GC/MS characterization and computational kinome-wide screening of pomegranate fruit extract identifies key phytochemicals interacting to CDK kinases implicated in acute myeloid leukemia cells.

Punica granatum (Pomegranate fruit) and its constituents are proven effective against various cancer types. However, a kinome-wide screening for the active phytochemicals against kinases is not reported. This study aims in validating pomegranate fruit extract (PFE) against acute myeloid leukemia (AML) cells, and computationally identifying the phytochemicals interacting with active kinases. PFE was made with Soxhlet extractor using absolute ethanol. Gas-chromatography-mass spectroscopy (GC-MS) for phytochemical identification and MTT assay for cytotoxicity in AML (THP-1, TF-1 and HL-60) cells were performed. Apoptosis, CDK5 and CDK8 were assessed with flow cytometry. Kinase profiling was performed using In silico kinome screening. GC-MS analysis revealed 38 bioactive phytochemicals in PFE including pyrazoles, aldehydes, phenols, esters, pyranosides, and octadecadienoic acids. The extract inhibited the AML cell proliferations with GI values of 195.5 μg/ml, 289.1 μg/ml, and 353.5 μg/ml in THP-1, THP-1, and HL-60 cells, respectively. PFE also exhibited a dose-responsive increase in apoptotic cell populations when treated to the AML cells. Computational screening and modeling predicted three critical constituents, viz., Deoxyartemisinin, 3-Methyl-3-phenyl-3H-indazole, and 8-fluoro-5,6-dimethoxy-3,4-dihydro-2H-naphthalen-1-one of pomegranate extract to interact mainly with cyclin-dependent kinases, including CDK5 and CDK8. Proteinand ligand docking predicted binding energies, and binding pose for top candidate lead molecules. In vitro assay exhibited the anticancer properties of PFE in AML cells. Computational kinome screening predicted top three PFE constituents targeting CDKs which may be responsible for the demonstrated anticancer efficacy of the extract against AML. This hypothesis further aligned with observed efficacy of PFE to inhibit CDK5 and CDK8 in all AML cells tested. PRACTICAL APPLICATIONS: Though Punica granatum (Pomegranate fruit) and its constituents are proven effective against various cancer types, a kinome-wide screening for the active phytochemicals against kinases is not reported. In this study, we have conducted GC/MS characterization of the active phytochemicals of PFE and have performed a kinome-wide screening for all the 38 identified compounds toward 310 active kinases commonly expressed in cancers. These observations warrant isolation and further evaluation of these phytochemicals or their analogues as effective CDK inhibitors against AML proliferation. Further, the computational methods used in this study will throw light on literature for new options of kinome panel screening of active phytochemicals or small molecules.