Abstract
The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.
Highlights
Cyclin-dependent kinase family (CDKs)3 [6, 7]
The structure of cdk2-cyclin A complexed with the CDK inhibitor proteins (CKIs) protein p27Kip1 has been resolved showing the interaction between the CKI substrate and the complex at the cyclin recruitment motif (CRM)
In an attempt to regulate aberrant cell cycle pathways, new synthetic agents that can restore the functions of altered tumor suppressor proteins, as CKIs, are central targets in current cancer research
Summary
Preparation of Synthetic Combinatorial Libraries and Individual Peptides—The dual defined position library, AcOOXXXX-NH2 was synthesized in an iterative format using the process of divide, couple, and recombine in conjunction with simultaneous peptide synthesis. The GST-pRb protein fragment, fpRb (amino acids 792–928), was used as a substrate In these cases, the reaction was stopped by adding 10 l of Laemmli buffer. The assay was carried out by adding 60 nM of the carboxyfluorescein NBI1-labeled peptide (CF-NBI1) to different concentrations of cyclin A-(171– 432) in PBS buffer and incubating the samples during 20 min at 30 °C before reading. Cells were fixed with 70% methanol for 2 h at room temperature, washed with PBS, and incubated with 50 g/ml of propidium iodide (Sigma) and 200 g/ml RNase for 10 min at room temperature. Cells were fixed with 70% methanol for 2 h at room temperature and washed with phosphate-buffered saline They were incubated with 50 g/ml propidium iodide (Sigma) and 200 g/ml RNase for 10 min. Membranes were blocked in 5% nonfat dry milk in TBS buffer for 1 h at room temperature and incubated with different primary anti-
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