) of glycocoll is rich in (1); (2) N-terminal repeat (NHR or HR1); (3) C-terminal repeat (CHR or HR2); (4) tryptophane is rich in district (TR).Analyze from structure, the hydrophobic amino acid 4-3 Tumor-necrosis factor glycoproteins that NHR and CHR contain [(abcdefg) _n] possibly form coiled coil (coied coil) structure, but definite frontier point is difficult to prediction.Up to nineteen ninety-five, Lu etc. ^[2]Adopt limited protease hydrolysis method to identify the protease resistant gp41 structure of forming by NHR polypeptide N51 (aa540-590) and CHR peptide C 43 (aa624-666).The result finds that synthetic N51 and C43 polypeptide can be combined together to form six stable aggressiveness helical bundle structures, points out them to comprise NHR and CHR hydrophobicity 4-3 Tumor-necrosis factor glycoproteins respectively.Afterwards, further adopt limited protease hydrolysis method digestion reorganization N51 (L6) C43 recombinant protein identify shorter NHR sequence (N36, aa546-581) with the CHR sequence (C34, aa628-661).N36 and C34 lay respectively at the mid-way of N51 and C43, also can form six stable aggressiveness helical bundle structures, are considered to the functional core texture of HIV-1 gp41.1997, Kim P research group of Massachusetts Polytechnics formed mixture with N36 and C34 polypeptide in vitro reactions and simulates HIV gp41, has taken the lead in solving the core texture of gp41 ^[3]Two other has independently also reported the crystalline structure based on six aggressiveness helical bundles of the synthetic polypeptide of gp41 in the laboratory subsequently ^[4-5], confirmed above discovery.Crystalline structure shows; HIV gp41 core texture is six strands of alpha-helix bundles; Wherein the N-spiral of three NHR compositions interacts through the amino acid in a and d position and forms the spiral tripolymer that is positioned at the center; The amino-acid residue of its e and g position then is exposed to the periphery of center convolution body, and a and the interaction of d position of the C-spiral of forming with three CHR.The C-spiral is combined in respectively in the groove of three N-spiralizations with antiparallel mode.Based on gp41 three-dimensional structure information; Scientist has proposed HIV-1 film syncretizing mechanism: significant conformational change takes place after the receptors bind on HIV envelope protein gp120 and the target cell; The fusion polypeptide of gp41 (FP) is exposed out and inserts target cell membrane, and the NHR on the three molecule gp41 with CHR takes place reverse the combination respectively, forms six stable helical bundle core textures; The distance of peplos and target cell membrane is furthered and merges, thereby mediation HIV gets in the target cell ^[3-5]Infusively be, crystalline structure also discloses NHR and contains tangible hydrophobic bag shaped structure (hydrophobic pocket), and being considered to might be the novel targets of inverase ^[6-7]

Nearest discovers, HIV-1gp41 CHR can be divided into the NHR hydrophobic pocket land that three functional zone (1) are positioned at the CHR N-terminal (PBD:pocket-binding domain, aa628-635); (2) NHR land (NBD:aa628-666) and (3) adipose membrane land (LBD:lipid-binding domain, aa666-673) ^[8]The big quantity research that comprises the rite-directed mutagenesis analysis shows the stability and the viral infection activity decisive role of the interaction partners gp41 core texture between NHR and CHR spiral ^[9-10]At gp41 six helical bundles is in the core texture forming process, and three amino acid of the last PBD of CHR insert the NHR hydrophobic pocket with high-affinity, and are most important to stablizing six helical bundle structures.Research is in the past inferred, CHR hydrophobic pocket land upstream sequence ⁶²³QIWNNMT ⁶²⁷Also possibly present α-Luo Xuanjiegou; Simultaneously, NHR downstream sequence ⁵⁸¹AVERYLKDQ ⁵⁹⁰Alpha-helix trend is also arranged ^[3-5]Infer that interaction between these two sites might influence NHR and CHR forms stable functional core texture, thereby influence the film fusion process of virus infection.Nearest discovers, contains ⁶²¹QIWNNMT ⁶²⁷Polypeptide (CP621-652) can form extremely stable six helical bundle structures (Tm=82 ℃) with NHR corresponding polypeptide (T21), be higher than the N36 that is considered to the gp41 core texture at present and the stability (Tm=64 ℃) of C34 complex body far away.This results suggest, in the HIV-1 course of infection, the CHR upper reaches ⁶²¹QIWNNMT ⁶²⁷Site and NHR downstream ⁵⁸¹AVERYLKDQ ⁵⁹⁰Between possibly interact and the film fusion process of gp41 mediation is had material impact, thereby the infection activity of decision virus.

The HIV fusion inhibitor research of target gp41

In theory, suppress any one link in the fusion process, just can suppress HIV and get into target cell, suppress the purpose that HIV infects and treat AIDS thereby reach.From the angle of viral syncretizing mechanism, the HIV entry inhibitor can be divided into three major types, and the one, disturb gp120 and CD4 receptors bind, be called adhesion inhibitors; The 2nd, the antagonism accessory receptor stops it to combine with the gp120-CD4 mixture, is called the accessory receptor antagonist; The 3rd type is the formation that stops the gp41 core texture, is called the HIV fusion inhibitor ^[11]The anti-HIV polypeptide drug T-20 (Enfuvirtide) that obtained U.S. FDA " fast passage " approval in 2003 plays a role to the forming process of gp41 core texture.T-20 is still effective to the virus that tolerates preceding two types of medicines, can be used for forming more " cocktail " scheme, is used for the treatment of AIDS.The drug target of HIV entry inhibitor both can be a virus envelope protein, like gp120 and gp41, also can be to get into proteins associated with HIV on the target cell, like acceptor molecules such as CD4 or CCR5, CXCR4.With CCR5 or CXCR4 is the accessory receptor antagonist of drug target; Can only effect be arranged to corresponding R5 or X4 virus; Can not suppress to use the R5X4 virus of two kinds of accessory receptors, also can't suppress the various accurate strain that HIV the infected carries fully, possibly cause the failure of treating and preventing.Simultaneously, act on human body protein and also possibly produce severe side effect.Therefore, as drug target, has significant meliority with HIV envelope protein itself aspect the security of medicine.Owing to the sequence of gp41 is more more conservative than gp120, and be viral distinctive albumen, thereby have more advantage as drug target research HIV entry inhibitor.

In the past ten surplus year in, the antiviral polypeptide of target gp41 is one of bright spot of HIV drug research, scientist adopts different strategies to design many efficient anti-HIV fusion inhibitors based on polypeptide or recombinant protein ^[11,13]As far back as 1992, wild etc. identified first anti-HIV peptide T 21 (DP-107) from the NHR district.Subsequently, more effective HIV merges the inhibition polypeptide to be identified from the CHR district, like SJ-2176 and T-20.Generally speaking, the prototype polypeptide that is derived from gp41 CHR has higher HIV-resistant activity than the prototype polypeptide that is derived from NHR, and wherein foremost is exactly T-20 and C34, and they can suppress HIV-1 cell fusion mediated and infection effectively.Based on the Fusion Model of HIV, the mechanism of action that the CHR polypeptide suppresses HIV entering target cell is considered to combine with gp41 NHR, thereby stops the CHR of gp41 itself to combine to form functional six helical bundle structures (dominant negative fashion) with NHR.Yet some scholars have different views to the mechanism of action of T-20 ^[14]Even it also might act on cytolemma gp120 simultaneously.T-20 and C34 comprise the NHR land on the structure, can interact with the NHR sequence.Though the most of sequence of these two polypeptide is overlapping, C34 comprises hydrophobic pocket land (PBD) and this site of T-20 shortage; Yet T-20 comprises the unexistent adipose membrane of C34 land (LBD), and this site is also very important to the antiviral activity of T-20.Research shows that NHR is last ⁵⁴⁷GIV ⁵⁴⁹Site (GIV motif) is closely related to the resistance of T-20 and C34 with HIV-1 ^[15]For the resistance problem that overcomes T-20 and improve pharmaceutical activity, designed s-generation fusion inhibitor T-1249.This peptide sequence has comprised HIV-1, and the CHR sequence of HIV-2 and SIV has higher anti-HIV ability, and is also very effective to the T-20 persister.But T-1249 is owing to existing problems on formulation have stopped clinical trial.T-20 is that first also is a present unique HIV-1 membrane fusion inhibitor that is approved for clinical treatment, but resistance has limited its application clinically owing to causing easily.Though C34 is active better, because its water-soluble problem also is difficult to develop into medicine.Based on the three-dimensional structure of HIV-1 gp41 core conformation and the sequence of C34, designing novel antiviral polypeptide is one of present research focus.In addition; Progress based on the theoretical research and development of HIV-1 gp41 core texture fusion inhibitor has also greatly promoted to seek enveloped virus (like sars coronavirus, respiratory syncytial virus and the H5N1 bird flu virus etc.) antiviral with similar fusion mechanism.

Reference:

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Summary of the invention

Technical problem to be solved by this invention provides a kind of polypeptide and verivate thereof that can efficiently suppress the HIV infection; This polypeptide is to invade the mechanism of target cell and design to HIV; Not only have high reactivity, and have target property, can greatly improve the antiviral effect of medicine; Reduce dosage, thereby reach the purpose that improves curative effect, reduces toxic side effect.

A polypeptide provided by the present invention, name is called P32, and its aminoacid sequence is shown in the sequence in the sequence table 1.

In order to improve its stability, the 1st amino acids residue of said sequence 1 is connected with aminoterminal protection base, and the 32nd amino acids residue of said sequence 1 is connected with carboxyl terminal protection base.

Another polypeptide provided by the present invention is the polypeptide of deriving of P32, and its aminoacid sequence is shown in the sequence in the sequence table 2.

In order to improve its stability, the 1st amino acids residue of said sequence 2 is connected with aminoterminal protection base, and the 33rd cysteine residues of said sequence 2 is connected with carboxyl terminal protection base.

Sequence 1 in the sequence table is made up of 32 amino-acid residues, and sequence 2 is made up of 33 amino-acid residues.Sequence 2 is compared with sequence 1, and only the carboxyl terminal in sequence 1 adds a cysteine residues, so that connect the SUV molecule.

Polypeptide derivative provided by the present invention; It is the SUV modifier of polypeptide of deriving shown in the sequence 2; Its aminoacid sequence is shown in the sequence in the sequence table 2; Wherein, said SUV modifier is that the 33rd cysteine residues of said sequence 2 is connected the compound that obtains with SUV through thioether bond.

In order to improve its stability, the 1st amino acids residue of said sequence 2 is connected with aminoterminal protection base, and SUV is connected with carboxyl terminal protection base.The name of this SUV modifier is called CHFI (Cholesterol-conjugated HIV fusion inhibitor).Its sequence and structure are Ac-WNEMTWEEWEKKIEEYTKKIEEILKKSEEQQKC+Cholesterol-NH ₂, at the C-terminal chemical coupling SUV molecule of synthetic polypeptide.

Wherein, the definition of the amino-acid residue of each letter character representative is following: W is that tryptophane, N are that l-asparagine, E are that L-glutamic acid, M are that methionine(Met), T are that Threonine, K are that Methionin, I are that Isoleucine, Y are that tyrosine, L are that leucine, S are that Serine, Q are that Stimulina, C are halfcystine; On behalf of SUV, Ac, Cholesterol represent ethanoyl, NH ₂Represent amino.

Above-mentioned aminoterminal protection base can be the arbitrary group in ethanoyl, amino, maleoyl, succinyl, tertbutyloxycarbonyl or benzyloxy or other hydrophobic groupings or the macromolecular carrier group; Said carboxyl terminal protection base can be NH ₂, the arbitrary group in carboxyl, carboxamido-group or tertbutyloxycarbonyl or other hydrophobic groupings or the macromolecular carrier group.

Contain the encoding sox of aforementioned polypeptides, or the recombinant vectors, reconstitution cell, reorganization bacterium or the recombinant virus that contain said encoding sox also belong to protection scope of the present invention.

The preparation method of SUV modifier of polypeptide derives shown in the sequence 2; May further comprise the steps: 1) the preparation aminoacid sequence is the polypeptide of sequence 2 in the sequence table, the 33rd cysteine residues of sequence 2 is connected through thioether bond with SUV obtain this SUV modifier.

Aforementioned polypeptides, above-mentioned polypeptide or the above-mentioned SUV modifier of deriving can be used for preparing following product:

A) product of anti-HIV, as resisting the product of following at least a strain: HIV gp41 spontaneous mutation strain comprises L33S, L33M, L33V, S35F, Q39R, L54M, Q56K, Q56R, L54M/Q56K, L54M/Q56R and L34M/L54M/Q56R; HIVgp41 induced mutation strain comprises I37Q/V38M, I37Q/V38Q, I37S/V38N and I37V/V38T; HIV gp41 wild strain; HIV strain NL4-3;

B) treat and/or prevent the product of AIDS, like medicine or vaccine.

The principle of design of CHFI polypeptide is based on the principle that the HIV film merges, and can combine interactional peptide sequence Ac-WNEMTWEEWEKKIEEYTKKIEEILKKSEEQQKC-NH between trans blocking-up CHR of viral gp41 molecule NHR and the NHR thereby at first design is synthetic ₂Through the high one-tenth thioether reactant of chemo-selective the C-terminal of polypeptide is carried out the SUV molecular modification then; Purpose is that polypeptide drugs can be interacted with the Lipid Rafts (Lipid raft) on the cell adipose membrane, thereby improves the target property and the bioavailability of polypeptide.CHFI makes it have cytolemma target property, and can prolong the transformation period of polypeptide simultaneously because terminal SUV is modified, to improve the antiviral activity of polypeptide drugs.

The CHFI peptide sequence designs according to 32 corresponding aminoacid sequences of HIV hypotype B standard virus strain NL4-3; 18 amino acid (56.25%) are wherein substituted, do in order to increase spiral stability and the polypeptide and the NHR bonded ability of polypeptide self through " salt bridge ".Simultaneously, introduce halfcystine (C), to connect the SUV molecule at the C-terminal of polypeptide.

Polypeptide of the present invention and verivate thereof have antiviral activity strong, have target property, long half time, good water solubility, significant advantage such as be easy to synthesize.

Description of drawings

Fig. 1 is the analysis mode high-efficient liquid phase chromatogram.

Fig. 2 is the mass spectrum of CHFI.

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Following preferential embodiment elaborates to the present invention, but does not mean that restriction content of the present invention.

Embodiment1. the synthetic and modification of polypeptide

1.1 material

Required chemical reagent uses preceding without being further purified all from main chemical reagent supplier.Rink resin (substituent constant is 0.44mmol/g) available from Tianjin with become company.

1.2 polypeptide is synthetic

The synthetic solid-phase polypeptide synthesis method (Fmoc/tBu strategy) that all adopts of all polypeptide, manually synthetic by carboxyl terminal to the aminoterminal direction.

The amino acid of employing N-fluorenylmethyloxycarbonyl (Fmoc) protection, employing Rink resin (substituent constant is 0.44mmol/g) are as solid phase carrier; DMF solution with 25% (volumn concentration) piperidines removes amino protecting group Fmoc; Removing step need carry out twice at every turn, and duration is respectively 8min and 10min.Connecing the method for condensing that reactive polypeptide selects for use is DIPC/HOBt method and PyBOP method, and amino acid and activating reagent all adopt 3 times of equivalents, and the reaction duration is 1 hour, adopts the qualitative colour developing of triketohydrindene hydrate (Kaiser method) monitoring reaction process.If the reaction of certain amino acid condensation not exclusively, proper extension reaction times or repeat condensation once is until obtaining required target peptide section Ac-WNEMTWEEWEKKIEEYTKKIEEILKKSEEQQKC-NH2.

1.3 cut and go side chain protected

Use cutting reagent (trifluoroacetic acid: 1: thioanisole: phenol: H ₂O: tri isopropyl silane=68.5: 10: 10: 5: 3.5: 1, v/v) target polypeptides cracking from the resin is got off and remove Side chain protective group (30 ℃ cutting 4 hours) down.Filtrating joins the polypeptide deposition is separated out, and is centrifugal.With ether washing several after drying, promptly obtain the polypeptide bullion.

1.4 the SUV modified polypeptide is synthetic

The synthetic document of publishing with reference to people such as Paolo Ingallinella of bromoacetic acid cholesteryl ester carries out (Paolo Ingallinella et al. Proc Natl Acad Sci U S A.2009; 106 (14): 5801-6).The bromoacetic acid cholesteryl ester is grafted on the polypeptied chain through the high one-tenth thioether reactant of chemo-selective, thereby obtains target molecule---the SUV modified polypeptide.Concrete grammar is: (sequence is Ac-WNEMTWEEWEKKIEEYTKKIEEILKKSEEQQKC-NH to 205 milligrams of thick peptides that obtain according to method in the step 1.3 ₂) be dissolved in 7.5 milliliters of DMSO, be dissolved in 68 milligrams of bromoacetic acid cholesteryl esters among 1 milliliter of THF (trifluoroacetic acid) to wherein adding, 500 microlitre DIEA (diisopropylethylamine) join in the mixing solutions subsequently.React 1 hour afterreaction and accomplish, obtain SUV polypeptide (Ac-WNEMTWEEWEKKIEEYTKKIEEILKKSEEQQKC+Cholesterol-NH2).

1.5 peptide purification and sign

Adopt HP1100 type (U.S. Agilent company) rp-hplc that the SUV polypeptide that step 1.4 prepares is carried out successfully purifying.Chromatographic column model: Cosmosil 5C ₄-AR (10 * 250mm).The chromatographic run condition: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is the aqueous solution of trifluoroacetic acid and acetonitrile, and the concentration expressed in percentage by volume of trifluoroacetic acid is 0.05%, and the concentration expressed in percentage by volume of acetonitrile is 2%.Mobile phase B is 90% (concentration expressed in percentage by volume) acetonitrile solution.To 40%B, 20 minutes time, elution flow rate is 25 milliliters of PMs to linear gradient elution, ultraviolet detection wavelength 220 nanometers by 20%B.Obtain the pure article of SUV polypeptide of fluffy state behind the freezing solvent, its chemical structure is characterized by the MALDI-TOF mass spectrum, and its purity is then by analysis mode high performance liquid chromatograph (flow velocity: 1 milliliter of PM) provide.Wherein, the model of analysis mode high performance liquid chromatograph: Tianjin, island CBM-10A VP PULS, the model of the chromatographic column that is adopted: Agela4.6*250mm C18.The chromatographic run condition: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is the aqueous solution of trifluoroacetic acid and acetonitrile, and the concentration expressed in percentage by volume of trifluoroacetic acid is 0.05%, and the concentration expressed in percentage by volume of acetonitrile is 2%.Mobile phase B is the aqueous solution of trifluoroacetic acid and acetonitrile, and the concentration expressed in percentage by volume of trifluoroacetic acid is 0.05%, and the concentration expressed in percentage by volume of acetonitrile is 90%.Linear gradient elution by 25%B to 45%B, 20 minutes time.

The result of table 1. analysis mode performance liquid chromatography

The mass spectrometric model of MALDI-T: Perseptive Biosystems (manufacturers) VOYAGER-DE STR (model)

Testing conditions: acceleration voltage 20000v

Grid 95％

Guide?wire 0.05

The result shows the SUV polypeptide that obtains via preparation type rp-hplc purifying, gets 13 milligrams of pure article, and yield is 6.34%.The pure article of SUV polypeptide that obtain characterize to such an extent that purity is 96.6% (Fig. 1 and table 1) by the analysis mode rp-hplc, the MALDI-TOF mass spectrum record its molecular weight be 4743.79Da (theoretical value is 4742.53Da) (Fig. 2).Should represent with CHFI by pure article.

T-20 among the following embodiment 2-4, C34, SFT, T1249, T267227, CP32M, P32 are not all modified by SUV; All synthetic according to the method described above; Their aminoterminal all is connected with ethanoyl, and their carboxyl terminal all is connected with amino, and their purity is all greater than 95%.

Embodiment2.CHFI the antiviral effect evaluation of polypeptide

2.1 polypeptide is to the restraining effect of HIV strain NL4-3: the molecular cloning plasmid pNL4-3 of coding HIV-1 virus strain NL4-3 provides (catalog number (Cat.No.): 114) by America NI H AIDS Research&Reference Reagent Program.Adopt the plasmid extraction kit of QIAGEN company to prepare the pNL4-3 plasmid, adopt the Lipofectamine of Invitrogen company ^TM2000 transfection reagents are with pNL4-3 plasmid transfection 293T cell, in 37 ℃ of 5%CO ₂Hatch in the cell culture incubator after 6 hours and change liquid, continue then to cultivate 48 hours.With the pipettor supernatant in collecting cell culturing bottle or the Tissue Culture Plate gently, cross the leaching supernatant through 0.45 μ m filter, add 20% foetal calf serum (FBS), be sub-packed in then in the PA tube, it is subsequent use or directly carry out titration of virus to place-80 ℃ of preservations.The step of titration of virus does, virus is done 5 times of dilutions in 96 orifice plates, and 8 gradients in 4 multiple holes are set, and final volume is 100 μ l.The TZM-bl cell is used trysinization, cell counting, with the DMEM perfect medium with cell dilution to 10 ⁵Individual/ml, every hole adds 100 μ l, and every porocyte is 10 ⁴Individual, add DEAE-dextran, final concentration is 15 μ g/ml.96 orifice plates are put into cell culture incubator, 37 ℃, 5%CO ₂Cultivated 48 hours.From cell culture incubator, take out 96 orifice plates then, from last appearance hole, inhale and abandon supernatant, add 30 μ l cell pyrolysis liquids, add 100 μ l luciferase detection reagent behind the placement 10min.With pipettor sucking-off 100 μ l liquid from every hole, be added in the corresponding 96 hole blanks, read luminous value in the microwell plate photometer.Calculate virus titer with the Reed-Muench method.For measuring the antiviral activity of anti-HIV polypeptide, polypeptide to be gone in 96 orifice plates by multiple proportions (2 times) dilution shop, final volume is 50 μ l, wherein substitutes polypeptide as negative control with 50 μ l DMEM substratum.Add TZM-bl cell 100 μ l (10 ⁵Individual cell/ml) contain the DEAE-dextran final concentration is 15 μ g/ml, adds acquired virus 50 μ l, and every hole is equivalent to 100TCID ₅₀After cultivating 48h, utilize luciferase detection reagent (Promega) to measure the relative fluorescence unit (RLU) in every hole.

2.2 detect the data analysis of anti-HIV-1 medicine: median effective dose is the dosage that can cause 50% maximum effect (quantitative response), with median effective dose (ED ₅₀) expression.It is relative light unit (RLU) numerical value that virus detects the data that obtain in the inverase experiment, can quantitative analysis medicine median effective dose (ED ₅₀), all drug levels all are converted into log ₁₀Concentration value, each concentration sample well all calculates average RLU value, calculates each concentration fluorescent inhibiting rate, calculation formula:

Data analysis utilizes GraphPad Prism Software

2.01 software, parameter S type dosage equation is analyzed the ED of every kind of medicine in the employing nonlinear regression analysis ₅₀Value and medicine

2.3HIV the preparation of pseudovirus:

Pseudovirus in the table 2 prepares according to following method.

(1) will express proteic plasmid pcDNA3.1-ENV of HIV ENV (the ENV encoding sox is inserted the recombinant expression plasmid that pcDNA3.1 (-) obtains) and HIV skeleton plasmid pSG3 ^{Δ ENV}(expressing all albumen except that ENV in the HIV genome) (provided by the NIH AIDS Research and Reference Reagent Program of NIH; Cat No:11051); In 1: 2 ratio transfection 293T cell of mass ratio, establish pSG3 simultaneously ^{Δ ENV}Contrast, i.e. the pSG3 Δ ENV of a transfection same amount.

(2) in 37 ℃, 5%CO ₂Hatched in the cell culture incubator 6 hours, and in cell culture incubator, continued to hatch 48 hours after changing liquid, pseudovirus is secreted to supernatant.

(3) with the pipettor supernatant in sucking-off Tissue Culture Flask or the Tissue Culture Plate as often as possible; Get supernatant through filtration of 0.45 μ m filter or the centrifugal 10min of 1000g; To make its final concentration be 20% to wherein adding FBS, is transferred in the PA tube in-80 ℃ to preserve subsequent use or directly carry out titration of virus.The measuring method of the titration method of pseudovirus and polypeptide antiviral activity is with HIV virus strain NL4-3.

2.4 antiviral activity detected result

At first; Adopt laboratory representative HIV-1 virus strain NL4-3 to estimate the antiviral activity of CHFI, carry out parallel detection with the s-generation antiviral polypeptide of reporting in the medicine T-20 of present listing and the document (C34, SFT, T1249, T267227, CP32M) as contrast simultaneously.The result finds (table 2), and CHFI suppresses the EC that NL4-3 infects ₅₀At 0.01nM, i.e. 10pM level is than the active (EC of T-20 ₅₀=74.71nM) high 7471 times; Than the active high 80-336 of the s-generation antiviral polypeptide of reporting in the document doubly.It is thus clear that CHFI has the antiviral activity of high degree.For further estimating the antiviral activity of CHFI; We have prepared 44 strain HIV pseudoviruss; Comprise the international present popular HIV strain of strain and China of representing, it is that 8 strains of A/E hypotype, B and C recombinant type are 18 strains of B/C hypotype, 1 strain of E hypotype that 2 strains of A hypotype, 10 strains of B hypotype, 5 strains of C hypotype, A and E recombinant type are wherein arranged.Adopt the antiviral experiment of these HIV pseudoviruss to show (table 3), CHFI has efficiently active to the various subtype virus (comprising A, B, C, AE, BC and E hypotype) of test, is significantly higher than T-20 contrast polypeptide and without the naked peptide P32 that modifies.Therefore, the HIV-resistant activity of CHFI is not only efficient, and wide spectrum.Presentation of results, SUV is modified the HIV-resistant activity that can significantly improve polypeptide.

The specific activity of the anti-HIV polypeptide of table 2.

Table 3.CHFI is to the restraining effect of different subtype HIV pseudovirus

Embodiment3.CHFI polypeptide has significant activity to the HIV persister

The amino acid variation in HIV fusion rotein gp41NHR zone can cause the resistance of virus to fusion inhibitor.For further estimating the HIV-resistant activity of CHFI, we test the restraining effect of CHFI to virus infection with cover HIV gp41 spontaneous mutation strain of one in the table 4 and induced mutation strain.The spontaneous mutation strain comprises L33S, L33M, L33V, S35F, Q39R, L54M, Q56K, Q56R, L54M/Q56K, L54M/Q56R and L34M/L54M/Q56R; Its preparation method is seen document (the Raghavan Chinnadurai that people such as Chinnadurai publish; Jan Mu ¨ nch and Frank Kirchhoff.Effect ofnaturally-occurring gp41 HR1 variations on susceptibility of HIV-1 to fusion inhibitors.AIDS 2005,19:1401-1405); The induced mutation strain comprises I37Q/V38M, I37Q/V38Q, I37S/V38N and I37V/V38T; Its preparation method is seen document (the Raghavan Chinnadurai that people such as Chinnadurai publish; Devi Rajan; Jan Mu ¨ nch; And Frank Kirchhoff.Human Immunodeficiency Virus Type 1 Variants Resistant to First-and Second-Version Fusion Inhibitors and Cytopathic in Ex Vivo Human Lymphoid Tissue.Journal of Virology, 2007; 81 (12): 6563-6572).Experimental result in the table 4 shows that anti-HIV peptide C HFI has high activity to the virus strain of these tests, explains that the sudden change of above-mentioned gp41 nature and inductive sudden change are very little to the antiviral activity influence of CHFI.T-20 as contrast presents significant resistance to above-mentioned most viruses; Wherein the resistance of L33M, L33V, S35F, Q39R, L54M, L34M/L54M/Q56R, I37Q/V38M, I37Q/V38Q, I37S/V38N and I37V/V38T mutant strain is especially obvious, explains that sudden change of these simple point mutations or two point or three point mutation can cause virus that resistance is appearred in T-20.Nearest research shows, the two sudden changes of the I37T of HIV gp41 and N43K point mutation or I37T/N43K can cause the remarkable resistance of HIV to s-generation HIV fusion inhibitor SFT and T-20, sees document (the Liu Z that people such as Liu publishes; Shan M, Li L, Lu L; Meng S, Cheng C, He Y; Jiang S; ZhangL.In Vitro selection and characterization of HIV-1 variants with increased resistance to Sifuvirtide, a novel HIV-1fusion inhibitor.Journal of Biological Chemistry, 2011; 286 (5): 3277-87).For this reason, our the antiviral activity influences of these sudden changes of having adopted the HIV pseudovirus system testings carried the two sudden changes of I37T, N43K point mutation or I37T/N43K to CHFI.Visible from the result of table 5, the two sudden changes of I37T and N43K point mutation or I37T/N43K are little to the activity influence of CHFI, and can cause the remarkable resistance to SFT and T-20.Comprehensive these experimental result explanations, CHFI has the high activity that suppresses to T-20 resistance strain, can be used for the treatment that the T-20 persister infects.

Table 4.CHFI is to the restraining effect of HIV mutant strain

Table 5.CHFI is to the restraining effect of HIV persister

Embodiment4.CHFI polypeptide is through combining target cell membrane performance efficient disease-resistance toxic action

For analyzing the mechanism of action of CHFI, whether we bring into play its antiviral activity efficiently through the effect target cell membrane with following experiment discussion CHFI.The basic step of experiment is: (1) with trysinization TZM-bl cell after and count, with the DMEM perfect medium with cell dilution to 10 ⁵Individual/ml, (every porocyte is 10 to add 100 μ l by every hole ⁴Individual) be layered in the 96 porocyte culture plates, put into 37 ℃, 5%CO ₂The cell culture incubator overnight cultures; (2) respectively peptide C HFI and P32 are carried out 3 times of dilutions from initial final concentration 125nM, establish 10 gradients.Add behind the cell in 37 ℃ 5%CO ₂Hatched in the cell culture incubator 30 minutes, and abandoned nutrient solution then,, add 150 μ l DMEM perfect mediums then and change liquid (washing group) to remove the polypeptide that does not combine cytolemma also with 1 * PBS washing 3 times.Simultaneously, be provided with without the cell hole that washs as contrast (not washing group); (3) two groups (washing group and do not wash group) adds 50 μ l respectively, and to contain final concentration be that pseudovirus SF162 or the NL4-3 of the DEAE-dextran of 15 μ g/ml (is equivalent to 100 TCID ₅₀).The negative cells control wells adds 50 μ l DMEM perfect mediums.96 orifice plates are put into cell culture incubator, 37 ℃, 5%CO ₂Cultivated 48 hours; (4) then, from cell culture incubator, take out 96 orifice plates, from last appearance hole, inhale and abandon supernatant, add 30 μ l cell pyrolysis liquids, place adding 70 μ l luciferase detection reagent (Promega) after 10 minutes.With pipettor sucking-off 70 μ l liquid from every hole, be added in the corresponding 96 hole blanks, in Modulus ^TMRead the relative fluorescence unit (RLU) in every hole in the multi-functional luminosity meter of II microwell plate, and calculate each concentration fluorescent inhibiting rate and ED by above-mentioned formula ₅₀Value.Experimental result in the table 6 shows that CHFI adds target cell after thorough washing still can keep its HIV-resistant activity efficiently.Activity to not washing group of virus strain NL4-3 CHFI is 0.15 ± 0.03nM, and the washing group is 0.27 ± 0.04nM, is 0.07 ± 0.01nM to not washing group of virus strain SF162 activity, and the washing group is 1.35 ± 0.06nM.And, be not 2.34 ± 0.56nM and 3.84 ± 0.50nM in scrubbed component not, but after washing, can not measure the antiviral activity of polypeptide during up to 250nM in concentration to NL4-3 and SF162 as the P32 polypeptide of contrast.This experimental result explanation; The CHFI that SUV is modified can stably combine cytolemma; The PBS thorough washing can not be removed CHFI; Suppress active so still can keep its powerful HIV, and can not effectively combine cytolemma without the corresponding naked peptide P32 that modifies, its antiviral activity is washed removal easily.Therefore, SUV modify can target polypeptide in surface of cell membrane, more can bring into play the antiviral activity of polypeptide effectively.This also is one of highly active molecular mechanism of CHFI.

Table 6.CHFI targeted cells film performance antiviral activity (EC ₅₀± SD, nM)

Claims (14)

1. polypeptide, its aminoacid sequence is shown in the sequence in the sequence table 1.

2. polypeptide according to claim 1 is characterized in that: the 1st amino acids residue of said sequence 1 is connected with aminoterminal protection base, and the 32nd amino acids residue of said sequence 1 is connected with carboxyl terminal protection base.

3. polypeptide according to claim 2 is characterized in that: said aminoterminal protection base is ethanoyl, amino, maleoyl, succinyl, tertbutyloxycarbonyl or benzyloxy; Said carboxyl terminal protection base is NH ₂, carboxyl, carboxamido-group or tertbutyloxycarbonyl.

4. the SUV modifier of the polypeptide of deriving of the said polypeptide of claim 1; Its aminoacid sequence is characterized in that shown in the sequence in the sequence table 2: said SUV modifier is that the 33rd cysteine residues of sequence 2 is connected the compound that obtains with SUV in the sequence table through thioether bond.

5. SUV modifier according to claim 4 is characterized in that: the 1st amino acids residue of said sequence 2 is connected with aminoterminal protection base, and SUV is connected with carboxyl terminal end protection base.

6. SUV modifier according to claim 5 is characterized in that: said aminoterminal protection base is ethanoyl, amino, maleoyl, succinyl, tertbutyloxycarbonyl or benzyloxy; Said carboxyl terminal protection base is NH ₂, carboxyl, carboxamido-group or tertbutyloxycarbonyl.

7. the encoding sox of the said polypeptide of claim 1.

8. express the recombinant vectors of the said encoding sox of claim 7.

9. express the reconstitution cell of the said encoding sox of claim 7.

10. express the reorganization bacterium of the said encoding sox of claim 7.

11. express the recombinant virus of the said encoding sox of claim 7.

12. the preparation method of the described SUV modifier of claim 4; May further comprise the steps: 1) the preparation aminoacid sequence is the polypeptide of sequence 2 in the sequence table, the 33rd cysteine residues of sequence 2 is connected through thioether bond with SUV obtain the described SUV modifier of claim 4.

13. claim 1 or 2 described polypeptide the preparation a) or b) in application:

A) medicine of anti-HIV-1,

B) medicine of the AIDS that causes by HIV-1 of treatment.

14. claim 4 or 5 or 6 described SUV modifiers the preparation a) or b) in application:

A) medicine of anti-HIV-1,

B) medicine of the AIDS that causes by HIV-1 of treatment.

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