Structure-based development of an osteoprotegerin-like glycopeptide that blocks RANKL/RANK interactions and reduces ovariectomy-induced bone loss in mice

Article abstract of DOI:10.1016/j.ejmech.2018.01.022

Osteoporosis is a metabolic bone disease characterized by low bone mass and micro-architectural deterioration of bone, for which the underlying mechanism is an imbalance between bone resorption and bone remodeling. The protein-protein interactions between receptor activator of nuclear factor-κB ligand (RANKL), RANK (its receptor), and osteoprotegerin (OPG), are known to mediate the development and activation of osteoclasts in bone remodeling, and are regarded as a pivotal therapeutic target for the treatment of osteoporosis. Herein, we disclose the successful development of a novel glycopeptide (OM-2), the structure of which is based on the key interacting sites of the reported RANKL and OPG crystal structure. OM-2 exhibited potent binding affinity with RANKL and resistance to degradation by protease enzymes. It also blocked RANKL/RANK interactions, and inhibited osteoclastogenesis in vitro. In vivo studies confirmed that OM-2 could effectively reduce bone loss and inhibit osteoclast activation in ovariectomized (OVX) mice at a dosage of 20.0 mg/kg/day. Accordingly, OM-2 is suggested as a therapeutic candidate for postmenopausal osteoporosis (PMOP) and osteoclastogenesis-related diseases like rheumatoid arthritis (RA). More importantly, its identification validates our structure-based strategy for the development of drugs that target the RANKL/RANK/OPG system.

Full text of DOI:10.1016/j.ejmech.2018.01.022

Accepted Manuscript
Structure-based development of an osteoprotegerin-like glycopeptide that blocks
RANKL/RANK interactions and reduces ovariectomy-induced bone loss in mice
Chao Liu, Xiao Chen, Xin Zhi, Weizong Weng, Quan Li, Xiang Li, Yan Zou, Jiacan Su,
Hong-Gang Hu
PII:
S0223-5234(18)30022-9
10.1016/j.ejmech.2018.01.022
EJMECH 10099
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2017
Revised Date: 3 January 2018
Accepted Date: 8 January 2018
Please cite this article as: C. Liu, X. Chen, X. Zhi, W. Weng, Q. Li, X. Li, Y. Zou, J. Su, H.-G. Hu,
Structure-based development of an osteoprotegerin-like glycopeptide that blocks RANKL/RANK
interactions and reduces ovariectomy-induced bone loss in mice, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.022.
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ACCEPTED MANUSCRIPT
Graph Abstract

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Structure-based Development of an Osteoprotegerin-like Glycopeptide That Blocks
RANKL/RANK Interactions and Reduces Ovariectomy-induced Bone Loss in Mice
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Chao Liu , Xiao Chen , Xin Zhi , Weizong Weng , Quan Li , Xiang Li , Yan Zou *, Jiacan
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Su *, Hong-Gang Hu *
1
. Department of Organic Chemistry, College of Pharmacy, Second Military Medical
University, Shanghai 200433, China
. Department of Orthopedics Trauma, Shanghai Changhai Hospital, Second Military Medical
University, Yangpu District, Shanghai 200433, China
. Graduate Management Unit, Shanghai Changhai Hospital, Second Military Medical
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University, Yangpu District, Shanghai 200433, China
Chao Liu and Xiao Chen contributed equally to this work.
*
Corresponding to Prof. Yan Zou, Email: zouyan@smmu.edu.cn; Prof. Jiacan Su,
Email:drsujiacan@163.com; Prof. Hong-Gang Hu, Email: hhu66@smmu.edu.cn.
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Abstract
Osteoporosis is a metabolic bone disease characterized by low bone mass and micro-
architectural deterioration of bone, for which the underlying mechanism is an imbalance
between bone resorption and bone remodeling. The protein-protein interactions between
receptor activator of nuclear factor-κB ligand (RANKL), RANK (its receptor), and
osteoprotegerin (OPG), are known to mediate the development and activation of osteoclasts in
bone remodeling, and are regarded as a pivotal therapeutic target for the treatment of
osteoporosis. Herein, we disclose the successful development of a novel glycopeptide (OM-2),
the structure of which is based on the key interacting sites of the reported RANKL and OPG
crystal structure. OM-2 exhibited potent binding affinity with RANKL and resistance to
degradation by protease enzymes. It also blocked RANKL/RANK interactions, and inhibited
osteoclastogenesis in vitro. In vivo studies confirmed that OM-2 could effectively reduce bone
loss and inhibit osteoclast activation in ovariectomized (OVX) mice at a dosage of 20.0
mg/kg/day. Accordingly, OM-2 is suggested as a therapeutic candidate for postmenopausal
osteoporosis (PMOP) and osteoclastogenesis-related diseases like rheumatoid arthritis (RA).
More importantly, its identification validates our structure-based strategy for the development
of drugs that target the RANKL/RANK/OPG system.
Keywords: Osteoprotegerin; Mimics; Glycopeptide; RANKL; Osteoclastogenesis
1
. Introduction
Diseases caused by the excessive activation of osteoclasts such as postmenopausal
osteoporosis (POMP) and rheumatoid arthritis constitute a worldwide threat to public health
1,2]. Osteoporosis is characterized by low bone mineral density (BMD) and poor bone
[
quality, leading to high risk of fragility fractures [3]. Worldwide, more than 8.9 million
people have experienced osteoporosis-stimulated fractures over the past decade, and the
World Health Organization (WHO) has identified osteoporosis as a major public health
concern [4].
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The use of therapeutic agents that inhibit osteoclastogenesis and restore the balance
between bone resorption and formation is an important strategy for the treatment of
osteoporosis [5]. Bisphosphonates are widely used to rectify osteoclast-induced bone
resorption in osteoporosis, but their use is severely limited due to their poor bioavailability
and gastrointestinal tolerability, renal toxicity, and tendency to cause osteonecrosis of the jaw
[
6-9]. Possible alternative anti-resorption drugs include selective estrogen receptor
modulators (SERM) and strontium ranelate, but the side-effects of these include
thromboembolic disease, drug rash with eosinophilia systemic syndrome, and abdominal
discomfort [6,7]. Bone-anabolic drugs are limited to teriparatide, an N-terminal fragment of
parathyroid hormone (PTH 1-34), which stimulates bone formation by regulating osteoblast
numbers and activity. Side-effects of this drug include back pain, nausea, leg cramps,
dizziness and osteosarcoma, which limit the duration of therapy [8,9]. Accordingly, novel
treatments for osteoporosis are urgently needed [10,11].
The RANKL/RANK/OPG system plays a vital role in osteoclastogenesis [12-18]. RANK is
expressed on osteoclast precursors and can be activated by its native ligand RANKL, which
leads to osteoclast differentiation, maturation, and bone resorption [12,13,17,19-24].
Meanwhile, by acting as a soluble decoy receptor to RANKL, OPG competes with RANK for
RANKL binding to inhibit osteoclastogenesis [12,18,19,25]. Various osteolytic bone diseases
such as primary osteoporosis, rheumatoid arthritis and osteolytic bone metastases result from
an imbalance between osteogenesis and osteoclastogenesis [26-28], and over-activated
osteoclasts play a leading role in disease pathogenesis [17,18,29,30]. Accordingly, blocking
the RANKL/RANK interaction to inhibit osteoclastogenesis is a promising therapeutic
strategy [31,32].
RANK-Fc, Fc-osteoprotegerin and anti-RANKL antibodies have been previously used to
block the RANKL/RANK interaction [33,34]. Denosumab is a RANKL monoclonal antibody
inhibitor and has been used for osteoporosis patients at a high risk of fracture, bone loss due
to certain medications, and bone metastases [35-39]. However, it is very expensive, and
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causes hypocalcemia side effect [40,41]. Moreover, low stability, poor bioavailability, high
cost and difficulties in administration were the major disadvantages hindered the appication
of large macromolecules for therapeutic intervention [42]. Therefore, small peptides or
peptidomimetic-based treatments are another option, and their unique chemical properties,
proper molecular weight, improved biological activity, low toxicity, and immunogenicity
could overcome these drawbacks [43,44]. Also, the specific nature of their target, weak drug-
drug interactions, and reduced propensity to accumulate in tissue, reduce the risk of
complications associated with their use [43]. In previous study, disulfide bond bearing
peptides from RANK/RANKL interaction such as WP9QY and L3-3 were developed and
their inhibitory activity was confirmed in vivo [45,46]. Effective OPG-like peptide mimics
were limited to disulfide bond peptide OP3-4 and linear peptide YR-11. They have been
shown to inhibit RANKL-induced osteoclastogenesis both in vitro and in vivo, prevent
osteolytic bone disease in myeloma and down regulate inflammatory cytokines [42,47,48].
Nevertheless, these designs were only based on the putative contacts derived from the co-
crystal structures of TNF-β·TNFR1 or deduced OPG binding sites to RANKL, with no
authentic crystal structure-based evidence.
In 2012, the structure of human RANKL ectodomain complexed with the N-terminal of
human OPG cysteine-rich TNFR homologous domains (OPG-CRD) was clarified, providing
precise molecular details of the RANKL/OPG interactions, and a structural basis for the
rational design of OPG-like peptides or peptidomimetics for the purpose of interrupting
ligand-receptor interaction [49]. Herein, we disclose the development of several OPG-like
peptides or peptidomimetics, guided by the crystal-structure of RANKL/OPG complex (PDB:
3
URF). RANKL-induced osteoclastogenesis inhibition was verified in vitro, and OM-2 was
identified as the most potent inhibitor. The circular dichroism (CD) spectroscopy, binding
affinity, protease resistance and effect on downstream signaling pathways of OM-2 were then
investigated, and its in vivo effects were evaluated in ovariectomized mice.
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. Results
.1. Structural-based design and synthesis of OPG-like mimics
The binding interface of RANKL/OPG-CRD presents two binding sites (Figure S1A) [49].
The first, binding site I, consists of small and separate interactions, is located on the OPG
4
7
65
"
50s loop" (His -Leu ) of the CRD-2 domain, and extends along and parallel to the groove
9
0
98
of RANKL. Binding site II, located at the OPG "90s loop" (Arg -Leu ) of the CRD-3
domain, is deeply within the groove, and flanked by AA' and CD loops of RANKL. Binding
9
5
site II uses the Glu of the OPG "90s loop" to form a hydrophilic interaction network with
2
23
241
257
RANKL residues Arg , Tyr and Lys , and is more important than binding site I in
RANKL/OPG-CRD binding. Furthermore, this location is also the major binding determinant
of RANKL/RANK interaction. Accordingly, peptides or peptidomimetics whose sequences
are derived from the OPG "90s loop" are hypothesized to inhibit protein-protein interactions
(
PPIs). Moreover, multiple disulfide bridges are separated on the OPG CRD-3 sequence, and
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the disulfide bonds between Cys and Cys are distributed around the OPG "90s loop"
sequence. This fragment is vital for its interaction with the AA' and CD loop of RANKL.
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98
Initially, we designed disulfide peptide OM-1 (Figure 1) based on the Cys –Leu
fragment, to obtain the shortest peptide with definite secondary structure. OM-1 was
synthesized via a solid phase coupling/solution phase cyclization strategy. The on-resin
peptide 1 was cleavaged from the solid support to obtain the free peptide 2. The following
oxidative folding [6 M Gn·HCl/100 mM NaH PO PBS buffer (pH=7.4) containing 10 %
2
4
DMSO] of 2 provided the disulfide peptide OM-1 in an overall yield of 40.0 % (Scheme 2).
However, given the propensity of disulfide bond-bearing peptides to degrade in the
physiological environment through reduction, polymerization or enzymatic cleavage of the
disulfide bond [50-52], we next sought to optimize OM-1 by chemical means, such as by
glycosylation or by stabilization of the disulfide-bond.
It was found that multiple N-linked N-acetyl-glucosamine glycosylation sites are present on
the OPG backbone [48,49]. Although the functions of these glycosylation sites remains
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unclear, glycosylation has been demonstrated to exert a multifaceted influence on the
chemical properties and function of peptides, including improved hydrophilicity and
bioavailability, better conformation restriction and maintenance, increased protease resistance,
and weaker immunogenicity [53]. Furthermore, chondroitin sulfate combined with
glucosamine sulfate could increase the expression ratio of OPG/RANKL, indicating a
potential regulative function in the RANK/RANKL/OPG system of carbohydrates [54].
However, it was suggested that an inappropriate spatial location of the carbohydrate block
might impart significant structural and conformational changes upon the peptide, and perturb
the interactions between the peptide and target protein [53]. In order to design a reasonable
glycopeptide mimic, we undertook a hypothetical geometry comparison and molecular
docking study to investigate the proper location for the carbohydrate block. The results
suggested that, compared with the non-glycosylated form, arrangement of the carbohydrate
group at the tail of peptide back bone has little impact on the peptide secondary structure and
didn’t disturb the mutual binding mode. As shown in Figure S1B, the locations of all the
amino acid side chains matched the non-glycosylated form. Furthermore, upon binding to the
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96
active sites of RANKL, the Ile and Leu residues were found to form hydrophobic and van
der Waals interactions with the surrounding hydrophobic residues of RANKL. More
9
5
importantly, Glu was found to be the key interaction residue, forming multiple H-bonds
2
57
241
with Lys and Tyr of RANKL (Figure S1C). It was with these observations in mind,
glycopeptide mimic OM-2 (Figure 1) was designed.
The synthesis of OM-2 was based on the key glycoamino acid building block Fmoc-
Asn[GlcNAc(Ac )]-OH (5). N-acetyl-β-D-glucosamine was selected as the starting material,
3
and after acetylation followed by reaction with sodium azide catalyzed by the phase transfer
catalyst tetrabutylammonium iodide, compound 3 was obtained in an 82.4 % yield. Reduction
t
of the azide group followed by coupling with Fmoc-Asp-O Bu through 1-
hydroxybenzotriazole (HOBt)/N,N'-diisopropylcarbodiimide (DIC) led to the protected
t
glycoamino acid building block 4, which underwent Bu deprotection using a TFA/DCM
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solution to give the target compound 5 in good yield (95.0 %, Scheme 1A). Compound 5 was
readily incorporated into the peptide backbone through standard Fmoc SPPS using O-(7-
azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) as the
coupling reagent, to obtain on-resin peptide 6. The following on-resin deprotection of the
acetyl protection group utilizing hydrazine hydrate gave the on-resin intermediate 7. After
acidic cleavage, global deprotection and oxidative folding, OM-2 was obtained in a yield of
2
5.2 % (Scheme 2).
We considered two alternatives to the disulfide bond of peptide OM-1. Firstly, diselenide
peptides have been shown to impart greater biological activity than their disulfide
counterparts, perhaps due to a different bond connectivity and/or conformational structure
resulting from the diselenide substitution, which is more metabolically stable than the
disulfide linkage [55,56]. Accordingly, peptide OM-3 (Figure 1) containing a diselenide
bridge was synthesized, with view to improving the biological activity and pharmacokinetic
properties of peptide OM-1. By incorporating (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-3-((4-ethoxybenzyl)selanyl) propanoic acid [Fmoc-Sec(Mob)-OH] into standard
SPPS, we assembled the protected on-resin peptide 8. After cleavage from the solid support,
the Mob protected peptide 9 was treated with TFA/DMSO solution (1:1, v/v) to complete the
final deprotection and folding step, giving the diselenide peptidomimetic OM-3 in 27.4 %
yield (Scheme 2).
Secondly, in view of its chemical orthogonality and its imperviousness to isomerases and
proteases, the triazole bridge was also selected to replace the disulfide-bond. Thus, the 1,4-
disubstituted 1,2,3-triazole bridge mimic OM-4 (Figure 1) was obtained using a
diaminodiacid-based synthetic strategy, according to our previous report [57]. Briefly, Boc-
Dap-OH was transformed into 3-azido-N-Boc-L-alanine (10) via a Cu(II)-catalyzed diazo
transfer reaction. Then, the tert-butoxycarbonyl (Boc) group of 10 was easily cleaved by
trifluoroacetic acid, and the free amino acid was further protected with allyloxycarbonyl
t
(
Alloc) and allyl groups to afford 11. Installation of the tert-butyl ( Bu) protective group
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followed by capping of 9H-fluoren-9-ylmethoxy)carbonyl (Fmoc) group on 2-propargyl-L-
t
glycine (H-Pra-OH) provided Fmoc-Pra-O Bu (12). Compound 13 could be obtained through
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between 11 and 12 directly
in a high chemical yield. Finally, the diaminodiacid building block 14 was prepared from 13
t
by removing the Bu protection group using trifluoroacetic acid (Scheme 1B). Next, 14 was
successfully attached to resin through standard Fmoc SPPS using HATU as the coupling
reagent, to obtain on-resin peptide 15. After cleavage of the protective allyl, Alloc and Fmoc
groups via [Pd(PPh ) ]/PhSiH and 20 % piperidine/DMF solution, macrocyclization was
3
4
3
successfully accomplished with (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyAOP), N-methylmorpholine (NMM) and 1-hydroxy-7-
azabenzotriazole (HOAt) as lactamization reagents, to achieve cyclic peptide 16. Finally, one
step global deprotection and cleavage with the TFA cocktail delivered OM-4 in a yield of
3
2.1 % (Scheme 2). To the best of our knowledge, this is the first time that glycopeptide,
disulfide, diselenide, and triazole-bridged derivatives of the same peptide have been
systematically synthesized and tested in a RANK/RANKL/OPG system study.
2
.2. Biological test in vitro and cytotoxicity study
OPG mimics were evaluated via tartrate-resistant acid phosphatase (TRAP) staining to
screen for biological activity against RANKL-induced osteoclast differentiation on bone
marrow mononuclear cells (BMMCs) [46]. Each mimic (30.0 µM) was co-cultured with
RANKL and macrophage colony-stimulating factor (M-CSF) in BMMCs, and were found to
rd
exhibit inhibitory effects on the TRAP activity ranging from 24.35 % to 59.20 % on the 3
day of induction (Figure 2A). OM-3 and OM-4, with 40.65 % and 42.15 % respectively,
were more potent inhibitors than OM-1 (24.35 %). Glycopeptide OM-2 exhibited greater
inhibitory potency than the positive control peptide OP3-4, a previously reported OPG-like
peptide mimic (58.20 % for OM-2; 55.57 % for OP3-4). Further tests at different
concentrations of OM-2 and OP3-4 were then carried out by TRAP staining. OM-2 was found
to reduce the number of TRAP-positive multinucleated cells in a dose-dependent manner
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(
Figure 2B and Table S1). The IC50 was 28.51 µM for OM-2 and 25.27 µM for OP3-4,
implying that OM-2, an OPG-like glycopeptide mimic, could effectively suppress
osteoclastogenesis in vitro. Subsequently, cell counting kit-8 (CCK-8) analysis was
performed on BMMCs to test the potential toxicity. The results showed that even at 100.0µM
treatments, these peptide mimics did not exhibit obvious cytotoxic effects, indicating the
inhibitory activities were not due to their cytotoxicity (Figure S6).
2
.3. CD spectroscopy of peptides and kinetic binding ability of OM-2
The secondary structures of the peptide mimics were analyzed by CD spectroscopy (Figure
A). All the peptides showed a broad negative peak around 215 nm, characteristic of a β-sheet
3
structure. This result suggested that the carbohydrate group on OM-2 has little impact on the
secondary structure compared to OM-1. The binding affinity of OM-2 to RANKL was next
investigated via surface plasmon resonance (SPR). OM-2 was found to bind to RANKL in a
dose-dependent manner. The apparent association (k ) and disassociation constants (k ) of
on
off
-1 -1
-1
OM-2 were estimated to be 608.7 M s and 0.0027 s respectively, and the relative binding
affinity (K ) to RANKL was 4.54 µM (Figure 3B); weaker than the affinity of RANKL to
d
RANK (RANKL to RANK K =13.9 nM) or OPG (RANKL to OPG K =17.6 nM), but
d
d
comparable than that between RANKL and the positive OP3-4 peptide (Figure S7, OP3-4 to
RANKL K =2.75 µM), suggesting the complex between RANKL and OM-2 to be relatively
d
stable. The improved binding ability compared with OM-1 (Figure 3C, OM-1 to RANKL
K =30.91 µM), is speculated to be attributable to the presence of carbohydrate structure into
d
peptide backbone. Although the mechanism for binding between each peptide and RANKL is
not clear without the crystal-structure of the OM-2/RANKL complex, the data obtained
definitively establishes that glycopeptides OM-2 possess superior chemical and biological
properties for the purposes of RANKL inhibition than normal peptides.
2
.4. Inhibition of NF-κB by OM-2
The differentiation of osteoclast precursors into mature osteoclastsis associated with
RANKL-stimulated NF-κB activation [23]. The activation of the NF-κB signal pathway is
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indicated by the procedural activation of its inhibitory subunit IκB, including phosphorylation,
ubiquitination, degradation, and nuclear localization signaling exposure [58]. To evaluate the
effects of OM-2 on RANKL induced NF-κB activation, a western blotting study was carried
out. The results showed that OM-2 could significantly inhibit RANKL-stimulated NF-κB
activation as indicated by the lack of IκB phosphorylation (Figure 3D). This study shows that
OM-2 effectively inhibits osteoclast formation, in part by interruption of the RANK/RANKL
protein-protein interaction and suppression of the NF-κB signaling pathway. .
2
.5. α-chymotrypsin resistance of OM-2
α-chymotrypsin is a protease that preferentially cleaves peptide amide bonds where the
side-chain of the amino acid N-terminal to the scissile amide bond is a large, hydrophobic
amino acid such as Trp, Phe and Leu, and is used for peptide protease stability studies [59].
OM-1 and OM-2 were subjected to the α-chymotrypsin mediated degradation test and
monitored by HPLC. Even after 8 hours' protease exposure, more than 60.70 % OM-2
remained intact with a superior protease resistance compared with OM-1 (38.47 %),
suggesting greater protease stability over normal peptides (Figure 3E). The improved in vitro
inhibitory activity could partly result from the greater protease resistance [53].
2
.6. Effects of OM-2 in OVX mice
The effects of OM-2 on ovariectomy-induced bone loss in mice were investigated. Six
weeks after the surgery, OVX mice treated with normal saline exhibited a significant loss of
trabecular bone in the distal femur. Intraperitoneal injection of OM-2 (20.0 mg/kg/day) in
OVX mice markedly inhibited trabecular bone loss compared with OVX mice, as shown by
hematoxylin and eosin (H&E) staining (Figure 4A). The number of osteoclasts was examined
by TRAP staining. OM-2 significantly decreased the proportion of TRAP positive cells in
femur bone (Figure 4B). These results were corroborated by micro-computed tomography
(
Micro CT). Figure 4C and Figure 4D depict the two-dimensional and three-dimensional
structures of the bone, as measured by trabecular bone volume expressed per unit total
volume (BV/TV), trabecular bone surface area expressed per unit total volume (BS/TV),
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trabecular number (Tb. N) and BMD. Compared with the OVX group, OVX+OM-2 group
displayed decreased serum interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), serum
C-terminal telopeptide of type I collagen (CTX-1) and tartrate-resistant acid phosphatase 5B
(
TRAcp5B) (P< 0.05) levels, suggesting that OM-2 inhibited bone loss in OVX mice by
inhibition of osteoclastogenesis (Figure 4E).
3
. Discussion
Four OPG-like peptides OM-1, OM-2, OM-3 and OM-4 or peptidomimetics have been
designed based on the crystal structure of human RANKL/OPG-CRD complex and
synthesized by SPPS, in order to test their ability to perturb RANK receptor signaling. In vitro
studies showed that glycopeptide OM-2 was the most potent inhibitor of RANKL-induced
osteoclastogenesis of all the peptides tested. It was able to effectively inhibit RANKL-
mediated NF-κB signaling pathway in BMMCs and displayed improved binding affinity and
superior protease resistance compared to OM-1. In vivo studies showed that OM-2 reduced
OVX induced bone loss and osteoclast formation.
The RANKL/RANK/OPG axis is well known to regulate osteoclast differentiation and play
a vital role in bone remodeling. Furthermore, RANKL is involved in various osteolytic
pathogenesis processes such as primary osteoporosis, rheumatoid arthritis, bone metastases,
etc., and protein therapeutics such as RANK-Fc, Fc-osteoprotegerin and RANKL monoclonal
antibodies that block RANKL activity are proven treatments for these diseases [33-39].
Denosumab was approved in 2010 for the treatment of postmenopausal women with
osteoporosis at high risk of fracture. Small peptides derived from either RANK or OPG were
also demonstrated to inhibit osteoclastogenesis both in vitro and in vivo [42,45-48]. However,
prior to 2012, when the crystal structure of human RANKL/OPG-CRD complex was first
revealed [49], there was no structural basis to inform OPG-like design. The crystal structure
study suggests that RANKL employs distinct binding modes to engage RANK and the OPG
receptor, and OPG binds to RANKL with a nearly 500-fold higher affinity and 150 times
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greater inhibitory activity on RANKL-stimulated osteoclastogenesis than RANK [60]. Hence,
we hypothesized that peptides derived from the key interacting site of OPG with RANKL
would be more effective at disrupting RANKL/RANK interactions than from RANK.
Accordingly, we designed and synthesized a disulfide peptide based on the OPG "90s loop"
sequence, regarded as the critical binding site for OPG/RANKL interactions [49], to obtain
the shortest fragment which could simulate its secondary structure. Thereafter, we carried out
an in vitro osteoclastogenesis inhibitory study. BMMCs were isolated, and induced with
RANKL (300.0 ng/mL) and M-CSF (30.0 ng/mL). Peptide OM-1 exhibited only weak
inhibitory activity, possibly due to the susceptibility of disulfide bond present in its structure.
To overcome this problem, we strategically modified the peptide structure. Many natural
macrocyclic peptide antibiotics and physiologically active proteins are glycosylated,
indicating the critical role of carbohydrates in sustaining physiological functions [61,62].
Additionally, glycosylation can improve chemical properties of peptides such as
hydrophilicity, oral bioavailability, protease resistance, and conformation stability [53]. Also,
the diselenide bridge is a well-known disulfide bond replacement strategy, widely used in
peptide and protein folding studies, to provide greater conformation stability without
adversely affecting biological activities [55,56]. Finally, the triazole bridge has been
extensively investigated in peptide chemistry too, for the purpose of mimicking and adding
rigidity to the amide backbone, and as a linking group in carbohydrate chemistry and vaccine
preparation [63-66]. More importantly, triazole substitution has already been used as an
effective replacement for the disulfide-bond [67,68]. Accordingly, three potential candidate
peptides OM-2 (bearing a disulfide bond), OM-3 (bearing a diselenide bond) and OM-4
(
bearing a triazole bridge) were synthesized and tested. Of these, OM-2 was found to exhibit
the strongest inhibitory activity, as measured in a TRAP assay at 30.0 µM concentration,
comparable to positive control OP3-4. Further studies found the IC of OM-2 and OP3-4 to
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0
be equal. Computational docking results suggested that the glycosylation did not affect the
secondary structure of the peptide, and CD spectroscopy study confirmed our incorporation of
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a carbohydrate block on OM-2 did not have a deleterious effect on its β-sheet structure.
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5
Furthermore, we believe that the Glu of OM-2 was essential for good biological activity,
due to the formation of hydrogen bonding network with active sites of RANKL.
Next, OM-2 was subjected to protease resistance and SPR experiments. The data suggested
OM-2 to have greater protease stability and stronger binding ability to RANKL compared
with OM-1 (its non-glycosylated form), a possible explanation for its improved activity. An
immunoblotting study found that both OM-2 and OP3-4 significantly suppressed the
RANKL-induced NF-κB signaling pathway, as indicated by a reduction in IκB
phosphorylation.
In vivo, an OVX mice model was used to observe the effects of OM-2 on OVX induced
bone loss. We found that OM-2 significantly reduced bone loss in OVX mice, by H&E
staining of the distal femur, and Micro CT analysis. For TRAP staining, OM-2 was found to
significantly reduce the number of activated osteoclasts around the trabecula. The serum level
of the bone resorption markers TRAcp5B and CTX-1 were also significantly reduced. In a
previous study, inflammation was found to play an important role in osteoclast differentiation
[
69]. Therefore, the serum levels of TNF-α and IL-6 also imply that OM-2 significantly
decreased the osteoclastogenesis in vivo. More importantly, in vivo experiments suggested
that OM-2 could impart bone protection when administered via intraperitoneal injection [70],
which is preferable to the intravenous mode of administration required by OP3-4.
In conclusion, the novel glycopeptide OM-2, the design of which was informed by the
crystal structure of the RANKL/OPG complex, has been synthesized and found to inhibit
osteoclastogenesis in vitro, with an IC of 28.51 µM, and reduce bone loss in OVX mice, at a
5
0
dosage of 20.0 mg/kg/day. This study establishes OM-2 as a promising lead for the
development of novel therapeutics for PMOP, as well as a variety of diseases caused by the
over-expression of RANKL, such as rheumatoid arthritis, periodontal diseases and bone
metastasis. Also, it constitutes a useful chemical tool for furthering our understanding of the
molecular mechanisms and biological functions of the RANK/RANKL/OPG system. Further
1
3

ACCEPTED MANUSCRIPT
optimization of OM-2 is ongoing in our laboratory, and the results will be disclosed in due
course.
4
. Experimental protocols
.1. Materials
All chemical reagents were purchased from Acros, Sigma-Aldrich, Alfa Aesar, Adamas
4
and InnoChem. Amino acids were obtained from GL Biochem Shanghai Co. Ltd; and all
solvents were from Sinopharm Chemical Reagent Co. Ltd. Dichloromethane (DCM) and N,N-
Dimethylformamide (DMF) were distilled over calcium hydride (CaH ) under an argon
2
atmosphere, and stored in flask containing 4 Å molecular sieves. All reactions vessels were
oven-dried before use. Reactions were monitored by thin-layer chromatography (TLC) and
visualized by UV (254 nm), ninhydrin and/or phosphomolybdic acid. Recombinant sRANKL,
RANK and M-CSF were purchased from Sigma.
4
.2.
Peptides were analyzed and purified by reverse phase HPLC. A C18 analytic column
Shimadzu Shim-pack VP-ODS, 4.6×250 mm, 5 µm particle size, flow rate 1 mL/min) was
HPLC, Mass spectrometry and NMR
(
used for analytical RP-HPLC, and a C18 column (Shimadzu Shim-pack PRC-ODS, 50×250
mm, 15 µm particle size, flow rate 13 mL/min) was used for semi-preparative RP-HPLC. The
solvent systems were buffer A (0.1% TFA in CH CN) and buffer B (0.1% TFA in water).
3
Data was recorded and analyzed using the software system LC Solution. High resolution mass
spectra were measured on a Waters Xevo G2 QTOF mass spectrometer. ESI-MS was
1
13
measured with a Bruker Esquire 3000 Plus Ion Trap mass spectrometer. H-NMR and C-
NMR spectra were recorded on a Bruker Avance 300 MHz instrument. Chemical shifts (δ)
1
13
are reported relative to TMS (0 ppm) for H-NMR and C-NMR spectra. Coupling constants
J) were given in Hertz (Hz); and the splitting patterns are abbreviated as follows: singlet (s);
broad singlet (s, br); doublet (d); doublet of doublet (dd); triplet (t); quartet (q); multiplet (m).
.3. Chemistry
(
4
1
4

ACCEPTED MANUSCRIPT
4
.3.1. (2R,3S,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-
diyldiacetate (3)
To N-acetyl-β-D-glucosamine (10.00 g, 45.24 mmol) was added acetyl chloride (30.00
mL) dropwise over 15 min at 0 ºC. The reaction mixture was stirred vigorously at room
temperature for 4 days. This mixture was diluted with DCM (100.00 mL) and saturated
NaHCO (100.00 mL) aqueous solution. The organic phase was separated, washed with
3
saturated NaHCO and brine (3×100.00 mL), dried over Na SO and concentrated, and used
3
2
4
without further purification. A mixture of commercially available NaN (8.82 g, 135.70 mmol)
3
and tetrabutylammonium iodide (16.68 g, 45.23 mmol) in DCM/water (1:1, 200.00 mL) was
stirred for 2 hours at room temperature. The organic layer was separated, washed with brine
(
(
(
3×100.00 mL), dried over Na SO , concentrated and purified by column chromatography
2 4
1
6:1-2:1, petro ether/EtOAc) to give 3 as a white powder (13.80 g, 82.4 %, 2 steps). H-NMR
300 MHz, CDCl ): δ 5.72 (d, J= 3.0 Hz, 1H), 5.25-5.22 (m, 1H), 5.10-5.07 (m, 1H), 4.75 (d,
3
J= 3.0 Hz, 1H), 4.27-4.24 (m, 1H), 4.17-4.14 (m, 1H), 2.09 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H),
1
3
1
7
3
.97 (s, 3H). C-NMR (300 MHz, CDCl ): δ 172.40, 172.08, 171.84, 170.67, 89.83, 75.42,
3
3.57, 69.48, 63.27, 55.61, 24.64, 22.12, 22.03, 21.98. ESI-MS m/z calcd for C H N O
8
1
4
20
4
+
72.13; found [M+H] 373.29.
4
.3.2. (2R,3S,4R,5R,6R)-6-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-
oxobutanamido)-5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (4)
To a solution of 3 (10.00 g, 26.88 mmol) in MeOH (200.00 mL) was added Pd/C catalyst
(
1.00 g). Then the mixture was stirred overnight at room temperature under hydrogen. After
the Pd/C catalyst was filtered and the MeOH was removed under vacuum, the residue was
used without further purification. To a solution of the intermediate in DMF (50.00 mL) was
t
added Fmoc-Asp-O Bu (12.14 g, 29.56 mmol), HOBt (3.99 g, 29.56 mmol) and DIC (3.72 g,
2
9.56 mmol) mixture of DCM/DMF (1:1, 100.00 mL) solution. The reaction was stirred
overnight at room temperature. Then, the reaction was filtrated and the filtrate was washed
1
5

ACCEPTED MANUSCRIPT
successively with 1 M HCl (3×100.00 mL), saturated NaHCO (3×100.00 mL) and brine
3
(
3×100.00 mL). The organic phase was dried over Na SO , filtered, concentrated and purified
2 4
by column chromatography (100:1-50:1, DCM/MeOH) to give 4 as a white powder (14.10 g,
1
7
2
5
1
4
1
7
1.2 %, 2 steps). H-NMR (300 MHz, CDCl ): δ 7.75 (d, J= 3.0 Hz, 2H), 7.60 (d, J= 3.0 Hz,
3
H), 7.40-7.37 (m, 2H), 7.36-7.26 (m, 2H), 7.17 (s, 1H), 6.07 (s, 1H), 5.94-75.92 (m, 1H),
.12-5.05 (m, 3H), 4.42-4.30 (m, 2H), 4.29-4.21 (m, 3H), 4.06-4.04 (m, 2H), 3.74-3.73 (m,
H), 2.85-2.82 (m, 1H), 2.71-2.69 (m,1H), 2.56 (s, 2H), 2.06-2.03 (m, 8H), 1.96 (s, 2H), 4.42-
1
3
.30 (m, 2H), 1.44 (s, 9H). C-NMR (300 MHz, CDCl ): δ 173.86, 173.44, 172.52, 172.07,
3
71.35, 170.64, 157.55, 145.34, 145.22, 142.70, 129.12, 128.49, 126.59, 121.39, 83.66, 81.74,
5.02, 74.33, 68.97, 68.60, 63.08, 54.97, 52.43, 48.57, 39.43, 29.33, 22.12, 21.99. ESI-MS
+
m/z calcd for C H N O 739.30; found [M+H] 740.19.
3
7
45
3
13
2
4
4
.3.3. N -(((9H-fluoren-9-yl)methoxy)carbonyl)-N -((2R,3R,4R,5S,6R)-3-acetamido-4,5-
diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)asparagines (5)
(10.0 g, 13.53 mmol) was dissolved in TFA/DCM (1:1, 20.00 mL) and stirred for 2 hours
4
at room temperature. The reaction mixture was concentrated in vacuo to yield 5 as a white
1
powder (8.77 g, 95.3 %) which was used directly in SPPS without further purification. H-
NMR (300 MHz, d-DMSO): δ 8.55 (d, J= 6.0 Hz, 1H), 7.88-7.85 (m, 3H), 7.69 (d, J= 6.0 Hz,
2
5
2
H), 7.47 (d, J= 3.0 Hz, 1H), 7.41-7.36 (m, 2H), 7.32-7.29 (m, 2H), 5.17-5.14 (m, 1H), 5.09-
.06 (m, 1H), 4.91-4.78 (m, 1H), 4.27-4.24 (m, 1H), 4.23-4.15 (m, 5H), 3.94-3.85 (m, 3H),
1
3
.66-2.62 (m, 1H), 1.97-1.94 (m,7H), 1.88 (s, 3H), 1.70 (s, 2H). C-NMR (300 MHz, d-
DMSO): δ 174.82, 171.90, 171.67, 171.37, 171.19, 157.71, 145.67, 142.56, 129.51, 128.96,
1
2
27.13, 121.99, 79.97, 75.25, 74.17, 70.28, 67.58, 65.72, 54.00, 51.87, 48.48, 38.73, 24.46,
+
2.40, 22.38, 22.25. ESI-MS m/z calcd for C H N O 683.23; found [M+Na] 706.54, [M-
3
3
37
3
13
-
H] 682.39.
4
.3.4. (S)-3-azido-2-((tert-butoxycarbonyl)amino)propanoic acid (10)
1
6

ACCEPTED MANUSCRIPT
To a solution of NaN (9.75 g, 154.00 mmol) in DCM/water (2:1, 75.00 mL) was added
3
Tf O (5.00 mL, 31.00 mmol) dropwise over 15 min at 0 ºC. The reaction mixture was stirred
2
vigorously at 0 ºC for 2 hours. The organic phase was separated, washed with saturated
Na CO (3×100.00 mL) and used without further purification. To a mixture of commercially
2
3
·
available Boc-Dap-OH (5, 3.18 g, 15.6 mmol), K CO (4.30 g, 31.20 mmol) and CuSO 5H O
2
3
4
2
(
25.00 mg, 0.10 mmol) in water/MeOH (2:1, 135.00 mL) was added freshly prepared TfN3
solution dropwise at 0 ºC, then more MeOH was added to homogeneity. The mixture was
allowed to warm to room temperature and stirred overnight. The DCM and MeOH was
removed under vacuum and the aqueous solution was acidified by 10 % HCl to pH=2. After
dilution with EtOAc (100.00 mL), the organic layer was separated, washed with brine
(
(
3×100.00 mL), dried over Na SO , concentrated and purified by column chromatography
2 4
1
80:1-20:1, DCM/MeOH) to give 10 as a colorless oil (2.62 g, 73.1 %). H-NMR (300 MHz,
1
3
d-DMSO): δ 7.24 (d, J= 6.0 Hz, 1H), 4.14 (s, 1H), 3.58 (m, 2H), 1.39 (s, 9H). C-NMR (300
MHz, d-DMSO): δ 171.32, 155.37, 78.53, 53.51, 50.94, 28.20. ESI-MS m/z calcd for
-
C H N O 230.10; found [M-H] 229.24.
8
14
4
4
4
.3.5. allyl(S)-2-(((allyloxy)carbonyl)amino)-3-azidopropanoate (11)
0 (3.00 g, 13.00 mmol) was dissolved in 4 M HCl/1,4-dioxane (30.00 mL) at 0 ºC. The
1
reaction mixture was stirred for 3 hours at room temperature. Then the resulting mixture was
filtrated and the solid was washed with EtOAc twice. After dried over vacuum, the solid was
used directly in the next step. To a solution of the resulting amino acid hydrochloride and
sodium carbonate (2.75 g, 26.00 mmol) in water/acetonitrile (2:1, 75.00 mL) was added allyl
chloroformate (1.37 mL, 13.00 mmol) dropwise at 0 ºC. The reaction was allowed to warm to
room temperature and stirred under Ar for 18 hours. The solvents were removed and the
residue was diluted with dimethylformamide (DMF, 20.00 mL). Then sodium bicarbonate
(
1.09 g, 13.00 mmol) and allyl bromide (1.12 mL, 13.00 mmol) were added. The
heterogeneous mixture was stirred for 48 hours at room temperature [additional allyl bromide
1
7

ACCEPTED MANUSCRIPT
(
0.56 mL, 6.50 mmol) was added at 22 hour]. The reaction was concentrated, diluted with
EtOAc (100.00 mL), washed with saturated NaHCO , 0.1 M KHSO , and brine. The organic
3
4
layer was dried over Na SO , filtered, concentrated and purified by column chromatography
2
4
1
(
50:1-10:1, petro ether/EtOAc) to give 11 as a yellow oil (2.43 g, 73.6 %, 3 steps). H-NMR
(
300 MHz, CDCl ): δ 5.94 (m, 2H), 5.62 (d, J= 6.0 Hz, 1H), 5.32 (m, 4H), 4.65 (d, J= 6.0 Hz,
3
1
3
2
H), 4.57 (m, 3H), 3.79 (d, J= 3.0 Hz, 2H). C-NMR (300 MHz, CDCl ): δ 169.15, 155.53,
3
1
32.33, 131.06, 119.45, 118.08, 66.71, 66.11, 53.95, 52.63. ESI-MS m/z calcd for
+
C H N O 254.10; found [M+H] 255.26.
10
14
4
4
4
.3.6. tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pent-4-ynoate (12)
To a solution of commercial available H-Pra-OH (5.00 g, 44.24 mmol) in water/1,4-
dioxane (1:1, 500.00 mL) was added NaHCO (18.58 g, 221.20 mmol) and FmocOSu (74.61
3
g, 221.20 mmol) successively at 0 ºC. Then the mixture was stirred overnight at room
temperature. After the 1,4-dioxane was removed under vacuum, the aqueous phase was
acidified with 1 M HCl to pH=1-2 and extracted with EtOAc (2×100.00 mL). The organic
layer was washed with brine (3×100.00 mL), dried over Na SO and concentrated to provide
2
4
crude Fmoc-Pra-OH which was used directly without further purification. To a solution of the
crude Fmoc-Pra-OH, tert-Butanol (8.10 ml, 88.48 mmol) and DMAP (1.08 g, 8.85 mmol) in
DCM (500.00 mL) was added DCC (10.03 g, 48.66 mmol, solved in 500.00 mL DCM)
dropwise at 0 ºC. The mixture was stirred for 1 hour at 0 ºC and 4 hours at room temperature.
Then, the reaction was filtrated and the filtrates were washed successively with 1 M HCl
(
3×100.00 mL), saturated NaHCO (3×100.00 mL) and brine (3×100.00 mL). The organic
3
phase was dried over Na SO , filtered, concentrated and purified by column chromatography
2
4
1
(
50:1-10:1, petro ether/EtOAc) to give 12 as a white powder (12.11 g, 70.3 %, 2 steps). H-
NMR (300 MHz, CDCl ): δ 7.79 (m, 2H), 7.44 (m, 2H), 7.38 (m, 2H), 7.32 (m, 2H), 5.70 (d,
3
1
3
J= 6.0 Hz, 1H), 4.40 (m, 3H), 4.25 (m, 1H), 2.79 (s, 2H), 1.53 (s, 9H). C-NMR (300 MHz,
CDCl ): δ 169.31, 155.61, 143.87, 143.80, 141.30, 127.73, 127.08, 125.17, 120.00, 78.56,
3
1
8

ACCEPTED MANUSCRIPT
8
2.88, 71.54, 67.20, 52.65, 47.14, 27.98, 22.99. ESI-MS m/z calcd for C H NO 391.17;
2
4
25
4
+
found [M+H] 392.35 .
4
.3.7. allyl (S)-3-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)- 3-
oxopropyl)-1H-1,2,3-triazol-1-yl)-2-(((allyloxy)carbonyl)amino)propanoate (13)
To a solution of 11 (100.00 mg, 0.255 mmol), 12 (64.77 mg, 0.255 mmol) and CuI (72.20
mg, 0.38 mmol) in dry DMF (1.40 mL) was added DIPEA (0.44 mL, 2.55 mmol) under Ar
atmosphere. Protected from light, the reaction mixture was stirred for 14 hours at room
temperature. The resulting mixture is diluted with EtOAc (100.00 mL) and water (100.00 mL).
The organic phases are filtered, washed with brine, dried over Na SO , filtered, concentrated
2
4
and purified by column chromatography (10:1-2:1, petro ether/EtOAc) to give 13 as a white
1
powder (138.00 mg, 84.1 %). H-NMR (300 MHz, CDCl ): δ 7.76 (d, J= 6.0 Hz, 2H), 7.61
3
(
d, J= 6.0 Hz, 2H), 7.40 (t, J= 3.0 Hz, 2H), 7.32 (m, 3H), 5.87 (m, 2H), 5.75 (d, J= 6.0 Hz,
H), 5.66 (d, J= 6.0 Hz, 1H), 5.30 (m, 3H), 5.28 (m, 1H), 4.84 (m, 3H), 4.76 (m, 2H), 4.66 (d,
1
1
3
J= 6.0 Hz, 3H), 4.37 (d, J= 6.0 Hz, 2H), 4.23 (m, 1H), 3.24 (m, 2H), 1.45 (s, 9H). C-NMR
300 MHz, CDCl ): δ 170.12, 168.43, 155.90, 155.65, 143.92, 143.84, 143.03, 141.31,
(
3
1
6
32.22, 131.06, 127.72, 127.11, 125.21, 123.40, 123.27, 119.98, 119.65, 118.17, 82.64, 67.01,
6.22, 54.15, 53.84, 50.95, 50.81, 47.16, 29.71, 28.58, 27.98. ESI-MS m/z calcd for
C H N O 645.27; found [M+Na]+ 668.51.
34
39
5
8
4
.3.8. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-((S)-3-oxo-2-(((tert-pentyloxy)
carbonyl)amino)-3-(((E)-prop-1-en-1-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)propanoic acid
14)
(
1
3 (138.00 mg, 0.21 mmol) was dissolved in TFA/DCM (1:1, 5.00 mL) and stirred for 2
hours at room temperature. The reaction mixture was concentrated in vacuo to yield 14 as a
white powder (120.00 mg, 95.1 %) which was used directly in SPPS without further
1
9

ACCEPTED MANUSCRIPT
1
purification. H-NMR (300 MHz, CD OD): δ 7.78 (d, J= 6.0 Hz, 2H), 7.74 (m, 1H), 7.63 (m,
3
2
5
2
1
1
H), 7.38 (t, J= 6.0 Hz, 2H), 7.29 (t, J= 6.0 Hz, 2H), 5.88 (m, 2H), 5.32 (d, J= 6.0 Hz, 1H),
.24 (m, 2H), 5.13 (m, 1H), 4.83 (m, 3H), 4.72 (m, 2H), 4.64 (m, 2H), 4.48 (m, 3H), 4.32 (m,
1
3
H), 4.20 (m, 1H), 3.29 (m, 1H). C-NMR (300 MHz, CD OD): δ 174.42, 174.32, 170.28,
3
58.45, 158.06, 145.26, 144.81, 144.49, 142.61, 134.08, 133.03, 128.84, 128.22, 126.33,
25.40, 120.97, 119.12, 117.78, 68.10, 67.50, 66.86, 55.61, 55.27, 55.11, 51.50, 28.82. ESI-
-
MS m/z calcd for C H N O 589.21; found [M-H] 588.19.
3
0
31
5
8
4
.3.9. General procedures for the Fmoc solid phase peptide synthesis
The amino acid residues were attached to the Rink amide resin (loading capacity= 0.33
mmol/g) with a single coupling procedure. All peptides were synthesized with a scale of 0.10
mmol.
(
(
(
a) Standard pre-activation of resin protocol: The resin was swollen in DCM/DMF mixture
solvent for 10 min.
b) Standard Fmoc-deprotection protocol: After treatment with 20 % piperidine/DMF (15
min twice) the resin was washed with DMF (5×), DCM (5×), and DMF (5×).
c) Standard coupling of natural amino acids protocol: After pre-activation of 4.00 equiv of
Fmoc-protected amino acid in DMF for 5 min using 3.80 equiv of HCTU and 8.00 equiv
of DIPEA, the solution was added to the resin. After 30 min, the resin was washed with
DMF (5×), DCM (5×), and DMF (5×). The coupling reaction was monitored with the
ninhydrin test.
(
d) Standard coupling of glycoamino acid or diaminodiacid protocol: After pre-activation of
1
2
.50 equiv of glycoamino acid or diaminodiacid building block in DMF for 15 min using
.00 equiv of HATU, 2.00 equiv of HOAt and 8.00 equiv of DIPEA, the solution was
added to the resin. After 2 hours, the resin was washed with DMF (5×), DCM (5×), and
DMF (5×). The coupling reaction was monitored with the ninhydrin test.
(
e) Standard capping protocol: Ac O/DIPEA/DMF (1:1:8) was added to the resin. After
2
2
0

ACCEPTED MANUSCRIPT
mechanically stirring for 15 min, the resin was washed with DMF (5×) and DCM (10×)
f) Standard deprotection of acetyl group protocol: To the peptide resin was added a solution
of 10 % hydrazine hydrate/DMF solution (10 mL). The resin was stirred for 12 hours.
Then, the resin was washed with DCM (5×) and DMF (5×).
(
(
g) Standard deprotection of Alloc/allyl protocol: To the peptide resin was added a solution
of 24.00 equiv of PhSiH in 2.00 mL DCM in the presence of argon and the resin was
3
manually stirred for 2 min. Subsequently, a solution of 0.25 equiv of Pd (PPh ) in 6.00
3
4
mL DCM was added. The reaction was mechanically stirred for 3 hours under argon.
Then, the resin was washed with DMF (5×), DCM (5×) and DMF (5×).
(
(
h) Standard cyclization protocol: After removal of Alloc/allyl and N-terminus Fmoc
successively, a solution of 5.00 equiv of PyAOP, 5.00 equiv of HOAt and 10.00 equiv of
NMM in NMP was added to the resin. After overnight reaction, the resin was washed
with DMF (5×), DCM (5×) and DMF (5×).
i) Standard cleavage protocol: The cleavage cocktail (TFA/TIPs/EDT/water= 95: 2: 2: 1,
v/v/v/v) was added to the resin. After stirring for 2 hours, the cleavage cocktail was
collected. The solution was bubbled with argon for concentration and the chilled diethyl
ether was added to precipitate the crude peptides. The peptide suspensions were
centrifuged for 3 min at 3000 rpm and then the clear solution was decanted. The step of
precipitation, centrifugation and decantation operations was repeated three times. The
resulting white residues were dissolved in CH CN/water, analyzed and purified by RP-
3
HPLC.
(
j) Standard oxidative folding protocol: Peptide in the reduced form was dissolved in the
oxidation buffer [0.50 mg/mL peptide in 6.00 M guanidine hydrochloride and 100.00 mM
sodium dihydrogen phosphate PBS buffer, pH=7.40, with 10.00 % dimethylsulfoxide
(
DMSO)]. This mixture was allowed to stir for 24 hours at room temperature. Then it was
2
1

ACCEPTED MANUSCRIPT
analyzed and purified by RP-HPLC.
(
k) Standard deprotection of 4-methoxybenzyl (Mob) group and oxidative folding protocol:
Peptide with Mob protection was dissolved in TFA/DMSO solution (1:1, v/v). This
mixture was allowed to stir for 20 min at room temperature. Then it was analyzed and
purified by RP-HPLC.
4
.4. Molecular docking
All the molecular modeling calculations were performed using SYBYL version 6.9.
Structures of compounds were assigned with Gasteiger–Huckel partial atomic charges.
Energy minimization was performed using the Tripos force field, Powell optimization
method, and MAXIMIN 2 minimizer, with a convergence criterion of 0.001 kcal/mol Å.
Simulated annealing was then performed. The system was heated to 1000 K for 1.0 ps and
then annealed to 250 K for 1.5 ps. The annealing function was exponential; 50 such cycles of
annealing were run and the resulting 50 conformers were optimized using methods described
above. The lowest energy conformation was selected. All the other parameters were default
values
4
.5.
Kinetic binding studies by surface plasmon resonance
Binding experiments were carried out on BiaCORE T200 (BiaCORE, Uppsala, Sweden)
instruments at 25 ℃. Recombinant sRANKL were immobilized to the dextran gydrogel on
the sensor surface (BiaCORE CM5 sensor chip) with a surface density of 3500 resonance
units. The surface regeneration was carried out among binding cycles using Gly-HCl buffer
(
pH=2.5). The apparent rate constants (k and k ) and the equilibrium binding constant (K )
on off d
for the binding interaction were estimated from the kinetic analysis of sensorgrams, using the
BIA evaluation software (BiaCORE).
4
.6. CD spectroscopy study
2
2

ACCEPTED MANUSCRIPT
CD measurements were recorded on a JASCO J-820 spectropolarimeter (JASCO Corp.,
Ltd). Peptides were dissolved in 50.0 % TFE aqueous solution with a concentration of 0.1
mg/mL. UV spectra were recorded at 20 ℃ in a quartz cell of 10.0 mm path length.
4
.7. Immunoblotting study
5
RAW264.7 cells (1×10 /well) were cultured in 6-well plates for 12 hours, treated with or
without the peptides for 2 hours, and stimulated with sRANKL at 300 ng/mL for the indicated
periods. Cells were then washed with PBS buffer and lysed with lysis buffer. Cell lysates (20
µg) were separated by SDS-PAGE, electroblotted onto nitrocellulose membranes and probed
with anti-phospho-IκBα, anti-IκBα, anti-ERK2 antibodies (Abcam, UK). The membranes
were then measured by the enhanced chemiluminescence (ECL) system.
4
.8. Effect on sRANKL-induced osteoclase formation in vitro
Murine osteoclast precursors from 8-week-old C57BL/6 male mice were cultured in 96
5
well plates (1×10 /mL, 200 µL/well) for 3 days co-cultured with 30 ng/mL murine CSF-1 and
3
00 ng/mL sRANKL with or without treatment of designed peptides at the indicated
concentration. Then, the fixed cells were stained using tartrate-resistant acid phosphatase kit
TRAP kit, Sigma). TRAP-positive multinucleated cells (with more than three nuclei) were
observed and counted by a microscope (OLYMPUS-BX53).
.9. CCK-8 assay
The CCK-8 assay was performed according to the manufacturer’s instructions. The CCK-8
(
4
reagent (Sigma) was dissolved with PBS solution in a concentration of 5 mg/mL. BMMCs
5
were cultured on a 96-well plate (1 × 10 /mL, 100 µL/well) for 24 hours incubation. Cells
were then cultured with various concentrations of OM-1, OM-2, OM-3 and OM-4 (0, 12.5,
2
5.0, 50.0, 100.0 µM) for 48 hours. The CCK-8 solution was added at 10 µL per well and
incubated for 2 hours. Absorbance was measured at 450 nm using ELISA plate reader.
4
.10. In vivo experiments
All experiments were performed in the SPF laboratory of Changhai Hospital, Shanghai.
Female 6-week-old C57BL/6 mice were supplied by Slack (Shanghai, China). All animal
2
3

ACCEPTED MANUSCRIPT
procedures were in accordance with the standards of the Ethics Committee at Second Military
Medical University. We divided the mice into three groups of six mice each: a control group;
ovariectomized mice treated with saline (OVX group); and OVX mice treated with OM-2
dissolved in saline (OVX+OM-2 group). The treated mice were injected intraperitoneally (i.p.)
with 20.0 mg/kg of OM-2 or vehicle every day. After 6 weeks of intervention, all mice were
anesthetized with chloral hydrate. Samples of femur bone and blood were obtained. Blood
o
was centrifuged with 3000 r for 5 minutes to give the supernatant, which was stored at -80 C.
Finally, the mice were euthanized.
4
.11. Bone histomorphometry analysis
Femur bones were fixed with 4 % paraformaldehyde for 4 days, and then decalcified for 2
weeks using 10% tetrasodium-EDTA. Paraffin-embedded sections (4 µm) from each femur
were processed for histologic observation of the metaphysis below the spongiosa by
hematoxylin and eosin (H&E) staining, and TRAP staining. Histologic measurements and
images were observed by a microscope (OLYMPUS-BX53). Trabecular bone was shown in
the H&E-stained sections, and its area was measured by Image-Pro Plus software. The
numbers of osteoclasts in the region of the metaphysis were counted within the sections by
TRAP staining.
4
.12. Bone structure analysis
The femur bone structure was analyzed by micro-computed tomography (Micro CT)
(
Huaiyu biological technology limited company, Shanghai, China). Structural parameters for
the metaphyseal region and the trabecular bone were analyzed using the built-in software of
the Micro CT. The evaluated trabecular parameters were total bone mineral density (BMD),
bone volume expressed per unit total volume (BV/TV), trabecular number (Tb. N) and bone
surface area expressed per unit total volume (BS/TV).The built-in software was used to
reconstruct two-dimensional and three-dimensional bone structure images.
4
.13. Serum biochemistry
2
4

ACCEPTED MANUSCRIPT
Serum was collected by the above methods. Serum levels of IL-6, TRAPcp5B, CTX-1 and
TNF-α were measured using ELISA kits (Anogen, Canada), according to the manufacturer’s
instructions.
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 21402235,
3
1370958 and 81461148033), PLA Youth Medical Science and Technology Youth
Development Program (No.16QNP086), and Instrumental Analysis Center of our university
for NMR spectroscopic and mass spectrometric analyses.
Conflict of Interest: The authors have no conflicts of interest to disclose.
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