Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells
Introduction
Cyclin-dependent kinases (CDK) are classic Ser/Thr kinases with molecular weights of 30–40 kDa. This family of enzymes plays an important and well-defined role in cell cycle regulation and proliferation. CDK enzymatic activation requires the binding of specific regulatory subunits, termed cyclins, CDK-cyclin complexes are necessary for phosphorylation of key proteins that regulate the orderly progression through the cell cycle.1, 2, 3, 4 Abnormal activation of various CDKs can ultimately lead to deregulated cell cycle progression, a common feature in many cancers.5, 6 Given the pivotal role that dysregulation of CDK activity plays in cancers, targeting the CDKs is a viable strategy for blocking and/or interfering with tumor cell proliferation.7, 8, 9
Thirteen CDK’s and at least 29 cyclins have been discovered from the human genome10 and have been extensively characterized in regards to controlling cell cycle. Mutations in CDK proteins can result in the overexpression and altered function of CDK’s and specific cyclins. In human tumors, mutations have been reported which result in the specific overexpression of cyclins D1 and E1 and CDK4 and CDK6.11, 12
Mutations in either CDK4 or cyclin D1 are implicated in primary solid tumors including breast, lung, pancreatic, gastrointestinal, head and neck, liver, and prostate. CDK6 is mutated in a smaller number of solid human cancers such as lymphomas, sarcomas and gliomas. CDK2 is rarely mutated in cancer. Aberrant activation of CDK1 has also been observed in a number of primary tumors including breast, colon, prostate, oral and lung.10, 13
Approximately 24 CDK inhibitors are currently in development or clinical trials. These molecules can be classified in at least four general categories including the purines, alkaloids, butyrolactones, and flavonoids.14, 15, 16, 17, 18, 19, 20 Despite differences in chemical structures, most CDK inhibitors competitively inhibit ATP binding at the catalytic site.21, 22 The specificity of the inhibitors is a major concern due to a high degree of sequence similarity within the active site of the CDKs and a large number of other protein kinases. In addition, the ability to monitor intracellular delivery and intratumoral distribution remains difficult. Therefore, development of potent CDK inhibitors that provide an imageable ‘readout’ may be advantageous in the development of clinically effective therapeutic drugs.
The 2,6,9-trisubstituted purine analogs (olomoucine and roscovitine) and the purvalanols have been shown to selectively inhibit a subset of CDK’s.15 These inhibitors act by selectively competing with ATP at its binding site in targeted CDK’s, however purvalanol B has been shown to target CDK2 and CDK1 complexes at submicromolar concentrations.16 Importantly, the carboxylic acid of the 6-anilino group of the purvalanols (Fig. 1A) can be modified without negatively affecting their CDK-inhibitory activity,23, 24 providing a strategy for the development of modified purvalanol B analogs.
Dansyl fluorochromes have been used in tracking small molecules intracellularly.25, 26, 27, 28 By synthetically coupling the fluorescent compound dansyl-ethylenediamine to purvalanol B, two fluorescent analogs of purvalanol B, termed VMY-1-103 and VMY-1-101 were developed (Fig. 1B). These compounds were evaluated for their CDK inhibitory activity and for their in vitro effects in two human breast cancer cell lines (p53-mutated, estrogen-independent MDA-MB-231 cells and p53 wild type, estrogen dependent MCF-7 cells).
Section snippets
Chemistry
The CDK2–purvalanol B crystal structure (PDB ID: 1CKP) revealed that the carboxylic acid of the 6-anilino substituent points outside of the ATP-binding pocket, and does not interfere with interior interactions of the kinase active site.13, 27, 28 Taking advantage of this structural arrangement, we synthetically coupled a fluorescent tag, dansyl ethylenediamine to the carboxylic acid of the 6-anilino substituent of purvalanol B. The newly synthesized fluorescent compounds VMY-1-101 and VMY-1-103
Inhibition of Cdk isoforms activity by VMY-1-101 and VMY-1-103
The inhibitory activities of newly synthesized compounds were evaluated against several cell-free cyclin-dependent kinases. Interestingly, VMY-1-101 and VMY-1-103 were found to be potent competitive inhibitors of ATP as tested against various cell cycle kinases compared to roscovitine. In fact VMY-1-101 and VMY-1-103 showed a greater percent inhibition of CDK2/cyclin E complex as compared to other kinases (Table 1).
VMY-1-101 and VMY-1-103 compounds inhibit the in vitro growth of breast cancer cells or multidrug resistant cells
Based on the potency in the in vitro cell-free enzyme kinase assay, we further
Discussion
Aberrant expression and activation of cell cycle regulatory proteins is a common feature of many cancers. Components of the cell cycle machinery, such as the CDKs, may therefore represent excellent molecular targets for the design of anticancer agents.32 Several classes of CDK inhibitors, including both natural33 and chemically synthesized agents11, 12 have been reported and several are in clinical trials.32 However as because the target specificity of CDK inhibitors is challenging, and in the
Materials
All reagents and solvents were commercially available and used without further purification. Chromatography was performed for purification of final compounds using a Biotage SP-1 system with silica gel cartridges. NMR spectra were recorded on a Varian 400 MR spectrometer at 400 MHz for H-1 and 100 MHz for C-13. Chemical shifts (δ) are given in ppm downfield from tetramethylsilane, and coupling constants (J-values) are provided in hertz (Hz). The purity of final compounds was evaluated by
Disclosure of potential conflicts of interest
A patent application has been filed by Georgetown University on behalf of the inventors that are listed as authors in this article.
Acknowledgements
We thank Dr. Karen Creswell for flow cytometry and Dr. Susette Mueller for microscopy imaging expertise, the core facilities of the Lombardi Comprehensive Cancer Center and the Drug Discovery Program at Georgetown University Medical Center for financial support. We thank Deborah L. Berry for histological staining of the human breast tissue. We thank Dr. Chris Albanese for his valuable scientific discussions.
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