Dataset Information

Identification of a small molecule inhibitor of importin ? mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries.

ABSTRACT: In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin ? with high nanomolar affinity and specifically inhibits importin ?/? mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin ? and the GTPase Ran. As a selective inhibitor of the importin ?/? import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.

SUBMITTER: Hintersteiner M

PROVIDER: S-EPMC2956136 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of a small molecule inhibitor of importin β mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries.

Hintersteiner Martin M Ambrus Géza G Bednenko Janna J Schmied Mario M Knox Andrew J S AJ Meisner Nicole-Claudia NC Gstach Hubert H Seifert Jan-Marcus JM Singer Eric L EL Gerace Larry L Auer Manfred M

ACS chemical biology 20101001 10

In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to id ...[more]

PMID: 20677820

Similar Datasets

Project description:A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Dataset Information

Identification of a small molecule inhibitor of importin ? mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries.

Publications

Identification of a small molecule inhibitor of importin β mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets