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\begin{document}

\title{NVP-BHG712 against postmenopausal osteoporosis in mice by targeting
Cathepsin K}



\author[1]{Yueran Zhao}%
\author[1]{Xin Liu}%
\author[1]{Shuang Liu}%
\author[1]{Huanxin Sun}%
\author[1]{Jing Zhang}%
\author[1]{Ming Li}%
\author[1]{Wendi Chen}%
\author[1]{Yuxuan Shi}%
\author[1]{Yongzhi Cao}%
\author[2]{Gang  Lu}%
\author[1]{Yunna Ning}%
\affil[1]{Shandong University}%
\affil[2]{The Chinese University of Hong Kong School of Biomedical Sciences}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose Recent evidence suggests that postmenopausal
osteoporosis is associated with increased bone resorption function in
osteoclasts, of which Cathepsin K is a key molecule. In this study, we
aimed to screen small molecule compounds targeting Cathepsin K and
evaluate whether they affect osteoclast bone resorption function.
Experimental Approach We screened out the small-molecule compound
NVP-BHG712 targeting CTSK by molecular docking, and studied its
pharmacological effect on bone resorption function of osteoclasts. To
this end, we evaluated bone mass changes in postmenopausal mice by \selectlanguage{greek}μ\selectlanguage{english}CT,
ELISA, and H\&E staining. In addition, we also investigated the effects
of NVP-BHG712 on osteoclast differentiation, bone resorption function
and expression of osteoclast differentiation related factors in vitro.
Key Results Surprisingly, we found that oral NVP-BHG712 treatment
significantly reduced bone loss in postmenopausal mice. In vitro
osteoclast culture, it was found that this effect was achieved by
inhibiting osteoclast differentiation and bone resorption. Meanwhile,
NVP-BHG712 significantly decreased the expression of genes related to
osteoclast differentiation, including CTSK, MMP9, CTR, IP3R1, IP3R3, and
OC-STAMP. Conclusion and Implications The present findings suggest that
NVP-BHG712 reduces bone resorption function by inhibiting osteoclast
differentiation, and is a potential drug for preventing and treating
postmenopausal osteoporosis and other diseases.%
\end{abstract}%



\sloppy


\section*{NVP-BHG712 against postmenopausal osteoporosis in mice by
targeting Cathepsin
K}

{\label{nvp-bhg712-against-postmenopausal-osteoporosis-in-mice-by-targeting-cathepsin-k}}

\textbf{Running title}

NVP-BHG712 against osteoporosis

\textbf{Authors}

Xin Liu\textsuperscript{1,2,3}, Shuang Liu\textsuperscript{1,2,3},
Huanxin Sun\textsuperscript{1,2,3}, Jing Zhang\textsuperscript{1,2,3},
Ming Li\textsuperscript{1,2,3}, Wendi Chen\textsuperscript{1,2,3},
Yuxuan Shi\textsuperscript{1,2,3}, Yongzhi Cao\textsuperscript{1,2,3},
Gang Lu\textsuperscript{4}, Yunna Ning\textsuperscript{1,2,3,*}, Yueran
Zhao\textsuperscript{1,2,3,*}

\textsuperscript{1}Center for~Reproductive Medicine, Shandong
University, Jinan, Shandong, 250012, China

\textsuperscript{2}Key laboratory~of~Reproductive Endocrinology of
Ministry of Education, Shandong University, Jinan, Shandong, 250012,
China

\textsuperscript{3}National Research Center for Assisted Reproductive
Technology and Reproductive Genetics, Shandong University, Jinan,
Shandong, 250012, China

\textsuperscript{4}CUHK-SDU Joint Laboratory on Reproductive Genetics,
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong
Kong SAR, China

\textsuperscript{*}Address correspondence to: Yunna Ning, Dr, Center for
Reproductive Medicine, Shandong University, Jinan, Shandong, 250012,
China. Email: yunna\_ning@163.com

Yueran Zhao, PhD, Center for Reproductive Medicine, Shandong University,
Jinan, Shandong, 250012, China. Email: \emph{yrzhao@sdu.edu.cn}

\textbf{Word count}

4737.

\textbf{Acknowledgements}

Authors received funding for this work from The National Key Research
and Development Program of China (No. 2017YFC1001002). All of these
authors have no disclosures in relations to this manuscript. We
acknowledge the Center for Reproductive Medicine for supporting the
experiment.

The research reported here was not pre-registered in an independent,
institutional registry.

\textbf{Data Availability}

All data presented in this article will be shared upon request.

\textbf{Conflict of interest statement}

The authors declare that they do not have any conflict of interest.

\textbf{Abstract}

\textbf{Background and Purpose}

Recent evidence suggests that postmenopausal osteoporosis is associated
with increased bone resorption function in osteoclasts, of which
Cathepsin K is a key molecule. In this study, we aimed to screen small
molecule compounds targeting Cathepsin K and evaluate whether they
affect osteoclast bone resorption function.

\textbf{Experimental Approach}

We screened out the small-molecule compound NVP-BHG712 targeting CTSK by
molecular docking, and studied its pharmacological effect on bone
resorption function of osteoclasts. To this end, we evaluated bone mass
changes in postmenopausal mice by \selectlanguage{greek}μ\selectlanguage{english}CT, ELISA, and H\&E staining. In
addition, we also investigated the effects of NVP-BHG712 on osteoclast
differentiation, bone resorption function and expression of osteoclast
differentiation related factors in vitro.

\textbf{Key Results}

Surprisingly, we found that oral NVP-BHG712 treatment significantly
reduced bone loss in postmenopausal mice. In vitro osteoclast culture,
it was found that this effect was achieved by inhibiting osteoclast
differentiation and bone resorption. Meanwhile, NVP-BHG712 significantly
decreased the expression of genes related to osteoclast differentiation,
including CTSK, MMP9, CTR, IP3R1, IP3R3, and OC-STAMP.

\textbf{Conclusion and Implications}

The present findings suggest that NVP-BHG712 reduces bone resorption
function by inhibiting osteoclast differentiation, and is a potential
drug for preventing and treating postmenopausal osteoporosis and other
diseases.

\textbf{Keywords}

Osteoporosis; Osteoclast differentiation; Cathepsin K; NVP-BHG712

\subsection*{Introduction}

{\label{introduction}}

The dynamic balance between bone formation and resorption is necessary
to maintain bone homeostasis\textsuperscript{(Chen, Wang, et al., 2018);
Matsuoka, Park, Ito, Ikeda, and Takeshita (2014); (Zaidi, 2007)}. Due to
internal and external factors, the disturbance of this dynamic balance
leads to an increase in bone resorption activity and the occurrence of
compensatory bone diseases, such as postmenopausal osteoporosis (PMPO)
and rheumatoid arthritis (RA)\textsuperscript{(Eastell et al., 2016)}.
Therefore, inhibition of osteoclast resorption activity may be an
effective method for treating pathological bone loss in these
diseases\textsuperscript{(Chen, Zhi, Cao, et al., 2017; Chen, Zhi, Pan,
et al., 2017; Chen, Zhi, et al., 2018; Zhi et al., 2018)}. Osteoclasts
are multinucleated giant cells that differentiate from mononuclear
macrophages; osteoclasts are located on the surface of bone tissue and
perform bone resorption functions by synthesizing and secreting a
variety of organic acids and proteases to dissolve bone salts and
degrade organic matter\textsuperscript{(Boissy, Saltel, Bouniol, Jurdic,
\& Machuca-Gayet, 2002)}. In the process of osteoclast differentiation,
they are stimulated by two essential cytokines, namely, macrophage
colony-stimulating factor (M-CSF) and receptor activator of nuclear
factor-\selectlanguage{greek}κ\selectlanguage{english}B ligand (RANKL)\textsuperscript{(Fumoto, Takeshita, Ito, \&
Ikeda, 2014; Park, Lee, \& Lee, 2017)}. The binding of the cytokine
M-CSF to the receptor c-Fms maintains the survival and proliferation as
well as the differentiation of BMMs into osteoclast precursor cells. The
cytokine RANKL binds to the receptor RANK to promote the differentiation
of osteoclast precursor cells into mature
osteoclasts\textsuperscript{(Fumoto et al., 2014)}, and this interaction
induces osteoclast differentiation and the expression of
function-related genes, including CTSK, MMP9 and CTR.

CTSK is an important osteoclast enzyme that degrades type I collagen,
and CTSK inhibitors have been used in the treatment of osteoporosis for
many years\textsuperscript{(Ho et al., 2009; Hou et al., 1999; Reiser,
Adair, \& Reinheckel, 2010; Schilling et al., 2007)}. Odanacatib, which
is a drug that inhibits CTSK function in osteoclasts, has been abandoned
by the Desert East Company because it increases the occurrence of
cardiovascular events\textsuperscript{(McClung et al., 2019; Mullard,
2016; Saag et al., 2021)}. A large amount of evidence has shown that
CTSK inhibitors can effectively inhibit bone resorption by
osteoclasts\textsuperscript{(Lotinun et al., 2013; Mukherjee \&
Chattopadhyay, 2016)}. Therefore, the identification of potential CTSK
inhibitors will facilitate the development of treatments for bone
metabolic diseases.

NVP-BHG712, which is as a specific EphB4 inhibitor, has attracted much
attention because of its effects in inhibiting tumor
metastasis\textsuperscript{(Becerikli et al., 2015; Li et al., 2021;
Troster et al., 2018)}. Angiogenesis plays a key role in bone formation
and osteoporosis\selectlanguage{polish}\textsuperscript{(Filipowska, Tomaszewski, Niedźwiedzki,
Walocha, \& Niedźwiedzki, 2017; Kusumbe, Ramasamy, \& Adams, 2014;
Ramasamy, Kusumbe, Wang, \& Adams, 2014)}\selectlanguage{english}. A recent study reported that
NVP-BHG712 inhibited angiogenesis\textsuperscript{(You et al., 2017)}.
However, the role of NVP-BHG712 in osteoclast formation remains unclear.
In our study, the results suggested that NVP-BHG712 may act as a potent
inhibitor of osteoclast formation to improve bone loss in ovariectomized
mice.

\subsection*{Methods}

{\label{methods}}

\textbf{Molecular docking} . To screen CTSK-targeting compounds, we
downloaded crystal conformations from the Protein Data Bank database and
screened CTSK-targeting compounds from more than 9,000 Selleckchem
compounds. The binding pocket of CTSK (Fig. 1A) to which the compounds
bind was identified according to the resolution, and the conformation of
CTSK while bound to the known ligands 5TDI, 4DMY, 3KWZ, and 4X6H was
selected according to the binding strength. According to the physical
characteristics of the compounds, the compound NVP-BHG712, which binds
to the CTSK molecule, was selected.

\textbf{Animals} . All animals care, ethical principles and experimental
procedures were approved by the Cheeloo College of Medicine of Shandong
University (Shandong, China). Animal studies are reported in compliance
with the ARRIVE guidelines and with the recommendations made by
the\emph{British Journal of Pharmacology} . C57BL/6JN mice (6-8 weeks of
age) were purchased from Beijing Vital River Laboratory Animal
Technology Co., Ltd. (Beijing, China). To evaluate the effect of
NVP-BHG712 on bone mass in OVX females, OVX females underwent
ovariectomy at 8 weeks of age. The mice were randomly divided into 6
groups (5 mice in each group): the Sham group, OVX group, and 5 mg
kg\textsuperscript{-1}, 10 mg kg\textsuperscript{-1}, 20 mg
kg\textsuperscript{-1}, and 40 mg kg\textsuperscript{-1} NVP-BHG712
groups. In the Sham group, the ovaries of the mice were only exposed to
the surrounding adipose tissue, while in the OVX group and different
NVP-BHG712 groups, both ovaries were removed from the mice. After one
week of postoperative recovery, the Sham and OVX groups were treated
with 0.5\% CMC-Na, and the NVP-BHG712 group was treated with NVP-BHG712
dissolved in 0.5\% CMC-Na. After 6 weeks, bilateral femurs and blood
were collected from the mice for analyses.

\textbf{Cell culture} . The RAW264.7 cell line was purchased from Zhong
Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China, RRID:
CVCL\_0493) and authenticated by STR profiling. The cells were cultured
in DMEM medium (Gibco, MA, USA) supplemented with 10\% fetal bovine
serum (Gibco) and 1\% penicillin-streptomycin (Solarbio, Beijing, China)
at 37\selectlanguage{ngerman}°C in a humidified atmosphere with 5\% CO\textsubscript{2}.

BMMs were obtained as described previously\textsuperscript{(Gooi, Chia,
Vrahnas, \& Sims, 2019; Maridas, Rendina-Ruedy, Le, \& Rosen, 2018;
Stern et al., 2012)} and were cultured in \selectlanguage{greek}α-\selectlanguage{english}MEM (Gibco, MA, USA)
supplemented with 10\% fetal bovine serum (Gibco) and 1\%
penicillin-streptomycin (Solarbio, Beijing, China) at 37\selectlanguage{ngerman}°C in a
humidified atmosphere with 5\% CO\textsubscript{2}.

\textbf{CCK-8} . According to the reagent standard, BMMs were incubated
in 96 well plates (1x10\textsuperscript{4} cells per well) for 24 hours.
BMMs were cultured in induction medium containing M-CSF (30 ng/ml) (R\&D
systems, Mn, USA) and RANKL (50 ng/ml) (R\&D systems). Then, different
concentrations of NVP-BHG712 (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, or 3.2
\selectlanguage{greek}μ\selectlanguage{english}M) were added and incubated for 72 hours. Then, CCK-8 solution
(Beyotime Biotechnology, Shanghai, China) was added to each well and
incubated for another 2 h before the absorbance was measured.

\textbf{TRAP staining} . TRAP staining for the detection of mature
osteoclasts was performed using a tartrate resistant acid phosphatase
kit (Servicebio) according to the manufacturer's instructions. The
number of TRAP+ cells per mm\textsuperscript{2} was counted.

\textbf{F-actin ring staining} . BMMs were fixed in 4\% paraformaldehyde
and stained with F-actin (Beyotime Biotechnology). DAPI was used to
stain the nuclei. Images were acquired using light and fluorescence
microscopes.

\textbf{Bone resorption} . BMMs were seeded on bone plates (Corning,
NYC, USA) and treated with M-CSF, RANKL and NVP-BHG712. The cells were
removed from the bone plates on day 7. Images were acquired using light
and fluorescence microscopes.

\textbf{Western blotting} . BMMs were harvested and subjected to Western
blotting assays, which were performed as described
previously\textsuperscript{(Hu et al., 2019; Sun et al., 2019)}, and the
following antibodies were used for immunoblotting: anti-CTSK (Abcam,
ab19027, RRID: AB\_2261274), anti-MMP9 (Abcam, ab76003, RRID:
AB\_1310463), anti-CTR (Abcam, ab11042, RRID: AB\_297696), anti-\selectlanguage{greek}β-\selectlanguage{english}actin
(Proteintech, 66009-1-Ig, RRID: AB\_2687938), anti-rabbit IgG (Cell
Signaling Technology (CST), \#7074, RRID: AB\_2099233), and anti-mouse
IgG (CST, \#7076, RRID:AB\_330924).

\textbf{RNA isolation and qPCR} . BMMs were harvested, and total RNA was
isolated using a RNeasy kit (Spark Jade). An aliquot of 1 \selectlanguage{greek}μ\selectlanguage{english}g of total
RNA was subjected to reverse transcription with a SPARK script II
reverse transcription PCR kit (Spark Jade) according to the
manufacturer's instructions. Quantitative PCR was performed using SYBR
Green Master Mix (Roche). mRNA expression levels were calculated by the
method using GAPDH as an internal control and further normalized to the
mean expression level of the control group. The primers were as follows:
mouse-MMP9: forward, 5'-CGTCGTGATCCCCACTTACT-3', and reverse,
5'-AACACACAGGGTTTGCCTTC-3'; mouse-CTR: forward, 5'-CCTGCAGA
TGCCCAGTGAAG-3', and reverse, 5'-TGTGTAGTAGCCCTGCTCCC-3'; mouse-IP3R1:
forward, 5'-AACTGTGGGACCTTCACCAG-3', and reverse,
5'-AACTCTCGCCAGTTTCTGG-3'; mouse-IP3R2: forward,
5'-GTTACAGGATGTCGTGGCCT-3', and reverse, 5'-ATTCGCCGTAATGTGCTACC-3';
mouse-IP3R3: forward, 5'-CAATGAGCACCACGAGAAGA-3', and reverse,
5'-AACTTGACAGGGGTCACCAG-3'; mouse-NFATc1: forward,
5'-GGGTCAGTGTGACCGAAGAT-3', and reverse, 5'-GGAAGTCAGAAGTGGGTGGA-3';
mouse-OC-STAMP: forward, 5'-ATGAGGACCATCAGGGCAGCCACG-3', and reverse,
5'-GGAGAAGCTGGGTCAGTAGTTCGT-3'; mouse-DC-STAMP: forward,
5'-AAAACCCTTGGGCTGTTCTT-3', and reverse, 5'-GGCTGCTTTGATCGTTTCTC-3';
mouse-Atp6v1c1: forward, 5'-CACGTTGGCTGTCTTGGTTG-3', and reverse,
5'-CTTCGCGTTAGCAAACTTCC-3'; mouse-\selectlanguage{greek}α\selectlanguage{english}v-integrin: forward,
5'-ACAAGCTCACTCCCATCACC-3', and reverse, 5'-ATATGAGCCTGCCGACTGAC-3';
mouse-GAPDH: forward, 5'-TCAAGAAGGTGGTGAAGCAG-3', and reverse,
5'-AGTGGGAGTTGCTGTTGAAGT-3'.

\textbf{ELISA and microenzyme labeling method} . Mouse serum was
collected, and the tartrate-resistant acid phosphatase 5b (TRACP-5b)
level was measured with a TRACP-5b ELISA Kit (Elabscience, Wuhan,
China). Alkaline phosphatase (ALP) activity was measured with an ALP
assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

\textbf{Microcomputed tomography} . Bone density and bone volume of the
right femur were measured using a Micro-CT SkyScan 1176 (Bruker,
Germany). Briefly, samples were fixed in 4\% paraformaldehyde and
incubated in 70\% ethanol. Scanning was performed using a voxel size of
9 mm, X-ray tube potential of 58 kV and X-ray intensity of 431 \selectlanguage{greek}μ\selectlanguage{english}A. For
specimen scanning, the volumes of interest were evaluated using CT
Analyzer software. Representative 3D images created using NRecon
software.

\textbf{H\&E staining} . Tissues were collected, cleaned to remove
excess tissue, fixed in 4\% paraformaldehyde and demineralized in 10\%
EDTA for 10--14 days before being embedded in paraffin wax. Sections
were cut to 5 \selectlanguage{greek}μ\selectlanguage{english}m thicknesses using a Leica RM2165 and subsequently
stained with H\&E or TRAP.

\textbf{Statistical Design} . SPSS version 26.0 (IL, USA) and GraphPad
Prism 9 (CA, USA) were used for statistical analysis. Two-tailed
unpaired Student's t tests were used for comparisons of two groups, and
one-way analysis of variance (ANOVA) with Tukey's multiple comparisons
test was used for comparisons of more than two groups. P values less
than 0.05 were considered statistically significant.

\subsection*{Results}

{\label{results}}

\textbf{3.1 NVP-BHG712 is a small molecule compound that targets CTSK.}

We used a novel strategy to identify potential active small molecule
compounds that target CTSK, and we ultimately identified NVP-BHG712 as a
potential bioactive compound from the Selleckchem compound library.

As mentioned above in the Methods, the molecular docking method was used
to search for small molecule compounds that target CTSK. First, we
downloaded crystal conformations of human CTSK from the PDB database.
According to the binding of the CTSK active pocket with known ligands,
the ligands 7AS and OLC bind to CTSK in an L-shape, with part in the
pocket and part extending upward, and the binding of the ligands KWZ and
I37 to CTSK occurs in both upward and downward conformations (Fig. 1A,
B). Analysis showed that I37 had the weakest binding strength among the
four ligands because the molecule was small and the interaction with
CTSK was weak, but its binding strength could reach an order of nM.
Therefore, we selected these four molecular conformations of CTSK for
molecular docking to search for new small molecule compounds that bind
to CTSK. According to the results of molecular docking analysis with
molDockTools software, a small molecule compound was identified to bind
to the four conformations of CTSK (Fig. 1C), and the compound was
NVP-BHG712 (Fig. 1D). Moreover, through a literature review, we did not
find any reports of an effect of NVP-BHG712 on CTSK and osteoclast
formation. Therefore, we next focused on the effects of NVP-BHG712 on
CTSK and osteoclast formation.

\textbf{3.2 NVP-BHG712 inhibited RANKL-induced osteoclastogenesis and
bone resorption in vitro.}

To investigate the cytotoxicity of NVP-BHG712, we observed the effects
of NVP-BHG712 at 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 \selectlanguage{greek}μ\selectlanguage{english}M on the
proliferation of BMMs and RAW264.7 cells. After 72 hours of treatment
with 0\textasciitilde{}0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712, the ratio of BMMs and RAW264.7
cells was 138\% and 154\%, respectively. When the concentration was 0.8
to 1.6 \selectlanguage{greek}μ\selectlanguage{english}M, NVP-BHG712 promoted cell proliferation. However, when the
concentration was increased to 3.2 \selectlanguage{greek}μ\selectlanguage{english}m, NVP-BHG712 significantly
inhibited the proliferation of both cell lines and caused obvious
cytotoxicity. The results indicated that there was no significant
cytotoxicity when the concentration of NVP-BHG712 was lower than 1.6 \selectlanguage{greek}μ\selectlanguage{english}M
(Fig. 2A). To further assess the effects of NVP-BHG712 on
osteoclastogenesis, a RANKL-induced osteoclastogenesis assay was carried
out in BMMs that were exposed to various concentrations of NVP-BHG712
(0, 0.1, 0.2, 0.4, 0.8, and 1.6 \selectlanguage{greek}µ\selectlanguage{english}M). The numbers of TRAP-positive
multinucleated cells were counted to assess RANKL-induced osteoclast
differentiation. As the results showed, the number of TRAP-positive
multinucleated cells was increased when treated with NVP-BHG712 at
0\textasciitilde{}0.2 \selectlanguage{greek}µ\selectlanguage{english}M and dramatically decreased when treated with
NVP-BHG712 at 0.4\textasciitilde{}1.6 \selectlanguage{greek}µ\selectlanguage{english}M (Fig. 2B, C).

Furthermore, a pit-formation assay was carried out to determine whether
osteoclast functions were impaired. Bone resorption pits on bone slices
are the direct result of bone resorption and are also the most reliable
indicator of bone resorption function in vitro. BMMs exposed to RANKL
were treated with different concentrations of NVP-BHG712 (0, 0.1, 0.2,
or 0.4 \selectlanguage{greek}μ\selectlanguage{english}M), and then, the area of bone lacunae absorption was observed.
The results showed that bone resorption activity was attenuated by
NVP-BHG712 (Fig. 2D, E).

\textbf{3.3 NVP-BHG712 inhibited osteoclastogenesis and F-actin ring
formation in mature osteoclasts at the early phase in vitro.}

To further identify at which specific phase NVP-BHG712 exerts its
inhibitory effect on osteoclastogenesis, RANKL-treated BMMs were
incubated with NVP-BHG712 (0.4 \selectlanguage{greek}μ\selectlanguage{english}M) at different time points and for
different durations (days 1-3, days 3-5, days 5-6, and days 1-6). TRAP
staining illustrated that osteoclast differentiation was more strongly
inhibited by NVP-BHG712 treatments in the first several days than in the
later days, suggesting that NVP-BHG712 mainly suppressed osteoclast
differentiation in the early phase (Fig. 3A, B). F-actin rings are
considered the characteristic structure of mature osteoclasts. To
further determine the inhibitory effect of NVP-BHG712 on osteoclast
function, immunofluorescence staining of the F-actin ring was performed
in BMMs that were exposed to NVP-BHG712 treatments. Similar to the prior
inhibitory effects, NVP-BHG712 more significantly inhibited the
formation of F-actin ring structures by mature osteoclasts in the first
several days than in the later days, as demonstrated by a decrease in
F-actin numbers compared to total osteoclast numbers (Fig. 3C, D). In
conclusion, the results suggest that osteoclast function is mainly
inhibited by NVP-BHG712 in the early phase.

\textbf{3.4 NVP-BHG712 inhibited the RANKL-induced expression of
osteoclast differentiation-related genes.}

CTSK is a key enzyme in the process of bone resorption by osteoclasts.
Under acidic conditions, activated CTSK participates in the degradation
of collagen type I, osteopontin and other bone matrix proteins in the
bone matrix, and it promotes the bone resorption function of
osteoclasts. To observe whether the small molecule compound NVP-BHG712,
which targets CTSK, can inhibit the protein expression of CTSK, Western
blotting was used to measure the expression of CTSK in RANKL-treated
BMMs-derived osteoclasts. The results showed that 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712
could inhibit CTSK expression in RANKL-treated BMMs-derived osteoclasts.
(Fig. 4A, B). To investigate the effect of NVP-BHG712 on osteoclast
differentiation-related genes, Western blotting was employed to measure
the proteins levels of MMP9 and CTR. The results showed that the 0.4 \selectlanguage{greek}μ\selectlanguage{english}M
concentration of NVP-BHG712 could significantly inhibit the
RANKL-induced proteins expression of MMP9 and CTR in osteoclasts (Fig.
4A, C, D). Next, we measured the mRNA expression of MMP9 and CTR, the
results showed that the mRNA expression of MMP9 and CTR was
significantly inhibited by NVP-BHG712 (Fig. 4E, F).

Next, we measured the RANKL-induced protein expression of CTSK in
BMMs-derived osteoclasts after adding 0, 0.1, 0.2, and 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712
to determine the effect of the different concentrations of NVP-BHG712 on
the protein expression of CTSK in osteoclasts. The protein expression of
CTSK in BMMs-derived osteoclasts treated with RANKL was measured after
incubation with 0.4 \selectlanguage{greek}μ\selectlanguage{english}M VP-BHG712 for 1, 2, 3, and 4 days to determine
the effect of different treatment times on the protein expression of
CTSK in osteoclasts. The results revealed that the protein expression of
CTSK was significantly decreased when the concentrations of 0.1, 0.2,
and 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 were added to the BMMs culture system (Fig. 4G,
H). The expression of CTSK protein was not significantly changed after
the addition of 0.4 \selectlanguage{greek}μ\selectlanguage{english}M VMP-BHG712 to the osteoclast differentiation and
maturation culture system for 1 and 2 days; however, the protein
expression of CTSK decreased significantly after 3 days and 4 days of
culture (Fig. 4I, J). The mRNA expression levels of IP3R1, IP3R2, IP3R3,
NFATc1, OC-STAMP, DC-STAMP, \selectlanguage{greek}α\selectlanguage{english}V-integrin and Atp6v1c1 were measured by
qPCR. We found that the mRNA expression of MMP9, CTR, IP3R1, IP3R3 and
OC-STAMP was significantly inhibited by NVP-BHG712 (Fig. 5A-H).
Therefore, NVP-BHG712 could inhibit the RANKL-induced expression of
osteoclast differentiation-related genes in BMMs.

\textbf{3.5 NVP-BHG712 did not affect the expression of osteoclast
differentiation-related receptors.}

To further investigate whether NVP-BHG712 affected the expression of
receptors related to osteoclast differentiation and thus affected the
downstream signal in the cells, we observed the expression of the c-Fms
receptor and RANK receptor in BMMs-derived osteoclasts by
immunofluorescence assay. The immunofluorescence of the c-Fms receptor
did not change significantly after treatment of osteoclasts with
NVP-BHG712 at a concentration of 0.4 \selectlanguage{greek}μ\selectlanguage{english}M (Fig. 5I, J). In addition, the
immunofluorescence results showed that NVP-BHG712 had no significant
effect on the expression of the RANK receptor, which is related to
osteoclast differentiation (Fig. 5K, L).

\textbf{3.6 NVP-BHG712 attenuated bone loss in ovariectomized mice.}

The decrease in estrogen during postmenopausal osteoporosis leads to an
increase in osteoclast activity, bone resorption rates become greater
than bone formation rates, and the bone turnover rate increases, leading
to bone loss related to high turnover. Ovariectomized female mice are
widely used as a postmenopausal osteoporosis model in
research\textsuperscript{(C. Y. Chen et al., 2019; K. Chen et al.,
2019)}. Thus, we established OVX models in 8-week-old female mice and
tested whether the intragastric administration of NVP-BHG712 every 3
days for five weeks could attenuate bone mass loss in these OVX female
mice. (Fig. 6A). The effect of NVP-BHG712 on bone mass in ovariectomized
mice was confirmed by measuring the serum TRACP-5b levels by ELISA and
the serum ALP levels with a microplate reader. The results of TRACP-5b
measurement showed that the serum TRACP-5b levels in the mice in the OVX
group were significantly increased compared with those in the Sham group
(Fig. 6B), which indicated that the model of postmenopausal osteoporosis
was successfully established. Similar results were observed in the
measurement of ALP activity, which was lower in the OVX group than in
Sham group (Fig. 6C). Both of these results proved the success of the
operation. After administration of NVP-BHG712 at a concentration of
10-40 mg kg\textsuperscript{-1}, the serum TRACP-5b levels were
significantly decreased in the mice. The serum ALP levels of the mice
were significantly increased after the administration of NVP-BHG712 at a
concentration of 5\textasciitilde{}40 mg kg\textsuperscript{-1}.
Together, these results suggest that gavage of NVP-BHG712 can be used in
the treatment of high turnover osteoporosis.

As shown in Fig. 5D-F, the NVP-BHG712-treated OVX mice had the lower
trabecular bone mass phenotypes than the OVX mice, as revealed by the 2D
and 3D \selectlanguage{greek}µ\selectlanguage{english}CT reconstructed images of distal femurs, the much higher BMD,
BS/TV, BV/TV, Tb.N, Tb.Th values, and the lower BS/BV, Tb. Pf, Tb.Sp
values. These results revealed that NVP-BHG712 increased the bone mass
in OVX mice. The results of HE staining and quantitative analysis of the
distal femur showed that NVP-BHG712 increased the density and number of
trabecular bones in OVX mice (Fig. 6G, H). Together, these findings
indicate that NVP-BHG712 exerts a protective effect against bone loss in
ovariectomized mice, which is related to the decreased osteoclast
resorption induced by NVP-BHG712.

After the operation, we recorded the weight of the mice before each
feeding, and the results showed that the weight of the mice in the OVX
group, different NVP-BHG712 treatment groups and Sham group increased
gradually, and no significant weight loss was observed (Fig. 7A). The
organ index of the mice was calculated as the ratio of the organ weight
to the body weight of the mice. Compared with those of the Sham group,
the heart index, spleen index, kidney index and tibia index of the OVX
group did not change significantly, and gavage with different doses of
NVP-BHG712 did not cause significant differences compared with OVX alone
(Fig. 7B-E). No differences in the heart weight/tibia length results
were observed between the different NVP-BHG712 groups and the OVX group
(Fig. 7F). Based on these results, we found that NVP-BHG712 did not
cause toxicity in ovariectomized mice when administered by gavage at a
dose of 40 mg kg\textsuperscript{-1}.

In conclusion, our findings identified a novel compound for the
prevention of postmenopausal osteoporosis (Fig. 7). With skeletal aging
and menopause, osteoclastic bone resorption activity increases
relatively or absolutely. During osteoclast bone resorption, CTSK is
released from the bone matrix into the bone marrow, where CTSK and other
cytokines promote bone resorption. NVP-BHG712 inhibits osteoclast bone
resorption by inhibiting the function of the CTSK protein, thereby
slowing the loss of bone mass that is associated with aging or
postmenopausal bone.

\subsection*{Discussion}

{\label{discussion}}

Our investigation to understand the role of NVP-BHG712 in osteoclasts
establishes that CTSK is the target of NVP-BHG712 and promotes
RANKL-induced osteoclast differentiation and maturation. This assertion
stems from the strong binding of NVP-BHG712 to each of the four
molecular conformations of CTSK and its inhibitory effect on
RANKL-induced osteoclast differentiation and maturation. When compared
with other compounds that inhibit osteoclast differentiation and
maturation, several striking features emerge in our results. In
particular, to our knowledge, the magnitude with which NVP-BHG712
inhibits osteoclast differentiation and maturation is matched by OCN.
Through a literature search, it was found that NVP-BHG712 had not been
previously reported in studies on CTSK targets and osteoclasts.
EphrinB2/EphB4 signaling pathway was activated to inhibit osteoclast
differentiation, while decreased osteoclast maturation and
differentiation was observed when NVP-BHG712, an EphB4-specific
inhibitor, was added, suggesting that EphB4 inhibition was not the
primary role of NVP-BHG712 in this process. Therefore, in this study, we
did not continue to investigate the role of Ephrin signaling pathways.
Therefore, we did not continue to investigate the role of Ephrin
signaling pathways. We observed a decrease in osteoclast maturation and
differentiation with the addition of NVP-BHG712, so we speculate that
inhibition of EphB4 to promote osteoclast maturation is not the primary
effect of NVP-BHG712 in this process. Therefore, we did not continue to
investigate the role of Ephrin signaling pathways. Moreover, we found
that in vitro osteoclast proliferation can be promoted by NVP-BHG712,
but bone resorption can be inhibited by NVP-BHG712, indicating that
NVP-BHG712 can inhibit osteoclast resorption. When we explored the time
at which NVP-BHG712 affected osteoclasts, we found that NVP-BHG712
mainly inhibited osteoclast differentiation and maturation in the early
stage. Additionally, our findings show that the formation of the F-actin
loop, which is used to demonstrate osteoclast function, is mainly
inhibited in the early stage after NVP-BHG712 treatment at different
time periods. However, NVP-BHG712 could not reverse the differentiation
and maturation of osteoclasts because mature osteoclasts were still
formed when NVP-BHG712 was added at a later stage. We hypothesize that
once CTSK-dependent osteoclast maturation is disrupted, the formation of
the bone resorption microenvironment is blocked, and bone resorption
function is lost (Fig. 6G).

CTSK plays a role in bone resorption, the immune system, tumorigenesis
and invasion, the circulatory system, the cardiovascular system and
other systems and participates in extracellular matrix remodeling in
different organs. Under acidic conditions, CTSK can bind to collagen to
degrade it, leading to increased bone fragility\textsuperscript{(Gruber,
2015; Mujawar et al., 2009)}. Furthermore, because NVP-BHG712 is a
CTSK-targeting compound that was identified by molecular docking, we
hypothesized that NVP-BHG712 inhibits the function and expression of
CTSK. Interestingly, Western blotting results showed that NVP-BHG712
inhibited the protein expression of CTSK and inhibited the protein
expression of MMP9 and CTR, which are markers related to osteoclast
differentiation. In addition, we also found an inhibitory effect of
NVP-BHG712 on IP3R1, IP3R3 and OC-STAMP mRNA expression, among which
IP3R1 and IP3R3 are related to intracellular calcium oscillation, and
OC-STAMP is related to osteoclast maturation. c-Fms and RANK receptors
play an important role in the proliferation and differentiation of
osteoclast precursor cells. M-CSF participates in the proliferation of
osteoclast precursor cells by binding to the c-Fms receptor, and RANKL
participates in the differentiation of osteoclast precursor cells by
binding to the RANK receptor. Our immunofluorescence staining results
showed that NVP-BHG712 had no inhibitory effect on the expression of
c-Fms and the RANK receptor, which indicated that the regulation of
osteoclast proliferation, differentiation and maturation by NVP-BHG712
did not occur through the M-CSF/c-Fms and RANKL/RANK signaling pathways.

The pathogenesis of PMOP involves bone remodeling caused by an imbalance
in bone resorption and bone formation\textsuperscript{(Darby, 1981)}.
Estrogen withdrawal leads to increased osteoclast production, enhanced
bone resorption, and substantial bone loss\textsuperscript{(Kameda et
al., 1997; Mundy, 2007)}. Therefore, targeting overactivated osteoclasts
provides an effective therapeutic option for PMOP. Osteoclast
hyperactivation promotes excessive bone loss in the pathophysiology of
PMOP. We further confirmed our in vitro findings by modelling the
pathological state of PMOP in an ovariectomized mouse model. Before the
start of the experiment, we measured the bone mass of 8-week-old female
mice at 3-6 weeks after ovariectomy by H\&E staining of femur sections.
It was found that the trabecular bone density of mice was significantly
reduced 5 and 6 weeks after ovariectomy. Therefore, NVP-BHG712
administration for 5 weeks after the operation can be used to explore
its effect in vivo. By measuring the serum levels of TRACP-5b and ALP,
we found that NVP-BHG712 significantly inhibited osteoclast bone
resorption activity. Using microcomputed tomography and H\&E staining of
the distal femur, we found that NVP-BHG712 significantly attenuated the
loss of bone mass in mice after ovariectomy. In conclusion, NVP-BHG712
inhibited osteoclast maturation and bone resorption in vivo and
prevented bone loss caused by ovariectomy. Additionally, it was observed
that the body weights of the mice in the Sham group, OVX group and
different NVP-BHG712 treatment groups were continuously increased and
did not decrease. The cardiac index, spleen index, kidney index, tibial
index, and heart weight to tibial length ratio were not significantly
different in OVX mice treated with different concentrations of
NVP-BHG712 (Fig. 6B-E), suggesting that 0\textasciitilde{}40 mg
kg\textsuperscript{-1} NVP-BHG712 was not toxic to mice in vivo. Our
results suggest that NVP-BHG712 at 0\textasciitilde{}40 mg
kg\textsuperscript{-1} concentration has a protective effect on bone
loss in ovaries excised mice, but the effect and toxicity of NVP-BHG712
at higher concentration need further investigation.

In summary, our work identifies a previously unknown small molecule
compound with anti-osteoporosis effects, namely, NVP-BHG712, which
inhibits osteoclast activation by inhibiting CTSK function. In the
ovariectomized female mouse model, NVP-BHG712 slowed the loss of bone
mass.Although the molecular mechanism of the effect of NVP-BHG712 on
osteoclast differentiation needs further investigation, this study may
provide new insights into the effect of NVP-BHG712 on bone metabolism in
osteoporosis.

\par\null

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\subsection*{Figure legends.}

{\label{figure-legends.}}

\textbf{Figure 1. NVP-BHG712 is a small molecule compound that targets
CTSK.}

A, Chemical formulas of the ligands KWZ, OLC, I37 and 7AS for the
molecular conformation of CTSK. B, Images of molecular conformations of
CTSK (3KWZ, 4DMY, 4X6H and 5TDI) bound to ligands (KWZ, OLC, I37 and
7AS). C, Molecular docking diagram of the small molecule compound
NVP-BHG712 with the four molecular conformations of CTSK. D, Chemical
structure of NVP-BHG712.

\textbf{Figure 2.NVP-BHG712 inhibited RANKL-induced osteoclastogenesis
and bone resorption in vitro.}

A, CCK-8 analysis of the cytotoxicity of NVP-BHG712 in RAW264.7 cells
and BMMs. Experiments were repeated 3 times. B, C Formation of
TRAP-positive cells from RANKL-treated BMMs treated with or without
different concentrations of NVP-BHG712 (0, 0.1, 0.2, 0.4, 0.8, or 1.6
\selectlanguage{greek}μ\selectlanguage{english}M) for 7 days (B) and quantification of osteoclast numbers (C). D, E
Representative images of pit formation by osteoclasts derived from
RANKL-treated BMMs with or without different concentrations of
NVP-BHG712 (0, 0.1, 0.2, or 0.4 \selectlanguage{greek}μ\selectlanguage{english}M) (D) and quantification of pit area
(E). The data are presented as the mean \selectlanguage{ngerman}± SD (n=3) (***\emph{P}
\textless{}.001)

\textbf{Figure 3.NVP-BHG712 inhibited osteoclastogenesis and F-actin
ring formation in mature osteoclasts at the early phase in vitro.}

A, B Representative images of the formation of TRAP-positive cells
treated with 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 (Group 1: without NVP-BHG712) at
different time points and four durations (Group 2-5: day 1-3, day 3-5,
day 5-6, day 1-6, respectively) (A) and quantification of osteoclast
numbers (B). C, D Representative immunofluorescence images of the
F-actin ring structures of mature osteoclasts derived from RANKL-induced
BMMs treated with 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 (Group 1: without NVP-BHG712) at
different time points and for durations (Group 2-5: day 1-3, day 3-5,
day 5-6, day 1-6, respectively) (C) and quantification of F-actin ring
formation of osteoclasts (D). Group C:M-CSF, Group R: M-CSF+RANKL, Group
N: M-CSF+RANKL+NVP-BHG712. The data are presented as the mean \selectlanguage{ngerman}± SD (n=3)
(*\emph{P} \textless{}.05, **\emph{P} \textless{}.01, ***\emph{P}
\textless{}.001).

\textbf{Figure 4.NVP-BHG712 inhibited the RANKL-induced expression of
osteoclast differentiation-related genes.} A-D, Representative Western
blots of the protein expression of CTSK, MMP9 and CTR in osteoclasts
derived from RANKL-treated BMMs treated with or without 0.4 \selectlanguage{greek}μ\selectlanguage{english}M
NVP-BHG712 (A) and the quantification of CTSK (B), MMP9 (C) and CTR (D)
expression. E-F, Representative quantification of the mRNA expression of
MMP9 (E) and CTR (F) in osteoclasts derived from RANKL-treated BMMs
treated with or without 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 by qPCR.

G, H Representative Western blots of the protein expression of CTSK in
osteoclasts derived from RANKL-treated BMMs treated with or without
different concentrations of NVP-BHG712 (0, 0.1, 0.2, or 0.4 \selectlanguage{greek}μ\selectlanguage{english}M) (G) and
the quantification of CTSK (H) expression. I, J Representative Western
blots of the protein expression of CTSK in osteoclasts derived from
RANKL-treated BMMs treated with or without 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 for
different time periods (days 1, 2, 3, and 4) (I) and the quantification
of CTSK (J) expression. Group C:M-CSF, Group R: M-CSF+RANKL, Group N:
M-CSF+RANKL+NVP-BHG712. The data are presented as the mean \selectlanguage{ngerman}± SD (n=3)
(*\emph{P} \textless{}.05, *\emph{P} \textless{}.01, ***\emph{P}
\textless{}.001).

\textbf{Figure 5. NVP-BHG712 did not affect the expression of osteoclast
differentiation-related receptors.}

A-H, Representative quantification of the mRNA expression of IP3R1 (A),
IP3R2 (B), IP3R3 (C), NFATc1 (D), OC-STAMP (E), DC-STAMP (F),
\selectlanguage{greek}α\selectlanguage{english}V-integrin (G) and Atp6v1c1 (H) in osteoclasts derived from
RANKL-treated BMMs treated with or without 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 by qPCR. I,
J Representative immunofluorescence staining images of c-Fms in
RANKL-treated BMMs treated with or without 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 (I) and the
quantification of c-Fms (J) expression. K, L Representative
immunofluorescence staining images of RANK in RANKL-treated BMMs treated
with or without 0.4 \selectlanguage{greek}μ\selectlanguage{english}M NVP-BHG712 (K) and the quantification of RANK (L)
expression. The data are presented as the mean \selectlanguage{ngerman}± SD (n=3) (*\emph{P}
\textless{}.05, *\emph{P} \textless{}.01, ***\emph{P} \textless{}.001)

\textbf{Figure6. NVP-BHG712 attenuated bone loss in ovariectomized
mice.}

C, Serum ALP (B) and TRACP-5b (C) levels in the Sham group,
ovariectomized group and ovariectomized groups treated with different
concentrations of NVP-BHG712 (5, 10, 20, or 40 mg
kg\textsuperscript{-1}) for 5 weeks. C-E, Representative microcomputed
tomography (\selectlanguage{greek}μ\selectlanguage{english}CT) 2D (C) and 3D (D) reconstruction images of distal
femurs from the Sham group, ovariectomized group and ovariectomized
groups treated with different concentrations of NVP-BHG712 (5, 10, 20,
or 40 mg kg\textsuperscript{-1}) for 5 weeks. Quantitative morphometric
properties of distal femurs showing bone mineral density (BMD), bone
volume (BV), bone surface/volume ratio (BS/BV), bone surface density
(BS/TV), fractional bone volume (BV/TV), trabecular thickness (Tb.Th),
trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular
pattern factor (Tb.Pf) (F). n=5. G, H H\&E staining images (G) and
statistical analysis (H) of mouse femur slices. Scale bars = 40 \selectlanguage{greek}μ\selectlanguage{english}m. All
the experiments were performed with five biological replicates per group
without independent repetition. The data are presented as the mean \selectlanguage{ngerman}± SD.
Statistical significance was determined by one-way ANOVA. (*\emph{P}
\textless{}.05, *\emph{P} \textless{}.01, ***\emph{P} \textless{}.001)

\textbf{Figure 7. NVP-BHG712 was not toxic in ovariectomized mice.}

A-F, Changes in the body weights of mice before each feeding (A), heart
index (B), spleen index (C), kidney index (D), forelimb grip-strength
index (E), and heart weight/tibial length (F) in the Sham group,
ovariectomized group and ovariectomized groups treated with different
concentrations of NVP-BHG712 (5, 10, 20, or 40 mg
kg\textsuperscript{-1}) for 5 weeks. G, Schematic diagram of the
mechanism by which NVP-BHG712 inhibits osteoclast function. With
skeletal aging and menopause, osteoclastic bone resorption activity
increases relatively or absolutely. During osteoclast bone resorption,
CTSK is released (purple dots) from the bone matrix into the bone
marrow, where CTSK and other cytokines promote bone resorption.
NVP-BHG712 inhibits osteoclast bone resorption by inhibiting the
function of the CTSK protein, thereby slowing the loss of bone mass
associated with aging or postmenopausal bone. All the experiments were
performed with five biological replicates per group without independent
repetition. The data are presented as the mean ± SD. Statistical
significance was determined by one-way ANOVA.\selectlanguage{english}
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