WO2019085804A1

WO2019085804A1 - Methods and use for detecting and inhibiting cell-free mg53

Info

Publication number: WO2019085804A1
Application number: PCT/CN2018/111695
Authority: WO
Inventors: Rui-Ping Xiao; Yan Zhang; Fengxiang LV; Xinli HU
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2017-11-02
Filing date: 2018-10-24
Publication date: 2019-05-09
Anticipated expiration: 2020-05-02
Also published as: CN109932509A; CN109932509B; TW201923346A

Abstract

The present invention provides a method for detecting an MG53 related disease or predicting a risk of an MG53 related disease, comprising the following steps: a) obtaining a test sample; and b) detecting cell-free MG53 in the test sample. The present invention also provides a method for reducing or inhibiting activity of cell-free MG53 in a subject, comprising administrating an effective amount of MG53 inhibitor to a subject in need thereof. Also provided are an MG53 antibody, a nucleic acid encoding the antibody, a clone or expression vector including the nucleic acid, a host cell including the clone or expression vector, and a pharmaceutical composition including above.

Description

METHODS AND USE FOR DETECTING AND INHIBITING CELL-FREE MG53

FIELD OF THE INVENTION

The present disclosure relates to the art of biomedicine, specifically, the present disclosure relates to a method for detecting cell-free MG53 or detecting a related disease or predicting a risk of an MG53 related disease, and a method for reducing or inhibiting cell-free MG53 activity in a subject, and compositions used for the methods.

BACKGROUND

Mitsugumin 53 (MG53) , also known as TRIM72, is a member of the Tripartite motif-containing proteins (TRIM) family. MG53 comprises a TRIM motif at the N-terminus and a SPRY motif at the C-terminus, and the TRIM motif consists of successively linked Ring, B-box and coiled-coil domains (see Chuanxi Cai et al., the Journal of Biological Chemistry, Vol. 284 (5) , 3314-3322 (2009) ) . . MG53 plays a variety of roles throughout the body, but is mainly expressed in striated muscles, and is essential for maintaining the homeostasis of skeletal muscle and the heart. MG53 was previously found to have cell membrane repair function and cardioprotective function (see, e.g., Chuanxi Cai et al., Nature Cell Biology, Vol. 11, 56-64 (2009) ; CN101797375B) . In addition, further studies have found that MG53 also plays a protective role in ischemic preconditioning (IPC) and ischemic postconditioning (PostC) , by activation of the reperfusion injury salvage kinase (RISK) pathway. The N-and C-termini of the MG53 molecule can bind to Caveolin-3 and P85-PI3K kinases respectively to form a complex, which activates the RISK pathway to elicit cardiac protection (see Chun-Mei Cao et al., Circulation 121, 2565-2574, (2010) ) .

Previous studies found that MG53 has E3 ubiquitin ligase activity, which contributes tothe development of insulin resistance and metabolic syndrome. The Ring domain of the TRIM motif at the N-terminus of MG53 binds to insulin receptor (IR) and insulin receptor substrate-1 (IRS1) , and mediates the ubiquitination and subsequent degradation of these proteins by the proteasome, thereby blocking the insulin signaling pathway and leading to insulin resistance and associated metabolic diseases such as obesity, diabetes, hypertension, dyslipidemia, etc. (see, for example, R. Song et al. Nature 494, 375-379, (2013) ; J.S. Yi et al., Nature Communications 4, 2354 (2013) ) .

However, those skilled in the art commonly consider MG53 to have fuctions relating to insulin resistance and metabolic syndrome only within cells.

BRIEF SUMMARY OF THE INVENTION

The present disclosure relates to a method for detecting an MG53 related disease or predicting a risk of an MG53 related disease, and a method for reducing or inhibiting cell-free MG53 activity in a subject. Additionally, the present disclosure also relates to an MG53 antibody or antigen-binding fragment thereof, a nucleic acid encoding such antibody or antigen-binding fragment thereof, a clone or expression vector including such nucleic acid, a host cell including the clone or expression vector, and a pharmaceutical composition including the products above. Furthermore, the present disclosure also relates to use of a detection agent for detecting cell-free MG53 in the manufacture of a kit, and use of the MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting cell-free MG53 in a subject.

In one aspect, the present disclosure relates to a method for detecting an MG53 related disease or predicting a risk of an MG53 related disease, comprising the following steps: a) obtaining a test sample; and b) detecting cell-free MG53 in the test sample.

In certain embodiments, the test sample is body fluid. In certain embodiments, the test sample is selected from whole blood, plasma, serum, tissue fluid, urine and sweat. In certain embodiments, the test sample is selected from whole blood, plasma or serum. In certain embodiments, the test sample substantially does not contain any cells.

In certain embodiments, the step b) comprises contacting an MG53 detection agent with the test sample. In certain embodiments, the MG53 detection agent is an MG53 antibody or antigen-binding fragment thereof, or an MG53 ligand or an MG53-binding fragment thereof. In certain embodiments, the MG53 detection agent is a small molecule compound capable of binding MG53. In certain embodiments, the MG53 detection agent has a detectable label. In certain embodiments, the detectable label is luminescent, magnetic, radioactive, or enzymatically active. In certain embodiments, the step b) comprises conducting radioimmunoassay, Western blot analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation, chemiluminescence, immunohistochemistry assay, dot blot assay or slit blot method.

In certain embodiments, the method of the present disclosure further comprises: c) comparing the detected value of MG53 obtained from step b) with a reference value. In certain embodiments, the reference value is obtained from a reference sample. In certain embodiments, the reference sample and the test sample are from the same subject or different subjects. In certain embodiments, the reference sample and the test sample are from the same subject, but the time at which the reference sample was collected is earlier or later for a period of time than the test sample. In certain embodiments, during the period of time, the subject accepts treatment or the health condition thereof has changed. In certain embodiments, the reference sample is obtained from a healthy subject or a subject suffering from an MG53 related disease. In certain embodiments, the subject is a human or a non-human mammal.

In certain embodiments, the MG53 related disease is metabolic syndrome. In certain embodiments, the MG53 related disease is a glucose metabolism or lipid metabolism related disease. In certain embodiments, the glucose metabolism or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complication, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension and obesity.

In another aspect, the present disclosure relates to a method for reducing or inhibiting activity of cell-free MG53 in a subject, comprising administrating an effective amount of MG53 inhibitor to a subject in need thereof.

In certain embodiments, the MG53 inhibitor specifically binds to MG53.

In certain embodiments, the MG53 inhibitor is an MG53 antibody or antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof, or a nonfunctional protein fragment of MG53. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions comprised in a sequence of SEQ ID NOs: 1, 9, 17, 25 or 33. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises three light chain complementary determining regions comprised in a sequence of SEQ ID NOs: 2, 10, 18, 26 or 34. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain, and the heavy chain comprises a sequence of SEQ ID NOs: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a light chain, and the light chain comprises a sequence of SEQ ID NOs: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In certain embodiments, the MG53 inhibitor is a small molecule compound. In certain embodiments, the MG53 inhibitor is a compound having Formula Ia or Formula Ib, or a pharmaceutically acceptable salt thereof,

wherein, L is a chemical bond, or an optionally substituted C ₁-C ₁₂ alkylene; M is a chemical bond, or an optionally substituted C ₆-C ₁₂ arylene or a 5-12 membered heterocyclic subunit; X is a chemical bond, or an optionally substituted -CH=N-NH-C (O) -, -CH ₂-O-, -O-CH ₂-C (O) -NH-, -O-C (O) -or -O-C (O) -CH=CH-; Y is an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group; Q is an optionally substituted C ₁-C ₁₂ alkylene or C ₂-C ₁₂ alkenylene; T is an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group; each R ₁ is independently hydrogen, nitro, halogen, hydroxyl, cyano, or an optionally substituted C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy; n is any integral from 1 to 5.

In certain embodiments, L is a chemical bond or C ₁-C ₃ alkylene. In certain embodiments, M is a chemical bond, or an optionally substituted phenylene or furylene. In certain embodiments, Y is an optionally substituted phenyl, furyl, quinolinyl or benzo dioxolanyl. In certain embodiments, Q is an optionally substituted C ₁-C ₃ alkylene or C ₂-C ₃ alkenylene. In certain embodiments, T is an optionally substituted phenyl or 1, 3, 4-thiadiazolyl.

In certain embodiments, the MG53 inhibitor is a compound having Formula II or a pharmaceutically acceptable salt thereof,

wherein, A is hydrogen, or an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group; G is -C (O) -O-R ₃, wherein R ₃ is an optionally substituted C ₁-C ₁₂ alkyl; J is an optionally substituted C ₆-C ₁₂ aryl; or G and J, together with the carbon atom linked thereto, form

wherein each R ₄ is independently hydrogen, nitro, halogen, hydroxyl, cyano, or an optionally substituted C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy, n is any integral from 1 to 4; B is a chemical bond, or an optionally substituted C ₆-C ₁₂ arylene or 5-12 membered heterocyclic subunit; D is a chemical bond, or an optionally substituted -O-R ₅-, wherein R ₅ is optionally substituted C ₁-C ₁₂ alkylene; E is an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group.

In certain embodiments, A is an optionally substituted naphthyl, phenyl or thienyl. In certain embodiments, G is -C (O) -O-R ₃, wherein R ₃ is an optionally substituted C ₁-C ₃ alkyl; J is optionally substituted phenyl; or G and J, together with the carbon atom linked thereto, form

wherein each R ₄ is hydrogen. In certain embodiments, B is a chemical bond, or an optionally substituted phenylene or furylene. In certain embodiments, D is a chemical bond, or an optionally substituted -O-CH ₂-. In certain embodiments, E is an optionally substituted phenyl.

In certain embodiments, the MG53 inhibitor is a compound having Formula III or a pharmaceutically acceptable salt thereof,

wherein, A is hydrogen, or an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group; each Y is independently optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group; R ₇ is hydrogen, nitro, halogen, hydroxyl, cyano, or optionally substituted C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy.

In certain embodiments, A is an optionally substituted phenyl. In certain embodiments, Y is an optionally substituted phenyl. In certain embodiments, R ₇ is hydrogen, nitro, halogen, hydroxyl or cyano.

In certain embodiments, the “optionally substituted” mentioned in any of the embodiments above refers to not substituted by any substituent group or substituted by one or more substituent groups selected from the followings: nitro, halogen, hydroxyl, cyano, C ₁-C ₁₂ alkyl, C ₁-C ₁₂ alkoxy, benzamide group, or -C (O) -O-R ₆, wherein the C ₁-C ₁₂ alkyl, C ₁-C ₁₂ alkoxy or benzamide group can further be substituted by nitro, halogen, hydroxyl, cyano, C ₁-C ₃ alkyl or C ₁-C ₃ alkoxy, wherein R ₆ is hydrogen or C ₁-C ₃ alkyl.

In certain embodiments, the MG53 inhibitor is a compound of the followings or a pharmaceutically acceptable salt thereof.

In certain embodiments, the method of the present disclosure can be used to ameliorate, treat or predict an MG53 related disease or a symptom thereof. In certain embodiments, the MG53 related diseased is metabolic syndrome. In certain embodiments, the MG53 related diseased is a glucose metabolism or lipid metabolism related disease. In certain embodiments, the glucose metabolism or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complication, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension and obesity.

In certain embodiments, diabetic cerebrovascular disease includes cerebral arteriosclerosis, ischemic cerebrovascular diseases, cerebral hemorrhage, encephalatrophy and cerebral infarction.

In certain embodiments, diabetic ocular complication includes diabetic retinopathy, diabetic cataract, and uveitis and blindness associated with diabetes.

In certain embodiments, diabetic neuropathy includes diabetic peripheral neuropathy.

In another aspect, the present disclosure relates to an MG53 antibody or antigen-binding fragment thereof, comprising three heavy chain complementary determining regions comprised in a sequence of SEQ ID NOs: 1, 9, 17, 25 or 33. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises three light chain complementary determining regions comprised in a sequence of SEQ ID NOs: 2, 10, 18, 26 or 34. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain has an amino acid sequence of SEQ ID NOs: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises further comprises a light chain, wherein the heavy chain has an amino acid sequence of SEQ ID NOs: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In another aspect, the present disclosure relates to an antibody or antigen-binding fragment, competing for binding to MG53 with the antibody or antigen-binding fragment of any of the embodiments above.

In another aspect, the present disclosure relates to an isolated nucleic acid, encoding the antibody or antigen-binding fragment of any of the embodiments above. In certain embodiments, the isolated nucleic acid comprises a nucleotide sequence of SEQ ID NOs: 5, 6, 7, 8, 13, 14, 15, 16, 21, 22, 23, 24, 29, 30, 31, 32, 37, 38, 39 or 40, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In another aspect, the present disclosure relates to a clone or expression vector, comprising the isolated nucleic acid of any of the embodiments above.

In another aspect, the present disclosure relates to a host cell, comprising the clone or expression vector of any of the embodiments above.

In yet another aspect, the present disclosure relates to a pharmaceutical composition, comprising the antibody or antigen-binding fragment of any of the embodiments above, the clone or expression vector of any of the embodiments above, or the cell of any of the embodiments above, and a pharmaceutically acceptable excipient.

In another aspect, the present disclosure relates to use of a detection agent for detecting cell-free MG53 in the manufacture of a kit, which is used for detecting an MG53 related disease, predicting a risk or development of an MG53 related disease, identifying potential MG53 inhibitor, evaluating therapeutic effect for an MG53 related disease, or detecting activity of cell-free MG53. In certain embodiments, the detection agent for detecting cell-free MG53 can be the MG53 detection agent of any of the embodiments above. In certain embodiments, the MG53 related disease can be any MG53 related diseases of any of the embodiments above.

In another aspect, the present disclosure relates to use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting cell-free MG53 in a subject. In certain embodiments, the MG53 inhibitor can be the MG53 inhibitor of any of the embodiments above. In certain embodiments, the method can be used to ameliorate, treat or predict an MG53 related disease or a symptom thereof. In certain embodiments, the MG53 related disease can be any MG53 related diseases of any of the embodiments above.

DESCRIPTION OF THE DRAWINGS

Figure 1: Pattern diagram of the molecular structure of wild type MG53.

Figure 2: Chart showing results of SPR experiment for detecting affinity between an MG53 antibody and antigen protein. K _D of the antibody =17.7 nM.

Figure 3: a. Chart showing results of SPR experiment for detecting affinity between MG53 and insulin receptor extracellular region. K _D of MG53 and insulin receptor extracellular region =8.0 nM; b. Chart showing results of SPR experiment for detecting binding of MG53 to insulin receptor extracellular region blocked by an MG53 antibody. Ki of the antibody=265 nM.

Figure 4: Detection of MG53 content in serum of MG53 ^-/-mice (MG53 gene knocked out) and WT mice by Western blotting assay, using an antibody of the present disclosure as the primary antibody. The upper panel is the image of Western blots, and the lower panel is bar graph chared based on values calculated from the Western blot image, wherein **represents that P<0.01, and that the difference is significant. t-test was used for statistical tests.

Figure 5: Detection of MG53 content in serum of TG mice (MG53 gene overexpressed) and WT mice by Western blotting assay, using an antibody of the present disclosure as the primary antibody. The upper panel is the image of Western blots, and the lower panel is bar graph chared based on values calculated from the Western blot image, wherein **represents that P<0.01, and that the difference is significant. t-test was used for statistical tests.

Figure 6: Detection of recombinant mouse MG53 and recombinant human MG53 by Western blotting assay, using an antibody of the present disclosure as the primary antibody.

Figure 7: Detection of MG53 content in serum of Type II diabetes patients and normal people by Western blotting assay, using an antibody of the present disclosure as the primary antibody. The upper panel is the image of Western blots, and the lower panel is bar graph chared based on values calculated from the Western blot image, wherein **represents that P<0.01, and that the difference is significant. t-test was used for statistical tests.

Figure 8: Detection of MG53 content in serum of mice having high-fat diet and mice having normal diet by Western blotting assay, using an antibody of the present disclosure as the primary antibody. The upper panel is the image of Western blots, and the lower panel is bar graph chared based on values calculated from the Western blot image, wherein **represents that P<0.01, and that the difference is significant. t-test was used for statistical tests.

Figure 9: a. Content of serum MG53 of fasted normal rats and fasted ZDF rats; b. Chart showing correlation between concentration of serum MG53 and body weight in fasted normal rats and fasted ZDF rats; c. Chart showing correlation between concentration of serum MG53 and blood glucose level in fasted normal rats and fasted ZDF rats; d. Chart showing correlation between concentration of serum MG53 and serum insulin level in fasted normal rats and fasted ZDF rats. Wherein, **represents that P<0.01, and that the difference is significant. t-test was used for statistical tests.

Figure 10: Concentration of GAPDH (control) , Akt, and Akt post insulin-induced phosphorylation, detected in different tissues (skeletal muscle, liver, visceral fat, and the heart) in mice injected or not injected with insulin, and followed by injection of BSA or rhMG53. The upper panels are images of Western blots, and the lower panels are bar graphs chared based on values calculated from the Western blot images, wherein **represents that P<0.01, and that the difference is significant. One-way anova was used for statistical tests.

Figure 11: a. Content of serum MG53 in normal mice and diabetic model mice; b. Blood glucose levels in the control group (administered with IgG) and the treatment group (administered with MG53 antibodies) ; c. Blood glucose levels in the control group (administered with IgG) and the treatment group (administered with MG53 antibodies) in insulin tolerance test. Wherein, **represents that P<0.01, and that the difference is significant; *represents that P<0.05, and that the difference is significant. t-test was used for statistical tests.

Figure 12: IRS1 plasmid (IRS1-GFP) and MG53 plasmid (MG53-myc) with Myc label were co-transfected in HEK293T cells and HEK293A cells respectively, and fluorescence intensity of GFP was detected, wherein **represents that P<0.01, and that the difference is significant; *represents that P<0.05, and that the difference is significant. t-test was used for statistical tests.

Figure 13: Response values in SPR assays for selection of different candidate molecules

Figure 14: a. Response value for compound No. 1 in the SPR assay; b. Response value for compound No. 10 in the SPR assay; c. Response value for compound No. 26 in the SPR assay; d. Response value for compound No. 16 in the SPR assay.

Figure 15: IRS1 plasmid (IRS1-GFP) and MG53 plasmid (MG53-myc) with Myc label were co-transfected in HEK293T, and compounds Nos. 1, 10, 26 and 16 were added respectively, and then fluorescence intensity of GFP was detected. ***represents that P<0.001, and that the difference is significant. One-way anova was used for statistical tests.

Figure 16: This figure illustrates the amino acid sequence SEQ ID NO: 3 of heavy chain of Antibody #6 and its encoding nucleic acid sequence SEQ ID NO: 7. The sequences marked by shadow corresponds to the variable region of heavey chain of Antibody #6.

Figure 17: This figure illustrates the amino acid sequence SEQ ID NO: 4 of light chain of Antibody #6 and its encoding nucleic acid sequence SEQ ID NO: 8. The sequences marked by shadow corresponds to the variable region of light chain of Antibody #6.

Figure 18: This figure illustrates the amino acid sequence SEQ ID NO: 11 of heavy chain of Antibody #110 and its encoding nucleic acid sequence SEQ ID NO: 15. The sequences marked by shadow corresponds to the variable region of heavey chain of Antibody #110.

Figure 19: This figure illustrates the amino acid sequence SEQ ID NO: 12 of light chain of Antibody #110 and its encoding nucleic acid sequence SEQ ID NO: 16. The sequences marked by shadow corresponds to the variable region of light chain of Antibody #110.

Figure 20: This figure illustrates the amino acid sequence SEQ ID NO: 19 of heavy chain of Antibody #84 and its encoding nucleic acid sequence SEQ ID NO: 23. The sequences marked by shadow corresponds to the variable region of heavey chain of Antibody #84.

Figure 21: This figure illustrates the amino acid sequence SEQ ID NO: 20 of light chain of Antibody #84 and its encoding nucleic acid sequence SEQ ID NO: 24. The sequences marked by shadow corresponds to the variable region of light chain of Antibody #84.

Figure 22: This figure illustrates the amino acid sequence SEQ ID NO: 27 of heavy chain of Antibody #9 and its encoding nucleic acid sequence SEQ ID NO: 31. The sequences marked by shadow corresponds to the variable region of heavey chain of Antibody #9.

Figure 23: This figure illustrates the amino acid sequence SEQ ID NO: 28 of light chain of Antibody #9 and its encoding nucleic acid sequence SEQ ID NO: 32. The sequences marked by shadow corresponds to the variable region of light chain of Antibody #9.

Figure 24: This figure illustrates the amino acid sequence SEQ ID NO: 35 of heavy chain of Antibody #43 and its encoding nucleic acid sequence SEQ ID NO: 39. The sequences marked by shadow corresponds to the variable region of heavey chain of Antibody #43.

Figure 25: This figure illustrates the amino acid sequence SEQ ID NO: 36 of light chain of Antibody #43 and its encoding nucleic acid sequence SEQ ID NO: 40. The sequences marked by shadow corresponds to the variable region of light chain of Antibody #43.

Figure 26: This figure illustrates the amino acid sequence SEQ ID NO: 41 of human wild-type MG53, the amino acid sequence SEQ ID NO: 42 of monkey wild-type MG53, the amino acid sequence SEQ ID NO: 43 of rat wild-type MG53, and the amino acid sequence SEQ ID NO: 44 of mouse wild-type MG53.

Figure 27: This figure illustrates the amino acid sequence SEQ ID NO: 45 of swine wild-type MG53, the amino acid sequence SEQ ID NO: 46 of dog wild-type MG53, the amino acid sequence SEQ ID NO: 47 of rabbit wild-type MG53, the amino acid sequence SEQ ID NO: 47 of Marmosets wild-type MG53, and the amino acid sequence SEQ ID NO: 61 of Callithrix jacchus wild-type MG53.

Figure 28: This figure illustrates the sequences of MG53 mutant (SEQ ID NO: 48-50) .

Figure 29: This figure illustrates the sequences of MG53 mutant (SEQ ID NO: 51-53) .

Figure 30: This figure illustrates the sequences of MG53 mutant (SEQ ID NO: 54-57) .

Figure 31: This figure illustrates the sequence of insulin receptor (SEQ ID NO: 58) .

Figure 32: This figure illustrates high glucose-and insulin-induced MG53 release in Langendorff-perfused rodent hearts. Figure 32A shows representative Western blots (upper panels) and averaged data (lower panels) showing that high glucose increased MG53 levels in perfusate in a time-dependent manner in isolated perfused rat heart (n=12 hearts) . Figure 32B shows representative Western blots (upper panels) and averaged data (lower panels) showing that high glucose increased MG53 levels in perfusate in a concentration-dependent manner in isolated perfused rat heart (n=10 hearts) . Figure 32C shows perfusate MG53 in isolated rat heart perfused with high D-glucose (25 mM) or L-glucose (13.9 mM L-glucose in the presence of basal D-glucose at 11.1 mM) (n=8/group) ; data were obtained under basal conditions (0 min, 11.1 mM D-glucose) and at 30 min after treatment. Figure 32D is as in Figure 32A except with insulin stimulation (n=11) . Figure 32E is as in Figure 32B except with insulin stimulation (n=6) . Figure 32F shows metabolic stimulation with high glucose (25 mM) combined with insulin (10 U/L) (G+I) in isolated perfused rat heart (n=15) . Figure 32G and Figure 32H show that perfusate MG53 was more abundant in MG53 TG mouse heart than in wt control heart under basal and metabolically-stimulated conditions (G+I, 25 mM glucose combined with 10 U/L insulin) (Figure 32G) , but was undetectable in mg53 ^-/-mouse heart (Figure 32H) (n = 8/group) . In all bar graphs, data are normalized to the corresponding non-specific bands (NS) which were obtained through Brilliant Green staining, and are presented as mean±s.e.m. (*P <0.05, **P <0.01) ; one-way ANOVA for A-E, G and H and t-test for F. The control of each bar graph is the 1st lane of each gel.

Figure 33: This figure illustrates high glucose-and insulin-induced MG53 secretion in isolated skeletal muscle and in vivo. Figure 33A shows representative Western blots (upper panels) and averaged data (lower panels) showing MG53 level in the perfusate of mouse soleus muscle with or without a high concentration of glucose (25 mM) and insulin (10 U/L) (G+I) treatment. (n=12) . Figure 33B shows representative Western blots and averaged data showing hyperglycemia (glucose, 2 g/kg, i.p., 3 repeats at 40-min intervals) increased serum MG53 in wt mice (n = 8) . Figure 33C shows representative Western blots and averaged data showing serum MG53 from healthy humans before and after oral glucose administration (75 g twice at a 30-min interval) (n=8) . Figure 33D shows that BFA dose-dependently suppressed MG53 release in C2C12 cells. Cells were infected with Ad-b-gal or Ad-MG53 and incubated with high glucose (25 mM) for 3 h (n = 8) . Figure 33E shows that BFA (30 mM) blocked the increase of perfusate MG53 induced by high glucose (25 mM) combined with insulin (10 U/L) (G+I) in isolated perfused rat heart (n = 10) . Figure 33F shows that EDTA-Na (0.75 mM) blocked the increase of perfusate MG53 induced by metabolic stress (G+I, 25 mM glucose combined with 10 U/L insulin) in isolated perfused rat heart (n = 10) . Figure 33G shows that Metabolic stimulation (G+I, 25 mM glucose combined with 10 U/L insulin) elevated MG53 release from wt but not syt7 ^-/-mouse heart (n = 10) . Figure 33H shows that Hyperglycemia (glucose, 2 g/kg, i.p., 3 repeats at 40-min intervals) increased serum MG53 in wt but not syt7 ^-/-mice (n = 10) . In all lower panels, data are normalized to the corresponding non-specific bands (NS) which were obtained through Brilliant Green staining, and are presented as mean±s.e.m. (*P <0.05, **P <0.01) ; t-test for A, and one-way ANOVA for B-H.

Figure 34: This figure illustrates cardiac-specific overexpression of MG53 leads to systemic insulin resistance and metabolic syndrome. Figure 34A shows representative Western blots and averaged data showing serum MG53 levels in wt and transgenic mice with cardiac-specific overexpression of MG53 (mg53 h-TG) at 14 weeks of age (n = 12 for wt and 14 for mg53 h-TG) . Figure 34B shows fasting body weights of wt and mg53 h-TG mice from 3 to 38 weeks of age (n = 28 for wt and 22 for mg53 h-TG) . Figure 34C shows glucose tolerance tests in wt and mg53 h-TG mice at the indicated ages (14 weeks, n = 9 for both wt and mg53 h-TG; 30 weeks, n = 7 for wt and 6 for mg53 h-TG) . Figure 34D shows insulin tolerance tests in wt and mg53 h-TG mice at the indicated ages (14 weeks, n = 9 for wt and 10 for mg53 h-TG; 30 weeks, n = 8 for wt and 7 for mg53 h-TG) . Figure 34 E shows blood glucose at 30 weeks of age (n = 11 for wt and 13 for mg53 h-TG) . Figure 34F shows fasting serum insulin at 30 weeks of age (n = 11 for wt and 13 for mg53 h-TG) . Figure 34G shows fasting serum triglyceride levels at 30 weeks of age (n = 11 for wt and 13 for mg53 h-TG) . Figure 34H shows potographs taken during necropsy revealing fat deposit in the abdominal cavity of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34I shows subcutaneous and visceral fat weight of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34J shows brown fat weight of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34K shows body lean and fat mass of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34L shows body lean and fat mass as a percentage of body weight of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34M shows haematoxylin and eosin staining of white and brown fat of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34N shows haematoxylin and eosin staining of liver of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34O shows haematoxylin and eosin staining of pancreas of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Figure 34P shows haematoxylin and eosin staining of skeletal muscle of wt and mg53 h-TG mice at 30 weeks of age (n = 11 for wt and 14 for mg53 h-TG) . Scale bars for white fat, 50 μm; brown fat, 25 μm; liver, 100 μm; and pancreas 100 μm. Figure 34Q shows oxygen consumption of wt and mg53 h-TG mice at 30 weeks of age (n = 12) . Figure 34R shows CO ₂ production of wt and mg53 h-TG mice at 30 weeks of age (n = 12) . Figure 34S shows daily energy expenditure of wt and mg53 h-TG mice at 30 weeks of age (n = 12) . Data are mean±s.e.m. *P <0.05, **P <0.01 vs wt; t-test.

Figure 35: This figure illustrates high glucose-and insulin-induced release of MG53 from striated muscle. Figure 35A shows perfusate lactate dehydrogenase (LDH, n=10) from the perfusate of mouse heart with or without high glucose (25 mM) and insulin (10 U/L) stimulation for 30 min. Figure 35A shows creatine kinase (CK, n-8) from the perfusate of mouse heart with or without high glucose (25 mM) and insulin (10 U/L) stimulation for 30 min. Figure 35C shows LDH level from the perfusate of mouse skeletal muscle (soleus) with or without high glucose (25 mM) and insulin (10 U/L) treatment (n=10) . Figure 35D shows CK level from the perfusate of mouse skeletal muscle (soleus) with or without high glucose (25 mM) and insulin (10 U/L) treatment (n=10) . Data are mean±s.e.m.

DETAILED DESCRIPTION OF THE INVENTION

Although the present disclosure will disclose multiple aspects and embodiments below, without departing from the spirit and scope of the invention, a person skilled in the art may make various equivalent changes and modification thereon. The multiple aspects and embodiments disclosed by the present disclosure are illustrative only, and are not intended to limit the present disclosure. The actual scope of protection of the present disclosure is determined by reference to the claims. Unless indicated otherwise, all technological and scientific terminologies possess the meaning as an ordinarily skilled artisan in the art commonly understands. All reference literatures, patents and patent applications cited in the present disclosure are incorporated herein by reference in their entirety.

Method for detecting an MG53 related disease or predicting a risk of an MG53 related disease

MG53

The term “MG53” as used herein refers to naturally existing wild-type MG53 expressed in animals and an MG53 mutant.

MG53 protein exists in many animals, including but not limited to human, ape, monkey, swine, dog, rabbit, rat, mouse, and other mammals. MG53 protein is a multi-functional protein with a structure shown in Figure 1. The full-length MG53 proteins of different species are slightly different in length, but generally has about 477 amino acids, comprising a TRIM motif at the N-terminus and a SPRY motif at the C-terminus, with the TRIM motif consisting of successively linked Ring, B-box and coiled-coil domains (RBCC) . MG53 protein is one of the important components for membrane repair, which plays an essential role in the pre-conditioning and post-conditioning protection of ischemia-reperfusion injury. Meanwhile, the high expression of MG53 protein may also cause insulin resistance and metabolic syndrome. The structure and function of MG53, as well as its interaction with other proteins have been reported in detail in the art (see, e.g., Chuanxi Cai et al., Journal of Biological Chemistry, 284 (5) , 3314-3322, (2009) ; Xianhua Wang et al., Circulation Research 107, 76-83, (2010) ; Eun Young Park et al., Proteins, 790-795 (2009) ) .

In certain embodiments, the amino acid sequence of the wild-type MG53 is set forth in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 61, which corresponds to the human, Macaca mulatta /M. fascicularis, rat, mouse, swine, dog, rabbit, and Callithrix jacchus wild-type MG53, respectively. In certain embodiments, the term “MG53” as used herein refers to MG53 derived from the subject to be detected or treated.

Test sample

The term “test sample” as used herein refers to a sample obtained from a subject in vivo, or obtained from a subject in vitro, or from an in vitro sample, including any material from a subject which a person skilled in the art considers to contain, or which may contain, cell-free MG53. In certain embodiments, the test sample is a blood sample. In certain embodiments, the test sample is body fluid. In certain embodiments, the test sample is selected from whole blood, plasma, serum, tissue fluid, urine, or sweat. In certain embodiments, the test sample is selected from whole blood, plasma, or serum. In certain embodiments, the test sample may further comprise other substances, such as preservatives, anticoagulants, buffers, fixatives, nutrients and antibiotics, etc.

In certain embodiments, the test sample substantially does not contain any cells, e.g., does not contain any cardiac muscle cell, striated muscle cell, or blood cell. The term “substantially does not contain” refers to not containing a particular substance in the amount which is more than a particular amount, or more than the remaining amount after conducting common impurity removal technology of the art, or any particular substance substantially affecting functions of other substances. For example, that the sample substantially does not contain cells may refer to that the sample does not contain cells in excess of a particular amount (e.g. no more than 1000, 800, 500, 200, 100, 50, 20, 10, 5 or 1 cells per millimeter) ; or that the sample does not contain cells in the number of those generally remaining in the sample after conducting routine separating means (e.g., supernatant following centrifugation) . The sample may be obtained using any method known in the art. For example, a blood sample may be obtained using vacuum or normal pressure blood collection needle. In certain embodiments, prior to being tested, the collected blood sample may be further treated, e.g., centrifuged.

MG53 detection agent

In certain embodiments, the MG53 detection agent is an MG53 antibody or antigen-binding fragment thereof, or an MG53 ligand or an MG53-binding fragment thereof. Any MG53 antibody or antigen-binding fragment thereof, or MG53 ligand or MG53-binding fragment thereof described by the present disclosure, or a combination thereof, can be used as an MG53 detection agent.

In certain embodiments, the MG53 detection agent is a small molecule compound capable of binding MG53. Any small molecule compound capable of binding MG53 described by the present disclosure, or a combination thereof, can be used as an MG53 detection agent.

In certain embodiments, the MG53 detection agent further has a detectable label. In certain embodiments, the detectable label is luminescent, magnetic, radioactive, or enzymatically active. Examples for detectable labels include fluorescence labels, enzyme labels, radioisotope labels, chemiluminescence labels, electrochemiluminescence labels, metal particle labels and hapten labels.

Examples for fluorescence labels include 5- (6) -carboxy fluorescein, 5-or 6-carboxy fluorescein, 6- (fluorescein) -5- (6) -carboxyamide hexanoic acid, isothiocyanate fluorescein, rhodamine, tetramethyl rhodamine, dye (such as Cy2, Cy3 and Cy5) , optionally substituted coumarin (e.g., AMCA, PerCP) , phycobiliprotein (e.g., R-phycoerythrin (RPE) and allophycocyanin (APC) ) , Texas Red, Princeton Red, green fluorescent protein (GFP) and analogs thereof, R-phycoerythrin and allophycocyanin conjugate and inorganic fluorescence label (such as semiconductor material based particles, e.g., particles coated with CdSe nanocrystalline) .

Examples for enzyme labels include horseradish peroxidase (HRP) , alkaline phosphatase (ALP or AP) , β-galactosidase (GAL) , glucose-6-phsphate dehydrogenase, β-N-acetyl glucosaminidase, β-glucuronidase, saccharase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO) .

Examples of common substrates for horseradish peroxidase include 3, 3'-diaminobenzidine (DAB) , nickel intensified diaminobenzidine, 3-amino-9-ethyl carbazole (AEC) , benzidine dihydrochloride (BDHC) , Hanker-Yates reagent (HYR) , indophenol blue (IB) , tetramethyl benzidine (TMB) , 4-chloro-1-naphthol (CN) , α-naphthol pyronine (α-NP) , o-dianisidine (OD) , 5-bromo-4-chloro-3-indole phosphate (BCIP) , nitroblue tetrazolium (NBT) , 2- (iodophenyl) -3-p-nitrophenyl-l-5-phenyl tetrazole chloride (INT) , tetranitroblue tetrazolium (TNBT) , and 5-bromo-4-chloro-3-indoxyl-β-D-galactosidase/Fe-ferricyanide (BCIG/FF) .

Examples of common substrates for alkaline phosphatase include naphthol-AS-B 1-phosphate/fast red TR (NABP/FR) , naphthol-AS-MX-phosphate/fast red TR (NAMP/FR) , naphthol-AS-B1-phosphate/fast red TR (NABP/FR) , naphthol-AS-MX-phosphate/fast red FR (NAMP/FR) , naphthol-AS-B1-phosphat/new fuschin (NAMP/NF) , bromochloro indole phosphate/nitroblue tetrazolium (BCIP/NBT) and 5-bromo-4-chloro-3-indolyl-b-d-pyran galactosidase (BCIG) .

Examples for radioisotope labels include isotopes of iodine, cobalt, selenium, tritium, carbon, sulfur and phosphorus.

Examples of chemiluminescence labels include luminol, isoluminol, acridinium ester, 1, 2-dioxolane, and pyridopyridazine.

Examples of electrochemiluminescence labels include ruthenium derivatives.

Examples of metal particle labels include gold particles and gold coating particles, which can be transformed by silver staining.

Example of hapten labels include DNP, fluorescein isothiocyanate (FITC) , biotin and digoxin.

In certain embodiments, exemplary detectable markers have one or more of the following structures.

These detectable markers can be linked to the MG53 detection agent by means of covalent binding, affinity binding, embedding, complexing, binding, mixing or adding, etc.

Detecting methods

Step b) detecting cell-free MG53 in the test sample, can be conducted using conventional methods of the art. In certain embodiments, these methods include but are not limited to radioimmunoassay, Western blot analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation, chemiluminescence, immunohistochemistry assay, dot blot assay or slit blot method.

A person skilled in the art is familiar with ordinary technologies and other variants of the technologies in the conduction of the various of detecting methods above, and is also capable of using a method above alone or in combination or alternatively with nuclear magnetic resonance (NMR) , matrix assisted laser desorption ionization-time of flight (MALDI-TOF) , liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) , etc.

In one embodiment, enzyme immunoassay is a sandwich enzymoimmunoassay, which detects cell-free MG53 using antibodies that specifically bind to MG53. A typical sandwich enzymoimmunoassay includes the following steps: a) linking an antibodies that specifically bind to cell-free MG53 to solid phase supports, to form solid phase antibodies, and removing non-bound antibodies and impurities by washing; b) adding a test specimen, which is contacted with the solid phase antibodies for certain time to form solid phase antibody-antigen complexes, and removing other non-bound substances by washing; c) adding enzyme-labeled antibodies which can bind to cell-free MG53, which is contacted with the solid phase antibody-antigen complexes for certain time sufficient for binding to the solid phase antibody-antigen complexes, and removing non-bound enzyme-labeled antibodies by washing; d) adding the substrate for the enzyme label, which is catalyzed by the enzyme label under proper condition, and determining the amount of cell-free MG53 according to the extent to which the substrate reacts.

Data processing

The detecting method of the present disclosure may further comprise a step of data processing. In certain embodiments, the method of the present disclosure further comprises: c) comparing the detected value of MG53 obtained from step b) with a reference value.

In certain embodiments, the reference value comes from a reference sample. In certain embodiments, the reference sample and the test sample are from different subjects. For example, the reference sample may come from a healthy subject or a subject suffering from an MG53 related disease, while the test sample comes from a subject suffering from or suspected to suffer from an MG53 related disease.

In certain embodiments, the reference sample and the test sample are from the same subject, but the reference sample is collected at an earlier or later time period than the test sample. In certain embodiments, during the interval between the collection of the reference sample and test sample, the subject accepts treatment or the health condition thereof has changed. In some other embodiments, the reference sample and the test sample are collected from different parts of the same subject.

In certain embodiments, the reference value is obtained from a healthy subject or a subject confirmed to suffer from an MG53 related disease. In certain embodiments, the reference value is characterized in the form of concentration of MG53.

In certain embodiments, these reference values can be negative reference values, being equal to or lower than which indicates that the subject which the test sample derives from is not suffering from MG53 related diseases or is at low risk of suffering from an MG53 related disease. In certain embodiments, the negative reference value can be set according to the mean value of MG53 detection values obtained from certain number of healthy subjects, e.g., setting such mean value or 80%, 90%, 110%, 120%, 150%or 200%of such mean value as the negative reference value.

In some other embodiments, these reference values can be positive reference values, being equal to or higher than which indicates that the subject which the test sample derives from is suffering from an MG53 related disease or is at high risk of suffering from an MG53 related disease. In certain embodiments, the positive reference value can be set according to the mean value of MG53 detection values obtained from certain number of subjects confirmed to suffer from MG53 related diseases, e.g., setting such mean value or 80%, 90%, 110%, 120%, 150%or 200%of such mean value as the positive reference value.

Subject

The term “subject” as used herein refers to human and non-human animal. Non-human animals include all vertebrates, e.g., mammals and non-mammals. A “subject” may also be a livestock animal (e.g., cow, swine, goat, chicken, rabbit or horse) , or a rodent (e.g., rat or mouse) , or a primate (e.g., gorilla or monkey) , or a domestic animal (e.g., dog or cat) . A “subject” may be a male or a female, and also may be at different ages. A human “subject” may be Caucasian, African, Asian, Sumerian, or other races, or a hybrid of different races. A human “subject” may be elderly, adult, teenager, child or infant.

In certain embodiments, the subject is a human or non-human mammal, e.g., mouse, rat, rabbit, goat, sheep, guinea swine or hamster.

MG53 related disease

The term “MG53 related disease” as used herein refers to such disease whereby the generation, onset, and development, etc., of which is related to activity of MG53. A person skilled in the art can determine whether a disease is related to MG53 through ordinary technical means. For example, whether the disease is related to MG53 can be determined by overexpressing MG53 protein, knockouting MG53 or reducing the content of MG53 in a subject through e.g., antisense nucleic acid technology, CRISPR/Cas9 technology and Trim21-mediated proteasome degradation and then assessing the condition of the disease in the subject.

The term “metabolic syndrome” as used herein refers to disorders characterized by aggregation of lipid and non-lipid cardiovascular risk factors caused by metabolic disturbance. In certain embodiments, metabolic syndrome is identified by the presence of any three of the following risk factors: waist circumference of over 102 cm in a male or over 88 cm in a female; serum triglyceride of at least 150 mg/dL; high density lipoprotein-cholesterol of less than 40 mg/dL in a male or less than 50 mg/dL in a female; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL. These risk factors can be easily measured in clinical practice, for example, pursuant to the guidance of the literature Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) , JAMA, 2001, 285: 2486-2497, which is incorporated herein by reference in its entirety.

Use of the detecting method

The method for detecting an MG53 related disease or predicting a risk of an MG53 related disease of the present disclosure can be used for many purposes.

In certain embodiments, the detecting method can be used to detect whether a subject suffers from an MG53 related disease, or to predict the risk at which a subject suffers from an MG53 related disease. For example, as described above, that the content of cell-free MG53 in a subject is equal to or lower than the negative reference value indicates that the subject is not suffering from MG53 related diseases or is at low risk of suffering from an MG53 related disease; that the content of cell-free MG53 in a subject is equal to or higher than the positive reference value indicates that the subject is suffering from an MG53 related disease or is at high risk of suffering from an MG53 related disease.

In certain embodiments, the detecting method can be used to predict the development of an MG53 related disease in a subject. For example, to predict the condition of an MG53 related disease (e.g., improvement or deterioration) in a subject by determining the change in content and/or activity of cell-free MG53 in a subject, compared with the value of the same subject a period of time ago. In certain embodiments, reduced content and/or activity of cell-free MG53 in a subject compared with the value of the same subject a period of time ago, indicates improvement of an MG53 related disease in the subject; increased content and/or activity of cell-free MG53 in a subject compared with the value of the same subject a period of time ago, indicates deterioration of an MG53 related disease in the subject.

In certain embodiments, the detecting method can be used to identify potential cell-free MG53 inhibitors. In certain embodiments, the use usually includes contacting a test compound or reagent with cell-free MG53, followed by detecting cell-free MG53. In certain embodiments, a test compound or reagent capable of reducing the content of cell-free MG53 by at least 10%can be considered as a cell-free MG53 inhibitor. In certain embodiments, the content of cell-free MG53 can be reduced by at least 20%, 40%, 50%, 80%, 90%, 95%or more. These identified cell-free MG53 inhibitors can be used to treat, prevent or ameliorate an MG53 related disease. Test compounds or reagents include, e.g., small molecule organic and inorganic compounds (e.g., molecules obtained from artificially synthetic chemical libraries and natural product libraries) , antibodies or antigen-binding fragments thereof, ligands or binding fragments thereof, or nonfunctional protein fragments of MG53.

In certain embodiments, the detecting method can be used to determine whether a subject responds to a cell-free MG53 inhibitor. After contacting a cell-free MG53 inhibitor, the presence and level of cell-free MG53 in the test sample can indicate whether the subject which the test sample derives from responds to the cell-free MG53 inhibitor. For example, that the content and/or activity of cell-free MG53 in the test sample is reduced by at least 10%indicates that the subject which the test sample derives from may respond to a cell-free MG53 inhibitor. In certain embodiments, compared with the reference sample, the content of cell-free MG53 in the test sample can be reduced by at least 20%, 40%, 50%, 80%, 90%, 95%or more.

In certain embodiments, the detecting method can be used to assess therapeutic effect for an MG53 related disease. In these embodiments, the reference sample and the test sample are from the same subject, but the time at which the reference sample was collected is earlier for a period of time than the test sample, and the subject accepts treatment during the period of time. In certain embodiments, that the content of cell-free MG53 in the test sample is reduced by at least 10%compared to the reference sample indicates that the subject responds to the treatment currently being accepted. In certain embodiments, compared with the reference sample, the content and/or activity of cell-free MG53 in the test sample can be reduced by at least 20%, 40%, 50%, 80%, 90%, 95%or more. According to the result of the assessment on response, the treatment strategy for the subject can be sustained or altered.

In certain embodiments, the detecting method can be used to assess the change of content of cell-free MG53 or its mutants, after the treatment using MG53 or its mutatnts. In these embodiments, the reference sample and the test sample are from the same subject, but the time at which the reference sample was collected is earlier for a period of time than the test sample, and the subject accepts the treatment using MG53 or its mutatnts during the period of time, thereby the content and/or activity of cell-free MG53 or its mutants was changed. According to the result of the assessment, the treatment strategy for the subject can be sustained or altered.

Method for reducing or inhibiting activity of cell-free MG53 in a subject

In another aspect, the present disclosure relates to a method for reducing or inhibiting activity of cell-free MG53 in a subject, which comprises administrating an effective amount of MG53 inhibitor to a subject in need thereof.

MG53 inhibitor

MG53 inhibitors can be small molecule organic or inorganic compounds (e.g., molecules obtained from artificially synthetic chemical libraries and natural product libraries) , antibodies or antigen-binding fragments thereof, ligands or MG53 binding fragments thereof, or nonfunctional protein fragments of MG53. In certain embodiments, the MG53 inhibitor can reduce the activity of cell-free MG53 by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95%or more.

In the present disclosure, “activity” , when used together with increase or reduce, refers to the detected functional activity, which may be represented by a change in the content of cell-free MG53 or, in the absence of a change in the content of cell-free MG53, a change in the functional activity of cell-free MG53. In some embodiments, activity of the MG53 is related to metabolic syndrome. In some embodiments, activity of the MG53 is related to glucose metabolism or lipid metabolism. In some embodiments, activity of the MG53 is related to insulin resistance, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension or obesity. In some embodiments, activity of the MG53 refers to insulin receptor and insulin receptor substrate binding activity.

In certain embodiments, the MG53 inhibitor specifically binds to MG53.

The term “specific binding” or “specifically binds” as used herein, with reference to MG53 inhibitor, refers to that the MG53 inhibitor preferentially identifies MG53 in a complicated mixture, and the binding constant of the inhibitor to MG53 is at least 2 fold of that of the inihibitor to other non-specific binding proteins. In certain embodiments, the equilibrium dissociation constant of the MG53 inhibitor to MG53 is less than or equal to 10 ^-6 or 10 ^-7 M. In certain embodiments, the equilibrium dissociation constant of the MG53 inhibitor to MG53 is less than or equal to 10 ^-7 M or 10 ^-8 M.

In certain embodiments, the MG53 inhibitor is an MG53 antibody or antigen-binding fragment thereof, an MG53 ligand or MG53 binding fragment thereof, or a nonfunctional protein fragment of MG53. Antibodies or antigen-binding fragments thereof, ligands or MG53 binding fragments thereof, or nonfunctional protein fragments of MG53 that can be used in the present disclosure include but are not limited to any MG53 antibody or antigen-binding fragment thereof, MG53 ligand or MG53 binding fragment thereof, or nonfunctional protein fragment of MG53 described herein, or a combination thereof.

In certain embodiments, the MG53 inhibitor is a small molecule compound capable of binding to MG53. Small molecule compounds binding to MG53 that can be used in the present disclosure include but are not limited to any small molecule compound binding to MG53 described herein, or a combination thereof.

Use of reducing or inhibiting method

In certain embodiments, the reducing or inhibiting method described herein can be used for the amelioration, treatment or prevention of an MG53 related disease or a symptom thereof. The reducing or inhibiting method includes administrating a therapeutically effective amount of MG53 inhibitor to a subject. See preceding texts for definition of MG53 related disease.

“Amelioration” , “treatment” or “prevention” of a disease or symptom includes preventing or alleviating a disease or discomfort, slowing the onset or rate of development of a disease or discomfort, reducing the risk of developing a disease or discomfort, preventing or delaying the development of symptoms associated with a disease or discomfort, reducing or ending symptoms associated with a disease or discomfort, generating a complete or partial regression of a disease or discomfort, curing a disease or discomfort, or some combination thereof.

The term “therapeutically effective amount” as used herein refers to the amount of a drug capable of ameliorating or eliminating a disease or symptom of a subject, or of preventively inhibiting or preventing the occurrence of the disease or symptom. A therapeutically effective amount can be the amount of a drug that ameliorates one or more diseases or symptoms of a subject to certain extent; the amount of a drug capable of restoring one or more physiological or biochemical parameters associated with the cause of a disease or symptom, partly or completely back to normal; and/or the amount of a drug capable of reducing the possibility that a disease or symptom occurs.

The therapeutically effective dosage of the MG53 inhibitor provided herein is dependent on various factors known in the art, for example, body weight, age, pre-existing medical condition, therapy currently being received, health condition of the subject, and intensity, allergic, superallergic and side effect of drug interaction, and route of administration and the extent to which the disease develops. A skilled artisan (e.g., a physician or veterinarian) may reduce or increase dosage in accordance with these or other conditions or requirement.

In certain embodiments, the MG53 inhibitor provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) . In certain of these embodiments, the MG53 inhibitor is administered at a dosage of about 50 mg/kg or less. In certain of these embodiments, the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. A particular dosage can be divided and administered multiple times separated by interval, e.g., once every day, twice or more every day, twice or more every month, once every week, once every two weeks, once every three weeks, once a month or once every two months or more. In certain embodiments, the administered dosage may vary over the course of treatment. For example, in certain embodiments, the initially administered dosage can be higher than subsequently administered dosages. In certain embodiments, the administered dosages are adjusted in the course of treatment depending on the response of the administration subject.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single dose may be administered, or several divided doses may be administered over time.

The MG53 inhibitor disclosed herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, sublingual, rectal, or topical) routes. The MG53 inhibitor disclosed herein may be administered in the form of a pharmaceutical composition of the present disclosure.

In certain embodiments, the MG53 inhibitor disclosed herein can be combined with other agents or therapies to treat a related disease or disorder. In some embodiments, the MG53 inhibitor is administered simultaneously or sequentially with other agents or therapies. In some embodiments, the other agents or therapies are one or more agents or therapies used for treating insulin resistance, diabetic cerebrovascular disease, diabetic ocular complication, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension and obesity. In some embodiments, the other agents are hypoglycemic agents, e.g., PPAR agonists, dipeptidyl peptidase (IV) inhibitors, GLP-1 analogs, insulins or insulin analogs, insulin sercretogogues, SGLT2 inhibitors, human dextrin analogs, biguanide, α-glocusidase inhibitors or a combination thereof.

MG53 antibody and antigen-binding fragment thereof

Another aspect of the present disclosure further provides an MG53 antibody and antigen-binding fragment thereof.

In certain embodiments, the MG53 antibody and antigen-binding fragment thereof comprises three heavy chain complementary determining regions comprised in a sequence of SEQ ID NOs: 1, 9, 17, 25 or 33. In certain embodiments, the MG53 antibody and antigen-binding fragment thereof further comprises three light chain complementary determining regions comprised in a sequence of SEQ ID NOs: 2, 10, 18, 26 or 34. In certain embodiments, the MG53 antibody and antigen-binding fragment thereof comprises three heavy chain complementary determining regions and three light chain complementary determining regions, the six complementary determining regions comprised in a sequence of SEQ ID NOs: 1/2, 9/10, 17/18, 25/26 or 33/34.

In certain embodiments, the MG53 antibody and antigen-binding fragment thereof comprises heavy chain, the variable regions of the heavy chain of the MG53 antibody and antigen-binding fragment thereof have an amino acid sequence of SEQ ID NOs: 1, 9, 17, 25 or 33, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In certain embodiments, the heavy chain of the MG53 antibody and antigen-binding fragment thereof has an amino acid sequence of SEQ ID NOs: 3, 11, 19, 27 or 35, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In certain embodiments, the MG53 antibody and antigen-binding fragment thereof further comprises a light chain, the variable regions of the light chain of the MG53 antibody and antigen-binding fragment thereof have an amino acid sequence of SEQ ID NOs: 2, 10, 18, 26 or 34, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In certain embodiments, the light chain of the MG53 antibody and antigen-binding fragment thereof has an amino acid sequence of SEQ ID NOs: 4, 12, 20, 28 or 36, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.

In yet another aspect of the present disclosure, a competitive antibody or antigen-binding fragment thereof is also provided, which competes for binding to MG53 with the antibody or antigen-binding fragment above.

The term “compete for binding” as used herein refers to the ability of an antibody or antigen-binding fragment thereof to specifically inhibit the binding interaction between the antigen it is against and another antibody molecule (e.g. MG53 and another MG53 antibody) . In certain embodiments, a competitive antibody or antigen-binding fragment that blocks binding between two antigen-antibody molecules inhibits the binding interaction between the two antigen-antibody molecules by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain, wherein the first constant region of the heavy chain is linked to the second constant region via a hinge region. The variable regions of the light and heavy chains are responsible for antigen binding specificity. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, and HCDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (see Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991) for specifics) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the structure of the variable regions. The constant regions of the heavy and light chains are irrelevant to antigen binding specificity, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain) , IgG2 (γ2 heavy chain) , IgG3 (γ3 heavy chain) , IgG4 (γ4 heavy chain) , IgA1 (α1 heavy chain) , or IgA2 (α2 heavy chain) .

The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, but does not comprise an intact antibody structure. Examples of antigen-binding fragment include, without limitation, an Fab, an Fab', an F (ab') ₂, an Fv fragment, a single-chain antibody molecule (scFv) , an scFv dimer, a camelized single domain antibody, and a nanobody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.

“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.

“Fab'” refers to a Fab fragment that includes a portion of the hinge region.

“F (ab') ₂” refers to a dimer of Fab'.

An “Fv” fragment consists of the variable region of a single light chain and the variable region of a single heavy chain.

“Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (see e.g., Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988) for specific introduction) .

“scFv dimer” refers to a polymer formed by two scFvs.

“Camelized single domain antibody” , also known as “heavy chain antibody” or “HCAb” (heavy-chain-only antibody) , refers to an antibody that contains two heavy chain variable regions but no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (see Hamers-Casterman C. et al., Nature. 363 (6428) : 446-8 (1993) ; Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, ” Immunogenetics. 54 (1) : 39-47 (2002) ; Nguyen VK. et al., Immunology. 109 (1) : 93-101 (2003) , which are incorporated herein by reference in their entirety) .

A “nanobody” consists of a heavy chain variable region from a heavy chain antibody and two constant regions, CH2 and CH3.

In certain embodiments, the antibody provided herein is a fully human antibody, a humanized antibody, a chimeric antibody, a mouse antibody or rabbit antibody. In certain embodiments, the antibody provided herein is a polyclonal antibody, a monoclonal antibody or a recombinant antibody. In certain embodiments, the antibody provided herein is a monospecific antibody, a bispecific antibody or a multispecific antibody. In certain embodiments, the antibody provided herein may further be labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is a fully human antibody, which is optionally produced by a transgenic rat, e.g., a transgenic rat in which the expression of endogenous rat immunoglobin gene is inactivated, and carrying recombinant human immunoglobin locus with J loci deletions and C-kappa mutations, and which can also be expressed by an engineered cell (e.g., CHO cell) .

The term “fully human” as used herein, with reference to antibody or antigen-binding fragment, refers to that the amino acid sequences of the antibody or the antigen-binding fragment correspond to that of an antibody produced by a human or a human immune cell, or derived from a non-human source such as a transgenic non-human animal that utilizes human antibody repertoires, or other human antibody-encoding sequences.

The term “humanized” as used herein, with reference to antibody or antigen-binding fragment, refers to an antibody or the antigen-binding fragment comprising CDRs derived from non-human animals, FR regions derived from human, and when applicable, constant regions derived from human. A humanized antibody or antigen-binding fragment is useful as human therapeutics in certain embodiments because it has reduced immunogenicity. In certain embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea swine, or a hamster. In certain embodiments, the humanized antibody or antigen-binding fragment is composed of substantially all human sequences except for the CDR sequences which are non-human.

The term “chimeric” as used herein, with reference to antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In some embodiments, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human species, such as from mouse or rabbit.

In certain embodiments, the MG53 antibody and antigen-binding fragment thereof provided herein is capable of specifically binding to MG53 with a binding affinity of ≤10 ^-6 M (e.g., ≤5x10 ^-7 M, ≤2x10 ^-7 M, ≤10 ^-7 M, ≤5x10 ^-8 M, ≤2x10 ^-8 M) . The binding affinity can be represented by K _D value, which is calculated as the ratio of dissociation rate to association rate (k _off/k _on) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g. K _D) can be detected using suitable methods known in the art, including plasmon resonance binding assay using instruments such as Biacore (see, for example, Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006) .

In certain embodiments, the antibody and antigen-binding fragment thereof provided herein inhibits the binding between MG53 and the ligand thereof with an IC ₅₀ of 0.2 nM-2000 nM (e.g., 1 nM-1500 nM, 5 nM-1000 nM, 10 nM-900 nM, 20 nM-800 nM or 50 nM-800 nM) .

The term percent (%) sequence “homology” as used herein refers to, with respect to amino acid sequence, the percentage of amino acid in a candidate amino acid sequence that are identical to the amino acid in a reference amino acid sequence, calculated after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids; with respect to nucleic acid sequence, the percentage of nucleotides in a candidate nucleic acid sequence that are identical to the nucleotides in a reference nucleic acid sequence, calculated after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical nucleotides. Alignment for purposes of determining percentage of homology can be achieved through various methods known in the art, for example, using publicly available tools such as BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) : http: //blast. ncbi. nlm. nih. gov/Blast. cgi, see also, Altschul S.F. et al., J. Mol. Biol., 215: 403–410 (1990) ; Stephen F. et al., Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute: http: //www. ebi. ac. uk/Tools/msa/clustalw2/, see also, Higgins D.G. et al., Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A. et al., Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and TCoffee (available on the website of Swiss Bioinformatics Institute, see also, Poirot O. et al., Nucleic Acids Res., 31 (13) : 3503-6 (2003) ; Notredame C. et al., J. Mol. Boil., 302 (1) : 205-17 (2000) ) . It is within the scope of knowledge for a person skilled in the art, that when sequence alignment is conducted using a software, the default parameters provided by the software may be used, or the parameters may be customize as appropriate for the alignment.

In certain embodiments, the MG53 antibodies or antigen-binding fragments thereof provided herein also include those resulted from conservative substitution of the amino acid residues to the MG53 antibody or antigen-binding fragment thereof provided herein.

The term “conservative substitution” of amino acid residues as used herein refers to substitution between amino acids with similar properties, such as between polar amino acids (e.g., glutamine and asparagine) , between hydrophobic amino acids (e.g., leucine, isoleucine, methionine and valine) , and between similarly charged amino acids (e.g., argine, lysine, and histidine, or glutamine and aspartic acid) .

In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise a conjugate.

In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety or a cytotoxic moiety. A variety of conjugates may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . In certain embodiments, the MG53 antibodies or antigen-binding fragments thereof disclosed herein may be engineered to contain one or more conjugate binding sites that may be utilized for binding to one or more conjugates. For example, such site may include one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent linkage to a conjugate. In certain embodiments, the MG53 antibodies or antigen-binding fragments thereof may be linked to a conjugate indirectly, or through another conjugate. For example, the MG53 antibodies or antigen-binding fragments thereof may be conjugated to biotin, then indirectly conjugated to a second conjugate (e.g., avidin) that is conjugated to biotin. In certain embodiments, the conjugate can be a detectable label, for example, fluorescent label (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate label (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases, or β-D-galactosidase) , radioisotope (e.g. ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, and ³²P, or other lanthanide) , chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule, or gold. In certain embodiments, the conjugate can be PEG which helps increase half-life of the antibody. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. In certain embodiments, the conjugate can be a cytotoxic moiety, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) , cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin (AMC) ) , and anti-mitotic agents (e.g., vincristine and vinblastine) .

MG53 ligand and MG53 binding fragment thereof

One aspect of the present disclosure provides an MG53 ligand and MG53 binding fragment thereof. In the present disclosure, the MG53 ligand and MG53 binding fragment thereof can be used as MG53 detection agent or MG53 inhibitor. Without desire to be bound by theory, presence or concentration of cell-free MG53 can be determined by detecting binding of an MG53 ligand or MG53 binding fragment thereof to cell-free MG53; binding of MG53 to original effector ligand/receptor can also be blocked or reduced by administering MG53 ligand or MG53 binding fragment thereof to a subject to compete for binding to MG53.

In some embodiments, the MG53 ligand is an insulin receptor or a variant thereof, e.g., human insulin receptor of SEQ ID NO: 58. In some embodiments, the MG53 ligand is the extracellular region of an insulin receptor or a variant thereof.

Nonfunctional protein fragment of MG53

One aspect of the present disclosure provides a nonfunctional protein fragment of MG53. “Nonfunctional protein fragment of MG53” provided herein can be any protein fragment which is lack of one or more physiological functions compared with a native MG53. In certain embodiments, the nonfunctional protein fragment of MG53 can compete with a native MG53 for binding to an MG53 ligand/receptor. In certain embodiments, activity of the nonfunctional protein fragment of MG53 is reduced by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%or more compared with a native MG53. In certain embodiments, the aforementioned reduced activity is the activity relating to an MG53 related disease. In certain embodiments, the aforementioned reduced activity is the activity of MG53 activating AKT phosphorylation.

In certain embodiments, the nonfunctional protein fragment of MG53 is an MG53 mutant. The term “MG53 mutant” or “MG53 protein mutant” as used herein refers to an MG53 protein variant or fragment in which the natural amino acid sequence of a wild-type MG53 protein is modified. Such modifications include, but are not limited to, deletion and/or substitution of one or more amino acids. In certain embodiments, the MG53 mutant of the present disclosure is identical to the amino acid sequence of a wide-type MG53 except for at least one serine in the coiled-coil-SPRY region of the wild-type MG53 is deleted and/or mutated into any other non-serine or non-threonine amino acids.

In certain embodiments, the MG53 mutant is identical to the amino acid sequence of a wide-type MG53 except for at least one serine in the coiled-coil-SPRY region of the wild-type MG53 is mutated into a non-polar amino acid. In certain embodiments, the non-polar amino acid is selected from the group consisting of glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, methionine, and tryptophan. Preferably, in certain embodiments, the non-polar amino acid is alanine. In certain embodiments, the MG53 mutant is identical to the amino acid sequence of a wide-type MG53 except for at least one serine in the coiled-coil-SPRY region of the wild-type MG53 is mutated into any non-serine or non-threonine polar amino acids. In certain embodiments, the polar amino acid is selected from the group consisting of glutamine, cysteine, asparagine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Preferably, in certain embodiments, the polar amino acid is cysteine. In certain embodiments, the MG53 mutant has an amino acid sequence of any of SEQ ID NOs: 48-57. In certain embodiments, the amino acid sequence of the MG53 mutant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence homology to the amino acid sequence of any of SEQ ID NOs: 48-57.

Other information regarding MG53 mutant may refer to Chinese Patent Application No. 201610847346.4, which is incorporated herein by reference in its entirety.

Method for preparing MG53 antibody or antigen-binding fragment thereof, MG53 ligand or MG53 binding fragment hereof, or nonfunctional protein fragment of MG53

The MG53 antibody or antigen-binding fragment thereof, MG53 ligand or MG53 binding fragment hereof, or nonfunctional protein fragment of MG53 of the present disclosure can be prepared by e.g., chemical synthesis or genetic engineering.

Chemical synthesis mainly includes two methods which are solid phase synthesis and liquid phase synthesis. Solid phase synthesis includes, e.g., Merrifield solid phase synthesis, which has been described in detail in the literature, e.g. Merrifield, J. Am. Chem. Soc. 85: 2149-2154, M. Bodanszky et al., Peptide Synthesis, John Wiley & Sons, Second Edition, 1976, and J. Meienhofer, “Hormonal Proteins and Peptides” , Vol. 2, p. 46, Academic Press (New York) , 1983, which are incorporated herein by reference in their entirety. Merrifield solid phase synthesis mainly comprises the following steps: first the protected carboxyl terminal amino acid is linked to resin according to the amino acid sequence of the target polypeptide; the resin is washed after the linking; the protective group (e.g., t-butyloxy carbonyl) on alpha amino group of carboxyl terminal amino acid is removed, during which it must be ensured that the linkage bond between the amino acid and the resin is not broken; and then the penultimate carboxyl terminal protected amino acid is conjugated to the resulted resin, during which an amide bond is formed between the free carboxyl group of the second amino acid and the amino group of the first amino acid linked to the resin; the preceding reaction processes are repeated successively according to the sequence of amino acid of the target polypeptide, until all amino acids are linked to the resin; finally, the protected peptide is cut off from the resin, and the target polypeptide is obtained after the protective group is removed. The polypeptides of the present disclosure can also be prepared by liquid phase synthesis, e.g., by standard solution peptide synthesis, which has been described in detail in the literature E. Schroder and K. Kubke, The Peptides, Vol. 1, Academic Press (New York) , 1965, which is incorporated herein by reference in its entirety. Liquid phase synthesis mainly comprises coupling amino acid or peptide fragment step by step, utilizing amide bond forming chemical or enzyme method.

The MG53 antibody or antigen-binding fragment thereof, MG53 ligand or MG53 binding fragment hereof, or nonfunctional protein fragment of MG53 can be produced by genetic engineering through cell culture and expression. In this method, a clone or expression vector with a target gene encoding the target protein needed to be expressed, is used to transform host cells, and the transformed host cells are cultured in a nutrient medium modified to be suitable for promoter inducement, transformed cell selection or amplification of genes encoding the target sequence (see Sambrook et al. (eds. ) , Molecular Cloning: a Laboratory Manual, (Cold Spring Harbor, 1989) , for detailed description regarding this method) .

The host cells in the present disclosure used to produce the antibodies or antigen-binding fragments thereof, MG53 ligand or MG53 binding fragment hereof, or nonfunctional protein fragment of MG53 may be cultured in a variety of media. Commercially available media such as Ham's F10, Minimal Essential Medium (MEM) , RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) , which are produced by Sigma, are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979) , Barnes et al., Anal. Biochem. 102: 255 (1980) , U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30, 985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN ^TM drug) , trace elements (defined as inorganic compounds used to provide nutrients, usually present at final concentrations in the micromolar range, such as ferrous sulfate, cupric sulfate, zinc sulfate, or manganese chloride) , and glucose or an equivalent energy source. The medium may also contain any other necessary supplements at appropriate concentrations that would be known to those skilled in the art. Selection of conditions for host cell culture, such as temperature, pH, and the like selected for expression, is well known to an ordinarily skilled artisan in the art.

When the aforementioned target protein is expressed using genetic engineering, the target protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the host cell is subject to lysis, and then the lysed fragments or the particulate debris are removed, for example, by centrifugation or ultrasonic. If the antibody is produced in the periplasmic space, for example, the procedure described in Carter et al., Bio/Technology 10: 163-167 (1992) can be used to isolate the target protein. Briefly, cells are lysed in the presence of sodium acetate (pH=3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) for over about 30 min, and then cell debris is removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The product prepared from the cells can be purified using purification methods such as hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. Following any preliminary purification step (s) , the mixture comprising the product of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 to 4.5, preferably performed at low salt concentrations (e.g., from about 0 to 0.25 M salt) .

Isolated nucleic acid

The term “isolated” as used herein refers to that a substance (e.g., polypeptide or nucleic acid) is isolated from the environment where it normally exists in nature, or exists in an environment different from where it normally exists in nature.

An “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. In certain embodiments, the antibodies and antigen-binding fragments have a purity of at least 90%, 93%, 95%, 96%, 97%, 98%, 99%as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, or capillary electrophoresis) , or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) .

The term “nucleic acid” or “polynucleotide” as used herein refers to ribonucleic acid (RNA) , deoxyribonucleic acid (DNA) or ribonucleic acid-deoxyribonucleic acid mixture (e.g., DNA-RNA hybrid) . Nucleic acid or polynucleotide can be single chain or double chain DNA or RNA, or DNA-RNA hybrid. Nucleic acid or polynucleotide can be linear or cyclic.

The term “encode” or “encoding” as used herein refers to being able to be transcribed into mRNA and/or translated into peptide or protein.

In some embodiments, nucleotide including polynucleotide can be in a modification form. The modification includes base modification (e.g., bromouridine) , ribose modification (e.g., cytosine arabinoside and 2’, 3’-dideoxynucleotide) and internucleotide linkage modification (e.g., thiophosphate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoramidate) .

The isolated nucleic acid provided herein also includes nucleic acid substituted according to degeneracy of genetic code. The term “degeneracy of genetic code” as used herein refers to the fact that there are two or more codons for the same amino acid. For example, proline has 4 synonymous codons, i.e., CCU, CCC, CCA, and CCG. It is known in the art, that due to degeneracy of nucleic acid genetic code, nucleic acids in certain sites of a known nuclei acid sequence may be substituted without altering the encoded amino acid sequence. A person skilled in the art can readily make genetic code degeneracy substitution, e.g., through site-directed mutagenesis of bases. Preferences for different codons vary among different organisms. To express the antibody or antigen-binding fragment of the present disclosure in certain selected biological cell, the codons preferred by the biological cell can be selected to obtain the corresponding encoding gene, and the antibody or antigen-binding fragment of the present disclosure is thus generated through recombination and expression.

Clone or expression vector

In another aspect, the present disclosure relates to a clone or expression vector comprising the isolated nucleic acid of any of the embodiments above.

The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic material element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Exemplary animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.

Host cell

In yet another aspect, the present disclosure relates to a host cell, which comprises the clone or expression vector of any of the embodiments above.

The term “host cell” as uses herein refers to a cell into which nucleic acid sequence (s) encoding one or more antibodies, MG53 ligands or nonfunctional protein fragment of MG53 provided herein ( “selected genes” ) have been introduced or capable of being introduced, and which further expresses or is capable of expressing the selected genes of interest. This term includes progeny of the parent cells, as long as the selected genes are present, regardless of whether the progeny is identical to the patent cells in morphology or in genetic make-up.

A vector containing polynucleotides encoding selected genes can be introduced into a host cell for cloning or gene expression. In the present disclosure, a host cell suitable for cloning or expressing DNA in the vector is a prokaryotic cell or eukaryotic cell. Prokaryotic cells suitable for the present disclosure include eubacteria and archaeba, wherein eubacteria include gram negative bacteria, gram positive bacteria and actinomyces. Exemplary eubacteria include, e.g., Colibactilus, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella, Pseudomonas, etc.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast can also serve as host cells for cloning and expressing vectors of selected genes. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts (e.g., K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16, 045) , K. wickeramii (ATCC 24, 178) , K. waltii (ATCC 56, 500) , K. drosophilarum (ATCC 36, 906) , K. thermotolerans, and K. marxianus) ; yarrowia (EP 402, 226) ; Pichia pastoris (EP 183, 070) ; Candida; Trichoderma reesia (EP 244, 234) ; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi (e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus) .

The host cell provided herein can be a mammalian host cell, and culture of mammalian host cells has become a routine procedure. Examples of useful mammalian host cells are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned in suspension culture, see Graham et al., J. Gen Virol. 36: 59 (1977) ) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, see Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ) ; mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (see Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .

Small molecule compound

One aspect of the present disclosure provides a small molecule compound binding to MG53. In the present disclosure, a small molecule compound binding to MG53 can be used as an MG53 detection agent or MG53 inhibitor.

In certain embodiments, the small molecule compound binding to MG53 is a compound having Formula Ia or Formula Ib, or a pharmaceutically acceptable salt thereof,

In certain embodiments, the small molecule compound binding to MG53 is a compound having Formula II, or a pharmaceutically acceptable salt thereof,

In certain embodiments, the small molecule compound binding to MG53 is a compound having Formula III, or a pharmaceutically acceptable salt thereof,

For clarity, various features of the present disclosure described in the context of individual embodiments may also be provided separately or in any suitable sub-combination.

As used herein, the term “substituted” , when refers to a chemical group, means the chemical group has one or more hydrogen atoms that is/are removed and replaced by substituents.

As used herein, the term “substituent” has the ordinary meaning known in the art and refers to a chemical moiety that is covalently attached to, or if appropriate, fused to a parent group.

As used herein, the term “optionally substituted” means that the chemical group may have no substituents (i.e. unsubstituted) or may have one or more substituents (i.e. substituted) . It is to be understood that substitution at a given atom is limited by valency. In certain embodiments, illustrating examples of the substituent mentioned in any of the embodiments above are: nitro, halogen, hydroxyl, cyano, C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy, benzamide group, or -C (O) -O-R ₆, wherein the C ₁-C ₁₂ alkyl, C ₁-C ₁₂ alkoxy or benzamide group can further be substituted by nitro, halogen, hydroxyl, cyano, C ₁-C ₃ alkyl or C ₁-C ₃ alkoxy, wherein R ₆ is hydrogen or C ₁-C ₃ alkyl.

As used herein, the term “C _n-C _m” indicates a range of the carbon atoms numbers, wherein n and m are integers and the range of the carbon atoms numbers includes the endpoints (i.e., n and m) and each integer point in between. For examples, C ₁-C ₆ indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms.

As used herein, the term “alkyl” , whether as part of another term or used independently, refers to a saturated hydrocarbon group that may be straight-chain or branched-chain. The term “C _n-m alkyl” refers to an alkyl having n to m carbon atoms. In certain embodiments, the alkyl group contains 1 to 12, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of alkyl group include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n -butyl, tert-butyl, isobutyl, sec-butyl, 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1, 2, 2-trimethylpropyl, and the like.

As used herein, the term “alkenyl” , whether as part of another term or used independently, refers to an unsaturated hydrocarbon group that may be straight-chain or branched-chain having at least one carbon-carbon double bond. In certain embodiments, the alkenyl group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. In certain embodiments, the alkenyl group contains 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, chemical groups such as ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

As used herein, the term “alkylene” , whether as part of another term or used independently, refers to a divalent saturated hydrocarbon moiety that may be straight-chain or branched-chain, and is linked to two other moieties of a molecule. The term “C _n-C _m alkylene” refers to an alkylene having n to m carbon atoms. In certain embodiments, the alkylene group contains 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylene groups include, but are not limited to, chemical groups such as methylene, ethidene, 1-methyl-methylene, propylidene, butylidene, and the like.

As used herein, the term “alkenylene” , whether as part of another term or used independently, refers to a divalent unsaturated hydrocarbon moiety that may be straight-chain or branched-chain having at least one carbon-carbon double bond, and is linked to two other moieties of a molecule. The term “C _n-C _m alkenylene” refers to an alkylene having n to m carbon atoms. In certain embodiments, the alkenylene group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. In certain embodiments, the alkenylene group contains 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 carbon-carbon double bond.

As used herein, the term “aryl” or “aromatic” , whether as part of another term or used independently, refers to a mono-or poly-carbocyclic ring system radicals with alternating double and single bonds between carbon atoms forming the rings. The term “C _n-C _m aryl” refers to an aryl having n to m carbon atoms forming the ring. In certain embodiments, the aryl ring systems have 5 to 10, 5 to 8, or 5 to 6 carbon atoms in one or more rings. In certain embodiments, the aryl ring systems have two or more rings fused together. Examples of aryl groups include, but are not limited to, chemical groups such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. In certain embodiments, phenyls include fused phenyls, e.g., benzo dioxolanyl.

As used herein, the term “arylene” , whether as part of another term or used independently, refers to a divalent aromatic ring or ring system that is linked to two other moieties of a molecule, i.e., the aforementioned two moieties are bonded to the ring at two different ring positions. When the aromatic ring of an arylene is a monocyclic ring system, the two moieties are bonded at two ring positions of the same ring. When the aromatic ring of an arylene is a polycyclic ring system, the two moieties may be bonded at two ring positions of the same ring or different rings. The term “C _n-C _m arylene” refers to an arylene having n to m carbon atoms forming the ring. An arylene may be substituted or unsubstituted. An unsubstituted arylene has no other substituents apart from the two moieties of the molecule it is linked to. A substituted arylene has other substituents apart from the two moieties of the molecule it is linked to.

As used herein, the term “alkoxy” , whether as part of another term or used independently, refers to a group of the formula “-O-alkyl” . The term “C _n-C _m alkoxy” means that the alkyl moiety of the alkoxy group has n to m carbon atoms. In certain embodiments, the alkyl moiety has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxy groups include, but are not limited to, chemical groups such as methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy) , t-butoxy, and the like.

As used herein, the term “n membered” , wherein n is an integer, typically employed in combination with a ring system to describe the number of ring-forming atoms in the ring system. For example, piperidinyl is an example of a 6 membered heterocycloalkyl ring, pyrazolyl is an example of a 5 membered heteroaryl ring, pyridyl is an example of a 6 membered heteroaryl ring, and 1, 2, 3, 4-tetrahydro-naphthalene is an example of a 10 membered cycloalkyl group.

As used herein, the term “heteroaryl” refers to aryl group wherein at least one ring atom in the aromatic ring is a heteroatom, and the remainder of the ring atoms are carbon atoms. The term “n-m membered heteroaryl” refers to heteroaryl having n to m ring-forming members. Example heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like. In certain embodiments, heteroaryl can have 5 to 10, 5 to 8, or 5 to 6 ring-forming members. In certain embodiments, heteroaryl is 5 membered or 6 membered heteroaryl. Examples of heteroaryl include, but are not limited to, furanyl, thienyl, pyridyl, quinolyl, pyrrolyl, N-lower alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, indolyl and the like.

A 5 membered heteroaryl is a heteroaryl with a ring having five ring-forming atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms can be independently selected from N, O, P, and S. Examples of 5 membered heteroaryl include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1, 2, 3-triazolyl, tetrazolyl, 1, 2, 3-thiadiazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-triazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 4-oxadiazolyl, 1, 3, 4-triazolyl, 1, 3, 4-thiadiazolyl, 1, 3, 4-oxadiazolyl and the like.

A 6 membered heteroaryl is a heteroaryl with a ring having six ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms can be independently selected from N, O, P, and S. Examples of 6 membered heteroaryl include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, pyridazinyl and the like.

As used herein, the term “heteroarylene” , whether as part of another term or used independently, refers to a divalent heteroaryl that is linked to two other moieties of a molecule, i.e., the aforementioned two moieties are bonded to the ring at two different ring positions. When the aromatic ring of a heteroarylene is a monocyclic ring system, the two moieties are bonded at two ring positions of the same ring. When the aromatic ring of a heteroarylene is a polycyclic ring system, the two moieties may be bonded at two ring positions of the same ring or different rings. A heteroarylene may be substituted or unsubstituted. An unsubstituted heteroarylene has no other substituents apart from the two moieties of the molecule it is linked to. A substituted heteroarylene has other substituents apart from the two moieties of the molecule it is linked to.

As used herein, the term “heterocyclic alkyl” refers to cycloalkyl group wherein at least one ring atom in the ring systems is a heteroatom, and the remainder of the ring atoms being carbon atoms. The term “n-m membered heterocyclic alkyl” refers to heterocyclic alkyl having n to m ring-forming members. In addition, the ring may also have one or more double bonds, but not have a completely conjugated system. In certain embodiments, the heterocyclic alkyl is saturated heterocyclic alkyl. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like. In certain embodiments, heterocyclic alkyl has 3 to 8, 3 to 6, or 4 to 6 ring-forming carbons. Examples of heterocyclic alkyl include, but are not limited to, azetidine, aziridine, pyrrolidyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.

As used herein, the term “heterocyclic alkylene” , whether as part of another term or used independently, refers to a divalent heterocyclic alkyl that is linked to two other moieties of a molecule, i.e., the aforementioned two moieties are bonded to the ring at two different ring positions. The term “C _n-m heterocyclic alkylene” refers to a heterocyclic alkylene having n to m carbon atoms. In certain embodiments, the heterocyclic alkylene contains 3 to 12, 3 to 6, or 4 to 6 carbon atoms.

As used herein, the term “heterocyclic group” includes heteroaryl and heterocyclic alkyl.

As used herein, the term “heterocyclic subunit” includes heteroarylene and heterocyclic alkylene.

As used herein, the terms “halo” and “halogen” refer to an atom selected from fluorine, chlorine, bromine and iodine.

As used herein, the terms “cyano” refer to a group of the formula “-CN” .

As used herein, the term “hydroxyl” refers to a group of the formula “-OH” .

As used herein, the terms “nitro” refer to a group of the formula “-NO ₂” .

As used herein, the terms “benzamide group” refers to the formula “-NH-C (=O) -phenyl” .

As used herein, the term “compound” is meant to include all stereoisomers (e.g., enantiomers and diastereomers) , geometric iosomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

The compounds described herein can be asymmetric (e.g., having one or more stereocenters) . All stereoisomers, such as enantiomers and diastereomers, are intended to be included unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, carbon-carbon double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

In certain embodiments, the compounds described herein have the (R) -configuration. In certain embodiments, the compounds described herein have the (S) -configuration.

Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallizaion using a chiral resolving acid, which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the various optically active camphorsulfonic acids. Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of N-methyl

benzyl

amine, 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1, 2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine) . Suitable elution solvent composition can be determined by those skilled in the art.

Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H-and 3H-imidazole, 1H-, 2H-and 4H-1, 2, 4-triazole, 1H-and 2H-isoindole, and 1H-and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

Compounds of the present disclosure can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, deuterium and tritium.

Methods for obtaining small molecule compounds

In certain embodiments, the small molecule compounds of the present disclosure can be obtained through organic synthesis. Compounds of the present disclosure, including salts, esters, hydrates, or solvates thereof, can be prepared using any known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

The reactions for preparing compounds of the present disclosure can be carried out in suitable solvents, which can be readily selected by those skilled in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants) , the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by those skilled in the art.

Preparation of compounds of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999) , which is incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) , infrared spectroscopy, spectrophotometry (e.g., UV-visible) , mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC) , liquid chromatography-mass spectroscopy (LCMS) , or thin layer chromatography (TLC) . Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) (see, e.g., “Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6) , 874-883, which is incorporated herein by reference in its entirety) and normal phase silica chromatography.

In certain embodiments, the small molecule compound of the present disclosure can be purchased by commercial approaches. In certain embodiments, the small molecule compounds of the present disclosure can be commercially available compound libraries, e.g., SPECS compound library, Netherlands.

Pharmaceutical composition

In a further aspect, the present disclosure relates to a pharmaceutical composition comprising an active substance (e.g., the antibody, antigen-binding fragment, MG53 ligand or nonfunctional fragment of MG53 of any of the embodiments above, the clone or expression vector of any of the embodiments above, or the cell of any of the embodiments above or any of the small molecule compounds above) , and a pharmaceutically acceptable excipient.

These pharmaceutical compositions can be prepared in a manner known in the pharmaceutical art. In certain embodiments, the compounds of the present disclosure may be admixed with pharmaceutically acceptable excipient for the preparation of pharmaceutical compositions.

As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In certain embodiments, compounds, materials, compositions, and/or dosage forms that are pharmaceutically acceptable refer to those approved by a regulatory agency (such as U.S. Food and Drug Administration, China Food and Drug Administration or European Medicines Agency) or listed in generally recognized pharmacopoeia (such as U.S. Pharmacopoeia, China Pharmacopoeia or European Pharmacopoeia) for use in animals, and more particularly in humans.

The pharmaceutically acceptable carriers for use in the pharmaceutical compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or Ringer's injection of glucose and lactate) , non-aqueous vehicles (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil) , antimicrobial agents, isotonic agents (such as sodium chloride or dextrose) , buffers (such as phosphate or citrate buffers) , antioxidants (such as sodium bisulfate) , anesthetics (such as procaine hydrochloride) , suspending/dispending agents (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone) , chelating agents (such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) ) , emulsifying agents (such as Polysorbate 80 (TWEEN-80) ) , diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, or emulsifiers.

In certain embodiments, the pharmaceutical composition is an oral formulation. The oral formulations include, but are not limited to, capsules, cachets, pills, tablets, troches (for taste substrates, usually sucrose and acacia or tragacanth) , powders, granules, or aqueous or non-aqueous solutions or suspensions, or water-in-oil or oil-in-water emulsions, or elixirs or syrups, or confectionery lozenges (for inert bases, such as gelatin and glycerin, or sucrose or acacia) and /or mouthwash and its analogs.

In certain embodiments, the oral solid formulation (e.g., capsules, tablets, pills, dragees, powders, granules, etc. ) includes the active substance and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or the followings: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants such as glycerol; (4) cleaving agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) retarder solutions such as paraffin; (6) accelerating absorbers such as quaternary ammonium compounds; (7) lubricants such as acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium sulfate, and mixtures thereof; and (10) colorants.

In certain embodiments, the oral liquid formulation includes pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs, etc. In addition to the active substance, the liquid dosage forms may also contain conventional inert diluents such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzene (meth) acrylate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, olive, castor and sesame oils) , glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid sorbitol esters, and mixtures thereof. Besides inert diluents, the oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preserving agents.

In certain embodiments, the pharmaceutical composition may be an injectable formulation, including sterile aqueous solutions or dispersions, suspensions or emulsions. In all cases, the injectable formulation should be sterile and should be liquid to facilitate injections. It should be stable under the conditions of manufacture and storage, and should be resistant to the infection of microorganisms (such as bacteria and fungi) . The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, etc. ) and suitable mixtures and /or vegetable oils thereof. The injectable formulation should maintain proper fluidity, which may be maintained in a variety of ways, for example, using a coating such as lecithin, using a surfactant, etc. Antimicrobial contamination can be achieved by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. ) .

In certain embodiments, the pharmaceutical composition is an oral spray formulation or nasal spray formulation. Such spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposomal formulations, or solid particulate formulations, etc. Aqueous aerosols are formulated by combining an aqueous solution or suspension of the agent with a conventional pharmaceutically acceptable carrier and stabilizer. The carrier and stabilizer may vary according to the needs of specific compounds, but generally include nonionic surfactants (Tweens, or polyethylene glycol) , oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugar or sugar alcohol. Aerosols are usually prepared from isotonic solutions and can be delivered by nebulizers.

In certain embodiments, the pharmaceutical compositions may be used in combination with one or more other drugs. In certain embodiments, the composition comprises at least one other drug. In certain embodiments, the other drugs are cardiovascular drugs, drugs for treating kidney diseases, drugs for cell membrane repair, etc.

In certain embodiments, the pharmaceutical compositions may be delivered to the subject by suitable routes including, but not limited to, the oral route, injection route (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracardiac injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc. ) , mucosal route (e.g., intranasal administration, oral administration, etc. ) , sublingual route, rectal route, transdermal route, intraocular route, pulmonary route. In certain embodiments, the pharmaceutical compositions can be administered by injection route.

Kit

In another aspect, the present disclosure relates to a kit for detecting cell-free MG53.

In certain embodiments, the kit comprises an MG53 detection agent (e.g., the MG53 detection agent described in the present disclosure) . In certain embodiments, the kit further comprises one or more other components, e.g., MG53 standard solution, diluent, washing solution, stop solution and chromogenic agent, etc. The kit provided herein can be used in a method for detecting cell-free MG53 provided herein.

Reagents in the kit can be placed in any kind of container, such that each component in the reagent is preserved stably and will not be absorbed or altered by the material of the container. For example, lyophilized regulating substrates/molecules and /or buffers may be contained within a sealed glass ampoule which is packed under neutral, non-reactive gas such as nitrogen. The ampoule can be composed of any suitable material, e.g., glass, organic polymer such as polycarbonate or polystyrene and the like, ceramic, metal or any other material commonly used to store reagents. Other examples of suitable containers include simple bottles, which may be made from materials similar to ampoules, and envelopes, which may be composed of metal foil lining (e.g., aluminium or alloy) . Other containers include tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile inlet, such as a bottle having a stopper that can be punctured using hypodermic needle. Other containers may have two compartments separated by an easily removable a film, upon removal of which the components can be mixed. The removable film can be glass, plastic, rubber, and the like.

In certain embodiments, the kit also comprises an enzyme-labeled plate and sealing films.

In certain embodiments, the kit can also be provided together with instructions. The instructions can be printed on paper or other base materials, and/or provided in the form of digitally readable media, such as diskette, CD-ROM, DVD-ROM, zip disk, video tape, compact disk, tape, and the like. Detailed instructions may be not physically associated to the kit, but as an alternative, the users may be alternatively directed to a website designated by the manufacturer or distributor of the kit, or the instructions may be provided in the form of emails. Methods for detecting cell-free MG53 are recorded in the instructions.

Pharmaceutical use

In another aspect, the present disclosure relates to use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting cell-free MG53 in a subject. In certain embodiments, the MG53 inhibitor can be any of the MG53 inhibitor above. In certain embodiments, the method can be used to ameliorate, treat or predict an MG53 related disease or a symptom thereof. In certain embodiments, the MG53 related disease can be any of the MG53 related diseases above. In some embodiments, the medicament is a pharmaceutical composition of the present disclosure.

In yet another aspect, the present disclosure relates to use of an MG53 detection agent in the manufacture of a kit for detecting cell-free MG53. In certain embodiments, the kit can be used for detecting an MG53 related disease, predicting a risk or development of an MG53 related disease, identifying potential MG53 inhibitor, evaluating therapeutic effect for an MG53 related disease, or detecting activity of cell-free MG53. In some embodiments, the MG53 detection agent is provided in the form of a pharmaceutical composition of the present disclosure.

EXAMPLES

The following examples are provided to explain the present disclosure, which are not intended to limit the present disclosure in any way.

Example 1: preparation and characterization of MG53 antibodies

Preparation of MG53 antibodies

Total RNA extracted from mouse was used for reverse transcription, to synthesize cDNA. cDNA library was obtained by reverse transcription using OligodT as primer and reverse transcriptase. The nucleic acid encoding mouse MG53 (NP _001073401.1) was obtained by PCR method using specific primer and the synthesized cDNA library as template. PCR was performed as follows: denaturation, 98℃ for 3 min; circulation process, 98℃ for 30 seconds, 65℃ for 30 seconds, and 72℃ for 90 seconds, 30 cycles; followed by extension of 72℃ for 5 min, and then stored at 4℃. The sequence of the primer is as follows: upstream primer: 5’-ataggtaccg ccaccatgtc ggctgcaccc ggcct-3’ (SEQ ID NO: 59) ; downstream primer: 5’-atactcgagc ggcctgttcc tgctccggcc-3’ (SEQ ID NO: 60) ; carrying with KpnI and XhoI restriction site.

The PCR product was subject to double enzyme digestion using KpnI and XhoI, and meanwhile, empty vector plasmid pcDNA4/TO/myc-HisB was treated by the same double enzyme digestion, the gel was extracted and the PCR product was ligated into the vector using T4 ligase, forming a plasmid pcDNA4-mMG53-myc. The sequence was verified as correct by sequencing.

The expression plasmids pcDNA4-mMG53-myc were transiently transfected into HEK293 cells. 2 days later, supernatant harvested from the transiently transfected cell culture was used for protein purification. The protein was purified by SDS-PAGE and quantified to be used for immunization.

3 rabbits were respectively immunized with 100 μg MG53 protein by intraperitoneal injection. After the rabbits were immunized for three to five times, the blood of the rabbits were taken to collect the serum, and the titer of the antibody was measured by ELISA assay, and the rabbit with the highest titer was selected.

B lymphocytes were isolated from the spleen of the rabbit with the highest titer. The isolated B lymphocytes and myeloma cells were subject to cell fusion (by the ratio of 1: 1) . The specific experimental procedure of the fusion is as follows: the cell mixture was washed and suspended with 5-10 ml ECF solution. ECF solution was added to adjust the concentration to 2 ×10 ⁶ cells/ml. Upon electrofusion of the cells, the cell suspension in the fusion chamber was immediately transferred into a sterile tube containing medium of more volume. After culture at 37℃ for over 24 hours, the cell suspension was mixed and pipetted into a 96-well plate (0.5×10 ⁶ cells/plate) . The cells were cultured at 37℃, under 5%CO ₂. When the clone was large enough, 100 μl supernatant was transferred from the 96-well plate for antibody screening assay.

Binding of the hybridoma supernatant to MG53 protein was detected using ELISA. Briefly, the plate was coated with 1 μg/ml human MG53 protein overnight at 4℃. After sealing and washing, the hybridoma supernatant was diluted by different times, and then transferred to the coated plate and cultured at room temperature for 1 hour. Subsequently, the plate was washed and then cultured for 45 min with the secondary antibody of goat-anti-rabbit IgG (H+L) HRP (Goat Anti-Rabbit HRP (IgG H&L) (ab6721) | Abcam) . After the plate was washed, TMB substrate was added and reaction was terminated with 2M HCl. Absorption value at 450 nm was read using Molecular Device. Five monoclonal hybridoma cells which have the best binding properties and are most suitable for detection were selected according to the results. The binding data of the five monoclonal hybridomas are shown in the following table.

Wherein, the positive standard value (commercially available MG53 antibody) is 0.456; the negative standard value (irrelevant antibody) is 0.083.

The five hybridoma cell lines were subcloned. Briefly, for each hybridoma cell line, the cells were counted and diluted to 5 cells/well, 1 cell/well and 0.5 cell/well in the clone medium. The cells were plated into 96-well plates at 200 μl/well, among the plates, one plate of 5 cells/well, one plate of 1 cell/well and four plates of 0.5 cell/well. All plates were placed under 37℃, 5%CO ₂, and cultured until all cell lines can be tested by ELISA.

Sequencing was then conducted on the five monoclonal hybridoma cells, the result of which showed that the MG53 antibodies were Antibody #6 (heavy chain nucleotide sequence as shown in SEQ ID NO: 7, light chain nucleotide sequence as shown in SEQ ID NO: 8) , Antibody #110 (heavy chain nucleotide sequence as shown in SEQ ID NO: 15, light chain nucleotide sequence as shown in SEQ ID NO: 16) , Antibody #84 (heavy chain nucleotide sequence as shown in SEQ ID NO: 23, light chain nucleotide sequence as shown in SEQ ID NO: 24) , Antibody #9 (heavy chain nucleotide sequence as shown in SEQ ID NO: 31, light chain nucleotide sequence as shown in SEQ ID NO: 32) , Antibody #43 (heavy chain nucleotide sequence as shown in SEQ ID NO: 39, light chain nucleotide sequence as shown in SEQ ID NO: 40) .

Characterization of binding ability of MG53 antibodies

The ability of MG53 antibodies to bind MG53 was verified by surface plasmon resonance (SPR) . Recombinant human MG53 protein (rhMG53) was dissolved in 10 mM sodium acetate buffer (pH=4.5) , and then rhMG53 was immobilized on a CM5 chip via the primary amine groups by using Amine Coupling kit (GE Healthcare) , and the remaining active sites were blocked by 1M ethanolamine. A reference flow cell was activated and blocked in the absence of MG53. The immobilization level was fixed at 500 Biacore response units (RU) , and the concentration of MG53 Antibody #84 (amino acid heavy chain sequence as shown in SEQ ID NO: 19, light chain sequence as shown in SEQ ID NO: 20, the concentration of which ranging from 1.95 nM to 1000 nM) flowing over the chip surface was serially increased, the data results of which are shown in Figure 2. It can be inferred from the experimental results above, that the MG53 antibodies of the present disclosure are capable of binding rhMG53 with high affinity.

The ability of MG53 antibodies to block the binding of MG53 to insulin receptor extracellular region is verified by surface plasmon resonance (SPR) . Recombinant insulin receptor (IR) extracellular region was dissolved in 10 mM sodium acetate buffer (pH=4.5) , and then IR was immobilized on CM5 chip via the primary amine groups by using Amine Coupling kit (GE Healthcare) , and the remaining active sites were blocked by 1M ethanolamine. A reference flow cell was activated and blocked in the absence of IR. The immobilization level was fixed at 500 Biacore response units (RU) , and the concentration of the reactant flowing over the chip surface was serially increased (rhMG53 was used in the experiment shown in Figure 3a, the concentration of which ranged from 1.95 nM to 1000 nM; a mixture of rhMG53 and MG53 Antibody #84 was used in the experiment shown in Figure 3b, wherein the concentration of MG53 antibody was fixed at 500 nM, while the concentration of rhMG53 ranging from 0 nM to 2000 nM) .

The two binding experiments above were both conducted in PBS-P buffer (20 mM phosphate buffer, 2.7 mM NaCl, 137 mM KCl, 0.05%vol/vol surface active agent P-20, pH=7.4) at 25℃. Regeneration was conducted with 10 mM NaOH (pH=10) after each injection of sample. Biacore T200 was used for detection and the software thereof was used for data analysis.

Example 2: study on function of cell-free MG53

Western blotting in example 2 was conducted as follows:

(1) Gel preparation: Glass plates were washed and dried. Vertical electrophoresis apparatus was set up, and filled with 8%separation gel and 5%concentration gel subsequently.

(2) Preparation for electrophoretic buffer: The mother solution for 5×electrophoretic buffer was prepared with Tris and glycine, diluted to 1×, and 600 ml of which was added to the vertical electrophoresis tank.

(3) Sample loading: The prepared protein sample was cooled to room temperature, melted and evenly mixed by centrifugation. Protein marker and protein sample were loaded from left to right sequentially. If there were wells without any sample, the same amount of 1×loading buffer should be added to supplement for balance, leaving no blank lane, to avoid skew bands.

(4) Gel running: The electrophoresis device was connected to the power, and a voltage of 7 V/cm was applied. When the front edge of bromophenol blue entered the separation gel and the protein marker was separated into apparent bands, the voltage was raised to 11 V/cm. The electrophoresis lasted until bromophenol blue reaches the end of separation gel.

(5) Preparation for transfer buffer: The mother solution for 20×transfer buffer was prepared with Tris and glycine, diluted to 1×, added with 20%methanol and mixed evenly for subsequent use.

(6) Transfer: Wet electrophoretic transfer cell was used for transfer. Sponge, double-layer filter paper, gel, PVDF film, double-layer filter paper, and sponge were placed on a black plastic plate sequentially following a “sandwich” -like order, which was gently pressed tight and placed into the electrophoretic transfer cell (attention is required to make sure that no bubbles appear between the PVDF film and the gel, otherwise the effect of transfer will be affected) , 1000 ml transfer buffer was added, and the device was placed on ice, plugged in, and transferred under constant current 275 mA for 2 hours.

(7) Sealing: 10×TBS solution was first diluted to 1×, and prepared into 1×TBST solution by adding 0.1%Tween20, and 5% (w/v) skim milk powder was then added to 1×TBST solution to prepare sealing buffer. Upon completion of transfer, the PVDF film was carefully taken out and placed into the sealing buffer, and gently shaken on a shaker for 1 hour at room temperature for sealing.

(8) Primary antibody incubation: Primary antibody solution was prepared by adding 1‰primary antibody into TBST solution containing 5% (w/v) BSA. Upon completion of sealing, the film was washed three times with TBST, each time for 5 min, added with the corresponding primary antibody solution, and gently shaken on a shaker overnight at 4℃ for incubation.

(9) Secondary antibody incubation: Secondary antibody solution was prepared by diluting the secondary antibody by 1: 2000 in TBST containing 5%skim milk powder. Attention need to be directed to whether the secondary antibody corresponds to the primary antibody, i.e., whether it is mouse antibody or rabbit antibody, etc. The film incubated overnight with primary antibody was taken out, washed three times with TBST, each time for 5 min, and then added with the secondary antibody solution, and gently shaken on a shaker for 1 hour at room temperature for incubation.

(10) Chemiluminescent assay: The PVDF film incubated with the secondary antibody was taken out, washed three times with TBST, each time for 5 min, and the film was soaked in TBST for subsequent use. Chemiluminescence reaction substrates A and B were prepared, mixed by ratio of 1: 1 (vol/vol) , carefully operated to avoid light, and then added onto the PVDF film, reacted for 5 min, and chemiluminescent assay was performed on the bands by ChemiDocXRS of BIO-RAD.

(11) Re-incubating primary antibody: The film was washed twice with TBST, each time for 5 min. Elution buffer was added, and the film was washed for 30 min at 50℃, 50 rpm. The film was then washed 6 times with TBST, each time for 5 min. After the antibody was eluted, the protein could be further tested by incubating other corresponding primary antibodies-secondary antibodies. Gray value of blot image of the chemiluminescent assay on the bands was analyzed using ImageJ software.

Detecting cell-free MG53 using the antibodies of the present disclosure

Blood was drawn from the tail vein of 14-week-old WT mice and MG53 ^-/- (MG53 gene knocked out through gene knockout technology) mice, and serum was collected by centrifugation. Western blotting assay was performed using MG53 Antibody #84 as the primary antibody, HRP-anti-rabbit antibody as the secondary antibody, loaded with 0.5 μL serum. Results of the assay are shown in Figure 4. It can be inferred from the experimental results that samples expressing MG53 and samples not expressing MG53 can be clearly distinguished using the MG53 antibodies of the present disclosure.

Blood was drawn from the tail vein of 14-week-old WT mice and TG mice (MG53 gene overexpressed through genetic technology) , and serum was collected by centrifugation. Western blotting assay was conducted using MG53 Antibody #84 as the primary antibody, HRP-anti-rabbit antibody as the secondary antibody, loaded with 0.5 μL serum, and results of the assay are shown in Figure 5. It can be inferred from the experimental results that serum MG53 in different concentrations can be clearly distinguished by experiments performed using the MG53 antibodies of the present disclosure.

Using the same Western blotting assay as described previously, rmMG53-myc (recombinant mouse MG53 protein with myc label expressed by HEK293) and rhMG53 (purified recombinant human MG53 protein expressed by HEK293 cell) were tested using MG53 Antibody, the results of the assay are shown in Figure 6. It can be inferred from the experimental results that the MG53 antibodies of the present disclosure can be used to detect human and mouse cell-free MG53.

Verification for function of cell-free MG53

Serum of 37 Type II diabetes patients (T2D) and serum of 21 normal people (Control) were obtained. The serum samples above were tested using the same Western blotting assay as described previously, the results of the assay were shown in Figure 7. It can be inferred from the experimental results that the increase in MG53 content in the serum of Type II diabetes patients is statistically meaningful over the content in that of normal people (using t-test assay) , and thereby whether diabetes is being suffered from and risk of having diabetes can be determined based on content of MG53 in serum.

Serum of 18 mice fed high-fat diet only (using Cat. #D12492 feed purchased from ResearchDiets Inc., wherein 60%of the energy is from fat, fed for 35 weeks) and serum of 15 mice fed normal diet were obtained. The serum samples above were tested using the same Western blotting assay as described previously, the results of the assay were shown in Figure 8. It can be inferred from the experimental results that a high-fat diet will induce increase of serum MG53, and thereby risk of having fat metabolism diseases can be determined based on content of MG53 in serum.

Serum of 6 12-week-old ZDF rats (Zucker diabetic fatty rats, purchased from Vital River Laboratories (Beijing, China) , Cat# 123) and 6 normal SD rats were obtained. Content of MG53 in the serum was detected using ELISA kit (Cusbio) , content of blood glucose was detected using blood glucose meter (Roche) , and content of blood insulin was detected using ELISA kit (Millipore) , and body weight was measured. The data is shown in Figure 9. It can be inferred from the experimental results that serum MG53 content is linearly correlated with body weight, and content of blood glucose and blood insulin, and thereby risk and development of diabetes, as well as response to treatment can be determined based on content of MG53 in serum.

8-10-week-old C57 (purchased from Vital River) mice were selected, fasted overnight, and then recombinant human MG53 protein (rhMG53) or BSA was injected via tail vein in the dosage of 6 mg/kg. 10 min later, insulin was injected intraperitoneally in the dosage of 1 U/kg or not injected. After another 10 min, the mice were sacrificedand corresponding tissue materials were obtained. Phosphorylation level of akt, which is an important molecule of the insulin signaling pathway, was tested by western blotting in skeletal muscle, liver, fat, and heart tissues for each experimental group, thereby evaluating insulin sensitivity in each of these tissues. The sources of various antibodies therein are as follows: Anti-phospho Ser473 Akt (p-AktS473) , CST Cat# 4060; Anti-total Akt (t-Akt) , CST Cat# 9272; Anti-GAPDH, Bioeasy Technology, Cat# BE0023. Statistical analysis was conducted using One-way anova. **represents that P<0.01.

The results of the assay are shown in Figure 10. It can be inferred from the experimental results that serum MG53 will induce sensitivity of insulin, and therefore sensitivity of insulin can be improved by reducing the concentration or activity of serum MG53.

High glucose-and high insulin-induced MG53 release from isolated perfused rodent hearts

Adult Sprague-Dawley rats (250 to 300 g) or mice (20 to 30 g) were anesthetized with pentobarbital (70 mg/kg, i.p. ) . The rat or mouse heart was excised and perfused on a Langendorff apparatus at a constant pressure of 55 mmHg with Krebs-Henseleit solution (in mM: NaCl 118, KCl 4.7, CaCl ₂ 2.5, MgSO ₄·7H ₂O 1.2, KH ₂PO ₄ 1.2, and glucose 11.1) . The buffer was continuously gassed with 95%O ₂/5%CO ₂ (pH 7.4) and warmed by a heating bath/circulator. The heart temperature was continuously monitored and maintained at 37±0.5℃. The outlet perfusate was collected for different periods of time. For EDTA or BFA treatment, 0.75 mM EDTA-Na (without Ca ²⁺ in the perfusion solution) or 30M BFA were included in the perfusion solution. The collected perfusate samples were centrifuged at 3,000 rpm with Amicon Ultra-15 10K Centrifugal Filter Devices (Millipore, Cat# UFC801096) for 15 min, and this was repeated 3 times. The concentrated perfusate samples were used for subsequent analysis.

In the isolated rodent hearts under Langendorff perfusion with physiological saline, we detected robust MG53 release by Western blotting analysis of the outlet perfusate accumulated over 30-min periods in rat hearts and 60-min periods in mouse hearts (Figure 32) . Strikingly, metabolic challenge with high glucose (25 mM) augmented rat myocardial MG53 release by 3-fold in the first 30 min after stimulation (Figure 32A) . Elevated MG53 release continued during the second 30 min (30–60 min) of glucose challenge, and was graded in a glucose concentration-dependent manner (Figure 32A and 32B) . In sharp contrast, L-glucose, which cannot be utilized by organisms, failed to alter myocardial MG53 release (Figure 32C) . To mimic the conditions of metabolic syndrome and T2D, we investigated the effect of combinatorial stimulation by glucose and insulin. Insulin stimulation with a basal concentration of glucose (11.1 mM) augmented the myocardial MG53 release in isolated perfused rat hearts in a time-and concentration-dependent manner (Figure 32D and 32E) . The co-presence of high glucose and insulin stimulated MG53 release at a rate ～4-fold of baseline (Figure 32F) . Furthermore, using MG53 overexpression (mg53 TG) and knockout (mg53 ^-/-) mouse models 6, we demonstrated that MG53 release induced by combined glucose and insulin stimulation was more than doubled from the mg53 TG heart compared to the wild-type (wt) , but was undetectable in the mg53 ^-/-heart (Figure 32G and 32H) , validating the identity of the released protein detected by Western blotting. As shown in Figure 35A and 35B, the MG53 release triggered by high glucose plus insulin occurred in the absence of any change in myocardial lactate dehydrogenase (LDH) or creatine kinase (CK) release (indexes of loss of cell membrane integrity) in perfused rat hearts, indicating that metabolism-regulated MG53 release is not caused by myocardial damage.

High glucose and insulin trigger MG53 release in isolated skeletal muscle and in vivo

Adult male C57 mice (20 to 30 g) were anesthetized with pentobarbital (70 mg/kg, i.p. ) . The soleus muscles were isolated from the mice and continuously perfused in modified Krebs-Henseleit solution (in mM: NaCl 118, KCl 4.7, CaCl ₂ 2.5, MgSO ₄·7H ₂O 1.2, KH ₂PO ₄ 1.2, and glucose 5) gassed with 95%O ₂/5%CO ₂ at room temperature (22℃) at a rate of 1.0 ml/min. After 10-min control perfusion, the soleus muscles were treated with modified Krebs-Henseleit solution buffer with or without high glucose (25 mM) combined with insulin (10 U/L) for 2 h. The perfusate samples were analyzed with Western blotting after concentration by ultra-filtration.

In addition to myocardium, the combined treatment of high glucose (25 mM) and insulin (10 U/L) effectively triggered MG53 release from isolated perfused skeletal muscle (soleus) from wt mice (Figure 33A) . Glucose-induced MG53 release from skeletal muscle occurred in the absence of any increase in the perfusate LDH or CK concentration (Figure 35C and 35D) . This result indicates that metabolic stimulation enables skeletal muscle as well as myocardium to release MG53 in a physiological context. Since skeletal muscle accounts for approximately 40%body mass of lean individuals, we hypothesized that high glucose-induced MG53 release would be sufficient to elevate circulating MG53 levels in vivo. Hyperglycemia (glucose, 2g/kg, i.p., 3 repeats at 40-min intervals) increased serum MG53 in wt mice (Figure 33B) .

Oral delivery of glucose in healthy humans

The healthy participants were fasted overnight, then glucose (75 g) was orally delivered twice at a 30-min interval. A blood sample was taken before the glucose treatment, and at 30 and 90 min after the second dose. The blood samples were centrifuged at 3,000 rpm, and serum was collected for Western blotting

Oral administration of glucose significantly elevated serum MG53 levels (Figure 33C) , along with increased blood glucose levels in healthy humans, in the absence of change of serum LDH concentrations.

Metabolism-induced MG53 release is mediated by a regulated secretory pathway

Using pharmacological and genetic approaches, we then sought to decipher the mechanism underlying the metabolism-regulated MG53 release. First, to distinguish regulated MG53 secretion from passive leakage, we evaluated the effect of Brefeldin A (BFA) , a protein transport inhibitor. BFA suppressed high glucose induced MG53 release in a dose-dependent manner in HEK293 cells expressing MG53-myc (Figure 33D) . In isolated perfused rat hearts, BFA (30 μM) also abrogated glucose-induced MG53 release (Figure 33E) , suggesting that glucose-triggered MG53 release is mediated by a canonical secretory pathway. In most cases of regulated secretion, a rise in the cytosolic Ca ²⁺ concentration is a prerequisite for fusion of the secretory-vesicle membrane with the plasma membrane and the release of the vesicle contents by exocytosis. Indeed, buffering extracellular Ca ²⁺ with EDTA (0.75 mM) largely abolished the metabolism-regulated MG53 release (Figure 33F) . In the syt7 ^-/-mice, MG53 release stimulated by high glucose plus insulin was abolished in isolated perfused hearts and in vivo (Figure 33G and 33H) , indicating that Syt7 is required for metabolism-regulated MG53 secretion. It is noteworthy that neither EDTA treatment nor Syt7 deficiency altered basal MG53 release in the perfused rodent heart (Figure 33F and 33G) or in vivo (Figure 33H) , suggesting that basal release may be attributable to non-canonical secretion or leakage. Taking together, we conclude that metabolism-induced MG53 release is mediated by a Ca ²⁺ and SNARE binding protein-dependent secretory mechanism, as is the case for many types of cytokines.

Cardiac-specific overexpression of MG53 is sufficient to trigger systemic insulin resistance and metabolic syndrome

In order to further investigate a possible causal relationship between chronic elevation of MG53 in the blood and the development of metabolic disorders, we generated transgenic mice with heart-specific overexpression of MG53 (mg53 h-TG) . The myocardial MG53 secretion would provide a constant supply of circulating MG53, while the MG53 expression was unperturbed in skeletal muscle. Figure 34A shows that the basal serum MG53 level was obviously higher in young adult mg53 h-TG mice at 14 weeks of age than in wt littermates.

Fasting body weights of wt and mg53 h-TG mice from 3 to 38 weeks of age were measured, and glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed on wt and mg53 h-TG mice at 14 weeks and 30 weeks of age, respectively. Briefly, for GTTs, mice were fasted overnight (for 16 h) and then injected intraperitoneally (i.p. ) with D-glucose (2 g/kg, Sigma-Aldrich) . For ITTs, mice were randomly fed and challenged with bovine insulin (0.75 U/kg, Sigma-Aldrich, i.p. ) . Blood from tail vein were collected and blood glucose measured before injection and at different time-points after injection (as indicated in Figure 34C and 34D) .

The young mg53 h-TG mice exhibited moderate obesity (Figure 34B) , glucose intolerance, and insulin intolerance (Figure 34C and 34D) . At 30 weeks of age, cardiacspecific overexpression of MG53 was sufficient to trigger full-blown metabolic syndrome, as manifested by hyperglycemia, hyperinsulinemia, and dyslipidemia (Figure 34E-34G) , abdominal fat accumulation, increased white fat, brown fat, and fat-to-lean ratio (Figure 34H-34M) , together with hepatosteatosis and pancreatic islet hypertrophy (Figure 34N and 34O) . As systemic insulin resistance evolved, the mg53 h-TG mice displayed severe obesity, glucose intolerance, and insulin intolerance at this time point (Figure 34B-34D) . Meanwhile, the daily energy expenditure of mg53 h-TG mice was significantly lower than that of the wt counterpart (Figure 34Q-34S) , but there was no difference in their daily food intake, core body temperature, or physical activity. At the later time point, mg53 h-TG mice also developed diabetic complications such as diabetic cardiomyopathy. Hence, a chronic elevation of circulating MG53 primarily impairs the whole-body insulin response, and secondarily leads to systemic metabolic syndrome that contributes to obesity, diabetes and various cardiovascular complications.

Example 3: study on therapeutic effect of the antibodies

20 8-10-week-old db/db mice (diabetes model mice, purchased from Jackson Laboratory (Bar Harbor, ME) Cat# 000642) were selected, wherein 10 mice were used as the control group, injected with IgG (Sigma, I5381) only; the other 10 were used as the therapeutic group, injected with MG53 Antibody #84. Meanwhile, 5 db/+ mice of the same age were ordered. db/+ mice were only used for the final material collection, and were not injected.

After the mice arrived, they were stabilized for 1 week, and then the body weight and blood glucose of the mice were measured, and the condition thereof were checked. On the day of the experiment, the animals were not fasted, and body weight and blood glucose were measured at 8 a.m. The mice were then grouped according to body weight and blood glucose meausurement, and injection was conducted according to grouping. Subsequently, injection was conducted at 9 a.m., at 1 mg/mouse/time, volume of injection: 0.24ml /mouse/time. Later the mice were fasted, and blood glucose was measured at 3 p.m. and 7 p.m. on the day of the experiment respectively, and body weight was measured at 9 a.m., and blood glucose was measured at 9 a.m. and 3 p.m. on every following day for 10 days.

On the 8th day after injection, insulin tolerance test (ITT) was performed (see, Song et al., Central role of E3 biquitin ligase MG53 in insulin resistance and metabolic disorders, Nature, 494, 375-381, 2013 for description of the experimental condition) .

The results of the assay are shown in Figure 11. It can be inferred from the experimental results that MG53 antibodies can effectively reduce blood glucose and can improve sensitivity of insulin, and thereby can be used to treat diabetes.

Example 4: study on MG53 small molecule inhibitor

All small molecule compounds used in example 4 were purchased from the SPECS compound library in Netherlands, and MG53 proteins were synthesized and prepared by ORIGENE, Inc.

Computer simulated screening

First, homology modeling was conducted on MG53 proteins. Existing protein structure with high homology in primary structure of protein, i.e., amino acid, and the analyzed structure of which has high resolution, was selected as reference structure, and then prediction was conducted on small molecule binding pockets using Cavity 2.0. Pockets with relatively large pocket volume, relatively high dissociation constant (pKD) and bi-affinity were selected as target pockets.

The principle for screening was to screen by different angles and different approaches. Each screening approach was scored by strength of binding between small molecule compounds and proteins, and each time the molecules with higher scores were selected, and finally the intersection of the screening results by different approaches was adopted. The specific operation is as follows: first, each group in the protein pocket was fixed, which were freely joined with small molecule compounds, and 20,000 compounds with high scores were selected; then, the small molecule compounds and groups were all allowed to freely rotate and join, and four thousand compounds with high scores were selected. Next, pymol software was used for manual screening, five standards for which are as follows: a. the molecular weight of the compound is to be greater than 300 Da, specific response is reduced if the molecular weight is too small; b. at least three hydrogen bonds interact between the compound and the group in the pocket, which is for increasing the interaction between the compound and the protein; c. the compound is to occupy 80%or more of the protein pocket, which is also for reducing non-specificity; d. the compound cannot be a polypeptide, which is to avoid non-specific binding of polypeptides in an organism; e. the compound does not contain any metal atom. Ultimately, 140 small molecule compounds were obtained.

Cell screening

Next, the 140 molecules were subject to cell level screening. A system specific for cell screening was constructed so as to conduct a large amount of screening experiments. First, IRS1 plasmid (IRS1-GFP) with GFP fluorescence label and MG53 plasmid (MG53-myc) with Myc label were constructed, and these two plasmids were simultaneously transfected into cells to allow them to be highly expressed. Under normal circumstances, MG53 will exercise its activity as E3 ligase and mediate ubiquitination degradation of IRS1, so that the intensity of fluorescence it produces is significantly reduced compared with the control group, and thereby the cellular insulin signaling pathway is destroyed. Another small molecule compound was added at the same time when these two plasmids were co-transfected into cells. If the small molecule compound has inhibiting effect to activity of MG53 protein, and impedes exercising of its normal function within the cells, fluorescence intensity of IRS1 will be restored to certain extent, according to which small molecule compounds with inhibiting to MG53 were identified. To select a cell with better controllability and easy to operate, a comparison experiment was conducted between HEK293T and HEK293A cells, i.e., MG53-Myc and IRS1-GFP were co-transfected in these two cells respectively. The results of selection are shown in Figure 12. It was found that in HEK293T cells, MG53 can greatly attenuate fluorescence intensity of IRS1. To enlarge the operation window for subsequent screening experiments, HEK293T cells were selected for subsequent experiments to perform in.

After the screening system was built, screening on the 140 small molecule compounds was conducted in HEK293T cells. First, HEK293T cells were cultured using 10 ml cell culture dish. Medium were changed once every two days, and cells in good growing and health state were selected, and MG53-Myc and IRS1-GFP were co-transfected by the time the cells covered 80%of the entire cell culture dish, and at the same time, pcDNA4 empty vector and IRS1-GFP were co-transfected for the control group. The cells were cultured for 24 hours after transfection, under fluorescence microscope, strong expression of GFP could be found in the control group, while fluorescence intensity in the experimental group was attenuated by more than 50%. The cells were then detached using 0.125%pancreatin, collected by low speed centrifugation, evenly mixed and then spread onto 96-well enzyme-labeled plates respectively, and continued to be cultured. 24 hours later, the cells were subject to change of medium and addition of small molecule compounds to be tested. Culture continued for another 24 hours, and at last the cells were washed 3 times with PBS, and subject to 485nm/528nm fluorescence signal assay. The compound was selected in concentrations of 100 μM, 10 μM and 1 μM respectively. A total of 48 candidate compound molecules were selected.

SPR screening

Initial screening for SPR assay was performed on the 48 candidate compound molecules. Based on theoretical calculation, the theoretical response of mutual binding between MG53 protein and small molecule compounds is 5, and therefore one of the standards for screening was that response is greater than or equal to 5. Two concentrations, 50 μM and 100 μM respectively, were selected for screening, and the control group was PBS solution. As shown in Figure 13, among these 48 small molecule compounds, a total of 15 molecules had a response of no less than 5 (wherein response of No. 10, No. 14, No. 16, No. 17, No. 25, No. 68, No. 13, No. 16, and No. 28 were relatively higher; response of No. 1, No. 3, No. 26, No. 28, No. 56 and No. 10 are relatively lower) . These compounds were the candidate molecules for the next step of screening.

Next, kinetic characteristics of binding of the 15 candidate molecules to MG53 were explored through concentration dependent SPR. Standards for screening include dissociation constant less than 50 μM, specific binding, and high molecular purity. Finally, 4 candidate molecules were obtained from screening (see Figure 14 for data, No. 1, No. 10, No. 26 and No. 16 respectively) . These 4 molecules all have dissociation constant of less than 50 μM (see the corresponding S-shaped curves on the right, dissociation constant being the compound concentration when response reaches half of the maximum response) , as well as very low minimum response concentration in the nanomolar range, indicating higher specificity and better druggability of the compounds. The S-shaped curves indicate that these compounds have favorable kinetic characteristics. The other 11 compound molecules were poor candidates due to insufficient molecular purity (as measured by means of small molecule mass spectra) , or overly high dissociation constant (dissociation constant being over 50 μM) , or bad kinetic characteristics exhibited (kinetic curve being linear or not S-shaped, indicating that binding between the molecule and MG53 protein has strong non-specificity) , etc. Among all 4 of these candidate molecules, molecule No. 26 is most noteworthy, the minimum response concentration of which is only 1.2 nM, and the dissociation constant of which is 100 nM, indicating that the molecule has excellent specific binding to MG53 protein, and the SPR response curve of which is of a “slow up, slow down” type, which indicates that, if the molecule is used in administration and treatment, it will exhibit many advantages such as low dosage, long and stable efficacy, long intervals between dosings, etc., and therefore is highly druggable.

Experimental verification for degradation

Next, intracellular activity of the 4 candidate molecules was further verified. HEK293T cells under good condition in culture were divided into 6 groups which were transfected with IRS1 and MG53-myc respectively, 4 groups of cells transfected with IRS1 and MG53-myc were respectively treated by adding compounds, concentration of the added compounds were all 50 μM. 24 hours after the addition of those compounds, extraction of cell total protein and SDS-PAGE gel vertical electrophoresis experiment were performed. Expression of IRS1 and MG53 protein were detected respectively upon exposure, using GAPDH as internal reference. All these 4 compound molecules were found to have different levels of recovery effect on MG53 mediated ubiquitylation degradation of IRS1 (see Figure 15) , indicating that each of these compound molecules demonstrates a certain level of inhibitive effect on MG53 exercising E3 ligase function in cells.

Structures of the 4 selected compounds are as follows:

Claims

A method for detecting an MG53 related disease or predicting a risk of an MG53 related disease, comprising the following steps:

a. obtaining a test sample; and

b. detecting cell-free MG53 in the test sample.
The method according to claim 1, wherein the test sample is selected from whole blood, plasma, serum, and tissue fluid.
The method according to claim 1, wherein the test sample substantially does not contain any cells.
The method according to claim 1, wherein the step b) comprises contacting an MG53 detection agent with the test sample.
The method according to claim 4, wherein the MG53 detection agent is an MG53 antibody or antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof.
The method according to claim 4, wherein the MG53 detection agent is a small molecule compound capable of binding MG53.
The method according to claim 4, wherein the MG53 detection agent has a detectable label.
The method according to claim 7, wherein the detectable label is luminescent, magnetic, radioactive, or enzymatically active.
The method according to claim 1, wherein the step b) comprises conducting radioimmunoassay, Western blot analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation, chemiluminescence, immunohistochemistry assay, dot blot assay or slit blot method.
The method according to claim 1, further comprising:

c. comparing the detected value of MG53 obtained from step b) with a reference value.
The method according to claim 10, wherein the reference value is obtained from a reference sample.
The method according to claim 11, wherein the reference sample and the test sample are from the same subject or different subjects.
The method according to claim 11, wherein the reference sample and the test sample are from the same subject, but the time at which the reference sample was collected is earlier or later for a period of time than the test sample.
The method according to claim 13, wherein during the period of time, the subject accepts treatment or the health condition thereof has changed.
The method according to claim 10, wherein the reference sample is obtained from a healthy subject or a subject suffering from an MG53 related disease.
The method according to any of claims 12-15, wherein the subject is a human or a non-human mammal.
The method according to claim 1, wherein the MG53 related disease is metabolic syndrome.
The method according to claim 1, wherein the MG53 related disease is a glucose metabolism or lipid metabolism related disease.
The method according to claim 18, wherein the glucose metabolism or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complication, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension and obesity.
A method for reducing or inhibiting activity of cell-free MG53 in a subject, comprising administrating an effective amount of MG53 inhibitor to a subject in need thereof.
The method according to claim 20, wherein the MG53 inhibitor specifically binds to MG53.
The method according to claim 20, wherein the MG53 inhibitor is an MG53 antibody or antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof, or a nonfunctional protein fragment of MG53.
The method according to claim 22, wherein the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain, and the heavy chain comprises a sequence of SEQ ID NOs: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.
The method according to claim 22, wherein the MG53 antibody or antigen-binding fragment thereof comprises a light chain, and the light chain comprises a sequence of SEQ ID NOs: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.
The method according to claim 20, wherein the MG53 inhibitor is a small molecule compound.
The method according to claim 20, wherein the MG53 inhibitor is a compound having Formula Ia or Formula Ib, or a pharmaceutically acceptable salt thereof,

wherein,

L is a chemical bond, or an optionally substituted C ₁-C ₁₂ alkylene;

M is a chemical bond, or an optionally substituted C ₆-C ₁₂ arylene or a 5-12 membered heterocyclic subunit;

X is a chemical bond, or an optionally substituted -CH=N-NH-C (O) -, -CH ₂-O-, -O-CH ₂-C (O) -NH-, -O-C (O) -or -O-C (O) -CH=CH-;

Y is an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group;

Q is an optionally substituted C ₁-C ₁₂ alkylene or C ₂-C ₁₂ alkenylene;

T is an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group;

each R ₁ is independently hydrogen, nitro, halogen, hydroxyl, cyano, or an optionally substituted C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy;

n is any integral from 1 to 5.
The method according to claim 26, wherein L is a chemical bond or C ₁-C ₃ alkylene.
The method according to claim 26, wherein M is a chemical bond, or an optionally substituted phenylene or furylene.
The method according to claim 26, wherein Y is an optionally substituted phenyl, furyl, quinolinyl or benzo dioxolanyl.
The method according to claim 26, wherein Q is an optionally substituted C ₁-C ₃ alkylene or C ₂-C ₃ alkenylene.
The method according to claim 26, wherein T is an optionally substituted phenyl or 1, 3, 4-thiadiazolyl.
The method according to claim 20, wherein the MG53 inhibitor is a compound having Formula II or a pharmaceutically acceptable salt thereof,

wherein,

A is hydrogen, or an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group;

G is -C (O) -O-R ₃, wherein R ₃ is an optionally substituted C ₁-C ₁₂ alkyl; J is an optionally substituted C ₆-C ₁₂ aryl; or G and J, together with the carbon atom linked thereto, form
wherein each R ₄ is independently hydrogen, nitro, halogen, hydroxyl, cyano, or an optionally substituted C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy, n is any integral from 1 to 4;

B is a chemical bond, or an optionally substituted C ₆-C ₁₂ arylene or 5-12 membered heterocyclic subunit;

D is a chemical bond, or an optionally substituted -O-R ₅-, wherein R ₅ is optionally substituted C ₁-C ₁₂ alkylene;

E is an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group.
The method according to claim 32, wherein A is an optionally substituted naphthyl, phenyl or thienyl.
The method according to claim 32, wherein G is -C (O) -O-R ₃, wherein R ₃ is an optionally substituted C ₁-C ₃ alkyl; J is optionally substituted phenyl; or G and J, together with the carbon atom linked thereto, form
wherein each R ₄ is hydrogen.
The method according to claim 32, wherein B is a chemical bond, or an optionally substituted phenylene or furylene.
The method according to claim 32, wherein D is a chemical bond, or an optionally substituted -O-CH ₂-.
The method according to claim 32, wherein E is an optionally substituted phenyl.
The method according to claim 20, wherein the MG53 inhibitor is a compound having Formula III or a pharmaceutically acceptable salt thereof,

wherein,

A is hydrogen, or an optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group;

each Y is independently optionally substituted C ₆-C ₁₂ aryl or 5-12 membered heterocyclic group;

R ₇ is hydrogen, nitro, halogen, hydroxyl, cyano, or optionally substituted C ₁-C ₁₂ alkyl or C ₁-C ₁₂ alkoxy.
The method according to claim 38, wherein A is an optionally substituted phenyl.
The method according to claim 38, wherein Y is an optionally substituted phenyl.
The method according to claim 38, wherein R ₇ is hydrogen, nitro, halogen, hydroxyl or cyano.
The method according to any of claims 26-41, wherein the optionally substituted refers to not substituted by any substituent group or substituted by one or more substituent groups selected from the followings: nitro, halogen, hydroxyl, cyano, C ₁-C ₁₂ alkyl, C ₁-C ₁₂ alkoxy, benzamide group, or -C (O) -O-R ₆, wherein the C ₁-C ₁₂ alkyl, C ₁-C ₁₂ alkoxy or benzamide group can further be substituted by nitro, halogen, hydroxyl, cyano, C ₁-C ₃ alkyl or C ₁-C ₃ alkoxy, wherein R ₆ is hydrogen or C ₁-C ₃ alkyl.
The method according to claim 20, wherein the MG53 inhibitor is a compound of the followings or a pharmaceutically acceptable salt thereof
The method according to claim 20, which is used to ameliorate, treat or predict an MG53 related disease or a symptom thereof.
The method according to claim 44, wherein the MG53 related diseased is metabolic syndrome.
The method according to claim 44, wherein the MG53 related diseased is a glucose metabolism or lipid metabolism related disease.
The method according to claim 46, wherein the glucose metabolism or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complication, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension and obesity.
An MG53 antibody or antigen-binding fragment thereof, comprising three heavy chain complementary determining regions comprised in a sequence of SEQ ID NOs: 1, 9, 17, 25 or 33.
The MG53 antibody or antigen-binding fragment thereof according to claim 48, comprising three light chain complementary determining regions comprised in a sequence of SEQ ID NOs: 2, 10, 18, 26 or 34.
The MG53 antibody or antigen-binding fragment thereof according to claim 48, comprising a heavy chain, wherein the heavy chain has an amino acid sequence of SEQ ID NOs: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.
The MG53 antibody or antigen-binding fragment thereof according to claim 50, further comprising a light chain, wherein the heavy chain has an amino acid sequence of SEQ ID NOs: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.
An antibody or antigen-binding fragment, competing for binding to MG53 with the antibody or antigen-binding fragment of any of claims 48-51.
An isolated nucleic acid, encoding the antibody or antigen-binding fragment of any of claims 48-52.
The nucleic acid according to claim 53, comprising a nucleotide sequence of SEQ ID NOs: 5, 6, 7, 8, 13, 14, 15, 16, 21, 22, 23, 24, 29, 30, 31, 32, 37, 38, 39 or 40, or a sequence having at least 80%, 85%, 90%, 95%or 99%sequence homology thereof.
A clone or expression vector, comprising the isolated nucleic acid of claim 53 or 54.
A host cell, comprising the clone or expression vector of claim 55.
A pharmaceutical composition, comprising the antibody or antigen-binding fragment of any of claims 48-52, the clone or expression vector of claim 55, or the cell of claim 56, and a pharmaceutically acceptable excipient.
Use of a detection agent for detecting cell-free MG53 in the manufacture of a kit, which is used for detecting an MG53 related disease, predicting a risk or development of an MG53 related disease, identifying potential MG53 inhibitor, evaluating treatment effect for an MG53 related disease, or detecting activity of cell-free MG53.
Use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting cell-free MG53 in a subject.