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(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-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 5~40 mg kg-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 µCT 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-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.

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(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(Darby, 1981). Estrogen withdrawal leads to increased osteoclast production, enhanced bone resorption, and substantial bone loss(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 0~40 mg kg-1 NVP-BHG712 was not toxic to mice in vivo. Our results suggest that NVP-BHG712 at 0~40 mg kg-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.

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Figure legends.