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

Article abstract of DOI:10.1016/j.bmc.2010.08.038

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.

Full text of DOI:10.1016/j.bmc.2010.08.038

Bioorganic & Medicinal Chemistry 18 (2010) 7611–7620
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecule peptidomimetic inhibitors of importin
nuclear transport
a/b mediated
Géza Ambrus a, Landon R. Whitby b, Eric L. Singer a, Oleg Trott c, Euna Choi a, Arthur J. Olson c,
Dale L. Boger b, Larry Gerace a,
⇑
a Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd., La Jolla, CA 92037, USA
b Department of Chemistry, The Scripps Research Institute, 10550 N Torrey Pines Rd., La Jolla, CA 92037, USA
c Department of Molecular Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd., La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 17 August 2010
Accepted 18 August 2010
Available online 21 August 2010
Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Transloca-
tion of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors
of the b-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhib-
itor 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 perme-
abilized cell screen for small molecule inhibitors of the importin
metic libraries, we identified b-turn and -helix peptidomimetic compounds that selectively inhibit
nuclear import by importin /b 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.
Ó 2010 Elsevier Ltd. All rights reserved.
a/b pathway. By analyzing peptidomi-
Keywords:
a
High content screening
Nucleocytoplasmic transport
Importin b
Small molecule inhibitor
Peptidomimetics
a
1. Introduction
to importin b frequently is mediated by various isotypes of the adap-
tor importin a
, which recognizes the cargo NLS.6 Although importin
In eukaryotes the nuclear envelope (NE ) segregates the nucleus
from the cytoplasm.1 Communication between these compartments
involves trafficking of macromolecules through large proteinaceous
channels that span the NE, called nuclear pore complexes (NPCs).2,3
The NPC is made up of multiple copies of approximately 30 different
proteins, collectively known as nucleoporins. Macromolecular trans-
port through the NPC is mediated by nuclear transport receptors
(also called importins and exportins), most of which are members
of the b-karyopherin family.4–6 The nuclear transport receptors in
complex with their cargoes bind to repeats of the Phe-Gly (FG)
amino acid motif that occur in natively disordered domains of ‘FG-
nucleoporins’ that line the transport channel of the NPC. These inter-
actions enable the receptors to penetrate the NPC and ferry cargoes
into and out of the nucleus.7 The best characterized import receptors
are importin b (also known as karyopherin b1) and transportin (or
karyopherin b2). The binding of protein cargoes to import receptors
is typically mediated by short amino acid stretches in the cargoes,
called nuclear localization sequences (NLSs). The binding of cargoes
b and transportin are structurally similar, they recognize structurally
different NLSs. The ‘classical’ NLS that is recognized by the importin
a/b pathway contains one or two short basic amino acid clusters. By
contrast, the well-characterized transportin NLS contains the con-
sensus sequence R/H/KX(2–5)PY in addition to an upstream basic or
hydrophobic motif.8
CRM1, also known as exportin-1, is the major karyopherin that
exports many cellular proteins from the nucleus to the cytoplasm,
typically through binding to a short leucine rich nuclear export se-
quence (NES) found on cargoes.9–13 Multiple mechanisms can regu-
late nuclear trafficking, including anchoring of cargoes in the
nucleus or cytoplasm, and exposure or masking of nuclear import
andexport signals byprotein–proteininteractionsor by posttransla-
tional modifications. A key component of all karyopherin-mediated
import and export pathways is the small GTPase Ran, which uses the
energy of GTP hydrolysis to specify transport directionality.14
The identification of leptomycin B (LMB), the first small mole-
cule inhibitor of CRM1, has greatly facilitated investigation of
nucleocytoplasmic transport.15 LMB has been used in hundreds
of studies to uncouple nuclear export from import and to analyze
nucleocytoplasmic shuttling proteins. In addition to being an ex-
tremely useful research tool, LMB has antibiotic and anti-tumor
activities although it is cell-toxic. In addition to LMB, several other
small molecule inhibitors targeting CRM1 and nuclear export have
⇑
Corresponding author. Tel.: +1 858 784 8514; fax: +1 858 784 9132.
E-mail address: lgerace@scripps.edu (L. Gerace).
Abbreviations: NE, nuclear envelope; NLS, nuclear localization sequence; NPC,
nuclear pore complex; NES, nuclear export sequence; LMB, leptomycin B; WGA,
wheat germ agglutinin; BSA, bovine serum albumin.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.038

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G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
been identified,16–21 as have been affinity-optimized peptide car-
goes that may competitively inhibit the transportin and importin
a
inhibitor of nuclear import has been described up to now (Supple-
mentary Fig. S1).
plates, as they were released from the plates due to the digitonin
treatment followed by the washing steps (Fig. 1A).30 We examined
multiple cell lines to identify ones suitable for screening, including
HaCaT, Cos-7, CV1, NIH 3T3, HeLa, NRK, FGM. We found that only
two of these cell lines, FGM and HaCaT, remained reproducibly at-
tached to plate wells by the end of the assay under optimal condi-
tions (data not shown, and below). For screening we have chosen
the FGM cell line, a metastatic variant of a pancreatic carcinoma cell
line with a relatively uniform nuclear morphology (Fig. 1B).31 In the
presence of GTP, Alexa555 labeled SV40 T antigen NLS peptide con-
jugated bovine serum albumin cargo (Alexa555-BSA-NLS) was effi-
ciently imported into the nucleus, whereas in the presence of
GMP-PNP, an unhydrolyzable GTP analogue nuclear import was
inhibited, as expected. The retention of FGM cells attached to the as-
say wells was enhanced by using precoated multiwell plates, such as
/b nuclear import pathways.22–24 However, no small molecule
In this study we report the identification and characterization of
small molecules that selectively inhibit importin a/b mediated nu-
clear import. We identified these compounds by screening pepti-
domimetic libraries, which previously have proven useful for
finding compounds that inhibit protein–protein interactions and
functions with IC50 values in the nanomolar to low micromolar
range.25–29 We have used a high-content screen involving a modi-
fied version of a digitonin permeabilized cell nuclear import assay.
This yielded multiple different but related inhibitors, with IC50 val-
ues for transport inhibition in the 100 lM range, only ca. 100-fold
less potent than the multivalent macromolecular inhibitor wheat
germ agglutinin (WGA). Some of these compounds may have sub-
stantially stronger binding affinities for individual nuclear trans-
port factors than reflected by their IC50 values, and could be
useful for future investigations of the nuclear transport machinery.
They also could provide the basis for development of more potent
import inhibitors.
Nunc CC2 coated plates or Greiner poly-
D-Lys coated plates. The ini-
tial cell seeding density also was a critical factor, and 40,000 cells/
well proved to be optimal under our conditions. Edge effects were
effectively eliminated by incubating the seeded plates for 1 h at
room temperature before transferring them to a CO2 incubator.32
CytosolandAlexa555-BSA-NLS concentrations, aswellasincubation
times were optimized, and the assay exhibited small well-to-well,
plate-to-plate and day-to-day variations. To further confirm the
validity of our assay we have tested the concentration dependence
for transport inhibition by WGA, a known macromolecular inhibitor
of nuclear import.33 The dose–response curve showed a good fit and
resulted in consistent IC50 values across experiments (Fig. 1C).
2. Results
2.1. Adaptation of the nuclear import assay to 96-well plate
format
2.2. Screening of 29,067 compounds in the form of compound
mixtures
We carried out screening of peptidomimetic libraries using a
permeabilized cell nuclear import assay, which involved incuba-
tion of digitonin-permeabilized cells with a fluorescently labeled
import cargo and exogenous cytosol as a source of nuclear trans-
A library of 29,067 peptidomimetic small molecules designed to
inhibit protein–protein interactions was screened in the permeabi-
lized cell nuclear import assay and subjected to a series of valida-
tion steps (Fig. 2A). The library contained single compounds as well
as mixtures with typically 10–20 compounds and occasionally up
to 100 compounds.25 Screening concentrations of DMSO were kept
at a constant 1%, and due to varying stock concentrations for sep-
arate sublibraries, the total compound/compound mixture concen-
port factors (including importins
a/b, transportin, and Ran), fol-
lowed by cell washing and fixation. The accumulation of cargo in
the nucleus was determined by light microscopy with automated
image acquisition, which allowed analysis of hundreds of cells
for each data point. Decrease in nuclear accumulation of fluores-
cently labeled cargo molecule was the readout for transport inhibi-
tion in the primary screen.
trations ranged from 10
l
m to 500
lM, with final individual
The adherent cell lines described by Adam et al. in the original nu-
clear transport assay were not suitable for screening with 96-well
compound concentrations never exceeding 25
lM. In the primary
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
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 m. (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.
a/b mediated nuclear import. Negative control wells contained GTP and for positive control wells, GMP-PNP was
l

G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
7613
say.35 We considered compounds or compound mixtures primary
hits if the average nuclear import levels from the respective wells
were below 50% of the negative control value (Fig. 2C). Our primary
screen returned 65 such primary hits.
Primary Screen with
Compound Mixtures
HIT
IDENTIFICATION
Confirmation
2.3. Validation of primary hits
Fluorescence
Quenching Assay
HIT
V
ALIDATION
The primary hits all came from compound mixtures of two clas-
ses of peptidomimetics: b-turn mimetics and
a-helix mimetics
(Supplementary Fig. S7). Each of these compound mixtures con-
Nuclear Integrity Test
tained 20 structurally related compounds. The primary hits were
rescreened three independent times at 500
lM total compound
Nuclear Transport Assays
for Pathway Specificity
C
OUNTER-SCREEN
ECONVOLUTION
concentrations (25 M for each compound). Compound mixtures
l
that gave an average level of nuclear import less than 50% were
considered confirmed hits and were further tested to exclude pos-
sible artifacts (Fig. 2A). One potential artifact is quenching of the
Alexa555 fluorophore present on the cargo molecule by a particu-
lar compound. This would cause a loss of fluorescence signal from
cargo and could lead to the false conclusion that nuclear import
was inhibited. To eliminate such compounds, fluorescence intensi-
ties of the nuclear import reaction mixtures containing the fluores-
cent cargo plus compounds were measured using a benchtop
fluorimeter. Three of the compound mixtures caused fluorescence
quenching of the Alexa555 dye, and were eliminated from further
analysis.
Screen with Deconvoluted
Compounds
D
Dose-Response
Measurements
150%
100%
50%
0%
A second potential artifact could be caused by disruption of the
permeability barrier of the NE upon exposure to certain types of
compounds, which might allow release of fluorescent cargo mole-
cule from the nucleus during the washing steps or during the nu-
clear import reaction. Once again this could be mistaken for
nuclear import inhibition due to the loss of intranuclear signal.
To test for such effects, an anti-lamin A/C antibody was included
in the nuclear import reaction, and was detected by immunofluo-
rescence microscopy after the completion of the reaction, cell
washing and fixation. The anti-lamin A/C antibody, which is too
large to diffuse across the NE, only yields nonspecific cytoplasmic
background when the NE is intact. However, when the NE integrity
is compromised (e.g., by detergents, Fig. 3A, lower middle panel),
the antibody diffuses into the nucleus and binds to the lamina,
which lines the inner nuclear membrane, leading to a characteris-
tic nuclear rim staining. Two compound mixtures were eliminated
from analysis by this validation test (58 E9 shown in Fig. 3A, right
panel). The final number of compound mixtures with confirmed
inhibitory effects and without detectable artifacts was 46 (Supple-
mentary Fig. S5).
z'=0.5 0.2
NLS cargo
NLS cargo + GMP-PNP
-50%
control wells
200%
150%
100%
50%
0%
primaryhits
compound wells
2.4. Counter-screen with M9 cargo identifies compound
mixtures that also inhibit transportin mediated nuclear import
-50%
Figure 2. Hit identification and validation. (A) Flowchart for the identification and
validation of nuclear import inhibitors of the importin /b 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
To assess the import receptor specificity of inhibition by the 46
compound mixtures, we subjected them to a transport assay to
measure transportin-mediated import (Fig. 3B). Instead of the
a
importin a/b specific cargo (Alexa555-BSA-NLS), we used a fluores-
primary screens was 0.5
0.2. (C) Relative nuclear import levels of screened
cently labeled GST fusion of the M9 sequence of hnRNP A1
(Alexa555-GST-M9), a well-described NLS for transportin.8 The
46 compound mixtures were screened three independent times
compound mixtures. Compound mixtures with over 50% inhibition were considered
primary hits and are highlighted in black.
at 500 lM total compound concentrations (25 lM for each com-
screen, each plate contained ten negative control replicates, in
which DMSO was added in lieu of the compounds, and six positive
controls replicates, in which GMP-PNP was added to a 2 mM final
concentration to inhibit nuclear import of the NLS cargo by block-
ing GTP hydrolysis by Ran (Fig. 1B).34 A compilation of average
fluorescence intensity values from individual control wells was
used to calculate the z0 factor values for the primary screen
(Fig. 2B). Z0 factor values are descriptive of the robustness of an as-
say and were around 0.5 in our case indicative of an excellent as-
pound). Most of these mixtures showed no significant inhibition
of transportin mediated nuclear import, but four of the mixtures
did significantly inhibit import of the M9 cargo as well (to less than
50% of control) (Supplementary Fig. S5). These could act by block-
ing a component common to the two import receptor pathways,
such as components of the Ran system. The remaining 42 com-
pound mixtures, which selectively inhibited the importin
way by our criteria, included 16 b-turn mimetics and 26
mimetics (Figs. 4A and 5A, Supplementary Figs. S5 and S6).
a
/b path-
a
-helix

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G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
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
lm. (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
l
m.
Although these compound mixtures did not detectably inhibit im-
port of the M9 cargo with our conditions, it is possible that they
could affect this pathway at higher concentrations. However, we
were unable to test this, because of the need to keep the DMSO
concentration at or below 1%. Validated hits that had no detectable
chain were listed as Class 2 compounds. In order to further inves-
tigate the potentially dominant roles of certain side chains in the
inhibition by the b-turn mimetics, we took the summed average
over the 20 mixtures containing a specified side chain substituent
in the Ri+2/Ri+3 position (Fig. 4B). This analysis revealed further that
among the aryl side chains the bicyclic aromatics Trp and Nap were
favored, with Trp by far being the dominant side chain for achiev-
ing inhibition. Furthermore, the basic side chains His and Lys also
showed significant activity, with the His being second only to Trp.
Not surprisingly, the most potent mixture contained both Trp and
His at the Ri+2/Ri+3 positions (Supplementary Figs. S9 and S10).
inhibition of transportin mediated import at 500
tration were titrated to 50 M and still showed statistically
significant inhibition of importin /b mediated nuclear import,
/b pathway inhi-
lM total concen-
l
a
indicating an at least 10ꢀ selectivity of importin
a
bition over transportin pathway inhibition (data not shown).
2.5. Structure–activity relationship analysis of the compound
mixtures
Although the
a-helix mimetics that selectively inhibited impor-
tin
a
/b mediated nuclear import were structurally more varied
than the b-turn mimetic hits, aromatic and His residues again
played key roles in inhibition (Fig. 5A). Analogous to the b-turn
mimetics, we classified the 10 members that contained a His side
chain as Class 1 compounds, while the remaining 16 members
which contained an aromatic residue were grouped as Class 2 com-
pounds. Interestingly, when both Ri and Ri+4 positions are occupied
by aromatic groups, HoPhe is a preferred side chain in position Ri of
the hits, whereas the structure of the aromatic residue in position
Ri+4 is less restricted (Fig. 5A). Bulky aliphatic groups appear at
both Ri and Ri+4 of the identified inhibitors, with Leu and Ile most
prevalent in position Ri of Class 2 hits, whereas Abu, Val, and Leu
are found in combination with His in Class 1 (Fig. 5A). The average
inhibitory positional analysis (described above for b-turn mimet-
ics) underscored the importance of the aromatic and His residues
The library construction of b-turn mimetic (Whitby, L.R., Ando,
Y., et al. soon to be published results) and
a-helix mimetic com-
pound mixtures covered all possible combinations of the 20 side
chains.36 This provided a straightforward opportunity for struc-
ture–activity relationship (SAR) analysis from our primary screen-
ing data. Compounds from both classes were tripeptide mimetics
and contained variable positions that were substituted with 20 dif-
ferent side chains (Supplementary Fig. S2). For the a-helix mimet-
ics, all possible 20 ꢀ 20 ꢀ 20 = 8000 compounds were synthesized
in the form of 400 compound mixtures where each mixture con-
tained specific Ri and Ri+4 groups and an equimixture of the 20 side
chains in position Ri+7 (Fig. 5A). Similarly, all possible b-turn mi-
metic compounds were synthesized in the form of 20 compound
mixtures, but due to the C2 symmetry present in the scaffold, the
total number of mixtures was 210 instead of 400 (Fig. 4A, Supple-
mentary Figs. S3 and S4).
for inhibition by the a-helix mimetics (Fig. 5B and C). For position
Ri, the unsubstituted aromatics Nap and HoPhe showed the great-
est activity across their respective 20 mixtures, with His and the
small aliphatic Abu also displaying strong effects (Fig. 5B). For
All importin a/b selective, b-turn mimetic hits with the excep-
tion of one compound mixture contained at least one aromatic side
chain in position Ri+2/Ri+3 (Ri+2 and Ri+3 are interchangeable due to
C2 symmetry) and in eight out of the 16 cases, the other side chain
also was an aromatic residue (Fig. 4A). Another prevalent residue
was histidine, with a total number of five occurrences among the
inhibitor mixtures. We grouped these compound mixture hits into
two classes (Fig. 4A). All hits that contained at least one His residue
formed Class 1 and the remaining hits with at least 1 aromatic side
Ri+4, the mixtures with a His side chain at this position displayed
the greatest average% inhibition (Fig. 5C).
2.6. Deconvolution of selected hits identifies individual
inhibitor structures
To identify the compounds active in selectively inhibiting
importin
a/b mediated nuclear import, we chose 3 validated

G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
7615
β-turn mimetics
Ri+2
aliphatic
aromatic
polar
0
0
1
1
0
0
0
0
2
2
2
2
2
3
0
0
0
1
0
0
16
Ala
Val
1
Leu
1
Ile
Met
Phe
HoPhe
HoPhe(4OH)
HoPhe(4Cl)
Phe(4Cl)
Tyr
1
1
1
1
1
1
1
1
1
i3+
1
Tyr(Me)
Trp
1
1
1
Ala(2-Nap)
Asn
Ser
Thr
1
His
Asp
Lys
0
0
0
0
0
0
0
0
0
0
1
0
5
4
0
0
0
5
0
1
Ri+2/Ri+3 side chain preferences
50
40
30
20
10
0
Figure 4. Structural features and activity of b-turn mimetic compound mixtures. (A) Scaffold structure of b-turn mimetics and classification of b-turn mimetic compound
mixture inhibitors of importin /b mediated nuclear import. Each b-turn mimetic compound mixture contains a specific Ri+2 and Ri+3 group and a mixture of 20 different
a
structures at the Ri position. Due to the scaffold’s C2 symmetry the Ri+2 and Ri+3 groups are interchangeable. Confirmed and validated b-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
Supplementary Figure S2 for side chain nomenclature. (B) Average% inhibition over the 20-compound mixtures of the complete b-turn mimetic library, which contain the
specified side chain in the Ri+2/Ri+3 position (the average of all 20 variations in the Ri+3/Ri+2 position which contain the specified Ri+2/Ri+3 side chain fixed).
20-compound mixtures and synthesized all their constituent indi-
vidual compounds separately (60 compounds in total). In addition,
we also deconvoluted two compound mixtures from each of the
two libraries that showed inhibition of both transportin dependent
port inhibition, we decided to test the deconvoluted compounds at
500 M, as well, in the nuclear import assay for inhibition of
l
Alexa555-BSA-NLS nuclear import (Supplementary Fig. S8). For
the b-turn mimetics, the screening of the 20 individual compounds
in the best performing mixture (Trp-His) revealed that aryl substit-
uents were highly preferred at the Ri position, with virtually all
compounds containing an aryl substituent showing significantly
greater inhibition versus those with non-aryl substituents
and importin
tal). Considering that the original compound mixtures that were
screened at 500 M total concentration contained structurally sim-
ilar compounds that could all potentially contribute to nuclear im-
a/b dependent nuclear import (80 compounds in to-
l

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G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
α-helix mimetics
Ri+4
small
bulky
aromatic
polar
aliphatic
aliphatic
0
0
2
1
4
4
1
0
4
0
0
0
1
2
1
0
0
5
0
1
26
Gly
Ala
1
1
1
1
1
1
Abu
Val
1
1
1
1
1
Leu
1
1
Ile
1
Met
Phe
1
1
HoPhe
Phe(4Cl)
Tyr
i
Tyr(Me)
Trp
1
1
1
Ala(2-Nap)
Asn
1
Ser
Thr
1
1
1
1
1
His
Asp
1
Lys
0
0
1
1
2
1
0
3
1
2
0
2
2
3
1
0
1
5
1
0
Ri side chain preferences
Ri+4 side chain preferences
50
40
30
20
10
0
50
40
30
20
10
0
Figure 5. Structural features and activity
mixture inhibitors of importin /b mediated nuclear import. Each
structures at the Ri+7 position. Confirmed and validated -helix mimetic compound mixture inhibitor hits are represented by a black square on the heat map. Please, refer to
Supplementary Figure S2 for side chain nomenclature. (B) Average% inhibition over the 20-compound mixtures of the complete -helix mimetic library which contain the
a-helix mimetic compound mixtures. (A) Scaffold structure of
a-helix mimetics and classification of a-helix mimetic compound
a
a-helix mimetic compound mixture contains a specific Ri and Ri+4 group and a mixture of 20 different
a
a
specified side chain at the Ri position (the average of all 20 variations of Ri+4 containing the specified Ri fixed). (C) Average% inhibition over the 20 mixtures of the
mimetic library, which contain the specified side chain at the Ri+4 position (the average of all 20 variations of Ri containing the specified Ri+4 fixed).
a-helix
(Supplementary Fig. S10). Among the aryl side chains, Trp, Nap,
and Tyr derivatives (Tyr, TyrMe, HoPhe(4OH)) performed the best
(Supplementary Fig. S10).
NLS, but even at 500
bition of Alexa555-GST-M9, indicating an at least 15-fold selectiv-
ity of inhibition of the importin /b over transportin mediated
lM compound concentration it gave no inhi-
a
From the
characterized the most potent inhibitor, 58H5-6 (Ri = HoPhe,
i+4 = Phe, Ri+7 = Ile, Fig. 6C). At as low as 31 M concentration
58H5-6 detectably inhibited the accumulation of Alexa555-BSA-
a
-helix mimetic deconvolutions, we identified and
nuclear trafficking in vitro (Fig. 6A and B). Dilution series of
58H5-6 gave highly reproducible dose–response curves of nuclear
import inhibition of Alexa555-BSA-NLS cargo accumulation in the
R
l
nucleus. These yielded an IC50 value of 106 lM for 58H5-6 in the

G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
7617
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 M 58H5-6. Scale bar is 10 m. (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 M 58H5-6. Scale bar is 10 m. (C)
Structure of 58H5-6. (D) Dose–response curve of 58H5-6 in the cell permeabilized nuclear import assay using the importin /b cargo Alexa555-BSA-NLS. (E) The
l
l
l
l
a
thermodynamically most stable conformation of 58H5-6 docked into an FxFG binding site on importin b. The 58H5-6 inhibitor (red) docked to importin b (green) is
superimposed on the crystallographic structure of an FxFG peptide fragment (yellow).
permeabilized cell nuclear import assay (Fig. 6D). We used surface
plasmon resonance to analyze whether 58H5-6 affects the binding
of the FxFG peptide. This analysis supports the possibility that 58H5-
6 may act by binding to FxFG binding pockets of importin b.
of importin b to the IBB domain of importin a, to RanGTP or to the
FG-domain of Nup153, but detected no observable effects (Supple-
mentary Fig. S11). Nor were we able to observe any in vivo inhib-
3. Discussion
itory effect of 58H5-6 on the importin a/b pathway by analysis of
the nucleocytoplasmic shuttling protein GFP-NFAT (Supplemen-
tary Fig. S12).
This work, along with another recent study from our labora-
tory,39 describes the first small molecule inhibitors of importin
a/b mediated nuclear import. To identify inhibitors in the study
described here, we have adapted a permeabilized cell nuclear im-
port assay, pioneered in our laboratory two decades ago, to a 96-
well plate format. This in vitro setup provides some advantages
over previously described in vivo nuclear import screens that mon-
itor the nuclear localization of endogenous or transfected proteins
in cells.40,41 First, since the cargo consists of an ectopic NLS conju-
gated to or fused to a carrier protein, it is not subject to regulation
that may occur for a normal cellular protein (involving NLS activa-
tion or protein anchoring). Thus, our assay is more likely to reveal
hits that directly target components of the nuclear transport
machinery rather than other regulatory elements such as kinases.
Second, cell-impermeable inhibitors are not discarded in the
screening process since the assay involves permeabilized cells.
Compound properties such as cell-permeability can later be opti-
mized by structure scans or selective chemical modifications that
remove or suitably replace charged functional groups and/or en-
hance lipophilicity. Moreover, the permeabilized cell assay can be
used to analyze hits emerging from other in vitro protein based
assays, which screen for inhibitors of a specific protein–protein
2.7. Molecular docking reveals a potential binding site of 58H5-
6 on importin b
The structure of 58H5-6, our most potent nuclear import inhibi-
tor, is superficially similarto FxFG repeats found in nucleoporins. We
hypothesized that 58H5-6 might inhibit nuclear import by interact-
ing with FxFG binding pockets on importin b to competitively
exclude endogenous FG-nucleoporins. To test the plausibility of this
hypothesis, we docked 58H5-6 into the region of a previously deter-
mined FxFG binding site in the importin b/FxFG crystal complex
using AutoDock Vina.37,38 From this analysis, we found that 58H5-
6 docks into this pocket without any steric clashes. Moreover, when
58H5-6 is docked in a fully flexible mode to the rigid FxFG binding
pocket of importin b, the thermodynamically most stable conforma-
tion among the various predicted binding modes is one that is very
similar to that of the FxFG peptide (Fig. 6E). In this conformation,
the buried phenyl group (Ri = HoPhe) and the central benzene group
ofthe 58H5-6inhibitoralignwell with thephenylalanineside chains

7618
G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
interaction or for binding of a compound to a member of the nucle-
ar transport machinery, such as our recent screen for importin b
binding compounds.
inhibitor wheat germ agglutinin (WGA). It is also worth noting that
larger protein fragments, such as the 211 amino acid long
Nup153-FG construct that contains nine FG repeats49 and the
119 amino acid long Nsp1p derived FF5 construct that contains five
FG repeats50 inhibited in vitro nuclear import at a concentration of
25 lM and 10 lM, respectively. Considering the size of these frag-
ments and the probable avidity effects of the multiple FG repeats,
these concentration ranges are in line with the IC50 value of 58H5-
6. No inhibition of importin a/b mediated nuclear import could be
observed in vivo, possibly due to insufficient cell permeability of
58H5-6. We cannot formally rule out an enzymatic activity in cells
or medium that modifies the peptidomimetic, although this seems
very unlikely. Initial experiments to identify the target of 58H5-6
through a series of surface plasmon resonance tests were unsuc-
cessful. Further chemical modification and optimization of 58H5-
6 could lead to a more soluble and/or more potent compound that
could facilitate target identification.
Another important feature of our screen was its focus on pepti-
domimetic libraries, which have proven useful for obtaining inhib-
itors of protein–protein interactions. Throughout the nuclear
import cycle from cargo binding to recycling of the receptor, at
least 6 types of protein–protein interaction govern nuclear trans-
port but only one enzymatic reaction occurs, the hydrolysis of
GTP by Ran. We obtained over 60 independent compound mixtures
with significant inhibitory activity by screening with a cargo for
the importin
braries: b-turn and
a
/b pathway. All hits identified came from two subli-
-helix tripeptide mimetics. In three indepen-
a
dent rescreenings of the primary hits, we eliminated compound
mixtures that did not reproduce the inhibition, and in two addi-
tional tests we excluded false positives that yielded nonspecific ef-
fects on the transport signal. This left us with 46 compound
mixtures that were specific hits. In an additional counterscreen,
we determined that 4 of these confirmed hits inhibited transportin
In summary, along with a separate very recent study from our
laboratory,39 we have provided the first description of small mole-
mediated nuclear import as well as importin
compounds most probably act on components common to the
importin /b and transportin mediated pathways, such as compo-
nents of the Ran machinery or certain FG-nucleoporins. Given the
high conservation of the nuclear transport machinery among high-
er eukaryotes,42–45 it is very likely that all of the above inhibitors—
a
/b import. These
cule inhibitors selectively targeting importin a/b mediated nuclear
import. We describe the prototype of these inhibitors and offer a
hypothesis for mode of action. These compounds could provide
the structural basis for the development of higher affinity and/or
cell permeable inhibitors in the future.
a
both importin a/b specific and nonspecific ones—are efficacious in
all human and most probably in all mammalian cell lines. This
4. Experimental
hypothesis, however, remains to be tested.
4.1. Library construction
Since both the b-turn and
a-helix peptidomimetic sublibraries
were constructed to contain all possible combinations of groups
representing 20 side chains used in the library synthesis, the corre-
lation of structure with inhibition could be comprehensively char-
acterized. Interestingly, confirmed hits from both sublibraries
contained either a histidine or an aromatic residue in one of their
two positions. On the one hand, the clustering of specific hits is a
validation of our screening and substantiates the structural fea-
tures of this class of nuclear import inhibitors. On the other hand,
the relatively restricted structural diversity of the hits that we
obtained was not expected, because of the large number of
protein–protein interfaces involved in nuclear protein import that
could potentially serve as targets. Thus, our screen may have iden-
tified inhibitors of only a subset of these interactions, such as the
binding of importin b to nucleoporins. Nucleoporin binding of nu-
clear transport receptors occurs through FxFG amino acid repeats
present in nucleoporins and many of our aromatic residue contain-
ing hits may mimic such an FxFG structure, particularly the stron-
gest individual inhibitor we obtained, 58H5-6 (Fig. 6C).
The a-helix mimetic and b-turn mimetic libraries are part of a
general small-molecule library designed to selectively modulate
protein–protein interactions by targeting the three main recogni-
tion motifs which mediate protein–protein interactions (
a
-helix,
b-turn, b-strand).36 The
a-helix mimetic library was designed
and synthesized as previously described36 to yield an 8000-mem-
ber library (20 ꢀ 20 ꢀ 20) composed of 400 mixtures of 20 struc-
turally related compounds which vary at the N-terminal position.
The b-turn mimetic library was constructed around a trans-pyrrol-
idine-3,4-dicarboxamide scaffold, which closely satisfies the geo-
metric constraints provided by a C -triplet analysis51 for the
a
display of triplets of amino acid side chain moieties in a manner
similar to a majority of the classes of b-turns (Supplementary
Fig. S3). The library was formatted and synthesized as outlined in
Supplementary Figure S4 to comprise a 4200-membered library
composed of 210 mixtures of 20 structurally related compounds,
which vary only at the R3 position. Notably, all triplet combinations
of groups which represent the 20 natural amino acid side chains
(or suitable replacements, see Supplementary Fig. S2) are compre-
hensively represented in the b-turn mimetic library with only 4200
compounds due to the simplifying C2 symmetry of the trans-pyr-
rolidine-3,4-dicarboxamide scaffold. See Supplementary data for
NMR quality control measurements on compound 58H5-6.
We synthesized individual compounds from a total of seven
compound mixtures and rescreened them in our primary assay. A
compound from Class 2 of the
58H5-6, proved to be the most potent individual compound that
inhibited the importin /b pathway. 58H5-6 showed an IC50 value
of 106 M for importin /b mediated nuclear import in vitro, and
no inhibition of transportin mediated nuclear import by 58H5-6
could be detected up to 500 M. Our docking study confirmed
a-helix peptidomimetic sublibrary,
a
l
a
4.2. Nuclear import assay
l
the steric and energetic plausibility of 58H5-6 binding to FxFG
pockets on importin b. While this offers an attractive mode of ac-
tion, i.e. the inhibition of importin b binding to nucleoporins (prob-
ably other than Nup153); docking predictions are not guaranteed
to reflect physiological processes. The relatively weak IC50 value
of 58H5-6 may simply be a consequence of the large structural
flexibility of importin b46,47 and/or its multiple, distinct binding
pockets for FG repeats,48 if 58H5-6 indeed acts by inhibiting FG-
nucleoporin binding. Although this potency of inhibition may ap-
pear modest, it is worth highlighting that this is only approxi-
mately 100-fold less potent than the tetravalent macromolecular
Nuclear import assays using permeabilized cells were carried
out as previously described52 with the following modifications.
FGM cells were seeded into poly-D-Lys coated clear bottom 96-well
plates (Greiner) at 40,000 cells per well. Plates were left at room
temperature for 1 h before transferring them to 37 °C in a CO2
incubator to avoid edge effects.32 Forty-eight hours after seeding
cells were gently washed three times with transport buffer
(20 mM HEPES pH 7.4, 110 mM KOAc, 2 mM Mg(OAc)2, 2 mM
DTT, 1 mM EGTA, and 1
lg/ml of each leupeptin, aprotinin, pepsta-
tin) in an automated plate washer (Bio-Rad). Cells were then

G. Ambrus et al. / Bioorg. Med. Chem. 18 (2010) 7611–7620
7619
permeabilized with 0.005% (50
for 5 min on ice. Cells were gently washed five times with trans-
port buffer and 50 l reaction mixtures were added to each well.
Reaction mixtures contained compounds at indicated concentra-
tions or equivalent amounts of DMSO, 3 mg/ml HeLa cytosol,
l
g/ml) digitonin in transport buffer
was used for docking calculations. The ‘search space’ was chosen
to be an XYZ-coordinates aligned box with 5 Å margins around
where the peptide fragment would have been. The rest of the
parameters were left at their default values. The receptor was trea-
ted as rigid, and the inhibitor fully flexible during docking.
l
0.5 lM cargo, an ATP regenerating system (0.8 mg/ml creatine
phosphate, 10 U/ml creatine phosphokinase, 1 mM ATP), 0.2 mM
GTP, in transport buffer. Compounds and compound mixtures dis-
solved in DMSO were directly diluted 100ꢀ into reaction mixtures
to yield a 1% DMSO final concentration. For the duration of the nu-
clear import assay this level of DMSO was still tolerable for cells.
Generally, no detectable precipitation of compounds occurred in
almost all cases, and the few cases where this was encountered
the data points were discarded. Total stock concentrations for indi-
Acknowledgment
This work was supported by NIH Grant R21NS059460 to L.G.
and CA078045 to D.B.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.038.
vidual compounds and compound mixtures ranged from 10
lM to
500 M. Cytosol, Alexa555 labeled BSA-NLS and GST-M9 cargoes
l
were prepared as described earlier.52 Wheat germ agglutinin
(WGA) was added at 1 mg/ml and GMP-PNP was added at 2 mM
to control reaction mixtures to inhibit import reactions. After the
addition of reaction mixtures plates were incubated at 30 °C for
30 min. Then the transport reactions were stopped by the addition
of ice cold transport buffer and the plates were left on ice for
10 min. Cells were gently washed 10 times with ice cold transport
buffer and fixed with 4% formaldehyde in transport buffer for
30 min at room temperature. Fixed cells were washed three times
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