7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent.

Here, we have identified the interaction site of the contraceptive drug gamendazole using computational modeling. The drug was previously described as a ligand for eukaryotic translation elongation factor 1-α 1 (eEF1A1) and found to be a potential target site for derivatives of 2-phenyl-3-hydroxy-4(1 H)-quinolinones (3-HQs), which exhibit anticancer activity. The interaction of this class of derivatives of 3-HQs with eEF1A1 inside cancer cells was confirmed via pull-down assay. We designed and synthesized a new family of 3-HQs and subsequently applied isothermal titration calorimetry to show that these compounds strongly bind to eEF1A1. Further, we found that some of these derivatives possess significant in vitro anticancer activity.

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Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the "bench" scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.

Currently, we are testing chemical libraries of quinolinone derivatives, compounds with high selective cytotoxic activity. These compounds, derived from quinolone antibiotics, are well known for their antibacterial, antiprotozoal, cytotoxic and immunosuppressive effects. 2-phenyl-3-hydroxy-4(1H)-quinolinones were tested by MTT assay for cytotoxic activity and some of them proved IC50 values in sub-micromolar concentrations. Cell cycle and DNA/RNA synthesis were further analysed by flow cytometry. The accumulation of cells was observed predominantly in G1 phase together with significant DNA and RNA synthesis inhibition. The most active derivative was tested in in vivo cancer mice model with using hollow fibres. Hollow fibres were filled by leukemic CEM cell line and were implanted subcutaneously and intraperitoneally to mice. Afterwards, mice were treated by tested compound. Hollow fibres were after 14 days taken from mice and surviving cells were tested by cytotoxic MTT assay. activity of tested quinolinone was comparable to doxorubicin. For protein targets identification, we have synthesized biotinylated molecules of original derivatives. Biotinylated derivatives were immobilized on the surface of streptavidin coated magnetic beads and whole complex (magnetic bead-streptavidin-biotin-quinolinone) was incubated with total cellular lysate of the CEM cell line. Thereafter non-bonded proteins were washed-out and specifically bonded proteins were separated on SDS-PAGE gel. Following silver-staining of the gel we identified sample lines with specifically bonded proteins to biotinylated compound and negative control. Specific proteins were cut off from the gel and trypsinized to get peptides for protein identification by MS analysis (LC-MS/MS and LC-MALDI). Identified targets are proteins associated with translational regulation, glucose metabolism and cytoskeleton. Potential targets were validated by western blot and by other assays (reporter cell models, kinase and enzymatic assays, evaluation in vitro transcription and translation). The work was supported by grants: LC07017, CZ.1.07/2.3.00/20.0009 coming from European Social Fund. Infrastructural part of this project (Institute of Molecular and Translational Medicine) was supported from the Operational Programme Research and Development for Innovations (project CZ.1.05/2.1.00/01.0030). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4748. doi:1538-7445.AM2012-4748