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Project titleUsing the OSMAC approach (one strain - many compounds) to search for promising "leading molecules" from marine micromycetes Penicillium thomii

Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-103 - Synthesis, structure, and properties of natural and physiologically active substances; medical chemistry and prediction of various types of biological activity

Keywordsbioactive compounds, secondary metabolites, marine-derived fungi, anticancer activity, antimicrobial activity, OSMAC, NMR, HPLC

Annotation
Cancer and bacterial infections cause the death of millions of people every year, both soon and in Europe. According to statistics, cancer is the second death in the world, claiming more than 9 million lives every year. Despite significant progress in the diagnosis of the disease in recent years, at the moment, the treatment of oncopathology still remains a serious problem, both because of the validity of an effective drug, and because of the presence of serious side effects in drugs. One of the main problems of modern medicine is the emergence of antibiotic-resistant strains of pathogenic strains of microorganisms. The ever-increasing use of antibiotics in medicine and agriculture is leading to an increase in the rate of development of antimicrobial resistance and the emergence of strains resistant to a wide range of antibiotic drugs. Thus, the search for new antibacterial and antifungal drugs, especially effective against resistant microorganisms, is an extremely urgent task. It is known that more than 50% of drugs are based on compounds isolated from natural sources. One of the approaches to the creation of new drugs is the search for "leading molecules" among natural compounds that exhibit the desired biological activity. Therefore, the isolation of new biologically active natural compounds is an important fundamental task on the way to the search for "leading molecules" - the basis for the creation of drugs. Marine microorganisms have turned out to be a source of new natural compounds with a wide range of biological activity applicable in various industries. The ever-increasing number of sequenced microbial genomes has revealed a discrepancy between the number of gene clusters potentially encoding the production of metabolites and the actual number of chemically characterized metabolites isolated from these microorganisms. Homologous and heterologous expression of these biosynthetic genes, which are often inactive under conditions of experimental laboratory cultivation, can lead to the discovery of "hidden" natural compounds of medical and biotechnological interest. The OSMAC (one strain, many compounds) approach based on modification of growth conditions has proven to be a powerful strategy for discovering new potentially active natural compound. In the project, as objects of research, we will use previously studied fungi-micromycetes Penicillium thomii, isolated from marine brown algae and sea grass, which have previously proven themselves as producers of antitumor and anti-inflammatory compounds. The biosynthetic potential of these fungi has not been revealed in the framework of the primary chemical studies carried out along the "classical" path. Thus, this project provides for the identification of "leader" molecules from these objects for further development of new antibiotics and antitumor compounds based on them. For the secondary metabolites of fungi isolated under the project, data will be obtained for the first time on their antimicrobial activity, as well as cytotoxicity against human urogenital tumors and a number of non-tumor cells, which will make it possible to select the most promising compounds for further drug development. The results of studies planned within the framework of this project will significantly expand the arsenal of natural compounds with new variants of chemical structures, many of which have no analogues among bioactive metabolites isolated from terrestrial sources. This will allow us to characterize the biosynthetic capabilities of marine fungi and purposefully use them as producers to obtain new variants of bioactive compounds. Thus, we will have at our disposal an affordable and renewable source of new natural compounds with antimicrobial and antitumor activity.

Expected results
The project provides for the selection of optimal cultivation conditions according to the OSMAC strategy for the previously studied fungi Penicillium thomii KMM 4674 and P. thomii KMM 4667 associated with the sea grass Zostera marina (Sea of Japan), P. thomii KMM 4675 (brown seaweed Sargassum pallidum, Sea of Japan) and P. thomii KMM 4645 (brown seaweed Sargassum miyabei, Sea of Japan) to increase the yield of bioactive compounds and/or activate dormant genes responsible for the synthesis of "hidden" metabolites. According to the OSMAC strategy, this project plans to change the chemical conditions in the cultivation system by varying the salinity (osmotic stress) and adding metal cations. The project involves the isolation and determination of the structure of individual compounds as a result of the preparative cultivation of the fungus Penicillium thomii on a specially selected modified medium according to the OSMAC strategy. Data will be obtained on the effect of osmotic stress (salinity) and metal cations (Mg2+, Zn2+, Fe3+, Ni2+) on the biosynthesis of low molecular weight secondary metabolites. Data will be obtained on the antimicrobial effect of the isolated compounds both directly against the most typical representatives of gram-positive and gram-negative bacteria and yeast-like fungi (Staphylococcus aureus, Escherichia coli, Candida albicans), and through the inhibition of some specific microbial enzymes (sortase, urease). Data will be obtained on the antitumor activity of the isolated compounds against a number of human prostate cancer cell lines, including drug-resistant ones. The research results will significantly expand the arsenal of natural antibiotic compounds, as well as compounds with antitumor activity against human prostate cancer. Some of these compounds can become the basis for the creation of drugs.

Annotation of the results obtained in 2023
The metabolic profiles of eight ethylacetane extracts of the microscopic fungus Penicillium thomii KMM 4679 (seagrass Zostera marina, Sea of Japan) obtained as a result of cultivation under various conditions according to the OSMAC strategy were studied. Studies of antimicrobial activity have shown that the control extract, as well as extracts obtained under hypersalt conditions, do not affect the growth of gram-negative bacteria E. coli, gram-positive bacteria S. aureus and yeast-like fungi C. albicans. At the same time, S. aureus and C. albicans turned out to be sensitive to the action of extracts obtained during cultivation with metal salts. All the studied extracts turned out to be slightly toxic to both normal human renal embryonic cells NEK 298 and human prostate cancer tumor cells PC-3. The extract of the fungal culture obtained during cultivation with MgCl2*6H2O turned out to be the least toxic to normal NEK 298 cells, while exhibiting cytotoxicity against tumor cells by 12.2%. The mushroom culture extract obtained during cultivation with Ni(NO3)3*6H2O turned out to be the most toxic to normal cells, reducing the viability of NEK 298 cells by 27.9%, while the viability of tumor cells under the influence of this extract was reduced by 15%. While the control extract reduced the viability of normal cells by 24.4% and tumor cells by 8.9%, Extracts of mushroom cultures obtained with the addition of 10 g/l of sea salt and ZnCl2 turned out to be the least toxic against all cell types. The cytotoxicity of extracts obtained with the addition of 20 and 30 g/l of sea salt, as well as Fe(NO3)3*9H2O, ranged from 12.5% to 15% with respect to normal and tumor cells. Using mathematical methods for studying HPLC-UV chromatograms, it was shown that changes in cultivation conditions significantly affected the metabolism of the fungus KMM 4679, while changes were observed both in hypersalt conditions and in samples obtained during cultivation with metal salts. The greatest changes compared to the control sample are observed when metal salts are added. All the analyzed extracts contained the same number of new peaks of molecular ions compared to the control sample, however, their intensity varied. The most intense peaks of molecular ions were found in samples cultured with magnesium and zinc, as well as in samples obtained under hypersalt conditions (with the addition of 20 and 30 g/l of natural sea salt to the culture medium). Due to the use of mathematical approaches for the analysis of HPLC-UV and HPLC-MS chromatograms of extracts of microscopic fungi, a significant effect of cultivation conditions on the metabolism of microscopic fungi has been shown. Changing the cultivation conditions according to the OSMAC strategy allows you to influence the metabolism of microscopic fungi. The metabolic profiles of eight ethylacetane extracts of the microscopic fungus P. thomii KMM 4645 - producer of meroterpenoids, austalides with antitumor and enzyme-inhibiting activity (marine brown algae Sargassum miyabei, Sea of Japan) obtained as a result of cultivation under various conditions according to the OSMAC strategy were studied. The obtained HPLC-MS chromatograms were analyzed by mathematical methods, tables were constructed reflecting the release time of molecular ions and their intensity. It has been shown that the strain of the fungus P. thomii KMM 4645, cultured under various conditions, mainly produces compounds with a molecular weight of up to 500, the largest number of peaks of molecular ions was found in extracts obtained by adding magnesium and nickel salts to the culture medium. The most intense peaks of molecular ions contained an extract obtained by cultivation with the addition of nickel salt, however, the molecular weight of these peaks does not correspond to the meroterpenoids previously isolated from this fungus. The metabolic profiles of ethyl acetate extracts of three microscopic P. antarcticum fungi, including the control strain of P. antarcticum KMM 4685 – producer of meroterpenoids, meroantarctins – inhibitors of p-glycoprotein (marine brown algae S. miyabei, Sea of Japan), obtained on rice medium with the addition of natural seawater, were studied. The obtained HPLC-MS chromatograms were analyzed by mathematical methods, tables were constructed reflecting the release time of molecular ions and their intensity. The analysis of HPLC-MS chromatograms revealed significant differences between the studied extracts of P. antarcticum mushroom strains, it was shown that the KMM 4685 strain is the most promising in terms of the number of molecular ion peaks, the Sm-1328 strain is in second place, and the Sm-1324 strain is of least interest in order to search for new representatives of citreohybridione-type meroterpenoids. Based on the combined results of screening 19 obtained ethyl acetate extracts of the studied four fungal strains, the planned scaled cultivation of the microscopic fungus P. thomii KMM 4679 was carried out on rice medium with the addition of natural seawater and on rice medium with the addition of magnesium salt (MgCl2*6H2O). Also, in excess of the planned, scaled cultivation of the fungus P. thomii KMM 4679 was carried out on a rice medium with the addition of natural seawater and the addition of 20 g/l of natural sea salt. Among the fungus P. antarcticum strain Sm-1328 (S. miyabei) was also selected for further work as a promising producer of new meroterpenoids. The fungus P. thomii KMM 4645 was not selected for further study after the study of the metabolic profiles of its ethyl acetate extracts by HPLC-MS method. 14 secondary metabolites, ten of which turned out to be new representatives of decalin-type polyketides, nine of which were classified as zosteropenillines: 11-α-zosteropenilline M, 8-hydroxy-11-β-zosteropenilline M, zosteropenillines N-S, 8-hydroxy-zosteropenilline G, and one derivative of pallidopenilline A: 13-dehydroxy-pallidopenilline A was isolated from the fungus P. thomii KMM 4679. In addition to the new metabolites, four known ones have been identified: zosteropenillins J and G, as well as pallidopenillin A and its derivative 1-acetyl-pallidopenilline A. The structures of the compounds of all metabolites have been established by single and two-dimensional NMR spectroscopy and high-resolution mass spectrometry. Relative connection configurations are set based on ROESY data and spin-spin constants. The absolute configurations of the asymmetric centers of zosteropenillin Q were established as 4R,5S,8S,9R,10R,13R using the modified Mosher method. To establish the absolute configurations of the new 11-α-zosteropenillin M (4R,5S,8S,10S,11S) and zosteropenillin N (4R,5S,8S,10S,11R), an approach based on the comparison of experimental spectra of electron circular dichroism (ECD) with theoretical calculated averaged spectra using nonstationary theory was applied density functional (TD-DFT). 1-acetyl-pallidopenilline A exhibits cytotoxic activity against human prostate cancer cells PC-3 (IC50 94.20 ± 1.13), inhibits the formation of colonies of these cells by more than 50% in a non-toxic concentration (2.5 μM) and suppresses their proliferation, but does not affect the migration of these cells. When combined with 1-acetyl-pallidopenillin A (20 μM) and the commercial drug doxorubicin (10 μM), the cytotoxic effect on human prostate cancer cells PC-3 increases significantly. 8-hydroxy-11-β-zosteropenillin M (IC50 = 87.29 ± 6.32 μM), zosteropenillin O (IC50 = 82.49 ± 6.51 µm) and zosteropenillin G (79.68 ± 0.30) reduced the viability of hormone-dependent cervical cancer cells Hela to 44.12 ± 5.13 – 34.35 ± 1.90%. 8- hydroxy-11-β-zosteropenillin M and 1-acetyl-pallidopenillin A (IC50 = 71.98 ± 2.48 μM) reduced cell viability of hormone-dependent human breast cancer cells MCF-7 to 57.46 and 23.71%, respectively. The new compounds 11-α-zosteropenillin M, zosteropenillin O, zosteropenillin R and zosteropenylin S at concentrations of 10-20 microns had a cytoprotective effect on human embryonic kidney cells HEK-293 in a model of hypoxia caused by cobalt (II) chloride, increasing cell viability by 30-50% and reducing the level of lipid peroxidation in mitochondria. 11-α-zosteropenillin M, zosteropenillin P, zosteropenillin Q and 1-acetyl-pallidopenillin A (100 μM) inhibited the growth of S. aureus by 30.08, 28.99, 29.95 and 31.65%, respectively. Zosteropenillin O inhibited the growth of C. albicans by 35.31% at 100 μM. Thus, the fungus P. thomii KMM 4679 (seagrass Zostera marina) is a producer of bioactive polyketides of the decalin type. These polyketides can be attributed to the lovastatin type, however, zosteropenilines and pallidopenilines do not have a methyl group at C-4 and were first isolated and published from P.thomii fungi from the Collection of Marine Microorganisms (KMM) of the PIBOC FEB RAS and were no longer isolated from microorganisms from other collections. A detailed study of the pathways of biosynthesis and active biosynthetic clusters in various cultivation conditions will allow the controlled production of the most active metabolites. Two new piperazine derivatives of chelvamides B and C, together with two known metabolites, thio-diketopiperazine saroclazine A and sesquiterpene guaiane type, were isolated from the fungus P. velutinum. The structures of the compounds were determined using a combination of single- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry. X-ray diffraction analysis data were obtained for helvamide B, which allowed us to confirm the assumed relative configuration. The absolute configuration of this compound was determined by comparing the calculated averaged electron circular dichroism (ECD) spectra with experimental spectra. Comparison of the ECD spectra calculated for R,R- and S,S- stereoisomers with the experimental ECD spectrum of the compound made it possible to reliably establish the configuration of helvamide B as R,R. Thanks to computational methods, it was possible to determine helvamide C as a mixture of stereoisomers (R,R and S,S). Helvamide B reduced the viability of hormone-dependent human prostate cancer cells PC3 by 34.03%, while the viability of normal HEK-293 cells decreased by only 16.64%. Helvamide C reduced the viability of PC3 cells by 39.41% and HEK293 cells by 28.42%. These compounds statistically significantly bound DPPH radicals by 4.92% and 4.54%, respectively. Helvamide C significantly suppressed the growth of yeast-like fungi Candida albicans by 31.54%. Yeast-like mushroom C. albicans, which causes nosocomial infection, significantly complicates the treatment of other diseases and recovery after surgical interventions. The data on new anti-candida compounds with low toxicity to human cells are promising. For five extracts of P.velutinum, a heat map was constructed reflecting the content of metabolites in the extracts. The P. velutinum strain from the Collection of Marine Microorganisms (PIBOC FEB RAS) is capable of producing alkaloids in sufficient quantities if there are sufficient nitrogen sources in the nutrient medium.

Publications

1. Borkunov G.V., Popov R.S., Khudyakova Y.V., Leshchenko E.V., БИОТЕХНОЛОГИЧЕСКИЙ ПОТЕНЦИАЛ МОРСКИХ ГРИБОВ РОДА PENICILLIUM 6-Я РОССИЙСКАЯ КОНФЕРЕНЦИЯ ПО МЕДИЦИНСКОЙ ХИМИИ, - (year - 2024)

2. Borkynov G.V., Shlyk N.P., Chingizova E.A., Leshchenko E.V. Новые зостеропениллины и паллидопениллины из морского микроскопического гриба Penicillium yezoense КММ 4679 Издательство ДВФУ, г. Владивосток, - (year - 2024) https://doi.org/https://doi.org/10.24866/7444-5806-5.

3. Leshchenko E.V., Chingizova E.A., Antonov A.S., Shlyk N.P., Borkunov G.V., Berdyshev D.V., Chausova V.E., Kirichuk N.N., Khudyakova Y.V., Chingizov A.R., Kalinovsky A.I., Popov R.S., Kim N.Yu., Chadova K.A., Yurchenko E.A., Isaeva M.P., Yurchenko A.N. New Zosteropenillines and Pallidopenillines from the Seagrass-Derived Fungus Penicillium yezoense KMM 4679 Marine Drugs, Mar. Drugs 2024, 22, 317 https://doi.org/10.3390/md22070317 (year - 2024) https://doi.org/10.3390/md22070317

4. Solovova M.A., Shlyk N.P., Borkunov G.V., Leshchenko E.V. Вторичные метаболиты морского микроскопического гриба Penicilium yezoense КММ 4679 культивированного согласно стратегии OSMAC Издательство ДВФУ, - (year - 2024) https://doi.org/doi.org/10.24866/7444-5806-5

5. Borkunov G.V., Leshchenko E.V., Berdyshev D.V., Popov R.S., Chingizova E.A., Shlyk N.P., Gerasimenko A.V., Kirichuk N.N., Khudyakova Y.V., Chausova V.E., Antonov A.S., Kalinovsky A.I., Chingizov A.R., Yurchenko E.A., Isaeva M.P., Yurchenko A.N. New Piperazine Derivatives Helvamides B–C from the Marine-Derived Fungus Penicillium velutinum ZK-14 Uncovered by OSMAC (One Strain Many Compounds) Strategy Natural Products and Bioprospecting, - (year - 2024) https://doi.org/10.1007/s13659-024-00449-9

6. Borkunov G.V., Chingizova E.A., Leshchenko E.V. Подбор условий культивирования микроскопического морского гриба Penicillium velutinum KMM 4674 с помощью стратегии OSMAC Микробные биотехнологии: фундаментальные и прикладные аспекты: материалы XIII Международной научной конференции, 2023. 466 с. (year - 2023)

7. Leshchenko E.V. Метаболиты морских грибов, ассоциированных с водорослями и травами, и перспективы их дальнейшего изучения при помощи стратегии OSMAC ГЕНЕТИЧЕСКИЕ ТЕХНОЛОГИИ В ИССЛЕДОВАНИЯХ ПРИРОДНЫХ СОЕДИНЕНИЙ. ВСЕРОССИЙСКАЯ НАУЧНАЯ ШКОЛА-КОНФЕРЕНЦИЯ МОЛОДЫХ УЧЕНЫХ И СТУДЕНТОВ, 2023, с. 111 (year - 2023) https://doi.org/10.24866/7444-5579-8

8. Leshchenko E.V., Borkynov G.V., Shlyk N.P., Khudyakova Y.V., Popov R.S. Использование подхода OSMAC с целью поиска новых вторичных метаболитов морских микроскопических грибов XX Всероссийская молодежная школа-конференция по актуальным проблемам химии и биологии, 2023, с. 51 (year - 2023) https://doi.org/10.47471/20_2023_09_04_08_0.-

Annotation of the results obtained in 2022
During our ongoing investigation for the project "Using the OSMAC approach (one strain - many compounds) to search for promising "leading molecules" from marine micromycetes Penicillium thomii" No. » №22-73-00190 from on 20 July 2022 to 15 May 2023 the following studies were carried out: The chromatographic profiles of 146 extracts of the previously studied marine-derived strains of the genus Penicillium obtained via cultivation under osmotic stress and with the addition of metal salts according to the OSMAC strategy were analyzed. The fungal strain P. thomii KMM 4674 (seagrass Zostera marina, Sea of Japan) was reidentification as Penicillium velutinum based on two molecular genetic markers: ITS and β-tubulin regions. BLAST search showed that the gene sequences were 100% identical with the sequences of the ex-type of strain Penicillium velutinum NRRL 2069. Fungal extracts were analyzed by thin layer chromatography and high-performance liquid chromatography with UV detection. It was found that strain KMM 4674 significantly increases the biosynthesis of mid-polar compounds when cultivated with the addition of iron (III), zinc (II), and nickel (II) salts. At the same time, during cultivation with the same salts, the chromatographic profile of P. thomii strain KMM 4645 becomes depleted in the middle polar part, while an increase in the intensity of the peaks in the nonpolar region is observed. Conditions for preparative cultivation of P. velutinum were selected according to the OSMAC strategy. Screening of the 54 selected extracts for their antimicrobial activity against gram-positive bacteria Staphylococcus aureus, gram-negative bacteria Escherichia coli and yeast-like fungi Candida albicans was carried out, and the cytotoxic effect of the 10 selected extracts against tumor cells of the human prostate cancer PC-3 and normal human embryonic kidney cells HEK 293T were assayed. As a result of the cultivation in hyposaline conditions and showing significant antimicrobial activity against S. aureus and E. coli (46.2% and 35.2%, respectively) the extract of the fungus Penicillium thomii KMM 4645 was discover. At the same time, an increase in the cultivation time of this sample to 1.5 months made it possible to record a stronger inhibition of the growth of S. aureus than for three weeks cultivating time. Discover of the 12 extracts of the P. velutinum KMM 4674 and one extract of the P. thomii KMM 4645 were identified with the most pronounced antimicrobial activity against C. albicans, which showed selective inhibition of the growth of yeast-like fungi, while not inhibiting the growth of bacteria. The cytotoxic effect assay of the some obtained extracts showed that they did not have a pronounced cytotoxic effect on cell lines PC-3 and HEK-293 at a concentration of 100 μg/ml. Most extracts reduced the viability of PC-3 and HEK-293 cells by 35-45%. The extract of the P. velutinum KMM 4674 obtained via cultivation in hypersaline conditions reduced the viability of PC-3 cancer cells by 55%, while the viability of normal HEK-293 cells decreased by 85%. The extract obtained via cultivation with iron(III) salt unexpectedly showed minimal cytotoxicity against both cell lines. Chromatographic separation of the ethylacetate extract of the Penicillium velutinum KMM 4674, obtained by cultivation with an iron (III) salt, was carried out to isolate the known guaian-type sesquiterpene together with five unidentified alkaloids, the structure determination of which is planned in 2023–2024. Selected extracts and isolated individual compounds were analyzed by high performance liquid chromatography with mass spectrometric detection. As a result, four known fungal metabolites were identified in the extracts. The extracts also contained previously isolated metabolites from this strain during cultivation on the standard rice medium. Based on the obtained data for all analyzed extracts of the fungus Penicillium velutinum KMM 4674 a heat map was formation, showing the relative content of metabolites in each extract. As a result of the study, an effective large-scale screening technique was mastered to identify promising objects of study, without the preparative cultivation and chromatographic separation of all extracts.

Publications