In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica

Abstract

Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development.

Keywords: Alpinia mutica; UCK2; alpinetin; amino acid active site residues; flavokawain B; in silico; nucleoside analogues.

Figure 1

Percentage cell viability of HT 29 cells treated with 5FU, FKB, and APN. MTT assay was used to determine the IC₅₀ of the drugs at different concentrations in µM for 72 h.

Figure 2

In silico redocking of UCK2 protein in complex with inhibitor CTP. (a) Complete x-ray structure of UCK2 receptor protein shown as a cartoon; (b) Amino acid residues in the active site of UCK2; (c) Interactions of CTP with UCK2 as identified by in silico docking analysis. CTP shown in ball-and-stick model with purple indicating carbon atoms, white for hydrogen, blue for nitrogen, red for oxygen, and phosphorus in orange.

Figure 3

(a) Interactions of ligands with UCK2 protein as identified by in silico docking analysis; (b) Ligands interacting with the amino acids residues in the active sites of UCK2 protein; (c) Surface representation of the hydrophobic contacts of bound ligand and the ligand binding pocket of UCK2 protein shown as translucent blue surface. Ligands shown as a ball-and-stick model with purple indicating carbon atoms, white for hydrogen, and red for oxygen Hydrogen bonds shown in green dotted lines, electrostatic in yellow, and hydrophobic in purple.

Figure 3

(a) Interactions of ligands with UCK2 protein as identified by in silico docking analysis; (b) Ligands interacting with the amino acids residues in the active sites of UCK2 protein; (c) Surface representation of the hydrophobic contacts of bound ligand and the ligand binding pocket of UCK2 protein shown as translucent blue surface. Ligands shown as a ball-and-stick model with purple indicating carbon atoms, white for hydrogen, and red for oxygen Hydrogen bonds shown in green dotted lines, electrostatic in yellow, and hydrophobic in purple.

Figure 4

Comparison of the binding mode of UCK2 inhibitors in the ligand binding pocket of UCK2 surface. (a) FKB shown in purple color and CTP in maroon color; (b) APN shown in purple color and CTP in maroon color.

Figure 5

UCK2 enzyme activity was measured based on the amount of ADP produced from the enzyme reaction (a) Cell lysate incubated in the presence of FKB (b) Cell lysate incubated in the presence of APN. Florescence intensity (λ_ex = 450 nm/λ_em = 590) was monitored using a fluorescence plate reader. Results were expressed as mean ± SD of three independent experiments, * p < 0.05, ** p < 0.01, ns: non-significant when compared to the control.

Figure 6

Skeletal structures of flavokawain B and alpinetin.