Specific inhibition of cyclin-dependent kinases and cell proliferation by harmine

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Abstract

As key regulators of the cell proliferation cycle, cyclin-dependent kinases (CDKs) are attractive targets for the development of anti-tumor drugs. In the present study, harmine was identified from a collection of herbal compounds to be a specific inhibitor of Cdk1/cyclin B, Cdk2/cyclin A, and Cdk5/p25 with IC50 values at low micromoles. It displayed little effect on other serine/threonine and tyrosine kinases tested. The CDK inhibition by harmine is competitive with ATP-Mg2+, suggesting that it binds to the ATP-Mg2+-binding pocket of CDKs. In cytotoxicity assays, harmine exhibited a strong inhibitory effect on the growth and proliferation of carcinoma cells whereas it had no significant effect on quiescent fibroblasts. Further, harmine was found to block DNA replication in the carcinoma cells. Taken together, harmine is a selective inhibitor of CDKs and cell proliferation.

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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.

1

These authors contributed equally to this work.

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