RU2663111C1 - Strain of asidiomycete daedaleopsis confragosa, containing proteins, exhibiting anticancer and antiviral activity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Strain of basidiomycete Daedaleopsis confragosa K-1326, which has cytostatic anticancer activity against human marrow myeloma (IM-9), human lymphadenoma (Namalva) and inhibitory activity against herpes simplex virus type 2, influenza virus A / H1N1 / California / 2009, is deposited in the Collection of bacteria, bacteriophages and fungi of the Federal State-Funded Institution SSC SRB WB “Vektor” of Federal Supervision Agency for Customer Protection and Human Welfare under the registration number F-1368. Strain of basidiomycete Daedaleopsis confragosa can be used to produce anticancer and antiviral drugs.

EFFECT: invention allows to increase yield of proteins exhibiting activity against tumor cells and viruses that are pathogenic to human.

1 cl, 4 dwg, 4 tbl, 6 ex

Description

The invention relates to a strain of the basidiomycete Daedaleopsis conf ragosa (de daleopsis rough), the total proteins from the mycelium of which exhibit antitumor and antiviral activity and can be used in biotechnology and the microbiological industry to obtain antitumor and antiviral drugs.

Fruit bodies of Dedaleopsis Rough Daedaleopsis confragosa fan-shaped, sessile broad, thicken toward the center. The upper surface is radially wrinkled or smooth from light gray, woody to reddish, brownish, ocher-brown with concentric zones. It fades when dried. The lower surface has large pores, which in maturity lengthen and become labyrinth-like (almost lamellar), first pale in color — grayish, cream, then brown (Fig. 1, 2).

A large group of biologically active substances contained in mushrooms are polypeptides, including those with antiviral activity. So, Frank Pirano [Pirano F. F. The Development of the Antiviral Drug RC 28 from Rozites caperata (Pers .: Fr.) P. Karst. (Agaricomycetideae). International Journal of Medicinal Mushrooms. Vol. 7 (2005), P. 356] isolated from an aqueous extract of the fungus Rozites coperata a new antiviral drug of protein nature that impedes the replication of herpes simplex viruses of types 1 and 2, cytomegaloviruses, respiratory syncytial virus, and type A influenza virus.

Antiviral protein RC28 with antiherpetic activity was purified from PBS extract of Rozites caperata (Cortinarious caperata), precipitated with acetone, followed by gel filtration and ion exchange chromatography. The molecular weight of this 28.251 kDa protein was measured by MALDI-TOF mass spectrography. The drug RC28 inhibits the replication of HSV-1 in vitro (IC50) at a concentration of 0.078 mg / ml and therapeutic index> 32. The complete peptide chain of the antiviral protein RC28 from Rozites caperata had 235 amino acid residues and does not belong to any of the known protein families [Gonga M., Pirainoc F., Yanb N., Zhanga J., Xiaa M., Mab J., Chenga J ., Liub X. Purification, partial characterization and molecular cloning of the novel antiviral protein RC28 // Peptides. V. 30. - I. 4. - 2009. - P. 654-659].

The closest analogue (prototype) is the strain Daedaleopsis confragosa 2266 which was isolated in pure culture in 2011 from spores of fruiting bodies collected in Altai Mountains. The strain was transferred to the mycology laboratory for studies of its antiviral activity. The strain is stored in the Collection of the Botanical Institute of St. Petersburg (LE-BIN). The aqueous extract from the deep mycelium of the strain Daedaleopsis confragosa 2266 showed an antiviral effect against influenza virus of different subtypes [Teplyakova TV, Psurtseva NV, Kosogova TA, Mazurkova NA, Vlasenko VA Antiviral activity of basidiomycetes of the Altai Mountains // Problems of Botany in Southern Siberia and Mongolia: Collection of scientific articles based on the materials of the IX International Scientific and Practical Conference (October 25-27, 2010, Barnaul). - Barnaul: ARTHKA, 2010 .-- S. 245-246 .; Teplyakova TV, Psurtseva NV, Kosogova TA, Mazurkova NA, Khanin VA, Vlasenko VA. Antiviral activity of Polyporoid mushrooms (higher Basidiomycetes) from Altai Mountains (Russia). International Journal of Medicinal Mushrooms. 2012; 14 (1): 37-45].

The aqueous extract from the deep mycelium of the same strain exerted an antitumor effect against the laryngeal carcinoma cells Hep-2 (the solids concentration of the extract, inhibiting cell growth by 50% (IC50), was μg / ml [Teplyakova T.V., Kanaeva O. I., Kosogova T.A., Kostina N.E., Bardasheva A.V., Troshkova G.P. Selection of producers of antitumor compounds among basidiomycetes // Science and Modernity - 2011: Proceedings of the XII International Scientific and Practical Conference: in 3 parts, Part 1 / Under the general editorship of S. S. Chernov. - Novosibirsk: Izda NSTU, 2011. - P. 217-223]. Protein fractions isolated from the deep mycelium of Daedaleopsis confragosa 2266 showed an antiviral effect against herpes simplex virus type 2 and human immunodeficiency [Gileva IP, Bardasheva AV, Gashnikova N.M., Balakhnin S.M., Kazachinskaya E.I., Kosogova T.A., Teplyakova T.V. Antiviral proteins from the basidiomycete fungus Daedaleopsis confragosa II Successes in medical mycology. - T. 12. -M .: National Academy of Mycology, 2014. - S. 302-303].

The technical result of the claimed invention is to obtain a strain of the basidiomycete Daedaleopsis confragosa producing proteins that exhibit higher (compared with the prototype) activity against tumor cells and some viruses pathogenic for humans.

The specified technical result is achieved by obtaining from the natural environment a new strain of the fungus Daedaleopsis confragosa K-1326 (author's name), the total proteins of which contain compounds with antiviral and antitumor (cytostatic) activity. The strain Daedaleopsis confragosa K-1326 was isolated from the fruiting body of the tinder fungus Dedaleopsis rough Daedaleopsis confragosa in the vicinity of the river Koltsovo, Novosibirsk region in September 2014 and deposited in the Collection of bacteria, bacteriophages and fungi of the Federal State Budget Scientific Center of the VB "Vector" Rospotrebnadzor under the number F-1368 (a certificate of deposit is attached).

The invention is illustrated by the following graphic materials. In FIG. 1 shows the fruiting bodies of Daedaleopsis confragosa on a birch, and FIG. 2 - the same, the lower surface of the fruiting body. In FIG. 3 shows a colony of the strain Daedaleopsis confragosa K-1326, and FIG. 4 shows hyphae and single buckles of the strain Daedaleopsis confragosa K-1326, uv. 400.

Cultural and morphological features of Daedaleopsis confragosa K-1326. In culture, it forms dense colonies, first white, then with brown mycelium. The mycelium is weakly branched, septate, with numerous single-type buckles. Generative hyphae are colorless (Fig. 3, 4).

Biological activity is determined against viruses and tumor cells. To determine it, total proteins isolated from the biomass of the fungus grown in deep culture can be used.

Activity is determined by the ability in vitro to inhibit the growth of tumor cells or to exert a virucidal effect (inhibit the process of virus replication) in cell cultures (USSR State Pharmacopoeia. General methods of analysis. Medicinal raw materials. - M .: Medicine, 1990. - II ed. - Issue 2. - T. 2. - P. 187-209; Manual on the experimental (preclinical) study of new pharmacological substances / Edited by the corresponding member of the Russian Academy of Medical Sciences, Professor R.U. Khabriev. - 2-ed., Revised. and add. - M.: Publishing House "Medicine", 2005. - 832 c .; Ek experimental evaluation of antitumor drugs in the USSR and the USA, under the editorship of ZP Sof'ina, A. Goldin. M: Medicine, 1980. - P. 105-106).

The strain is stored in the form of cultures grown on KHA agarized medium (potato glucose agar) in test tubes at a temperature of 2-6 ° C for 12 months, and also in the form of agar blocks in sterile water at 2-6 ° C for 12 months.

Optimum media for reseeding:

- liquid based on molasses and corn extract (MK), g / l: molasses -20, corn extract -10, KH ₂ PO ₄ - 3, MgSO ₄ × 7 N ₂ 0 - 0.2 g / l ;, tap water - up to 1 liter

The strain is not genetically modified and does not contain the genes of other organisms, transferred resistance genes, genetic changes associated with the use of genetic technical methods.

The strain is not toxic, since strains of this species in the USSR were considered as producers of food protein. For example, 1 kg of protein product from the mycelium of the strain Daedaleopsis confragosa G-115 contained as much protein as 1 kg of meat [I.A. Reshetnikova. Mycelium of mushrooms as a source of feed and dietary protein. - M.: Publishing House of Moscow State University, 1989. - 55 p.].

The inventive strain Daedaleopsis confragosa K-1326 differs from the prototype in a higher growth rate on an agar medium. The growth coefficient (PK) for the strain Daedaleopsis confragosa K-1326 when growing mushrooms on an agarized potato-glucose medium (KGA) in surface culture was 66, and when deep cultivated on a glucose-tryptone medium (GTS) containing 1 liter of water: glucose - 35 g; tryptone - 12 g; yeast extract - 5 g, KH ₂ PO ₄ - 1 g; MgSO4 × 7 H2O - 0.5 g, the dry matter biomass yield was 11.0 ± 0.5 g / l, while for the Daedaleopsis confragosa 2266 strain the growth coefficient was 48, and the dry biomass yield on the GTS was 10, 5 ± 0.7

[Bardasheva A.V., Kosogova T.A., Teplyakova T.V. Selection of natural strains of basidiomycetes - producers of biologically active substances by biomass productivity // Modern Mycology in Russia. Volume 5. Ed .: Yu.T. Dyakov, Yu.V. Sergeev. Materials of the III International Mycological Forum. Moscow. Apr 14-15 2015 Moscow: Nat. Acad. mycol. 2015. Volume 5. P. 284-285]. Solids samples were determined by the gravimetric method according to GOST R 52838-2007 “Feed. Methods for determination of dry matter content. "

An advantage of the claimed strain is the ability of total proteins to inhibit the development of pathogenic viruses and human tumor cells.

Example 1. The cultivation of the biomass of the fungus based on the inventive strain Daedaleopsis confragosa K-1326 deep method. To obtain biomass in deep conditions, a nutrient medium was prepared based on molasses and corn extract (MK), g / l: molasses -20, corn extract -10, KH ₂ PO ₄ - 3, MgSO ₄ - 0.2. The medium was sterilized in an autoclave at 1.2 atm for 30 minutes. Immersed cultivation was carried out in 500 ml flasks with 100 ml of medium on a rocking chair. At the first stage, seed was grown. For sowing flasks with inoculum, a culture grown at 28 ° C for 7 days on oat agar was used. To prepare it, 50 g of oat grains were boiled for 1 hour in 1 liter of water, insisted for 12 hours, agar-agar was added to the resulting solution at the rate of 20 g per 1 liter. The medium was autoclaved at 1.2 atm for 30 min and poured into Petri dishes, for 3 days in 500 ml flasks with 75 ml of culture medium MK. Inoculation was carried out by carving sterile blocks with a diameter of 5 mm, 10 blocks were placed in each bottle. The growth of the seed crop lasted 3 days. The cultivation of the fungus to isolate proteins from the culture fluid was carried out on MK medium seeded with seed medium at the rate of 10% by volume of the medium. The culture was grown on a circular shaker (190 rpm) at a temperature of 26-28 ° C for 10 days.

Example 2. Obtaining total proteins from the culture fluid of Daedaleopsis confragosa K-1326. Liquid culture

Daedaleopsis confragosa K-1326 in a volume of 800 ml was filtered through a sterile gauze filter (4-6 layers of gauze), placed in a funnel, in a glass flask with a capacity of 1 l. The edges of the filter were interconnected and twisted for the most complete extraction of the liquid. To the obtained extract, with stirring, dry ammonium sulfate was added portionwise to a saturation of 70% (472 g / l). The resulting suspension was left at 4-6 ° C for 16-18 hours to form a precipitate. The precipitate was collected by centrifugation (7000 g - 15 min) and dissolved in 15 ml of water.

The obtained biological material was purified by gel chromatography on a column (1.5 × 28 cm, V = 50 ml) with Sephadex G-25, collecting an absorbing solution at a wavelength of 280 nm in 3 ml fractions of 3 ml. The analysis of the contents of the fractions was carried out by electrophoresis in 15% PAAG. Protein-containing fraction solutions were pooled and dialyzed against 500 ml of a solution containing 20 mM Tris-HC1, pH 7.5, 50 mM NaCl.

The yield was 10-11 ml, total protein 12-13 mg.

On the electrophoregram, when analyzing the solution by electrophoresis in 15% SDS page, major bands of proteins with molecular weights of about 38000-40000 and 16000-18000 Da and minor strips with molecular weights of 34000, 28000 and 26000 Da were visualized.

The drug was packaged in 0.5 or 1 mg vials and freeze-dried in the regimen: 4 h — freezing at minus 70-75 ° С; 16 h - drying at 0.1 mm RT. Art.

Example 3. Determination of the cytotoxic activity of the total proteins of the inventive strain Daedaleopsis confragosa K-1326 in relation to human bone marrow myeloma cells (IM-9). An MTT test was used to study cytotoxic activity. The studied compounds were tested in three parallel measurements at 6 concentrations - 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 , 10 ^-4 and 10 ^-3 . Then, according to the curve of the dependence of cell growth on the concentration of the compound, IC _{50 was} determined, that is, the concentration of the drug, causing a 50% inhibition of cell growth. After determining the IC ₅₀ , additional testing was carried out at 4-5 concentrations close to the IC ₅₀ .

Criterion for assessing the cytotoxic effect. A compound is considered to be cytotoxic active at an IR of ₅₀ ≤10 ^-4 ; an analogue of a known antitumor drug is evaluated as cytotoxic if its IR is ₅₀ ≤IR ₅₀ of the reference drug.

The MTT test is based on the enzymatic reduction of the unpainted tetrazolium salt (3- [4,5-dimethylthiosol-2-yl] -2,5-diphenyltetrosolium bromide, MTT) in living metabolically active cells with the formation of blue formazan crystals.

The setting of the experiment. A culture of human bone marrow myeloma cells (IM-9) at a concentration of 5 × 10 ³ in 100 μl of Medium Needle MEM (FBUN SSC WB “Vector” Rospotrebnadzor) containing 10% blood serum of cow fruits (Gibco, USA) was placed in 96- well plate and incubated 24 hours at 37 ° C. Then the test compounds were added at various concentrations and incubated for 48 hours under the same conditions. After that, 5 μl of the prepared MTT solution (Sigma, USA) (initial concentration 5 mg / ml) was added to each well of the plate and further incubated for 4 hours. At the end of the incubation, 100 μl of dimethyl sulfoxide (DMSO) was added to each well of the tablet to dissolve blue formazan crystals formed as a result of the reaction, and the absorbance values of the substrate in the wells were evaluated on a Tecan Sunrise microplate reader (Tesap, Austria) at a wavelength of 492 nm.

As a negative control, IM-9 cells were used without the addition of the test drug. An anticancer drug Cisplatin (Lance-Pharm, Russia) was used as a reference drug (positive control). The results are shown in table 1.

Table 1

The viability of the culture of human bone marrow myeloma cells (IM-9) under the influence of total proteins of Dedaleopsis rough (strain Daedaleopsis confragosa K-1326)

A sample of total proteins (strain Daedaleopsis confragosa K-1326) showed a cytotoxic effect (table 1) at concentrations of 10 ^-3 , comparable to the positive control (Cisplatin) at the same concentration (74.96 and 75.07%, respectively). The total protein of the strain of the prototype Daedaleopsis confragosa 2266 did not suppress, but stimulated the growth of human bone marrow myeloma cells. Cisplatin at a concentration of 10 ^-3 inhibited the growth of tumor cells by 24.93%. Thus, we can conclude that a sample of total Dedaleopsis rough proteins (strain Daedaleopsis confragosa K-1326) exerted a cytotoxic effect on human bone marrow myeloma cell culture (IM-9) in the MTT test, comparable to the anticancer drug Cisplatin.

Example 4. Determination of the cytotoxic activity of the claimed strain of Daedaleopsis confragosa K-1326 against human lymphoma cells (Namalva). In accordance with the methodology described in example 3, studies were conducted on the cytotoxicity of the total proteins of the strain Daedaleopsis confragosa K-1326 against human lymphoma (Namalva). The results indicating the cytostatic activity of total proteins from the deep culture of Daedaleopsis confragosa K-1326 are presented in table 2.

table 2

The viability of the culture of human lymphoma cells (Namalva) under the influence of total proteins Dedaleopsis rough (strain Daedaleopsis Canfragosa K-1326)

Table 2 shows that the total protein of the strain Daedaleopsis confragosa 2266 does not inhibit, but stimulates the growth of human lymphoma cells, while the protein of the strain Dae daleopsis with onfragosa K-1326 reduces the viability of tumor cells at concentrations of 0.001-0.0001 g / l per 27.1-33.66%.

Example 5. The study of the toxicity and antiviral activity of total proteins Daedaleopsis confragosa K-1326 against herpes simplex virus of the second type (HSV-2). Evaluation of the activity of total proteins from the deep culture of Daedaleopsis confragosa K-1326 against herpes simplex virus type 2 was carried out in a culture of Vero cells. A strain of MS herpes simplex virus type 2 was used (obtained from the American Type Culture Collection). Prepared dilutions of the preparations were applied to a two-day culture (3 replicates for each dilution), except for control wells (virus control and cell control). To assess the toxicity of the samples, the cells were stained with a gentian violet solution. Staining results were recorded using a Zemfira 680 photometer with a BioRed RU Zemf control computer in accordance with the manufacturer's instructions. Received optical density (OD) for each of the wells of the tablet at a wavelength of 570 nm. The average OD value was calculated for each group of three wells with different concentrations of the sample. Based on the obtained values, a graph was constructed of the dependence of OD on the concentration of the sample. 50% toxic concentration (TC ₅₀ ) was determined from the graphs.

To determine the antiviral activity, a virus was added to the wells at a dose of 2 × 10 ^-5 PFU / cell (the volume of the working mixture was 100 μl). The reaction was taken into account on day 4. Assessment of activity was carried out using the method of inhibition of plaque formation. Cells were stained with gentian violet. The percentage of inhibition of plaque formation (% IB) was determined by the formula:% IB = [1- (number of plaques in the test / number of plaques in the virus control)] × 100. The concentration capable of inhibiting the development of 50% of plaque forming units (PFU) of the number of PFU in the control was determined (50% inhibitory concentration - IC ₅₀ ). The average% IB value was calculated for each group of three wells with different concentrations of the sample. Based on the obtained values, a graph of the dependence of% IB on the concentration of the sample was plotted. According to the graphs, IC _{50 was} determined. The selectivity index (IS) or therapeutic index (TI) was defined as the ratio of a 50% toxic dose to a 50% inhibitory dose.

Drugs with TI> 10 were considered highly active, and drugs with TI <2 inactive, drugs with TI between 2 and 10 were considered medium active (Paragas J. et al., 2004). The results are shown in Table 3.

Table 3

Antiviral activity of total proteins of different strains of Daedaleopsis confragosa against herpes simplex virus type II, strain MS.

Note: TS ₅₀ -50% toxic dose: the dose of the drug that inhibits cell growth by 50%.

IC ₅₀ - inhibitory dose: the dose of the drug that inhibits the reproduction of the virus by 50%>.

IS - selectivity index, or therapeutic index: equal to the ratio of 50% of the toxic dose of the drug to 50%> the inhibitory dose (TC ₅₀ / IC ₅₀ ).

As can be seen from table 3, the greatest antiviral efficacy was shown by the strain Daedaleopsis confragosa K-1326, both in inhibitory effective dose (IC ₅₀ = 0.18 μg / ml) and in selectivity index.

Example 6. The study of the toxicity and antiviral activity of total proteins Daedaleopsis confragosa K-1326 against influenza virus A / California / 07/09 (H1N1pdm09) in an MDCK cell culture (dog kidney cells). MDCK cells were scattered in 96-well plates with an inoculum concentration of 300,000 cells / ml, 100 μl per well. Dilutions of the test samples were prepared in a support medium. In the work, serial binary dilutions of the samples were used. Prepared dilutions of the preparations were applied to the daily culture (3 replicates per dilution), except for control wells (virus control and cell control). To determine antiviral activity, a virus was added to the wells at a dose of 100 TCID ₅₀ / well. The reaction was taken into account on the 3rd day. Evaluation of the activity of the samples was carried out using a neutral red dye. The test results were recorded using a 680 Zemfira photometer with a BioRed RU Zemf control computer in accordance with the manufacturer's instructions. Indicators of optical density at a wavelength of 490 nm were obtained for each of the wells of the tablet. For each dilution, the average OD value was calculated. Based on the obtained values, graphs of the dependence of OD on the concentration of the drug were built. TC ₅ о and 1С _{50 were} determined from the graphs. The selectivity index (IS) was calculated as the ratio of TC ₅₀ to IC ₅₀ - The results are presented in table 4.

As can be seen from table 4, the effective dose for influenza virus in the studied strains of IC ₅₀ (μg / ml) is almost the same (0.2 and 0.18 μg / ml).

Thus, the data given in examples 3-6 confirm the claimed technical result, namely, that the strain of the fungus Daedaleopsis confragosa K-1326 produces total proteins isolated from the downstream culture of the fungus, exhibiting inhibitory activity against tumor cells and some pathogenic viruses for a person.

Table 4

Antiviral activity of total proteins of the fungus of Daedaleopsis confragosa strains against influenza virus A / California / 07/09 (H1N1 pdm09)

Claims (1)

The strain of the basidiomycete Daedaleopsis confragosa K-1326 containing proteins in the culture fluid of the deep culture that exhibit cytostatic antitumor activity against human bone marrow myeloma (IM-9), human lymphoma (Namalva) and inhibitory activity against herpes simplex virus type 2, influenza virus A / H1N1 / California / 2009, and deposited in the Collection of Bacteria, Bacteriophages and Fungi of the Federal State Budgetary Scientific Center of the World Health Center "Vector" of the Federal Service for Supervision of Consumer Rights Protection under the number F-1368

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