Use of ano5 gene as a target for osteoporosis treatment

Abstract

This invention relates to the biomedical field and discloses the application of the Ano5 gene as a therapeutic target for osteoporosis. This invention utilizes a previously constructed... Year 5 Gene knockout mouse model, construction Year 5 ‑ / ‑ The +OVX mouse model was used to clearly demonstrate the effects of inhibiting Ano5 on osteoporosis at the imaging, histological, biomechanical, serological, and molecular levels. Further research is needed on the inhibition... Year 5 The effects and molecular mechanisms of osteoporosis development, and using osteoclast ferroptosis as a starting point, reveal... Year 5 The mechanism by which gene defects inhibit bone resorption by promoting ferroptosis provides a theoretical basis for treating osteoporosis by targeting the chloride channel protein ANO5. This will contribute to the development of new targets for chloride channels in the treatment of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of the Ano5 gene as a therapeutic target for osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a systemic skeletal disease characterized by reduced bone mass and destruction of bone microstructure, leading to increased bone fragility and a significantly increased risk of fracture. It is a complex systemic metabolic disorder that can be primary or secondary. Primary osteoporosis includes age-related osteoporosis and postmenopausal osteoporosis, affecting more than 200 million people worldwide, with about one-third of women and one-fifth of men over the age of 50 having the disease [1]. With the aging of the global population, the incidence of osteoporosis has increased significantly, posing a serious public health challenge. The basic pathogenesis of OP involves an imbalance in bone remodeling, i.e., bone resorption exceeds bone formation, leading to a decrease in bone density and strength. In addition to basic treatment drugs such as calcium and vitamin D, the current treatment strategy for osteoporosis mainly includes: anti-bone resorption drugs: bisphosphonates and RANKL inhibitor denosumab [2, 3], and bone formation-promoting drugs: parathyroid hormone (PTH) analog teriparatide [4]. However, the above drugs have adverse reactions such as decreased bone density and liver and kidney damage after discontinuation [5, 6].

[0003] Recent studies have found that chloride channels, including members of the voltage-gated chloride channels (CLC) family and the cystic fibrosis transmembrane conductance regulator (CFTR), are closely related to bone remodeling [7, 8]. Chloride channels are mainly responsible for the transmembrane transport of chloride ions and play a key role in maintaining intracellular osmotic pressure, ion balance, and signal transduction. By maintaining the chloride ion concentration gradient between the intracellular and extracellular spaces, chloride channels indirectly affect intracellular pH, calcium concentration, cAMP levels, and signal transduction, thereby regulating the proliferation, differentiation, and apoptosis signals of osteoblasts and osteoclasts, playing an important role in bone remodeling.

[0004] Anoctamin5 (ANO5) is a member of the ANO transmembrane protein family, all of which possess 10 transmembrane regions. As calcium-dependent chloride ion channels, they are primarily involved in various biological processes, including bone remodeling. ANO1 activates calcium... 2+ / NFATc1 and RANKL / RANK signaling promote osteoclast differentiation, while inhibiting ANO1's ion transport function has a significant rescue effect on osteoporotic bone loss [9, 10]. ANO6 promotes osteoblast differentiation and matrix mineralization by regulating calcium ion signaling, while inhibiting ANO6 leads to bone loss

[11] . ANO5 is highly expressed in muscle and bone, and its subcellular location is in the cell membrane and endoplasmic reticulum. Its mutation can lead to the rare genetic disease jaw diaphysis dysplasia (GDD). The team previously constructed a... Ano5 Gene knockout mouse models successfully replicated the phenotypes of jaw enlargement, tibial curvature, and increased bone fragility observed in GDD patients, further suggesting that Ano5 deficiency can lead to enhanced bone formation. A deeper understanding of the relationship between the chloride ion channel ANO5 and the development of osteoporosis will not only help elucidate the pathogenesis of bone metabolic diseases but may also provide new insights into developing novel treatment strategies for bone diseases.

[0005] Currently, first-line osteoporosis medications mainly include bisphosphonates, calcitonin, parathyroid hormone analogs, RANKL inhibitors, and estrogens. Although these drugs are effective in increasing bone density and reducing fracture risk, they still have drawbacks such as safety risks (e.g., osteonecrosis of the jaw, fracture, cancer risk), poor administration convenience, low compliance, and limited efficacy. Further in-depth research into the molecular mechanisms regulating bone homeostasis may provide us with new perspectives for treating osteoporosis.

[0006] Although there have been preliminary studies on the relationship between chloride ion channels and bone remodeling, the focus has been mainly on ANO1 and CLC family members. Ano5 The gene defect replicated the clinical phenotype of patients with jawbone dysplasia in both in vivo and in vitro, characterized by abnormally increased bone mass, thickened cortical bone, and increased bone fragility. However, the relationship between ANO5 and the development of osteoporosis and the mechanism by which it regulates bone homeostasis remains unclear. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the Ano5 gene as a therapeutic target for osteoporosis.

[0008] In order to achieve the objectives of this invention, in a first aspect, this invention provides the application of the Ano5 gene as a drug target for the prevention and / or treatment of osteoporosis.

[0009] The Ano5 gene is located on human chromosome 11p14.3-15.1 and encodes the chloride ion channel protein ANO5, which belongs to the anoctamin protein family (formerly named TMEM16 family). The reference sequence number of the human Ano5 gene in NCBI is NCBI ID: 233246 (NM_177694.6).

[0010] Secondly, the present invention provides the use of an inhibitor of the Ano5 gene in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

[0011] Furthermore, the inhibitor prevents and / or treats osteoporosis by inhibiting osteoclast activity and / or promoting osteoblast activity.

[0012] Furthermore, the inhibitor suppresses osteoclast activity by inducing ferroptosis in osteoclasts.

[0013] Furthermore, the osteoporosis mentioned is postmenopausal osteoporosis or senile osteoporosis.

[0014] Furthermore, the inhibitor is a substance that specifically inhibits the transcription or translation of the Ano5 gene, or can specifically inhibit the expression or activity of the ANO5 protein.

[0015] The inhibitor may be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, etc.

[0016] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of the Ano5 gene and a pharmaceutically acceptable carrier or excipient; said pharmaceutical composition is used for the prevention and / or treatment of osteoporosis.

[0017] Fourthly, the present invention provides a method for screening candidate drugs for the prevention and / or treatment of osteoporosis, comprising the following steps: (a) Construct a system expressing the Ano5 gene; (b) Contact the system with the candidate material; (c) Detect the effect of the candidate substance on the expression level of the Ano5 gene or the activity of the ANO5 protein at the transcriptional or translational level; (d) Screen for substances that can inhibit the expression level of the Ano5 gene or the activity of the ANO5 protein.

[0018] Furthermore, the system is a cell expressing the Ano5 gene.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention utilizes previously constructed Ano5 Gene knockout mouse model, construction Ano5 - / - The +OVX mouse model was used to clearly demonstrate the effects of inhibiting Ano5 on osteoporosis at the imaging, histological, biomechanical, serological, and molecular levels. Further research is needed on the inhibition... Ano5The effects and molecular mechanisms of osteoporosis development, and using osteoclast ferroptosis as a starting point, reveal... Ano5 The mechanism by which gene defects inhibit bone resorption by promoting ferroptosis provides a theoretical basis for treating osteoporosis by targeting the chloride channel protein ANO5. This will contribute to the development of new targets for chloride channels in the treatment of osteoporosis. Attached Figure Description

[0020] Figure 1 In a preferred embodiment of the present invention Ano5 The effect of gene knockout on serum estradiol levels. .

[0021] Figure 2 Knockout in a preferred embodiment of the present invention Ano5 Effects on bone phenotype in osteoporotic mice. AB: μCT was used to observe the microstructure of the femur (A) and tibia (B) in mice, and to quantitatively analyze the bone mineralization density and bone mineral content of the cortex; C: μCT was used to detect the cortical bone thickness of the femur and tibia in mice; D: HE staining and quantitative analysis of cortical bone thickness. .

[0022] Figure 3 Knockout in a preferred embodiment of the present invention Ano5 Effects on the biomechanical properties of bone tissue in osteoporotic mice. The tibial fragility was observed using a three-point bending stress test, and displacement-load curves were plotted to analyze the failure load, failure stress, fracture load, fracture stress, and elastic modulus. .

[0023] Figure 4 Knockout in a preferred embodiment of the present invention Ano5 Effects on osteogenic activity in osteoporotic mice. A: ELISA was used to detect ALP levels in mouse serum; B: qRT-PCR was used to detect ALP levels in mouse femoral tissue. Ocn and Col1α1 The expression. .

[0024] Figure 5 Knockout in a preferred embodiment of the present invention Ano5 Effects on osteoclast activity in osteoporotic mice. A: ELISA was used to detect the level of β-CTx in mouse serum; B: TRAP staining was used to observe the number of TRAP-positive mature osteoclasts in the femur and to quantify them; C: Western blot was used to detect the protein expression of NFATC1, cFOS, and CTSK in tibial tissue; D: qRT-PCR was used to detect the expression of β-CTx in mouse femur tissue. Nfatc1 and Ctsk The expression. .

[0025] Figure 6 In a preferred embodiment of the present invention, the effect of inhibiting Ano5 on osteoclast ferroptosis levels was described. A: Flow cytometry was used to detect the PI levels of un-RANKL-induced BMMs and mature osteoclasts (OCs) on day 5 of RANKL induction. + A: Cell proportion and quantitative analysis; B: Flow cytometry was used to detect the effects of Fer-1, Z-VAD-FMK, Nec-1, CQ, BCS and 2-ME small molecule reagents on... Ano5 Knockout group osteoclast PI + Effects on cells; C: Biochemical detection of MDA levels in un-RANKL-induced BMMs and mature osteoclasts (OCs) on day 5 of RANKL induction; D: TRAP staining to observe the number of TRAP-positive mature osteoclasts (≥3 nuclei) on day 5 of RANKL induction. . Detailed Implementation

[0026] This invention provides a novel target and biomarker for molecular targeted therapy of osteoporosis.

[0027] This invention is based on the previously constructed Ano5 Gene knockout mouse models (based on the CRISPR-Cas9 system, with sgRNA targeting exons 11 and 12 of the Ano5 gene; the DNA sequences of the sgRNA targeting sites are 5'-GCAAGCCAGATAACATCCCAGG-3' and 5'-GCCTAGTGTGCACACATCCCTGG-3'; 5'-CCTGGGATGTTATCTGGCTTGC-3' and 5'-CCAGGGATGTGTGCACACTAGGC, precisely knocking out...) Ano5 The gene contains 833 bases; the method for constructing a mouse model can be found in CN109694881A. Further construction was then carried out... Ano5 - / - The OVX model, through imaging, biomechanical, histological, serological, and molecular studies, clearly demonstrated that inhibiting the chloride ion channel Ano5 effectively prevents osteoporotic bone loss and reduces fracture risk. Furthermore, it revealed that the inhibitory effect of Ano5 knockout on osteoclast function depends on its regulation of ferroptosis.

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] Example 1 Construction Ano5 - / - OVX mouse model Twelve 8-week-old wild-type mice were selected. Ano5 + / + Female C57BL / 6 mice, weighing 19.5 ± 0.5 g, were randomly divided into groups. Ano5 + / + Sham group (sham surgery group) and Ano5 + / + The OVX group (ovarian removal group) included 12 eight-week-old females of the same age and weight. Ano5 Gene knockout type ( Ano5 - / - C57BL / 6 female mice ( Ano5 The method for constructing gene knockout mouse models can be found in CN109694881A), and the mice are randomly divided into groups. Ano5 - / - sham group and Ano5 - / - OVX group. Six mice per group were anesthetized, confirmed by the loss of the pinch reflex. After anesthesia, the mice were fixed in a prone position, and the fur on their backs was shaved and disinfected with iodine. A longitudinal incision (0.5 cm in length) was made 1 cm on each side of the spine, and the muscle layer was bluntly dissected to locate the fat pad surrounding the ovary. In the OVX group, the fallopian tube-uterus junction was ligated, and the ovary was completely removed. In the sham group, only the abdominal fat surrounding the ovary was removed. Mice were fed routinely in individual cages. Mice reached puberty 4 weeks post-surgery, and were sacrificed at 12 weeks. Tibial and femoral bones were collected. Whole blood was also collected using ocular blood sampling, and serum was extracted after standing at 4°C for 1 hour and centrifuging at 3000 rpm for 5 minutes. ELISA testing according to the kit instructions revealed a significant decrease in serum estradiol levels in the OVX group compared to the sham group. Figure 1 The system indicated that modeling was successful.

[0030] Example 2 Knockout Ano5 Preventing bone loss in OVX mice Collected tibiae and femurs were fixed in paraformaldehyde for 24-48 hours. Micro-CT was used to observe the microstructure and morphology of the bone tissue. Mid-shafts of long bones were selected for cortical bone observation. Regardless of... Ano5 - / - The BMD and BMC of the femoral cortex in the sham group mice were higher than those in the sham group mice. Ano5 + / + The sham group; importantly, Ano5 - / - The bone mineral density (BMD) and bone vascular mass (BMC) of the femoral cortex in the OVX group mice were significantly higher than those in the OVX group. Ano5 + / + OVX group ( Figure 2 A). The BMD and BMC of the tibial tissue show the same trend of change ( Figure 2 B). We measured the cortical thickness of the tibia and femur and found... Ano5- / - OVX mice have a cortical bone thickness (Ct.Th) greater than Ano5 + / + OVX group ( Figure 2 C). In addition, mouse femurs were collected, fixed with paraformaldehyde for 24-48 hours, decalcified with 10% EDTA until needle insertion was unimpeded, dehydrated, embedded, and 5μm paraffin sections were prepared for HE staining. Ano5 - / - OVX mice had significantly higher femoral Ct.Th levels than wild-type OVX mice. Figure 2 D). The above results indicate that inhibiting Ano5 can prevent bone loss in osteoporosis.

[0031] Example 3: Inhibiting Ano5 enhances the biomechanical properties of bone tissue in OVX mice Tibial tissue was collected and preserved in saline. Its biomechanical properties were assessed using a three-point bending stress test. The tibia was gently fixed to a lower support with a span of 1 cm, and its position and orientation were adjusted so that the stress point was near the midpoint of the tibia. The displacement speed was adjusted to mm / min, and the force was slowly applied automatically until the bone fractured. Displacement-load curves were collected, and data on elastic modulus, maximum load, and fracture load were obtained. The diameter of the tibial fracture ends was measured using calipers, with three measurements taken and the average value. The maximum stress and fracture stress were calculated using the formula S=F×L / [D^3×3.1416 / 32] (S: stress, F: load, D: diameter). We found that ovariectomy significantly reduced the fracture resistance of wild-type mice, while... Ano5 - / - The elastic modulus, fracture stress, fracture load, maximum stress, and maximum load of the tibia in OVX mice were all significantly higher than those in OVX mice. Ano5 + / + OVX mice ( Figure 3 This indicates that inhibiting Ano5 can effectively reduce the risk of fractures caused by osteoporosis.

[0032] Example 4: Inhibition of Ano5 enhances osteogenic activity in OVX mice Mouse serum was collected using the same method described above, and the level of alkaline phosphatase (ALP), an early marker of osteogenic differentiation, was detected by ELISA. It was found that ovariectomy led to a decrease in ALP levels, but... Ano5 + / + Compared to the OVX group Ano5 - / - Serum ALP levels were significantly elevated in OVX mice, suggesting enhanced osteogenic activity in vivo. Figure 4 A). In addition, femoral tissue was collected, and RNA was extracted using the Trizol method after grinding with liquid nitrogen. qRT-PCR detection revealed… Ano5- / - OVX is a component of bone markers. Ocn (Gene ID: 12096, NM_007541.3) and Col1a1 The expression of (Gene ID: 12842, NM_007742.4) was significantly higher than that of other genes. Ano5 + / + OVX group ( Figure 4 B), indicating knockout Ano5 It has a significant enhancing effect on bone formation activity in OVX mice.

[0033] Example 5 Knockout Ano5 Inhibits osteoclast function in OVX mice ELISA was used to detect serum levels of β-carboxy-terminal cross-linked telopeptide of type I collagen (β-CTx), a marker of bone resorption. Results showed that ovariectomy led to an increase in β-CTx levels, but compared to... Ano5 - / - Serum β-CTx levels in OVX mice were significantly lower than those in mice with OVX mice. Ano5 + / + OVX group ( Figure 5 A). Mouse femoral tissue was collected, routinely decalcified, dehydrated, embedded, sectioned, and then stained with TRAP. The nuclei were stained with hematoxylin. + Multinucleated cells (≥3 nuclei) are mature osteoclasts. Quantitative analysis results show that, compared with... Ano5 + / + Compared to the OVX group Ano5 - / - The number of mature osteoclasts in OVX mice was significantly reduced. Figure 5 B). Molecular level detection revealed Ano5 - / - The expression of osteoclast-related molecules Nfatc1 (Gene ID: 18018, NM_001164111.1), cFos (Gene ID: 14281, NM_010234.3), and Ctsk (Gene ID: 13038, NM_007802.4) in the bone tissue of OVX mice was lower than that of... Ano5 + / + OVX group ( Figure 5 C), further indicating knockout Ano5 It can prevent the development of osteoporosis by inhibiting osteoclast activity.

[0034] Example 6 Knockout Ano5 Inhibits osteoclast activity by promoting ferroptosis. To further reveal the knockout Ano5To investigate the mechanism of osteoclast inhibition, we used flow cytometry. Ano5 + / + and Ano5 - / - Apoptosis levels in mouse-derived BMMs (bone marrow macrophages) and mature osteoclasts induced by RNAKL for 5 days were detected, revealing that knockout... Ano5 This led to a significant increase in the proportion of cell death. Figure 6 A). The addition of apoptosis inhibitor Z-VAD-FMK, necroptosis inhibitor Nec-1, autophagy inhibitor CQ, copper death inhibitor BCS, and disulfide death inhibitor 2-ME failed to inhibit [the growth of the plant]. Ano5 - / - Osteoclast death occurred, but after adding 1 μM of the ferroptosis inhibitor Fer-1, Ano5 - / - Osteoclast death decreased significantly later, suggesting that the cell death mechanism was ferroptosis. Figure 6 B). Among them, the apoptosis inhibitor Z-VAD-FMK (HY-16658B), the necroptosis inhibitor Nec-1 (HY-15760), the autophagy inhibitor CQ (HY-17589A), the copper death inhibitor BCS (HY-W034953), the disulfide death inhibitor 2-ME (HY-12033), and the ferroptosis inhibitor Fer-1 (HY-100579) were all purchased from MCE (MedChemExpress).

[0035] Meanwhile, the levels of malondialdehyde (MDA), a biochemical marker of ferroptosis, were detected using a biochemical reagent kit, revealing that knockout... Ano5 This leads to a significant increase in osteoclast MDA levels ( Figure 6 C). Importantly, TRAP staining results showed that the addition of the ferroptosis inhibitor Fer-1 effectively rescued ferroptosis. Ano5 - / - Cell differentiation and maturation capacity ( Figure 6 D). The above data shows that knocking out Ano5 It inhibits the differentiation and maturation of osteoclasts by promoting ferroptosis.

[0036] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0037] References: [1] Sözen T, Özışık L, Başaran N. An overview and management ofosteoporosis. European journal of rheumatology .4, 46-56 (2017). [2] Kavanagh KL, Guo K, Dunford JE, Wu X, Knapp S, Ebetino FH, et al.The molecular mechanism of nitrogen-containing bisphosphonates asantiosteoporosis drugs. Proceedings of the National Academy of Sciences of the United States of America .103, 7829-7834 (2006). [3] Rinotas V, Liepouri F, Ouzouni MD, Chalkidi N, Papaneophytou C,Lampropoulou M, et al. Structure-Based Discovery of Receptor Activator ofNuclear Factor-κB Ligand (RANKL)-Induced OsteoclastogenesisInhibitors. International journal of molecular sciences .24, (2023). [4] Martin TJ, Sims NA, Seeman E. Physiological and PharmacologicalRoles of PTH and PTHrP in Bone Using Their Shared Receptor, PTH1R. Endocrine reviews .42, 383-406 (2021). [5] Dömötör ZR, Vörhendi N, Hanák L, Hegyi P, Kiss S, Csiki E, et al.Oral Treatment With Bisphosphonates of Osteoporosis Does Not Increase theRisk of Severe Gastrointestinal Side Effects: A Meta-Analysis of RandomizedControlled Trials. Frontiers in endocrinology .11, 573976 (2020). [6] Dito G, Lugaresi M, Degradi C, Guabello G, Longhi M, Corbetta S.Efficacy of switching from teriparatide to zoledronic acid or denosumab onbone mineral density and biochemical markers of bone turnover in olderpatients with severe osteoporosis: a real-life study. Endocrine .82, 181-189(2023). [7] Zifarelli G. The Role of the Lysosomal Cl(-) / H(+) Antiporter ClC-7 in Osteopetrosis and Neurodegeneration. Cells .11, (2022). [8] Dumortier C, Frauenpreis A, Hoarau A, Ryan AL, Gangloff SC,Danopoulos S, et al. CFTR mutation is associated with bone differentiationabnormalities in cystic fibrosis. J Cyst Fibros . (2025). [9] Sun W, Guo S, Li Y, Li J, Liu C, Chen Y, et al. Anoctamin 1controls bone resorption by coupling Cl(-) channel activation with RANKL-RANKsignaling transduction. Nat Commun .13, 2899 (2022).

[10] Li S, Sun W, Li S, Zhu L, Guo S, He J, et al. Tamsulosinameliorates bone loss by inhibiting the release of Cl(-) through wedging intoan allosteric site of TMEM16A. Proc Natl Acad Sci USA .122, e2407493121(2025).

[11] Ousingsawat J, Wanitchakool P, Schreiber R, Wuelling M, VortkampA, Kunzelmann K. Anoctamin-6 controls bone mineralization by activating thecalcium transporter NCX1. J Biol Chem .290, 6270-6280 (2015).

Claims

1. Application of the Ano5 gene as a drug target for the prevention and / or treatment of osteoporosis.

2. The use of Ano5 gene inhibitors in the preparation of drugs for the prevention and / or treatment of osteoporosis.

3. Use according to claim 2, characterized in that, The inhibitors prevent and / or treat osteoporosis by inhibiting osteoclast activity and / or promoting osteoblast activity.

4. Use according to claim 3, characterized in that, The inhibitor suppresses osteoclast activity by inducing ferroptosis in osteoclasts.

5. Use according to any one of claims 2 to 4, characterized in that, The inhibitor is a substance that specifically inhibits the transcription or translation of the Ano5 gene, or that can specifically inhibit the expression or activity of the ANO5 protein.

6. Use according to claim 5, characterized in that, The inhibitor is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies.

7. The use according to any one of claims 1 to 6, characterized in that, The osteoporosis mentioned refers to postmenopausal osteoporosis or senile osteoporosis.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises a therapeutically effective amount of an inhibitor of the Ano5 gene and a pharmaceutically acceptable carrier or excipient; the pharmaceutical composition is used for the prevention and / or treatment of osteoporosis.

9. A method for screening a candidate drug for prevention and / or treatment of osteoporosis, characterized by, Includes the following steps: (a) Construct a system expressing the Ano5 gene; (b) Contact the system with the candidate material; (c) Detect the effect of the candidate substance on the expression level of the Ano5 gene or the activity of the ANO5 protein at the transcriptional or translational level; (d) Screen for substances that can inhibit the expression level of the Ano5 gene or the activity of the ANO5 protein.

10. The method of claim 9, wherein, The system is a cell expressing the Ano5 gene.

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