WO2013075594A1 - Polypeptide anti-infection par le vih conçu artificiellement, composition et utilisation associées - Google Patents

Polypeptide anti-infection par le vih conçu artificiellement, composition et utilisation associées Download PDF

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Publication number
WO2013075594A1
WO2013075594A1 PCT/CN2012/084433 CN2012084433W WO2013075594A1 WO 2013075594 A1 WO2013075594 A1 WO 2013075594A1 CN 2012084433 W CN2012084433 W CN 2012084433W WO 2013075594 A1 WO2013075594 A1 WO 2013075594A1
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seq
polypeptide
formula
stereoisomer
seelikk
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PCT/CN2012/084433
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English (en)
Chinese (zh)
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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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中国人民解放军军事医学科学院毒物药物研究所
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Publication of WO2013075594A1 publication Critical patent/WO2013075594A1/fr

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    • A—HUMAN NECESSITIES
    • 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
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the invention belongs to the field of biomedicine and relates to an artificially designed, non-natural sequence anti-HIV infection polypeptide, in particular to a polypeptide represented by formula I, a derivative thereof, a stereoisomer thereof, or a physiologically inactive thereof salt.
  • the present invention also relates to a pharmaceutical composition comprising the above-described polypeptide of the formula I, a derivative thereof, a stereoisomer thereof, or a salt thereof which is not physiologically toxic, and a polypeptide of the formula I, a derivative thereof, a stereoisomer thereof, or The use of non-physiological salts in the treatment or prevention of diseases associated with HIV infection, especially acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • HIV-1 Human immunodeficiency virus type 1
  • HIV fus ion inhibitors are a new class of anti-HIV drugs that interfere with the entry of viruses into target cells. Unlike traditional enzyme inhibitors, they act outside the cell and cut off the spread of the virus at the initial stage of infection. Prevention and control of HIV-1 infection has special significance, and thus has become a hot spot for new mechanisms of anti-HIV drug research.
  • HIV fusion inhibitors target the HIV-1 fusion protein gp41, a functional protein that mediates fusion of HIV-1 to target cell membranes. HIV fusion inhibitors inhibit the formation of core hexamer structures (6-HB) by membrane fusion by inhibiting gp41, thereby inhibiting virus entry into target cells.
  • T-20 is the first and currently marketed HIV-1 fusion inhibitor, a native sequence 36 peptide (referred to as C-peptide) derived from the HIV-1 gp41 CHR domain, which was approved by the FDA in 2003.
  • T20 binds to gp41, which competitively inhibits the formation of functional core structures mediated by gp41 and blocks the fusion of HIV-1 with target cell membranes. Inhibition of viral infection of target cells.
  • T20 Although the discovery of T-20 opened up a new field of using peptide drugs to control HIV, its limitations and limitations have limited the widespread use of T20.
  • the first is drug resistance. Since T20 is completely derived from the gp41 native sequence, it is highly sensitive to target mutations. A single amino acid residue mutation in the target sequence results in a 10-fold decrease in T20 activity, and two residue mutations reduce its activity. 100 times. Therefore, although the native sequence of T20 retains the structure of the natural ligand to the utmost extent, it is also susceptible to T20 resistance due to the high mutation of the target HIV-1 gp41 sequence. Secondly, T20 is easily degraded by proteases and has low bioavailability in vivo. Therefore, how to overcome drug resistance and improve the stability of enzymatic hydrolysis is the main direction of research on a new generation of HIV fusion inhibitors.
  • HIV-1 peptide fusion inhibitors are based primarily on the optimization and modification of active natural ligand sequences.
  • the first generation fusion inhibitors T-20, C34, etc. are the natural sequences directly derived from gp41 CHR; the second generation T1249, Sifuvirtide, T1144, etc. are based on the natural sequence, and the non-conservative amino acid residues The base is obtained by deleting and replacing. Due to the high sensitivity of natural origin sequences to drug resistance mutations, new design ideas need to be explored.
  • the present inventors have proposed an anti-HIV active peptide design idea based on a non-naturally occurring sequence of a target structure: based on the three-dimensional crystal structure of the target gp41 NHR spiral trimer, a computer-aided rational design method is used to design a non-natural sequence oc spiral from the beginning.
  • the peptide which mimics the active conformation of the native C-peptide, binds to the target to produce the corresponding biological function, thereby designing a novel structure of the anti-HIV active polypeptide.
  • the use of non-native sequences and natural sequences is completely non-homologous, exploring new ideas for inhibiting drug resistance. The present invention has thus been completed. Summary of the invention
  • the object of the present invention is to artificially design a new completely non-native sequence of an anti-HIV-infected polypeptide according to the target structure, to achieve an HIV infection-inhibiting activity comparable to or higher than the native sequence, and to inhibit the T20-resistant HIV strain.
  • One aspect of the invention relates to a polypeptide of formula I, a derivative thereof, a stereoisomer thereof, or a salt thereof which is not physiologically toxic, XalAAXdlXelBB- X a 2 AXd2Xe2BB ⁇ XasAAXdsXesBB- X a 4 AXd4Xe4BB ⁇ XasAAXdsXesBB
  • X al, X a2, X a3 , X a4, X a5 each independently a D or L natural or non-natural hydrophobic or hydrophilic amino acid;
  • A is a D or L natural or non-natural acidic amino acid
  • X dl , X d2 , X d3 , X d4 , X d5 are each independently a D- or L-type natural or non-natural hydrophobic amino acid;
  • Xei. X e2 , X , X e X e5 are each independently a natural or non-natural hydrophobic acid of type D or L;
  • X is a natural or non-natural basic amino acid of the D or L form; as used herein, the term “stereoisomer” refers to the D- or L-stereo configuration of a polypeptide of Formula I.
  • X isoleucine (I ), cineine (V), norleucine (N le ), leucine (L), alanine (A), Serine (S), threonine (T), asparagine (N), and glutamine (Q);
  • A is selected from D-type or L-form glutamic acid (E) or aspartic acid (D);
  • X dl , X d2 , X d3 , X d4 , X d5 are each independently selected from the following amino acids of type D or L: glutamine (Q), tryptophan (W), naphthyl alanine (N al ), 3-quinoline alanine (Q al ), isoleucine (I ), cineine (V), norleucine, leucine (L), alanine (A), asparagine ( N), serine (S), threonine (T), and tyrosine (Y);
  • Xei. X e2 , X , X , X e5 are each independently selected from the following amino acids of type D or L: isoleucine (I ), cineine (V), norleucine, leucine (L) , alanine (A), serine (S), glutamine (Q), asparagine (N), threonine (T), and tyrosine (Y);
  • polypeptide of Formula I is selected from lysine (K) or arginine (R) of type D or type L.
  • polypeptide of Formula I a derivative thereof, a stereoisomer thereof, or a salt thereof that is not physiologically toxic, wherein the polypeptide of Formula I is selected from the group consisting of:
  • N al EEN al N IEELIKK SEELIKK IEEQIKK QEESIKK (SEQ ID NO: 10)
  • NEEQIKK Q al EEQ al D IEELIKK SEELIKK IEEQIKK (SEQ ID NO: 68); another aspect of the invention relates to a pharmaceutical composition comprising at least one of the above-described polypeptides of formula I, derivatives thereof, A construct, or a salt thereof that is not physiologically toxic, and a pharmaceutically acceptable carrier or adjuvant.
  • a further aspect of the invention relates to an HIV fusion inhibitor comprising at least one of the above-described polypeptides of formula I, derivatives thereof, stereoisomers thereof, or salts thereof which are not physiologically toxic.
  • a further aspect of the invention relates to the use of a polypeptide of the above formula I, a derivative thereof, a stereoisomer thereof, or a salt thereof which is not physiologically toxic, for the preparation of an HIV fusion inhibitor.
  • a further aspect of the invention relates to the use of a polypeptide of the above formula I, a derivative thereof, a stereoisomer thereof, or a salt thereof which is not physiologically toxic for the preparation of a medicament for the treatment or prevention of a disease associated with HIV infection, in particular AIDS.
  • a further aspect of the invention relates to a method of treating or preventing a disease associated with HIV infection, in particular AIDS, comprising administering an effective amount of a polypeptide of formula I, a derivative thereof, or a stereoisomer thereof to a subject to be treated or prevented. Body, or a salt thereof that is not physiologically toxic.
  • the invention also relates to a method of using the polypeptide of the formula I, a derivative thereof, a stereoisomer thereof, or a salt thereof which is not physiologically toxic, comprising an effective amount of the polypeptide of the formula I, a derivative thereof The stereoisomer, or a salt thereof that is not physiologically toxic, is administered to a subject in need thereof.
  • the term "natural” refers to a naturally occurring, unmodified, such as the amino acid sequence of HIV-1 gp41 itself.
  • non-native means artificially designed, different from nature, such as modified, modified sequences of directly derived natural sequences, and the like.
  • hydrophobic amino acid means an amino acid whose side chain is a hydrophobic group, and includes: Ala, Val, Leu, He, Met, Phe, Trp and the like.
  • hydrophilic amino acid means an acid having a hydrophilic group capable of forming a hydrogen bond with water, and includes: Ser, Thr, Cys, Asp, Asn, Glu, Gin, Arg , Lys, His, Tyr, etc.
  • the term "acidic amino acid” means an amino acid having a gas group in a side chain, and includes: Glu, Asp and the like.
  • basic acid means an acid having an amino group or a mercapto group in a side chain, and includes: Lys, Arg and the like.
  • Env envelope glycoprotein
  • HOBt 1-Hydroxylbenzotriazole anhydrous
  • NHR N-terminal heptad repeat
  • HIV Human Immunodeficiency Virus
  • HIV-1 Human Immunodeficiency Virus Type I
  • Trp Tryptophan
  • Rink Amide resin used in the DIEA N,N-diisopropylethylamine example is the product of Tianjin Nankai Synthetic Co., Ltd.; the natural amino acid protected by HBTU, H0BT, DIEA and Fmoc is Shanghai Jill Biochemistry. The company and Chengdu Chengnuo New Technology Co., Ltd.
  • Fmoc-protected naphthylalanine and quinoline alanine are laboratory synthetic products; N-methylpyrrolidone (leg P) is ACR0S company product; trifluoroacetic acid ( TFA) is a product of Beijing Bomaijie Technology Co., Ltd.; DMF and DCM are products of South Korea's Samsung; chromatographic pure acetonitrile is Fisher's product. Other reagents are domestically produced pure products if they are not described.
  • a standard Fmoc solid phase peptide synthesis method was employed. All peptide sequences were amidated at the C-terminus and acetylated at the N-terminus.
  • Rink Amide resin is used, the amino acid is in the L configuration, and the peptide chain is extended from the C terminal to the N terminal.
  • the activator is a DMF solution containing HBTU and HOBt, and the activated base is a leg P solution of DIEA.
  • the deprotecting agent is a DMF solution of piperidine.
  • the cleavage agent is trifluoroacetic acid (TFA), and the crude peptide is dissolved in water and stored by lyophilization.
  • the peptide was synthesized using a CEM fully automated microwave peptide synthesizer.
  • the synthesis conditions were as follows: J ⁇ acid: 0. 2M DMF solution,
  • Activator 0. 45M HBTU/HOBt DMF solution (where the molar ratio of HBTU to HOBt is 1: 1),
  • Blocking reagent 20% v/v DMF solution of acetic anhydride.
  • Rected Rink Amide resin 0. 5g (0. 25mmol) was placed in the CEM microwave peptide synthesizer reactor, and then the amino acid, activator, activated base, deprotection reagent, blocking reagent were arranged at the above concentration, and then CEM microwave was used. Automated peptide synthesizer for synthesis. After completion, the peptide resin was washed with DMF for 3 times, then shrunk with anhydrous methanol, and dried under vacuum at room temperature to obtain a peptide resin of 2.05 g.
  • Pyrolysis of Peptide Resin Weigh the peptide resin synthesized by the above microwave synthesizer 2. 05g, put it into a 250ml eggplant-shaped bottle, water bath, and electromagnetic stirring. The lysate was prepared by adding 1 gram of the peptide resin to 10 ml. TFA needs to be cooled in a water bath for 30 minutes or pre-stored in a water tank; the prepared lysate is added to the peptide resin under water bath conditions, electromagnetic stirring, the resin turns orange-red, and the reaction is carried out in a water bath for 30 minutes, then, the water bath is removed, room temperature The reaction was further stirred for 90 min and the reaction was completed.
  • the resulting crude peptide was purified by medium or high pressure preparative color transfer.
  • the color column is a C8 column, and the eluent is acetonitrile, water and a small amount of trifluoroacetic acid.
  • the color column was previously equilibrated with 200 ml of an aqueous solution containing 15% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid solution.
  • the mixture was washed with 200 ml of an aqueous solution containing 15% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid in 0-15 minutes, and the fraction of the eluate was detected by high performance liquid chromatography. 1% ⁇ / ⁇ The acetonitrile content of the eluate was increased to 65% (v / v) in 10-30 minutes (the content of trifluoroacetic acid was unchanged, maintaining 0. 1% ⁇ / ⁇ ) until the main peak of the purified polypeptide is eluted. The eluates were combined, and the organic solvent was removed by rotary evaporation, and the peptide was freeze-dried to obtain a pure peptide content of more than 95% by HPLC.
  • Example 69 Inhibition of HIV-1 biological activity assay
  • HIV-1 chronically infected with H9 cells.
  • H9 cells the HIV-1 virus gene is integrated into the DNA strand of H9 cells and expressed together with H9 cells:
  • the viral membrane protein GP160 is expressed in H9 cells. surface.
  • H9 cells become a huge HIV-1 virus.
  • the whole process can accurately reflect the fusion process of HIV-1 virus particles with target cells.
  • the test compound polypeptide inhibits fusion of HIV-1 infected H9 cells with MT-2 target cells, thereby judging the anti-fusion activity of the test compound.
  • HIV-1 IIIB- infected H9 cells (H9/HIV-1 IIIB , prepared by the laboratory, see Jiang, S., Radigan, L for specific methods) . and Zhang, L. (2000) Proc. SPIE 3926, 212-219) were labeled with a fluorescent reagent, Calcein_AM (Molecular Probes, Inc., Eugene, OR), and then 50 ⁇ l per well was added to a 96-well plate. Labeled H9/HIV-l mB cells (2 X10 5 cells/ml), and then add 50 ⁇ l of different concentrations of test samples (test samples are examples)
  • test compounds Compounds 1-68 of SEQ ID NOS: 1-68 prepared in Examples 1-68) all showed high inhibition of HIV-1 mediated cell fusion activity.

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  • Health & Medical Sciences (AREA)
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Abstract

La présente invention concerne un polypeptide et le dérivé et stéréo-isomère ou sel sans toxicité physiologique de celui-ci, tels que présentés dans la formule 1. Le polypeptide est un polypeptide anti-infection par le VIH conçu artificiellement. La présente invention concerne également des compositions pharmaceutiques contenant le polypeptide et le dérivé et stéréo-isomère ou sel sans toxicité physiologique de celui-ci, et l'utilisation du polypeptide et du dérivé et stéréo-isomère ou sel sans toxicité physiologique de celui-ci, dans le traitement ou la prévention de maladies associées provoquées par l'infection par le VIH.
PCT/CN2012/084433 2011-11-21 2012-11-12 Polypeptide anti-infection par le vih conçu artificiellement, composition et utilisation associées WO2013075594A1 (fr)

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CN201110368738.XA CN103122024B (zh) 2011-11-21 2011-11-21 人工设计的抗hiv感染多肽、组合物以及用途

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Publication number Priority date Publication date Assignee Title
WO2019223642A1 (fr) * 2018-05-23 2019-11-28 中国人民解放军军事科学院军事医学研究院 Polypeptide antiviral, composition pharmaceutique et utilisation associées

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CN104292303B (zh) * 2013-07-16 2017-03-01 国家纳米科学中心 与gp41蛋白结合的多肽、多肽芯片、其制备方法和应用
CN106317209B (zh) * 2015-07-02 2020-03-13 中国人民解放军军事医学科学院毒物药物研究所 共价交联的n肽抑制剂

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WO2011110049A1 (fr) * 2010-03-12 2011-09-15 中国人民解放军军事医学科学院毒物药物研究所 Polypeptide de fusion anti-vih et son utilisation

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WO2011110049A1 (fr) * 2010-03-12 2011-09-15 中国人民解放军军事医学科学院毒物药物研究所 Polypeptide de fusion anti-vih et son utilisation

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SHI, WEIGUO ET AL.: "Design of highly potent HIV fusion inhibitors based on artificial peptide sequences", CHEMCOMM., vol. 48, 12 October 2012 (2012-10-12), pages 11579 - 11581, XP055069835 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019223642A1 (fr) * 2018-05-23 2019-11-28 中国人民解放军军事科学院军事医学研究院 Polypeptide antiviral, composition pharmaceutique et utilisation associées

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