Metabolomic profiling and cytotoxic potential of three endophytic fungi of the genera Aspergillus, Penicillium and Fusarium isolated from Nigella sativa seeds assisted with docking studies

Abstract The main aim of our study is to investigate the anticancer potential of our cultivated entophytic fungal strains from Nigella sativa seeds. The strains were identified by sequencing of the partial 18S rRNA gene and the internal transcribed spacer (ITS) region as Aspergillus sp. (SA4), Penicillium sp. (SA5), and Fusarium sp. (SA6). We carried out metabolic profiling for three fungal strains to investigate their metabolites diversity. Profiling of the different extracts revealed their richness in diverse metabolites and consequently fourteen compounds (1–14) were annotated. In addition, the obtained extracts were examined against three cell lines HepG2, MCF-7 and Caco-2 showed activity with IC50 values in the range of 1.95–39.7 μg/mL. Finally, molecular docking study was performed showing questinol as the lowest glide binding score value (-5.925 kcal/mol) among all identified compounds. Our results showed Nigella sativa-associated endophytes as a promising source for further studies to look for anticancer secondary metabolites. Graphical Abstract


Introduction
Cancer is one of the serious problems lead to high mortality rates in the world (Arbyn et al. 2020). It was reported that the total world death from cancer is about 7.6 million and this number is predicted to increase to 13.1 million in 2030 (Latosi nska and Latosi nska 2013). Treatment by herbal plants potentially can be used as an alternative medicine without severe side effects with a wide range of pharmacological applications (Srivastava et al. 2019). Endophytic fungi can be considered as a source of chemically novel bioactive secondary metabolites belonging to different chemical classes (Attia et al. 2020;El-Hawary et al. 2021;Sayed et al. 2022) such as steroids, terpenoids, quinones, alkaloids, flavonoids, phenolic acids and others with unique structures and numerous pharmacological properties (Gouda et al. 2016). Nigella sativa is an annual flowering plant which belongs to the family Ranunculaceae and is innate in Southwest Asia, Southern Europe, North Africa but it is cultured and used in different parts of the world (Datta et al. 2012;Gholamnezhad et al. 2015). Bioactive metabolites from Nigella sativa seeds have beneficial effects against various cancer types, including blood cancer (El-Mahdy et al. 2005), hepatic cancer (Thabrew et al. 2005), colon cancer (Salim and Fukushima 2003), pancreatic cancer (Ahmad et al. 2021), prostate cancer (Ansary et al. 2021), and breast cancers (Korak et al. 2020). Cancer and various infectious diseases are threatened human health over the worldwide (Guo et al. 2008;Vasundhara et al. 2019). Endophytic fungi able to produce bioactive compounds that have promise as potential tools that could be useful in regards to safety and human health concerns, although there is still a significant demand from discovery for promising anticancer agents from natural source. The current study describes the isolation and identification of three endophytic fungi, strains SA4, SA5, and SA6 from Nigella sativa seeds. The strains were identified as Aspergillus sp., Penicillium sp. and Fusarium sp. using phylogenetic marker genes. The isolated fungal strains were cultivated and their chemical profiles were explored by LC-MS-based metabolomics. Moreover, the anticancer potential of our strains against three cancer cell lines (HepG-2, MCF-7, and Caco-2) was evaluated. In order to explore the possible anticancer mechanism, an in silico study was performed. Docking scores and interactions were compared to the cocrystallized ligands.

Results and discussion
The three fungal strains SA4, SA5 and SA6 were isolated from the Nigella sativa seeds as endophytes and identified based on partial 18S rRNA gene sequences (approx. 1000 nt) and the internal transcribed spacer (ITS) region as Aspergillus sp., Penicillium sp., and Fusarium sp., respectively (see supplementary data). To investigate the chemical diversity of metabolomes of the three strains against different media conditions, the crude culture extracts were subjected to the high-resolution mass spectrometry (HR-MS) analysis. Metabolic profiling was carried out for all extracts of the three strains results in annotation of 14 compounds as shown in Table S1, Figure S2 (1-4), (5-11) and (12-14) from strains SA4, SA5 and SA6, respectively. Moreover, investigating the cytotoxic potential of different extracts against a number of cancer cell lines (HepG-2, MCF-7, and Caco-2) using the MTT viability assay showed that the crude extract of strains SA4 and SA6 cultured on PDA medium exhibited the highest inhibitory activity against (HepG-2, MCF-7, and Caco-2) cells, with IC 50 values of 5.69, 8.09, and 1.95 mg/mL 16.9, 3.13, and 10.4 mg/mL respectively, while the crude extract of strain SA5 cultured on PDA medium displayed moderate cytotoxic effects against the studied cell lines with IC 50 values of 39.7, 22.7, and 5.78 mg/mL, respectively. To analyze the significant antiproliferative effect of isolated compounds from strains SA4 and SA6, the X-ray crystal structure of human Su(var)3-9, enhancer of Zeste, Trithorax (SET)/inhibitor 2 of protein phosphatase 2 A (I2PP2A) oncoprotein; (PDB: 2E50) (Shady et al. 2021) was selected for molecular modeling studies using computational program Schr€ odinger Small Drug Discovery Suite 2021-2 software (Hisham et al. 2022). SET is a multifunctional oncoprotein that plays a role in cell cycle progression, cell migration, apoptosis, transcription, and DNA repair, among other things (Gadallah et al. 2022). Overexpression of SET oncoprotein contributes to cancer progression (which is also known as an inhibitor to tumor suppressor protein phosphatase 2 A (PP2A) (Gadallah et al. 2022). As a result, inhibiting SET would serve a significant and effective role in inhibiting the growth of cancerous cells. D-erythro(e)-C18 ceramide was used as a flexed ligand in induced fit docking because to its better affinity for binding to SET protein (De Palma et al. 2019). The glide scores of compounds isolated from endophytic fungi species against the SET oncoprotein active site are summarized in Table S3.
From data presented in the table, fortunately all isolated compounds' glide score values are higher than reference D-e-C18 ceramide. Questinol showed the lowest glide binding score value (-5.925 kcal/mol) among all isolated compounds. It revealed four hydrogen bond interaction through two hydroxyl groups via Glu 57 (1.71 Å), Arg 64 (2.46 Å) and Glu 114 (1.92 Å) amino acid residues. Additionally, oxygen atom of methoxy group formed H-bond with Trp 213 amino acid residue (1.87 Å). In addition, it had a hydrophobic contact via Arg 64 via the phenyl moiety of questinol (Figure 1). Strain SA6 participated to have catenarin that has the lower glide energy À5.653 kcal/ mol among other isolated compounds and may possess their potent antiproliferative activity against HepG2, MCF7 and Caco-2.

Experimental
All experimental information are discussed in detail in the supplementary information.

Conclusion
Metabolic profiling of the crude extracts of the fungal endophytic strains Aspergillus sp. SA4, Penicillium sp. SA5 and Fusarium sp. SA6 isolated from the Nigella sativa seeds showed their richness in several classes of metabolites, e.g. polyketides, benzenoids, quinones, and alkaloids. Exploring the metabolic differences of these fungi cultivated on different media revealed the evident potential of the applied OSMAC approach (one strain many compounds) is used to find out the most convenient medium to induce the production of diverse microbial secondary metabolites. OSMAC approach as a powerful tool for the discovery of new microbial secondary metabolites. Moreover, different extracts from all fungal strains exhibited different in vitro inhibitory potencies against HepG-2, Caco-2, and MCF-7 tumor cells. Molecular modelling of identified dereplicated compounds on SET active site showed that questinol and catenarin have lower glide energy and may possess their potent antiproliferative activity against HepG2, MCF7 and Caco-2 cell lines.