High troughput screen for small molecule inhibitors of Ran regulated functions

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A number of the spindle assembly factors (SAFs) which are regulated by Ran GTPase during mitosis have variety of well recognized and intensively studied (but often not well understood) connections to cancer: BRCA1, HURP, TPX2, Aurora A, TACC3, survivin, RHAMM, cdk11. Also the levels of Ran are highly increased in many human tumors and unlike in non-cancer derived cells, the growth of cancer-derived cells is inhibited by Ran RNAi. Arguably the best characterized mechanism potentially linking Ran to cancer is the RanGTP, importin beta and importin alpha1 regulating the activation of Aurora A through its binding to TPX2. RanGTP is required for the release of TPX2 from its inhibitory complex with importin alpha1-importin beta. The importin-freeTPX2 binds Aurora A, protecting it from dephosphorylation and thus supporting sustained Aurora A kinase activity towards is many mitotic targets. More than 30 Aurora A kinase inhibitors are being developed as potential cancer therapeutics and several of them entered phase I and II clinical trials. However, the deletion of Aurora A gene is lethal in embryos and the heterozygous mice develop significantly higher number of tumors and aneuploidy. Complete inhibition of the Aurora A could therefore potentially induce de novo aneuploidy and cancer. Instead, we propose developing inhibitors which would prevent local activation of TPX2/Aurora A pathway by RanGTP gradient, thus potentially decelerating Aurora A activation and mitosis progression. Because such inhibitors would likely have similar effect on many other Ran-regulated mitotic pathways, their effect could be sufficiently strong to prevent mitosis in cells displaying dependence on steep mitotic RanGTP gradient. In 2010 we obtained competitive R03 funding from NIH Roadmap Initiative to carry two quantitative HTS format screens (qHTS) screens for small molecule inhibitors of Ran-regulated importin alpha1/importin beta interaction. The primary screens among 340 000 compounds, each at 5 different concentrations were completed in May 2011 at NIH Chemical Genomics Center (NCGC), Rockville, MD. In Assay A, we were screening for inhibitors of importin alpha1- importin beta binding and in Assay B for inhibitors of RanGTP-induced dissociation of the importin alpha from importin beta. After bioinformatics analysis and confirmation screens, the initial set of 3500 primary hits was narrowed down to a subset of highest quality candidate inhibitors: 104 in Assay A and 242 candidate inhibitors in Assay B. Cherry picking among the primary hits increased this set of inhibitors worthy further analysis to approximately 1000. Currently we are working on orthogonal secondary chemiluminescence screens to identify specifically acting inhibitors. In progress are also screens for biological activity of the candidate inhibitors where we are monitoring their effects on mitotic spindle assembly in tissue culture cells and on nuclear import of calcium-regulated NFAT- GFP nuclear transport reporter. The secondary assays are currently being optimized in a low throughput format in our lab at NCI and we then plan to carry them at a higher throughput format at NCGC with the entire set of 1000 candidate inhibitors. Our objective is to identify specifically acting inhibitory compounds which display the expected biological activity. This data is required to secure R03 funding for the second year of the project where we will focus on the best identified inhibitor(s) and analyze their mechanism of action and optimize their performance based on structure-activity relationship analysis.