The use of the white biotechnology toolkit to edit natural purines for studying their anticancer activity via mTOR pathway.

Purine alkaloids were proven to have significant cytotoxic activity against different cancer cell lines via modulating several cellular pathways, leading to inhibition of cell proliferation and increasing cell death. The search for new potential cytotoxic compounds produced by natural eco-friendly means is of great importance. The microbial transformation of natural purine alkaloids, caffeine (Cf), theophylline (Tp), theobromine (Tb), and theacrine (Tc) via filamentous fungi was explored using Aspergillus versicolor (AUMC 4807), Aspergillus niger (NRRL 328), Cunninghamella echinulata (ATCC 1382), and Penicillium chrysogeneum (ATCC 9480). Nine metabolites were isolated via demethylation, and oxidation reactions, namely; 1.3.7-trimethyl uric acid (M1), theacrine (M2), theobromine (M3), paraxanthine (M4), theophylline (M5), 3-methylxanthine (M6), caffeine (M7), 7-methylxanthine (M8) and 3,7,9-trimethyl uric acid (M9). The structure elucidation of the metabolites was based primarily on 1D, 2D-NMR analyses and HRMS. In vitro cytotoxic activity of metabolites was evaluated against CNS (SNB-75) and melanoma (MDA-MB-435) cancer cell lines. Based on the pharmacophore and structural similarity, mTOR enzyme inhibition assay was carried out, and results were confirmed by molecular docking and molecular dynamic studies using mTOR as the target enzyme. Furthermore, the binding mode of M9 with mTOR was investigated using docking computations. The steadiness and binding affinities of compound M9 in complex with mTOR were estimated and compared to caffeine (M7) over the 100 ns MD course. Results confirmed that M9 has great potential as a cytotoxic agent with experimentally proved safety that can be produced by biotransformation.