GLUL deubiquitase targeting chimera and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a GLUL deubiquitinase targeting chimera and application thereof. The invention focuses on the function of GLUL in osteogenic differentiation of BMSCs, discovers a new target for intervening in osteogenic differentiation of T2DM jaw bone BMSCs, and reveals the molecular mechanism of T2DM mediated GLUL ubiquitination modification, damage of GLUL protein homeostasis and inhibition of osteogenic differentiation of jaw bone BMSCs. The invention focuses on the ability of GLUL-DUBTAC to maintain the steady state of the GLUL protein, directly targets the GLUL ubiquitin chain, does not influence the programmed degradation of the GLUL non-ubiquitination pathway, realizes the resynthesis of the GLUL protein ligand-target protein, can complete the inverse degradation of necessary GLUL protein by using the catalyst amount of GLUL-DUBTAC, is innovatively used for enhancing the osteogenic differentiation ability of T2DM jaw bone BMSCs, and has the advantages of high stability, high efficiency, high stability and the like. The bottleneck problem that the T2DM reduces the bone combination rate in the healing stage of the implant is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to GLUL deubiquitinase-targeted chimeras and their applications. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a complex systemic metabolic disease characterized by hyperglycemia and insulin resistance, and is considered a relative contraindication for dental implantation, seriously affecting the oral health of patients. Implant osseointegration is a complex process. When an implant is implanted into the alveolar bone, a blood clot rich in a fibrous network is first formed around the implant as a matrix. Subsequently, bone marrow mesenchymal stem cells (BMSCs) derived from the jawbone microenvironment begin to accumulate on the surface of the implant and the original bone interface, and migrate, proliferate, differentiate into osteoblasts under the action of cytokines, chemokines, and chemokine receptors, secrete bone matrix, promote the formation of new bone, and cooperate with osteoclasts to jointly complete the remodeling of the bone tissue around the implant. During the implant healing period, the implant gradually achieves a stable bond with the patient's jawbone through this process. However, T2DM inhibits the osteogenic differentiation ability of BMSCs, affects the regeneration and repair of the bone tissue around the implant, interferes with implant-bone bonding during the healing period, making the time required for the dental implant to achieve effective bone bonding during the healing period longer and the effect worse.

[0003] Previous strategies mainly included implant surface modification or direct reprogramming of the implant microenvironment. However, the above methods have limited effects on improving bone integration. At the same time, focusing on endogenous treatment targeting BMSCs is not ideal, and there are potential hazards of damage to the immune system or digestive system. Antidiabetic drugs, including sitagliptin, glimepiride, and insulin, have been proven to enhance bone regeneration in T2DM, but they may exhibit adverse side effects, including liver and gastrointestinal damage.

[0004] How to reduce the failure rate of oral implants during the healing period in patients with T2DM? Currently, the main method proposed by scholars is to control blood sugar, but some studies have shown that the efficacy of blood sugar control is limited. ① Clinically, the implant stability of T2DM patients with good blood sugar control during the healing stage is still lower than that of non-diabetic patients; ② Cell experiments have shown that the proliferation, migration, adhesion, and osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) derived from the jaws of T2DM patients with good blood sugar control are lower than those of non-diabetic patients; ③ Animal experiments have shown that although insulin has been used to reduce the blood sugar level in T2DM rats, and to some extent improved the microstructure of trabecular bone around the implant, it still cannot reach the level of the control group. In addition, studies have shown that the implant stability of T2DM patients with good blood sugar control during the healing stage is still lower than that of non-diabetic patients, indicating that high glucose is not the only factor inhibiting the osteogenic differentiation ability of jaw-derived BMSCs in T2DM and reducing the success rate of implants during the healing period. Only controlling blood sugar cannot effectively rescue the inhibitory effect of T2DM on the osteogenic differentiation of BMSCs.

[0005] The above studies have shown that high glucose is not the only factor causing an increased failure rate of implant healing in T2DM. Only controlling blood sugar cannot completely reduce the failure rate of oral implant healing in T2DM patients, and more effective methods need to be found. Summary of the Invention

[0006] In view of this, the present invention designed and synthesized a GLUL deubiquitinase-targeted chimera (GLUL-DUBTAC), which connects the target protein ligand and the deubiquitinase (DUB) ligand through a linker, so as to bring the DUB near the ubiquitin-labeled protein to remove the ubiquitin chain, thereby preventing the target protein from being degraded.

[0007] In order to achieve the above invention object, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides the use of GLUL overexpression or its promoter or activator in the preparation of a drug for promoting osteogenic differentiation ability;

[0009] The GLUL is positively correlated with the osteogenic differentiation ability of T2DM patients.

[0010] In some specific embodiments of the present invention, the promoting osteogenic differentiation ability includes acting in a high-glucose environment;

[0011] Preferably, the high-glucose environment includes the in-vivo environment of T2DM patients;

[0012] Preferably, the positive correlation between the GLUL and the osteogenic differentiation ability of T2DM patients includes:

[0013] (I). Knocking out GLUL inhibits the osteogenic differentiation of BMSCs;

[0014] (II). GLUL inhibits the osteogenic differentiation of BMSCs and / or implant osseointegration in a dose-dependent manner;

[0015] More preferably, the knocking out of GLUL to inhibit the osteogenic differentiation of BMSCs includes:

[0016] (i). Knocking out GLUL reduces the ALP and ARS staining of BMSCs;

[0017] (ii). Knocking out GLUL reduces the ALP activity and calcium ion quantification of BMSCs;

[0018] (iii). Knocking out GLUL reduces the expression of osteogenic differentiation markers Runx2, ALP, and / or OCN;

[0019] More preferably, the inhibition of the osteogenic differentiation of BMSCs and / or implant osseointegration by GLUL in a dose-dependent manner includes:

[0020] MSO reduces the GLUL protein level in the per-implant bone tissue and inhibits implant osseointegration in a dose-dependent manner.

[0021] In a second aspect, the present invention also provides the use of an inhibitor of SYVN1 to mediate the ubiquitination modification of GLUL in the preparation of a drug for promoting the osteogenic differentiation of BMSCs;

[0022] SYVN1 interacts with GLUL;

[0023] SYVN1 reduces the stability of GLUL protein by mediating the ubiquitination and degradation of GLUL.

[0024] In some specific embodiments of the present invention, the interaction between SYVN1 and GLUL includes:

[0025] The mRNA level of GLUL is low and the mRNA level of SYVN1 is high in BMSCs of T2DM;

[0026] The expression of GLUL is decreased and the expression of SYVN1 is increased in BMSCs of T2DM, and with the increase of glucose concentration, the level of SYVN1 increases while the level of GLUL decreases;

[0027] If the expression level of SYVN1 is high in BMSCs of T2DM, the expression level of GLUL is low;

[0028] When the expression level of SYVN1 increases or decreases, the mRNA level of GLUL decreases or increases accordingly, and there is an obvious negative correlation between the two;

[0029] Overexpression of the SYVN1 gene was found to downregulate the protein level of GLUL;

[0030] SYVN1 has an inhibitory effect on the in vitro osteogenic differentiation of BMSCs, which is opposite to the function of GLUL;

[0031] Preferably, the protein levels of osteogenic markers RUNX2, ALP, and OCN increased after knocking down SYVN1 and decreased after overexpressing SYVN1.

[0032] In some specific embodiments of the present invention, the ubiquitination modification sites of GLUL include K259 / 334A;

[0033] The osteogenic differentiation functions under a high-glucose environment;

[0034] The ligand of GLUL includes HY-126351. The English name of HY-126351 is PROTAC ERRα ligand 2, the chemical formula is C 20 H 13 F6NO4, which has no Chinese name for the time being, is produced by MedChemExpress, CAS number: 2306388-57-6, and the structure is shown in Formula V:

[0035]

[0036] In a third aspect, the present invention also provides a GLUL deubiquitinase-targeted chimera (DUBTAC), which includes a GLUL ligand, a deubiquitinase (DUB) ligand, and a linker, and the GLUL ligand and the deubiquitinase (DUB) ligand are connected by a linker.

[0037] In some specific embodiments of the present invention, the GLUL deubiquitinase-targeted chimera (DUBTAC) brings the deubiquitinase near ubiquitinated GLUL, removes the ubiquitin chain, and prevents the target protein GLUL from being degraded.

[0038] In some specific embodiments of the present invention, the GLUL ligand includes HY-126351.

[0039] In a fourth aspect, the present invention also provides the application of the GLUL deubiquitinase-targeted chimera (DUBTAC) in maintaining the protein stability of GLUL in BMSCs of T2DM;

[0040] Preferably, maintaining the protein stability of GLUL in BMSCs of T2DM includes:

[0041] The GLUL deubiquitinase-targeted chimera maintains the endogenous protein homeostasis of GLUL;

[0042] The GLUL deubiquitinase-targeted chimera maintains the targeting ability of the GLUL protein;

[0043] The GLUL deubiquitinase-targeted chimera reduces the ubiquitin level of exogenous GLUL;

[0044] More preferably, the GLUL deubiquitinase-targeted chimera reduces the ubiquitin level of exogenous GLUL elevated by SYVN1.

[0045] In a fifth aspect, the present invention also provides the use of the described GLUL deubiquitinase-targeted chimera (DUBTAC) in the preparation of a drug for promoting osseointegration or osteogenesis of diabetic implants.

[0046] In some specific embodiments of the present invention, the promotion of osseointegration of diabetic implants includes: improving the osseointegration effect of the implanted bone in T2DM mice.

[0047] The present invention focuses on the function of GLUL in the osteogenic differentiation of BMSCs, discovers a new target for intervening in the osteogenic differentiation of T2DM jaw BMSCs, reveals the molecular mechanism by which T2DM mediates GLUL ubiquitination modification, disrupts GLUL protein homeostasis, and inhibits the osteogenic differentiation of jaw BMSCs, and has innovation in mechanism research.

[0048] The present invention focuses on the ability of "GLUL-DUBTAC" to maintain GLUL protein homeostasis, directly targets the GLUL ubiquitin chain using "GLUL-DUBTAC", and does not affect the programmed degradation of the GLUL non-ubiquitination pathway, realizes the resynthesis of the GLUL protein ligand-target protein, and a catalytic dose of "GLUL-DUBTAC" can complete the reverse degradation of the essential GLUL protein. It is innovatively used to enhance the osteogenic differentiation ability of T2DM jaw BMSCs and attempts to solve the bottleneck problem of the reduced osseointegration rate of implants during the healing period in T2DM, and has innovation in the intervention strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0050] Figure 1It is shown that knocking out GLUL inhibits the osteogenic differentiation of BMSCs; among them, a) shows that the ALP staining of the GLUL-KO group after osteogenic induction for 10 days is weaker than that of the GLUL-WT group; b) shows that the ARS staining of the GLUL-KO group after osteogenic induction for 14 days is weaker than that of the GLUL-WT group; c) shows that the ALP quantification of the GLUL-KO group is lower than that of the GLUL-WT group, and the difference is statistically significant (n = 6); d) shows that the calcium ion quantification of the GLUL-KO group is lower than that of the GLUL-WT group, and the difference is statistically significant (n = 6); e) Western results show that after knocking out GLUL, the protein expression levels of GLUL, RUNX2, ALP, and OCN decrease; f) PCR shows that in the GLUL-KO group at 3 days and 14 days of osteogenic induction, the mRNA expression level of RUNX2 decreases, and the difference is statistically significant (n = 6); g) shows that in the GLUL-KO group at 3 days, 7 days, and 14 days of osteogenic induction, the mRNA expression level of ALP decreases, and the difference is statistically significant (n = 6); h) shows that in the GLUL-KO group at 14 days of osteogenic induction, the mRNA expression level of OCN decreases, and the difference is statistically significant (n = 6); i) shows that the micro-CT results show that the effect of heterotopic osteogenesis of the GLUL-KO group is verified by the nude mouse replantation experiment. It is found that the high-density purplish red around the replant is less than that of the GLUL-WT group from the axial, sagittal, and coronal positions of the replant, scale bar 2000 μm; j) shows that the bone quality statistical indicators show that the BV / TV, Tb.Th, Tb.N, and BMD values of the GLUL-KO group are lower than those of the GLUL-WT group, and the Tb.Sp value is higher than that of the GLUL-WT group, and the difference is statistically significant (n = 6); k) shows that HE staining shows that the area of osteoid tissue (marked by black dotted line) is smaller in the GLUL-KO group and larger in the GLUL-WT group, and osteocytes and bone marrow cavities (black arrows) can be found; l) shows that the area of osteoid tissue in the GLUL-KO group is lower than that of the GLUL-WT group, and the difference is statistically significant (n = 6); m) shows that in a high-glucose environment, the effect of knocking out GLUL on the osteogenic differentiation of BMSCs is further verified, and it is found that the ARS staining of the GLUL-KO group is further weakened in a high-glucose environment; n) shows that in a high-glucose environment, the ALP activity of the GLUL-KO group is lower than that of the GLUL-WT group, and the difference is statistically significant (n = 6); (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using one-way ANOVA and Student Newman-Keuls test);

[0051] Figure 2It is shown that GLUL inhibits osteogenic differentiation of BMSCs and implant osseointegration in a dose-dependent manner; among them, a) shows a schematic diagram of the inhibitory effect of MSO (GLUL inhibitor) on BMSCs at different concentrations; b) shows that ALP staining shows that as the concentration of MSO increases, the ALP staining degree of BMSCs decreases; c) shows that ARS staining shows that as the concentration of MSO increases, the ARS staining degree of BMSCs decreases; d) ALP quantification shows that the ALP activity decreases under the action of 1 mM and 10 mM MSO concentrations, and the difference is statistically significant (n = 6); e) calcium ion quantification shows that the calcium ion quantification decreases under the action of 1 mM and 10 mM MSO concentrations, and the difference is statistically significant (n = 6); f) q-PCR shows that the mRNA expression level of GLUL gradually decreases under the action of 0.1 mM, 1 mM and 10 mM MSO concentrations, and the difference is statistically significant (n = 6); g) shows that the mRNA level of RUNX2 decreases at 0.1 mM and 1 mM MSO concentrations, and there is no obvious difference between 10 mM and 1 mM concentrations (n = 6); h) shows that the mRNA level of ALP decreases at 0.1 mM, 1 mM and 10 mM MSO concentrations, and the difference is statistically significant (n = 6); i) shows that as the concentration of MSO increases, the mRNA level of OCN gradually decreases, and the difference is statistically significant (n = 6); j) WB shows that as the concentration of MSO increases, the protein level of GLUL is lower, and the protein levels of osteogenic markers RUNX2, ALP, and OCN are lower; k) protein quantification shows that as the concentration of MSO increases, the protein level of GLUL decreases, and the difference is statistically significant (n = 6); l) protein quantification shows that as the concentration of MSO increases, the protein level of RUNX2 decreases, and the difference is statistically significant (n = 6); m) protein quantification shows that as the concentration of MSO increases, the protein level of ALP decreases, and the difference is statistically significant (n = 6); n) protein quantification shows that as the concentration of MSO increases, the protein level of OCN decreases, and the difference is statistically significant (n = 6); o) shows the time points of local injection of mice with different concentrations of MSO to establish an implant model; p) histology shows that the higher the concentration of MSO, the less mature bone around the implant. The black in the figure is the pure titanium implant, and the scale bars are 25 μm and 250 μm; q) shows that detecting the GLUL protein level in the bone tissue around the implant reveals that GLUL gradually decreases as the concentration of MSO increases, and the difference is statistically significant (n = 10); r) shows that as the concentration of MSO increases, the implant osseointegration rate decreases, and the difference is statistically significant (n = 10); (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using one-way ANOVA and Student-Newman-Keuls test);

[0052] Figure 3Under T2DM and high glucose conditions, the expressions of SYVN1 and GLUL were verified. Among them, a) showed a volcano plot marking low expression of GLUL and high expression of SYVN1 in the T2DM group; b) showed that the docking score of SYVN1 and GLUL predicted by molecular docking was 0.97; c) q-PCR found that the mRNA level of GLUL in BMSCs of T2DM was low, and the difference was statistically significant (n = 5); d) q-PCR found that the mRNA level of SYVN1 in BMSCs of T2DM was high, and the difference was statistically significant (n = 5); e) WB showed that the protein level of SYVN1 in the T2DM group and the high glucose-induced group was higher than that in the control group, while the protein level of GLUL was lower than that in the control group; f) Protein quantification in T2DM patients found that the expression level of SYVN1 increased, and the difference was statistically significant (n = 5); g) Protein quantification in T2DM patients found that the expression level of GLUL decreased, and the difference was statistically significant (n = 5); h) Protein quantification in the high glucose-induced group found that the expression level of SYVN1 increased with the increase in glucose concentration (G0: 0 mM glucose, G+: 5 mM glucose, G++: 25 mM glucose), and the difference was statistically significant (n = 5); i) Protein quantification in the high glucose-induced group found that the expression level of GLUL decreased with the decrease in glucose concentration, and the difference was statistically significant (n = 5); j) IHC showed that after the BMSCs of T2DM patients were replanted, the expression level of SYVN1 in the T2DM group was high, while the expression level of GLUL was low. Yellow arrows indicated positive cells, and the scale bars were 100 μm and 50 μm; k) IHC statistics showed that the expression level of SYVN1 in the T2DM group was high, and the difference was statistically significant (n = 5); l) IHC statistics showed that the expression level of GLUL in the T2DM group was low, and the difference was statistically significant (n = 5); (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using one-way ANOVA and Student Newman-Keuls test).

[0053] Figure 4It shows that SYVN1 mediates GLUL ubiquitination modification to regulate the osteogenic differentiation of BMSCs; among them, a) shows the lentiviral transfection efficiency of SYVN1 knockdown and overexpression. Sh nc represents the control group for SYVN1 knockdown, Sh SYVN1 represents the group with SYVN1 knockdown, Vector represents the control group for SYVN1 overexpression, and SYVN1 represents the group with SYVN1 overexpression; b) shows that after knocking down SYVN1, the ALP staining is enhanced, and after overexpressing SYVN1, the ALP staining is weakened; c) shows that after knocking down SYVN1, the ARS staining is enhanced, and after overexpressing SYVN1, the ARS staining is weakened; d) ALP quantification shows that after knocking down SYVN1, the ALP activity is enhanced, and after overexpressing SYVN1, the ALP activity decreases, and the difference is statistically significant (n = 3); e) calcium ion quantification shows that after knocking down SYVN1, the calcium ions increase, and after overexpressing SYVN1, the calcium ions decrease, and the difference is statistically significant (n = 3); f) Wb shows that after knocking down SYVN1, RUNX2, ALP, and OCN increase, and after overexpressing SYVN1, RUNX2, ALP, and OCN decrease; g) shows that BMSCs are treated with the protein synthesis inhibitor CHX and the lysosome inhibitor MG132 for 8 h, and there is no obvious decrease in GLUL in the group treated with both simultaneously, while obvious reduction in the GLUL protein level can be seen in the group treated with CHX alone; h) shows that when the expression of SYVN1 is downregulated with the SYVN1 inhibitor LS-102, the expression level of GLUL is upregulated, while when the expression of SYVN1 is upregulated with the SYVN1 activator zonisamide, the expression level of GLUL is downregulated in a dose-dependent manner, and when using the SYVN1 enzyme-inactivated mutant, the expression level of GLUL remains basically unchanged; i) shows that there is a negative correlation between the expression of GLUL and SYVN1. As the expression level of SYVN1 increases or decreases, the GLUL mRNA level decreases or increases accordingly, and there is a negative correlation, and the difference is statistically significant (n = 15); j) shows the verification of the expression efficiency of the F-GLUL plasmid; k) shows that with the gradient expression of SYVN1, the expression of GLUL decreases, and after the action of Mg132, the expression level of GLUL increases slightly; l) CO-IP shows that exogenous SYVN1 binds to the GLUL protein; m) shows that after overexpressing SYVN1, the ubiquitin level of GLUL increases with the increase of SYVN1; n) shows that after the K259 / 334A point mutation, the ubiquitin level of GLUL decreases; o) shows that after the K259 / 334A double mutation, the ubiquitin level of GLUL further decreases; p) shows that after the K259 / 334A mutation of GLUL, the ALP staining decreases; q) shows that after the K259 / 334A mutation of GLUL, the ARS staining decreases; r) shows that after the K259 / 334A mutation of GLUL, the ALP activity decreases, and the difference is statistically significant (n = 3); s) shows that after the K259 / 334A mutation of GLUL, the calcium ion quantification decreases, and the difference is statistically significant (n = 3); t) shows that under high glucose conditions, when SYVN1 is knocked down, the ARS staining increases;u) It shows that under high glucose conditions, the ARS staining in the WT-GLUL group is higher than that in the control group and the K259 / 334A mutant group; v) It shows that under high glucose conditions, the calcium ion quantification increases after SYVN1 knockdown, and the difference is statistically significant (n = 6); w) It shows that under high glucose conditions, the calcium ion quantification in the WT-GLUL group is higher than that in the control group and the K259 / 334A mutant group, and the difference is statistically significant (n = 6);

[0054] Figure 5 It shows the screening and preparation of GLUL-DUBTAC ligands; among them, a) shows the crystal structure of GLUL protein, and the potential allosteric sites are predicted using the Allostite server (http: / / mdl.shsmu.edu.cn / AST / ). The red color indicates the predicted allosteric sites; b) MST finds that the docking scores of HY-N0026 and HY-126351 are 9.9561E-06 and 1.2291E-06 respectively. c). c) shows that the number of active groups of HY-N0026 is 8, and the docking score is -9.089; d) shows that the number of active groups of HY-126351 is 1, and the docking score is -7.011; e) shows the molecular docking score of GLUL when only the linker and HY-126351 are present, which is -5.67; f) shows the predicted molecular docking score of EN523 and GLUL, which is -6.1, close to the molecular docking score of -5.67 between HY-126351 and GLUL, ensuring the binding of HY-126351 and GLUL; g) shows GLUL-DUBTAC, and displays its molecular structural formula, chemical formula, exact molecular weight of 775.24, molecular weight of 775.69, and is named HY-X3369; h) shows the synthetic intermediates of HY-X3369, HM-2027_1, HM-406B_7, HM-2027_2, HM-2027_3, HM-2027_4;

[0055] Figure 6It shows that GLUL-DUBTAC targets to maintain the balance of endogenous and exogenous protein homeostasis of GLUL and promotes the osseointegration of diabetic implants; among them, a) shows that under the action of CHX, WB shows that 10 μmol of HY-X3369 can maintain the protein level of GLUL; b) shows that under the action of CHX, WB shows that after the addition of HY-X3369, the protein level of P53 still decreases, while MG132 can still restore the protein level of P53; c) shows that HY-X3369 reduces the ubiquitin level of exogenous GLUL; d) shows that HY-X3369 reduces the ubiquitin level of exogenous GLUL elevated by SYVN1; e) shows that HY-X3369 enhances the ALP staining of T2DM jaw-derived BMSCs and BSMCs under high glucose; f) shows that HY-X3369 enhances the ARS staining of T2DM jaw-derived BMSCs and BSMCs under high glucose; g) shows that HY-X3369 enhances the ALP quantification of T2DM jaw-derived BMSCs and BSMCs under high glucose, and the difference is statistically significant (n = 3); h) shows that HY-X3369 enhances the calcium ion quantification of T2DM jaw-derived BMSCs and BSMCs under high glucose, and the difference is statistically significant (n = 3); i) shows that at 8 weeks, there is more mature bone (MB, light blue) around the implants in the HY-X3369 group, while there is still new bone (NB, dark blue, white dotted line) in the control group that has not matured. The scale bars are 100 μm, 200 μm, and 2 mm respectively; j) shows that the osseointegration rate of implants in the HY-X3369 group is higher than that in the DMSO group, and the difference is statistically significant (n = 6); k) shows that the bone quality around the implants in the HY-X3369 group is more mature and there is less new bone than in the DMSO group, and the difference is statistically significant (n = 6); l) shows that at 8 weeks, there is more mature bone (green) around the implants in the HY-X3369 group, while there are still a large number of bone resorption lacunae in the control group, and osteoid (Os, marked by white arrows) can be observed. The scale bars are 100 μm, 200 μm, and 2 mm respectively; m) shows that the osteoid area in the HY-X3369 group is lower than that in the DMSO group, and the difference is statistically significant (n = 6); n) shows that micro-CT shows that the bone density around the implants in the HY-X3369 group is high; o) shows that the BV / TV in the HY-X3369 group is higher than that in the DMSO group, and the difference is statistically significant (n = 6); p) shows that the Tb.Th in the HY-X3369 group is higher than that in the DMSO group, and the difference is statistically significant (n = 6); q) The Tb.Sp in the HY-X3369 group is lower than that in the DMSO group, and the difference is statistically significant (n = 6); r) shows that the Tb.N in the HY-X3369 group is higher than that in the DMSO group, and the difference is statistically significant (n = 6);

[0056] Figure 7 It shows the chemical formula of GLUL-DUBTAC

[0057] Figure 8 It shows the preparation of HM-2027_2;

[0058] Figure 9 Show the nuclear magnetic resonance detection results of HM-2027_2;

[0059] Figure 10 Show the preparation of HM-2027_3;

[0060] Figure 11 Show the liquid chromatography-mass spectrometry detection results 1 of HM-2027_3;

[0061] Figure 12 Show the liquid chromatography-mass spectrometry detection results 2 of HM-2027_3;

[0062] Figure 13 Show the preparation of HM-2027_5;

[0063] Figure 14 Show the liquid chromatography-mass spectrometry detection results 1 of HM-2027_5

[0064] Figure 15 Show the liquid chromatography-mass spectrometry detection results 2 of HM-2027_5;

[0065] Figure 16 Show the nuclear magnetic resonance detection results of HM-2027_5;

[0066] Figure 17 Show the high performance liquid chromatography detection results of HM-2027_5. Detailed implementation manners

[0067] The present invention discloses GLUL deubiquitinase-targeted chimeras and their applications. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0068] Glutamine synthetase (GLUL) is a mitochondrial enzyme that plays a key regulatory role in cell metabolism. Its main function is to catalyze the glutamate-dependent condensation of ammonia to synthesize glutamine. In the skeletal system, GLUL mainly regulates the function of BMSCs by maintaining mitochondrial homeostasis, activating the mammalian target of rapamycin (mTOR) signaling pathway, and inhibiting the production of reactive oxygen species (ROS). GLUL is downregulated in BMSCs derived from the jaws of T2DM. Among the strategies for intervening in the function of BMSCs, maintaining the protein homeostasis of GLUL is a previously undiscovered and potentially significant method.

[0069] Deubiquitinase-targeting chimera (DUBTAC1) has emerged as a new and promising modality that draws inspiration from natural regulatory mechanisms and promotes the stabilization of endogenous proteins targeted for maintenance in cells. It links a target protein ligand and a deubiquitinase (DUB) ligand via a linker, thereby bringing the DUB near ubiquitinated proteins to remove ubiquitin chains and prevent the target protein from being degraded. Its advantages of protein resynthesis, broad target selection range, and high biosafety play an important role in the field of targeted protein stabilization. In this study, we explored the role of GLUL in the osteogenic differentiation of T2DM BMSCs and developed a novel GLUL-DUBTAC named HY-X3369 against the GLUL ubiquitination degradation mechanism. The osteogenic effect of HY-X3369 and the bone-bonding repair effect in diabetic mice were evaluated to explore the precise regulation of the bone-bonding repair process in T2DM and provide a theoretical basis and candidate methods for conducting clinical trials for the treatment of bone bonding in T2DM patients.

[0070] The present invention has developed a small molecule drug called GLUL deubiquitinase-targeting chimera (DUBTAC). The principle of DUBTAC is to link a target protein ligand and a deubiquitinase (DUB) ligand via a linker, thereby bringing the DUB near ubiquitinated proteins to remove ubiquitin chains and prevent the target protein from being degraded ( Figure 5)。DUBTAC has three major advantages. ① Protein resynthesis can be recycled multiple times, and only a small amount of drug is required to achieve the stability of substrate proteins; ② The target selection has a wide range of effects. For proteins without active sites, as long as they can bind to their ubiquitin chains, DUBTAC can induce the degradation of the relevant protein ubiquitin chains; ③ High biological safety. DUBTAC is specifically designed based on the ligand of the target protein. During the action process, it only targets the target protein. When the target protein is maintained at a certain level or is degraded programmatically through non-ubiquitination pathways, DUBTAC only binds to the substrate protein as a normal ligand structure and does not affect the programmed degradation of the substrate protein through non-ubiquitination pathways.

[0071] Experimental method:

[0072] The primary culture method of human jaw-derived BMSCs. During the process of preparing the hole for implant surgery, the discarded bone chips were collected and stored in phosphate-buffered saline (Gibco, Grand Island, NY, USA) containing antibiotics (10000 U / ml penicillin and 10 mg / ml streptomycin; Gibco). After centrifugation at 1100 rpm, the bone chips were resuspended in mesenchymal stem cell medium (MSCM; ScienCell, Carlsbad, CA, USA), then inoculated in a culture dish, and cultured in a humidified cell incubator containing 5% CO2 at 37 °C for 7 days without moving. A single-cell suspension was obtained using a filter with a pore size of 70 μm (Falcon, BD Labware, Wilmington, DE, USA), and then cultured in MSCM. The medium was changed every three days. The primary cells obtained by low-speed drilling technology and cultured by the above method showed the characteristics of BMSCs

[29] 。HEK293T was purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (SIBS, CAS, China).

[0073] Osteogenic induction and detection of BMSCs: The BMSCs were seeded at 5×10 per well 5Cells were seeded at a density of [number of cells] in 6-well culture dishes and cultured in osteogenic medium for 14 days using the StemPro Osteogenic Differentiation Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Osteogenic differentiation was induced after the cells reached 70 - 80% confluence. According to the manufacturer's instructions, on the 10th day after osteogenic induction, the intracellular ALP activity in BMSCs was evaluated using an ALP activity assay kit (Nanjing Jiancheng Bioengineering Institute, China) and normalized based on protein concentration. The optical density (OD) was recorded at 520 nm. After 14 days of osteogenic induction, the cells were fixed in 70% ethanol and stained with 2% ARS (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes. Then, 1 ml of isopropanol was added to each well to dissolve the red perylenequinone derivative in the calcium nodules, and the OD value was measured at 550 nm using a spectrophotometer (51119700DPC, Thermo Fisher Scientific, Waltham, USA).

[0074] Prediction of potential ubiquitination modification sites of GLUL and virtual screening Potential isomerization sites were predicted using the Allosite server (http: / / mdl.shsmu.edu.cn / AST / ) based on the structure of GLUL (PDB ID: 2752). Grid-based ligand docking services for virtual screening of 735,62 compounds in the MCE 50K Diversity Library and MCE Bioactive Compound Library were performed using the Glide software ( Maestro 11.4). Seven of the highest-scoring compounds purchased from MedChemExpress were used for experimental purposes.

[0075] Microscale thermophoresis (MST) To determine the binding affinity of the ligand to GLUL, microscale thermophoresis (MST) was performed using a Monolith NT.115 (NanoTemper Technologies GmbH, Munich, Germany) and standard capillaries. The GLUL recombinant protein was obtained from Proteintech (Cat. Ag4471). According to the manufacturer's instructions, the GLUL recombinant protein was labeled with the fluorescent dye Red-NHS using NanoTemper's labeling kit MO-L011 Monolith TM and the NanoPhotometer TM(Implen GmbH, Munich, Germany) measured the absorbance at 260 and 546 nm and a fluorophore / conjugate ratio of 0.5 was determined. 200 nM labeled GLUL protein was incubated with 1 μM to 62.5 μM compounds. The analysis was performed in binding buffer including 0.05% Tween 20 at 25°C. The fluorescence signal was normalized and the Hill equation was fitted using MO Affinity Analysis v2.1.3 software (NanoTemper).

[0076] Surgical procedures: 30 5-week-old male BALB / C homozygous nude mice (nu / nu) BALB / C, 60 8-week-old male C57BL / 6 and 20 GK male rats (SPF Biotechnology Co., Ltd., Beijing, China) were used in this experiment. GK rats were supplemented with a diabetic diet, and blood glucose values ​​were measured by tail vein blood sampling every 3 days. When the average blood glucose value was greater than 16.7 mM / L, the modeling standard was reached. In the nude mouse reimplantation step, a single cell suspension of BMSCs was obtained after trypsin digestion, and the cell density was adjusted to 4×106 cells / ml. The cells were incubated with 40 mg of hydroxyapatite bone powder at 37°C for 1 hour and centrifuged at 150g for 5 minutes at 37°C. Treated cells and control cells were implanted into the subcutaneous space of the back of 5-week-old male BALB / Chomozygous nude mice (nu / nu) (SPF Biotechnology Co., Ltd., Beijing, China), and the mice were placed in cages (6 per cage) in a controlled environment (20-25°C, 40-60% relative humidity). The animals were fed a sterile diet (SPF Biotechnology Co., Ltd.), and they had free access to water; each group was implanted in two symmetrical positions. The implantation procedures of C57BL / 6 and GK rats were the same as described previously. In brief, C57BL / 6 and GK rats were anesthetized by intraperitoneal injection of ketamine and xylazine. Each left leg was shaved and disinfected. To provide sterile conditions during the surgery. The distal metaphysis of the left femur was exposed through a skin incision, and then a hole with a diameter of 1 mm and a depth of 2 mm was drilled in the lateral metaphysis of the femur by hand drill using a water-cooled minimally invasive method. Before implantation in the hole, a pure titanium rod (Xi'an Qiyue Biological Co., Ltd.) was implanted in the hole on the left femur. The wound healed layer by layer. The injection site of MSO and GLUL-DUBTAC was local to the wound. After surgery, all animals received a subcutaneous injection of 0.5 mg kg-1 ketoprofen. According to each preset time point, animals were killed by intraperitoneal injection of excessive sodium pentobarbital.

[0077] Synthesis of GLUL-DUBTAC: Catalog NO. (HY-X3369), easily soluble in DMSO

[0078] Dissolve compound HM-2027_1 (500 mg, 1.12 mmol, 1.0 eq.) in DMF (10 mL). Add HM-406B_7 (313 mg, 1.80 mmol, 1.6 eq.), HATU (768 mg, 2.02 mmol, 1.8 eq.), and DIEA (726 mg, 5.61 mmol, 5 eq.) under an ice bath. After addition, stir at room temperature for 2 h. LCMS shows that the reaction is complete. The reaction solution is directly passed through a column (RP-HPLC, ACN in water, 60%, TFA condition), and the product is obtained by lyophilization to get 310 mg of a pale yellow solid, yield: 45.9%. Dissolve compound HM-2027_2 (120 mg, 0.199 mmol, 1.0 eq.) in DCM (5 mL). Add TFA (1.0 ml) under an ice bath. After addition, stir at room temperature for 2 h. LCMS shows that the reaction is complete. The reaction solution is concentrated at low temperature, taken through DCM once, and dried with an oil pump. The crude product is not purified and is directly used for the next step to obtain 100 mg of a pale yellow solid, yield 100%. Dissolve compound HM-2027_3 (100 mg, 0.199 mmol, 1.0 eq.) in DMF (4.0 ml). Add HM-2027_4 (76 mg, 0.259 mmol, 1.3 eq.), T3P (190 mg, 0.299 mmol, 1.5 eq, 50% m / m), and DIEA (103 mg, 0.798 mmol, 4.0 eq) under an ice bath. After addition, stir at room temperature for 2 h. LCMS shows that the reaction is complete. The reaction solution is directly sent for reverse-phase preparation (RP-HPLC, FA condition), and the product is obtained by lyophilization to get 25 mg of a white solid, yield 16%, purity 98%.

[0079]

[0080] In the present invention, HM-2027_5, GLUL-DUBTAC, and HY-X3369 are all compounds represented by Formula I, and they are only named differently at different stages of compound research and development, but they are essentially the same.

[0081] The raw materials and reagents used in the GLUL deubiquitinase-targeted chimeras and their applications provided by the present invention can all be purchased from the market.

[0082] The present invention will be further described below in conjunction with the examples:

[0083] Example 1 Preparation of HM-2027_2 (as Figure 8 shown)

[0084] Dissolve the compound HM-2027_1 (500 mg, 1.12 mmol, 1.0 eq.) from MCE in DMF (10 mL). Under an ice bath, add HM-406B_7 (313 mg, 1.80 mmol, 1.6 eq.), HATU (768 mg, 2.02 mmol, 1.8 eq.), and DIEA (726 mg, 5.61 mmol, 5 eq.). After addition, stir at room temperature for 2 h. LCMS shows that the reaction is complete. The reaction solution is directly passed through a column (RP-HPLC, ACN in water, 60%, TFA condition), and the product is obtained by lyophilization. 310 mg of a pale yellow solid is obtained, and the yield is 45.9%. (All the above compounds are from MCM). HNMR detection: XGJ33022-1-P.

[0085] The detection results are as Figure 9 shown.

[0086] Example 2 Preparation of HM-2027_3 (as Figure 10 shown)

[0087] Dissolve the compound HM-2027_2 (120 mg, 0.199 mmol, 1.0 eq.) in DCM (5 mL). Under an ice bath, add TFA (1.0 ml). After addition, stir at room temperature for 2 h. LCMS shows that the reaction is complete.

[0088] The reaction solution is concentrated at low temperature, taken through DCM once, and dried with an oil pump. The crude product is not purified and directly used for the next step. 100 mg of a pale yellow solid is obtained, and the yield is 100%. LCMS detection: XGJ33022-3-R1.

[0089] The detection results are as Figure 11 and Figure 12 shown.

[0090] Example 3 Preparation of HM-2027_5 (as Figure 13 shown)

[0091] Dissolve the compound HM-2027_3 (100 mg, 0.199 mmol, 1.0 eq.) in DMF (4.0 ml). Under an ice bath, add HM-2027_4 (76 mg, 0.259 mmol, 1.3 eq.), T3P (190 mg, 0.299 mmol, 1.5 eq, 50% m / m), and DIEA (103 mg, 0.798 mmol, 4.0 eq). After addition, stir at room temperature for 2 h. LCMS shows that the reaction is complete. The reaction solution is directly sent for reverse-phase preparation (RP-HPLC, FA condition), and 25 mg of a white solid is obtained by lyophilization. The yield is 16%, and the purity is 98%. LCMS detection: XGJ33022-5-P1.

[0092] The detection results are as Figures 14 - 17 shown below.

[0093] Example 4: Knockout of GLUL inhibits osteogenic differentiation of BMSCs

[0094] To further clarify the function of GLUL in the osteogenic differentiation of BMSCs from the jaws of T2DM patients, we constructed a GLUL-KO lentiviral transfection system using the Caspas9 gene editing technology. The lentivirus with GLUL knockout was used to transfect human BMSCs from the jaws, and the osteogenic induction medium was used to induce the osteogenic differentiation of BMSCs. It was found that knocking out GLUL reduced the ALP and ARS staining of BMSCs ( Figure 1 a, b) and the ALP activity and calcium ion quantification ( Figure 1 c, d).

[0095] Western blot analysis showed that knocking out GLUL also decreased the protein levels of the osteogenic differentiation markers Runx2, ALP, and OCN ( Figure 1 e).

[0096] In addition, the results of real-time RT-PCR showed that compared with the GLUL-WT group, knocking out GLUL decreased the expression of the osteogenic markers Runx2 ( Figure 1 f), ALP ( Figure 1 g), and OCN ( Figure 1 h). This indicates that the lack of GLUL affects the in vitro osteogenic differentiation ability of BMSCs.

[0097] To further verify the effect of GLUL-KO on osteogenesis in vivo, after replanting in nude mice, micro-CT observation showed that in the axial, sagittal, and coronal planes of the replanted tissues, the purple-red areas representing high density around the replanted tissues were less than those in the GLUL-WT group ( Figure 1 i). The bone mass statistical indicators showed that the BV / TV, Tb.Th, Tb.N, and BMD values of the GLUL-KO group were lower than those of the GLUL-WT group, and the Tb.Sp value was higher than that of the GLUL-WT group ( Figure 1 j).

[0098] In the histological detection of heterotopic osteogenesis, the area of osteoid tissue (marked by black dotted lines) was smaller in the GLUL-KO group and larger in the GLUL-WT group, with statistical differences. Osteocytes and bone marrow cavities (black arrows) could be found ( Figure 1 k, Figure 1 l). Moreover, under the high-glucose induction condition of diabetes, the ARS and ALP activities in the GLUL-KO group were still lower than those in the GLUL-WT group ( Figure 1 m, Figure 1n). The above research results indicate that the deletion of GLUL can significantly inhibit the osteogenic differentiation ability of T2DM patients and can also exert its effect under high glucose conditions.

[0099] Example 5 GLUL Inhibits Osteogenic Differentiation of BMSCs and Implant Osseointegration in a Dose-Dependent Manner

[0100] To further clarify the dose-effect relationship of GLUL, we used

[0101] L-Methionine-DL-sulfoximine (MSO, MedChemExpress, Shanghai, China), which is a highly specific and irreversible inhibitor of Glutamine synthetase and has been used to inhibit glutamine-dependent ammonia-stimulated neuronal toxicity in vitro and enhance glutamine deficiency-dependent depression. MSO was prepared at concentration gradients of 0, 10 -2 , 10 -1 , 10 for dose-effect studies ( Figure 2 a). It was co-incubated with BMSCs induced to osteogenesis for 10 days respectively. ALP staining showed that as the MSO concentration gradient increased, the ALP staining gradually decreased ( Figure 2 b). After osteogenic induction for 14 days, ARS staining showed that as the MSO concentration gradient increased, the ARS staining gradually decreased ( Figure 2 c). After osteogenic induction for 3 days, ALP quantification showed that the ALP activity decreased under the action of 1 mM and 10 mM MSO ( Figure 2 d). After osteogenic induction for 10 days, calcium ion quantification showed that the ALP activity decreased under the action of 1 mM and 10 mM MSO ( Figure 2 e). q-PCR showed that the mRNA levels of GLUL and RUNX2 decreased under the action of 0.1 mM, 1 mM and 10 mM MSO ( Figure 2 f, Figure 2 g), while the mRNA levels of ALP and OCN decreased significantly after using MSO ( Figure 2 h, Figure 2 i). At the protein level, it was also found that GLUL, RUNX2, ALP, and OCN decreased significantly with the increase in MSO concentration, and the difference was statistically significant ( Figure 2 j-n). It indicates that the in vitro osteogenic differentiation ability of BMSCs depends on the dose effect of GLUL.

[0102] To verify the correlation between GLUL expression level and implant osseointegration, we established a mouse femoral implant model and locally injected different doses of MSO 2 weeks before implant surgery, 2 weeks, 4 weeks, and 6 weeks after implant surgery ( Figure 2o). Specimens were obtained after 8 weeks, and histological grinding slices with implants were prepared. It was observed that under normal conditions, mature bone tissue was present around the Ti implants. As the MSO dose increased, the mature bone gradually decreased ( Figure 2 p). The GLUL protein levels in the bone tissue around the implants at different MSO concentrations were statistically analyzed, and it was found that the expression of the GLUL protein was inhibited by high-concentration MSO ( Figure 2 q). The implant bone-bonding rate also decreased as the MSO dose increased ( Figure 2 r). It was shown that MSO could reduce the GLUL protein level in the bone tissue around the implants and inhibit implant bone bonding in a dose-dependent manner.

[0103] Example 6. Under T2DM and high-glucose conditions, the expressions of SYVN1 and GLUL were verified

[0104] The applicant studied the mechanism of the downregulation of GLUL in T2DM BMSCs. The ubiquitin-lysosome pathway is an important channel for enzyme degradation in cells. Abnormal modification of ubiquitination often causes the degradation of key proteins in bone metabolism, thereby triggering bone metabolic dysfunction. Therefore, we collected BMSCs from the jaws of 5 T2DM patients and matched them with BMSCs from the jaws of 5 non-T2DM patients for proteomics research. The proteomics data have been deposited to the ProteomeXchange Consortium with dataset identifier PXD016489. We found that the E3 ubiquitin ligase - synovial apoptosis inhibitor 1 (SYVN1), which is closely related to the ubiquitination pathway, was significantly upregulated at the protein level in the T2DM group. SYVN1 is a six-transmembrane endoplasmic reticulum membrane protein belonging to the ubiquitin ligase E3 family. It plays a role in delivering ubiquitin molecules during the ubiquitination process of proteins, determines the substrate specificity by selectively recognizing and polyubiquitinating substrate proteins, and thus regulates the substrate protein homeostasis. It is a key enzyme in the protein ubiquitination reaction. We made a volcano plot to label SYVN1 and GLUL ( Figure 3 a). Subsequently, molecular docking prediction was performed, and the docking score was 0.97, indicating a strong interaction potential between GLUL and SYVN1 ( Figure 3 b). q-PCR found that the mRNA level of GLUL was low and the level of SYVN1 was high in the BMSCs of T2DM ( Figure 3 c, Figure 3 d). At the protein level, it was found that the expression of GLUL decreased and the expression of SYVN1 increased in the BMSCs of T2DM. Moreover, as the glucose concentration increased, the level of SYVN1 increased and the level of GLUL decreased, and the difference was statistically significant (Figure 3 e-i). After re-implantation in nude mice, IHC showed that SYVN1 had a higher positive expression level and GLUL had a lower positive expression level in the T2DM group ( Figure 3 j-l). The above results indicated that SYVN1 had a higher expression and GLUL had a lower expression in BMSCs of T2DM.

[0105] Example 7 SYVN1 mediates GLUL ubiquitination modification to regulate osteogenic differentiation of BMSCs

[0106] In view of the correlation between the expressions of SYVN1 and GLUL in the above experiments, but the function and its regulatory mechanism of SYVN1 were still unclear. We then used lentiviruses with SYVN1 knockdown and overexpression to study the function of SYVN1 in regulating osteogenic differentiation of BMSCs. First, the transfection efficiency of lentiviruses with SYVN1 knockdown and overexpression was verified ( Figure 4 a). After 10 days of osteogenic induction, ALP staining showed that after knocking down SYVN1, the ALP staining was stronger than that of the control group, and after overexpressing SYVN1, the ALP staining was weaker than that of the control group ( Figure 4 b). After 14 days of osteogenic induction, ARS staining showed that the SYVN1 knockdown group was stronger than the control group, and the SYVN1 overexpression group was weaker than the control group ( Figure 4 c). ALP and calcium ions also indicated that the SYVN1 knockdown group was stronger than the control group, and the SYVN1 overexpression group was weaker than the control group ( Figure 4 d, Figure 4 e). WB showed that the protein levels of osteogenic markers RUNX2, ALP, and OCN increased after knocking down SYVN1 and decreased after overexpressing SYVN1 ( Figure 4 f). The above research results indicated that SYVN1 had an inhibitory effect on the in vitro osteogenic differentiation of BMSCs, which was opposite to the function of GLUL. Next, we studied the degradation mechanism of GLUL. We used the protein synthesis inhibitor Cycloheximide (CHX, MedChemExpress, Shanghai, China) as an import barrier and the proteasome inhibitor MG132 (MedChemExpress, Shanghai, China) as an export barrier to treat BMSCs for 8 h to study the degradation of endogenous GLUL. No obvious decrease in GLUL was seen in the group treated with both, and a significant decrease in the GLUL protein level was seen in the group treated with CHX alone, indicating that most of GLUL was degraded through the proteasome pathway ( Figure 4g). When the expression of SYVN1 was downregulated using the SYVN1 inhibitor LS-102 (MedChemExpress, Shanghai, China), the expression level of GLUL was upregulated. When the expression of SYVN1 was upregulated using the SYVN1 activator Zonisamide (MedChemExpress, Shanghai, China), the expression level of GLUL was downregulated in a dose-dependent manner. When the SYVN1 enzyme-inactivated mutant (Shanghai GenePharma Co., Ltd.) was used, the expression level of GLUL remained basically unchanged, indicating that SYVN1 did regulate the expression level of GLUL ( Figure 4 h). The q-PCR experiment further demonstrated that when the expression level of SYVN1 increased or decreased, the GLUL mRNA level decreased or increased accordingly, and there was an obvious negative correlation between the two, with r = -0.5319 and p = 0.0413. Figure 4 i). Subsequently, we constructed the overexpression plasmid Flag-GLUL with a Flag tag and its control vector PC3.1, and transfected them into 293T cells respectively. After 48 h, samples were prepared for WB to verify that the plasmid vectors had good transfection efficiency. Figure 4 j). Subsequently, the SYVN1 gene was overexpressed in 293T cells in a gradient manner, and it was found that the protein level of GLUL was downregulated, and this effect could be reversed after treatment with MG132. Figure 4 k). To further determine the interaction relationship between exogenous SYVN1 and GLUL, we transfected Flag-GLUL and the conventional SYVN1 overexpression vector alone or in combination in 293T. Flag IP found that SYVN1 could directly interact with GLUL, suggesting that SYVN1 could directly bind to GLUL and might be the enzyme mediating the ubiquitination and proteasomal hydrolysis of GLUL. Figure 4 l). To further determine whether SYVN1 could mediate the ubiquitination and degradation of GLUL, we co-transfected the HA-Ubi and Flag-GLUL plasmids into 293T cells, and simultaneously transfected the PC3.1-SYVN1 and its control PC3.1 vectors, and treated the cells with MG132. After the Flag IP experiment, the ubiquitination level of GLUL was detected using the HA antibody. It could be seen that after overexpressing SYVN1 for ubiquitination IP, the ubiquitination level of GLUL increased with the increase of SYVN1, suggesting that SYVN1 could reduce the stability of GLUL protein by mediating the ubiquitination and degradation of GLUL. Figure 4m). In the present invention, a deubiquitination point mutation plasmid was constructed. HA-Ubi, GLUL-WT, and the K259A, K276A, and K334A point mutation plasmids were transiently transfected into 293T cells respectively. After 36 h, MG132 was added. After 48 h of transfection, cells were harvested to prepare protein samples and subjected to IP experiments. The results showed that ubiquitination signals could be detected for GLUL-WT, while among the ubiquitination site mutants, the ubiquitination levels of K259A and K334A decreased, and the ubiquitination level of K276A remained basically unchanged, indicating that K259A and K334A might be the main ubiquitination modification sites of GLUL. Figure 4 n). To further clarify the effect of the double-site mutation of K259A and K334A on the ubiquitination level of GLUL, we constructed a K259 / 334A double mutation plasmid. After transfection, it was found that the degree of ubiquitination degradation of K259 / 334A was further enhanced. Figure 4 o). The above results proved that K259 / 334A was the main regulatory site for GLUL ubiquitination modification. After this site was mutated, its ubiquitination degradation level was significantly affected. Functionally, through ALP Figure 4 p) and ARS Figure 4 q) staining, it was found that the staining degree of the K259 / 334A mutation group was significantly lower than that of the GLUL-WT group, while ALP quantification Figure 4 r) and calcium ion quantification Figure 4 s) showed that the quantification results of the K259 / 334A mutation group were also significantly lower than those of the GLUL-WT group. In addition, under high glucose conditions, we verified the osteogenic differentiation function of the SYVN1 and GLUL-K259 / 334A mutation groups. ARS staining found that the staining degree was significantly increased after knocking down SYVN1 under high glucose Figure 4 t), and the staining degree was significantly decreased after the K259 / 334A mutation Figure 4 u). ALP activity detection found that the ALP activity of the group with SYVN1 knocked down under high glucose was significantly increased Figure 4 v), and the ALP activity was significantly decreased after the K259 / 334A mutation Figure 4 w). The above research results showed that inhibiting SYVN1 under high glucose conditions could significantly promote the osteogenic differentiation ability of T2DM patients, while the mutation of the ubiquitination site K259 / 334A of GLUL could significantly inhibit the osteogenic differentiation ability of T2DM patients and also play a role under high glucose conditions.

[0107] Example 8 Screening and Preparation of GLUL-DUB TAC Ligands

[0108] Given the ubiquitination degradation mechanism of GLUL protein with K259 / 334A as the main site in T2DM, in order to identify potential ubiquitination modification sites, we first reconstructed the crystal structure of GLUL and used the Allostite server to predict potential allosteric sites ( http: / / mdl.shsmu.edu.cn / AST / ), and the predicted allosteric sites are shown in red ( Figure 5 a). Then, we predicted potential small molecule ligands of GLUL through virtual screening, and selected nearly 735,62 compounds from the MCE 50K Diversity Library and the MCE Bioactive Compound Library to dock to the predicted sites. Based on the GLUL compound binding model with a high predicted score, we selected and purchased 7 compounds, namely 7 small molecule ligands with a high score and the possibility of DUBTAC synthesis, HY-N0026, HY-F0002, HY-N2497, HY-126351, HY-N0033, HY-128851, and HY-N4085 (all purchased from MCE), and verified the affinity of the small molecule ligands using MST.

[0109] Among them:

[0110] HY-N0026: 2'-Acetylacteoside Synonyms: 2-Acetylsyringoside; 2'-AA), chemical formula C 31 H 38 O 16 , CAS number: 94492-24-7 , and the structure is shown in Formula II:

[0111]

[0112] HY-F0002: Nicotinamide impurity 3 sodium salt, chemical formula C 21 H 27 N7NaO 17 P3, CAS number: 1184-16-3 , and the structure is shown in Formula III:

[0113]

[0114] HY-N2497: Apioglucosylisoliquiritigenin, chemical formula: C 26 H 30 O 13 , CAS number: 120926-46-7 , and the structure is shown in Formula IV:

[0115]

[0116] HY-126351: Chemical formula C 20 H13 F6NO4, CAS No.: 2306388-57-6 , and the structure is as shown in Formula V:

[0117]

[0118] HY-N0033: Chinese name: Chrysoeriol-7-O-glucoside; Chrysoeriol glucoside; Chemical formula C 35 H 46 O 19 , CAS No.: 94079-81-9 , and the structure is as shown in Formula VI:

[0119]

[0120] HY-128851: Chinese name: Coenzyme A, Chemical formula C 21 H 36 N7O 16 P3S, CAS No.: 85-61-0 , and the structure is as shown in Formula VII:

[0121]

[0122] HY-N4085: Chinese name: Vitexin-2″-O-glucoside, Chemical formula C 27 H 30 O 15 , CAS No.: 38950-94-6 , and the structure is as shown in Formula VIII:

[0123]

[0124] It was found that HY-N0026, HY-126351 and GLUL had obvious binding, and the docking scores were 9.9561E-06 and 1.2291E-06 respectively ( Figure 5 b), the KD value represents the concentration of the Ligand molecule when 50% of the Target molecule is bound, and the unit is M. The larger the KD value, the lower the affinity. On the contrary, the smaller the KD value, the higher the affinity between the Target molecule and the Ligand molecule. And the docking mode diagram and docking score of the binding site were constructed. Among them, the docking score of HY-N0026 was -9.089. In the 3D map, the C skeleton of the GLUL protein is shown in green and blue, the N atom is shown in blue, the O atom is shown in bright red, the H atom is shown in white, and HY-N0026 is shown as a light blue stick. The hydrogen bond length is shown as a red dotted line, and the longer the bond length, the weaker the hydrogen bond interaction. HY-N0026 can form 2 hydrogen bond interactions and 1 π-π interaction with the target protein: a phenolic hydroxyl group acts as a hydrogen bond acceptor to form a hydrogen bond interaction with SER257, and the distance is A hydroxyl group on the sugar ring acts as a hydrogen bond donor to form a hydrogen bond with ALA191 at a distance of In addition, a benzene ring can form a π-π interaction with TYR336 ( Figure 5 c). The docking score of HY-126351 is -7.011. In the 3D image, the Human GLUL protein C skeleton is shown in green, N atoms are shown in blue, O atoms are shown in bright red, H atoms are shown in white, and HY-126351 is shown as a green stick. The hydrogen bond length is shown as a red dotted line. The longer the bond length, the weaker the hydrogen bond. HY-126351 can form two hydrogen bonds and one π-π interaction with the target protein: the carboxyl group can form two hydrogen bonds with ARG262 and LYS334, with distances of In addition, a benzene ring can form a π-π interaction with TYR336 ( Figure 5 d). The docking score of HY-F0002 was -8.916, the docking score of HY-N2497 was -8.813, the docking score of HY-N0033 was -8.782, the docking score of HY-128851 was -8.723 and the docking score of HY-N4085 was -8.557. In order to further clarify which compound is suitable for the small molecule ligand of DUBTAC, we analyzed the molecular structures of HY-N0026 and HY-126351 and found that the number of active groups of HY-N0026 is 8, while the number of active groups of HY-126351 is 1. Too many active groups will reduce the success rate of DUBTAC synthesis and reduce the purity of DUBTAC. In addition, HY-N0026 is a natural product and is not suitable as a ligand. Therefore, HY-126351 was selected as the ligand of GLUL-DUBTAC. However, adding a linker from the carboxyl end may reduce the effect of one hydrogen bond and affect the binding force. To further clarify, the molecular docking score of GLUL with only the linker and HY-126351 is -5.67, indicating that although the binding force is indeed reduced after the hydroxyl group binds to the linker, it is not reduced too much, and the binding of HY-126351 and GLUL can still be guaranteed ( Figure 5 e). In addition, in order to prevent the excessive binding of OTUB1 ligand EN523 to the target protein, which would lead to the weakening of GLUL-DUBTAC function, we predicted the molecular docking score of EN523 and GLUL to be -6.1, which is close to the molecular docking score of HY-126351 and GLUL, -5.67, which further ensures the binding of HY-126351 and GLUL, indicating that HY-126351 is a good ligand for GLUL ( Figure 5 f). Based on this, we designed and synthesized GLUL-DUBTAC, and displayed its molecular structure, chemical formula, precise molecular weight of 775.24, molecular weight of 775.69, and named it HY-X3369 (Figure 5 g). The synthesis process of HY-X3369 and its intermediates is shown, Figure 5 h), mainly including HM-2027_1, HM-406B_7, HM-2027_2, HM-2027_3, HM-2027_4, and the LCMS, HPLC and chromatogram of HY-X3369 were detected.

[0125] Example 9 GLUL-DUBTAC targets to maintain the balance of endogenous and exogenous protein homeostasis of GLUL and promotes osseointegration of diabetic implants

[0126] To further evaluate the effect of HY-X3369 on maintaining the endogenous protein homeostasis of GLUL, we evaluated the protein level of GLUL after 8 h with 0, 1, 10, 50, 100 μmol of HY-X3369 under the action of CHX. The endogenous GLUL protein level was restored at 10 μmol. Figure 6 a). To further evaluate the targeting ability of HY-X3369 to maintain GLUL protein, under the action of CHX, when using HY-X3369, the protein level of P53 was still decreased, while MG132 could still restore the protein level of P53. Figure 6 b). To further evaluate the effect of HY-X3369 on maintaining the exogenous GLUL protein homeostasis, it was found that HY-X3369 decreased the ubiquitin level of exogenous GLUL. Figure 6 c), and HY-X3369 could decrease the ubiquitin level of exogenous GLUL elevated by SYVN1. Figure 6 d). To further evaluate the effect of HY-X3369 on the osteogenic differentiation ability of BMSCs in T2DM, it was found that HY-X3369 enhanced the ALP staining of BMSCs derived from the jaws of T2DM and BSMCs under high glucose. Figure 6 e), HY-X3369 enhanced the ARS staining of BMSCs derived from the jaws of T2DM and BSMCs under high glucose. Figure 6 f), enhanced the ALP quantification of BMSCs derived from the jaws of T2DM and BSMCs under high glucose. Figure 6 g) and calcium ion quantification. Figure 6 h). The osseointegration function of HY-X3369 was studied. At 8 weeks, there was more light blue mature bone around the implants in the HY-X3369 group, while there was still dark blue new bone in the control group. Figure 6 i), the osseointegration rate of implants in the HY-X3369 group was higher than that in the DMSO group. Figure 6 j), the bone quality around the implants in the HY-X3369 group was more mature, and the new bone was less than that in the DMSO group. Figure 6k). At 8 weeks, there was more mature bone (green) around the implants in the HY-X3369 group, while there were still a large number of bone resorption lacunae in the control group, and osteoid could be observed. Figure 6 l). The osteoid area in the HY-X3369 group was lower than that in the DMSO group. Figure 6 m). Micro-CT showed a high bone density around the implants in the HY-X3369 group. Figure 6 n). BV / TV, Tb.Th, and Tb.N in the HY-X3369 group were higher than those in the DMSO group, while Tb.Sp was lower than that in the DMSO group. Figure 6 o - r). The above research results indicate that HY-X3369 can maintain the protein stability of GLUL in BMSCs of T2DM and has an improving effect on the bone-implant bonding effect in T2DM mice.

[0127] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of GLUL overexpression or its promoter or activator in the preparation of a drug for promoting osteogenic differentiation ability; The GLUL is positively correlated with the osteogenic differentiation ability of T2DM patients.

2. The application according to claim 1, characterized in that, The promoting osteogenic differentiation ability includes functioning in a high glucose environment; Preferably, the high glucose environment includes the in vivo environment of T2DM patients; Preferably, the GLUL being positively correlated with the osteogenic differentiation ability of T2DM patients includes: (I), knocking out GLUL inhibits the osteogenic differentiation of BMSCs; (II), GLUL inhibits the osteogenic differentiation of BMSCs and / or implant osseointegration in a dose-dependent manner; More preferably, the knocking out GLUL inhibits the osteogenic differentiation of BMSCs includes: (i), knocking out GLUL reduces the ALP and ARS staining of BMSCs; (ii), knocking out GLUL reduces the ALP activity and calcium ion quantification of BMSCs; (iii), knocking out GLUL reduces the expression of osteogenic differentiation markers Runx2, ALP and / or OCN; More preferably, the GLUL inhibits the osteogenic differentiation of BMSCs and / or implant osseointegration in a dose-dependent manner includes: MSO reduces the GLUL protein level in the bone tissue around the implant and inhibits implant osseointegration in a dose-dependent manner.

3. Use of an inhibitor of SYVN1-mediated GLUL ubiquitination modification in the preparation of a drug for promoting the osteogenic differentiation of BMSCs; The SYVN1 interacts with GLUL; SYVN1 reduces the stability of GLUL protein by mediating the ubiquitination and degradation of GLUL.

4. The application according to claim 3, characterized in that The SYVN1 interacting with GLUL includes: In BMSCs of T2DM, the mRNA level of GLUL is low and the mRNA level of SYVN1 is high; In BMSCs of T2DM, the expression of GLUL is reduced and the expression of SYVN1 is increased, and as the glucose concentration increases, the level of SYVN1 increases while the level of GLUL decreases; In BMSCs of T2DM, the expression level of SYVN1 is high, then the expression level of GLUL is low; When the expression level of SYVN1 increases or decreases, the mRNA level of GLUL decreases or increases accordingly, and there is an obvious negative correlation between the two; Overexpressing the SYVN1 gene, it is found that the protein level of GLUL is down-regulated; SYVN1 has an inhibitory effect on the in vitro osteogenic differentiation of BMSCs, which is opposite to the function of GLUL; Preferably, the protein levels of osteogenic markers RUNX2, ALP, and OCN increase after knocking down SYVN1 and decrease after overexpressing SYVN1.

5. The application according to claim 3 or 4, characterized in that, The ubiquitination modification site of the GLUL includes K259 / 334A; The osteogenic differentiation includes functioning in a high glucose environment; The ligand of the GLUL includes HY-126351.

6. GLUL deubiquitinase-targeting chimeras (DUBTACs), characterized in that, Including a GLUL ligand, a deubiquitinase (DUB) ligand and a linker, the GLUL ligand and the deubiquitinase (DUB) ligand are connected by a linker.

7. The GLUL deubiquitinase-targeting chimera (DUBTAC) according to claim 6, wherein, The GLUL ligand includes HY-126351.

8. Use of the GLUL deubiquitinase-targeted chimera (DUBTAC) according to claim 6 or 7 in maintaining the stability of GLUL protein in BMSCs with T2DM; Preferably, maintaining the stability of GLUL protein in BMSCs with T2DM includes: The GLUL deubiquitinase-targeted chimera maintains the endogenous protein homeostasis of GLUL; The GLUL deubiquitinase-targeted chimera maintains the targeting ability of GLUL protein; The GLUL deubiquitinase-targeted chimera reduces the ubiquitin level of exogenous GLUL; More preferably, the GLUL deubiquitinase-targeted chimera reduces the ubiquitin level of exogenous GLUL elevated by SYVN1.

9. Use of the GLUL deubiquitinase-targeted chimera (DUBTAC) according to claim 6 or 7 in the preparation of a drug for promoting osseointegration or osteogenesis of diabetic implants.

10. The application according to claim 9, characterized in that, The promotion of osseointegration of diabetic implants includes: improving the osseointegration effect of implanted bone in T2DM mice.

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