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Importazole, a small molecule inhibitor of the transport receptor importin-?.

ABSTRACT: During interphase, the transport receptor importin-? carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. Importin-? and RanGTP have been implicated in additional cellular processes, but the precise roles of the Ran/importin-? pathway throughout the cell cycle remain poorly understood. We implemented a FRET-based, high-throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin-? and identified importazole, a 2,4-diaminoquinazoline. Importazole specifically blocks importin-?-mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. When added during mitosis, importazole impairs the release of an importin-? cargo FRET probe and causes both predicted and novel defects in spindle assembly. Together, these results indicate that importazole specifically inhibits the function of importin-?, likely by altering its interaction with RanGTP. Importazole is a valuable tool to evaluate the function of the importin-?/RanGTP pathway at specific stages during the cell cycle.

SUBMITTER: Soderholm JF

PROVIDER: S-EPMC3137676 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Importazole, a small molecule inhibitor of the transport receptor importin-β.

Soderholm Jonathan F JF Bird Stephen L SL Kalab Petr P Sampathkumar Yasaswini Y Hasegawa Keisuke K Uehara-Bingen Michael M Weis Karsten K Heald Rebecca R

ACS chemical biology 20110421 7

During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. Importin-β and RanGTP have been implicated in additional cellular processes, but the precise roles of the Ran/importin-β pathway throughout the cell cycle remain poorly understood. We implemented a FRET-based, high-throughput small molecule screen for compoun ...[more]

PMID: 21469738

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Project description:Lipoprotein transport from the inner to the outer membrane, carried out by the Lol machinery, is essential for the biogenesis of the Gram-negative cell envelope and, consequently, for bacterial viability. Recently, small molecule inhibitors of the Lol system in Escherichia coli have been identified and shown to inhibit the growth of this organism by interfering with the function of the LolCDE complex. Analysis of the transcriptome of E. coli treated with one such molecule (compound 2) revealed that a number of envelope stress response pathways were induced in response to LolCDE inhibition. However, Pseudomonas aeruginosa is refractory to inhibition by the same small molecule, but we could demonstrate that E. colilolCDE could be substituted for the P. aeruginosa orthologues, where it functions in the correct transport of Pseudomonas lipoproteins, and the cells are inhibited by the more potent compound 2A. In the present study, we took advantage of the functionality of E. coli LolCDE in P. aeruginosa and determined the P. aeruginosa transcriptional response to LolCDE inhibition by compound 2A. We identified key genes that responded to LolCDE inhibition and also demonstrated that the same genes appeared to be affected by genetic depletion of the native P. aeruginosa LolCDE proteins. Several of the major changes were in an upregulated cluster of genes that encode determinants of alginate biosynthesis and transport, and the levels of alginate were found to be increased either by treatment with the small molecule inhibitor or upon depletion of native LolCDE. Finally, we tested several antibiotics with differing mechanisms of action to identify potential specific reporter genes for the further development of compounds that would inhibit the native P. aeruginosa Lol system.IMPORTANCE A key set of lipoprotein transport components, LolCDE, were inhibited by both a small molecule as well as genetic downregulation of their expression. The data show a unique signature in the Pseudomonas aeruginosa transcriptome in response to perturbation of outer membrane biogenesis. In addition, we demonstrate a transcriptional response in key genes with marked specificity compared to several antibiotic classes with different mechanisms of action. As a result of this work, we identified genes that could be of potential use as biomarkers in a cell-based screen for novel antibiotic inhibitors of lipoprotein transport in P. aeruginosa.

Dataset Information

Importazole, a small molecule inhibitor of the transport receptor importin-?.

Publications

Importazole, a small molecule inhibitor of the transport receptor importin-β.

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