High troughput screen for small molecule inhibitors of Ran regulated functions

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Background and rationale In the interphase, Ran GTPase acts as a key regulator of the nucleo-cytoplasmic transport through the nuclear pore channel which is mediated by RanGTP interaction with NTRs of the importin beta superfamily. The loading of nuclear localization signal (NLS) protein cargos on the NTR adaptor protein importin alpha requires that the N-terminus of importin alpha binding to importin beta. The importin alpha-importin beta-NLS cargo complex then transports to the nucleus where its dissociation is induced by RanGTP binding to importin beta. In mitosis, the dynamic RanGTP-regulated loading and unloading of cargos continues after the nuclear envelope is disassembled. NLS cargos that at the same time serve as spindle assembly factors (SAFs) are inhibited by importin a/b binding and locally activated by RanGTP gradient surrounding chromosomes. Thus, the RanGTP gradient has an essential role as the regulator of mitotic spindle assembly. Out of 5 importins alpha isoforms in humans, only importin alpha 1 is known to participate in mitotic SAF regulation. Interestingly, majority of proteins that are known to be involved in importin alpha1 mitotic regulation (acting downstream or required for function) are at the same time recognized cancer-related proteins (BRCA1, HURP, hTOG, TACC, TPX2, Eg5), some are suspected to function as cancer mitogens (TPX2, Aurora A) and at least one of them, Aurora A (subjected to RanGTP- importin alpha1/importin beta- TPX2- dependent activation) is considered as promising cancer treatment target (phase II clinical trials for Aurora A inhibitors were reported). The available molecular structures suggest that the interface of importin alpha 1-importin beta in their complex provides a unique molecular surface, supporting the feasibility of importin isotype-specific compound development. Project summary We propose to develop compounds specifically targeting RanGTP-regulated function of importin alpha 1 using previously published and well characterized FRET-based biosensor for RanGTP-induced importin alpha 1 - importin beta dissociation. Towards this goal, we redesigned this biosenso to obtain a 5 times wider on-off FRET signal amplitude. Next, we developed a 1536-well plate- based assay protocol for quantitative high throughput screening (qHTS) in small molecule libraries of the NIH Chemical Genomics Center (NCGC). The validation assays at NCGC suggested that our assay is sufficiently robust (z= 0.67) and suitable for screening the complete set of chemical libraries available at NCGC. Presently we are developing a modified version of the assay to search for compounds targeting several steps in the regulated importin alpha 1- importin beta interaction in one screen. The identified compounds will be subjected to a battery of secondary screens to define their molecular mechanism of action. Further optimization of the compounds will be guided by the structure-activity relationship identification achieved by the analysis of the screen data. We plan to analyze the effects of the identified compounds on mitosis progression and cell growth of cancer-derived cells (NCI-60). The key aspect of importin alpha1/importin beta complex targeting in cancer cells would be dynamic suppression of a group of cooperatively acting mitotic activators, as compared to their complete individual inhibition. We expect that the growth of cancer cells that are dependent on Ran would be suppressed. At the same time the residual levels of Aurora A activity which is required for ploidy maintenance in normal cells would be preserved. In the future, we expect that the strategy of our assay could be used to develop compounds that would specifically target all existing importin alpha isoforms separately.