WO2020032894A1 - Peptide immunomodulateur dérivé d'une séquence précurseur de terminal amino cxcl8 - Google Patents

Peptide immunomodulateur dérivé d'une séquence précurseur de terminal amino cxcl8 Download PDF

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Publication number
WO2020032894A1
WO2020032894A1 PCT/TR2019/050621 TR2019050621W WO2020032894A1 WO 2020032894 A1 WO2020032894 A1 WO 2020032894A1 TR 2019050621 W TR2019050621 W TR 2019050621W WO 2020032894 A1 WO2020032894 A1 WO 2020032894A1
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WIPO (PCT)
Prior art keywords
cxcl8
peptide
immune system
amino acid
acid sequence
Prior art date
Application number
PCT/TR2019/050621
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English (en)
Inventor
Güneş ESENDAĞLI
Original Assignee
Hacettepe Üni̇versi̇tesi̇
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Publication date
Application filed by Hacettepe Üni̇versi̇tesi̇ filed Critical Hacettepe Üni̇versi̇tesi̇
Publication of WO2020032894A1 publication Critical patent/WO2020032894A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is related to a short peptide sequence derived by being originated from the precursor sequence (CXCL8-(1 -30)) located at the amino-terminal of the CXCL8 chemokine molecule and detected to have functions that affect immune system responses.
  • Cytokines are protein and peptide groups which enable communication between cells that have been produced by animal and plant cells. In inflammation and immune reactions, active lymphocytes, endothelium, epithelium and connective tissue cells including macrophages are involved. Acting through specific receptors, cytokines stimulate cell proliferation, differentiation and adjustment of cell responses.
  • Cytokines emit signals to immune system cells such as macrophages when pathogens are present and they enable said signals to be transferred to the infected region.
  • Chemokines are a specific group of cytokines which mediate chemotaxis between cells. Chemokines have four sub families which are CXC, CC, CX3C and XC.
  • CXC chemokines Two N-terminal cysteine residues have been separated with an amino acid in CXC chemokines. In mammals there are 17 different CXC chemokines, and they are divided into two categories being either ELR-positive or ELR-negative in relation to comprising a specific amino sequence (ELR pattern), the glutamic acid-leucine-arginine structure.
  • ELR-positive CXC chemokines induce migration of neutrophils specifically besides some other immune cells and they interact with CXCR1 and CXCR2 chemokine receptors.
  • Other CXC chemokines which do not comprise ELR pattern usually act as chemo-attractants for lymphocytes.
  • Patent numbered CN102816209 describes a peptide (C20) which has been derived from the chemotactic peptide chemerin and which shows similar functions.
  • WO20121 18124 (A1 ) is related to a novel cyclic peptide sequence which antagonizes the CXCL12 chemokine that has been bonded to CXCR4.
  • Patents, JP2010047598, US6875738 (B1 ), US6613742 (B1 ), W00009152 (A1 ) and CA2322764 (A1 ) describe peptide sequences which have the ability to antagonize the CXCL12 chemokine that has been bonded to CXCR4.
  • the US2004152634 (A1 ) document describes peptide sequences which have the capacity to bind to CXCR1 and which have the ability to antagonize IL-8.
  • Patent numbered US2014154249 (A1 ) defines peptides which can directly bind with chemokines and prevent the activity of said molecules and by this means which can change the processes related to inflammation.
  • W02005056581 (A2) describes peptide sequences which bind to CCR5, and which have the ability to antagonize CCL5 chemokines and to block the entrance of the HIV virus which infects the cell via said receptor.
  • WO0222657 is related to peptide sequences which antagonize the activity of chemokines by binding to various chemokine receptors.
  • the small molecules having peptide structures play a role in arranging several physiological processes and providing communication between cells.
  • Peptides having a simple structure and low molecular weight are easily stabilized and they reach the target tissue easily.
  • This invention related to determining and obtaining a small peptide molecule derived from said sequences by examining said precursor (CXCL8-(1 -30)) sequence located at the amino terminal of the known CXCL8 chemokine molecule with a rational and unique algorithm.
  • Pep8 A small peptide molecule has been derived from this sequence, the peptide which can be synthesized and which is suitable for in vitro use has been selected and it has been named Pep8.
  • said small peptide molecule does not have a toxic effect on the peripheral blood mononuclear cells and research has been carried out to understand the activation, increase of the molecule under activation of various stimulants and how said molecule effected cytokine synthesis and it has been determined that said molecule shows immune regulatory effects.
  • Pep8 has been synthesized by being fluorescently marked and research has been carried out to understand with which immune cell types it interacts more with.
  • a small peptide molecule has been derived such that it can be synthesized and is suitable to be used in vitro by carrying out an examination with a rational algorithm on the precursor sequence (CXCL8-(1 -30)) at the amino terminal of CXCL8.
  • the chemical formula belonging to the peptide molecule that has been designed has been calculated using an isoelectric point, solubility in polar medium (hydrophobic aa. %), molecular weight parameters and bioinformatics software.
  • the related parameters have been compared for both the total peptide sequences comprised by the precursor protein regions and the small peptide molecule that has been derived from said regions (Table-1 a, b).
  • hydrophobic regions (usually the first 24-28 aa.) which match the signal sequence providing transfer to organelle and cell membranes following the translation of proteins are excluded and different peptide sequences have been selected to have at least 8aa. length and the net charge in neutral pH has been analyzed in order for the peptides to have good interaction with the cell surface ( Figure-1 , graphic C).
  • the determined Pep8 sequence has been synthesized to be amino terminal acetylated by LifeTein (USA) company and the HPLC (Purity, %91.09), mass spectrophotometer analyses have been carried out to confirm the purity and accuracy of the peptide sequence.
  • the lyophilized form of Pep8 has been dissolved to obtain 5 mM stock concentration with sterile dH 2 0.
  • the peptide stock solutions have been diluted in the cell culture medium such as to have final concentrations of 500 mM, 50 mM, 5 pM, 0.5 pM and 50 nM and working solutions have thereby been obtained.
  • the dissolved peptides have been aliquot and stored at -86° C.
  • PBM peripheral blood mononuclear
  • Pep8 affected the proliferation of PBM cells, the levels of the activation markers and cytokine synthesis.
  • Pep8 has affected the proliferation ability of immune cells. Cytokine production also showed change in the presence of Pep8.
  • Immune reciulatory effects It increases were at low levels in terms of the activation markers on mononuclear leukocytes under the stimulation of Lipopolysaccharide (increase in CD86; no change in the levels of HLA-DR, CD154, CD25 CD69, CD80). It reduces pro-inflammatory IFN-g cytokine synthesis; but does not affect IL-10, IL-4, TGF-b, and TNF-a levels.
  • THP-1 stimulation it does not affect the activation levels of mononuclear leukocytes under the stimulation of CD3/allogenic mononuclear cell THP-1 stimulation. It increases the proliferation of T lymphocytes. It increases pro-inflammatory cytokines TNFa, IFN-g and anti-inflammatory cytokines TGF-b and IL-10 d levels; it does not change IL-4 levels.
  • FITC fluorescence
  • Pep8 has the capacity to affect immune responses with different aspects and with different sensitivity.
  • the molecule obtained can be used in immune system biology studies, the diagnosis and treatment of all immune diseases and targeting other tissue and cells that interact with the immune system.
  • this peptide derivative that has been obtained has the capacity to affect immune responses with different aspects and with different sensitivity.
  • the findings obtained indicate that the small peptide molecule which manifests during the proteolytic processing of CXCL chemokines, may have the capacity to influence immune responses.
  • the small peptide molecules are useful for therapeutic applications. Since the peptide synthesized by the present invention has been shown to have altering effects on immune responses, there may be therapeutic use of the peptide molecule in inflammatory diseases or cases including the immunologic or immune system in a variety of applications in the fields of health sciences and biology.
  • the Pep8 molecule in order to prepare a drug or agent to be used in diagnosing and treating of diseases related with the immune system, in targeting and/or marking immune system cells. Accordingly, the Pep8 molecule not only can be used alone directly, it can also be used by obtaining various biological and chemical modifications.
  • the Pep8 molecule having the mentioned amino acid sequence can be used in the preparation of a drug or agent for therapeutic purposes mentioned below.
  • FIG. 1 Hydrophobicity (A) and isoelectric point (B and C) graphics drawn at the designing stage of the Pep8 peptide.
  • the isoelectric point graphic has been provided of the total precursor peptide sequences belonging to CXCL8 (Region from the methionine aa at the 1 st position to the region of CXC or ELR motif). The net charge value observed at pH 7 has been illustrated in the box.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne la séquence "Gly Ala Val Leu Pro Arg Ser Ala Lys" dérivée de la séquence précurseur de chimiokine CXCL8 humaine (CXCL8-(1-30)). Elle a été caractérisée par les fonctions de liaison à des cellules du système immunitaire et de déclenchement de réponses immunitaires. Ce peptide court fonctionnel est plus long que 8aa, il présente une charge positive à pH neutre et il est amphiphile.
PCT/TR2019/050621 2018-08-10 2019-07-25 Peptide immunomodulateur dérivé d'une séquence précurseur de terminal amino cxcl8 WO2020032894A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2018/11689 2018-08-10
TR2018/11689A TR201811689A2 (tr) 2018-08-10 2018-08-10 Cxcl8 ami̇no ucu öncül di̇zi̇si̇nden türevlenen i̇mmünmodülatör pepti̇t

Publications (1)

Publication Number Publication Date
WO2020032894A1 true WO2020032894A1 (fr) 2020-02-13

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PCT/TR2019/050621 WO2020032894A1 (fr) 2018-08-10 2019-07-25 Peptide immunomodulateur dérivé d'une séquence précurseur de terminal amino cxcl8

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TR (1) TR201811689A2 (fr)
WO (1) WO2020032894A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090022657A1 (en) * 2003-10-22 2009-01-22 Applied Research Systems Ars Holding N.V. Novel CXCL8 antagonists
WO2014184384A1 (fr) * 2013-05-17 2014-11-20 Centre National De La Recherche Scientifique (Cnrs) Anticorps anti-cxcl1, cxcl7 et cxcl8 et leurs applications

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090022657A1 (en) * 2003-10-22 2009-01-22 Applied Research Systems Ars Holding N.V. Novel CXCL8 antagonists
WO2014184384A1 (fr) * 2013-05-17 2014-11-20 Centre National De La Recherche Scientifique (Cnrs) Anticorps anti-cxcl1, cxcl7 et cxcl8 et leurs applications

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt 1 July 1989 (1989-07-01), "Interleukin-8, [Homo sapiens (Human", Database accession no. P10145 *
YOYEN-ERMIS, D. ET AL.: "CXCL kemokinlerden turevlenen küçük peptit molekullerinin immun duzenleyici etkileri", ULUSAL IMMUNOLOJI KONGRESI, vol. 23, 30 April 2015 (2015-04-30), pages 62 *

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