TR201811689A2 - IMMUNMODULATOR PEPTITIS PRODUCED FROM CXCL8 AMINO TIP PREVIOUS SERIES - Google Patents

Abstract

The present invention relates to the functional short peptide - Gly Ala Val Leu Pro Arg Ser Ala Lys - sequence derived from the human CXCL8 chemokine precursor peptide sequence (CXCL8- (1-30)). It is characterized by its effect on immune system responses and binding to immune system cells. This functional short peptide is longer than 8 aa, has a positive charge at neutral pH and is amphiphilic.

Description

DESCRIPTION Immunmodulatory peptide derived from the CXCL8 amino terminal prec'L'II region Technical Area: This invention is derived from the proto-sequence located in the amino-terminal region of the CXCL8 chemokine molecule. (CXCL8-(1-30)) inspired function and influencing immune system responses It is related to the short peptide sequence found to be

State of the Technique: Cytokines are produced by animal and plant cells, enabling cells to communicate with each other. It is a group of proteins and peptides. Active in inflammation and immune reactions by lymphocytes, immune cells including macrophages, endothelium, epithelium and connective tissues they are created. Cytokines bind to receptors in cells, leading to cell proliferation, They stimulate differentiation and regulation of cellular responses.

In the presence of a pathogen, cytokines attack immune system cells such as T cells and macrophages. They signal and allow them to go to the infection site.

A specific group of cytokines is chemokines, which mediate chemotaxis between cells.

There are four subfamilies of chemokines: CXC, CC, CX3C and XC.

In CXC chemokines, two N-terminal cysteines are separated by an amino acid. 17 different mammals Contains CXC chemokine, specific amino acid in glutamic acid-leucine-arginine structure sequence (ELR motif), ELR-positive and ELR-negative divided into categories.

ELR-positive CXC chemokines specifically target neutrophils as well as some other immune cells. They induce migration and interact with CXCR1 and CXCR2 chemokine receptors. ELR other CXC chemokines that do not contain the motif often function as chemoattractants for lymphocytes. a peptide (020), which performs similar functions.

W has the ability to antagonize the CXCL12 chemokine that binds to CXCR4. It encompasses a novel cyclic peptide sequence with have the ability to antagonize the CXCL12 chemokine that binds to CXCR4 includes peptide sequences.

U has the capacity to bind CXCR1' and antagonize IL-8 encompasses peptide sequences demonstrating the ability.

The activity of this molecule by binding directly to US patent chemokines It identifies peptides that inhibit inflammation, thereby altering processes associated with inflammation.

W no.Iu patent Antagonizing the CCL5 chemokine that binds to CCRS and the ability to block the entry of the HIV virus, which infects the cell through this receptor. contain peptide sequences.

W, this patent binds to various chemokine receptors, encompasses peptide sequences that antagonize its activity.

These patents have one of the following features: 1- The chemokine from which the related peptide originates ability to mimic (agonist) its function, 2- The function of the chemokine from which the peptide originates Ability to antagonize, 3- From the mature protein region of the chemokine from which the relevant peptide originates being engineered (outside of the progenitor site), 4- A specific CXCL or CCL chemokine molecule specificity.

From the amino terminal precursor region of the CXCL8 chemokine mentioned in this patent application The function of the short peptide sequence (Pep8) derived from (CXCL8-(1-30)) of any chemokine There is no antagonizing or agonizing. In this invention, the immune system answers The peptide sequence capable of affecting

In the state of the art, from a sequence that overlaps with the peptide sequence covered by the application, or from this information that speaks directly to the specific functions of the small peptide we have described not found. “Gly Ala Val” derived from the precursor sequence of the CXCL8 chemokine (CXCL8-(1-30) The sequence “Leu Pro Arg Ser Ala Lys” is unknown in the state of the art.

In the state of the art, peptides originating from CXCL8 also contain carboxy termini. (does not originate from precursor sites as in our invention) and what CXCL8 does It is evaluated in terms of antagonizing or agonizing its functions. This patent from the amino-terminus precursor sites of the CXCL8 chemokine mentioned in its application. antagonize or agonize any chemokine of the derived short peptide sequence (Pep8) function is not available. Influencing immune system responses in this invention The peptide sequence with the same capacity has been determined.

Description of the invention Small molecules with peptide structure are involved in the regulation of many physiological processes and intercellular plays a role in communication. Peptides of simple structure and low molecular weight stabilizes more easily and reaches the target tissue more easily.

This invention is a precursor (CXCL8-(1-30)) located at the amino terminus of the known CXCL8 chemokine molecule. derived from these sequences by examining the sequence with a rational and unique algorithm. It includes the identification and recovery of the small peptide molecule.

Small peptide molecule derived from this sequence will be suitable for in vitro use and synthesized. The possible peptide was selected and named Pep8.

Then peripheral blood mononuclear cells of the small peptide molecule It has been determined that it has no toxic effect on the immune cells and the molecule It has been investigated how it affects its activation, proliferation and cytokine synthesis under has been found to exert a regulatory effect.

Pep8 is also synthesized as fluorescently labeled and with which immune cell types it is more common. interaction has been investigated.

Detailed description of the invention The precursor sequence (CXCL8-(1-30)) located at the amino end of CXCL8 was determined by a rational algorithm. A small peptide molecule from this sequence will be suitable for in vitro use. and has been derived so that it can be synthesized.

The design and derivation criteria used in this step are given below: Being longer than 0 8 mm, . positive charge transport at neutral pH, . amphipathic, . It is dissolution in polar environment (hydrophobic aa. ratio being 555%).

Chemical formula of the designed peptide molecule, isoelectric point, solubility in polar environment (hydrophobic wt%), molecular weight parameters using bioinformatics software calculated. The relevant parameters are both the total peptide sequences contained in the precursor protein regions. as well as for the small peptide molecule derived from these regions (Table-1a,b).

Amino terminal precursor peptide sequence used as template Small peptide molecule (Pep) CXCL8 Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu With Pep8= Gly Ala Val Leu Pro Arg Ser Ala Lys Ser Ala Ala Leu Cys Glu Gly Ala Val Leu Pro Arg Ser Ala Lys CXCL8 Pep8 Length mm. 30 9 Net load (pH7) +2 +2 Hydrophobic a. 56.67% 44.44% Solubility in water POOR GOOD In the design phase of the small peptide molecule, first of all, the National Center for Biotechnology has been made. Characteristic CXC or ELR for mature chemokines that have gained biological activity Janin hydrophobicity curves [SIB 7] by selecting protein regions up to the Bioinformatics Resource Portal (ExPASy); Lifetein peptide property calculator; Innovagen using peptide property calculator software] (Figure-1, A graph). a) Using these curves, the progenitor chemokine region (CXCL8-(1-30)) is amphipathic. owners have not been determined. The isoelectric points of these regions are still the same. The graph was prepared using (Figure-1, Chart B). b) Transfer of proteins to organelles and cell membranes after translation The hydrophobic regions (usually the first 24-28 aa.) overlap with the signal sequence that provides by keeping at least 8 mm. Different peptide sequences were selected to have length and In order for the peptides to interact well with the cell surface, their net charge at neutral pH (Figure-1, C graph) was analysed.

The determined Pep8 sequence will be amino-terminal acetylated by LifeTein (USA). The peptide was synthesized and analyzed by HPLC (Purity, 91.09%), mass spectrophotometry. The purity and accuracy of the sequence has been confirmed.

Pep8 in lyophilized form to a stock concentration of 5 mM with sterile dH2O. has been resolved. Final concentrations of peptide stock solutions are 500 µM, 50 pM, 5 µM, 0.5 µM and Working solutions were diluted to 50 nM in cell culture medium. has been created. The dissolved peptides were aliquoted and stored at -86°C.

Then peripheral blood mononuclear cells of the small peptide molecule It has been determined that it has no toxic effect on the immune cells and the molecule It was investigated how Pep8 affects its activation, proliferation and cytokine synthesis under It has been determined that it is a small peptide molecule that can show an immune regulatory effect.

Effects of Pep8 on activation of peripheral blood mononuclear (PKM) cells has been examined. Effect of CXCL precursor peptide derivatives on activation of PKM cells. analyzed and evaluated.

The effects of Pep8 on the proliferation of PKM cells were investigated by flow cytometric and PKM The effects on the proliferation of CFSE dyes are determined by the dilution level of the CFSE dye. has been evaluated. Related analyzes were made by flow cytometry method.

The effects of Pep8 on cytokine levels secreted from PKM cells were determined by ELISA experiments. researched.

In conclusion, Pep8's proliferation of PKM cells, the level of activation markers and It has been determined that it has an effect on cytokine synthesis. proliferation of Pep8 immune cells affect his ability. Cytokine production also changed in the presence of Pep8.

Immune regulation effect: Mononuclear leukocytes under lipopolysaccharide (LPS) stimulation In terms of markers, it affects a low level of increase. Pro-inflammatory IFN-y cytokine reduces its synthesis; It does not change IL-10, IL-4, TGF-ß, TNF-oi levels.

Activation level of mononuclear leukocytes under CDB/CD28 stimulation (CD86 increase; HLA- DR, CD154, CD25 CD69, CD80 does not change the level) are low in terms of markers effects in the direction of the art. It does not affect the proliferation of T lymphocytes.

Activation of mononuclear leukocytes under CD3/allogeneic mononuclear cell THP-1 stimulation does not affect the level. It increases the proliferation of T lymphocytes. Pro-inflammatory cytokines TNF-oi and increases the level of lFN-γ; increases the levels of anti-inflammatory cytokines TGF-ß and IL-1O; IL-4 does not change the level.

As a result of labeling this peptide sequence with fluorescent (FITC), monocytes, NK and B has not been determined.

Results Pep8 has the capacity to influence immune responses from different angles and with varying sensitivity. in hand of the molecule; Immune system biology studies show that all inflammatory diseases diagnosis and treatment, and the immune system or other tissues and cells that interact with the immune system. There may be uses for targeting.

Immune responses of this peptide derivative obtained from different angles and with different sensitivity. understood that it can affect When considered in terms of immune system biology, the relevant small molecule is likely to be released during inflammatory responses. This Although isolation or detection of peptides from inflammatory sites has not been performed, The findings revealed during proteolytic processing of CXCL chemokines are small. indicates that the peptide molecule may have the capacity to influence immune responses. is doing.

On the other hand, small peptide molecule is suitable for therapeutic applications. known. The effects of altering immune responses of the peptide synthesized by the present invention immunological or immune system involved or inflammatory of the peptide molecule found in diseases or events in the fields of health sciences and biology. It may have therapeutic use in various applications.

Amino acid from or adjacent to the CXCL8 chemokine precursor sequence specified in the application specific labeling of immune cells by a small peptide molecule derived from capacity can be used. In this way, especially myeloid origin and monocytic Identification of cells may be possible with the use of the relevant peptide molecule.

In the diagnosis and treatment of diseases related to the immune system or the cells of the immune system of a drug or agent to be used in targeting and/or marking It is possible to use Pep8 molecule in its preparation. For this purpose, the Pep8 molecule It can be used directly as well as obtaining various biological and chemical modifications. may also be used.

The Pep8 molecule with the indicated amino acid sequence is used for the following therapeutic purposes. It can be used in the preparation of the drug or agent. o To influence the response of immune system cells by interacting with immune system cells o Diagnosis and treatment of diseases related to the immune system o targeting immune system cells o Marking of immune system cells Description of figures: Figure 1. The hydrophobicity (A) and isoelectric point (B) plotted during the design phase of the Pep8 peptide and C) graphs A) Janin hydrophobicity curve of total precursor peptide sequences of CXCL8 chemokines. small peptide The regions determined to be the most suitable for the design algorithm are marked in the box. selected The amino acid sequence of the small peptide is indicated at the bottom of the box. 8) Total precursor peptide sequences of CXCL8 (from methionine aa. at position 1 to CXC or The isoelectric dot plot (region up to the ELR motif) is given. At pH 7 in the box The monitored net charge value is shown.

C) The isoelectric dot plot of the small peptide sequences of CXCL8 is given. pH in the box The net charge value monitored is shown in Fig. <110> ESENDAGLI, SUNES <120> AN IMMUNOMODULATOR PEPTIDE DERIVED FROM CXCL8 AMINO TERMINAL PRECURSOR SEQUENCE <160> 13 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <220> <223> A peptide molecule derived from CXCL8 chemokine amino terminal precursor sequence <400> Gly Ala Val Leu Pro Arg Ser Ala Lys hydrophobicity Janin hydrophobicity MTSKLAVALLAAFLlSAALCEGAVLPRSAK all pro-peptide

Claims (6)

REQUESTS 1-Small peptide molecule derived from the human CXCL8 chemokine precursor peptide sequence (CXCL8-(1-30)), 0 Gly Ala Val Leu Pro Arg Ser Ala Lys (Pep8) 2- It is a peptide molecule with the amino acid sequence as claimed in claim 1, and has the following characteristics: It is longer than 0 8 aa, it carries a positive charge at neutral pH, it is amphipathic, and . hydrophobic aa to dissolve in polar environment. rate is 55% or less. 3- Use of a peptide molecule having the amino acid sequence specified in Claim 1 in the preparation of a drug or agent to be used to affect the responses of immune system cells by interacting with immune system cells. 4- Use of the peptide molecule with the amino acid sequence specified in Claim 1 in the preparation of a drug or agent to be used in the diagnosis and treatment of diseases related to the immune system. 5- Use of a peptide molecule having the amino acid sequence specified in Claim 1 in the preparation of a drug or agent to be used in targeting immune system cells. 6- Use of a peptide molecule with the amino acid sequence specified in Claim 1 in the preparation of a drug or agent to be used in the labeling of immune system cells.