Small molecule peptidomimetic inhibitors of importin α/β mediated nuclear transport

Abstract

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Translocation of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors of the β-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhibitor of the exportin CRM1, has proved to be an invaluable tool for cell biologists, but up to now no small molecule inhibitors of nuclear import have been described. We devised a microtiter plate based permeabilized cell screen for small molecule inhibitors of the importin α/β pathway. By analyzing peptidomimetic libraries, we identified β-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/β but not by transportin. Structure-activity relationship analysis showed that large aromatic residues and/or a histidine side chain are required for effective import inhibition by these compounds. Our validated inhibitors can be useful for in vitro studies of nuclear import, and can also provide a framework for synthesis of higher potency nuclear import inhibitors.

Figure 1. Permeabilized cell nuclear import assay adapted to 96-well plate format

A. Schematic diagram of permeabilized cell nuclear import assay. Soluble cytosolic components are released after the plasma membrane is perforated by the glycoside digitonin, which leaves the NE and ER membranes intact. An ATP regenerating system, GTP, nuclear transport factors and labeled cargo is added exogenously. The fluorescence accumulated in the nucleus is quantified by light microscopy. B. Fluorescence microscopy images of permeabilized cells following importin α/β mediated nuclear import. Negative control wells contained GTP and for postitive control wells, GMP-PNP was added to achieve complete inhibition of import. Nuclei were stained with Hoechst 33342, and the DNA dye and Alexa555-BSA-NLS cargo were visualized after cell fixation. Scale bar is 10 μm. C. Dose response curve of wheat germ agglutinin (WGA), a well-characterized protein inhibitor of nuclear import, as measured in the permeabilized cell nuclear transport assay.

Figure 2. Hit identification and validation

A. Flowchart for the identification and validation of nuclear import inhibitors of the importin α/β pathway from compound mixtures. B. Relative nuclear import values of a compilation of negative and positive control wells taken from all of the plates screened (10 negative control wells and 6 positive control wells per plate). The average z′ factor derived from the primary screens was 0.5 ± 0.2. C. Relative nuclear import levels of screened compound mixtures. Compound mixtures with over 50% inhibition were considered primary hits and are highlighted in black.

Figure 3. Counter-screen for pathway specificity

A. Nuclear integrity test of confirmed hits. Control wells were either treated with 0.005% digitonin permeabilizes the plasma membrane but leaves the NE intact, or with 0.1% digitonin that permeabilizes both. Confirmed hits were incubated with cells permeabilized with 0.005% digitonin together with anti-lamins A/C. Immunofluorescence using antibodies against lamins A/C detects cells where the NE integrity is breached, as seen by strong nuclear rim labeling. Only a nonspecific “haze” of cytoplasmic background was obtained in cells with intact nuclei. Compounds such as 58E9 (right panels) were eliminated from further investigation. Scale bar is 10 μm. B. Fluorescence microscopy images of permeabilized cells following transportin mediated nuclear import. Positive control wells contained WGA to inhibit import. Nuclei were stained with Hoechst 33342 and Alexa555-GST-M9 cargo was visualized directly. Scale bar is 10 μm.

Figure 4. Structural features and activity of β-turn mimetic compound mixtures

A. Scaffold structure of β-turn mimetics and classification of β-turn mimetic compound mixture inhibitors of importin α/β mediated nuclear import. Each β-turn mimetic compound mixture contains a specific R_i+2 and R_i+3 group and a mixture of 20 different structures at the R_i position. Due to the scaffold's C₂ symmetry the R_i+2 and R_i+3 groups are interchangeable. Confirmed and validated β-turn mimetic compound mixture inhibitor hits are represented by a black square on the heat map. Redundant structures resulting from the scaffold's C₂ symmetry are in light gray. Please, refer to Supplemental Figure S2 for side chain nomenclature. B. Average % inhibition over the 20-compound mixtures of the complete β-turn mimetic library, which contain the specified side chain in the R_i+2/R_i+3 position (the average of all 20 variations in the R_i+3/R_i+2 position which contain the specified R_i+2/R_i+3 side chain fixed).

Figure 5. Structural features and activity α-helix mimetic compound mixtures

A. Scaffold structure of α-helix mimetics and classification of α-helix mimetic compound mixture inhibitors of importin α/β mediated nuclear import. Each α-helix mimetic compound mixture contains a specific R_i and R_i+4 group and a mixture of 20 different structures at the R_i+7 position. Confirmed and validated α-helix mimetic compound mixture inhibitor hits are represented by a black square on the heat map. Please, refer to Supplemental Figure S2 for side chain nomenclature. B. Average % inhibition over the 20-compound mixtures of the complete α-helix mimetic library which contain the specified side chain at the R_i position (the average of all 20 variations of R_i+4 containing the specified R_i fixed). C. Average % inhibition over the 20 mixtures of the α-helix mimetic library, which contain the specified side chain at the R_i+4 position (the average of all 20 variations of R_i containing the specified R_i+4 fixed).

Figure 6. Structure and activity of compound 58H5-6

A. Nuclear import of Alexa555-BSA-NLS cargo in the cell permeabilized nuclear import assay in the presence of 1% DMSO (negative control), 1% DMSO and 2 mM GMP-PNP (positive control) or 500 μM 58H5-6. Scale bar is 10 μm. B. Nuclear import of Alexa555-GST-M9 cargo in the cell permeabilized nuclear import assay in the presence of 1% DMSO (negative control), 1% DMSO and 1 mg/ml WGA (positive control) or 500 μM 58H5-6. Scale bar is 10 μm. C. Structure of 58H5-6. D. Dose-response curve of 58H5-6 in the cell permeabilized nuclear import assay using the importin α/β cargo Alexa555-BSA-NLS. E. The thermodynamically most stable conformation of 58H5-6 docked into an FxFG binding site on importin β. The 58H5-6 inhibitor (red) docked to importin β (green) is superimposed on the crystallographic structure of an FxFG peptide fragment (yellow).