Small-Molecule Antagonist Targeting Exportin-1 via Rational Structure-Based Discovery

Article abstract of DOI:10.1021/acs.jmedchem.9b01663

Exportin-1 (also named as CRM1) plays a prominent role in autoimmune disorders and has emerged as a potential therapeutic target for colitis. Here we report on the rational structure-based discovery of a small-molecule antagonist of exportin-1, LFS-829, with low-range nanomolar activities. The co-crystallographic structure, surface plasmon resonance binding assay, and cell-based phenotypic nuclear export functional assay validated that exportin-1 is a key target of LFS-829. Moreover, we demonstrated that the C528S mutation or the knockdown on exportin-1 can abolish the cellular activities of LFS-829. Strikingly, oral administration of LFS-829 can significantly reverse the pathological features of colitis model mice. We revealed that LFS-829 can attenuate dual NF-κB signaling and the Nrf2 cytoprotection pathway via targeting exportin-1 in colitis mice. Moreover, LFS-829 has a very low risk of cardiotoxicity and acute toxicity. Therefore, LFS-829 holds great promise for the treatment of colitis and may warrant translation for use in clinical trials.

Full text of DOI:10.1021/acs.jmedchem.9b01663

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Article
Small-molecule antagonist targeting exportin-1
via rational structure-based discovery
Xibao Tian, Jiali Gao, Meishuo Liu, Yuqin Lei, Fangjun Wang, Jin Chen,
Peng Chu, Jiujiao gao, Feida Long, Minzhi Liang, Xiangyu Long, Huiying
Chu, Cuixia Liu, Xueliang Li, Qingxiang Sun, Guohui Li, and Yongliang Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01663 • Publication Date (Web): 30 Mar 2020
Downloaded from pubs.acs.org on March 30, 2020
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Small-molecule antagonist targeting exportin-1 via rational structure-based discovery
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Xibao Tian1, ‡, Jiali Gao1, ‡, Meishuo Liu1, ‡, Yuqin Lei2, ‡, Fangjun Wang3,4, ‡, Jin Chen3,4, Peng Chu1,
Jiujiao Gao1, Feida Long1, Minzhi Liang1, Xiangyu Long1, Huiying Chu5, Cuixia Liu6, Xueliang Li6,
Qingxiang Sun2,*, Guohui Li5,* and Yongliang Yang1, *
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1Laboratoy of Innovative Drug Discovery, School of Bioengineering, Dalian University of Technology,
Dalian, 116023, China;
2Department of Pathology and State Key Laboratory of Biotherapy, West China Hospital, Sichuan
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University, Chengdu, 610041, China;
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3CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic
R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian
116023 Liaoning, PR China;
4University of Chinese Academy of Sciences, Beijing 100049, China;
5Laboratory of Molecular Modeling and Design, State key Laboratory of Molecular Reaction
Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Rd.
Dalian 116023, China;
6Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University,
Nanjing, China
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Key Words: colitis; exportin-1; NF-kB signaling; Nrf2 pathway; drug discovery
‡These authors contributed equally to the work.
*To whom correspondence may be addressed:
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Yongliang Yang, E-mail: everbright99@foxmail.com (lead contact)
Guo-hui Li, E-mail: ghli@dicp.ac.cn
Qingxiang Sun, E-mail: qingxiang.sun@scu.edu.cn
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Abstract
Exportin-1 (also named as CRM1) plays a prominent role in autoimmune disorders and has emerged
as a potential therapeutic target for colitis. Here we report on the rational structure-based discovery of
a small-molecule antagonist of exportin-1, LFS-829, with low-range nanomolar activities. The co-
crystallographic structure, surface plasmon resonance binding assay and cell-based phenotypic nuclear
export functional assay validated that exportin-1 is a key target of LFS-829. Moreover, we
demonstrated that the C528S mutation or the knockdown on exportin-1 can abolish the cellular
activities of LFS-829. Strikingly, oral administration of LFS-829 can significantly reverse the
pathological features of colitis model mice. We revealed that LFS-829 can attenuate dual NF-kB
signaling and Nrf2 cytoprotection pathway via targeting exportin-1 in colitis mice. Moreover, LFS-
829 has a very low risk of cardiotoxicity and acute toxicity. Therefore, LFS-829 holds great promise
for the treatment of colitis and may warrant translation for use in clinical trials.
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Introduction
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The incidence of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and
Crohn’s disease (CD), has been increasing very rapidly during the past decade worldwide, affecting
more than two million individuals in North America, more than three million individuals in Asia and
around two million individuals in Europe1. The chronic inflammatory disorders of colitis also harbor
a huge risk of colorectal cancer development2. Unfortunately, to date, there is no known cure for colitis
and available first-line treatment options are aimed towards controlling symptoms for mildly patients3.
Therefore, new therapeutic strategies are urgently needed to combat colitis.
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Exportin-1 (also named as CRM1) is responsible for the nuclear export of critical transcription
factors and tumor suppressor proteins 4-8. There is a considerable amount of evidence implicating that
the dysregulation of CRM1 contributes to the pathogenesis of a panel of human cancer and
autoimmune disorders. In particular, prior reports demonstrated that CRM1 is highly upregulated in
the intestinal epithelial cells (IEC) of both CD patients and model animals9. Therefore, CRM1 has
emerged as a viable target for developing new therapeutics against colitis. Previously, we found that
sulforaphene, the major active chemical component from the seeds of Raphanus sativus, can alleviate
colitis in model mice and protect the epithelial barrier by covalently targeting a conserved cysteine
residue in the NES (nuclear export signal) binding cleft of CRM15, 10
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Herein, we reported the rational structure-based discovery and extensive evaluation of the
synthetic analogue of sulforaphene, LFS-829, in colitis model mice. Importantly, the co-
crystallographic structure, cell-based phenotypic nuclear export functional assay, SPR (surface
plasmon resonance) binding assay firmly establish that CRM1 is a major cellular target of LFS-829.
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Furthermore, oral treatment of LFS-829 can reverse the pathological development of colitis in model
mice. Our proteomics analysis study also showed that LFS-829 can reverse the disease-affected
signatures in colitis mice. Remarkably, LFS-829 exhibits excellent safety profiles and pharmacological
properties compatible for treating colitis. Our results not only provide the mechanistic insights to
explain the activities of LFS-829 in reversing colitis but also open new avenues of developing novel
pharmacological treatment of colitis.
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Results
CRM1 is significantly upregulated in the IEC of colitis patients
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To investigate the intestinal expression of CRM1 in colitis patients, we performed analysis on the
endoscopic mucosal biopsy tissues from the inflamed and non-inflamed areas of UC patients and
normal individuals. We employed Western blot experiments and confirmed that CRM1 was
significantly overexpressed in the intestinal epithelial cells (IEC) of colitis patients as compared with
normal controls (Figure S1). Hence, our results indicated that CRM1 is closely associated with the
progression and pathogenesis of colitis.
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Structure-based discovery of small molecule antagonist LFS-829
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In our prior work, we identified that sulforaphene (LFS-01) is a natural antagonist of CRM1 and
the isocyanate group of LFS-01 can trap with the cysteine residue in the nuclear export signal (NES)
binding cleft of CRM1 via a covalent bond5. We revealed that oral administration of LFS-01 can
reverse colitis in model mice10. Herein we wanted to develop a potent antagonist based on the scaffold
of LFS-01 and evaluate its efficacy in colitis model. First, to increase the non-covalent interactions
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with the NES binding groove of CRM1, we installed a phenyl ring next to the sulfoxide moiety in the
parent structure of LFS-01, which leads to compound LFS-06 that inhibits nuclear export with an IC50
value of 1.5 µM, 3 folds better than that of LFS-01 determined by cell-based phenotypic nuclear export
functional assay as previously described5. We next sought to optimize the scaffold of LFS-06 with
better potency and drug-like properties for colitis. To this end, we generated a virtual combinatorial
library based on the phenyl ring of LFS-06. In total, 45 substitution moieties were used in the
generation of a virtual library consisting of 2025 analogue molecules with disubstituted groups on the
phenyl ring (see Dataset_S1). Furthermore, we used the ADME/T module as well as the Drug-likeness
module implemented in the DiscoveryStudio suite to filter out top 100 analogue molecules from the
combinatorial library with acceptable ADME/T and drug-like properties. Subsequently, we employed
covalent docking to predict the binding of the analogue molecules with the nuclear export signal (NES)
cleft of CRM1. Lastly, we selected LFS-829 from top 15 analogue molecules to prepare on the basis
of binding energy, drug-like properties, ADME/T properties, solubility and easy synthesis (see
Scheme 1, also see Dataset_S1).
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Scheme 1. Discovery scheme of small-molecule antagonist LFS-829.
Crystallographic structure of LFS-829 bound with CRM1
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To establish the precise mode of actions of LFS-829, we determined its co-crystal structure
complexed with humanized yeast (Saccharomyces cerevisiae) CRM1 protein, yeast RanBP1 and
human RanGTP at 2.60 Å resolution (PDB code: 5ZPU; Figure 1; Figure S2-S3). The co-crystal
structure clearly shows that LFS-829 forms a covalent bond with the sulfur atom of Cys539 located in
the NES binding cleft of CRM1, similar to that of existing CRM1 inhibitor KPT-33011. The di-
trifluoromethyl-phenyl moiety fits well into a subpocket in the NES binding cleft of CRM1 formed by
Ile555, Met556, Thr575, Val576, Lys579 and Leu580 (Figure 1B and 1E). Importantly, the two
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trifluoromethyl substituent groups form strong hydrogen bonding with Leu536, Lys548, Lys579 and
Leu580 (Figure 1C and 1E). Moreover, the phenyl ring of LFS-829 enjoys hydrophobic contacts with
Phe583 and Phe572 (Figure 1E). This co-crystal structure provides a solid structural basis for the
specific and covalent binding of LFS-829 with Cys539 in the NES-binding cleft of CRM1. Additionally,
our mass spectroscopic analysis and pull-down assay experiments also showed that LFS-829 is a potent
and covalent CRM1 inhibitor (Figure S4). In particular, we showed by pull-down assay that LFS-829
binds to both human CRM1 and humanized yeast CRM1 strongly by inhibiting the binding of GST-
NESMVM to the NES binding groove (Figure 1D). In addition, C528S mutation on CRM1 completely
abolished LFS-829’s binding, suggesting that the covalent bond formation is essential for its function
(Figure 1D and Figure S4, C528 in human CRM1 is equivalent to C539 in yeast CRM1).
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Figure 1. Crystallographic structure of compound LFS-829 bound to CRM1. (A) Co-
crystallographic structure of compound LFS-829 (green) complexed with CRM1 (cyan) at 2.60 Å
resolution (PDB code: 5ZPU); (B) A covalent bond between Cys539 and LFS-829 was detected; (C)
Electrostatic potential surface display of LFS-829 bound in the nuclear export signal (NES) cleft of
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CRM1; (D) SDS-PAGE analysis of GST-NESMVM pull-down assay of various CRM1 in the presence
or absence of 50 µM compound LFS-829; (E) Schematic representation shows the interaction of LFS-
829 with the NES-binding cleft of CRM1 (the red dash line indicates the hydrogen bonding and the
dark dash line indicates the hydrophobic pocket).
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CRM1 is a major cellular target of LFS-829
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In addition to the co-crystal structure, we revealed that CRM1 is a major target of LFS-829 through
a series of experiments. First, the incubation of CRM1 protein with LFS-829 caused an increase in
molecular weight of 373 Da as determined by LC-MS/MS via LTQ Orbitrap method, corresponding
to the covalent addition of the LFS-829 moiety (Figure S4). We demonstrated that the activities
towards HCT-15 cells by LFS-829 was significantly abrogated when CRM1 was knockdown by
siCRM1, implicating that CRM1 is a main target responsible for the cellular activities of LFS-829
(Figure 2B). Next, we tried to evaluate the binding specificity of LFS-829 with CRM1 as compared
to other possible protein targets with reactive cysteine in the binding pocket. Here, we chose IkBa and
Keap1 as two control probes to investigate because both of them are reactive for covalent inhibitors
and thereby frequently used for testing selectivity. Our SPR (Surface Plasmon Resonance, Biacore
T200) results revealed that LFS-829 binds strongly with CRM1 (Kd~26.95nM, Figure 2A) as
compared to KPT-330 (Kd~18.74nM). In contrast, LFS-829 doesn’t bind with either two control probes
IkBa or Keap1 in SPR binding assay experiments.
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Importantly, we investigated the time course of CRM1-LFS829 complex degradation by cell-
based phenotypic nuclear export functional assay (Figure 2C). Here, KPT-330 was used as control
compound because prior studies have shown that KPT-330 is a reversible CRM1 inhibitor. Briefly,
NES-GFP was quickly accumulated in the nucleus upon the treatment of KPT-330 or LFS-829.
Subsequently, cells (293T cell line) were washed with PBS to remove the KPT-330 or LFS-829 and
we monitored the localization of NES-GFP per hour by confocal microscopy. Only after two hours,
NES-GFP in cells incubated with LFS-829 was almost completely translocated to the cytoplasm,
whereas in cells incubated with KPT-330, NES-GFP was still localized in the nucleus of the cells. This
result implicates that LFS-829 binds with CRM1 in a more reversible fashion as compared to that of
KPT-330, indicating that LFS-829 may exert a lower toxicity profile. Moreover, we used GSH/GSSG
detection assay kit to show that the GSH:GSSG ratio keeps constant upon the treatment of LFS-829
and therefore we ruled out the possibility that LFS-829 is a general redox modulator (see Figure S7).
Collectively, our results revealed that CRM1 is a major cellular target of LFS-829 and it appears that
LFS-829 is a reversible inhibitor of CRM1.
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Figure 2. CRM1 is a major cellular target of LFS-829. (A) The SPR (Surface Plasmon Resonance)
binding assay between LFS-829 and CRM1 performed on Biacore T200 with KPT-300 as positive
control (LFS-829, Kd=26.95 nM; KPT-330, Kd=18.74 nM); (B) The cellular activities of LFS-829 on
siCRM1-HCT-15 and wild type HCT-15 cell line with different concentrations of treatment for 24 h;
(C) The time course of CRM1-LFS829 complex degradation determined by cell-based phenotypic
nuclear export functional assay. The localization of NES-GFP per hour was monitored by confocal
microscopy after cells (293T cell line) were washed with PBS to remove the compounds. Only after
two hours, NES-GFP in cells incubated with LFS-829 was almost completely translocated to the
cytoplasm, indicating its reverse binding.
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LFS-829 exhibits superior efficacy than first-line therapy in colitis model
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Next, we assessed the therapeutic efficacy of LFS-829 in DSS (dextran sodium sulfate) induced
colitis mice as compared to 5-ASA (5-aminosalicylicacid), the first-line therapy for colitis patients12,
13. Oral treatment of LFS-829 significantly attenuated the severity of DSS-induced colitis in model
mice (Figure 3). The dosage of 30mg/kg that we used in the murine colitis model was translated from
the clinical dosage of 5-ASA for human. Remarkably, LFS-829 treatment can reduce body weight loss
(Figure 3A), reverse colon shortening (Figure 3B), decreased diarrhea (Figure 3C) and improved
colonic histology (Figure 3D). On the other hand, mice subjected to seven days’ administration of
DSS supplemented in drinking water developed an acute colitis characterized by bloody diarrhea,
ulcerations and infiltrations with granulocytes and showed severe weight loss resulting in a mortality
of 20% (Figure 3A). Furthermore, we observed an extensive destruction of the colon mucosal layer
and severe colonic damage in the untreated mice group characterized by ulceration, epithelial cell loss,
reduction of the density of the tubular glands and disseminated fibrosis. In contrast, the LFS-829-
treated group showed a preserved mucosal architecture, reduced swelling, inflammation and ulceration
and more complete re-epithelialization (Figure 3D). Noteworthy, oral treatment of 5-ASA failed to
prevent DSS-induced colitis at the same dose. Importantly, we demonstrated that LFS-829 can
attenuate colitis in a dose-dependent fashion and 3mg/kg dose of LFS-829 could lead to increased
severity of colitis. These data provide strong evidences that LFS-829 oral treatment can reverse the
course of DSS-induced acute colitis.
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Figure 3. Oral administration of LFS-829 significantly ameliorates DSS-induced colitis mice as
compared to 5-ASA. Oral treatment of LFS-829 was assessed by (A) Survival rate. (B) Body weight.
(C) Diarrhea. (D) The colon after sacrifice for each group. (E) H&E-stained results for the sections of
mouse colon. (F) The colon length determined for each group after sacrifice. (G) DAI score for each
group. (H) Histopathological score for the H&E-stained sections. Statistical analysis of the quantitative
multiple group comparisons was performed using the one-way analysis of variance (ANOVA)
followed by Tukeyʼs test, * p < 0.05, ** p < 0.01, *** p < 0.001.
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The comparison of LFS-829 with clinical candidate compound APD334
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APD334 (also known as Etrasimod) is the next generation small-molecule modulator of
sphingosine-1-phosphate (S1P) currently in the evaluation of Phase 3 clinical trials for the treatment
of ulcerative colitis (UC)14. Of note, APD334 achieves rather positive results in clinical remission for
colitis patients (2mg per day for patients which can be translated into 10mg/kg dosage in colitis murine
model). Therefore, we wanted to compare the therapeutic potential of LFS-829 with APD334 in colitis
model. In brief, the group of mice treated with LFS-829 after DSS administration recovered more body
weight and achieved better disease activity index score (DAI score) as compared to the APD334
treatment group under the same dose (Figure 4A-B). Moreover, animals treated with LFS-829
exhibited much less incidence of diarrhea over the course of treatment as compared with the APD334
treatment group (Figure 4C). In addition, oral treatment of LFS-829 to colitis model mice significantly
restored colon length as compared to APD334 treatment group (Figure 4D-E). Noteworthy, we
observed a much better recovery of swelling, inflammation and ulceration in LFS-829 treatment group
as compared to the APD334 treatment group within one week. This is consistent with the results that
LFS-829 treatment can reduce more proinflammatory cytokines levels in the serum of colon tissues as
compared to APD334 treatment group as measured by ELISA assay (Figure 7D). We found that LFS-
829 treatment clearly restored the damaged colon mucosal layer in DSS group as observed by the
histopathological staining analysis (Figure 4E). Together, our results indicate that LFS-829 treatment
promotes better efficacy and mucosal healing in colitis model mice than APD334 in one-week dosing
schedule. However, we wanted to remind the reader that our results don’t infer that APD334 is not
efficacious for colitis. Our results only implicate that LFS-829 can exert its biological effects against
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colitis in a relatively short period of dosing time as compared to APD334. More studies may be
warranted to compare the two compounds in a long period of dosing time for colitis treatment.
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Figure 4. LFS-829 exerts superior efficacy than APD334 in colitis mice in one-week dosing
schedule. (A) The change of body weight during the course of treatment by LFS-829 and APD334.
(B) DAI score during the course of treatment by LFS-829 and APD334. (C) The percentage of diarrhea
during the course of treatment. (D) Colon length among different treatment groups. (E) The colon after
sacrifice for LFS-829 treatment group and APD334 treatment group. From left to right: control group,
DSS model group, APD334 treatment group (10mg/kg) and LFS-829 treatment group (10mg/kg). (F)
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H&E staining of colon section for different animal groups. (G) Immunohistochemical analysis for
Occuludin expression in intestinal epithelial layer for different animal groups.
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Proteomics analysis of the expression of disease signatures
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To dissect the molecular pathways underlying the pathogenesis of colitis, we assessed the gene
expression signatures determined by RNA-seq techniques in both UC patients and DSS animal model
derived from previous literatures15, 16. Of 92 IBD-associated genes, functional enrichment analysis
revealed that gene signatures involved in host defense, inflammatory response, NF-kB signaling,
immune response and gastrointestinal disease are mostly dysregulated in both UC patients and DSS-
induced animal model (Figure 5, Dataset_S2). Next, we employed proteomics technique to detect the
global protein expression change in the intestinal epithelial cells (IEC) of the DSS animal model group
and LFS-829 treatment group, respectively. Comparative inspection of the protein expression in DSS
model group and LFS-829 treatment group revealed the expected antithetical expression patterns
between the two groups (Figure 5). Interestingly, functional enrichment analysis implicated that
protein signatures involved in epithelial barrier, wound healing, immune response, inflammatory
response, host defense, gastrointestinal disease and NF-kB signaling were reciprocally expressed
between the LFS-829 treatment group and the colitis model group. These results implicated that LFS-
829 treatment can restore the disturbed disease signature patterns in colitis model mice.
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Figure 5. Proteomics analysis of protein signatures between DSS-induced animal group and
LFS-829 treated group. Comparison of the LFS-829 treatment group signature (right) to the DSS-
induced animal group protein signature profile (left) determined by proteomics techniques, showing a
generally anticorrelated pattern, with genes at the top- and bottommost ends of the expression pattern
showing generally oppositional expression patterns. Protein signatures that are up-regulated are shown
in green; Protein signatures that are downregulated are shown in red. An adjusted [Adj] p-value ≤ 0.05
was considered significant. The pathways and functional groups significantly enriched (enrichment P
< 0.05) in the top and bottom 25% of genes are shown on the right-hand side.
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LFS-829 attenuates both NF- B signaling and Nrf2 cytoprotection pathway in colitis model
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Previous work has suggested that NF-kB signaling and host defense pathway play predominant
roles in the pathogenesis of colitis. In accordance, our proteomics results also revealed that gene
signatures involved in host defense and NF-kB signaling were dysregulated in DSS model group.
Therefore, we wanted to investigate the biological effects of LFS-829 towards NF-kB signaling and
host defense pathway in colitis model. First, we observed a significant accumulation of IkBa and Nrf2
in the nucleus by confocal microscopy upon treatment with LFS-829 (Figure 6). Consequently, the
accumulated IkBa in the nucleus can bind to NF-kB and inhibit its transcriptional activities.
Analogously, the accumulated Nrf2 in the nucleus can induce the cellular defense pathway. Next, as
assessed by Western blots, we found a significant upregulation of p65 levels in the intestinal epithelial
cells obtained from the DSS-treated group as compared to the control group (see Figure 7A). This
constitutive activity of NF-kB p65 was accompanied by the increased production of a few
proinflammatory cytokines including Cox-2, TNF-a, IL-6, IL-1b and IL-23 as measured by ELISA
assay (see Figure 7D). Remarkably, when colitis mice were treated with LFS-829, a significant
reduction of NF-kB p65 as well as the proinflammatory cytokines mentioned above were observed
(Figure 7A and 7D). Accordingly, we found that oral treatment of LFS-829 can abrogate the clinical
signs of established intestinal inflammation in colitis mice. Moreover, LFS-829 treated colitis mice no
longer had diarrhea and started to gain weight (Figure 3-4).
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On the other hand, prior reports have shown that a few important antioxidant and anti-
inflammatory enzymes regulated by Nrf2 cellular defense pathway play protective roles in the
pathogenesis of colitis17, 18. As mentioned before, the nuclear retention of Nrf2 by LFS-829 treatment
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may activate the cytoprotection pathway in the colon of colitis mice. Hence, we evaluated the
expression levels of several antioxidant and phase II detoxifying enzymes among different animal
groups. As expected, we found that DSS-induced colitis group was associated with increased
expression of Nrf2-regulated enzymes such as NQO1 and reduced expression of Nrf2 and phase II
detoxifying enzyme HO-1 as compared to the control group (Figure 7B). In contrast, in LFS-829
treatment group, we found a significant reduction of expression levels of NQO1and increase of
expression levels of Nrf2 and HO-1 as compared to the DSS model group (Figure 7B). Of note, the
overexpression of MRP8 (S100A8) and MRP14 (S100A9), which is called as the calprotectin complex,
has been regarded as a very valuable biomarker for monitoring the colonic inflammatory conditions in
colitis patients19. Interestingly, we observed that the upregulation of MRP8 (S100A8) and MRP14
(S100A9) in DSS model group as compared to the control group was restored upon the treatment of
LFS-829 as measured by Western blot (Figure 7C). Tight junction protein occludin is crucial to the
integration of the intestinal epithelial barrier and therefore often used as a critical pathological marker
for colitis patients. Our immunohistochemical analysis results revealed that occludin expression was
significantly reduced in colitis mice (Figure 4F). Conversely, LS-829 treatment restored the
dysregulaiton of occludin which suggested that LFS-829 protects the epithelial barrier during intestinal
damage of colitis. Taken together, our results implicate that LFS-829 can reverse the pathogenetic
course of colitis by regulating both NF-kB signaling and Nrf2 cellular defense pathway.
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Figure 6. LFS-829 treatment causes nuclear accumulation of IkBa and Nrf2 observed by confocal
microscopy. (A) Nuclear retention of IkBa upon treatment with LFS-829; (B) Nuclear retention of
IkBa upon treatment with LFS-829; (C-D) Quantification of nuclear IkBa and Nrf2 ratio by
fluorescence intensity per cell.
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Figure 7. LFS-829 treatment attenuates downstream targets of NF- B signaling and Nrf2
cytoprotection pathway. (A) LFS-829 treatment downregulates the expression of p65 and Cox-2. (B)
LFS-829 treatment attenuates the expression of HO-1 and NQO1. (C) LFS-829 treatment regulates the
expression of MRP8 and MRP14. (D) ELISA detection of cytokine production after treatment.
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Encapsulation of LFS-829 in nanoparticles
IBD is a chronic relapsing disease and patients may require a rather long-term medication.
Therefore, oral colon-specific drug delivery system can enhance the drug efficacy and reduce adverse
effects for patients20. In our prior work, we have prepared a water-soluble nanoparticle encapsulated
form of sulforaphene by a-Cyclodextrin10. Analogously, to enhance the oral bioavailability, stability,
solubility and other therapeutic properties of LFS-829, we prepared a nanoparticle encapsulated form
of LFS-829 by a-cyclodextrin as previously described (named as CD-LFS-829 or Cyclone-829,
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Figure 8). Molecular modeling and scanning electron microscopy (SEM) study demonstrates that
LFS-829 can be well stabilized in the cavity of a-CD (Figure 8A-B). In brief, we examined the
therapeutic potential of CD-LFS-829 in DSS-induced colitis rodent model. Notably, oral treatment of
CD-LFS-829 (30mg/kg/day) significantly restored the colon length and histological score as compared
to the untreated DSS group (Figure 8C-D-E). Taken together, our study provides a demo
encapsulation system for the potential use of LFS-829 in clinical treatment of colitis.
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Figure 8. Oral administration of encapsulated form of LFS-829 ( -cyclodextrin-LFS-829, also
named as CD-LFS-829) ameliorate DSS-induced colitis in model mice. (A) Modeling structure of
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LFS-829 encapsulated in a-cyclodextrin as a host-guest complex. (B) The scanning electron
microscopy (SEM) data for water-soluble formulation CD-LFS-829. (C) Survival rate of different
animal groups. (D) Percentage of diarrhea among different animal groups. (E) Histopathological score
for the H&E-stained sections.
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Permeability and stability properties of LFS-829
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The Parallel Artificial Membrane Permeability Assay (PAMPA) study and the Caco-2 cell
permeability study determined that the permeability index of LFS-829 was high with approved drugs
as control (SI Table 2-3), suggesting that LFS-829 may achieve good bioavailability in human.
Moreover, we employed artificial gastric juice stability assay and artificial intestinal juice stability
assay to determine the stabilities of LFS-829 in alimentary system, critical properties for IBD new
drug. LFS-829 is rather stable in both artificial gastric juice (half-life ~24hrs) and artificial intestinal
juice (half-life ~8hrs), suggesting a pharmacokinetic profile compatible with once-daily dosing
(Figure 9A-B).
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Figure 9. Evaluation of acute toxicity, gastrointestinal tract stability and cardiotoxicity of
compound LFS-829. (A) Stability test of LFS-829 in artificial gastric juice (Omeprazole is an FDA-
approved drug for GI disorders and routinely used as a control in pharmacokinetic study); (B) Stability
test of LFS-829 in artificial intestinal juice (Chlorambucil is an FDA-approved chemotherapy for
chronic leukemia and routinely used as a control in pharmacokinetic study). (C) H&E-stained results
for the sections of mouse colon in vehicle-treated group and LFS-829 treated-group; (D) The
concentration dependence curve of LFS-829 blockade of hERG currents and the inhibition constant
IC50 of LFS-829 is above 30µM.
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Assessment of acute toxicity profile of LFS-829
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To determine the suitability of LFS-829 in clinical practice, we assessed the acute toxic effects of
LFS-829 in mice. Ten C57BL6 mice were exposed to oral administration of LFS-829 at an increasing
dose of 300mg/kg to 600mg/kg over the course of one day whereas another ten mice were exposed to
vehicle only (70% Ethanol, n=10). The LFS-829-treated animals didn’t show lethargy, weight loss or
other physical indications of sickness. The mice were sacrificed after the 14-day washout period to
assess delayed toxicity. Colon, spleen and other tissues were examined and no toxic effects or tissue
damage (either macroscopic or microscopic) were observed (Figure 9C and Figure S10).
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Evaluation of cardiotoxicity profile of LFS-829
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Cardiotoxicity is a crucial factor and major safety concern for preclinical or clinical evaluation of
pharmacological therapeutics for the treatment of chronic relapsing diseases such as IBD21. Therefore,
it is necessary to evaluate the potential cardiotoxicity for LFS-829. To this end, we used a hERG
potassium channel patch-clamp assay to test whether LFS-829 would cause QT interval prolongation
as a result of hERG potassium channel inhibition. LFS-829 did not inhibit hERG even at a high
concentration, suggesting a very low risk for cardiotoxicity (Figure 9E-F, Figure S9-10, SI_Table 3).
These studies clearly established the excellent safety profiles of LFS-829 for its potential use in clinical
treatment of colitis.
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Discussion and Conclusions
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There are unmet medical needs for disease-modifying therapies for IBD with excellent response
rates and safety profile due to the relative inefficacy of the current maintenance therapies. It is rather
challenging to develop therapeutics for colitis because the pathogenesis of colitis is extremely
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complicated and remains elusive. For instance, two recent clinical trials with IL-23 antibodies,
Secukinumab22 and Brodalumab23, showed no efficacy or even higher rates of adverse events for colitis
patients. Moreover, in the past decades, no therapeutics have been introduced in clinical practice for
the treatment of colitis. Of note, a few small-molecule compounds for colitis are currently under
clinical development including JAK inhibitors and sphingosine-1-phosphate receptor 1 (S1P1)
modulator such as Etrasimod (also named as APD334)14.
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In light of this, here we designed a rational structure-based strategy to discover a potent small-
molecule antagonist for exportin-1 based on the scaffold of sulforaphene with distinct mode of actions.
Our previous work has found that sulforaphene, a natural antagonist of CRM1, exhibits therapeutic
effects towards colitis. To improve the potency, we first introduced a hydrophobic phenyl ring next to
the sulfoxide moiety in the parent structure of LFS-01. Next, through the screening a virtual
combinatorial library, we identified LFS-829 with the replacement of two trifluoromethyl group at R3
and R5 position, resulting in a potency nearly 30-fold higher than that of the parent compound as
determined by cell-based phenotypic nuclear export functional assay. Our strategy has the advantages
of considering affinity, ADME/T properties and other properties. LFS-829 is amenable to large-scale
synthesis and has low cost of chemicals, which may be advantageous as a therapy for colitis.
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We revealed that LFS-829 can inhibit CRM1 in the low nanomolar range (~26.95 nM) through
SPR binding assay. We revealed that the cellular activities by LFS-829 was significantly diminished
when CRM1 was suppressed by siCRM1, implicating that CRM1 is a main cellular target of LFS-829.
The study of the time course of CRM1-LFS829 complex degradation suggest that LFS-829 is a more
reversible inhibitor as compared to KPT-330. This indicates that LFS-829 may exert a lower toxicity
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profile which is advantageous because colitis patients typically require a very long-term medication.
Strikingly, our study showed that LFS-829 was very efficacious in mouse model of colitis and exhibits
superior therapeutic potential than first-line therapy 5-ASA as well as APD334, a promising candidate
compounds under clinical trials for colitis. Importantly, our study provides the co-crystallographic
structure of LFS-829 bound to CRM1 which also firmly establish the working mechanism of LFS-829.
Therefore, our study may provide insights to guide functional elucidation of the mechanism of action
for LFS-829 and may open new avenues for future drug development based on similar chemical
scaffolds.
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It has been well recognized that colitis is a complex and heterogeneous disease with several
molecular pathways contributing to the disease pathophysiology. Therefore, the next generation
therapeutics for colitis may allow the simultaneous modulation of multiple disease-causing cytokines
or pathways. Our study, for the first time, demonstrated that a small-molecule antagonist of exportin-
1 can reverse the pathogenesis of colitis by regulating both NF-κB signaling and Nrf2 cellular defense
pathway (Scheme 2). The activation of NF-κB signaling plays a dominant role in the inflamed gut of
colitis patients. In particular, it has been found that the upregulation of NF-κB (p65 or p50) is
significant in the macrophages and epithelial cells isolated from the inflamed gut specimens of IBD
patients24. Moreover, there has been mounting evidences suggesting that NF-kB signaling in the gut
epithelium plays as a critical regulator of epithelial integrity and intestinal immune homeostasis and
thereby is closely associated with the pathogenesis of colitis25, 26. We revealed that LFS-829 treatment
can lead to significant nuclear accumulation of IkBa which consequently strongly suppresses NF-κB
signaling and its downstream targets. In particular, we found that the exposure of colitis mice with
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LFS-829 leads to the suppression of a few critical downstream targets of NF-kB signaling including
Cox-2, IL-6, TNF-a, IL-1b and IL-23, all of which are hallmarks in the pathogenesis of colitis. On the
other hand, Nrf2 plays a fundamental role in cellular defense pathway against inflammation and
intestinal integrity through regulation of proinflammatory cytokines and induction of Phase II
detoxifying enzymes. Of note, Nrf2 deficient mice were more susceptible to DSS-induced colitis27.
Our data clearly show that LFS-829 treatment can lead to the nuclear retention of Nrf2 and enable the
activation of Nrf2 cellular defense pathway. Particularly, we found that LFS-829 administration
restored the dysregulation of detoxifying enzymes HO-1 and NQO1 in the colon tissues of colitis mice,
both of which are essential for the cytoprotective effects during inflammation and the maintenance of
mucosal homeostasis in the gastrointestinal tract of colitis patients. Moreover, one of the current
focuses in colitis research attends to a group of immunogenic endogenous damage-associated
molecular patterns (DAMPs)28. The calprotectin, also named as S100A8/S100A9 complex, belongs to
DAMPs and has been recognized to play a key role in the etiology of colitis during colon tissue damage.
Moreover, the complex S100A8/S100A9 proteins are present at high levels in the serum of patients
and therefore S100A8/S100A9 has been established as sensitive and specific biomarkers for colitis19.
Notably, we observed that S100A8/S100A9 was upregulated in colitis mice and the expression level
of S100A8/S100A9 was reduced upon the administration of LFS-829, indicating that LFS-829
reverses the pathogenesis of colitis. Collectively, we concluded that LFS-829 exerted its therapeutic
effects towards colitis through dual targeting of NF-κB signaling and Nrf2 cellular defense pathway.
Moreover, it has been documented that there exists a functional crosstalk between NF-kB signaling
and Nrf2/ARE pathway17, 29, 30. Therefore, we argued that the dual attenuation of both NF-kB signaling
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and Nrf2 cytoprotection pathway by LFS-829 may have synergistic effects to prevent inflammation
and restore intestinal homeostasis (Scheme 2).
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Scheme 2. LFS-829 attenuates dual NF-kB signaling and Nrf2 cytoprotection pathway
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The pathological characteristics of colitis determines that the onset of the disease may translate
into longstanding disease activity and thereby require a very long-term medication. Hence, the safety
profile of a candidate compound for treating colitis is of utmost importance. Noteworthy, our results
suggest that LFS-829 has a rather low risk of both cytotoxicity and cardiotoxicity. Furthermore, we
evaluated the acute toxicity of LFS-829 in animals and our results suggest that LFS-829 barely shows
any acute toxicity at dose of 600mg/kg. Moreover, we showed that LFS-829 is quite stable in artificial
gastric juice and intestinal juice with excellent permeability index. Hence, LFS-829 may hold great
potentials as colitis therapy with good safety profile. Finally, there is an increasing risk for colitis
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