CN1908010B - Furin enzyme inhibitor of resisting HIV-1 and preparation method thereof - Google Patents

Abstract

The invention discloses a new Furin enzyme inhibitor and preparing method of HIV-1 in the biochemical and medical domain, which is characterized by the following: changing some amino acids at feature position based on inhibiting segment of original green gram tryptone; obtaining rough sample to produce high-purity sample through step-by-step reduction-oxidation way; certifying the effect of Furinenzyme inhibitor (10<-9>M Ki value); possessing key role for Furin enzyme from HIV-1 gp160 into gp120 and gp41; providing a new path to treat AIDS.

Description

The Furin enzyme inhibitors and the method for making thereof of anti-HIV-1

Technical field

The invention belongs to biological chemistry, field of pharmacology, relate to a kind of hiv inhibitor, more specifically, the present invention relates to a kind of Furin enzyme inhibitors and method for making thereof of new anti-HIV-1.

Background technology

The AIDS full name is AIDS (acquired immunodeficiency syndrome; AIDS), this disease is infected by human immunodeficiency virus (human immunodeficiency virus, HIV or hiv virus) and causes; Cause by the part of the infected's immunologic function or completely lose; The CD4+ cell number reduces, generator opportunistic infections, tumour etc. then, and clinical manifestation is varied.This disease velocity of propagation is fast, case fatality rate is high, and can't cure at present, has caused the concern of national governments with society.

The infection of HIV is that the processing by adventitia precursor protein gp160 begins; Become two fragments after the processing of gp160 through precursor processive enzyme (for example Furin enzyme): surface glycoprotein (surface glycoprotein; SU) gp120 and transmembrane protein (transmembrane protein, TM) gp41.HIV-1 at first combines with acceptor CD4 on the target cell through the gp120 among its membrane glycoprotein mixture gp120-gp41; Make the conformation of gp120 change; And then with target cell on auxilliary receptor CXCR 4 or CCR5 combine, cause the configuration of gp41 to change, expose its fusogenic peptide and be inserted into host cell membrane; Thereby start the fusion of viromembrane and cytolemma, accomplish the course of infection that virus gets into host cell.The processing of gp160 precursor is the first step of phagocytic process, if can suppress the invasion that this step just can stop virus, is important drug target.The processing of gp160 mainly is the effect that relies on precursor processive enzymes such as Furin enzyme.At Thomas; People's such as Gary patent (Thomas; Gary et al, UnitedStates Patent:5604201, February 18,1997) in point out that Furin is the sequence (P that contains RXXR to the requirement of cleavage site ₁And P ₄The site is R, and P ₂And P ₃The site then is an arbitrary amino acid), and research in recent years shows and just contains such cleavage site among the outer membrane glycoprotein gp160 of HIV-1: site 1:Arg508-Glu-Lys-Arg511; Site2:Lys500-Ala-Lys-Arg503 has proved the significance of Furin to the processing of HIV-1 virus precursor albumen, further specifies the effect that suppresses the Furin enzyme and invades the feasibility of human body host cell to suppressing HIV-1 virus.(McCune?et?al.，1988，Cell?53：55-67；Hallenberger?et?al.，1992，Nature?360：358-361；Etienne?Decroly?etal，1994，the?journal?of?biological?chemistry?vol.269No.16pp：12240-12247；RominaOliva?et?al，2002，Chem.Eur.J.，8，No.6：1467-1473；Ilia?Tikhonov?et?al，2004，FEBS?Letter565：89-92)

The research that is directed against the suppressor factor inhibition HIV of Furin enzyme at present has the report of some, mainly contains two kinds: stoichiometric peptidyl suppressor factor (decanoyl-Arg-Val-Lys-Arg-CH ₂Cl) and alpha1-antitrypsin Portland (α 1-PDX).Decanoyl-Arg-Val-Lys-Arg-CH wherein ₂The alkylation attribute limits of Cl its use, when suppressing the Furin enzyme, have lower activity.α 1-PDX suddenlys change through the reactive behavior site to alpha1 Anti-trypsin it to be comprised generally believe it is minimum Furin cleavage site-Arg-Ile-Pro-Arg-.α 1-PDX has highly selective (Ki=600pM) to Furin in the middle of in vitro tests, but when inhibitor concentration is brought up to high density, also will suppress other PCs.In the middle of cell and zooscopy, find that α 1-PDX is effective Furin enzyme inhibitors.(Komiyama，T.et?al.，2000，Biochemistry?39：15156-15165；Cameron.A.et?al.，2000，J.Biol.Chem.275：36741-36749；FeiHao?et?al.，2001，ActaBiochimica?et?Biophysica?Sinica，33(6)：591-599；Jean，F.et?al.，1998，Proc.Nati.Acad.Sci.U.S.A.95：7293-7298；Anderson，E.D.et?al.，1993，J.Biol.Chem.268：24887-24891；Molloy，S.S.et?al.，Academic?Press?San?Diego，CA，2001，The?Enzymes199-235；Gary?Thomas，Nat?Rev?Mol?Cell?Biol，2002Oct；3(10)：753-66；Pierre?Cordelieret?al.Jul?2003，Biochim?Biophys?Acta，1638(3)：197-207；Bouchaib?BAHBOUHI，2001，Biochem.J.360：127-134)

Make up on the basis that these existing Furin enzyme inhibitorss generally all are natural macromoleculars, this area presses for the little and active high artificial constructed Furin enzyme inhibitors of molecule, can become more effective anti-HIV-1 suppressor factor.

Summary of the invention

Deficiency based on above prior art exists the object of the present invention is to provide a kind of molecular weight little, active high anti-HIV-1 suppressor factor.

Another object of the present invention just provides sequences Design, method for making and the purposes of said anti-HIV-1 suppressor factor.

In first aspect of the present invention, a kind of new Furin enzyme inhibitors fragment is provided, have following aminoacid sequence:

P wherein ₁The site is R; P ₂The site is K; P ₄The site is R; P ₆The site is X ₁, can be R or K; P ₇The site is X ₂, can be all basic aminoacidss such as Ala; The pairing situation of C is C4-19, C9-17; C pointed in the building-up process obtains intermediate product by the protection of ethanamide methyl (Acm) protection base.

The structure of described aminoacid sequence is following:

In second aspect of the present invention, a kind of anti-HIV-1 suppressor factor is provided, contain described Furin enzyme inhibitors, have following aminoacid sequence:

P wherein ₁The site is R; P ₂The site is K; P ₄The site is R; P ₆The site is X ₁, can be R or K; P ₇The site is X ₂, can be all basic aminoacidss such as A; The pairing situation of C is C4-19, C9-17.

In the third aspect of the invention, a kind of method for preparing this suppressor factor is provided, comprise step:

A. utilize polypeptide synthetic method, synthesize needed Furin enzyme inhibitors fragment, institute's synthetic crude product has following aminoacid sequence:

Wherein, P ₁The site is R, P ₂The site is K, P ₄The site is R, P ₆The site is R, P ₇The site is A, and C pointed in the building-up process obtains intermediate product by the protection of Acm protection base;

B. will synthesize crude product reduction, obtain reducing the product of form and carry out separation and purification after the desalination with HPLC;

C. with the product of the reduction form of purifying with 2-PDS oxidation 3h after freeze-drying, with carrying out the HPLC purifies and separates after the oxidation products desalination, obtain purer oxidation intermediary product;

D. the mesomorphic product of oxidation is carried out the iodine oxidation, thereby the Acm protection of sloughing C makes the C correct pairing, the purifies and separates of process desalination and HPLC obtains purer anti-HIV-1 suppressor factor.

In the third aspect of the invention, the application of described Furin enzyme inhibitors fragment in the medicine of preparation treatment AIDS is provided.

In fourth aspect of the present invention, provide a kind of and infected drug composition effective for HIV, contain claim 1 described Furin enzyme inhibitors fragment and pharmaceutically acceptable carrier or vehicle.Can Furin enzyme inhibitors fragment be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium; Wherein pH is about 5-8 usually; Preferably pH is about 6-8, although the pH value can change with being prepared Substance Properties and illness to be treated to some extent.The pharmaceutical composition for preparing can carry out administration through conventional route, comprising but be not limited to: intraperitoneal, intravenously or topical.

Pharmaceutical composition of the present invention can directly be used for the treatment of AIDS.In addition, also can simultaneously or be used in combination the other treatment agent.

Pharmaceutical composition of the present invention contains Furin enzyme inhibitors fragment and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount, is mixed with the mode that is fit to deliver medicine to the HIV infected patient.Said carrier includes, but are not limited to: salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical prepn should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, pharmaceutical composition of the present invention also can use with the other treatment agent.

When using pharmaceutical composition of the present invention; Be that immune conjugate with safe and effective amount is applied to human body; Wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight; And in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.

As used herein, term " M3 " refers to the described anti-HIV-1 suppressor factor of this patent.

As used herein will be understood by those skilled in the art that, when aminoacid sequence involved in the present invention is described; Described E is L-glutamic acid (Glu), and described P is proline(Pro) (Pro), and described S is Serine (Ser); Described C is halfcystine (Cys), and described A is L-Ala (Ala), and described R is l-arginine (Arg); Described K is Methionin (Lys), and described I is Isoleucine (Ile), and described Q is Stimulina (Gln); Described H is Histidine (His), and described N is aspartic acid (Asn).

Description of drawings

Fig. 1, the HPLC collection of illustrative plates of the M3 of purifying, the C18 post (Agilent, 9.4 * 250mm), Mobile phase B is 70% acetonitrile that contains 0.08%TFA, mobile phase A is the water that contains 0.1%TFA, gradient (20-40%B/10-40min).Flow velocity is a M3 reduction form peptide for the 2ml/min. main peak.X-coordinate is the elution time, and ordinate zou is A230nm.

Fig. 2, the mass-spectrogram of the M3 of purifying.

Fig. 3, M3 is to the inhibition constant Dixon mapping of Furin; X-coordinate is a M3 concentration, and ordinate zou is a fluorescent absorption; Inhibition constant K i=3.26x10 to Furin ^-9M.

Fig. 4, the external restraining effect of M3 to HIV.

Embodiment

The inventor is support through extensive and deep discovering with the mung bean trypsin fragment, introduces to generally believe it is minimum Furin enzyme cleavage site-Arg-X-X-Arg-(wherein X represents amino acid arbitrarily) at the active function region mutation, simultaneously at P ₆R/K (Arg/Lys), P are introduced in the site ₇Neutral amino acids is introduced in the site, suppresses active to strengthen it, and with its suppressor factor that is configured to the furin enzyme, reaches the processing of inhibition to the outer membrane glycoprotein gp160 of HIV-1, stops the intrusion of HIV-1 virus.

As used herein, term " M3 " refers to the described anti-HIV-1 suppressor factor of this patent, and its substruction is following:

Wherein, P ₁The site is R; P ₂The site is K; P ₄The site is R; P ₆The site is X ₁, can be R or K; P ₇The site is X ₂, can be all basic aminoacidss such as A; The pairing situation of K is as shown in the figure: C4-19, C9-17.

Research shows, for the cleavage site major requirement P of Furin ₁The site is R and P ₄The site is R, and for P ₂The site can be K or R, P ₂Booster action is played in the site, and for P ₃The site then can be an arbitrary amino acid.P ₂And P ₆Basic aminoacids has played and has strengthened the active effect that suppresses, and in view of P ₆The site is R or K, P ₇Site mutation alkalize amino acid is for P ₆Stabilization is played in the site, and the inhibition that has also strengthened M3 simultaneously is active.Consider the segmental structure of support mung bean trypsin in the present invention, P ₃The site keeps original C to come the conformation of stabilize proteins.

As used herein, " polypeptide of the present invention " is meant the intermediate product in M3 and the preparation process thereof.

Polypeptide of the present invention is synthetic polypeptide.Polypeptide of the present invention is the product that the method with chemosynthesis obtains.

The method of reducing that the present invention adopted is to adopt the system of the thick peptide of 2mg/ml, 8M urea, 10mg/ml to react; Used reduction damping fluid can be tart 1 ‰ TFA systems; It also can be the damping fluid (50mM Tris, 1mM EDTA, pH 8.0) of alkalescence.

The method for oxidation that the present invention adopted is to make disulfide linkage form method of matching with the 2-PDS oxidation; The concrete grammar that adopts is that 1.5mg2-PDS is dissolved in the 1ml methyl alcohol; What dropwise add 100ml comprises in the 0.1%TFA system of the phthalin of HPLC purifying oxidation at room temperature 3 hours.The effect of 2-PDS is the carrying out of accelerating oxidizing reaction, but does not influence the effect of oxidation.In practice process of the present invention, adopt the method effect of 2-PDS oxidation good.

The method of taking off the oxidation of Acm iodine that the present invention adopted is 19mgI ₂/ mg oxidation gained peptide is dissolved in 80% acetic acid of 5ml/mg oxidation gained peptide, and the dissolving back directly adds in the oxidation gained polypeptide, adds 80% acetic acid of 4 times of volumes simultaneously, and it is best reacting about 1 hour.The termination of reaction relies on ascorbic interpolation to accomplish.Gained solution is done with the mode of rotary evaporation is dense, and freeze-drying after the post desalination is carried out purifying with HPLC excessively.

What the desalination of mistake post was adopted among the present invention is the desalting column of the filler filling of molecular sieve Sephadex G-15.

What the peptide section purifying work among the present invention after the desalination was adopted is the HPLC purification technique.

Major advantage of the present invention is:

(1) to have a molecular weight little for peptide section of the present invention, and structure is simple relatively, is prone to carry out the advantage of actually operating

(2) peptide section of the present invention is processed to form on the segmental basis of mung bean trypsin, has good biological activity, contains very high bioavailability.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used to the present invention is described rather than be used to limit scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, the condition of perhaps advising according to normal condition usually according to manufacturer.

The preparation of embodiment 1 anti-HIV-1 suppressor factor of the present invention

Synthesizing of 1M3 peptide section

Get BOC-Asn-OCH ₂-Pam resin (0.742mmol/g) is pressed the sequence of peptide and is extended to the N end one by one by the C end.Whenever, take turns circulation beginning and remove BOC with 50%TFA, again with the 10%DIEA neutralization, one takes turns loop ends detects the synthetic quality of monitoring with ninhydrin reaction.Side chain protective group is: and Asp (OCHex), Ser (Bzl), Cys (4-Meb, Acm), Lys (ClZ), Arg (Tos).Remove N with 50%TFA/DCM earlier after the synthetic completion of peptide and hold the BOC protection basic, moisture content is removed in peptide resin vacuum-drying.

The preparation of the cutting of 2 resins and the thick peptide of M3

Get the 100mg peptide resin and add the 0.5ml p-cresol, a little phenol and Thioanisole, processing is 1.5 hours in 0 ℃ of dry hydrogen fluoride (HF) in, polypeptied chain acidolysis from the resin is got off, and remove various Side chain protective groups.Cracking is accomplished the back with 5X20ml exsiccant ETHYLE ACETATE (DTT that contains 10mg/100ml) washing, contains the thick peptide of 20% acetonitrile solution (DTT that contains 10mg/100ml) extracting, merging extract and lyophilize with the acetic acid of the 1N of 40+40+20ml.Get the thick peptide of M3.

The preparation of 3M3 reduction form

In the reaction tubes of 7ml, react with the ratio of the thick peptide of 2mg/ml M3,8M urea, 10mg/ml DTT.Actual in the present invention operation is to add the thick peptide of 12mg, 2.88g urea, 60mg DTT to be dissolved in the 1 ‰ TFA solution of 6ml, and insulation reaction is 3 hours in 37 ℃ of water-baths, and freeze-drying after Sephadex G15 desalination obtains also ortho states of M3.

The mesomorphic preparation of 4 M3 oxidations

Go back in the 1 ‰ TFA solution that ortho states M3 is dissolved in 100ml freeze dried; Simultaneously 1.5mg 2-PDS is dissolved in the 1ml methyl alcohol; 2-PDS solution is dropwise added comprising in 1 ‰ TFA systems of the phthalin of HPLC purifying of 100ml; Oxidation at room temperature 3 hours, freeze-drying after Sephadex G15 desalination obtains purer M3 oxidation intermediate state through the HPLC purifying again.

The preparation of the final oxidation form of 5 M3

Freeze dried M3 oxidation intermediate state is sloughed last protection base to the Acm on the Cys with the method for iodine oxidation.19mg iodine is dissolved in the middle of the acetic acid of 5ml 80%; Join then in the middle of the freeze dried M3 oxidation intermediates; The acetic acid that adds 20ml 80% in addition; Reacted 1 hour, and dropwise added the ascorbic saturated solution for preparing in advance then and decorporate up to the reddish-brown safety of reaction solution, termination promptly reacts completely.The mode of reaction solution through rotary evaporation concentrated.Treat that the acetic acid that adds 1ml 1N dissolves after dense the doing, freeze-drying after Sephadex G15 desalination obtains the final oxidation state of purer M3 (Fig. 1) through the HPLC purifying again.

Fig. 2 has shown the mass-spectrogram of the M3 of purifying.Identify that through mass spectrum the main peak molecular weight is 2339.5Da, meets the theoretical molecular 2339.71Da of M3final, confirms as the final of M3.

The active mensuration of inhibition of embodiment 2 anti-HIV-1 suppressor factor of the present invention

1. the mensuration of enzyme activity

Furin passes through fluorescence spectrophotometry appearance The real time measure to the hydrolysis vigor of fluorogenic substrate pGlu-Arg-Thr-Lys-Arg-MCA.An amount of furin is added 1ml survey damping fluid alive, and (100mM Hepes, pH 7.5,1mM CaCl ₂, 0.5%Triton X-100,1mM beta-mercaptoethanol), 37 ℃ of insulations add an amount of fluorogenic substrate (50-80 μ M) after 3 minutes again.Excitation wavelength and the emission wavelength of surveying luminoscope when living are respectively 370nm and 460nm, and slit width is 10nm.

2. the mensuration of suppressor factor kinetic constant

Use dixon mapping (1/V is to I) (Dixon.1953) to obtain the Ki constant of different mutants.In the mensuration to furin, the concentration of substrate of use is respectively 50,80 μ M.IC ₅₀Mensuration then carry out two fit method reciprocal and confirm suppressing curve with software.From the IC that obtains ₅₀, also can release the Ki constant by following formula:

$\mathrm{Ki}=\frac{I_{50}}{1+\frac{\left[s\right]}{\mathrm{Km}}}$

S is a fluorogenic substrate concentration, and Km is Michaelis-Menton constant (pERTKR-MCA is 2.7 μ M to furin).

Fig. 3 has shown the inhibition constant Dixon mapping of M3 to Furin; X-coordinate is a M3 concentration, and ordinate zou is a fluorescent absorption; Inhibition constant K i=3.26 * 10 to Furin ^-9M.

3.HIV inhibition active

Ficoll-Paque density gradient centrifugation prepares fresh PMBC (PBMC), cultivates with RPMI 1640 training liquid (containing 10% heat-inactivated fetal bovine serum, 100 μ g/ml Streptomycin sulphates, 100U/ml penicillium mould).After 3 days, 37 ℃ with 1000 TCID through the phytohemagglutinin of 1 μ g/ml and 10ng/ml IL-2 activation ₅₀HIV compare 1X10 ^-5The ratio of individual PBMC was with HIV infection of PBMCs 1 hour.PBS washes and removes the HIV that does not infect for twice, and cell is resuspended in the RPMI 1640 training liquid (containing 10ng/ml IL-2), with 1 * 10 ^-5Individual/hole adds and contains in 96 orifice plates of different concns suppressor factor, and additional TV is 200 μ l.After 48 hours, draw 20 μ] training liquid, add 20 μ l suppressor factor, make training liquid keep the suppressor factor of same concentrations.HIV infected after 5 days, with the antigenic amount of ELISA inspection HIV p24, only added training liquid and did a contrast for.The infection rate of HIV is the ratio of sample well and control wells HIV p24 antigen burst size, and the concentration that M350% suppresses the HIV infection of PBMCs is 20 μ M.

Fig. 4 has shown the external restraining effect to HIV of M3, and X-coordinate is peptide concentration (μ g/ml), and ordinate zou is inhibiting rate (%), and the rising along with peptide concentration is described, inhibiting rate also raises.

Those of ordinary skills should be understood that the technological line that is equal to the present invention also can be used for the structure and the exploitation of similar proteinase inhibitor.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (4)

1. a Furin enzyme inhibitors fragment is characterized in that, the segmental aminoacid sequence of said Furin enzyme inhibitors is:

Wherein, P ₁The site is R; P ₂The site is K; P ₄The site is R; P ₆The site is R; P ₇The site is A; The pairing situation of C is C4-19, C9-17.

2. the suppressor factor of an anti-HIV-1 is characterized in that, contains the described Furin enzyme inhibitors of claim 1 fragment.

3. the application of the described Furin enzyme inhibitors of claim 1 fragment in the medicine of preparation treatment AIDS.

4. one kind is infected drug composition effective for HIV, it is characterized in that, contains claim 1 described Furin enzyme inhibitors fragment and pharmaceutically acceptable carrier or vehicle.

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