Abstract #5353: MKC-1 interacts with multiple protein targets: Characterization by induced fluorescence

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO MKC-1 is a clinical-stage orally active cell-cycle inhibitor that exhibits antiproliferative activity through G2/M arrest, apoptosis, and antiangiogenic effects. MKC-1 binds to tubulin and inhibits microtubule formation and, in addition, binds to members of the importin-\#946; family. Binding has been shown by the property of induced fluorescence, whereby MKC-1 exhibits no appreciable fluorescence properties in aqueous solution but fluoresces intensely when dissolved in organic solvents or when bound by target proteins. Here, MKC-1 induced fluorescence has been exploited to determine the binding site of the compound to target proteins. MKC-1 was shown to bind to the colchicine site of tubulin, through competition with colchicine and other colchicine-site binding drugs such as 2-methoxyestradiol and combretastatin A4, and the absence of competition with vincristine. Importin-\#946;s belong to a superfamily of proteins containing HEAT-repeats, which are tandemly repeated sequences with helical topology approximately 50 amino acids in length. The MKC-1 binding region of importin-\#946;1 was determined by taking advantage of this modular architecture and generating GST-tagged recombinant fragments of the protein. MKC-1 binds to the C-terminal portion of importin-\#946;1, between residues 500-876, as a GST-tagged fragment comprised of these residues induced MKC-1 fluorescence at 1.3-fold the intensity of the full-length protein, whereas recombinant fragments not containing these residues induced MKC-1 fluorescence at levels 0.25-fold or lower of that of full-length GST-importin-\#946;1. Furthermore, these results, along with fluorescence titration experiments, show that MKC-1 binds to importin-\#946;1 at a single, distinct site, despite importin-\#946;1 being comprised of 19 HEAT-repeat units. Primary sequence homology amongst HEAT-repeat family members suggested that MKC-1 may bind other HEAT-repeat proteins, including the key oncological target mammalian target of rapamycin (mTOR). Indeed, MKC-1 does inhibit signaling through the PI3K-Akt-mTOR signaling pathway. Analogous to the importin-\#946;1 experiments, the binding of MKC-1 to mTOR is being investigated through recombinant fragments of the protein; MKC-1 unequivocally does not bind to the FK506/rapamycin binding domain of mTOR. The fluorescence properties of MKC-1 are also exploited to determine the cellular localization of the compound, suggesting the nature of intracellular targets. This property of induced fluorescence could also be used to search for new MKC-1 targets. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5353.