Development of Small-Molecules Targeting Receptor Activator of Nuclear Factor-κB Ligand (RANKL) - Receptor Activator of Nuclear Factor-κB (RANK) Protein-Protein Interaction by Structure-Based Virtual Screening and Hit Optimization

Article abstract of DOI:10.1021/acs.jmedchem.8b02027

Targeting RANKL/RANK offers the possibility of developing novel therapeutic approaches to treat bone metabolic diseases. Multiple efforts have been made to inhibit RANKL. For example, marketed monoclonal antibody drug Denosumab could inhibit the maturation of osteoclasts by binding to RANKL. This study is an original approach aimed at discovering small-molecule inhibitors impeding RANKL/RANK protein interaction. We identified compound 34 as a potent and selective RANKL/RANK inhibitor by performing structure-based virtual screening and hit optimization. Disruption of the RANKL/RANK interaction by 34 effectively inhibits RANKL-induced osteoclastogenesis and bone resorption. The expression of osteoclast marker genes was also suppressed by treatment of 34. Furthermore, 34 markedly blocked the NFATc1/c-fos pathway. Thus, our current work demonstrates that the chemical tractability of the difficult PPI (RANKL/RANK) target by a small-molecule compound 34 offers a potential lead compound to facilitate the development of new medications for bone-related diseases.

Full text of DOI:10.1021/acs.jmedchem.8b02027

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Article
Development of Small Molecules Targeting Receptor Activator
of Nuclear Factor-#B Ligand (RANKL) - Receptor Activator
of Nuclear Factor-#B (RANK) Protein-Protein Interaction
by Structure-Based Virtual Screening and Hit Optimization
Min Jiang, lei peng, Kai Yang, tianqi wang, Xueming Yan, Tao Jiang, Jianrong Xu, jin qi,
hanbing zhou, niandong qian, qi zhou, bo chen, xing xu, Lianfu Deng, and Chunhao Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b02027 • Publication Date (Web): 13 May 2019
Downloaded from http://pubs.acs.org on May 13, 2019
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Development of Small Molecules Targeting
Receptor Activator of Nuclear Factor-κB
Ligand (RANKL) - Receptor Activator of
Nuclear Factor-κB (RANK) Protein-Protein
Interaction by Structure-Based Virtual
Screening and Hit Optimization
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Min Jiang a‡, Lei Peng b, c‡, Kai Yang a, Tianqi Wang a, Xueming Yan a, Tao Jiang a,
Jianrong Xu d, Jin Qi a, Hanbing Zhou a, Niandong Qian a, Qi Zhou a, Bo Chen a,
Xing Xu a*, Lianfu Deng a*, Chunhao Yang b, c
*
a Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of
Traumatology and Orthopaedics, Shanghai Ruijin Hospital, Shanghai Jiaotong
University School of Medicine, 197 2nd Ruijin Road, Shanghai 200025, China
b State Key Laboratory of Drug Research, Department of Medicinal Chemistry,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu
Chong Zhi Road, Shanghai 201203, China
c School of Pharmacy, University of Chinese Academy of Sciences,
No.19A Yuquan Road, Beijing 100049, China
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d Department of Pharmacology and Chemical Biology, Institute of Medical Sciences,
Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road,
Shanghai 200025, China.
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‡ These authors contributed equally to this work.
Abstract: Targeting RANKL/ RANK offers the possibility of developing novel
therapeutic approaches to treat bone metabolic diseases. Multiple efforts have been
made to inhibit RANKL. For example, marketed monoclonal antibody drug
Denosumab could inhibit the maturation of osteoclasts by binding to RANKL. This
study is an original approach aimed at discovering small-molecule inhibitors
impeding RANKL/RANK protein interaction. We identified compound 34 as a potent
and selective RANKL/RANK inhibitor by performing structure-based virtual
screening and hit optimization. The disruption of the RANKL/RANK interaction by
34 effectively inhibits RANKL-induced osteoclastogenesis and bone resorption. The
expressions of osteoclast marker genes were also suppressed by treatment of 34.
Furthermore, 34 markedly blocked the NFATc1/ c-fos pathway. Thus our current
work demonstrates that the chemical tractability of the difficult PPI (RANKL/RANK)
target by a small molecule compound 34 offers a potential lead compound to facilitate
the development of new medications for bone-related diseases.
Key words: Osteoclasts, RANKL, RANK, Therapeutics, Osteoclastogenesis
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INTRODUCTION
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Bone remodeling is crucial for maintaining the normal skeletal structure1. To
maintain bone homeostasis, bone metabolism consists of a life-long remodeling
process which involves a cross-talk between osteoclasts and osteoblasts2-5.
Osteoblasts are mononucleated bone-forming cells derived from mesenchymal stem
cells (MSCs). Osteoclasts are cells derived from bone marrow-derived macrophages
cells (BMMs) and play an important role in bone resorption. Enhanced
osteoclastogenesis is a common pathological feature in many age-associated bone
diseases, such as osteoporosis, rheumatoid joint destruction, cancer induced bone
metastasis and Paget's disease6-9. Receptor activator of NF-kB ligand (RANKL)
which belongs to the tumor necrosis factor (TNF) receptor-ligand family is expressed
by osteoblasts, stromal cells and osteocytes10. RANKL plays multiple functions in
immune system11, hormone-derived breast cancer development12 and especially in
bone remodeling. RANKL plays an essential role in osteoclast differentiation, acting
through its binding to receptor activator of NF-kB (RANK) and then associating with
TNF receptor associated family members to trigger downstream signaling events such
as NF-κB pathway and nuclear factor of activated T-cells, cytoplasmic 1
(NFATc1)/c-fos pathway13-15. Osteoprotegerin (OPG) functions as a decoy receptor
for RANKL and inhibits osteoclastogenesis by preventing RANKL/RANK
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interaction16, 17
.
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Since RANKL/RANK/OPG are recognized critical proteins for the bone
metabolism, targeting this system offers the possibility of developing novel
therapeutic approaches to treating bone metabolic diseases18-20. Denosumab, a human
monoclonal antibody against RANKL, is developed and indicated for treating
postmenopausal osteoporosis and bone metastasis from solid tumors21-24. It is the first
RANKL inhibitor approved by FDA and its total sales is more than 3 billion dollars in
2016. However, there are inadequate responses to denosumab in the treatment of
osteoporosis and bone-metastatic cancers25. In addition, its application is limited
partially for their high price. Besides, recombinant proteins such as Fc-OPG and
RANK-Fc have also been developed as therapeutics for osteoporosis26,
27. Alternatively, OPG (OP3-4 peptide) was developed based on the structure of the
loop in the third cysteine-rich domain of OPG (OP3 site) to mimic the soluble
osteoprotegerin (OPG) and showed inhibitory activity against the RANKL-induced
osteoclastogenesis and bone resorption28, 29. One of eRANK (ectodomain of mouse
RANK) mutants, the cyclic peptide L3-3 was proved as a potent RANKL inhibitor
which blocked RANKL-induced osteoclast differentiation more efficiently than
OP3-425. ITC (isothermal titration calorimetry) analysis of L3-3 and OP3-4 showed
that the binding affinity (KD) with RANKL were 17.3 μM and 284.4 μM
respectively25.
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Although multiple efforts have been made to inhibit RANKL, the therapeutic
applications of biologics in the clinic were hindered for some of drawbacks, including
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high cost, unfavorite intravenous administration etc. An alternative approach to inhibit
RANKL is to utilize nonpeptidic small organic molecules, which, in general, have
better pharmacokinetic properties. To date, there are just a handful of nonpeptidic
small molecular inhibitors targeting RANKL and RANK protein interaction. SPD304,
T8, T23 were proven to directly bind to RANKL with Kd values of 13.8 μM, 6.3 μM,
7.3 μM respectively. Despite their promising effectiveness, the agents are not specific
in inhibiting RANKL-RANK interaction. It has been demonstrated that SPD304, T8,
T23 have poor selectivity on TNF and RANKL. Among them, SPD304 and T23
displayed high toxicity30, 31. Development of small molecular inhibitors of RANKL
with low toxicity, high potency and selectivity remains inadequate. The binding
modes between RANKL/RANK and RANKL/OPG were well-characterized,
providing molecular foundations for the development of small molecules targeting
RANKL-RANK interface25, 32, 33. Here, based on virtual screening, we identified a
new scaffold compound 1 as a candidate small molecule that may target RANKL and
RANK protein interaction. The binding to RANKL/RANK and the
anti-osteoclastogenesis activity of 1 were confirmed in surface plasmon resonance
(SPR) analysis and RANKL-induced osteoclastogenesis assay respectively. Based on
these promising results, we considered 1 as a hit compound and further chemical
optimization resulted in four novel compounds (2, 7, 29, and 34) with good activities
against osteoclastogenesis. 2, 7, 29, and 34 were also determined by SPR binding
assays and displayed high affinity for RANKL with KD values of 2.7, 3.1, 3.5 μM
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and 614 nM respectively. Moreover, 34 was further validated to inhibit
RANKL-induced bone resorption by downregulating the c-fos/NFATc1 signaling
pathway and osteoclastogenesis marker genes. Thus our current work offers an
attractive starting point for facilitating the PPI (protein/protein interaction,
RANKL/RANK) target strategy by small molecular compounds and helps further
development of new medications for osteoporosis and bone-related diseases discovery
progress.
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RESULTS and DISCUSSION
Discovery of a Lead compound, through Virtual Screening
Structure based virtual screening is an effective method in hit compound
identification and has been widely used in the process of lead compound discovery. In
this study, we used a molecular docking method with Schrodinger (Schrödinger,
LLC.) standard precision mode to screen SPECS database (http://www.specs.net),
which contains more than 200,000 compounds, to identify the RANKL inhibitors.
Based on the analysis of the interaction surface of published RANKL crystal
structures, the crystal structure with concave pocket (Protein Data Bank PDB ID
3URF) mostly suitable molecular docking was chosen as receptor structure for virtual
screening. 3URF includes two parts, RANKL and its decoy receptor OPG. Three
amino acids, Glu93, Ile92 and Glu95 from OPG probe deeply into the concave
surface of RANKL that defines the main binding pocket for small molecular
inhibitors targeting RANKL/RANK interaction. The top 109 compounds were
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overlaid with complex structure for visual inspection based on the three criteria: (1)
Compounds must not have high strained motifs. (2) Compounds must occupy the
deep pocket surrounding residues Glu93, Ile94 and Glu95 of OPG. (3) Compounds
must have a certain ratio of hydrophobic fragments participating in key interactions.
Finally, 70 compounds were selected and purchased from SPECS (Fig. 1). These
virtual screening hits were also filtered for Pan Assay Interference Compounds
(PAINS)34. The ranking of prioritized 109 compounds is given in Supporting
Information.
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Inhibition of RANKL-induced Osteoclastogenesis and RANKL/RANK
interaction by Compound 1
Since tartrate-resistant acid phosphatase (TRAP) is an established marker for
osteoclast differentiation and bone resorption35, the 70 candidate molecules selected
by virtual screening were tested for TRAP activity assay on RANKL-induced
osteoclastic activation and differentiation from BMMs to quickly determine their
osteoclastogenesis inhibitory activity. According to TRAP activity assay, we chose
compound 1 as a lead compound since its inhibition on TRAP activity was >50%
(54.9 %) at the concentration of 5 μM (Fig. 2A). After RANKL binding to
its receptor RANK on osteoclast precursors, various downstream signalings are
triggered to stimulate osteoclast differentiation and activity. Then the inhibitory effect
of 1 was evaluated against RANKL-induced osteoclast differentiation on BMMs.
BMMs were induced to become mature osteoclasts by M-CSF and RANKL, and
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compound 1 with different indicated concentrations were incubated with BMMs for 5
days. As shown in Fig. 2B-C, treatment of 1 significantly reduced osteoclast
formation in a dose-dependent manner especially at the concentrations of 5 μM (50.0
%) and 10 μM (93.3 %). Compound 1 inhibited RANKL-induced osteoclast
differentiation with IC50 = 6.6 μM (Figure S1, Supporting Information). In response to
RANKL-RANK interaction, up-regulation of specific genes has been observed36. Real
time PCR was used to assess whether 1 can regulate RANKL-induced gene expression
levels during osteoclastogenesis. The results were consistent with TRAP activity and
osteoclast formation assays. The master regulators of osteoclast NFATc1, as well as bone
resorption-related genes TRAP and Cathepsin K were significantly and dose-dependently
inhibited by 1 at day 3 of culture (Fig. 2D-F). In addition, MTT assay against BMMs
showed the anti-osteoclastogenic effects of 1 are not attributable to cellular toxicity
(Fig. 2G). To further validate the inhibitory effect on osteoclastogenesis of 1 was
donated by targeting RANKL and RANK protein interaction, SPR analysis was
conducted for characterizing the binding properties of 1 for RANKL. According to
SPR analysis, 1 has higher binding affinity for RANKL (Kd = 9.8 μM) than OP3-4
and L3-3 (Kd = 284.4 μM and 17.3 μM respectively). The binding affinity of RANKL
for RANK was reported as 0.11 nM37. We retested this value (0.1 nM, Figure S2,
Supporting Information) and compound 1 inhibited RANKL binding to its receptor
RANK in a dose dependent manner with IC50 = 26.2 μM. The equilibrium binding
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curve fits are shown in Fig. 2H-I. This result indicated the direct binding of 1 for
RANKL and 1 has the property of impeding RANKL/RANK protein interaction.
Analysis of the Binding Mode of Compound 1
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The proposed binding mode of 1 was presented in Fig. 3. The PDB ID of crystal
structure used for molecular modeling is 3URF in Protein Data Bank. 1 binds partly in
the pocket of RANKL occupied by three residues from OPG (93-95) with a phenyl
group. The phenyl group is surrounded by hydrophobic side chains of His180,
Lys181, Tyr241 and Glu237. The indole and dimethylphenyl group bind in clefts
extending from the RANKL-OPG interaction site. The two bridging amides bind with
Leu236, Gln237 and Asn295 through strong hydrogen bonds. The indole NH interacts
with Asn295 amide through hydrogen bond too (Fig. 3).
Chemical Modification of Compound 1 and SAR Exploration
To explore the SAR and improve the potency of compound 1, 15 compounds
(compounds 2-16) were firstly synthesized to investigate the structure−activity
relationship (SAR) for the carbamate part of compound 1 (Scheme 1).
(Scheme 1)
Then we evaluated their osteoclastogenesis inhibitory activity by a tartrate-resistant
acid phosphatase (TRAP) activity assay. Testing compounds at 5 μM were added to
BMMs in the presence of RANKL (50 ng/mL) and M-CSF (20 ng/mL) and incubated
for 3 days. The activity results were summarized in Table 1. When the atom O in CBZ
group of compound 1 was replaced by methylene group (X position, compound 2),
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the inhibitory activity was remarkably increased. Then, para- or meta-substituents like
fluorine atom or methoxyl group on the benzene ring (R1 position) led to the reduced
inhibitory of TRAP activity (compounds 3-6, Table 1), while para-substituted chlorine
atom on the benzene ring in the R1 position (compound 7, Table 1) showed more
potent inhibitory activity than compound 1, and meta-substituted chlorine atom
displayed the same potency as compound 1 (compound 8, Table 1). The introduction
of heteroaromatic ring, such as pyridyl and thienyl, in the R1 position showed the
same potency or a little decrease (compounds 9-12, Table 1), while replacement by
2-thiazolyl in the R1 position (compound 13, Table 1) could increase the inhibitory
activity. As for cycloalkanes, the introduction of cyclohexyl, cyclopentyl, or
cyclopropyl in the R1 position decreased the inhibitory activity (compounds 14-16,
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Table 1). Thus compound 2 was the most potent for the first-round optimization.
(Table 1)
Next, modification was focused on 2,6-dimethyl aniline while 3-phenyl propanoic
acid amide was kept in the structure. Different substituents were introduced to the
benzene ring of aniline structure, like methyl, fluoride, methoxyl, dimethylamino,
morpholinyl, methylsulfonyl, trifluoromethyl (compounds 17-26, Table 2), however,
the inhibitory activities of these compounds generally decreased than that of
compound 2.
(Table 2)
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Afterwards, the substitution of the indole ring at the R5 and R6 position were
investigated (compounds 27-29, Table 3). Fluorine atom and methoxyl showed
comparable potency to compound 2. The results of compounds 30-32 showed that
increase or decrease of the chain length was unfavorable to the inhibitory potency
(compounds 30-32, Table 3). While replacement of carbonyl group with methylene
group in the Y position could increase the inhibitory activity of TRAP slightly (33 vs
30, 34 vs 2, 35 vs 31, 36 vs 32, Table 3). Compound 34 was slightly superior to
compound 2 against TRAP. Furthermore, replacement of carbonyl group with
methylene group in the Z position turned out that the inhibitory activity against TRAP
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decreased dramatically (37 vs 2).
(Table 3)
Inhibition of RANKL-induced Osteoclastogenesis by Compounds 2, 7, 29 and 34
Among all of the synthesized compounds, 2, 7, 29, and 34 performed better than
others as illustrated by more than 90% or close to 90% inhibition at 5 μM. Cell
survival test based on MTT assay suggested that antiosteoclastogenic effects of 2, 7,
29, and 34 are not attributable to cellular toxicity on BMMs (Table 4). The
enantiomers of compound 2 showed a similar inhibitory effect (Figure S3, Supporting
Information). In view of the relatively high inhibitory activity on TRAP and low
cytotoxicity, we further evaluated their effects on RANKL-induced
osteoclastogenesis. M-CSF and RANKL induced the formation of TRAP-positive
multinucleated cells, while treatment of compounds 2, 7, 29, and 34 (5.0 μM) even
completely inhibited osteoclast formation. In addition, BMMs challenged with or
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without compounds were double stained with rhodamine phalloidin and DAPI
(4',6-diamidino-2-phenylindole). The results showed that M-CSF and RANKL
induced the formation of typical osteoclasts morphology such as the formation of
F-actin ring and numerous nuclei. In contrast, treatment with compounds had smaller
size and fewer nuclei numbers of F-actin ring formation. The results above indicated
that compounds strongly inhibited osteoclast differentiation in a dose dependent
manner especially at the concentrations of 2.5 μM and 5.0 μM based on TRAP
staining and F-actin staining (Fig. 4A). Representative pictures of 34 were shown in
Fig. 4B-C. The binding activity to RANKL of compounds 2, 7, 29, and 34 were also
determined by SPR binding assays and they displayed much higher affinity for
RANKL with KD values of 2.7, 3.1, 3.5 μM and 614 nM respectively (Table 5).
Interestingly, 34 displayed weak affinity for TNF (93.6 μM, Figure S4, Supporting
Information). As to its highest affinity to RANKL, 34 was chosen as a prototype for
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further examination in our subsequent studies.
(Table 4)
(Table 5)
Inhibition of RANKL-induced Bone resorption by Compound 34
It is well known that bone resorption is a characteristic feature of osteoclasts during
bone remodeling. RANKL/RANK interaction could induce the bone resorption
activity of mature osteoclasts, thus we evaluated the effect of 34 on RANKL-induced
bone resorption subsequently. As shown in Fig. 5A, almost no resorption was
detected in bone slices treated with 5 μM 34 while obvious resorbing pits were
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formed in the RANKL-treated group as visualized by toluidine blue staining. 34 (1,
2.5 μM) also significantly decreased the percentage of resorbed area. Besides, the
calculated bone resorption shown in Fig. 5B is consistent with Fig. 5A.
Inhibition of osteoclastogenesis-related gene expression and c-Fos/NFATc1
signaling pathway by Compound 34
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We used RT-PCR to examine the effects of 34 on RANKL-induced
osteoclast-specific gene expressions during osteoclastogenesis. BMMs were treated
with different concentrations of 34 for 3 days. TRAP, cathepsin K and NFATc1 were
increased in response to RANKL stimulation, while 34 strongly decreased the
expressions of these genes even at 2.5 μM (Fig. 6A-C). c-fos/NFATc1 signaling
pathway serves as a major regulatory axis downstream of RANKL. They trigger
RANKL-induced osteoclast formation through regulating the expressions of related
genes such as TRAP and cathepsin K. We thus conducted WB to verify the effects of
34 on the protein expressions of c-fos and NFATc1. As shown in Fig. 6D, 34 could
effectively downregulate the c-fos/NFATc1 signaling in BMMs in a dose-dependent
manner.
CONCLUSION
The development of potent RANKL inhibitors is currently a hot topic of bone
metabolic diseases drug discovery. Promising results were obtained such as
recombinant proteins (RANK-Fc, Fc-OPG, and anti-RANKL antibodies) and small
peptides (OP3-4 and L3-3). However, the application of large macromolecules is
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limited partially by high cost and difficulties in administration. There are also some
drawbacks of small peptides such as poor tissue penetration, oral bioavailability.
Currently there is only a handful of nonpeptidic small molecular inhibitors targeting
RANKL and RANK protein interaction. Among them, most of compounds displayed
high toxicity, poor potency and selectivity. It thus seems urgent to explore small
molecular inhibitors with high potency and selectivity preventing RANKL/RANK
interaction. Here, based on structure-based virtual screening and hit optimization, we
reported 2-amino-3-(1H-indol-3-yl)-N-phenylpropanamides as a novel scaffold
possessing high binding affinity to RANKL targeting RANKL-RANK protein-protein
interaction. Four compounds exhibited high binding affinity to the RANKL (KD for
RANKL<10 μM). Among them, 34 was identified as a potent and selective RANKL
inhibitor (KD for RANKL = 614 nM and KD for TNF = 93.6 μM). These four
compounds were further measured in functional assays. They showed significant
inhibitory effects in vitro for osteoclastogenesis. The inhibition of RANKL-induced
osteoclastogenesis was not due to cytotoxic effects. Moreover, 34 exerted inhibitory
action on the bone resorption activity of osteoclasts. Using RT-PCR and WB
techniques, we also illustrated 34 decreased the mRNA levels and the expressions of
nuclear receptor NFATc1 and c-fos that controlled the expressions of bone resorption
enzymes such as TRAP and cathepsin K. To date, small molecules impeding
RANKL/RANK interaction is still an enormous challenge and our work will offer an
attractive starting point for rational design. This novel series of compounds also have
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high potential to be developed as novel therapeutic agents for treating bone metabolic
diseases in the future.
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EXPERIMENTAL SECTION
Virtual Screening
Protein preparation. Crystal structures of RANKL complexed with OPG was
downloaded from RCSB (Protein Data Bank PDB ID 3URF). This protein structure
contains two parts, RANKL and OPG. RANKL was prepared using default Protein
Preparation Wizard Workflow provided in the Maestro, with a pH of 7.0 ± 2.0.
Residues within a distance of 6 Å around the residues Glu93, Ile94 and Glu95 of OPG
were defined as the binding pocket for virtual screening. Other parameters were set as
the default.
Ligand Database Preparation. The Specs database (http://www.specs.net), which
contains ~200,000 compounds was used for the virtual screening. To refine the
database, we filtered it by Lipinski’s rule of five and removed pan-assay interference
compounds with Pipeline Pilot version 6.5 (Pipeline Pilot, Accelrys Software Inc.,
San Diego, CA). The remaining compounds were prepared using LigPrep to generate
all stereoisomers and protonation states with Epik. At last, each compound was
generated up to ten conformations using Confgen to produce a docking library with
most diversified starting conformation which usually will affect the docking results.
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Virtual Screening Procedure. First, Glide SP mode was used as the primary
screening engine. Then the top 50,000 conformations were subsequently docked by
Glide XP mode. After docking, top ranking compounds were selected based on their
highest scoring conformation. The best scored 109 compounds were overlaid with
complex structure for visual inspection. Finally, 70 compounds were selected and
purchased from Specs.
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Biological materials
Dimethyl sulfoxides (DMSO), MTT, formaldehyde, DAPI, Triton X-100, TRAP
staining kit were purchased from Sigma. ɑ-Modified eagle's medium (ɑ-MEM), fetal
bovine serum (FBS), penicillin and streptomycin (PS) were from Gibco. Rhodamine
phalloidin was from Molecular Probes. M-CSF, human RANK (hRANK), human
RANKL (hRANKL), human TNFα (hTNF) and RANKL (mRANKL) were obtained
from Novoprotein. Reverse transcript reagents and SYBR Green PCR Master Mix
were from Takara. TRAP activity assay kit and BCA protein assay kit were from
Beyotime Biotechnology. Antibodies were from Cell Signal Technology. C57BL/6
mice were purchased from SLAC laboratory and were maintained at 22 °C-24 °C and
55 %–60 % humidity in a room with a 12/12-h light/dark cycle. All animal
experiments were conducted according to the guidelines of the humane use and care
of laboratory animals and were approved by the Shanghai Jiao Tong University
School of Medicine Animal Study.
Surface Plasmon Resonance
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Studies were performed using a BIAcore T200 instrument (GE Healthcare).
Approximately 9000 response units (RU) of hRANKL, 300 RU of hRANK or 17000
RU of hTNF were covalently immobilized onto sensor CM5 chip by a standard
amine-coupling procedure. In binding assay, compounds were injected in a dilution
series using 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) Tween-20 as
running buffer. DMSO in running buffer [1% (v/v)] was used as a detergent control.
KD determination experiments were performed using single-cycle kinetics with 30 s
contact time, 30 μL/min flow rate at 20 °C. KD values were calculated using steady
state analysis at equilibrium and BIAcore T200 evaluation software version 1.0 (GE
Healthcare). In competition assay, compounds were incubated with indicated
concentration of hRANKL for 1 hour and then injected to the chip. Data
was corrected by subtraction of detergent control.
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Isolation and Culture of BMMs and Osteoclasts
BMMs isolated from 4-week-old male C57/BL6 mice were prepared and cultured
using our previously published protocol38. Briefly, all the bone marrow cells in the
femur and tibiae of mouse were flushed out by ɑ-MEM and washed for several times.
The cells were then incubated in full ɑ-MEM containing 10% FBS, 100 U/mL
penicillin and 100 μg/mL streptomycin overnight. To obtain BMMs, nonadherent
cells were collected and then cultured in full ɑ-MEM containing 30 ng/mL M-CSF for
proliferation. After reaching 90% confluence, the adherent cells classified as BMMs
were harvested by cell scraper. BMMs were induced to mature osteoclasts by full
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ɑ-MEM plus M-CSF (30 ng/mL) and mRANKL (50 ng/mL), which called as
osteoclast-inducing medium (OIM), for 5-6 days.
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Tartrate-resistant Acid Phosphatase (TRAP) Colorimetric Assay
BMMs were seeded in 96-well plates at a density of 3×103 cells/well and exposed
to OIM in combinations with various concentrations of compounds in triplicate for 3
days. TRAP activity was measured by TRAP activity assay kit according to the
manufacturer's instructions. Cell culture mediums were removed and cells were
washed by PBS for 3 times. Then cells were lysised by passive lysis buffer (Promega)
for 15 min at 37 ºC and the supernatant was collected and incubated with
para-nitrophenylphosphate (p-NPP) in the presence of disodium tartrate for 45 min.
To stop the reaction, the reaction mixtures were added by NaOH. The TRAP activity
was then quantified by measuring OD405 with an Infinite F200 PRO absorbance
microplate reader.
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Cell Viability by MTT Assay
BMMs were plated at a density of 3×103 cells/well in 96-well plates in full ɑ-MEM
plus 30 ng/mL M-CSF for proliferation. The next day, the cells were treated with
various concentrations of synthetic compounds in triplicate for 2 days. After
compound
treatment,
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tetrazolium
reagent
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) was
added to each well, and the plates were incubated at 37 ºC for an additional 2 h. Then,
the supernatant was removed, and the crystals were solubilized in 100 μL of DMSO
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per well. The optical density (OD) at 570 nm (OD570) was measured with an Infinite
F200 PRO absorbance microplate reader (Tecan). Cell viability was calculated
relative to the control.
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TRAP Staining
BMMs were cultured in 96-well plates in full medium containing M-CSF and
allowed to adhere overnight. The medium was replaced by OIM with various
concentrations of synthetic compounds for an additional 5-6 days. The cell culture
media was replaced every two days. TRAP staining was then performed using a
leukocyte acid phosphatase kit (Sigma) according to the manufacturer's instructions.
The red TRAP-positive multinucleated cells (>3 nuclei/cell) were visualized,
photographed and quantified as mature osteoclasts under an Olympus microscope.
F-actin Staining
BMMs were cultured in 6-well plates in full medium containing M-CSF and
allowed to adhere overnight. The medium was replaced by OIM with various
concentrations of synthetic compounds for an additional 5-6 days. The cell culture
media was replaced every two days. F-actin was visualized by means of phalloidin
staining. Cells were fixed with 3.7% (w/v) formaldehyde and permeabilized with
0.2% Triton X-100. The cells were then incubated with 2 units/mL rhodamine
phalloidin (Molecular Probes) at at 37 ºC for 20 min. Nuclei were visualized with
4,6-diamidino-2-phenylindole (DAPI, 1 μg/mL). Actin rings were then analyzed by
an Olympus microscope.
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Bone resorption assay
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BMMs were seeded in 96-well plates at a density of 3×103 cells/well in full medium
containing M-CSF and the next day cells were induced in OIM until osteoclasts began
to form. Bone slices from bovine femur were cut into 400 μM thick. The cells were
gently harvested using cell dissociation solution and counted, and equal numbers of
multinucleated cells were seeded onto bovine femur bone slices in 96-well plates. The
next day, the cells were induced with OIM combined with various concentrations of
synthetic compounds for 2 days. Bone slices were gently brushed and sonicated to
remove the extraneous cells. After fixed with 2.5% glutaraldehyde and stained by
0.5% toluidine blue, the slices were then imaged using a Zeiss confocal microscope
with 100 × magnification. Three fields of view were randomly selected for each bone
slice for further pit area analysis and were quantified using ImageJ software39.
RNA Isolation and Quantitative Real-time PCR (qRT-PCR)
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For the real-time RT-PCR analysis, total mRNA was extracted from cells with
TRIzol (Invitrogen) according to the manufacturer’s protocol and then subjected to
cDNA synthesis. qRT-PCR was performed using an ABI 7500 Sequencing detection
system according to the manufacturer’s instructions. The levels of NFATc1, TRAP,
cathepsin K and β-actin were quantified by qRT-PCR using specific primers. All
values are reported as the mean ± SD of triplicate measurements of each cDNA
sample. The mRNA levels were normalized with β-actin. The PCR reactions used
specific primers for detecting and quantifying the following genes: TRAP (forward 5’
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- TCCCCAATGCCCCATTC - 3’; reverse 5’ - CGGTTCTGGCGATCTCTTTG - 3’);
NFATc1 (forward 5’ - ACCACCTTTCCGCAACCA - 3’; reverse 5’ -
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TTCCGTTTCCCGTTGCA
-
3’);
Cathepsin
K
(forward
reverse
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5′-GAAGAAGACTCACCAGAAGCAG-3′;
5′-TCCAGGTTATGGGCAGAGATT-3′)
and
β-actin
(forward
5’
-
CTGTCCCTGTATGCCTCTG - 3’; reverse 5’ - ATGTCACGCACGATTTCC - 3’).
Western blot assays
BMMs were seeded into six-well plates and stimulated with mRANKL and M-CSF
for three days in the presence of various concentrations of synthetic compounds. The
cells were then washed and lysed for protein extraction using ice cold RIPA buffer.
The lysates were subsequently subjected to SDS-PAGE. Immunoblot analyses were
performed with the indicated primary antibodies (c-fos and NFATc1) at 4 ºC
overnight. The secondary antibodies were visualized using an Immobilon Western kit
(Millipore, Billerica, MA).
Chemistry Methods. All chemicals and solvents were purchased from commercial
sources and used without further purification. Column chromatography was
performed on silica gel (200 - 300 mesh). H NMR and 13C NMR spectra were
1
obtained on Varian-MERCURY Plus-400 or Bruker AVANCE III 500 or Bruker
AVANCE III 600 instruments at 400/100 MHz or 500/126 MHz or 600/151 MHz,
respectively. Chemical shifts were reported in parts per million (ppm, δ). Proton
coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q),
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multiplet (m), or broad (br). Low-resolution mass spectra (ESI) were obtained using
Agilent HPLC-MS (1200-6110). High resolution mass spectra (HRMS) were obtained
using Agilent 1290-6545 UHPLC-QTOF. Melting points (mp) were measured by
Büchi 510 melting point apparatus without further corrected.
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Synthesis of Compounds. Synthetic routes for 36 target compounds and all
intermediates were outlined in Scheme 1. The target compounds were produced
mainly by two different ways. For compounds 1-16 and 25-36, the synthesis started
from commercially available DL-tryptophan derivatives (37a-d). The introduction of
protecting groups by di-tert-butyl dicarbonate (Boc2O) yielded compounds 38a-d, and
key intermediates 39a-f were synthesized by condensation of compounds 38a-d and
commercially available anilines, the protecting groups of intermediates 39a-f were
released with trifluoroacetic acid (TFA) treatment, and the resultants were then
reacted with commercially available acids, or acyl chloride, or aldehyde, or different
methanesulfonates (gained by treatment with corresponding alcohols and MsCl) to
generate the target compounds. For compounds 17-24, commercially available
DL-tryptophan (37a) was treated with ethanol and thionyl chloride to give compound
40, condensation of compound 40 and 3-phenylpropionic acid yielded compound 41,
and key intermediate 42 was gained by alkaline hydrolysis of compound 41, and then
condensation of intermediate 42 and commercially available anilines produced target
compounds. The purity of the 36 synthesized compounds was determined to be
greater than 95% by HPLC analysis [HPLC: column, Eclipse XDB-C18, 4.6 x 150
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mm, 5 μm; mobile phase (A, water with 0.1% formic acid, and B, methanol);
gradient, 5% to 95% B in 6 min; flow rate, 1.0 mL/min; UV detection, 254 nm; oven
temperature, 35 °C. Agilent 1200, Agilent Technologies, California, USA].
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Synthesis of (±)-benzyl (1-((2,6-dimethylphenyl)amino)-3-(1H-indol-3-yl)-1
-oxopropan-2-yl)carbamate (1)
Compound 39a (94 mg, 0.23 mmol) was dissolved in 0.85 mL of dichloromethane,
and trifluoroacetic acid (TFA) (0.17 mL, 2.3 mmol) was added. The mixture was
stirred at room temperature for 2 h, and concentrated in vacuum. The resulting residue
was dissolved in 3 mL of dichloromethane and triethylamine (Et3N) (64 μL, 0.46
mmol) was added, the mixture was cooled to 0 °C, and benzyl carbonochloridate (49
μL, 0.35 mmol) was added, the reaction mixture was allowed to warm to room
temperature and stirred overnight. The reaction mixture was quenched with water and
extracted with dichloromethane; the organic layer was washed with water and brine
successively, dried over anhydrous sodium sulfate, and concentrated in vacuum. The
resulting residue was purified by silica gel chromatography (petroleum ether/ethyl
acetate, v/v, 10:1 to 2:1) to give the title product as a white solid (41 mg, 40%). mp =
179-181 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.84 (s, 1H), 9.46 (s, 1H), 7.71 (d, J
= 7.9 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.33 – 7.25 (m, 4H),
7.25 (d, J = 2.3 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.07 – 7.02 (m, 3H), 7.00 (t, J = 7.4
Hz, 1H), 5.04 (d, J = 12.7 Hz, 1H), 4.94 (d, J = 12.7 Hz, 1H), 4.52 (ddd, J = 9.9, 8.1,
5.0 Hz, 1H), 3.25 (dd, J = 14.6, 5.2 Hz, 1H), 3.04 (dd, J = 14.4, 9.7 Hz, 1H), 2.06 (s,
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6H). 13C NMR (126 MHz, DMSO-d6) δ 170.60, 155.95, 137.06, 136.10, 135.32,
134.89, 128.24, 127.64, 127.54, 127.48, 127.17, 126.35, 124.00, 120.86, 118.55,
118.20, 111.28, 110.07, 65.20, 55.77, 27.77, 17.91. HRMS (ESI) m/z Calcd for
C27H28N3O3 [M + H]+, 442.2125; Found, 442.2127. Purity 95.2%.
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(±)-N-(2,6-dimethylphenyl)-3-(1H-indol-3-yl)-2-(3-phenylpropanamido)propan
amide (2)
The title compound was prepared from 39a and 3-phenylpropanoic acid following
general procedure A (SI). Yellow solid (64%). mp = 213-215 °C. 1H NMR (600 MHz,
DMSO-d6) δ 10.84 (d, J = 2.3 Hz, 1H), 9.40 (s, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.67 (d,
J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.23 – 7.18 (m, 3H), 7.16 – 7.12 (m, 3H),
7.08 (t, J = 7.2 Hz, 1H), 7.06 – 7.01 (m, 3H), 7.00 (t, J = 7.9 Hz, 1H), 4.78 (td, J =
8.5, 5.9 Hz, 1H), 3.23 (dd, J = 14.5, 5.7 Hz, 1H), 2.99 (dd, J = 14.5, 9.1 Hz, 1H), 2.80
– 2.70 (m, 2H), 2.47 – 2.37 (m, 2H), 2.02 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ
171.42, 170.38, 141.29, 136.12, 135.34, 134.91, 128.20, 128.13, 127.53, 127.27,
126.36, 125.78, 123.68, 120.89, 118.54, 118.21, 111.27, 110.16, 53.72, 36.81, 31.03,
27.94, 17.91. HRMS (ESI) m/z Calcd for C28H30N3O2 [M + H]+, 440.2333; Found,
440.2323. Purity 95.6%.
The resolution of the enantiomers of 2 was performed on a Autoprep HPLC
(PDR-separations) system with a Superchiral S-AD (Chiralway) column (0.46 cm x
15 cm, 5 μm) as the stationary phase and 100% methanol as the mobile phase (flow
rate = 10 mL/min). The compounds were detected by UV absorption at 220 nm.
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Specific rotations were measured with a Rudolph Autopol VI polarimeter, while the
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concentrations (c) were given in g/100 mL. The (+)-2 with a retention time of 3.02
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min showed [α]20 = +45.26° (c = 0.366, MeOH), and the (-)-2 with a retention time
D
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of 4.87 min showed [α]20D = -44.16° (c = 0.351, MeOH).
(±)-2-(3-(4-chlorophenyl)propanamido)-N-(2,6-dimethylphenyl)-3-(1H-indol-3-
yl)propanamide (7)
The title compound was prepared from 39a and 3-(4-chlorophenyl)propanoic acid
1
following general procedure A (SI). White solid (51%). mp = 217-219 °C. H NMR
(500 MHz, DMSO-d6) δ 10.83 (d, J = 2.3 Hz, 1H), 9.38 (s, 1H), 8.25 (d, J = 8.0 Hz,
1H), 7.67 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.22
– 7.19 (m, 2H), 7.14 – 7.10 (m, 2H), 7.10 – 7.07 (m, 1H), 7.06 – 7.02 (m, 3H), 7.02 –
6.98 (m, 1H), 4.77 (td, J = 8.6, 5.8 Hz, 1H), 3.23 (dd, J = 14.4, 5.6 Hz, 1H), 2.99 (dd,
J = 14.4, 9.1 Hz, 1H), 2.79 – 2.69 (m, 2H), 2.41 (t, J = 7.6 Hz, 2H), 2.01 (s, 6H). 13C
NMR (126 MHz, DMSO-d6) δ 171.15, 170.33, 140.23, 136.12, 135.31, 134.86,
130.35, 130.00, 128.01, 127.51, 127.24, 126.34, 123.72, 120.87, 118.54, 118.19,
111.25, 110.12, 53.69, 36.47, 30.25, 27.95, 17.86. HRMS (ESI) m/z Calcd for
C28H29ClN3O2 [M + H]+, 474.1943; Found, 474.1935. Purity 96.0%.
(±)-N-(2,6-dimethylphenyl)-3-(6-fluoro-1H-indol-3-yl)-2-(3-phenylpropanamid
o)propanamide (29)
The title compound was prepared from 39d and 3-phenylpropanoic acid following
general procedure A (SI). White solid (51%). mp = 206-208 °C. 1H NMR (500 MHz,
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DMSO-d6) δ 10.90 (s, 1H), 9.38 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.65 (dd, J = 8.7,
5.4 Hz, 1H), 7.23 – 7.18 (m, 3H), 7.17 – 7.10 (m, 4H), 7.07 – 7.00 (m, 3H), 6.89 –
6.83 (m, 1H), 4.77 (td, J = 8.5, 5.8 Hz, 1H), 3.21 (dd, J = 14.5, 5.8 Hz, 1H), 2.98 (dd,
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J = 14.5, 9.0 Hz, 1H), 2.81 – 2.68 (m, 2H), 2.48 – 2.36 (m, 2H), 2.01 (s, 6H). C
NMR (126 MHz, DMSO-d6) δ 171.40, 170.24, 158.84 (d, J = 233.8 Hz), 141.26,
135.91 (d, J = 12.5 Hz), 135.30, 134.85, 128.17, 128.11, 127.51, 126.35, 125.76,
124.28 (d, J = 2.6 Hz), 124.13, 119.53 (d, J = 10.4 Hz), 110.44, 106.64 (d, J = 24.3
Hz), 97.18 (d, J = 25.3 Hz), 53.65, 36.76, 31.01, 27.87, 17.86. HRMS (ESI) m/z
Calcd for C28H29FN3O2 [M + H]+, 458.2238; Found, 458.2231. Purity 98.0%.
(±)-N-(2,6-dimethylphenyl)-3-(1H-indol-3-yl)-2-((3-phenylpropyl)amino)propa
namide (34)
The title compound was prepared from 39a and 3-phenylpropyl methanesulfonate
following general procedure C (SI). Yellow solid (22%). mp = 197-199 °C. 1H NMR
(400 MHz, Chloroform-d) δ 8.84 (s, 1H), 8.19 (s, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.39
(d, J = 8.1 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.19 – 7.13 (m, 3H), 7.13 – 7.05 (m,
5H), 6.91 (d, J = 7.2 Hz, 2H), 3.58 (dd, J = 10.2, 3.5 Hz, 1H), 3.52 (dd, J = 14.3, 3.6
Hz, 1H), 3.01 (dd, J = 14.4, 10.2 Hz, 1H), 2.68 (dt, J = 13.5, 7.2 Hz, 1H), 2.54 (dt, J =
12.3, 6.7 Hz, 1H), 2.50 – 2.35 (m, 2H), 2.17 (s, 6H), 1.66 (tt, J = 7.9, 7.6 Hz, 2H). 13C
NMR (151 MHz, Chloroform-d) δ 172.77, 141.50, 136.63, 135.23, 133.91, 128.45,
128.35, 128.33, 127.55, 127.14, 125.96, 123.06, 122.60, 119.96, 119.03, 111.87,
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111.49, 64.02, 48.54, 33.21, 31.56, 29.74, 18.73. HRMS (ESI) m/z Calcd for
C28H32N3O [M + H]+, 426.2540; Found, 426.2543. Purity 96.4%.
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Supporting Information
General synthetic procedures and characterization data for all target compounds and
intermediate compounds; NMR spectra for final compounds.
SMILES data for compounds 1-37.
Corresponding Author Information
Xing Xu: xuxing0510315@126.com
Lianfu Deng: lfdeng@msn.com
Chunhao Yang: chyang@simm.ac.cn
ACKNOWLEDGEMENTS
This work was supported by the NSFC (No. 81703576, 81502902), Shanghai
Municipal Health Bureau scientific research project (No. 201840185, No.
20154Y0005), Committee of Science and Technology young scientific research
foundation of Shanghai (No. 16ZR1431900), Guangci Distinguished Young Scholars
Training Program (No. GCQN-2017-A16, GCQN-2017-B22).
ABBREVIATIONS USED
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Boc2O,
di-tert-butyl
dicarbonate;
HATU,
6
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2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate;
8
9
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; TFA,
trifluoroacetic acid; DCM, dichloromethane; Et3N, triethylamine; DMAP,
4-Dimethylaminopyridine; NaBH3CN, sodium cyanoborohydride; MeCN,
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acetonitrile;
SOCl2,
thionyl
chloride;
EtOH,
ethanol;
EDCI,
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride;
HOBT,
1-Hydroxybenzotriazole; THF, tetrahydrofuran; MsCl, methanesulfonyl chloride.
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