Peptide showing growth stimulating activity of hepatocyte growth factor (hgf)

FIELD: medicine.

SUBSTANCE: peptide is described by general formula I: X1-X2-X3-X4-X5 (I) where XI represents Q or V; X2 represents P; X3 represents G or (β-A); X4 represents R, or X4 represents K with X1-Q or with X1-V and X3-(p-A); X5 represents G, where G is glycine, (β-A) is β-alanine, P is proline, V is valine, Q is glutamine, K is lysine, R is arginine.

EFFECT: invention presents extended range of effective therapeutic agents involved in liver regeneration.

4 dwg, 1 tbl, 3 ex

 

The invention relates to the field of Bioorganic chemistry, biochemistry and medicine, namely to biologically active substances peptide having growth promoting activity of the growth factor, hepatocyte, and may find application in medicine, veterinary medicine, as well as in experimental biochemistry.

The process of regeneration of the liver can be divided into three phases: initiation, proliferation and inhibition. At the stage of proliferation primarily functioning as a growth factor HGF hepatocyte [1], which provides the transition of hepatocytes from G1 into S-phase. It is shown that the growth factor HGF hepatocyte involved in liver regeneration in vivo and is a mitogen for hepatocytes [2]. Growth factor hepatocyte ("scattering factor, scatter factor, SF) is a glycoprotein consisting of two covalently linked abietinic with a molecular mass of 62 kDa and 34 kDa [3-4].

The inventive peptide has growth promoting activity of the growth factor HGF hepatocyte and stimulates the proliferation of hepatocytes.

The present invention is to expand the Arsenal of effective therapeutic agents that are involved in regeneration of the liver.

The main technical result that can be obtained by carrying out the present invention lies in the realization of this purpose is achieved through the creation of a new sin is micheskogo peptide, possessing growth promoting activity of the growth factor HGF hepatocyte, the General formula I:

,

where X1 represents Q or V;

X2 represents P;

X3 is a, G or β-A;

X4 represents R or K;

X5 is a, G,

where G is glycine, (β-A) - β-alanine, P - Proline, V - valine, Q - glutamine, K is lysine, R is arginine.

Thus, identified eight (8) sequences of oligopeptides, which are active growth factor hepatocyte.

The formula of the claimed peptide was revealed by the results of computer design binding sites growth factor HGF hepatocyte with receptors on the surface of stem cells. Computer design of binding sites growth factor hepatocyte HGF to its receptor was performed using software, performing computer modeling of the spatial structure of protein molecules and the design of low molecular weight compounds responsible for the biological function of a protein.

The following drawings form part of the description of the present invention and are included to further demonstrate certain aspects of the present invention. The present invention can be better understood by reference to one or more of these drawings in combination with the detailed description of PR is stavlennik here specific embodiments of the present invention.

Figure 1 shows the spatial structure of the growth factor HGF hepatocyte [5].

Figure 2 presents the spatial structure of a protein growth factor HGF hepatocyte with identified functional site functional site is highlighted).

Figure 3 presents the spatial structure of the complex of HGF/receptor (PDB ID 1SHY) [6].

Figure 4 presents the spatial structure of the complex of HGF/receptor (PDB ID 1SHY) with identified functional site of the protein G-CSF (functional website dedicated).

Example 1. In silico design of functional site of HGF on the basis of the spatial structure of the protein HGF.

To implement design and functional website HGF was used a computer program.

The source data for the work was the primary and the spatial structure of proteins imported from the database Protein Data Bank [7]. In the database of the Bank conducted a search of the spatial structure of the growth factor HGF hepatocyte. Result for computer design of selected spatial structure of the growth factor HGF hepatocyte [5] (figure 1).

Next conducted a computer simulation, which allowed us to identify amino acid residues of a protein growth factor HGF hepatocyte involved in the interaction of growth factor hepatocyte HGF to its receptor (figure 2)On the basis of the received data predicted the formula of the inventive peptide.

Example 2. In silico design of functional site of the growth factor HGF hepatocyte based on the spatial structure of the complex of HGF/receptor.

To implement design and functional website HGF was used a computer program.

The source data for the work was the primary and the spatial structure of proteins imported from the database Protein Data Bank [7]. In the database of the Bank conducted a search of the spatial structure of the complex of HGF/receptor. Result for computer design of selected spatial structure of the complex of HGF/receptor [6] (figure 3).

Next conducted a computer simulation, which allowed us to identify amino acid residues of a protein growth factor HGF hepatocyte involved in the interaction of growth factor hepatocyte HGF to its receptor (figure 4).

Presented in figure 2 and figure 4 data on computer simulation results, we can conclude that the proposed Oligopeptide is a functional site of the growth factor HGF hepatocyte involved in binding to receptors on the surface of stem cells and stimulating the proliferation of hepatocytes. This connection may find application in medical and experimental biochemistry.

As information confirming the possibility to implement the ia of the invention, investigated (Example 3) the effect of the polypeptide drug is created synthetically, on the explants of rat liver at the age of 14 days, obtained in accordance with the techniques described in the works of Havinson V.H. al. [8, 9]. The experiments were carried out on 90 samples of tissue explants. Methods of cultivation were described in detail previously [10].

Example 3. Study of biological activity of the synthesized peptides.

The studied peptides were added to the culture medium at concentrations of 20 ng/ml served as Control explants without adding peptides to the culture medium. Petri dishes with tissue explants were placed in a thermostat at 37°C and after 3 days were viewed by phase-contrast microscope. Then defined area index (PI), which was calculated in arbitrary units as the ratio of total Explant, together with area visulaise cells to the source area of the Explant. Values of PI expressed in percent of the control value of FE taken as 100%. The significance of differences of individual in the control and experimental samples was evaluated using a t-student test.

The results of biological testing of selected oligopeptides in the table.

ConnectionQPGRGQPKG VPGRGVPGKGQPβ-ARGQPβ-AKGVPβ-ARGVPβ-AKG
Area, % of control170175150145180185160155
Reliability********
* - P<0.05 compared with control

According to the research results we can conclude that when using the inventive peptides as growth factors increases cell proliferation of hepatocytes.

Sources of information

1. G.K. Michalopoulos, Frances De M.C. Liver regeneration. Science 1997; 276: 60-66.

2. G.K. Michalopoulos Liver regeneration: molecular mechanisms of growth control. // FASEB J., 4, 176-187, 1990. FASEB J. 1990.

3. Gherardi, E., Coffer, Purification and characterization of scatter factor. // Cell and Motilty factors, ed. I.D.Goldberg. Birkhauser Verlag, Cell and Motilty factors, ed. I.D.Goldberg. Birkhauser Verlag, Basel, 53-62, 1991.

4. Weidner C.M., Behrens J., Vandekerckhove J., Birchmeier W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. // J. Cell. Biol., 111, 2097-2108, 1990. J. Cell. Biol. 1990.

5. http://www.rcsb.org/pdb/explore/explore.do?structureld=1SI5

6. http://www.rcsb.org/pdb/explore/explore.do?structureld=1SHY

7. http://www.rcsb.org/pdb/home/home.do

8. Khavinson V.H., Malinin V.V., Chalisova NI, Grigoriev H. tissue-specific action of peptides in tissue culture in rats of different age // "Advances in gerontology", 2002, issue 9, Volume 3, s-285.

9. Khavinson V.H. tissue-specific action of peptides, Byull. the experimental. Biol. and medicine. - 2001. - T, N 8. - S-229.

10. Khavinson V.H., Morozov V.G., Chalisova NI, Okulov, V.B. have been the Effect of peptides on brain cells of the nervous tissue in vitro // Cytology. - 1997. - T, N 7. - S-575.

List of amino acid sequence (in single-letter code):

1. Q-P-G-R-G

2. Q-P-G-K-G

3. Q-P-(β-A)-R-G

4. Q-P-(β-A)-K-G

5. V-P-G-R-G

6. V-P-G-K-G

7. V-P-(β-A)-R-G

8. V-P-(β-A)-K-G

Peptide with growth promoting activity of the growth factor HGF hepatocyte, the General formula I:
,
where X1 represents Q or V;
X2 represents P;
X3 is a, G or (β-A);
X4 represents R or
X4 is K if X1 is Q or when X1 is V and X3 - (β-A);
X5 is a, G,
where G is glycine, (β-A) - β-alanine, P - Proline, V - valine, Q - glutamine, K is lysine, R is arginine.



 

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