CN103122024B - AntiHIV1 RT activity infection polypeptide, composition and the purposes of engineer - Google Patents

AntiHIV1 RT activity infection polypeptide, composition and the purposes of engineer Download PDF

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CN103122024B
CN103122024B CN201110368738.XA CN201110368738A CN103122024B CN 103122024 B CN103122024 B CN 103122024B CN 201110368738 A CN201110368738 A CN 201110368738A CN 103122024 B CN103122024 B CN 103122024B
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ieelikk
polypeptide
seelikk
ieeqikk
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CN103122024A (en
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刘克良
史卫国
王潮
蔡利锋
贾启燕
王昆
郑保华
张沙
白玉
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Institute of Pharmacology and Toxicology of AMMS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/18—Antivirals for RNA viruses for HIV

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Abstract

The invention belongs to biomedicine field, is related to the polypeptide that the AntiHIV1 RT activity of engineer a kind of infects, in particular it relates to Formula IXa1AAXd1Xe1BB‑Xa2AAXd2Xe2BB‑Xa3AAXd3Xe3BB‑Xa4AAXd4Xe4BB‑Xa5AAXd5Xe5The salt of polypeptide, its derivative, its stereoisomer or its physiological-toxicity-free shown in BB Formulas I.The invention further relates to the pharmaceutical composition of the salt containing above-mentioned Formulas I polypeptide, its derivative, its stereoisomer or its physiological-toxicity-free, and Formulas I polypeptide, its derivative, the salt of its stereoisomer or its physiological-toxicity-free are treating or preventing the purposes of the especially acquired immunodeficiency syndrome of relevant disease caused by HIV (AIDS).

Description

AntiHIV1 RT activity infection polypeptide, composition and the purposes of engineer
Technical field
The invention belongs to biomedicine field, a kind of AntiHIV1 RT activities that be related to engineer, Non-native sequences infect polypeptide, In particular it relates to the salt of the polypeptide, its derivative, its stereoisomer or its physiological-toxicity-free shown in Formulas I.The present invention also relates to And the pharmaceutical composition of the salt containing above-mentioned Formulas I polypeptide, its derivative, its stereoisomer or its physiological-toxicity-free, and Formulas I polypeptide, its derivative, the salt of its stereoisomer or its physiological-toxicity-free related disease caused by HIV is treated or prevented The purposes of disease especially acquired immunodeficiency syndrome (AIDS, i.e. AIDS).
Xa1AAXd1Xe1BB-Xa2AAXd2Xe2BB-Xa3AAXd3Xe3BB-Xa4AAXd4Xe4BB-Xa5AAXd5Xe5BB Formulas I.
Background technology
AIDS is worldwide popular at present, carrys out serious threat to mankind's health care belt.Human Mn superoxide dismutase (HIV-1) be cause AIDS infect main pathogens.Due to traditional RTI and protease inhibitors resistance Sex chromosome mosaicism becomes increasingly conspicuous so that developmental function turns into common recognition in HIV-1 life cycle novel targets medicine.HIV fusion inhibitors (HIV fusion inhibitors) is the new class inverase that viral interference enters target cell, with traditional enzyme inhibitor Difference, it act on it is extracellular, infection initial link cut-out virus propagation, this is for preventing and controlling HIV-1 to infect Acquire a special sense, thus as the focus of new mechanism inverase research.
For HIV fusion inhibitors using HIV-1 fusion proteins gp41 as action target, gp41 is mediation HIV-1 and target cell membrane The functional protein of fusion.The dimeric structure of core six (6-HB) that HIV fusion inhibitors promote film to merge by suppressing gp41 Formed, so as to suppress cell entry target cell.T-20 is the HIV-1 fusion inhibitors of first and currently the only listing, it It is the peptide of native sequences 36 (being referred to as C peptides) derived from HIV-1 gp41 CHR functional areas, was ratified in 2003 by FDA.T20 can Combined with gp41, be configured to so as to the functional core hearty cord of Reverse transcriptase gp41 mediated fusions, block HIV-1 and target cell membrane Fusion process, suppress viral target cell infection.
Although T-20 discovery opens the frontier using peptide medicament control HIV, one existing for itself A little defects and deficiency, limit T20 extensive use.It is drug resistance first, because T20 is derived from gp41 native sequences completely, High to the sensitiveness of target mutation, the mutation of target sequence single amino acids residue can cause T20 activity to reduce by 10 times, and two residual Base mutation can then make its activity reduce by 100 times.Therefore, although T20 native sequences maintain natural ligand to greatest extent Structure, but also cause virus easily to produce drug resistance to T20 due to the high mutability of target HIV-1 gp41 sequences.Secondly, T20 is easily easily degraded by proteases, and vivo biodistribution availability is low.Therefore, how to overcome drug resistance and improve enzymolysis stability, be new The Main way of generation HIV fusion inhibitors research.
Current HIV-1 peptides fusion inhibitors, are mainly based upon the optimization and transformation of active natural ligand sequence.Such as the A generation fusion inhibitor T-20, C34 etc. are the native sequences for being directly derived from gp41 CHR；Second generation T1249, sifuvirtide, T1144 etc. is then on the basis of native sequences, obtained from wherein nonconserved amino acid residues are carried out with deletion replacement.Due to The hypersensitivity of natural origin sequence pair resistance mutation is, it is necessary to explore new mentality of designing.
Present inventors have proposed the HIV-resistant activity peptide design philosophy of the non-natural derived sequence based on target construction：It is based on The three-dimensional crystalline structure of target gp41 NHR spiral tripolymers, the Rational design method of appliance computer auxiliary, is from the beginning designed non- The alpha helical peptides of native sequences, simulate the activity conformation of natural C peptides, with target with reference to and produce corresponding biological function, thus Design the HIV-resistant activity polypeptide of brand new.Using Non-native sequences and the complete non-homology feature of native sequences, explore and suppress The new approaches of drug resistance.Present invention is completed therefrom.
The content of the invention
The present invention seeks to according to target construction, the AntiHIV1 RT activity infection polypeptide of the new complete Non-native sequences of artificial design, reach To the suppression HIV activity equal or higher with native sequences, while T20 drug resistance HIV strains can be suppressed.
One aspect of the present invention be related to polypeptide shown in Formulas I, its derivative, its stereoisomer or its without physiology poison The salt of property,
Xa1AAXd1Xe1BB-Xa2AAXd2Xe2BB-Xa3AAXd3Xe3BB-Xa4AAXd4Xe4BB-Xa5AAXd5Xe5BB Formulas I.
Wherein,
Xa1、Xa2、Xa3、Xa4、Xa5It is separately D types or the natural or non-natural hydrophobicity or hydrophilic ammonia of L-type Base acid；
A is the natural or non-natural acidic amino acid of D types or L-type；
Xd1、Xd2、Xd3、Xd4、Xd5It is separately D types or the natural or non-natural hydrophobic amino acid of L-type；
Xe1、Xe2、Xe3、Xe4、Xe5It is separately D types or the natural or unnatural hydrophobic acidic amino acid of L-type；
B is the natural or non-natural basic amino acid of D types or L-type；
Term used herein " stereoisomer, refer to D- the or L- spatial configurations of Formulas I polypeptide.
In one embodiment of the invention, Xa1、Xa2、Xa3、Xa4、Xa5It is separately as follows selected from D types or L-type Amino acid：Tryptophan (W), naphthylalanine (Na1), 3- quinoline alanines (Qa1), isoleucine (I), valine (V), just bright ammonia Sour (Nle), leucine (L), alanine (A), serine (S), threonine (T), asparagine (N) and glutamine (Q)；
A is selected from the glutamic acid (E) or aspartic acid (D) of D types or L-type；
Xd1、Xd2、Xd3、Xd4、Xd5The separately following amino acid selected from D types or L-type：Glutamine (Q), tryptophan (W), naphthylalanine (Na1), 3- quinoline alanines (Qa1), isoleucine (I), valine (V), nor-leucine (N1e), leucine (L), alanine (A), asparagine (N), serine (S), threonine (T) and tyrosine (Y)；
Xe1、Xe2、Xe3、Xe4、Xe5The separately following amino acid selected from D types or L-type：Isoleucine (I), valine (V), nor-leucine (N1e), leucine (L), alanine (A) serine (S), glutamine (Q), asparagine (N), threonine And tyrosine (Y) (T)；
B is selected from the lysine (K) or arginine (R) of D types or L-type.
In one embodiment of the invention, described Formulas I polypeptide, its derivative, its stereoisomer or its without life The salt of toxicity is managed, wherein, the Formulas I polypeptide is selected from following compound：
1.WEEWDKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：1)；
2.WEEWRKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：2)；
3.WEEWSKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：3)；
4.WEEWIKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：4)；
5.WEEWAKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：5)；
6.Na1EENa1DKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：6)；
7.Na1EENa1RKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：7)；
8.Na1EENa1SKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：8)；
9.Na1EENa1QKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：9)；
10.Na1EENa1NKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：10)；
11.Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：11)；
12.Qa1EEQa1RKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：12)；
13.Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：13)；
14.Qa1EEQa1QKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：14)；
15.Qa1EEQa1NKK IEELIKK SEELIKK IEEQIKK QEESIKK(SEQ ID NO：15)；
16.SEEQIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：16)；
17.SEENIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：17)；
18.SEESIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：18)；
19.QEESIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：19)；
20.QEENIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：20)；
21.NEESIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：21)；
22.NEEQIKK WEEWSKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：22)；
23.SEEQIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：23)；
24.SEENIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：24)；
25.SEESIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：25)；
26.QEESIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：26)；
27.QEENIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：27)；
28.NEESIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：28)；
29.NEEQIKK WEEWDKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：29)；
30.QEESIKK WEEWSKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：30)；
31.QEENIKK WEEWSKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：31)；
32.NEESIKK WEEWSKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：32)；
33.NEEQIKK WEEWSKK IEELIKK QEELIKKIEESIKK(SEQ ID NO：33)；
34.SEEQIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：34)；
35.SEENIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：35)；
36.SEESIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：36)；
37.QEESIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：37)；
38.QEENIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：38)；
39.NEESIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：39)；
40.NEEQIKK WEEWDKK IEELIKK QEELIKK IEESIKK(SEQ ID NO：40)；
41.SEEQIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：41)；
42.SEENIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：42)；
43.SEESIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：43)；
44.QEESIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：44)；
45.QEENIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：45)；
46.NEESIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：46)；
47.NEEQIKK Na1EENa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：47)；
48.SEEQIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：48)；
49.SEENIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：49)；
50.SEESIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：50)；
51.QEESIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：51)；
52.QEENIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：52)；
53.NEESIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：53)；
54.NEEQIKK Na1EENa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：54)；
55.SEEQIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：55)；
56.SEENIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：56)；
57.SEESIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：57)；
58.QEESIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：58)；
59.QEENIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：59)；
60.NEESIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：60)；
61.NEEQIKK Qa1EEQa1SKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：61)；
62.SEEQIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：62)；
63.SEENIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：63)；
64.SEESIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：64)；
65.QEESIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：65)；
66.QEENIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：66)；
67.NEESIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：67)；
68.NEEQIKK Qa1EEQa1DKK IEELIKK SEELIKK IEEQIKK(SEQ ID NO：68)；
Another aspect of the present invention is related to a kind of pharmaceutical composition, and it contains at least one above-mentioned Formulas I polypeptide, it spreads out The salt of biology, its stereoisomer or its physiological-toxicity-free, and pharmaceutically acceptable carrier or auxiliary material.
The present invention still another aspect be related to a kind of HIV fusion inhibitors, its contain at least one above-mentioned Formulas I polypeptide, The salt of its derivative, its stereoisomer or its physiological-toxicity-free.
The present invention still another aspect be related to above-mentioned Formulas I polypeptide, its derivative, its stereoisomer or its without physiology Purposes of the salt of toxicity in HIV fusion inhibitors are prepared.
The present invention still another aspect be related to above-mentioned Formulas I polypeptide, its derivative, its stereoisomer or its without physiology The salt of toxicity is preparing the purposes in being used to treat or prevent the medicine of HIV relevant disease especially AIDS.
The still another aspect of the present invention is related to a kind of side for treating or preventing HIV relevant disease especially AIDS Method, described method include the Formulas I polypeptide, its derivative, its solid to giving the object effective dose for receiving treatment or prevention The salt of isomers or its physiological-toxicity-free.
In the present invention, term " natural " refers to naturally occurring, without artificial modification, as HIV-1 gp41 in itself Amino acid sequence etc..
In the present invention, term " non-natural " refers to what is artificially designed, from natural different, such as to directly derivative natural Sequence is engineered, sequence after modification etc..
In the present invention, term " hydrophobic amino acid " refers to the amino acid that side chain is hydrophobic group, including：Ala, Val, Leu, Ile, Met, Phe, Trp etc..
In the present invention, term " hydrophilic amino acid " refers to that side chain contains can act on the hydrophilic radical to form hydrogen bond with water Amino acid, including：Ser, Thr, Cys, Asp, Asn, Glu, Gln, Arg, Lys, His, Tyr etc..
In the present invention, term " acidic amino acid " refers to the carboxylic amino acid of side chain, including：Glu, Asp etc..
In the present invention, term " basic amino acid " refers to amino acid of the side chain containing amino or guanidine radicals, including：Lys, Arg Deng.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is The conventional products of acquisition purchased in market can be passed through.
The abbreviation used in the present invention has following implication：
AIDS (Acquired Immure Deficiency Syndrome) AIDS, acquired immunodeficiency syndrome
Ala (Alanine, A) alanine
Arg (Arginine, R) arginine
Asn (Asparagine, N) N
Asp (Aspartic acid, D) aspartic acid
DCM (Dichloromethane) dichloromethane
DMF (N, N-Dimethyl malonate) dimethylformamide
Env (Envelope glycoprotein) envelope glycoprotein
ESI-MS (Electronic spray ion mass spectroscopy) electrospray ionization mass spectrum
Fmoc (Fluorenylmethoxycarbonyl) fluorenylmethyloxycarbonyl
Gly (Glycine, G) glycine
Gln (Glutamine, Q) paddy acyl ammonia
Glu (Glutamic acid, E) glutamic acid
6-HB (six-helix bundle) six conveyor screws
The tetramethylurea hexafluorophosphoric acids of HBTU 2- (1H-1- hydroxybenzotriazoles) -1,1,3,3-
His (Histidine, H) histidine
HoBt (1-Hydroxylbenzotriazole anhydrous) 1- hydroxy benzo triazoles
NHR (N-terminal heptad repeat, NHR) N-terminal repetitive sequence
CHR (C-terminal heptad repeat, CHR) C-terminal repetitive sequence
HIV (Human Immunodeficiency Virus)) human immunodeficiency virus
HIV-1 human Mn superoxide dismutases
HPLC (High performance liquid chromatography) high performance liquid chromatography
Ile (Isoleucine, I) isoleucine
Leu (Leucine, L) leucine
N1eNor-leucine
Lys (Lysine, K) lysine
Phe (Phenylalanine, F) phenylalanine
Ser (Serine, S) serine
TFA (trifluoroacetic acid) trifluoroacetic acid
Thr (Threonie, T) threonine
Trp (Tryptophan, W) tryptophan
Tyr (Tyros ine, Y) tyrosine
Val (Valine, V) valine
Na1Naphthylalanine
Qa13- quinoline alanines
Solid-phase synthesized carrier Rink amide resins used in embodiment are that Tianjin Nankai synthesizes responsibility Co., Ltd product； The natural amino acid of HBTU, HOBT, DIEA and Fmoc protection has for the sincere promise new technology in Shanghai gill biochemical corp and Chengdu Limit responsible company's product；The naphthylalanine and quinoline alanine of Fmoc protections are this laboratory sintetics；N- crassitudes Ketone (NMP) is ACROS Products；Trifluoroacetic acid (TFA) is Beijing Bo Maijie Science and Technology Ltd.s product；DMF, DCM are Korea Spro Samsung of state product；Trifluoroacetic acid aqueous solution is Fisher Products.Other reagents are domestic analysis net product such as without explanation.
Embodiment 1：The preparation of compound 6
Using the Fmoc solid-phase peptide synthesis of standard.All equal C-terminals of peptide sequence are amidatioon, and N-terminal is acetylation.Choosing With Rink Amide resins, amino acid is L-configuration, and peptide chain is extended from C-terminal to N-terminal.Condensing agent is HBTU/HOBt/DIEA.Remove-insurance Shield agent is piperidines/DMF solution.Decomposition agent is trifluoroacetic acid (TFA), and thick peptide is freezed after being dissolved with water and preserved.With middle hydraulic fluid phase color Spectrum Flash or high pressure liquid phantom preparing chromatogram (HPLC) are isolated and purified, pure peptide content ＞ 95%.Matrix Assisted Laser Desorption flies Row time mass spectrum (MALDI-TOF-MS) confirms peptide sequence molecular weight.
Peptide systhesis is carried out using the full-automatic microwave Peptide synthesizers of CEM, synthesis condition is as follows：
Amino acid：0.2M DMF solution,
Activator：0.45M HBTU/HOBt DMF solution,
Activate alkali：2M DIEA nmp solution,
Deprotection agent：The DMF solution of 20% v/v piperidines,
Closed reagent：The DMF solution of 20% v/v acetic anhydrides.
Weigh Rink Amide resins 0.5g (0.25mmol) to insert in CEM microwave Peptide synthesizer reactors, then will Amino acid, activator activate alkali, and deprotecting regent is more with CEM microwave full-automatics after closed reagent is configured by above-mentioned concentration Peptide synthesizer is synthesized.After the completion of peptide resin wash 3 times with DMF after shunk with absolute methanol, room temperature in vacuo dry, obtain peptide tree Fat 2.05g.
Lysate (percent by volume)：Trifluoroacetic acid: dithioglycol: metacresol: water=82.5: 10: 5: 2.5.
The cracking of peptide resin：The synthetic peptide resin 2.05g of microwave synthesizer is weighed, is put into 250ml eggplant-shape bottles, ice Bath, electromagnetic agitation.The amount that 10ml is added by 1 gram of peptide resin prepares lysate.TFA needs advance ice bath cooling 30min or advance Deposit in refrigerator and use；The lysate prepared is added in the peptide resin under condition of ice bath, electromagnetic agitation, resin becomes orange Red, 30min is reacted under condition of ice bath, then, remove ice bath, room temperature is further continued for stirring reaction 90min, and reaction is completed.Acutely stir The lower addition cold diethyl ether 200ml into reactor is mixed, separates out white precipitate, continues to stir 30min；Funnel is filtered with G4 core to filter Go out precipitate, washed 3 times, dried repeatedly with cold diethyl ether.Distilled water 50ml is added, acetonitrile 5ml makes solid fully dissolve, filtered, Filtrate freezes to obtain thick peptide 1.03g.
The thick peptide of gained prepares chromatogram with medium-pressure or high pressure and purified.Chromatographic column is C8 posts, and eluant, eluent is acetonitrile, water and few Measure trifluoroacetic acid.Concrete operation step：Thick peptide 1.00g is weighed, adds water 20ml, acetonitrile 5ml to make solid dissolving, centrifuges 10min (3000 revs/min), take supernatant loading.Chromatographic column is put down with the trifluoroacetic acid solution 200ml of 15% acetonitrile/water/0.1% in advance Weighing apparatus.Continue to be rinsed with the trifluoroacetic acid solution 200ml of 15% acetonitrile/water/0.1% after loading, efficient liquid phase detection eluent composition. Ethane nitrile content is gradually risen according to liquid phase testing result, until purified polypeptide main peak is eluted out.Merge eluent, rotation Turn evaporative removal organic solvent, freeze to obtain pure peptide, HPLC detection levels are more than 95%.
Pure peptide is through its molecular weight of MALDI-TOF-MS or ESI mass spectrum confirmations.
Embodiment 2-68：Compound 1-5,7-68 preparation
Numbering 1- numberings 5, numbering, the compound of 7- numberings 68 are synthesized by embodiment (1) method, and the amino acid is L Configuration, simply corresponding amino acid residue replaced.
Embodiment 69：Suppress the detection of HIV-1 bioactivity
Compound suppresses the cell-cell fusion activity evaluation (IC of HIV-1 mediations50)：
Dye the Cell-Cell Fusion of transfer method detection HIV-1 mediations：HIV-1IIIBH9 cells (the H9/HIV- of infection 1IIIB) marked by a kind of fluorometric reagent Calcein-AM (Molecular Probes, Inc., Eugene, OR), then, 96 The marked H9/HIV-1 of 50 μ l are added in orifice plate per holemBCell (2 × 105/ ml), add the test samples of 50 μ l various concentrations Product (compound prepared by institute testing example 1-34 and 44-53, from 250 twice of gradient dilution of μ g/ml concentration), 37 DEG C of incubations 30min；Then MT-2 cells 100ul (1 × 10 is added per hole6/ ml), 37 DEG C of incubation 2h.Fusion and the Calcein marks not merged (MT-2 is a kind of conventional effector cell to the MT-2 cells of note HIV-1 infection, is used for the target for simulating HIV in this experiment Cell, commercially available) counted with reverse fluorescence microscope (Zeiss, Germany).Calculate IC50Value.
According to the method described above, the table 1 that determination of activity result is seen below.
Table 1：Polypeptide suppresses the cell fusion activity result of HIV mediations
The result of table 1 shows, test-compound compound 1-68 is to show cell fusion that high suppression HIV-1 is mediated Activity.Wherein compound 1-3,7,8,10,16,19-24,26,29,30,39,62,68 suppress the cell fusion that HIV-1 is mediated and lived Property level (IC50) value and T-20 and C34 it is substantially suitable.
Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, those details can be carried out with various modifications and replacement, these change in the guarantor of the present invention Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.

Claims (7)

1. it is selected from SEQ ID NO：The salt of polypeptide or its physiological-toxicity-free shown in 1-68.
2. it is selected from SEQ ID NO：1-3、SEQ ID NO：7-8、SEQ ID NO：10、SEQ ID NO：16、SEQ ID NO：19- 24、SEQ ID NO：26、SEQ ID NO：29-30、SEQ ID NO：39、SEQ ID NO：62 and SEQ ID NO：Shown in 68 Polypeptide or its physiological-toxicity-free salt.
3. a kind of pharmaceutical composition, it contains the salt of the polypeptide or its physiological-toxicity-free described at least one claim 1 or 2, And pharmaceutically acceptable carrier or auxiliary material.
4. a kind of HIV fusion inhibitors, it contains polypeptide described at least one claim 1 or 2 or its physiological-toxicity-free Salt.
5. purposes of the salt of the polypeptide or its physiological-toxicity-free described in claim 1 or 2 in HIV fusion inhibitors are prepared.
6. the salt of the polypeptide or its physiological-toxicity-free described in claim 1 or 2 is being prepared for treating HIV relevant disease Purposes in medicine.
7. the salt of the polypeptide or its physiological-toxicity-free described in claim 1 or 2 is being prepared for treating in the medicine of AIDS Purposes.
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