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Checkpoint inhibition and cell cycle effects of 13-hydroxy-15-oxozoapatlin, ent-kaur-16-en-15-oxo-18-9oic acid, and isogranulatimide Rundle, Natalie T.

Abstract

Cell cycle checkpoints are activated in response to DNA damage and cause arrest in G₁ and G₂ phase. Inhibitors of the G₂ checkpoint are sought because they can increase the effectiveness of DNA damaging cancer therapies against cells with mutant p53. These inhibitors also have potential value as tools for biochemical analysis of the checkpoint pathway. Our laboratory conducted a cell-based screen to identify chemical inhibitors of the G₂ DNA damage checkpoint in extracts from terrestrial plants, marine microorganisms, and marine invertebrates. Several new checkpoint inhibitors were identified, including 13-hydroxy-15-oxozoapatlin (OZ), entkaur- 16-en-15-oxo-18-oic acid (OKA), and isogranulatimide (IGR). The goals of my research were i) to study the cell cycle effects of IGR, OZ, and OKA, and ii) to identify their molecular targets, if possible. Most of the checkpoint inhibitors discovered in the cell-based screen were inhibitors of checkpoint protein kinases or protein phosphatases, but a small number acted against unknown targets. OZ is a member of the latter category.-OZ was a potent inhibitor of the G₂ checkpoint (IC₃₀ 6 μM), but did not inhibit the checkpoint kinases ATM, ATR, Chkl, Chk2, or Plkl in vitro. OZ also showed no activity against purified PP1, PP2A, or Ser/Thr protein phosphatases in MCF-7 cell extracts. OKA is very similar in structure to OZ, suggesting that it also does not inhibit protein phosphatases or checkpoint kinases. We hypothesized that OZ and OKA act on a new checkpoint protein. Unlike other checkpoint inhibitors, OZ and OKA are also antimitotic agents. OZ- and OKAtreated cells arrested in a stage resembling prometaphase, in which bipolar spindles formed and chromosomes failed to congress. In contrast to other antimitotic agents, OZ and OKA did not exert a strong effect on tubulin polymerization in vitro. Nevertheless, live cell microscopy demonstrated that chromosome movement was greatly reduced in OZ- and OKA-treated cells. Immunofluorescence microscopy revealed that the intracellular localization of the mitotic motor protein CENP-E and an associated kinase, hBubRl, were altered after OZ or OKA treatment. These results suggest that OZ and OKA interfere with the activity of a mitotic motor, causing a block in chromosome alignment and stalling progression through mitosis. Considering that a target of OZ and OKA could play a role in both the checkpoint pathway and in chromosome congression, we became interested in identifying the molecular target(s) of these compounds. A biotinylated analogue of OKA (B-OKA) was synthesized and retained the principal biological activities of OZ and OKA. B-OKA bound covalently to several target proteins. These were purified by streptavidin affinity precipitation and six B-OKA-binding proteins were identified using mass spectrometry. One of these, RanBP2, was chosen for more detailed studies. RanBP2 and biotinylated proteins were purified from Xenopus laevis egg extracts treated with B-OKA. A biotinylated protein that co-migrated with full-length RanBP2 was detected. Fragments of biotinylated RanBP2 were also precipitated by RanBP2 polyclonal antibody and by streptavidin agarose. These results are consistent with a direct interaction between B-OKA and RanBP2, suggesting that modulation of RanBP2 activity results in checkpoint inhibition and/or prometaphase arrest. Many checkpoint inhibitors block protein kinase activity. IGR shares structural similarity with the protein kinase inhibitor staurosporine. In vitro kinase assays revealed that IGR was a selective inhibitor of GSK-3 p kinase activity. I tested whether checkpoint inhibition by IGR is caused by its action on GSK-3 p. The kinase was over-expressed by transfection, and the response of transfected cells to ionizing radiation was examined by immunofluorescence microscopy and flow cytometry. The checkpoint effects of a closely related compound, didemnimide A, were also examined. The results did not support a role for GSK-3 P in the checkpoint pathway. Taken together, these studies describe the cell cycle effects of two different types of checkpoint inhibitors: i) a type that has antimitotic effects, including OZ and OKA, and ii) the protein kinase inhibitor IGR. This research also exemplifies two different strategies for identifying targets of small molecule inhibitors. In the case of OKA, chemical modification of the compound allowed unbiased screening for interacting proteins from amongst all available cellular targets in vivo. In the case of IGR, a candidate target was selected on the basis of structural information and in vitro data, and tested for checkpoint effects in vivo. This research represents a first and important step in the characterization of novel small molecule inhibitors.

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