Inhibitors of nuclear transport
David A Jans, Alexander J Martin and Kylie M Wagstaff

Central to eukaryotic cell function, transport into and out of the nucleus is largely mediated by members of the Importin (IMP) superfamily of transporters of a- and b-types. The first inhibitor of nuclear transport, leptomycin B (LMB), was shown to be a specific inhibitor of the IMPb homologue Exportin 1 (EXP1) almost 20 years ago, but it has only been in the last five or so years that new inhibitors of nuclear export as well as import have been identified and characterised. Of utility in biological research, these inhibitors include those that target-specific EXPs/IMPs, with accompanying toxicity profiles, as well as agents that specifically target particular nuclear import cargoes. Both types of inhibitors have begun to be tested in preclinical/clinical studies, with particular focus on limiting various types of cancer or treating viral infection, and the most advanced agent targeting EXP1 (Selinexor) has progressed successfully through >40 clinical trials for a range of high-grade cancers and is approaching FDA approval for a number of indications. Selectively inhibiting the nucleocytoplasmic trafficking of specific proteins of interest remains a challenge, but progress in the area of the host–pathogen interface holds promise for the future.

Address
Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia

Corresponding author: Jans, David A ([email protected])

Current Opinion in Pharmacology 2019, 58:50–60
This review comes from a themed issue on Cell nucleus Edited by Daniel R Larson and Naoko Imamoto

https://doi.org/10.1016/j.ceb.2019.01.001

0955-0674/ã 2018 Elsevier Ltd. All rights reserved.

Introduction
Much is now known regarding transport into and out of the nucleus of molecules >45 kDa mediated by members of the Importin (IMP) superfamily of transporters of ati and b-types, and how they facilitate the transport of proteins that possess the requisite nuclear targeting signals (nuclear localisation signal — NLS — and nuclear export sequence — NES — for nuclear import and export respectively) [1]. Nuclear protein import requires a cargo containing an NLS to be recognised by the IMPa/b1 heterodimer, or IMPb1
alone or one of the many homologues thereof, followed by translocation through the nuclear envelope-embedded nuclear pore complexes (NPCs) dependent on interactions between IMPb1 and hydrophobic repeats within the nucleoporins that make up the NPC [2ti ]. Once inside the nucleus, binding of RanGTP to the IMPb dissociates the complex to release cargo into thenucleoplasmto play its nuclear role [1]; the general scheme is highlighted in Figure 1a, illustrating the fact that this transport mode can also be hijacked by viruses to gain viral protein access to the nucleus to facilitate infection. Nuclear protein export occurs analogously, whereby an NES in the cargo protein is recognised in the nucleus by specific members of the IMPb family called exportins (EXPs) (of which EXP1/CRM1/
XPO1 is the best characterised) bound to RanGTP, before export to the cytoplasm and dissociation of the complex after GTP hydrolysis by Ran [2ti ,3ti ]; this is illustrated in Figure 1b, highlighting the example of tumour suppressor proteins that help control the cell cycle.

Nucleocytoplasmic transport is regulated in response to a range of stimuli in the short-term and during processes such as cell differentiation, development, transformation and in infection and disease [e.g. Refs. 3ti ,4ti ]. Post-trans- lational modification is an important modulator; although phosphorylation is the best understood mechanism of regulating nucleocytoplasmic transport, acetylation, methylation and various other modifications have also been shown to play a role. Protein–protein interaction is integrally involved, such as through proteins such as negative regulators of nuclear transport that inhibit trans- port through a direct binding/sequestration interaction with either specific cargoes (e.g. such as IkB — Inhibitor of kappa B — and SuFu — Suppressor of Fused — that target the transcription factors NFkB and Gli1, respec- tively), classes of cargoes (e.g. the BRCA1 binding protein BRAP2 that recognises various viral and host proteins upon phosphorylation at specific sites close to the NLS), or IMPs themselves (e.g. the etoposide-induced factor Ei24 that binds to and sequesters IMPa and IMPb1 [5]) and thereby impact entire nuclear transport pathways. Inhibitory forms of IMPs that bind cargo but prevent transport (e.g. of IMPa and IMP13 [6]) are also known. Finally, at least one endogenously expressed small mole- cule has been described in cells (the anti-inflammatory prostaglandin 15-deoxy-D12,14-prostaglandin J2) that can inhibit nuclear export mediated by EXP1 [7]. Clearly, the existence of all of these cellular mechanisms that can essentially switch IMP/EXP-mediated transport on or off in a physiological context implies that pharmacological manipulation of the cellular nuclear transport system is possible.

Figure 1

(a) CYTOPLASM
NUCLEUS
ATTENUATED

IMPα
NPC

DENV
ANTIVIRAL RESPONSE

NS5
DENV

NS5
HIV-1 Integrase

HIV-1 Integrase

(ai)
Ivermectin

HIV-1

IMPα

DENV
NS5

Ran IMPα
GTP

Inhibition of IMPα/β1- dependent transport

FULL CELLULAR ANTIVIRAL RESPONSE

HIV-1 integration

(aii)
Integrase
Flunisolide

IMPα

Inhibition of import of specific cargoes recognised by IMPα/β/1

NO HIV-1

HIV-1 Integrase
integration

DENV
NS5

4-HPR

(b)

Tumour

Tumour Suppressor

Suppressor Growth regulator

Ran Tumour GDP Growth
NPC
Growth regulator

Ran
GTP
Tumour Suppressor
Growth regulator

Inhibited Tumour Growth

Ran
GTP

Inhibition of EXP1- dependent transport
(bi)
Selinexor

Current Opinion in Cell Biology

Mode of action of different inhibitors blocking IMPa/b1-mediated nuclear import of viral proteins (a) and EXP1-mediated nuclear export of cancer regulatory proteins (b).
(a) In the absence of inhibitors, Dengue NS5 protein and HIV-1 Integrase (IN) are recognised by the host IMPa/b1 heterodimer in infected cells, where IMPa interacts with IMPb via the IBB domain of IMPa, followed by nuclear import to impact host antiviral responses/mRNA splicing (NS5)

Nuclear Export Inhibitors targeting EXP1
Since there are multiple distinct IMPs/EXPs performing often redundant or competing functions with respect to nuclear import and export of particular cargoes, the use- fulness of small molecule inhibitors specific to one or other IMP/EXP is immediately obvious; clearly, a set of inhibitors for all of the IMPs/EXPs would enable the role of the proteins in a biological process to be analysed directly. However, until relatively recently, the palette of nuclear transport inhibitors available to researchers has been limited to EXP1 and Leptomycin B (LMB). Dis- covered as a potent antifungal in 1983 [8], LMB was later shown to be a nuclear export inhibitor specifically target- ing EXP1 and in particular a specific cysteine residue (residue 529 in fission yeast/528 in mammalian systems) essential for NES recognition by EXP1 [9]. At low con- centrations, LMB blocks the nuclear export of various proteins, including signalling molecules such as mitogen activated protein kinase (MAPK) and NFkB/IkB, tumour suppressors such as BRCA1, and viral proteins such as Human Immunodeficiency Virus-1 (HIV-1) Rev protein [see Ref. 3ti ]. LMB’s pleiotropic effects on multiple different cargoes transported by EXP1 can cause G1 cell cycle arrest in mammalian cells, making it of interest as an anti-tumour agent, but LMB (elactocin) failed in Phase I clinical trials because of toxicity (effects of profound anorexia and malaise at all dose regimes) [10]. LMB, however, has proved to be an immensely useful tool in establishing the biological role of EXP1, highlighted by over 1000 publications. Since the discovery of LMB, a number of new inhibitors of nuclear export mediated by EXP1 (Table 1) have been described, as well as of nuclear import mediated by IMPs (Table 2), and those that target specific cargoes (see Table 3), rather than IMPs directly (see below). The EXP1 inhibitors all appear to mimic or approximate LMB’s action at the NES binding site, including covalent binding to Cys528 itself (see Figure 2a), implying that masking the NES binding site is the simplest and most efficient means to nullify EXP1’s role in nuclear export. Strikingly, EXP1 has been the only EXP targeted for pharmacological intervention, which of course underpins the impact that LMB has had in terms of directing research in this sphere, but also highlights the fact that strategies to identify and develop inhibitors of nucleocytoplasmic trafficking remain in their infancy (see, however, below).

Table 1 shows selected examples of EXP1 inhibitors, summarising some of their main properties and listing the

extent to which preclinical/clinical studies have estab- lished their utility in terms of treating human disease. Of these, the SINE (selective inhibitors of nuclear export) compounds are by far the most progressed, with a number of clinical trials completed successfully for a range of high-grade cancers. SINE inhibitors also target the LMB/
NES binding site on EXP1 (see Figure 2a) [11–15; see Refs. 16,17], but do so reversibly, in contrast to LMB, thus resulting in an apparent reduced toxicity, and enabling them to be used to block nuclear export in cancer cells, and hence contribute to cancer cell killing (see Figure 1bi) [18–23,24ti ]. The SINE Selinexor has been tested in more than 40 clinical trials in over 10 cancer types, with more than 2400 patients treated. Particular application appears to include in acute myeloid leukae- mia when used alone or in combination therapy with other agents (six out of 14 patients achieved remission) [21,22], and in sarcoma (dedifferentiated liposarcoma maintained in seven out of 15 patients >4 months after treatment) [23]. Excitingly, Selinexor has received Orphan Drug/Fast Track designation from the FDA for the treatment of patients with penta-refractory multiple myeloma [24ti ]. Other SINE compounds are currently in clinical trials (see Table 1), with Verdinexor of interest for viral disease, with influenza the main focus [25]. The work with SINE compounds, and Selinexor in particular, underlines the important progress that has been made in applying detailed knowledge of nucleocytoplasmic trans- port, especially through structural/modelling approaches, to pharmacological intervention in the clinic. The extent to which targeting EXP1 specifically may be inextricably linked with toxicity, and hence limit the use of EXP1- targeted therapy to high grade cancer [see also Ref. 26ti ]
and potentially acute viral infections where treatment would be only for a number of days, remains to be seen.

Nuclear Import Inhibitors targeting IMPs
A body of work in the last five or so years has described small molecules that target IMPs and, therefore, inhibit nuclear import (Table 2), derived through a range of approaches, including conventional high throughput screening [27–30], in silico screening [31titi ,32ti ], activity- based profiling [33] and structure-based drug design [see Ref. 34]. These largely include compounds targeting the two best characterised nuclear import proteins, IMPa and IMPb1 (see Figure 1a); as for EXP1 inhibitors, the simple fact that the inhibitors target transporters essential for cell function means that toxicity is an inevitable corollary of their use, limiting clinical application. Unlike the case of

(Figure 1 Legend Continued) or help mediate integration into the host genome (IN). (ai) The compound ivermectin [27,36] binds IMPa directly (binding site indicated in red) to prevent recogniton of the viral protein NLSs, and thereby nuclear import of the viral proteins. (aii) Viral protein- specific inhibitors, in contrast, can target the viral proteins NS5 or IN directly (binding sites indicated in red — see also Figure 2b) to prevent recognition by the IMPa/b1 heterodimer, and hence nuclear import [26ti,46,51]. (b) In the absence of inhibitors, EXP1 in complex with Ran-GTP mediates the nuclear export of NES-containing cargoes such as tumour suppressors to prevent them exerting control over cell proliferation in transformed cells. (bi) The specific EXP1 inhibitor selinexor targets the site on EXP1 responsible for binding export cargoes (binding site in
red — see also Figure 2a) [11–17] to prevent their nuclear export and inhibit tumour cell proliferation.

Table 1
Summary of the properties of selected inhibitors of nuclear export that target EXP1/CRM1

Inhibitor
Mode of action on EXP1
Biological effects
Clinical potential

Leptomycin B (LMB) [see Refs. 8,9]
Elactocin [10]
Targets NES-binding groove (Cys-528), preventing cargo binding [9]
ti Antifungal [8]
ti Cytotoxic/apoptotic effects in cancer cell lines
ti Phase I trial for advanced stage refractory cancers ended due to dose-limiting toxicity [10]

Goniothalamin [see Refs. 18,19]
Covalent binding
ti Inhibits Rio2 kinase nuclear export in HeLa cells
ti Induces cell cycle arrest/ apoptosis/DNA damage in breast, cervical, hepatocellular carcinoma, and non-small cell lung cancer lines
ti Studies in mice indicate inhibition of inflam- matory cytokine production in colon tissue and reduced tumour development in colitis- associated and colorectal cancer models

CBS9106
(and derivatives e.g. S109)
[see Ref. 20]

Reversible binding at Cys528; induces neddylation/
proteosome- dependent degradation

ti Decreases EXP1 protein levels through pro- teasome dependent degradation
ti Inhibition of NF-kB activity
ti Antiproliferative effects on a large number of human cancer cell lines (cell cycle arrest at G1 or G2)

ti Inhibits tumour growth and survival time in a mouse multiple myeloma model

SINEa series [see Ref. 3ti]:

Slowly reversible binding at Cys528

ti Reduced growth, increased apoptosis and cell cycle arrest in G1 phase in a vast number of cancer cell lines [large literature–see Refs. 3ti,26ti]

ti Large number of preclinical and clinical stu- dies (selected examples below)

Selinexor (KPT-330)

>40 Clinical Trials, including combination trials) [21–23,24ti]:
ti Phase I/II clinical trials for a variety of solid/
haematological cancers.
ti Phase III trials for relapsed/refractory multi- ple myeloma, liposcarcoma and endometrial cancer.
ti FDA orphan drug/Fast Track designation for penta-refractory multiple myeloma [24ti]

Verdinexor (KPT-335) [see 25]

ti Limited virus shedding, and reduced pul- monary pro-inflammatory cytokine expres- sion, and moderate leukocyte infiltration into the bronchoalveolar space in mice and fer- rets [25]
ti Well-tolerated in Phase I; to be trialled as anti-influenza agent.

Eltanexor (KPT-8602) [see 56]

ti Phase I/II trial for relapsed/ refractory multi- ple myeloma, metastatic colorectal cancer, metastatic castration resistant prostate can- cer and higher risk myelodysplastic syndrome

Plumbagin [see Ref. 57]

Covalent binding to Cys528

ti Prevents EXP1 interaction with FOXO1, p21, p53 and p73 in Hela cells.
ti Suppresses NK-kB signalling pathway in and apoptosis of breast cancer lines.

ti Mouse studies indicate inhibition of DU145 hormone-refractory prostate cancer cell tumours and 50% tumour regression of PTEN-P2 prostate cancer cell tumours when combined with castration.

Curcumin and metabolites (e.g. octahydro- curcumin)
[see Ref. 58]

Covalent binding to Cys528

ti Suppression of NK-kB, myeloid differenta- tion protein 2 (MD2), Akt/mTOR and STAT3 signalling
ti Nuclear retention of FOXO1 and upregulation of p27 and p73

ti Phase IIa trial indicates reduced aberrant crypt foci number in colorectal neoplasia

a SINE, selective inhibitor of nuclear export.

Table 2
Summary of the properties of selected inhibitors of nuclear import that target IMPs
Inhibitor Target Reported biological effects Clinical potential

Ivermectin
IMPa
ti Inhibits nuclear accumulation of a range of IMPa/b1- but not IMPb1-recognised car- goes [27,36]
ti Long-term treatment induces PARP degra- dation/apoptosis [31titi]
ti Inhibits nuclear accumulation of the IMPa/
b1-transported factor HIF1a and HIF1a- dependent transcriptional responses in hypoxia [40]
ti Inhibits replication of viruses such as HIV-1, DENV1-4, Influenza, VEEVa [36–39]
ti Broad-spectrum antiparasitic agent, includ- ing against parasitic worm infestations caus- ing river blindness/filariasis, strongyloidiasis/
ascariasis, as well as ectoparasites causing scabies, pediculosis and rosacea [35]

Gossypol

IMPa

ti Inhibits WNVb protein NS5 nuclear import, and virus production [59]
ti Inhibits Hendra Virus V protein nuclear import and virus production [60]
ti Reduces viability of prostate cancer cell lines, and induces apoptosis/activates p53 [61]

ti Abandoned as human male contraceptive due to side effects including hypokalaemic paralysis and irreversible testicular damage.
ti Inhibits tumour incidence in a mouse xeno- graft model for prostate cancer [61]

Bimax 1 Bimax 2

IMPa

ti Inhibits IMP a/b-mediated nuclear import and growth of yeast, NIH 3T3 cells [33]

ti No trials to date

NTMc cSN50.1 N50 peptide [see Refs. 33,41,42]

IMPa5; IMPa1

ti Reduced nuclear import of pro-inflammatory NF-k B, STAT1a and AP-1
ti When fused to a cell-penetrating peptide, can target to the nucleus of cancer lines to increase efficacy of coadministered doxoru- bicin in cell killing [44ti ]

ti Reduces hyperlipidaemia, atherosclerosis and fatty liver in LDL receptor-deficient mice [43]
ti Prevents development of Type 1 diabetes in NOD mouse model.
ti Improves bacterial clearance, reduces thrombocytopenia and extends survival (in combination therapy) in polymicrobial sepsis mouse model [42]

INI-43d [31titi]

IMPb1; targets site of IMPa RanGTP binding to IMPb1

ti Inhibits proliferation of cervical, oesopha- geal, breast cancer but not normal cells through cell cycle arrest (G2-M)/induction of PARP degradation/apoptosis
ti Sensitises cervical cancer cells to killing by cisplatin

ti Inhibits oesophageal and cervical tumour growth in a mouse xenograft model.

Karyostatin 1Ae IMPb1; disrupts RanGTP binding to IMPb1

ti Inhibits IMPa/b mediated nuclear import of NFAT in cancer cell lines [28]

ti No trials to date

Importazole
[see Refs. 29,62]

IMPb1; disrupts RanGTP binding to IMPb1

ti Inhibits nuclear import of SV40 T-Antigen and NFAT in immortalised cell lines
ti Inhibits proliferation of multiple myeloma cell lines, inducing apoptosis via inhibition of NF- kB nuclear import [62] and PARP degrada- tion [31titi]
ti Kills malignant more efficiently than normal cells in a breast cancer tumour progression model [45]

ti No trials to date

M9Mf [34]

IMPb2/Kapb2/
Transportin

ti Inhibits nuclear import of IMPb2 cargoes (e.g. hnRNP A1 and M and HuR), but not IMPa/b1 cargoes [34,46]

ti No trials to date

aVEEV, Venezuelan equine encephalitis virus.
bWNV, West Nile Virus.
cNTM, nuclear transport modifier; although predicted to target the NLS-binding site of IMPa5, NTM also targets IMPa1, albeit with lower affinity.
dINI-43, 3-(1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine; ^PARP, poly ADP ribose polymerase.
eLikely targets IMPb1 residues Trp430 and Glu530.
fDesigned to target the PY-NLS binding site on IMPb2.

Table 3
Summary of the properties of selected inhibitors of nuclear import that target specific cargoes
Inhibitor Target Biological Effects Clinical potential

Mifepristone [27,36,48titi]
HIV IN-IMPa/b1 Interaction (impacts IN NLS residues)
ti Inhibits nuclear accumulation of IN but not other cargoes in transfected cells and in an in vitro nuclear import assay [27,36,48titi ]
ti Inhibits HIV-1 infection in cell culture model [36,48titi ]
ti Approved for human use (in combination with prostaglandin analog) for medical abor- tion and emergency contraception, or to treat hyperglycaemia in Cushing’s disease, uterine leiomyomata, endometriosis and unresectable or malignant meningioma [47]
ti Did not proceed past Phase I/II trials for HIV-1 [49]

Budesonide and analogues
[see Ref. 48titi]

HIV IN-IMPa/b1 Interaction (impacts IN NLS residues)

ti Inhibits nuclear accumulation of IN but not other cargoes in an in vitro nuclear import assay [48titi ]
ti Inhibits HIV-1 infection in cell culture model [48titi ]

ti Approved for asthma, chronic obstructive pulmonary disease, allergic rhinitis and nasal polyps, oral and rectal forms (inflammatory bowel diseases); cross reaction with HIV protease inhibitor ritonavir [50,51]

Flunisolide

ti Approved for asthma and rhinitis [50,51], without toxicity in HIV-1 patients

4-HPR Fenretinide [see Ref. 30]

Flavivirus
NS5-Impa/b1 Interaction

ti Inhibits infection with DENV including severe (antibody dependent enhanced – ADE) dis- ease form as well as ZIKV and WNV in cell culture [30,52,53,59]

ti Safety demonstrated in clinical trials for pediatric, recurrent breast and bladder can- cer [see Refs. 54,55]
ti Inhibits infection with DENV ADE-disease form as well as Zika virus in mouse model [30,53]
ti Phase IIb trials planned for DENV

EXP1, where all inhibitors appear to target the well- defined NES-binding pocket, much less is known of the precise molecular detail of the sites of binding of these inhibitors. The best characterised inhibitor of IMPa is ivermectin, a compound that was FDA approved for parasitic infections such as river blindness in humans, as well as veterinary indications [see Ref. 35], long before its nuclear transport inhibitory properties were discov- ered. Ivermectin was identified in 2011 in a high through- put screen for inhibitors of HIV-1 Integrase (IN) recog- nition by IMPa/b1, whereby specific inhibitors targeting IN itself (see below) could be distinguished from those targeting IMPa/b directly using a nested counterscreen strategy [27]. Ivermectin was shown to inhibit nuclear import of IN, but also of simian virus SV40 large tumour antigen (T-ag) and other IMPa/b1-dependent (but not IMPb1-dependent) cargoes, consistent with the idea that IMPa rather than IN itself was the direct target [27,36]
(see Figure 1ai). Importantly, it was confirmed to inhibit HIV-1 replication in a cell system through preventing IN nuclear access to mediate integration of the HIV-1 genome in the form of the preintegration complex (PIC), a key step in infection [36] (see Figure 1ai). Additionally, because of the fact that many viruses rely on IMPa/b1-dependent nuclear import of specific viral proteins for robust infection, a number of other viruses have since been shown to be inhibited by ivermectin, including Dengue virus (DENV) and related flaviviruses, influenza and Venezuelan equine encephalitis virus

(VEEV) [37–39]. Further, since IMPa/b1-dependent nuclear transport is integral to so many cellular functions, it seems likely that more research and clinical applications of ivermectin, such as in hypoxic signalling [see Ref. 40]
and cancer therapy [see Ref. 31titi ], remain to be discovered.

The importance of the discovery of ivermectin as a general IMPa/b1 nuclear transport inhibitor that is widely available is demonstrated by the rapid uptake of its use by the scientific community as a laboratory agent to probe protein nuclear transport mechanisms with already close to 100 publications documenting its use. This highlights poignantly the urgent need for inhibitors of other IMPs/EXPs to be identified, especially as under- standing of most other IMPs, apart from IMPb1, and perhaps IMPb2/Kapb2/transportin remains somewhat superficial, due in large part to a dearth of suitable research tools. Whether ivermectin or indeed many of the agents shown in Table 2 targeting IMPa inhibit all IMPa isoforms to the same extent is not clear, the only reported IMPa isoform-specific agent (NTM — nuclear transport modifier) thus far being cSN50.1, a peptide based on the NLS of the NF-kB p50 subunit predicted to target the NLS-binding pocket [41]. NTM/cSN50.1 has been shown to be efficacious in several preclinical models [42,43,44ti ], including reducing atherosclerosis, plasma cholesterol, triglycerides and glucose along with liver fat and inflammatory markers, in a murine model of

Figure 2

(a)
Empty
HIV-1 Rev NES
peptide

KPT-251 KPT-276 KPT-8602 LMB

(b)

Budesonide Flunisolide

Current Opinion in Cell Biology

Prevention of nuclear target signal recognition through inhibitors of EXP1 occupying the NES-binding pocket (a) and altering conformation at the NLS in HIV-1 Integrase (b).
(a) The NES-binding pocket of EXP1 is shown as a surface fill model in grey, with no ligand (empty), or bound to the ligands HIV-1 Rev NES peptide, LMB, KPT-251, -276 or -8602 (in yellow). Residues lining the binding groove of EXP1 are highlighted in blue, with Cys528 in magenta. The panels were generated from PDBs 4HB2 [11], 4HAT [11], 3NC0 [12], 4GPT [13], 4WVF [14] and 5JLJ [15] respectively using Pymol software [see also Refs. 16,17]. (b) Sites on IN for binding of Budesonide (left) or Flunisolide (right), based on 15N-1H HSQC spectra of 15N-labelled

familial hypercholesterolemia when fused to the signal sequence of human fibroblast growth factor 4 [43].

Several small molecules with anti-tumour potential that target IMPb1 have been characterised [see also Refs. 26ti ,31titi ]; these include the small molecules importazole, to which malignant breast cancer cells are hypersensitive compared to isogenic normal cells [45; see also Ref. 31titi ], and INI-43, identified by in silico screening to target the overlapping binding sites on IMPb1 for IMPa and RanGTP [31titi ]. Significantly, INI-43 shows anticancer therapeutic potential against oesophageal and cervical cancer in a mouse xenograft model [31titi ]. Table 2 high- lights the fact that there appears to be only one IMP that has been targeted for inhibitor development other than IMPa and IMPb1. Peptide inhibitor M9M (a fusion of the N-terminal and C-terminal halves of the NLSs of the heterogeneous nuclear ribonucleoprotein particle – hnRNP – A1 and M proteins, respectively), was designed to target the PY-NLS binding site of IMPb2/Kapb2/
Transportin [34], and confirmed to inhibit nuclear import of IMPb2 cargoes (hnRNP A1 and M, as well as pre- mRNA-binding protein HuR), but not those recognised by IMPa/b1, implying robust specificity for IMPb2- dependent transport pathways [see also Ref. 46]. The work summarised in Tables 1 and 2 testifies to the success in identifying inhibitors of utility for research and poten- tially the clinic, as well as emphasising very clearly that although we now appear to have inhibitors for our best understood nuclear import/export pathways, inhibitors to the IMPs/EXPs thus far not represented would be invalu- able to help establish the importance of these transporters in mammalian cell biology, as well as provide potential new avenues for therapeutic intervention in the future.

Cargo-specific nuclear import inhibitors; targeting the host–pathogen interface
As alluded to above, because the nuclear transport inhi- bitors listed in Tables 1 and 2 target essential IMPs/EXPs and thereby general transport pathways of the cell, their use is likely to be inextricably linked with toxicity, likely limiting their clinical use beyond high-grade cancers [see Ref. 26ti ] and potentially acute viral infections where treatment would be only for a number of days. Inhibitors specific to particular cargoes, by contrast, are of great interest, since global effects on multiple cellular proteins/
functions are spared in this scenario. Progress has been made in the case of selective targeting of viral proteins (see also [42]), with application in developing therapeu- tics for viral disease; since viruses represent a huge burden of disease worldwide for human health, with treatments to combat them either preclusively expensive and limited by problems of resistance (e.g. HIV-1), or non-existence (e.g. flaviviruses), targeting the

host–pathogen interface to avoid issues of resistance and toxicity seems an attractive possibility [27,36]. Table 3 (and see also Figure 1aii) summarises progress in this area, highlighting the fact that specific inhibitors can be identified that ultimately are useful as antivirals, and showing the way for the future in terms of screening/
counterscreening for agents that can have exquisite selec- tivity [27].

The first cargo-specific nuclear transport inhibitor described was mifepristone as a specific inhibitor of recognition by IMPa/b1 of HIV-1 IN (see also above) but not other IMPa/b1-recognised cargoes [27]. Mifep- ristone is a steroid approved for use for several human indications through its progesterone/glucocorticoid recep- tor (GR) antagonistic activity (see Table 3) [47], but although able to inhibit HIV infection in cell models [see Refs. 36,48titi ], Phase I/II trials indicate insufficient efficacy against HIV-1 [49]. A recent follow-up study succeeded in confirming direct binding of mifepristone (but not ivermectin) to the core domain of IN in the vicinity of the NLS using NMR [48titi ]. Although mifep- ristone has also been reported to inhibit nuclear accumu- lation of VEEV Capsid that is mediated by IMPa/b1, as well as VEEV replication [see Ref. 38], it is unlikely that this occurs through direct impact of mifepristone on VEEV Capsid-IMPa/b1 interaction [27,36], but rather through effects on steroid hormone receptor signalling.

A second cargo-specific agent, budesonide, also able to inhibit recognition of HIV-1 IN by IMPa/b1 [48titi ] has been recently characterised, together with analysis of the perturbing effects of binding of budesonide and analo- gues thereof to the IN NLS region as visualised by NMR, and inhibitory effects on IN nuclear import, HIV-1 repli- cation [48titi ]. Most significantly, budesonide and its ana- logue flunisolide inhibit HIV PIC nuclear entry mediated by IN, consistent with the idea that IN is a key contribu- tor to nuclear transport of the HIV PIC; that is, in inhibiting IN nuclear import, budesonide/flunisolide pre- vents nuclear import of the PIC (see Figure 1aii) [see Refs. 27,48titi ]. NMR analysis showed that budesonide binding perturbs residues adjacent to, and even within the IN NLS, consistent with idea that it directly impacts IN recognition by IMPa/b1; structure–activity relation- ship analysis showed varying levels of binding of the budesonide analogues to the budesonide binding patch on IN (see Figure 2b), which correlated strongly with ability to inhibit both IN-IMPa/b1 binding and IN nuclear accumulation of IN in an in vitro reconstituted nuclear transport assay, as well as to decrease HIV viral replication. Significantly, the strongest acting analogue flunisolide was also shown to prevent IN/PIC nuclear import (Figure 1aii) and integration of the PIC.

(Figure 2 Legend Continued) INCORE123 in the absence and presence of ligand. High strength perturbations observed are shown in blue superimposed onto the IN core domain crystal structure (grey surface model, Pymol software)]; IN NLS residues are in pink [see Ref. 48titi].

Budesonide and flunisolide are FDA-approved for rhini- tis/asthma/Crohn’s disease/ulcerative colitis and asthma/
rhinitis respectively (Table 3); although they have not been used to treat HIV-1, budesonide interacts with HIV protease inhibitor drugs such as ritonavir and has other toxicity issues [50,51], but flunisolide is considered largely safe [51]. Thus, flunisolide may have potential as a HIV-1 specific agent in the clinic [48titi ,51].

A cargo-specific inhibitor with promise as an antiviral with respect to flaviviruses such as DENV and Zika virus (ZIKV) is N-(4-hydroxyphenyl) retinamide (4-HPR, also known as fenretinide) which was identified, using the high-throughput screening/counterscreening strategy used to identify mifepristone/budesonide (above), as a specific inhibitor of IMPa/b1-DENV NS5 interaction through targeting NS5 (see Figure 1aii) rather than IMPa/b1 [30]. 4-HPR was shown to inhibit all forms of DENV disease, including infection by all four serotypes of DENV, as well as the severe antibody-dependent enhanced (ADE) lethal DENV haemorrhaghic fever form

implementing state-of-the-art screening/counterscreen- ing strategies, together with structure-based approaches (see above) make this eminently realisable, ensuring that the area of nuclear transport will continue to prove a fertile avenue of research for the foreseeable future.

Conflict of interest statement
Nothing declared.

Acknowledgements
We are grateful to the National Health and Medical Research Council and National Breast Cancer Foundation for funding to DAJ (Senior Principal Research Fellow APP1103050) and KMW (Career Development Fellowship #ECF-17-007), respectively.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:
ti of special interest
titi of outstanding interest
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cleocyte cultures (which represent a model of human infection), and in a lethal mouse model [30]; it also appeared to have potential as a prophylactic. Importantly, 4-HPR can also protect against West Nile Virus (WNV) [30] and ZIKV [52,53]. Excitingly in this context, 4-HPR has an established safety profile, having been used exten- sively in an oral formulation in humans for various forms
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high doses to children for long periods [see Refs. 30,54,55]. Pharmacokinetic analyses indicate that clini- cally effective concentrations of 4-HPR are likely to be realistically achieved to combat DENV infection in humans, making it an exciting prospect for further devel- opment as an anti-DENV agent.

Outlook
There has been much progress made in the last five or so years in identifying and characterising nuclear transport inhibitors. Perhaps understandably, the main efforts have been directed at the best known nuclear transport path- ways (EXP1-mediated nuclear export and IMPa/b1- dependent nuclear import), but the value of the derived for research as well as clinical applications cannot be underestimated. It seems clear that a palette of selective inhibitors for all of the IMPs/EXPs would be ideal to aid in establishing their key roles in cell biology, as well as opening up new possibilities with respect to therapeutics.

In terms of clinical applications, the low toxicity of inhibitors of nuclear transport of specific cargoes (e.g. 4-HPR — see above) highlight the possibility of fine- tuning inhibitors to focused research, and importantly clinical applications. Delineating the fine detail of nucleo- cytoplasmic transport remains of key importance to understanding how cells function; the progress in
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Intriguing study that used in silico structure-based high-throughput screening based on the crystal structure of the VEEV Capsid protein NLS peptide-IMPa complex to derive IMPa targeting as well as Capsid specific inhibitors.
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Landmark clinical study leading to FDA ophan drug/Fast Track designa- tion of Selinexor to treat relapsed/refractory multiple myeloma.
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43.Liu Y, Major AS, Zienkiewicz J, Gabriel CL, Veach RA, Moore DJ, Collins RD, Hawiger J: Nuclear transport modulation reduces hypercholesterolemia, atherosclerosis, and fatty liver. J Am Heart Assoc 2013, 2 e000093.

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58.Zhang Z, Luo D, Xie J, Lin G, Zhou J, Liu W, Li H, Yi T, Su Z, Chen J:

High quality focused SAR study that correlated binding of budesonide analogues as well as mifepristone to the NLS region of HIV-1 IN by NMR with anti-HIV-1 activity, demonstrating inhibition of nuclear entry/integra- tion of the HIV-1 preintegration complex.
49.Para MF, Schouten J, Rosenkranz SL, Yu S, Weiner D, Tebas P, White CJ, Reeds D, Lertora J, Patterson KB et al.: Phase I/II trial of the anti-HIV activity of mifepristone in HIV-infected subjects ACTG 5200. J Acquir Immune Defic Syndr 2010, 53:491-495.
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Octahydrocurcumin, a final hydrogenated metabolite of curcumin, possesses superior anti-tumor activity through induction of cellular apoptosis. Food Funct 2018, 9:2005-2014.
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