Biochemical and Biophysical Research Communications
Specific inhibition of cyclin-dependent kinases and cell proliferation by harmine☆
Section snippets
Materials and methods
Protein kinase assay. The kinase activities of CDKs were measured using a protocol described previously [12]. Assay mixtures contained 30 mM Mops, pH 7.4, 10 mM MgCl2, 15 μM [γ-32P]ATP (∼500 dpm/pmol), and 100 μM of the substrate peptide HS(9–18) (Pro-Lys-Thr-Pro-Lys-Lys-Ala-Lys-Lys-Leu), harmine (Sigma) at concentrations as specified, and CDK enzymes. Reactions were performed at 30 °C for 10 min. Phosphate incorporation into the substrate peptide was measured by scintillation counting. Cdk5/p25 was a
Specific CDK inhibition by harmine
We conducted inhibitor screens to target Cdk2/cyclin A and Cdk5/p25 from compounds isolated from medicinal herbs that are used in cancer therapeutic formulas. From the screens, harmine (Fig. 1) was identified to exhibit strong inhibition towards the targets. The harmine effect was then carefully examined using Cdk1/cyclin B, Cdk2/cyclin A, and Cdk5/p25. It displayed dose-dependent inhibition of the tested CDKs (data not shown). Under the assay condition using 15 μM ATP-Mg2+, IC50 values are
Discussion
The importance of CDKs in cell proliferation has stimulated a great interest in the development of CDK inhibitors. To date, several classes of CDK inhibitors have been reported, including staurosporine, butyrolactone I, flavopiridol, indirubins, paullones, hymenialdisine, and a series of 2,6,9-substituted purine derivatives such as olomoucine, roscovitine, and purvalanol [17], [18]. These compounds exert their inhibitions by competing with ATP-Mg2+ for the ATP-binding pocket of CDKs. In the
Acknowledgements
We thank Dr. Nam Sang Cheung for his critical reading and comments of the manuscript. This work was supported in parts by the Research Grants Council of Hong Kong, the Area of Excellence Scheme established under the University Grants Committee of Hong Kong (AoE/B-15/01), and the Agency for Science, Technology and Research of Singapore.
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Abbreviations: CDK, cyclin-dependent kinase; ATP, adenosine 5′-triphosphate; Erk, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PKA, cAMP-dependent protein kinase; PKC, protein kinase C.
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These authors contributed equally to this work.