Identification of a Small Molecule Inhibitor of Importin β Mediated Nuclear Import by Confocal On-Bead Screening of Tagged One-Bead One-Compound Libraries

Abstract

In eukaryotic cells, proteins and RNA 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 β with high nanomolar affinity and specifically inhibits importin α/β 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 β and the GTPase Ran. As a selective inhibitor of the importin α/β import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.

Importin β1 Protein-mediated Nuclear Localization of Death Receptor 5 (DR5) Limits DR5/Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Cell Death of Human Tumor Cells

“…siRNA and shRNA have been reported to be effective in silencing the gene in vivo, and phase I-III clinical studies have already been initiated against various diseases including cancers (43). Therefore, tumor-specific delivery of importin ␤1 siRNA or a recently identified small molecule inhibitor of importin ␤-mediated nuclear import (44) may be useful in applications that will improve the antitumor effects of recombinant TRAIL (dulanermin) and anti-DR5 mAb (lexatumumab) for the therapeutic treatment of human cancers.…”

The G ProteinαChaperone Ric-8 as a Potential Therapeutic Target

“…At least two precedents have been set for blocking the Importin-b/Ran PPI with proven cellular effects (Hintersteiner et al, 2010;Soderholm et al, 2011). A small molecule inhibitor screen of the Importin-b PPI with Ran identified "karyostatin 1A" that blocked regulated nuclear import with micromolar affinity (Hintersteiner et al, 2010;Soderholm et al, 2011).…”

“…At least two precedents have been set for blocking the Importin-b/Ran PPI with proven cellular effects (Hintersteiner et al, 2010;Soderholm et al, 2011). A small molecule inhibitor screen of the Importin-b PPI with Ran identified "karyostatin 1A" that blocked regulated nuclear import with micromolar affinity (Hintersteiner et al, 2010;Soderholm et al, 2011). A second Importin-b/Ran small molecule-inhibition screening strategy identified 2,4-diaminoquinazoline, termed "Importazole", as an inhibitor of Importin-b-mediated nuclear import and mitotic spindle assembly (Soderholm et al, 2011).…”

Targeting the Nuclear Import Receptor Kpnβ1 as an Anticancer Therapeutic

“…Ivermectin is a broadspectrum antiparasitic reagent recently found to inhibit Kpna/b, but it does not appear to block import mediated by Kpnb1 alone (14). Karyostatin 1A (15) and importazole (16) are the first novel small molecule inhibitors of Kpnb1 to be identified, which inhibit RanGTP binding to Kpnb1; however, their off-target effects have not yet been examined, nor have their anticancer effects. There thus remains a need for the identification of novel effective Kpnb1 inhibitors and for these to be tested for their effects on cancer cells.…”