WO2015003581A1 - Polypeptide bivalent inhibant une infection au vih - Google Patents

Polypeptide bivalent inhibant une infection au vih Download PDF

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Publication number
WO2015003581A1
WO2015003581A1 PCT/CN2014/081616 CN2014081616W WO2015003581A1 WO 2015003581 A1 WO2015003581 A1 WO 2015003581A1 CN 2014081616 W CN2014081616 W CN 2014081616W WO 2015003581 A1 WO2015003581 A1 WO 2015003581A1
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WO
WIPO (PCT)
Prior art keywords
seq
formula
polypeptide
aca
pharmaceutically acceptable
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Application number
PCT/CN2014/081616
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English (en)
Chinese (zh)
Inventor
刘克良
凌彦博
姜喜凤
薛慧芳
王昆
蔡利锋
Original Assignee
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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Application filed by 中国人民解放军军事医学科学院毒物药物研究所 filed Critical 中国人民解放军军事医学科学院毒物药物研究所
Publication of WO2015003581A1 publication Critical patent/WO2015003581A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention belongs to the field of biomedicine and relates to a polypeptide against HIV infection. Specifically, the present invention relates to a bivalent polypeptide represented by Formula I or Formula II, a derivative thereof, a stereoisomer thereof, or no physiological toxicity thereof. Salt.
  • the invention further relates to a pharmaceutical composition comprising a bivalent polypeptide of formula I or formula II, a derivative thereof, a stereoisomer thereof, or a salt thereof which is not physiologically toxic, and a bivalent polypeptide of formula I or formula Use of a derivative thereof, a stereoisomer thereof, or a physiologically toxic salt thereof for the preparation of a medicament for treating or preventing a disease associated with HIV infection, particularly Acquired Immune Deficiency Syndrome (AIDS) .
  • AIDS Acquired Immune Deficiency Syndrome
  • HIV Human Immunodeficiency Virus
  • HIV-1 HIV-1
  • HIV-2 HIV-1 is one of the more popular ones.
  • Env The infection of human host cells by HIV-1 virus requires a process of fusion of the viral membrane with the target cell membrane. Such a process needs to be enveloped by a viral glycoprotein (Env).
  • Env consists of two parts, a surface subunit gpl20 that binds to the host cell, and a transmembrane subunit gp41 that binds to the host to mediate membrane fusion.
  • Gp41 is inserted into a host cell through its amino-terminal fusion peptide to promote the fusion process of the viral membrane with the target cell membrane.
  • the completion of the fusion state requires the viral gp41 N-terminal repeat (N-HR) and C-terminal repeat (C-HR) folding A six-helix bundle (6-HB) is formed.
  • Inhibitors of the glycoprotein gp41 acting on the HIV-1 virus surface are also known as fusion inhibitors, of which T-20 is currently the only polypeptide-based HIV fusion inhibitor for clinical use.
  • the peptide or small molecule fusion inhibitor of this fusion link competitively binds to the gp41 target exposed in the process to inhibit the formation of the virus itself 6-HB, thereby inhibiting the virus from invading the human immune cells.
  • the trivalent HIV inhibitor designed by Hirokazu et al., with a C34-based sequence, has a cell fusion IC 5Q value of 1.3 nm, which is about 34-fold more active than the starting sequence (W. Nomura, C Hashimoto, A. Ohya, K. Miyauchi, E. Urano, T. Tanaka, T. Narumi, T. Nakahara, J. a. Komano, N. Yamamoto, H. Tamamura, ChemMedChem 2012, 7, 205-208. ). Summary of the invention
  • the object of the present invention is to find a bivalent anti-HIV substance having a relatively high biological activity starting from a shorter sequence.
  • the bivalent polypeptide of the formula I or formula, or a derivative thereof, a stereoisomer thereof or a pharmaceutically acceptable salt thereof has good activity for inhibiting target cells infected with HIV, and thus can be used as a medicament for treating or preventing HIV. Infection, thus completing the present invention.
  • a first aspect of the invention relates to a bivalent polypeptide, derivative, stereoisomer or pharmaceutically acceptable salt thereof, of formula I or formula II: among them
  • C is L-form or D-type cysteine
  • Z is a polypeptide sequence of L-form or D-form
  • a disulfide bond is used between the two monovalent polypeptide chains.
  • the composition of the non-natural amino acid is selected from the group consisting of ⁇ -alanine (BAla), 6-aminocaproic acid (Aca), and triethylene glycol (ethylene group having one end amino group and one end carboxyl group).
  • BAla ⁇ -alanine
  • Aca 6-aminocaproic acid
  • triethylene glycol ethylene group having one end amino group and one end carboxyl group.
  • Teg NH 2 -CH 2 CH 2 -0-CH 2 CH 2 -0-CH 2 CH 2 -0-CH 2 CH 2 -0-CH 2 CH 2 -COOH.
  • Z is selected from the group consisting of:
  • the bivalent polypeptide of Formula I or Formula II is selected from the group consisting of:
  • a disulfide bond is used between the two monovalent polypeptide chains.
  • the natural amino acid includes alanine (Ala), cineine (Val), leuco acid (Leu), isoleucine (ne), proline (Pro), phenylalanine. (Phe), tryptophan (Trp), melitic acid (Met), saccharate (Gly), serine (ser), threonine (Thr), half; acid (Cys), tyrosine (Tyr ), Asn, Gin, Lys, Arginine (Arg), Histidine (His), Aspartic Acid (Asp), Acid (Glu).
  • the GGSTeg means glycine glycine threonine plus Teg.
  • the above compound number [(1) ⁇ (22)] represents the compound (bivalent polypeptide).
  • Another aspect of the invention relates to a pharmaceutical composition comprising at least one bivalent polypeptide of the formula I or formula ⁇ of any one of the first aspects of the invention, a derivative thereof, a stereoisomer, or a pharmaceutically acceptable An acceptable salt, and, optionally, a pharmaceutically acceptable carrier or excipient.
  • a further aspect of the invention relates to a bivalent polypeptide, derivative, stereoisomer, or pharmaceutically acceptable salt thereof of Formula I or Formula ⁇ of any one of the first aspects of the invention for the preparation of an HIV fusion inhibitor Use in.
  • a further aspect of the invention relates to an HIV fusion inhibitor comprising at least one A divalent polypeptide, derivative, stereoisomer, or pharmaceutically acceptable salt thereof of Formula I or Formula II according to any one of the first aspects of the invention.
  • a further aspect of the invention relates to a bivalent polypeptide, derivative, stereoisomer, or pharmaceutically acceptable salt thereof of Formula I or Formula ⁇ of any one of the first aspects of the invention, for use in the preparation of a therapeutic or Use in the prevention of HIV-related diseases, especially AIDS drugs.
  • Still another aspect of the present invention relates to a method of treating or preventing a disease associated with HIV infection, particularly AIDS, the method comprising administering to a subject receiving treatment or prevention an effective amount of Formula I or Formula 1 of any one of the first aspects of the present invention.
  • the HIV means HIV-1 or HIV-2, preferably HIV-1.
  • the monovalent polypeptide and the monomer of the bivalent polypeptide have the same meaning, meaning
  • the disulfide bond means -S - S -.
  • the (C-LrZ refers to 0 -L factory Z , where ⁇
  • the present inventors synthesized a bivalent polypeptide by a certain method, and a disulfide bond was used between two polypeptide chains of a bivalent molecule to synergistically act on two polypeptide sequences to enhance the target. Inhibition of activity, design of highly active HIV fusion inhibitors, explored new ideas for inhibiting HIV infection. detailed description
  • Env envelope glycoprotein
  • HIV Human Immunodeficiency Virus 1 human immunodeficiency virus
  • HIV-1 human immunodeficiency virus type I HIV-1 human immunodeficiency virus type I
  • TFA trifluoroacetic acid
  • Threonine Trp Tryptophan
  • the solid phase synthetic carrier used in the examples Rink-amide resin is the product of Tianjin Nankai Synthetic Co., Ltd.; HBTU, HOBT, DIEA and Fmoc protected natural acid based acid are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Chengnuo New Technology Co., Ltd. Trifluoroacetic acid (TFA), DMF, DCM are products of Beijing Bomaijie Technology Co., Ltd.; Chromatography Pure acetonitrile is Fisher's product. Other reagents are domestically produced pure products if they are not described.
  • Peptide synthesis using standard Fmoc solid phase methods Rink-Amide resin was selected and the peptide chain was extended from the C-terminus to the N-terminus.
  • the condensing agent is HBTU/HOBt/DIEA.
  • the deprotecting agent is a piperidine/DMF solution.
  • the cleavage agent is trifluoroacetic acid (TFA), and the crude peptide is dissolved in water and stored in lyophilization. It was isolated and purified by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC) with a pure peptide content of >95%.
  • Matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-TOF-MS was used to determine the molecular weight of the peptide sequence.
  • Rink Amide resin 0.51g (0.233mmol) of Rink Amide resin was weighed into the CS Biol36 peptide synthesizer reactor, then amino acid, activator, activated base, deprotecting reagent, blocking reagent according to the concentration (protected amino acid: 0.25M DMF solution, Activator: 0.2MHBTU/HOBt in DMF solution, activated base: 0.4MDIEA in DMF solution, deprotecting agent: 20% v/v, piperidine in DMF solution, blocking reagent: 20% v/v, acetic acid Sf in DMF solution After the configuration, synthesis was carried out using a CS Biol36 polypeptide synthesizer.
  • peptide resin was washed with DCM for 3 times, then shrunk with anhydrous methanol, and dried under vacuum at room temperature to obtain a peptide resin of 2.11.
  • the dried polypeptide was placed in a 500 ml eggplant-shaped flask, and a lysing agent (v/v) was added in a ratio of 10 ml per gram of the resin.
  • the compounds (2) - (22) were synthesized according to the above method, and T20 and C34 were synthesized as described above.
  • a 20 ⁇ !/well cell lysate was plated onto a 96-well phosphor plate.
  • the thawed luciferaseassay buffer (Promega, Fitchburg, WI, USA) was added to the luciferaseassay substrate (Promega, Fitchburg, WI, USA) and added 40 ⁇ !/well in a 96-well phosphor plate. The luminescence was detected immediately on the microplate reader. Calculate IC 5 . value.
  • the compounds (1)-(22) are corresponding to the compound numbers in the examples and the inventions of the present invention, and T20 and C34 are control drugs.
  • the structure of C34 is
  • the structure of WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL, T20 (Fuzeon or enfuvirtide) is YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF.
  • the experimental results show that most of the compounds of the present invention can be compared with their monomers. More effectively inhibit HIV-1 mediated cell fusion,

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Communicable Diseases (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • AIDS & HIV (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne un polypeptide divalent de formule I ou de formule II permettant de résister à une infection au VIH, et un dérivé, un stéréoisomère ou un sel pharmaceutiquement acceptable de celui-ci. La présente invention concerne également une composition pharmaceutique comprenant le polypeptide, ou un dérivé, un stéréoisomère ou un sel pharmaceutiquement acceptable de celui-ci, et leur utilisation dans la préparation d'un médicament destiné au traitement ou à la prévention de maladies résultant d'une infection au VIH. Formule I : (C-L1-Z)2 　Formule II : (Z-L1-C)2
PCT/CN2014/081616 2013-07-08 2014-07-04 Polypeptide bivalent inhibant une infection au vih WO2015003581A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310283591.3 2013-07-08
CN201310283591.3A CN104277113B (zh) 2013-07-08 2013-07-08 抑制hiv感染的二价多肽

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WO2015003581A1 true WO2015003581A1 (fr) 2015-01-15

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN110551179B (zh) * 2018-05-31 2022-03-15 中国科学院微生物研究所 一种经修饰的抗hiv多肽及其制备方法和用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101993485A (zh) * 2009-08-20 2011-03-30 重庆富进生物医药有限公司 促胰岛素分泌肽类似物同源二聚体及其用途
EP2377880A2 (fr) * 2008-08-13 2011-10-19 New York Blood Center Thérapie de combinaison d'inhibitors de fusion/entrée du VIH ciblant gp41

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2377880A2 (fr) * 2008-08-13 2011-10-19 New York Blood Center Thérapie de combinaison d'inhibitors de fusion/entrée du VIH ciblant gp41
CN101993485A (zh) * 2009-08-20 2011-03-30 重庆富进生物医药有限公司 促胰岛素分泌肽类似物同源二聚体及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIAO, JUNPENG ET AL.: "Synthesis of N-Terminally Linked Protein and Peptide Dimers by Native Chemical Ligation", BIOCONJUGATE CHEMISTRY, vol. 21, 25 October 2010 (2010-10-25) *

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CN104277113A (zh) 2015-01-14
CN104277113B (zh) 2017-09-26

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