A drug or pharmaceutical composition for inhibiting TRAF6 and its application

Abstract

This invention relates to a TRAF6 inhibitor, comprising any Traf6i-n compound or a salt thereof; and the use of the Traf6i-n compound or a salt thereof in the preparation of a medicament for treating diseases caused by abnormal TRAF6 expression. This application develops a small molecule inhibitor targeting TRAF6 and, by establishing a mutant mouse model lacking E3 ligase activity, obtains unexpected technical effects in the treatment of diseases related to Golgi-associated TRAF6.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a drug or pharmaceutical composition that enhances glucose metabolism and its application. Background Technology

[0002] Tumor necrosis factor receptor-associated factor 6 (TRAF6), a unique E3 ubiquitin ligase, plays a central regulatory role in the TLR / IL-1R signaling pathway. It activates downstream IKK complexes and TAK1 kinases through K63 ubiquitination, thereby mediating a cascade amplification effect in the NF-κB and MAPK signaling pathways. This mechanism has been well-documented in Nature Immunology (2008; 9:817). Recent studies have found a significant positive correlation between abnormal TRAF6 activity and autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease (Cell 2011; 147:290), and that it promotes osteoclast differentiation through the RANKL signaling axis during bone metastasis in breast cancer (Cancer Cell 2016; 29:763).

[0003] Traditional inhibitor development focuses on blocking the TRAF6-UBE2N interaction interface. For example, the natural product genistein can competitively inhibit the ubiquitin-binding domain (JBC 2007; 282:3683), but its poor selectivity leads to significant off-target effects. In recent years, C25-Hydroxyprotostemonine compounds discovered based on virtual screening have improved the IC50 to 0.8 μM (Eur J Med Chem 2020; 185:111822), but their rigid steroidal skeleton limits their druggability.

[0004] Inhibiting TRAF6 function has demonstrated unique value in disease treatment: for example, in tumor immunotherapy, it can block PD-L1 expression in MDSC cells to restore T cell killing activity (Nat Commun 2019; 10:179), and inhibit tumor cell invasion and metastasis by downregulating MMP-9 (Oncogene 2018; 37:4395). In the field of neurodegenerative diseases, animal experiments have shown that TRAF6 inhibitors can reduce Aβ-induced microglial overactivation and decrease Tau protein phosphorylation levels by 62% (J Neurosci 2020; 40:4245). Recent research has further discovered that TRAF6-mediated LC3 ubiquitination is a key node in the regulation of autophagic flux, suggesting its potential application in anti-infective therapy (Science 2021; 371:eabc3599).

[0005] Therefore, the screening of suitable TRAF6 inhibitors remains a problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention proposes the application of Traf6i-n compounds or their salts in the preparation of drugs for treating diseases caused by abnormal TRAF6 expression; wherein Traf6i-n is selected from Traf6i-1 to 14, and Traf6i-1 to 14 have the following structural formulas:

[0007] Traf6i-1: Traf6i-2: Traf6i-3: Traf6i-4: Traf6i-5: Traf6i-6: Traf6i-7: Traf6i-8: Traf6i-9: Traf6i-10: Traf6i-11: Traf6i-12: Traf6i-13: And Traf6i-14:

[0008] As described above, the TRAF6 expression abnormality is an increase in the expression level of the TRAF6 protein.

[0009] As described above, the diseases caused by abnormal TRAF6 expression include, but are not limited to, diabetes, abnormal glucose metabolism, tumors, rheumatoid arthritis, inflammatory bowel disease, breast cancer bone metastasis, neurodegenerative diseases, and infectious diseases.

[0010] As described above, Traf6i-n can inhibit the activity of TRAF6.

[0011] As described above, Traf6i-n can regulate protease activity.

[0012] As described above, protease activity is regulated by ubiquitination, phosphorylation, or methylation; preferably, protease activity is regulated by ubiquitination of the cytoplasmic domains of the protease.

[0013] In the applications described above, the protease is a PCSK family protein; preferably, the protease is a PCSK family protein having a transmembrane domain.

[0014] As described above, the PCSK family proteins include: FURIN, PCSK5B, PCSK7, PCSK8, and PCSK9.

[0015] In the application described above, the abnormal glucose metabolism is caused by decreased insulin sensitivity in the subject, reduced glucose uptake in the subject's muscles, or reduced hepatic gluconeogenesis in the subject.

[0016] As described above, the abnormal glucose metabolism is caused by a high-fat diet, inhibited FURIN activity, decreased INSRβ / pro-INSR, abnormal muscle glucose uptake, or abnormal hepatic gluconeogenesis.

[0017] In the application described above, the diabetes is type 2 diabetes.

[0018] This application relates to the use of any Traf6i-n compound and its salt in the preparation of an inhibitor of intracellular TRAF6 activity.

[0019] This application also relates to compositions comprising any Traf6i-n compound or a salt thereof, said compositions being capable of treating diseases caused by abnormal TRAF6 expression.

[0020] This application further relates to a TRAF6 inhibitor, which includes any Traf6i-n compound or a salt thereof.

[0021] This application relates to a drug or drug composition that enhances glucose metabolism in a subject, comprising one or more of an active substance that inhibits TRAF6, FURIN, and INSRβ.

[0022] The drug or drug composition described above, wherein the active substance is a small molecule compound; preferably, the small molecule compound includes any Traf6i-n compound or a salt thereof.

[0023] In the drug or drug composition described above, the active substance is a siRNA or shRNA that silences TRAF6; preferably, the sequence of the siRNA is shown in SEQ ID NO.7 or SEQ ID NO.8; and the sequence of the shRNA is shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13.

[0024] The drug or drug composition described above, wherein the active substance is a carrier comprising a site-directed mutagenesis-mediated TRAF6, wherein the site-directed mutagenesis does not include TRAF6. K67 and / or TRAF6 H87 Preferably, the site-directed mutation of TRAF6 is a mutation of TRAF6. Q54 TRAF6 S66 TRAF6C70 TRAF6 P71 TRAF6 I72 TRAF6 L74 TRAF6 L77 TRAF6 G86 TRAF6 R88 and / or TRAF6 C93 Site-directed mutation.

[0025] This application relates to the use of a drug or pharmaceutical composition in the preparation of a pharmaceutical composition that enhances glucose metabolism in a subject; wherein the drug or pharmaceutical composition comprises one or more of an active substance that inhibits TRAF6, FURIN, and INSRβ.

[0026] As described above, the pharmaceutical composition alters the C-terminal portion (Asn) of the TRAF-C / MATH domain. 358 (Terminal) activity.

[0027] As described above, the pharmaceutical composition alters the activity of the cytoplasmic domain portion of FURIN.

[0028] This application developed a small molecule inhibitor targeting TRAF6 and obtained unexpected technical effects in the treatment of diseases by establishing a mutant mouse model with E3 ligase activity deficiency. Attached Figure Description

[0029] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0030] Figure 1 This relates to the effect of a TRAF6 inhibitor on insulin sensitivity and glycemic control in obese patients according to an embodiment of the present invention; wherein,

[0031] Figure 1 a represents the molecular docking of Traf6i-13 and TRAF6; Traf6i-13 is represented by the green ball-and-stick model, and the potential residues for the interaction between TRAF6 and Traf6i-13 are represented by the cyan model;

[0032] Figure 1 b is to study the interaction between recombinant Flag-TRAF6 and Traf6i-13 using surface plasmon resonance (SPR) experiments;

[0033] Figure 1 c represents K, which combines Traf6i-13 and Flag-TRAF6. D ;

[0034] Figure 1d represents the interaction between Flag-TRAF6 and GFP-UBC13 when co-expressed in HEK293 cells treated with different concentrations of Traf6i-13;

[0035] Figure 1 e represents the fasting blood glucose levels of male DIO mice before and after administration of Traf6i-13 (100 mg / kg body weight / d) for 1 month; mice were administered Traf6i-13 orally after 5 months of HFD via gavage; where n = 7-9;

[0036] Figure 1 f represents glucose production in primary hepatocytes treated with a TRAF6 inhibitor; cells were pretreated with a specified concentration of Traf6i-13 for 16 hours, followed by glucose output measurement; where n = 5-6.

[0037] Figure 1 g- Figure 1 h represents the treatment of male DIO mice with Traf6i-13 (100 mg / kg body weight / d) for 5 weeks via PTT; among which, Figure 1 g represents blood glucose during the PTT (Pulse Test). Figure 1 h represents the area of ​​glucose under the curve; n = 6 - 9;

[0038] Figure 1 i- Figure 1 j represents the oral glucose tolerance test (OGTT) of male DIO mice after one month of administration of Traf6i-13 (100 mg / kg body weight / day); among which, Figure 1 i represents blood glucose levels during the OGTT; Figure 1 j represents the area under the blood glucose curve; n = 6 - 9;

[0039] Figure 1 k- Figure 1 The effect of Traf6i-13 (100 mg / kg body weight / day) treatment for 6 weeks on insulin sensitivity of primary hepatocytes isolated from male DIO mice; hepatocytes were stimulated with insulin for 30 minutes; among which, Figure 1 k represents immunoblotting; Figure 1 l represents the quantitative data for INSRβ, n = 6; Figure 1 m represents the quantitative data of PKB-T308 phosphorylation, n = 3; Figure 1 n represents the quantitative data of PKB-S473 phosphorylation, n = 3; Figure 1 o represents quantitative data for INSRβ / pro-INSR, n = 6;

[0040] Figure 1 g- Figure 1 j、 Figure 1 l、 Figure 1 Perform a t-test. Figure 1 e- Figure 1 f、 Figure 1 m- Figure 1 A two-way ANOVA was performed; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0041] Figure 2 Traf6i-13, according to an embodiment of the present invention, inhibits TRAF6; wherein,

[0042] Figure 2 a represents the molecular docking of Traf6i-13 (Traf6i-13) with TRAF6; the top image shows the N-terminal dimer motif of TRAF6 (3HCS.pdb), and the bottom image shows the N-terminal cavity of TRAF6, which is used for potential drug interactions;

[0043] Figure 2 b represents the Il-1β mRNA level (Traf6i-1 to Traf6i-14) in BMDMs treated with 10 ng / ml LPS (with or without 10 μg / ml compound); the red line indicates 80% inhibition; n = 3-5;

[0044] Figure 2 c represents the Tnf-α mRNA level in BMDMs treated with 10 ng / ml LPS [with or without 10 μg / ml compounds (Traf6i-1 to Traf6i-14)]; n = 3-5;

[0045] Figure 2 d represents the pharmacokinetics of Traf6i-13 in C57BL6 mice after acute gavage administration of 100 mg / kg Traf6i-13; gavage administration was used.

[0046] Figure 2 e represents the pharmacokinetic parameters of Traf6i-13 in C57BL6 mice; where administration was by gavage.

[0047] Figure 2 f shows a schematic diagram of the one-chamber model (top) and the fitting results (bottom);

[0048] Figure 2 g shows a schematic diagram of the two-compartment model (top) and the fitting results (bottom);

[0049] Figure 2 h represents the pharmacokinetic parameters of the Traf6i-13 two-compartment model in C57B6 mice; where gavage administration was used.

[0050] Figure 2 i represents the model prediction and actual experiment (100 mg / kg) on ​​day 27 of continuous administration;

[0051] Figure 2j represents the tissue distribution of Traf6i-13 after gavage, n = 6;

[0052] Figure 2 k represents the effect of Traf6i-13 on the body growth of male DIO mice; WT mice were fed HFD starting at 6 weeks of age and orally administered Traf6i-13 at 21 weeks of age; n = 7-9;

[0053] Figure 2 l- Figure 2 After treatment with Traf6i-13 (100 mg / kg body weight / d) for 6 weeks, the livers of male DIO mice ( Figure 2 l) or white adipose tissue ( Figure 2 Expression of inflammatory genes in m); n = 5-7;

[0054] Figure 2 n- Figure 2 q represents the effect of Traf6i-13 on insulin sensitivity in L6 myotubular cells; cells were deprived of serum for 16 hours in the presence or absence of Traf6i-13 (10 μM); subsequently, cells were stimulated with insulin for 30 minutes. Figure 2 n represents the quantitative data for INSRβ, n = 6; Figure 2 o represents quantitative data for INSRβ / pro-INSR, n = 6; Figure 2 p represents quantitative data on PKB-S473 phosphorylation, n = 3; Figure 2 q represents immunoblotting;

[0055] Figure 2 k- Figure 2 Perform a t-test. Figure 2 A two-way ANOVA was performed on p, where * indicates p < 0.05 and *** indicates p < 0.001;

[0056] Figure 3 This relates to the effect of TRAF6 inactivation on insulin signaling according to an embodiment of the present invention; wherein,

[0057] Figure 3 a- Figure 3 d represents the protein and mRNA levels of INSR in primary mouse hepatocytes with TRAF6 knockout; TRAF6 in primary hepatocytes was downregulated using siRNA; siNC and siRNA negative controls were also included. Figure 3 a represents immunoblotting; Figure 3 b represents the quantitative result of INSRβ, n = 4; Figure 3 c represents the quantitative result of INSRβ / pro-INSR, n = 4; Figure 3 d represents the mRNA level of Insr, and n = 5-6;

[0058] Figure 3 e- Figure 3 f represents the response of PKB phosphorylation to insulin in TRAF6 knockout primary mouse hepatocytes; among which... Figure 3 e represents a representative immunoblot; Figure 3 f represents the quantitative result of PKB phosphorylation, n = 4-5;

[0059] Figure 3 g- Figure 3 i represents the protein level of INSR in the insulin response of L6 myotubes with TRAF6 knockout; TRAF6 in L6 myotubes was downregulated using shRNA, shNC, and shRNA negative controls; among which... Figure 3 g represents immunoblotting; Figure 3 h represents the quantitative results of INSRβ and TRAF6; Figure 3 i represents the quantitative result of INSRβ / pro-INSR; n = 6;

[0060] Figure 3 j- Figure 3 k represents the response of the insulin signaling pathway in the TRAF6 knockout L6 myotubule of clone shTraf6-4 to insulin; where... Figure 3 j represents the representative immunoblot; Figure 3 k represents the quantitative result of PKB-S473 phosphorylation; n = 5-6;

[0061] Figure 3 l represents glucose uptake by insulin in the L6 myotube with TRAF6 knockout, n = 6;

[0062] Figure 3 m- Figure 3 p represents WT and TRAF6 I72A The protein levels of INSR in skeletal muscle and liver were knocked out; among them, Figure 3 m represents WT and TRAF6 I72A Immunoblot of INSR knocked out in gastrocnemius muscle (4-6 month old male mice); Figure 3 n represents wild type and TRAF6 I72A Immunoblotting of INSR knockout in the liver (6-month-old male mice), crosses indicate nonspecific bands; Figure 3 o represents the quantitative results of INSRβ and TRAF6;

[0063] Figure 3 p represents the quantitative result of INSRβ / pro-INSR, n = 4-5;

[0064] Figure 3 q- Figure 3 r represents female WT and TRAF6. I72AThe phosphorylation response of PKB to insulin in the soleus muscle of knockout mice (4 months old) was eliminated; soleus muscle was isolated from overnight fasted mice and stimulated with or without insulin; among them, Figure 3 q represents immunoblotting; Figure 3 r represents the quantitative result; n = 3-4.

[0065] Figure 3 s- Figure 3 t represents the phosphorylation of PKB and TRAF6 in WT. I72A Knockout of insulin response in primary hepatocytes; Figure 3 s represents the representative immunoblot; Figure 3 t represents the quantitative result; n = 5-6;

[0066] pass Figure 3 b- Figure 3 d、 Figure 3 h- Figure 3 i Figure 3 o- Figure 3 t-test for p and Figure 3 f、 Figure 3 k、 Figure 3 r and Figure 3 Statistical analysis was performed using a two-way ANOVA; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0067] Figure 4 This describes the effect of TRAF6 knockout on L6 insulin signaling according to an embodiment of the present invention; wherein,

[0068] Figure 4 a- Figure 4 c represents the protein levels of INSR and TRAF6 in L6 myotubes of clone shTraf6-17 lacking TRAF6; TRAF6 in L6 myotubes was downregulated using shRNA, shNC, and shRNA negative controls; among which... Figure 4 a represents immunoblotting; Figure 4 b represents the quantitative results of INSRβ and TRAF6; Figure 4 c represents the quantitative result of INSRβ on pro-INSR; n = 5;

[0069] Figure 4 d- Figure 4 e represents the response of the insulin signaling pathway to insulin in the L6 myotubule of the TRAF6 knockout clone shTraf6-17; among which, Figure 4 d represents immunoblotting; Figure 4 e represents the quantitative result of PKB phosphorylation; n = 3;

[0070] Figure 4 f- Figure 4h represents the protein levels of INSR and TRAF6 in the TRAF6 knockout L6 myotube of clone shTraf6-19; TRAF6 is downregulated in the L6 myotube via shRNA; shNC, shRNA negative control; Figure 4 f represents immunoblotting; Figure 4 g represents quantitative data for INSRβ and TRAF6; Figure 4 h represents the quantitative data from INSRβ to pro-INSR; n = 5;

[0071] Figure 4 i- Figure 4 j represents the response of the insulin signaling pathway to insulin in the L6 myotubule of clone shTraf6-19 TRAF6 knockout; among which, Figure 4 i represents immunoblotting; Figure 4 j represents the quantitative result of PKB phosphorylation; n = 3;

[0072] Using the t-test Figure 4 b- Figure 4 c and Figure 4 g- Figure 4 Statistical analysis was performed on h. Figure 4 e and Figure 4 Two-way ANOVA was performed; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0073] Figure 5 TRAF6 is an embodiment of the present invention. I72A The generation and characterization of mice; among which,

[0074] Figure 5 a is TRAF6 I72A A schematic diagram of the mouse generation strategy;

[0075] Figure 5 b is for TRAF6 Ile 72 Diagram of the locus;

[0076] Figure 5 c is a 5-month-old male TRAF6 I72A Knock-in the expression of TRAF6 in various tissues of mice;

[0077] Figure 5 d represents male WT and TRAF6. I72A Knock-in mouse (5-month-old) gastrocnemius muscle Insr mRNA levels, n=6;

[0078] Figure 5 e represents female WT and TRAF6 I72A Expression and phosphorylation of PKB in EDL muscle of knock-in mice (4 months old) under insulin action;

[0079] Figure 5 f represents male WT and TRAF6. I72A Insr mRNA levels in the livers of knock-in mice (5 months old), n=8;

[0080] Using the t-test Figure 5 d、 Figure 5 f. Perform statistical analysis;

[0081] Figure 6 TRAF6 is an embodiment of the present invention. I72A Mouse body growth, energy expenditure, and glucose homeostasis; among which,

[0082] Figure 6 a represents male WT and TRAF6. I72A Growth curves of knockout mice from weaning to 20 weeks of age, n = 9-19;

[0083] Figure 6 b represents a 12-week-old male WT and TRAF6. I72A The proportions of muscle mass and fat mass to body weight in knockout mice, n=8;

[0084] Figure 6 c represents male WT and TRAF6. I72A Food intake of knock-in mice (4 months old), n = 7-8;

[0085] Figure 6 d represents male WT and TRAF6. I72A Energy consumption of knock-in mice (4 months old), n = 7-8;

[0086] Figure 6 e represents male WT and TRAF6. I72A Physical activity of knock-in mice (4 months old); XAMB, number of continuous x-axis infrared beam breaks, n = 7-8;

[0087] Figure 6 f represents male WT and TRAF6. I72A Respiratory exchange rate (RER) of knock-in mice (4 months old); n = 7-8;

[0088] Figure 6 g- Figure 6 h represents male WT and TRAF6. I72A GTT was knocked into the peritoneal cavity of 9-week-old mice; among them, Figure 6 g represents the blood glucose level during the GTT; Figure 6 h is the area of ​​glucose under the curve, and n = 4;

[0089] Statistical analysis was performed using t-tests; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0090] Figure 7 TRAF6 is an embodiment of the present invention. I72A Improved glucose homeostasis in mice; among which,

[0091] Figure 7 a) WT and TRAF6 (for animals that are fasted at night (9 weeks old) or allowed to eat freely (6 weeks old) I72A Knock in the blood glucose levels of male mice, n=4-5;

[0092] Figure 7 b represents WT and TRAF6 animals that are fasted at night or allowed to eat freely. I72A Plasma insulin levels in knock-in male mice (11 weeks old), n = 6–8;

[0093] Figure 7 c- Figure 7 d represents male WT and TRAF6. I72A OGTT was knocked into mice (11 weeks old); among them, Figure 7 c represents blood glucose during the OGTT; Figure 7 d is the area of ​​glucose under the curve; n = 7-8;

[0094] Figure 7 e- Figure 7 f represents WT and TRAF6 I72A Glucose uptake in isolated skeletal muscle; among which, Figure 7 e represents the soleus muscle of a 4-6 month old male mouse; Figure 7 f represents the EDL muscle tissue of male mice aged 3-4 months; n = 6-8;

[0095] Figure 7 g- Figure 7 i represents male WT and TRAF6. I72A The response of cell surface and total GLUT4 levels in the soleus muscle of 6-month-old knock-in mice to insulin; among which... Figure 7 g represents immunoblotting; Figure 7 h is the quantitative value of total GLUT4, n = 6; Figure 7 i represents the quantification of GLUT4 on the cell surface, n = 3;

[0096] Figure 7 j refers to males aged 4-6 months (WT and TRAF6). I72A Knock-in mouse liver Pepck and G6pase mRNA levels, n=8-9;

[0097] Figure 7 k- Figure 7l represents female WT and TRAF6 I72A Knock-in PTT cells of 15-week-old mice; among them, Figure 7 k represents blood glucose levels during the PTT (Pulse Test). Figure 7 l is the area under the glucose curve; n = 7-9;

[0098] Figure 7 m- Figure 7 n represents WT and TRAF6 I72A Pepck (knock-in primary hepatocytes) Figure 7 m) and G6pase ( Figure 7 mRNA levels of n); n = 15-16;

[0099] Figure 7 a- Figure 7 d、 Figure 7 h、 Figure 7 j- Figure 7 l、 Figure 7 m- Figure 7 Perform a t-test on n. Figure 7 e- Figure 7 f is used for two-way ANOVA, where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001;

[0100] Figure 8 This describes the effect of TRAF6 on the expression of INSRα and INSRβ proteins according to an embodiment of the present invention; wherein,

[0101] Figure 8 a represents the protein level of INSRα-Flag when co-expressed with Myc-TRAF6 in HEK293 cells;

[0102] Figure 8 b represents the protein level when INSRβ-Flag and Myc-TRAF6 are co-expressed in HEK293 cells;

[0103] Figure 8 c represents the quantization of INSRβ in its approximately 100 kDa form, and... Figure 9 e is relevant, n=3;

[0104] Statistical analysis was performed using t-tests.

[0105] Figure 9 This refers to the inhibitory effect of TRAF6 on INSR processing according to an embodiment of the present invention; wherein,

[0106] Figure 9 a represents the expression of pro-INSR and mature INSRβ in the presence of TRAF6; INSR-HA with Flag vector, Flag-TRAF6, and Flag-TRAF6C70A Flag-TRAF6 I72A They were co-expressed in HEK293 cells; using HA antibody, pro-INSR and mature INSRβ from INSR-HA were detected in cell lysates by immunoblotting; red arrows indicate INSRβ and blue arrows indicate pro-INSR;

[0107] Figure 9 b shows the effect of lysosomal and proteasome inhibitors on TRAF6-induced decrease in INSRβ; in HEK293 cells treated with the lysosomal inhibitor bafilomycin-A1 or the proteasome inhibitor MG-132 for 18 hours, INSR-HA was co-expressed with Flag vector and Flag-TRAF6, respectively; using HA antibody, pro-INSR and mature INSRβ from INSR-HA were detected in cell lysates by immunoblotting; red arrows represent INSRβ, and blue arrows represent pro-INSR;

[0108] Figure 9 c- Figure 9 d represents the effect of TRAF6 on INSR processing; INSR-Flag is used in conjunction with HA carrier and HA-TRAF6 respectively. (Lazidohomoalaine) was used to treat HEK293 cells co-expressing the drug for a specified time; after immunoprecipitation with Flag beads, the drug was... The protein reaction buffer kit reacts with nascent pro-INSR and mature INSRβ, and is detected using an HRP-labeled avidin antibody; among which, Figure 9 c represents a representative immunoblot, with red arrows indicating INSRβ and blue arrows indicating pro-INSR; Figure 9 d represents the quantization of INSR processing; n = 4;

[0109] Figure 9 e- Figure 9 f represents WT and TRAF6 I72A Knock into newly generated INSRβ in primary hepatocytes; within a specified time, use 50 μM... Primary hepatocytes were treated with (L-azidohomoalaine); after immunoprecipitation with anti-INSRβ antibody, they were... The protein reaction buffer kit reacted with newly formed INSRβ and was detected using an HRP-labeled avidin antibody; Figure 9 e represents a representative immunoblot; the orange arrow highlights the newly formed INSRβ form of approximately 95 kDa; the red arrow highlights the newly formed INSRβ form of approximately 100 kDa. Figure 9f is the quantization of the newly generated INSRβ in the form of approximately 95 kDa; n = 3;

[0110] Statistical analysis was performed using t-tests. * indicates p < 0.05, and *** indicates p < 0.001.

[0111] Figure 10 This is a TRAF6-mediated lysosomal degradation of FURIN according to an embodiment of the present invention; wherein,

[0112] Figure 10 a- Figure 10 b represents WT and TRAF6 I72A The levels of FURIN protein knocked into skeletal muscle (from 5-month-old female mice) and liver (from 6-month-old male mice); Figure 10 a represents immunoblotting; Figure 10 b represents the quantitative value of FURIN; n = 4-6;

[0113] Figure 10 c represents the levels of WT and TRAF6 in the skeletal muscle and liver of 5-month-old female mice. I72A Knock-in FURIN activity, n=4;

[0114] Figure 10 d- Figure 10 e represents the protein levels of INSR and FURIN in primary hepatocytes from TRAF6 knockout mice. TRAF6 and FURIN were downregulated in primary hepatocytes using siRNA, with siNC serving as a negative control. Figure 10 d represents the representative immunoblot; Figure 10 e represents quantitative data for INSRβ and FURIN (left) and quantitative data for INSRβ on pro-INSR (right); n = 8;

[0115] Figure 10 f- Figure 10 g is FURIN in WT and TRAF6 I72A Stability of knock-in primary hepatocytes, and time indicator of primary hepatocyte treatment with 50 μg / ml CHX; among which... Figure 10 f represents a representative immunoblot; Figure 10 g represents the quantification of FURIN protein; n = 3;

[0116] Figure 10 h represents a decrease in FURIN's dependence on TRAF6 E3 ligase; FURIN-HA is associated with Flag, Flag-TRAF6, and Flag-TRAF6, respectively. I72A Co-expressed in HEK293 cells;

[0117] Figure 10i represents the effect of lysosomal and proteasome inhibitors on TRAF6-induced reduction of FURIN; in HEK293 cells treated with the lysosomal inhibitor bafimycin-A1 or the proteasome inhibitor MG-132 for 18 hours, FURIN-HA was co-expressed with the Flag vector and Flag-TRAF6, respectively.

[0118] Figure 10 j- Figure 10 k represents the interaction between TRAF6 and FURIN in HEK293 cells; TRAF6 and FURIN were co-expressed in HEK293 cells and immunoprecipitated using Flag beads;

[0119] Figure 10 l- Figure 10 m represents TRAF6 and FURIN in HEK293 cells ( Figure 10 l) and mouse liver ( Figure 10 Intrinsic interactions in m);

[0120] Figure 10 n represents the subcellular distribution of TRAF6 and FURIN; ER and Golgi apparatus were isolated from mouse liver and labeled Grp94 (ER) and GM130 (Golgi apparatus), respectively.

[0121] Figure 10 b- Figure 10 c. Figure 10 Perform a t-test on g. Figure 10 e Figure 10 One-way ANOVA was performed using h; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0122] Figure 11 Cells and TRAF6 according to an embodiment of the present invention I72A The TRAF6-FURIN axis in mice; where,

[0123] Figure 11 a represents male WT and TRAF6. I72A The levels of Furin mRNA in the skeletal muscle and liver of knock-in mice (5 months old), n=4-8;

[0124] Figure 11 b shows the expression of FURIN and TRAF6 proteins in two clones of the TRAF6 knockout L6 myotube;

[0125] Figure 11 c represents the expression of FURIN protein in primary hepatocytes of TRAF6 knockout mice;

[0126] Figure 11 d represents female WT and TRAF6I72A Expression of PCSK9 protein in the serum of 7-week-old mice;

[0127] Figure 11 e represents female WT and TRAF6 I72A Expression of FURIN protein in serum of 11-week-old mice;

[0128] Figure 11 f. Male WT and TRAF6 animals that are fed freely or fasted overnight. I72A Total cholesterol (TC) in the serum of knock-in mice (11 weeks old), n = 4-8;

[0129] Figure 11 g refers to 5-6 month old male WT and TRAF6 I72A The TC content in the liver of knock-in mice was n=5;

[0130] Figure 11 h represents the localization of GFP-TRAF6 in primary mouse hepatocytes using the Golgi apparatus marker (RCAS1), with cell nuclei stained with DAPI. The scale bar is 10 μm in length.

[0131] Statistical analysis was performed using t-tests. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0132] Figure 12 According to an embodiment of the present invention, TRAF6 degrades the ubiquitination of FURIN; wherein,

[0133] Figure 12 a is a schematic diagram of the TRAF6 functional domain;

[0134] Figure 12 b represents the interaction between FURIN and the TRAF-C / MATH domain. FURIN-HA interacts with the Flag vectors Flag-TRAF6 and Flag-TRAF6, respectively. M1-C357 Flag-TRAF6 N358-V530 The FURIN and TRAF6 domains were co-expressed in HEK293 cells, and the interaction between the FURIN and TRAF6 domains was determined by co-immunoprecipitation using Flag beads.

[0135] Figure 12 c is a schematic diagram of the FURIN functional domain;

[0136] Figure 12 d represents the interaction between TRAF6 and the cytoplasmic domain (CD) of FURIN; Flag-TRAF6 and mCherry-labeled full-length FURIN or CD were co-expressed in HEK293 cells, and immunoprecipitation was performed to measure the TRAF6 / FURIN interaction.

[0137] Figure 12 e represents TRAF6 ubiquitination of FURIN; in HEK293 cells containing HA-Ub, FURIN-Flag is associated with the Myc vectors Myc-TRAF6 and Myc-TRAF6, respectively. C70A Myc-TRAF6 I72A Co-expression; ubiquitination on the FURIN Flag immunoprecipitated by immunoblotting was detected using HA antibody;

[0138] Figure 12 f represents TRAF6 ubiquitination of FURIN under PA treatment; in the presence of HA-Ub, FURIN-Flag was co-expressed with Myc vector and Myc-TRAF6 in HEK293 cells, respectively.

[0139] Figure 12 TRAF6 induces Golgi-associated FURIN ubiquitination; in HEK293 cells containing Flag-Ub and TMEM115-3×HA, FURIN-GFP is co-expressed with Myc vector and Myc-TRAF6, respectively; Golgi TMEM115-3×HA in HEK293 cells is enriched by immunoprecipitation; and FURIN ubiquitination is measured in the Golgi apparatus.

[0140] Figure 12 h represents the cytoplasmic domain (CD) of TRAF6 ubiquitinated FURIN; in HEK293 cells with Flag-Ub present, FURIN-CD-GFP was co-expressed with Myc and Myc-TRAF6, respectively; the polyubiquitinated protein was immunoprecipitated from the lysate using Flag beads, and FURIN-CD-GFP was detected in the Flag immunoprecipitate.

[0141] Figure 12 i represents the effect of CD deficiency on TRAF6-mediated FURIN ubiquitination; in HEK293 cells with Flag-Ub present, FURIN-HA and FURIN-ΔCD-HA were co-expressed with Myc and Myc-TRAF6, respectively; polyubiquitinated proteins were immunoprecipitated from lysates using Flag beads, and FURIN-HA and FURIN-ΔCD-HA were detected in the Flag immunoprecipitate;

[0142] Figure 12 j- Figure 12 k represents the stability of FRUIN in HEK293 cells when Flag-TRAF6 is overexpressed with FURIN-GFP and FURIN-ΔCD-GFP, respectively; 50 μg / ml CHX treatment in HEK293 cells indicates the time. Figure 12 j represents the representative immunoblot; Figure 12k represents the quantitative value of FURIN protein; n = 5;

[0143] Figure 12 l represents the effect of Traf6i-13 (10 μM) on TRAF6-mediated FURIN ubiquitination;

[0144] Statistical analysis was performed using t-tests; * indicates p < 0.05, ** indicates p < 0.01;

[0145] Figure 13 This refers to the interaction between TRAF6 and PCSKs according to an embodiment of the present invention; wherein,

[0146] Figure 13 a represents the interaction between FURIN and TRAF-C / MATH domains and PA treatment; in HEK293 cells, FURIN-HA interacts with the Flag-labeled TRAF6 MATH domain (Flag-TRAF6). N358-V530 TRAF6 and FURIN were co-expressed, and their interaction was detected by immunoprecipitation.

[0147] Figure 13 b represents the interaction between TRAF6 and the cytoplasmic domain (CD) of FURIN; Flag-TRAF6 and GFP-labeled full-length FURIN or CD were co-expressed in HEK293 cells, and the interaction between TRAF6 and FURIN was determined by immunoprecipitation.

[0148] Figure 13 c represents the interaction between the FURIN cytoplasmic domain and TRAF6 with PA treatment. In HEK293 cells, Flag-TRAF6 was co-expressed with the GFP-labeled FURIN cytoplasmic domain, and the TRAF6 / FURIN interaction was detected by immunoprecipitation.

[0149] Figure 13 d- Figure 13 g represents the interaction between TRAF6 and PCSK5B in HEK293 cells. Figure 13 d) PCSK7 Figure 13 e), PCSK8 / SKI-1 ( Figure 13 f) and PCSK5A ( Figure 13 g) Interaction; TRAF6 and PCSKs co-expressed in HEK293 cells, immunoprecipitated by HA antibody or Flag beads;

[0150] Figure 13h represents the isolation of Golgi apparatus from HEK293 cells expressing TMEM115-3×HA by immunoprecipitation of TMEM115-3×HA; Golgi apparatus were labeled with GM130 and GRASP55, and cytoplasm was labeled with GAPDH;

[0151] Figure 13 i- Figure 13 k represents the expression levels of INSR-Flag protein and mRNA in HEK293 cells; Myc-TRAF6 co-expressed FURIN-GFP and FURIN-ΔCD-GFP in HEK293 cells with INSR-Flag labeling, respectively; among which... Figure 13 i is Insr-flag mRNA, n=4-5; Figure 13 j represents immunoblotting; the red arrow indicates INSRβ, and the blue arrow indicates pro-INSR; Figure 13 k represents the quantitative data of INSRβ / pro-INSR, n = 3;

[0152] Two-way ANOVA was used for statistical analysis. *** indicates p < 0.001.

[0153] Figure 14 This is an embodiment of the present invention for screening Traf6i-13 binding sites on TRAF6; wherein,

[0154] Figure 14 a. The effect of Traf6i-13 on TRAF22-mediated p62 ubiquitination;

[0155] Figure 14 b- Figure 14 d represents the alanine scan of 11 potential Traf6i-13 binding sites predicted by molecular docking; TRAF6-mediated FURIN ubiquitination was used as a readout to look for mutations that do not affect TRAF6 activity but are resistant to Traf6i-13.

[0156] Figure 14 e is for Traf6i-13 for Flag-TRAF6 and Flag-TRAF6 K67A The effects of mediated FURIN ubiquitination;

[0157] Figure 14 f is to study the recombinant Flag-TRAF6 through surface plasmon resonance (SPR) experiments. K67A Interaction with Traf6i-13;

[0158] Figure 14 g represents endogenous TRAF6 knockout and Flag-TRAF6 or Flag-TRAF6 K67AProtein levels of INSR and FURIN in L6 myotubes after PA treatment following overexpression;

[0159] Figure 15 According to an embodiment of the present invention, TRAF6 inactivation alleviates the HFD / PA-induced decrease in INSR; wherein,

[0160] Figure 15 a- Figure 15 d represents the effect of PA on the processing of FURIN and pro-INSR in primary hepatocytes; after 8 hours of PA treatment, primary hepatocytes were stimulated with insulin or without insulin for 30 minutes; among them, Figure 15 a represents immunoblotting; Figure 15 b represents the quantification of INSRβ and FURIN, = 6; Figure 15 c represents the quantitative determination of PKB-T308 phosphorylation, n = 3; Figure 15 d represents the quantization of INSRβ / pro-INSR, n = 6;

[0161] Figure 15 e- Figure 15 h represents the effect of PA treatment on L6 myotubular insulin signaling in TRAF6 knockout; among which... Figure 15 Quantization of INSRβ; Figure 15 f represents immunoblotting; Figure 15 g represents the quantitative determination of PKB-S473 phosphorylation; Figure 15 h represents the quantization of INSRβ / pro-INSR, n = 3;

[0162] Figure 15 i- Figure 15 k represents the levels of INSR, FURIN, and TRAF6 proteins in the skeletal muscle of male mice fed a high-fat diet for 6 months; among which, Figure 15 i represents immunoblotting; Figure 15 j represents the quantitative results of INSRβ, FURIN, and TRAF6; Figure 15 k represents the quantitative result of INSRβ / pro-INSR, n = 6;

[0163] Figure 15 l represents the correlation between human liver PKB-T308 phosphorylation and BMI, n=27;

[0164] Figure 15 m is the correlation between human liver TRAF6 protein and BMI, n=27;

[0165] Figure 15 n- Figure 15 p represents male WT and TRAF6 animals fed with HFD for 3 months. I72A INSRβ protein levels in mouse skeletal muscle; among which, Figure 15n represents the immunoblot; Figure 15 o represents the quantitative result of INSRβ; Figure 15 p represents the quantitative result of INSRβ / pro-INSR, n=3;

[0166] right Figure 15 b、 Figure 15 d、 Figure 15 j- Figure 15 Perform a t-test on k. Figure 15 c. Figure 15 e- Figure 15 h、 Figure 15 o- Figure 15 A two-way ANOVA was performed; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0167] Figure 16 This relates to the effect of TRAF6 inactivation on HFD-induced INSRβ reduction according to an embodiment of the present invention; wherein,

[0168] Figure 16 a- Figure 16 c represents the protein levels of INSR, FURIN, and TRAF6 in the liver of male mice fed HFD for 6 months. The cross indicates a non-specific band; where, Figure 16 a represents immunoblotting; Figure 16 b represents the quantitative results of INSRβ, FURIN, and TRAF6; Figure 16 c represents the quantitative result of INSRβ on pro-INSR, n = 6;

[0169] Figure 16 d represents the correlation between human liver INSRβ protein and BMI, n = 27;

[0170] Figure 16 e represents the correlation between human liver FURIN protein and BMI, n = 27;

[0171] Figure 16 f is the correlation between human liver INSRβ to pro-INSR protein and BMI, n=27;

[0172] Figure 16 g represents male mice WT and TRAF6 fed with HFD. I72A The growth curve of the knock-in, n = 7-12;

[0173] Figure 16 h refers to WT and TRAF6 infants fed HFD for 2 months. I72A Knock-in the body composition of male mice, n = 6-12;

[0174] Figure 16 i- Figure 16 j represents male WT and TRAF6 animals fed with HFD at 3 months of age. I72A OGTT was performed on knock-in mice; among them, Figure 16 i represents blood glucose levels during the OGTT; Figure 16 j represents the area under the blood glucose curve, and n = 4 - 7;

[0175] Figure 16 k- Figure 16 n refers to male WT and TRAF6 animals fed with HFD for 3 months. I72A Protein levels of INSRβ and TRAF6 in mouse liver; among which, Figure 16 k represents immunoblotting; Figure 16 l represents the quantitative result of INSRβ; Figure 16 m represents the quantitative result of FURIN; Figure 16 n represents the quantitative result of INSRβ on pro-INSR, n = 3;

[0176] Figure 16 b- Figure 16 c. Figure 16 g- Figure 16 j uses a t-test. Figure 16 l- Figure 16 Statistical analysis was performed using a two-way ANOVA; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; and

[0177] Figure 17 This is a palmitoylation of TRAF6 according to an embodiment of the present invention; wherein,

[0178] Figure 17 a- Figure 17 c represents the effect of PA treatment on the levels of INSRβ, FURIN, and TRAF6 proteins in L6 myotubules; among which... Figure 17 a represents immunoblotting; Figure 17 b represents the quantification of INSRβ, FURIN, and TRAF6; Figure 17 c represents the quantization of INSRβ / pro-INSR; n = 3;

[0179] Figure 17 d represents the palmitoylation of the C-terminal MATH domain and the N-terminal portion (ΔMATH) of Flag-TRAF6 WT;

[0180] Figure 17 e- Figure 17 f represents the TRAF6 protein associated with the Golgi apparatus in the livers of CD or HFD mice; among which... Figure 17 e represents immunoblotting; Figure 17 f represents the quantitative result of Golgi-related TRAF6; n = 3;

[0181] Statistical analysis was performed using t-tests; ** indicates p < 0.01, *** indicates p < 0.001;

[0182] Figure 18 This is a model of the TRAF6-FURIN axis in the regulation of INSR maturation according to an embodiment of the present invention; the figure illustrates the model controlling INSR maturation via the TRAF6-FURIN axis; under normal dietary conditions, TRAF6 is inactive, and FURIN is active to promote the processing of pro-INSR into mature INSR; under HFD conditions, PA activates TRAF6, thereby ubiquitinizing the cytoplasmic domain of FURIN on the Golgi apparatus; TRAF6 ubiquitination promotes the degradation of convertase via lysosomes; the reduction of FURIN inhibits the processing of pro-INSR into mature INSR, thereby leading to insulin resistance; gene or drug inhibition of TRAF6 can prevent PA / HFD-induced reduction of mature INSR, thereby alleviating insulin resistance; and

[0183] Figure 19 This invention relates to the effect of Traf6i-13 on the proliferation rate of tumor cell lines according to an embodiment of the present invention. Traf6i-13 reduces the relative proliferation rate of tumor cell lines; wherein,

[0184] Figure 19 Traf6i-13 reduces the relative proliferation rate of HepG2 tumor cell lines;

[0185] Figure 19 b indicates that Traf6i-13 reduces the relative proliferation rate of the Huh7 tumor cell line;

[0186] Figure 19 c represents Traf6i-13 reducing the relative proliferation rate of the HCT116 tumor cell line. Detailed Implementation

[0187] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0188] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments may also be utilized, or structural, logical, or electrical changes may be made to the embodiments of the present application.

[0189] Obesity is a significant pathological factor that induces insulin resistance and leads to type 2 diabetes through multiple mechanisms. Insulin receptor (INSR) inactivation contributes to the development of insulin resistance, and its protein levels are downregulated in obesity, the mechanism of which remains unclear. This application demonstrates that the E3 ligase TRAF6 is a key regulator of INSR maturation, and its inactivation prevents palmitic acid or high-fat diet-induced INSR reduction. Therefore, TRAF6 gene inactivation enhances insulin signaling, further increasing muscle glucose uptake and inhibiting hepatic gluconeogenesis. TRAF6 inactivation increases the proton exchange enzyme FURIN, which controls the processing of pro-INSR into mature INSR. Mechanistically, TRAF6 binds to the Golgi apparatus, ubiquitinates the cytoplasmic tail of FURIN, leading to its lysosomal degradation. The TRAF6-FURIN axis also regulates cholesterol metabolism through processing in the circulating PCSK9. This application develops a small molecule inhibitor of TRAF6 that improves insulin sensitivity and glycemic control. In summary, this application reveals the crucial role of TRAF6 in regulating proton exchange processing and has therapeutic implications for metabolic control.

[0190] The technical terms used in this application have the following meanings:

[0191] The term "mouse" in this document refers to mice kept at the Institute of Model Animals, Nanjing University. All animal experiments were conducted in accordance with the Animal Protocol (MARC-CS16) approved by the Institute of Animal Protection and Use, Nanjing University (IACUC). Mice were housed in designated pathogen-free animal facilities with a 12-hour light / dark cycle and free access to food and water, unless otherwise specified. Males and female heterozygous mutants were mated to produce homozygous mutants and wild-type littermates.

[0192] The term "WT" in this document refers to wild-type mice. For example, the WT mice mentioned in this application are experimental mice that have not undergone editing of the genes related to this application, carry a natural genetic background, and are used as a control group. In this application, WT mice and corresponding experimental group mice (such as TRAF6 mice) I72AThe strains and substrains of the mice are completely identical, and the methods of obtaining them are also the same as those of the corresponding experimental group mice. The difference is that no specific genes of this application were edited, such as the absence of TRAF6. I72A mutation.

[0193] The term "C57BL6 mouse" used in this article refers to one of the most commonly used inbred laboratory mouse strains in biomedical research. Developed by Clarence Little through hybridization in 1921, the C57BL6J and C57BL6N branches were derived in 1951, and it is now one of the most widely used mouse strains globally. Through gene modification or induction, it is used in research on obesity, diabetes, atherosclerosis, neurodegenerative diseases (such as Alzheimer's disease), and cancer.

[0194] Among them, the C57BL6J mouse carries a spontaneous mutation in the Nnt gene (affecting insulin secretion) and is widely used in metabolic research; the C57BL6N mouse has a normal Nnt gene and is often used as a gene editing model.

[0195] The term "TRAF6" in this article refers to a member of the TRAF protein family, a multifunctional adaptor protein and E3 ubiquitin ligase that plays a central regulatory role in multiple signaling pathways, especially in immune responses, inflammatory responses, cell survival, and metabolism.

[0196] TRAF6 may play opposite roles in different organs. For example, in breast cancer, lung cancer, and colorectal cancer, TRAF6 promotes tumor cell proliferation, invasion, and metastasis by activating NF-κB; while in prostate cancer, TRAF6 inhibits tumor progression by ubiquitinizing and degrading oncogenic proteins (such as c-Myc).

[0197] The term "TRAF6" in this article I72A "This refers to altering the mouse TRAF6 gene, replacing isoleucine (Ile, I) at position 72 with alanine (Ala, A). For example, in some embodiments, TRAF6..." I72A Methods for generating knock-in mice include: generating TRAF6 in a C57BL6N background. I72A Knock into the mouse, then knock into TRAF6 I72A In mice, the isoleucine residue Ile72 (surrounded by the sequence SEQ ID NO.1: KYECPiCLMAL) on TRAF6 was mutated to alanine. I72ASimultaneously with the mutation, a SphI restriction site was introduced at the TRAF6 site via synonymous mutation to facilitate genotyping. The mutant region (491 bp) was amplified using primers (SEQ ID NO.2: 5'-GAAGATCGACTGACAACAGAGC-3' and SEQ ID NO.3: 5'-CATACCGTAGTACAAACTGGGA-3'), and then SphI (244 / 247 bp TRAF6) was used for amplification. I72A The knockout product was digested with enzymes to digest TRAF6. I72A Genotyping was performed on knockout mice. TRAF6 I72A Knockout mice were backcrossed into the C57BL6J background for at least 5 generations before the experiment.

[0198] The term "TRAF6" in this article C70A "This refers to altering the mouse TRAF6 gene, replacing cysteine ​​(Cys, C) at position 70 with alanine (Ala, A)."

[0199] Inhibitors of TRAF6 activity include small molecule inhibitors, such as any of the Traf6i-n compounds or their salts as described in this application, such as compound 6877002, C25-140, etc.; natural compounds, such as curcumin, triptolide, etc.; gene-silencing siRNA / shRNA, CRISPR-Cas9 technology, etc.; and protein degrading agents (PROTACs), such as XY-12, etc.

[0200] The term "Traf6i-n" in this article refers to the 14 compounds identified from a search of 3 million compounds in the ChemDiv chemical library in order to find candidate compounds that may bind to the n-terminal cavity of TRAF6, which are potential TRAF6 inhibitors.

[0201] According to one embodiment of this application, the Traf6i-n compound is one of Traf6i-1 to 14, wherein the structural formulas of Traf6i-1 to 14 are shown below:

[0202] Traf6i-1: Traf6i-2: Traf6i-3: Traf6i-4: Traf6i-5: Traf6i-6: Traf6i-7: Traf6i-8: Traf6i-9: Traf6i-10: Traf6i-11: Traf6i-12: Traf6i-13: And Traf6i-14:

[0203] TRAF6 is structurally highly similar to its family members such as TRAF2, and inhibitors are prone to off-target effects. According to one embodiment of this application, Traf6i-3, Traf6i-10, or Traf6i-13 are used as TRAF6 inhibitors, especially Traf6i-13, which can inhibit TRAF6 activity without causing off-target effects.

[0204] The term "Traf6i-13" in this article refers to an inhibitor of TRAF6 activity, synthesized by Nanjing Pharmaceutical Technology Co., Ltd., China.

[0205] The term "HEK293 cell" in this article, also known as human embryonic kidney cell 293, is a cell line derived from human embryonic kidney cells. It has the characteristics of high transfection efficiency, easy culture, very little expression of endogenous receptors required for extracellular ligands, and is relatively easy to transfect. It is a commonly used cell line for expressing and studying exogenous genes.

[0206] The term "UBC13" in this article refers to a ubiquitin-conjugating enzyme (E2 enzyme), belonging to the E2 ubiquitin-conjugating enzyme family. It is responsible for receiving ubiquitin molecules from E1 ubiquitin activators and transferring them to target proteins (usually working in conjunction with E3 ubiquitin ligases). It is a core component of the ubiquitin-proteasome system (UPS). It participates in regulating protein ubiquitination by catalyzing the covalent linkage of ubiquitin molecules to target proteins. UBC13 relies on E3 ubiquitin ligases (such as RNF8, RNF168, and TRAF6) to recognize specific substrates and complete the ubiquitination reaction.

[0207] The term "DIO mouse" in this article refers to the diet-induced obesity mouse, an animal model that simulates human obesity and related metabolic syndromes through a long-term high-calorie diet (such as a high-fat, high-sugar diet). This model is widely used in research on metabolic diseases (such as type 2 diabetes, fatty liver, and cardiovascular diseases) and drug development.

[0208] The term "HFD" in this document refers to a high-fat diet or feed in which fat typically provides 45%-60% of the body's energy. Common fat sources include lard, palm oil, or hydrogenated vegetable oil. In this application, the high-fat diet (60 kcal% fat, Cat. No. 12492) was obtained from Research Diets (USA).

[0209] When used in this application as mouse feed, the term "CD" refers to a typical diet or feed in which fat typically provides 10%-15% of the energy.

[0210] Chronic inflammation in diet-induced obesity is a key factor in the development of insulin resistance in obese individuals. Circulating endotoxins and fatty acids activate Toll-like receptor 4 (TLR4), which signals through tumor necrosis factor receptor-associated factor 6 (TRAF6) to promote the expression of pro-inflammatory molecules. TRAF6 acts as both an adaptor protein and an E3 ubiquitin ligase, catalyzing the formation of a Lys63-linked ubiquitin chain. This ubiquitin chain activates protein kinase TAK1 to phosphorylate IKK-β and MAP kinase 6. The latter two kinases further transduce signals, ultimately activating transcription factors NF-κB and AP-1, respectively, to induce pro-inflammatory cytokine genes. Germline- and hematopoietic-specific loss of TLR4 can ameliorate obesity-related insulin resistance. In addition to TLR signaling, TRAF6 also signals through the co-stimulatory receptor CD40, which plays a crucial role in obesity-induced inflammation. Pharmacological disruption of the CD40-TRAF6 interaction can reduce adipose tissue inflammation and prevent obesity-induced insulin resistance. These studies indicate that TRAF6 E3 ligase plays a key regulatory role in the production of pro-inflammatory cytokines in immune cells, indirectly regulating insulin sensitivity in target cells such as skeletal muscle cells and hepatocytes.

[0211] The term "gavage" in this article refers to a method of drug administration or feeding to mammals, which involves the direct delivery of a specified composition into the digestive tract of a mammal using experimental methods.

[0212] The term "PTT" in this article refers to the pyruvate tolerance test, an important experiment for evaluating hepatic gluconeogenesis. Gluconeogenesis is the metabolic process of converting non-carbohydrate substances into glucose, and the liver is the primary site of this process. In the PTT, by administering pyruvate exogenously and monitoring changes in blood glucose levels, we can understand the animal's ability to convert pyruvate into glucose, thereby assessing its metabolic status and potential glucose metabolism disorders.

[0213] The term "GTT" in this article refers to the glucose tolerance test, a laboratory test used to diagnose diabetes. Specifically, the term "OGTT" refers to the oral glucose tolerance test.

[0214] The term "PKB" in this article refers to protein kinase B (PKB), also known as the Akt signaling pathway, which is a serine / threonine protein kinase with a molecular weight of approximately 60 kDa. The main phosphorylation sites of AKT are threonine and serine residues, located in the kinase domain (T305 / T308 / T309) and regulatory domain (S472 / S473 / S474), respectively.

[0215] The term "PKB-T308" in this article refers to the phosphorylation of PKB or Akt at the T308 site.

[0216] The term "PKB-S473" in this article refers to the phosphorylation of PKB or Akt at the S473 site.

[0217] The term "LPS" in this article refers to lipopolysaccharide, which can induce macrophages to secrete inflammatory factors, including tumor necrosis factor-α (TNF-α) and interleukin-1β (Il-1β). In some embodiments, TRAF6 is required for LPS-induced expression of TNF-α and Il-1β in macrophages.

[0218] The term "BMDM" in this article refers to a type of macrophage extracted from bone marrow and differentiated through in vitro culture. Its core value lies in providing an experimental model for studying the biological functions and immune regulation mechanisms of macrophages.

[0219] The term "INSR" in this article refers to an antibody that specifically recognizes the insulin receptor (INSR), a homodimeric receptor tyrosine kinase composed of two extracellular α subunits and two transmembrane β subunits. These subunits are derived from the insulin proprotein (pro-INSR) via cleavage by the proprotein convertase FURIN. Upon binding to the INSR, insulin activates the receptor kinase, thereby stimulating the insulin receptor substrate (IRS)-PI3 kinase (PI3K)-protein kinase B (PKB, also known as Akt) pathway to phosphorylate multiple downstream targets. Following insulin stimulation, the IRS is recruited to the plasma membrane, where it connects the receptor and the downstream PI3K-PKB signaling pathway. Furthermore, the IRS can form condensates in the cytoplasm via liquid-liquid phase separation (LLPS), acting as a cytoplasmic signaling center to mediate insulin action. The IRS is not only a core component mediating insulin signaling but also a key factor in insulin resistance. Multiple serine residues on the IRS are phosphorylated by serine / threonine kinases such as PKC, mTOR, and IkB kinase (IKK)-β, thereby antagonizing INSR-catalyzed IRS tyrosine phosphorylation and leading to insulin resistance. Furthermore, impaired formation of the IRS condensate has been reported to also result in palmitate (PA)-induced insulin resistance. In addition to these IRS-centric mechanisms of insulin resistance, decreased INSR expression also contributes to the development of insulin resistance in diet-induced obesity. However, the molecular mechanisms underlying decreased INSR expression remain unclear.

[0220] The term "pro-INSR" in this article refers to the immature precursor protein of the insulin receptor (INSR), which requires post-translational processing to form the functionally mature INSR. It plays a crucial role in the biosynthesis, folding, and membrane localization of the insulin receptor. Pro-INSR is a single-chain polypeptide, composed of a single polypeptide chain (approximately 190 kDa) encoded by a gene, containing precursor sequences of α and β subunits initially linked by disulfide bonds. Pro-INSR is primarily located in the endoplasmic reticulum and Golgi apparatus. The processed mature INSR is transported to the cell membrane surface to perform its signal reception function.

[0221] The term "insulin resistance" in this article refers to a weakened state in diet-induced obesity where insulin's role in controlling postprandial metabolic responses to maintain metabolic health is diminished. Insulin resistance impairs systemic metabolic homeostasis, which is fundamental to the development of type 2 diabetes (T2D). The increasing global prevalence of T2D over the past few decades necessitates a better understanding of the underlying mechanisms of insulin resistance in order to develop new and effective therapies.

[0222] The term "PCSKs" in this article refers to the PCSK protein family, a key class of serine proteases primarily responsible for cleaving inactive proproteins into their mature, active forms. Members of this family play crucial roles in physiological processes such as hormone regulation, lipid metabolism, and immune responses, and are closely associated with various diseases, including hypercholesterolemia, diabetes, and cancer. The PCSK protein family influences a wide range of physiological processes, from metabolic homeostasis to immune defense, by precisely regulating the maturation of proproteins.

[0223] The PCSK family comprises nine members, namely PCSK1 through PCSK9:

[0224] PCSK1, also known as PC1 or PC3, is mainly distributed in neuroendocrine cells and pancreatic β cells, and processes pro-hormones (such as proinsulin → insulin, proglucagon → glucagon).

[0225] PCSK2, also known as PC2, is mainly distributed in pancreatic α cells and the central nervous system, and works with PCSK1 to process pro-hormones (such as generating glucagon).

[0226] PCSK3, also known as FURIN, is widely expressed in various tissues. It extensively cleaves precursor proteins (such as growth factors, viral proteins, and coagulation factors) and is an important proprotein convertase that mediates the conversion of pro-INSR into mature INSR.

[0227] PCSK4, also known as PC4, is mainly distributed in the testes and ovaries and is involved in the processing of reproductive system proteins (such as sperm maturation-related proteins).

[0228] PCSK5, also known as PC5 or PC6, is mainly distributed in the placenta and embryonic tissues, regulating extracellular matrix proteins (such as BMPs and TGF-β). It produces two main isoforms through different splicing mechanisms: PCSK5A (PC5A) and PCSK5B (PC5B). The main differences between the two lie in their domain composition, cellular localization, and function.

[0229] PCSK6, also known as PACE4, is mainly distributed in embryonic development and tumor tissues, and processes growth factors and membrane proteins (such as Notch receptors).

[0230] PCSK7, also known as PC7 or PC8, is mainly distributed in the liver and intestines and regulates lipid metabolism-related proteins (such as PCSK9 precursor cleavage).

[0231] PCSK8, also known as SKI-1 or S1P, is mainly distributed in the liver and adipose tissue. It cleaves sterol regulatory element-binding proteins (SREBPs) and regulates cholesterol synthesis.

[0232] PCSK9, also known as NARC-1, is mainly distributed in the liver, intestines, and kidneys. It binds to the low-density lipoprotein receptor (LDLR) and promotes its degradation, thereby regulating blood cholesterol levels.

[0233] The terms "HA" and "HA antibody" in this article refer to antibodies that specifically recognize the HA tag (Hemagglutinin Tag). These antibodies are widely used in molecular biology and biomedical research for the detection, purification, or localization of recombinant proteins carrying the HA tag. The HA tag is typically a short, artificially designed peptide sequence, as shown in SEQ ID NO.4: YPYDVPDYA (9 amino acids). It readily elicits strong antibody responses, causes minimal interference with the structure and function of the fusion protein, and can be fused to the N-terminus, C-terminus, or interior of the protein.

[0234] The term "Flag" in this article refers to FLAG proteins, a type of tag widely used in molecular biology and biotechnology, consisting of artificially designed short peptide tags. The amino acid sequence of a Flag is typically as shown in SEQ ID NO.5: DYKDDDDK (8 amino acids), containing hydrophilic residues (such as aspartic acid D and lysine K) to minimize its impact on protein structure. Anti-FLAG antibodies can be used to perform experiments such as Western blot, immunofluorescence, and co-immunoprecipitation (Co-IP).

[0235] Among them, Flag-TRAF6, Flag-TRAF6 C70A Flag-TRAF6 I72A These are TRAF6 and TRAF6 with the Flag tag, respectively. C70A TRAF6 I72AProteins such as...

[0236] The term "Myc" in this article refers to an artificial short peptide tag widely used in molecular biology research, commonly used for labeling, detecting, or purifying recombinant proteins. The core sequence of Myc is shown in SEQ ID NO.6: EQKLISEEDL (10 amino acids). It has the characteristics of small molecular weight, minimal interference with the structure and function of fusion proteins, high immunogenicity, easy activation of antibody responses, suitability as an epitope tag, and flexible position, allowing insertion into the N-terminus, C-terminus, or interior of proteins.

[0237] Among them, Myc-TRAF6 and Myc-TRAF6 C70A Myc-TRAF6 I72A "etc." refers to TRAF6 and TRAF6 with the Myc tag, respectively. C70A TRAF6 I72A Proteins such as...

[0238] The term "GFP" in this document refers to Green Fluorescent Protein, which emits green fluorescence under illumination from blue light to ultraviolet light and is used for molecular labeling. Using GFP as a tag to express specific proteins allows for the study of subcellular localization of those proteins and their interactions with other molecules. GFP is a commonly used fluorescent protein in this field, and any GFP amino acid sequence can be used in this application. Furthermore, the nucleotide and amino acid sequences of GFP are not within the scope of this application and will not be described further here.

[0239] The term "mCherry" in this document refers to a red fluorescent dye with excellent photostability and photostability of monomeric molecules. It is commonly used in biotechnology as a tracer for molecular labeling and localization of cellular components. In molecular biology, it is often used in conjunction with GFP. mCherry is also a commonly used fluorescent protein in this field. Any mCherry amino acid sequence can be used in this application, and the nucleotide and amino acid sequences of mCherry are not within the scope of this application; therefore, their sequences will not be described here.

[0240] Compared to the tags involved in this application, the HA tag sequence, as shown in SEQ ID NO.4: YPYDVPDYA, has high sensitivity and low interference, but should be avoided from being confused with virus research; the FLAG tag sequence, as shown in SEQ ID NO.5: DYKDDDDK, has high specificity and is easy to purify, but may affect the protein charge properties; the Myc tag sequence, as shown in SEQ ID NO.6: EQKLISEEDL, has the characteristics of broad compatibility and high sensitivity, but some mammalian cells have endogenous Myc, which may interfere with the detection results; fluorescent protein sequences such as GFP and mCherry are longer, which have the characteristics of visualizing the detection results and not requiring antibodies, but the large molecular weight may interfere with protein function.

[0241] The term "siRNA" in this document refers to Small interfering RNA, also known as small interfering RNA or silencing RNA, which is a double-stranded RNA molecule approximately 21-25 nucleotides in length. siRNA inhibits the expression of a specific gene by binding to the mRNA of its target gene, leading to mRNA degradation. Typically, siRNA is produced by the cleavage of long RNA chains by the Dicer enzyme, then binds to a RISC complex to form a RISC complex, which in turn degrades the target mRNA. In this application, siTraf6, siFurin, etc., refer to small interfering RNAs used to silence Traf6 and Furin expression, respectively.

[0242] The term "shRNA" in this article refers to short hairpin RNA. After entering the cell, shRNA can be cleaved into siRNA by the Dicer enzyme, thereby inhibiting the expression of target genes. Specifically, shTraf6 and other shRNAs mentioned in this article refer to shRNAs that can inhibit the Traf6 gene.

[0243] The term "respiratory exchange ratio" or "RER" in this article refers to the ratio of the amount of oxygen inhaled to the amount of carbon dioxide exhaled by an animal during respiration. The respiratory exchange ratio (RER) is an important parameter for measuring respiratory gas exchange and is commonly used in studies of energy metabolism and nutrient utilization.

[0244] The terms “Pepck” and “G6pase” in this article refer to phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), respectively, both of which are key enzymes in gluconeogenesis.

[0245] The term "Grp94" in this article refers to glucose regulatory protein 94, a member of the heat shock protein 90 (HSP90) family that resides in the endoplasmic reticulum (ER). As a molecular chaperone, it assists in the correct folding of proteins and maintains ER homeostasis.

[0246] In this application, cDNAs encoding mouse TRAF6 (NP_033450.2), INSR (NP_001316985.1), FURIN (NP_035176.1), PCSK5A (NP_001156616.1), PCSK5B (NP_001177412.1), PCSK7 (NP_001268863.1), and PCSK8 (NP_062683.3) were cloned into the vectors pcDNA5-FRT / TO-HA, pcDNA5-FRT / TO-Flag, or pCS2-FRT / TO-6Myc and expressed in mammalian cells. Point mutations were introduced using site-directed mutagenesis using a standard procedure. All plasmids were sequenced using GENEWIZ / Azenta (Suzhou, Jiangsu, China).

[0247] In some embodiments, human studies were conducted in accordance with the research protocol (2018-SR-054) approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. Liver tissue sections were flash-frozen in liquid nitrogen and subsequently analyzed by immunoblotting.

[0248] In some embodiments, in a glucose or pyruvate tolerance test, mice were fasted for 16 hours and then given glucose (1.5 g / kg by gavage) or pyruvate (2 g / kg by intraperitoneal injection). Blood glucose levels were measured at specified time points using a Contour-TS blood glucose meter (Bayer) via tail bleeding.

[0249] In some embodiments, blood glucose levels were measured using a contour TS glucometer (Bayer). Serum total cholesterol (TC) was measured using a laboratory cholesterol assay kit (Cat. No. 294-65801, Wako Chemicals USA). A rat / mouse insulin space-protected (Bulk) enzyme-linked immunosorbent assay kit (Cat. No. EZRMI-13BK, Millipore) was used. For liver TC measurement, lipids were first extracted from liver blocks using an organic solvent consisting of chloromethane:isopropanol:NP-40 (7:11:0.1). After the organic solvent evaporated, TC in the extract was measured using a LabAssay cholesterol kit (Cat. No. 294-65801, Wako Chemicals USA).

[0250] In some embodiments, the muscle culture and in vitro glucose uptake method includes: isolating the soleus and extensor digitorum longus (EDL) muscles, stimulating them with or without insulin, collecting and measuring muscle glucose. L6 muscle tubes are cultured in serum-free DMEM for 16 hours, followed by 30 minutes of insulin stimulation with or without insulin. Cells are then incubated for 10 minutes in glucose uptake buffer containing 2-deoxy-D-[1,2-3h(N)] glucose, with or without insulin. Isotopes in cell lysates are measured using a Tri-Carb 2800TR scintillation counter (PerkinElmer), with protein concentration as a normalization value.

[0251] In some embodiments, the biotinylation method of membrane proteins includes: stimulating isolated soleus muscle with or without insulin for 50 minutes, followed by oxidative stimulation with EZ-link. TM Incubate in Sulfo-NHS-SS biotin (Cat. No. 21331, Thermo Fisher Scientific) Krebs Ringer bicarbonate buffer (with or without insulin). Use Pierce TM NeutrAvidin TM Biotinylated surface proteins were precipitated from the lysate using agarose (Cat. No. 29200, Thermo Fisher Scientific), and the surface level of GLUT4 was determined by Western blotting.

[0252] In some embodiments, the method for isolating and detecting palmitoylated proteins includes: isolating and detecting palmitoylated proteins using a resin-assisted capture of S-acylated proteins (acyl-RAC) assay. Briefly, in the acyl-RAC assay, the free thiol group on the cysteine ​​residue in the protein is first blocked, then hydroxylamine specifically releases the palmitoylated cysteine ​​residue, which is subsequently captured by thiopropyl microspheres. The eluted fraction is then treated with SDS buffer for Western blotting.

[0253] In some embodiments, the subcellular division method for the endoplasmic reticulum and Golgi apparatus includes: taking the liver of a male mouse euthanized by cervical dislocation, and rapidly homogenizing it using a Dounce homogenizer in 3 ml of an ice-cold solution containing 10 mM HEPES, pH 7.4, 150 mM sucrose, 0.5 mM DTT, and a mixture of protease inhibitors (Cat. No. HYK0010, MCE) until no tissue fragments are visible. Centrifuging at 1900 g for 10 min at 4 °C, and transferring the supernatant to a centrifuge tube (Cat. No. 328874, Beckman). Centrifuging at 100,000 g for 90 min at 4 °C in an OPTIMA XPN-100 centrifuge (SW60Ti rotor, Beckman). Microspheres were resuspended in 800 μL of 8.58% sucrose solution at the top of a sucrose density gradient (from bottom to top): 56% (160 μL), 50% (320 μL), 45% (480 μL), 40% (800 μL), 35% (800 μL), 30% (480 μL), and 20% (160 μL). After centrifugation for 18 h at 43,900 rpm in a Beckman SW60Ti rotor at 4 °C, 12 fractions (approximately 333 μL each) were collected from top to bottom of each centrifuge tube. Western blot analysis was performed using GRP94 (ER), GM130 (Golgi apparatus), and RCAS1 (Golgi apparatus) as markers to detect the ER and Golgi subcellular compartments.

[0254] In some embodiments, the cell culture, transfection, and stimulation methods include: human embryonic kidney HEK293 cells derived from the Cell Resource Center of the Chinese Academy of Medical Sciences and Peking Union Medical College (China), and rat L6 myoblasts derived from Dr. Amira Klip (University of Toronto, Canada). Cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% (v / v) fetal bovine serum. L6 myoblasts differentiated into myotubes for 5 days in DMEM containing 2% (v / v) horse serum. Primary hepatocytes were isolated from adult mouse livers perfused with collagenase digestion buffer and then cultured in M199 medium supplemented with 10% (v / v) fetal bovine serum.

[0255] Cells were transfected with plasmids or siRNA using a Lipofectamine-3000 (Thermo Fisher Scientific) mediated method. The siRNA sequences targeting TRAF6 are shown in SEQ ID NO.7: 5'-CACAAGUGCCCAGUUGACAAUGAAA-3' (siTraf6-1) and SEQ ID NO.8: 5'-CCUGACGGUAAAGUGCCCAAAUAAA-3' (siTraf6-2). The siRNA sequences targeting FURIN are shown in SEQ ID NO.9: 5'-CAUCCCGACCUAGCAGGCAAUUAUG-3' (siFurin-1) and SEQ ID NO.10: 5'-CAGACCACAUGACUACUCUGCUGAU-3' (siFurin-2).

[0256] shRNA was delivered into L6 myoblasts using lentivirus-mediated gene transfer. The shRNA sequences targeting TRAF6 are shown in SEQ ID NO.11: 5'-GCAAAGCGAGAGATTCTTTCC-3' (shTraf6-4), SEQ ID NO.12: 5'-GCACGAATGTATTGTCCATTG-3' (shTraf6-17), and SEQ ID NO.13: 5'-GCATCTTCCCGGTTTGTAAGA-3' (shTraf6-19).

[0257] For insulin stimulation, cells were cultured in serum-free DMEM for 16 hours (control), followed by 30 minutes of stimulation with or without insulin. For inhibitor treatment, cells were treated with 5 μM MG132 (Cat. No. S2619, Selleck) or 200 nM bafimycin-A1 (Cat. No. S1413, Selleck) for 18 hours, or 50 μg / mL cycloheximide (Cat. No. S7418, Selleck) at the indicated time points. PA treatment was as follows: 400 μM for 24 h in L6 myotubes and 600 μM for 8 h in primary hepatocytes.

[0258] In some embodiments, the glucose production assay includes: primary hepatocytes at 1.5 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 12-well plates and cultured overnight in M199 medium. Cells were then washed twice with DPBS, and the medium was changed to glucose-free DMEM (Cat. No. 11966025, Thermo Fisher Scientific) containing 10 mmol / L sodium lactate, 1 mmol / L sodium pyruvate, and 100 nmol / L dexamethasone. Hepatocytes were stimulated for 8 hours with or without 100 μmol / L 8-bromo-cAMP (Cat. No. HY12306, MCE). To assess the effect of insulin on gluconeogenesis, insulin (600 nM) was added to the medium two hours after the addition of 8-bromo-cAMP.

[0259] In some embodiments, the protein extraction and concentration determination method includes: extracting proteins from cells or mouse tissues. During cell lysis, the cells are washed after aspirating the culture medium and then lysed with lysis buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 10 mM sodium β-glycerophosphate, 50 mM NaF, 5 mM NaPPi, 1% (v / v) Triton X-100, 0.27 M sucrose, 0.1% (v / v) β-ME, 1 mM benzomididine and Na3VO4, 0.2 mM PMSF, 1 μg / ml leucopeptide, aprotinin, pepsin inhibitor, and microcystin-LR). To detect protein ubiquitination, HEK293 cells are collected with 100 μL of lysis buffer containing 2% SDS and boiled at 100°C for 10 minutes. 900 μL of lysis buffer is added to bring the final SDS concentration to 0.2%. Mouse tissues were collected, flash-frozen in liquid nitrogen, and stored at -80°C. Frozen tissue blocks were homogenized in lysis buffer using a Polytron homogenizer (Kinematica, Luzern, Switzerland). Cell or tissue debris was removed by centrifugation, and the protein concentration in the resulting homogenate was determined using Bradford reagent (Thermo Fisher Scientific).

[0260] In some embodiments, the immunoprecipitation and immunoblotting method comprises: incubating cell lysate with anti-GFP affinity beads (Cat. No. SA070005, Smart-Lifesciences) or anti-Flag affinity beads (Cat. No. SA042005, Smart-Lifesciences) overnight at 4°C. After intensive washing to remove non-specific binding proteins from the beads, the immunoprecipitate is denatured in loading buffer containing SDS and collected for subsequent analysis. Proteins are separated by SDS-PAGE electrophoresis and blotted onto a nitrocellulose membrane. After blotting, the membrane is plugged with 5% skim milk and then incubated overnight at 4°C with a primary antibody. The membrane is then further incubated with a horseradish-peroxidase-conjugated secondary antibody, and the chemiluminescent signal is recorded using a gel recording system (Tanon, China) after incubation with ECL substrate (GE Healthcare). Each channel in the immunoblotting assay represents a biological replication. The immunoblotting signal is quantified using ImageJ (version 1.46) and normalized using internal loading controls.

[0261] In some embodiments, the assay of the nascent protein includes: transfecting HEK293 cells with C-terminal labeled INSR for 48 hours. Then, at specified time points, using... (L-azidohomoalaine, Cat. No. C10102, Thermo Fisher Scientific) labeled the neonatal protein. Cells were then lysed, and total INSR-Flag was immunoprecipitated from the cell lysate by incubation overnight at 4°C with anti-Flag affinity beads (Smart-Lifesciences). Immunoprecipitation of proteins was performed using a protein reaction buffer kit (Cat. No. C10276, Thermo Fisher Scientific). Naïve INSR-Flag (precursor) and mature INSRβ-Flag were determined by measuring the biotinylated portion of the immunoprecipitated proteins using Western blotting with an HRP-labeled NeutrAvidin antibody (Cat. No. A2664, Invitrogen). INSR treatment was defined as the ratio of mature INSRβ to total INSR.

[0262] In some embodiments, the furfural activity assay comprises: homogenizing frozen mouse tissue blocks with 5× lysis / reaction buffer (500 mM HEPES (pH 7.0), 2.5% (v / v) Triton X-100, 5 mM CaCl2, and 5 mM β-ME), and then incubating on ice for 30 min. Immunoprecipitation with FURIN antibody was performed overnight at 4°C. The FURIN immunoprecipitate was incubated with 180 μL of 1.1× lysis / reaction buffer at 37°C for 15 min. Then, 20 μL of preheated 1 mM Pyr-Arg-Thr-Arg-AMC trifluoroacetate (Cat. No. 4018149, Bachem) was added, and the mixture was incubated at 37°C for 90 min. Fluorescence intensity (ex: 355 nm; em: 460 nm) was measured using a Bio-TEK Synergy H1 hybrid reader.

[0263] In some embodiments, the Golgi immunoprecipitation method includes: transfecting HEK293 cells with TMEM115-3×HA. Cells are collected with 1 ml of frozen KPBS and homogenized using a Dounce homogenizer. The cell homogenate is centrifuged at 1000 g for 2 min at 4°C. After centrifugation, the supernatant is transferred to a new centrifuge tube and incubated with anti-HA magnetic beads at room temperature for 10 min. Then, the centrifuge tube is placed on a magnet for 30 seconds to remove the supernatant. The anti-HA magnetic beads are washed three times with 1 ml of ice-cold KPBS. The Golgi apparatus on the immunoprecipitated beads is lysed directly in lysis buffer containing 0.2% SDS and used for subsequent assays.

[0264] In some embodiments, the surface plasmon resonance (SPR) analysis method includes: performing SPR experiments using a Biacore T200 optical biosensor (Cytiva Lifesciences). Recombinant Flag-TRAF6 WT and mutant proteins were purified from HEK293 cells and immobilized on an SCM5 series sensor chip. A solution of n-ethyl-NO-(3-dimethylaminopropyl)carbodiimide (2 mM) / N-hydroxysuccinimide (4 mM) (pH 7.0) was injected at a flow rate of 5 μL / min for 400 seconds to activate the carboxymethylated dextran gold surface. After chip activation, the purified recombinant protein (50 μg / mL) was injected into the activated surface of the sample channel at a flow rate of 5 μL / min in sodium acetate solution at pH 4.0 for 120 seconds. Kinetic titration was performed using PBS-P +1X (pH = 7.4, Cytiva Lifesciences) was used as the run buffer, and the experiment was conducted at 25°C. The drug was gradually diluted starting at 2 μM at a flow rate of 20 μL / min for multi-cycle kinetic analysis of association and dissociation. The kinetic trace was calculated by first subtracting the response of the reference channel in each cycle, and then subtracting the blank signal in the zero-concentration cycle. The kinetic constant K was determined using Biacore T200 evaluation software (Cytiva Lifesciences). D .

[0265] In some embodiments, the pharmacokinetic and tissue distribution analysis methods include: blood collection at specified time points after administration of Traf6i-13. Plasma samples are mixed with acetonitrile (1:5) and tamsulosin (TAM) is used as an internal standard (IS, 5 μg / mL). The mixture is then centrifuged at 18,000 rpm for 10 minutes at 4°C for 30 seconds. The supernatant is collected, and the previous step is repeated. Finally, 80 μL of the supernatant is taken for quantitative analysis using HPLC-triple quadrupole MS / MS.

[0266] Mouse tissue (approximately 0.1 g) was minced and thoroughly homogenized in physiological saline (10 times the tissue mass volume) using a high-speed tissue homogenizer. 50 μL of the tissue homogenate and 10 μL of TAM were added, bringing the final concentration to 5 μg / mL. The mixture was vortexed for 5 min, followed by the addition of 1 mL of methyl tert-butyl ether and a further vortexing for 5 min. The mixture was centrifuged at 18000 rpm for 5 min, and 800 μL of the supernatant was collected and evaporated using a nitrogen blower. The dried residue was redissolved in 100 μL of methanol, centrifuged at 18000 rpm for 5 min, and 80 μL of the supernatant was transferred to a sample vial. 10 μL of the sample was injected and analyzed using HPLC-Triple Quadrupole MS / MS.

[0267] The quantitative method for determining the concentration of Traf6i-13 in mouse plasma and tissues included: analysis of samples using a Sciex API4000+ triple quadrupole mass spectrometer (Redwood City, CA, USA) with an ESI source. The chromatographic column was a Kinetex C18100A (2.6 μm, 50 × 2.1 mm, USA). The mobile phase was water (0.1% formic acid) and acetonitrile (B). Gradient elution was performed as follows: 0–0.01 min (30% B), 0.01–0.5 min (30% B), 0.5–1 min (30%–85% B), 1–3 min (85% B), 3.0–4.0 min (85%–30% B), 4.0–7.0 min (30% B); the temperature was 40 °C, and the injection volume was 10 μL. The ESI parameters are as follows: ion spray voltage, 5500V; ion source gas 1, 50psi; coal gas 2, 60psi; ion spray temperature, 500℃; collision gas, 4Pa; curtain gas, 20psi. Tamsulosin at m / z 499.3→109.0 (M13) and m / z 409.2→228.2 (tamsulosin) were used for positive multiple reaction monitoring (MRM) fragmentation transitions. Traf6i-13 and IS were set to CE, 60 (eV) and DP, 120 (V). The entire system was controlled using Analyst 1.5.1 software (LC-MS / MS).

[0268] In some embodiments, the PK model construction method includes: pharmacokinetic modeling for multi-dose drug regimens, using Phoenix WinNonlin software to construct a partitioned model in its PK model module. This process first involves fitting the partitioned model to preliminary experimental data, then comparing and analyzing the fitting results of various models to determine the most suitable model.

[0269] A suitable model was determined, and simulation parameters were derived based on the partitioning model. These parameters were then used to predict pharmacokinetic characteristics after multiple dosings. Finally, the predicted results were juxtaposed with actual observation data to evaluate the accuracy of the model. The relevant parameters of the compartment model were calculated as follows.

[0270] Single-chamber model:

[0271]

[0272] k a is the absorption rate constant, and F is the bioavailability.

[0273] Two-room mode:

[0274] C(t)=Ae -αt +Be -βt (2)

[0275]

[0276]

[0277]

[0278] C(t) represents the plasma drug concentration at time t, A and B are constants related to the initial dose and distribution volume, α is the distribution rate constant, and β is the elimination rate constant. 12 It is the transfer rate constant from the central compartment to the peripheral compartment, k 10 Vc is the elimination rate constant of the central compartment, and D0 is the apparent volume of the central compartment distribution. C0 is the administered dose and C0 is the immediate blood drug concentration after administration.

[0279] In some embodiments, the method for RNA isolation and real-time quantitative PCR includes: extracting total RNA from mouse tissues or cells using RNAiso Plus (Cat. No. 9109, TaKaRa). The kit (RR047A, TaKaRa) reverse transcribed it into cDNA. This was then processed using Applied... StepOnePlus TM A real-time PCR system (Life Technologies) was used to perform real-time quantitative PCR to determine the expression level of target genes. Primers are shown in Table 1:

[0280] Table 1. qPCR primers used for target gene analysis

[0281]

[0282] In some embodiments, data are expressed as mean ± SEM for statistical analysis. Student's t-test and two-way ANOVA were performed on both groups using Prism software (GraphPad, San Diego, CA, USA). P < 0.05 was considered statistically significant.

[0283] The recombinant human insulin mentioned in this article was purchased from Novo Nordisk (Bagsvaerd, Denmark).

[0284] The cyclohexylimide (Cat. No. S7418), bafilomycin-A1 (Cat. No. S1413), and MG132 (Cat. No. S2619) mentioned in this article were purchased from Selleck (Shanghai, China).

[0285] The organic reagents (methanol and acetonitrile) mentioned in this article were supplied by Merck (Darmstadt, Germany). HPLC-grade formic acid was supplied by Sigma-Aldrich Chemicals (St. Louis, MO).

[0286] In this application, all other chemicals are derived from Sigma-Aldrich (Shanghai, China), Thermo Fisher Scientific (Shanghai, China) or Sangon Biotech (Shanghai, China).

[0287] The commercially sourced antibodies used in this application are shown in Table 2:

[0288] Table 2: List of Commercial Antibodies

[0289]

[0290]

[0291] This invention proposes the use of Traf6i compounds or salts thereof in the preparation of medicaments for treating diseases caused by abnormal TRAF6 expression; wherein the Traf6i is selected from Traf6i-1 to 14, and the Traf6i-1 to 14 have the following structural formulas:

[0292] Traf6i-1: Traf6i-2: Traf6i-3: Traf6i-4: Traf6i-5: Traf6i-6: Traf6i-7: Traf6i-8: Traf6i-9: Traf6i-10: Traf6i-11: Traf6i-12: Traf6i-13: And Traf6i-14:

[0293] In some embodiments, abnormal TRAF6 expression is defined as an increase in the expression level of TRAF6 protein; in other embodiments, diseases caused by abnormal TRAF6 expression include, but are not limited to, diabetes, abnormal glucose metabolism, tumors, hyperlipidemia, rheumatoid arthritis, inflammatory bowel disease, breast cancer bone metastasis, neurodegenerative diseases, and infectious diseases.

[0294] The Traf6i compound or its salt can inhibit the activity of TRAF6. In some embodiments, the Traf6i compound or its salt can regulate protease activity. Further, the Traf6i compound or its salt can regulate protease activity through ubiquitination, phosphorylation, or methylation; preferably, it regulates protease activity by ubiquitinizing the cytoplasmic domain of the protease. In some embodiments, the protease is a PCSK family protein; preferably, the protease is a PCSK family protein with a transmembrane domain; specifically, PCSK family proteins include, but are not limited to: FURIN, PCSK5B, PCSK7, PCSK8, and PCSK9.

[0295] In some embodiments, abnormal glucose metabolism is caused by decreased insulin sensitivity, reduced muscle glucose uptake, or decreased hepatic gluconeogenesis in the subject. Specifically, abnormal glucose metabolism is caused by a high-fat diet, inhibited FURIN activity, decreased INSRβ / pro-INSR, abnormal muscle glucose uptake, or abnormal hepatic gluconeogenesis. Preferably, the diabetes is type 2 diabetes.

[0296] This application relates to the use of any Traf6i compound or a salt thereof in the preparation of an inhibitor of intracellular TRAF6 activity.

[0297] This application also relates to compositions comprising any Traf6i compound or a salt thereof, said compositions being effective in treating diseases caused by abnormal TRAF6 expression.

[0298] This application further relates to a TRAF6 inhibitor, which includes any Traf6i compound or a salt thereof.

[0299] This application relates to a drug or drug composition that enhances glucose metabolism in a subject, comprising one or more of an active substance that inhibits TRAF6, FURIN, and INSRβ.

[0300] In some embodiments, the active substance is a small molecule compound; preferably, the small molecule compound includes any Traf6i compound or its salt.

[0301] In some embodiments, the active substance includes siRNA or shRNA that silences TRAF6; preferably, the sequence of the siRNA is shown in SEQ ID NO.7 or SEQ ID NO.8; and the sequence of the shRNA is shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13.

[0302] In some embodiments, the active substance comprises a vector for site-directed mutagenesis of TRAF6, wherein the site-directed mutagenesis does not include TRAF6. K67 and / or TRAF6H87 Preferably, the site-directed mutation of TRAF6 is a mutation of TRAF6. Q54 TRAF6 S66 TRAF6 C70 TRAF6 P71 TRAF6 I72 TRAF6 L74 TRAF6 L77 TRAF6 G86 TRAF6 R88 and / or TRAF6 C93 Site-directed mutation.

[0303] The aforementioned drugs or pharmaceutical compositions can be used to prepare drugs or pharmaceutical compositions that enhance glucose metabolism in subjects. In some embodiments, the subject's glucose metabolism can be enhanced by increasing insulin signaling, increasing muscle glucose uptake, or inhibiting hepatic gluconeogenesis.

[0304] In some embodiments, glucose metabolism in subjects can be enhanced by regulating protease activity, wherein methods for regulating protease activity include, but are not limited to, ubiquitination, phosphorylation, or methylation. In some embodiments, protease activity can be regulated by ubiquitination; further, in some embodiments, protease activity can be regulated by ubiquitination of the membrane-localized cytoplasmic portion of the protein.

[0305] In some embodiments, the protease is a PCSK family protein, preferably including: FURIN, PCSK5B, PCSK7, PCSK8, and PCSK9.

[0306] In some embodiments, the pharmaceutical composition alters the C-terminal portion (Asn) of the TRAF-C / MATH domain. 358 The activity of the terminal domain. In other embodiments, the pharmaceutical composition alters the activity of the cytoplasmic domain portion of FURIN.

[0307] The aforementioned drugs or drug compositions can be used to treat subjects with abnormal glucose metabolism. These abnormalities may be caused by a high-fat diet; or by inhibition of FURIN activity and a decrease in INSRβ / pro-INSR; or by abnormal muscle glucose uptake or hepatic gluconeogenesis, etc.

[0308] The above composition can be used in the preparation of pharmaceuticals for treating diseases caused by abnormal glucose metabolism, leading to elevated blood sugar levels. In some embodiments, the disease can be diabetes. Further, the disease can be type 2 diabetes.

[0309] Example 1: TRAF6 inhibitors can increase INSR levels and improve insulin sensitivity.

[0310] To develop inhibitors targeting TRAF6, the inventors used the TRAF6 n-terminal dimer motif (3HCS.pdb) for molecular docking. Figure 1 a, Figure 2 a). To find candidate compounds that might bind to the n-terminal cavity of TRAF6, a search of 3 million compounds in the ChemDiv chemical library was conducted, from which 14 compounds were identified ( Figure 2 (b) It is well known that TRAF6 is essential for LPS-induced expression of interleukin-1β (Il-1β) in macrophages. The inventors then used a bone marrow-derived macrophage (BMDM) system to examine the bioactivity of these 14 compounds in inhibiting TRAF6-dependent Il-1β expression under LPS.

[0311] When Traf6i-3, Traf6i-10, and Traf6i-13 are used to treat BMDM, they can inhibit >80% LPS-induced Il-1β expression. Figure 2 b). These three compounds also significantly inhibited LPS-induced Tnf-α expression in BMDM. Figure 2 c).

[0312] This application selected the most potent compound, Traf6i-13, to further investigate its effect on TRAF6. In surface plasmon resonance (SPR) experiments, Traf6i-13 can interact with K... D The purified recombinant TRAF6 bound at 64.7 nM ( Figure 1 bc). Furthermore, Traf6i-13 treatment reduces the interaction between TRAF6 and its homologous E2 ubiquitin-binding enzyme UBC13 ( Figure 1 d). Therefore, the inventor renamed Traf6i-13 to Traf6i-13. Unless otherwise specified, Traf6i-13 as mentioned below refers to Traf6i-13.

[0313] Plasma pharmacokinetic profiles of mice after oral administration of Traf6i-13 (100 mg / kg) showed that, at T... max Its concentration in plasma reached C at 0.5 h. max (1010±218.57ng / mL)( Figure 2 Through comparative analysis of various compartmental models, Traf6i-13 was determined to be the drug that conforms to the two-compartment absorption model. Figure 2 fg).

[0314] The inventors induced obese (DIO) mice by gavage administration of Traf6i-13 (100 mg / kg / day) to investigate whether the drug could reduce metabolic inflammation and thus improve glycemic control. After 4 weeks of continuous administration, plasma Traf6i-13 concentrations were measured at different time points following oral administration of Traf6i-13 (100 mg / kg). Parameters obtained through data fitting were used... Figure 2 The inventors predicted the results of the drug administration and juxtaposed these predictions with experimental observations, which showed good agreement. Figure 2 i). Tissue distribution analysis showed that Traf6i-13 was enriched in the liver, kidney, ileum, and skeletal muscle, but not in white adipose tissue (WAT), heart, and pancreas. Figure 2 j). During the experiment, oral administration of Traf6i-13 had no effect on the body weight of DIO mice. Figure 2 Contrary to the inventors' expectations, Traf6i-13 did not alleviate metabolic inflammation in the liver and WAT of DIO mice. Figure 2 (lm). It is very likely that the drug cannot reach immune cells such as macrophages in these tissues. Notably, the fasting blood glucose level of DIO mice treated with Traf6i-13 was significantly reduced (lm). Figure 1 e). Hepatic glucose output via gluconeogenesis is a major determinant of fasting blood glucose levels. Traf6i-13 treatment can inhibit 8-Br-cAMP-induced primary hepatocyte gluconeogenesis in a dose-dependent manner. Figure 1 f). The pyruvate tolerance test was used to evaluate hepatic gluconeogenesis in vivo. Notably, Traf6i-13 treatment significantly reduced hepatic glucose output in pyruvate-stimulated DIO mice (f). Figure 1 Insulin inhibition of hepatic gluconeogenesis is a key determinant of postprandial glucose clearance. In oral glucose tolerance tests, administration of TRAF6 was consistent with a reduction in hepatic glucose output, thus improving glucose clearance. Figure 1 ij).

[0315] Measurements of insulin signaling in primary hepatocytes isolated from DIO mice treated with Traf6i-13 revealed increased NSRβ protein and insulin-stimulated PKB phosphorylation in the primary hepatocytes of Traf6i-13-treated DIO mice. Figure 1 The inventors then measured the ratio of INSRβ to pro-INSR and found that the ratio of INSRβ to pro-INSR increased in primary hepatocytes from Traf6i-13 treated DIO mice (kn). Figure 1The inventors further investigated the effects of Traf6i-13 on insulin signaling in the L6 myotubules. Traf6i-13 treatment of the L6 myotubules increased INSRβ and the ratio of INSRβ to pro-INSR, and enhanced insulin-stimulated PKB phosphorylation (…). Figure 2 nq).

[0316] Example 2: TRAF6 gene inactivation increases INSR levels and enhances insulin signal transduction.

[0317] Given the effects of Traf6i-13 / Traf6i-13 on insulin signaling in muscle and hepatocytes, this application further investigates whether TRAF6 has a direct effect on INSRβ in these cells. The inventors first used primary hepatocytes as a model to study the effect of TRAF6 knockout on insulin signaling. Knocking out TRAF6 using small interfering RNA (siRNA) did not change Insr mRNA levels, but it increased INSRβ protein levels and the ratio of INSRβ to pro-INSR in mouse primary hepatocytes. Figure 3 Meanwhile, compared with the control group, insulin-stimulated PKB phosphorylation was enhanced in TRAF6 knockout mouse primary hepatocytes (ad). Figure 3 To determine whether this phenomenon is specific to hepatocytes, the inventors examined the effect of TRAF6 downregulation on insulin signaling in L6 muscle cells. TRAF6 expression in L6 myotubes was downregulated using short hairpin RNA (shRNA) technology. Figure 3 gl, Figure 4 Notably, compared to control cells, insulin-induced PKB phosphorylation was significantly increased in three independent shTRAF6 clones (j). Figure 3 jk, Figure 4 de, Figure 4 Furthermore, the level of INSRβ protein and the ratio of INSRβ to pro-INSR were significantly increased in TRAF6 knockout muscle cells. Figure 3 gi, Figure 4 ac, Figure 4 Consistent with enhanced insulin signaling, in TRAF6 knockout muscle cells, control glucose uptake was moderately increased, while insulin-stimulated glucose uptake was significantly increased. Figure 3 l).

[0318] TRAF6 expression is crucial for perinatal and postnatal survival; its absence in mice leads to premature death shortly after birth. In contrast, TRAF6 with E3 ligase inactivity... L74HMutant mice exhibited normal early survival rates and body weights comparable to their littermates, indicating that TRAF6's scaffold function as an adaptor protein is also crucial. Similar to Leu74, Ile72 on the TRAF6 loop domain is one of the seven residues required for E2 interaction; a mutation in its alanine residue inhibits E3 ligase activity. Thus, the inventors obtained a TRAF6... I72A Knock-in mutant mice, in which Ile72 on TRAF6 is replaced by alanine ( Figure 5 ab) Investigate whether and how the E3 ligase of TRAF6 regulates insulin sensitivity and metabolism in vivo. TRAF6 I72A The mice were born normally and had a normal survival rate after birth. TRAF6 was found in the examined tissues. I72A The TRAF6 protein levels in mice and wild-type (WT) littermates were comparable. Figure 5 c). Similar to TRAF6 knockout L6 muscle cells, TRAF6 I72A The levels of INSRβ protein and the ratio of INSRβ to pro-INSR in mouse skeletal muscle were significantly increased. Figure 3 m, op), while Insr mRNA remained normal in these animals ( Figure 5 d). It is worth noting that, compared to WT muscle, from TRAF6 I72A Increased insulin-stimulated PKB phosphorylation in isolated soleus and EDL muscle from mice ( Figure 3 qr, Figure 5 e). Similar to skeletal muscle, TRAF6 I72A The level of INSRβ protein in the liver was significantly elevated. Figure 3 (no), while Insr mRNA levels did not increase ( Figure 5 f), and at the same time, the ratio of INSRβ to pro-INSR also increases ( Figure 3 p). Furthermore, compared to WT cells, insulin-stimulated TRAF6... I72A Enhanced PKB phosphorylation in primary hepatocytes ( Figure 3 st).

[0319] In summary, these data indicate that TRAF6 inactivation enhances INSRβ protein and simultaneously enhances insulin signaling in muscle and hepatocytes.

[0320] Example 3 TRAF6 I72A Mutant inactivation can improve glycemic control in mice.

[0321] The inventors then studied TRAF6 with E3 ligase inactivation. I72A The effect of mutations on glucose homeostasis. Although TRAF6 I72AThe mice had normal weight at weaning, but gained far less weight in the first 6 weeks after birth, and thereafter their weight was significantly lower than that of their WT littermates. Figure 6 TRAF6 I72A In adult mice, the proportion of fat mass in body weight decreases, while the proportion of muscle mass increases. Figure 6 b). Despite TRAF6 I72A Spontaneous physical activity in mice was significantly reduced, but compared to WT littermates, TRAF6... I72A Mice have a higher food intake and expend more energy. Figure 6 ce). TRAF6 I72A The respiratory exchange rate (RER) of mice was higher than that of the WT control group. Figure 6 f) indicates that they utilize more carbohydrates to generate energy. TRAF6 I72A Control mice had lower blood glucose and serum or plasma insulin levels. Figure 7 ab). When small amounts of glucose are administered orally or intraperitoneally, TRAF6 I72A The glucose clearance rate of the mice was higher than that of their WT littermates. Figure 7 cd Figure 6 gh).

[0322] Insulin increased glucose uptake in the WT soleus muscle and EDL muscle by approximately 155% and 75%, respectively. Figure 7 Consistent with PKB activation, insulin-stimulated glucose uptake at TRAF6 was higher than that in WT muscle. I72A It is also enhanced in the soleus muscle and EDL muscle. Figure 7 GLUT4 is a major glucose transporter that mediates insulin-stimulated glucose uptake in skeletal muscle; it is expressed in WT and TRAF6. I72A The mice were comparable ( Figure 7 Under the influence of insulin, GLUT4 is translocated from its intracellular storage site to the cell membrane, mediating glucose transport. Insulin increases GLUT4 levels on the surface of WT soleus muscle cells; this insulin-induced GLUT4 translocation occurs at TRAF6. I72A Significant enhancement in muscles ( Figure 7 g and 7i).

[0323] The liver is another insulin-sensitive organ; insulin controls postprandial blood glucose by inhibiting hepatic gluconeogenesis. PEPCK and G6Pase are two key gluconeogenic enzymes, and their mRNA levels are high in the TRAF6 region. I72A Reduced in the liver ( Figure 7 j) suggests that loss of hepatic TRAF6 E3 ligase activity may reduce hepatic gluconeogenesis. Subsequently, a pyruvate tolerance test revealed that TRAF6I72A Mice injected with pyruvate did indeed produce lower glucose levels than their littermates in the wild-caught (WT) mice. Figure 7 Primary hepatocytes were isolated and the expression of Pepck and G6pase mRNA was measured under 8-bromo-cAMP (8-Br-cAMP) and insulin stimulation. 8-Br-cAMP treatment increased the expression of these two gluconeogenic genes in WT hepatocytes, while TRAF6... I72A The expression of these two genes in cells was significantly suppressed. Figure 7 mn). Compared with WT cells, insulin has a greater effect on TRAF6. I72A The inhibition of 8-Br-cAMP-induced Pepck and G6pase expression in hepatocytes was more pronounced. Figure 7 mn).

[0324] In summary, these data indicate that inactivation of the TRAF6 E3 ligase increases insulin sensitivity and improves overall glycemic control.

[0325] Example 4: TRAF6 inhibits pro-INSR processing into mature INSRβ

[0326] INSR expression is a precursor that first undergoes glycosylation, then cleavages into N-terminal α-chains and C-terminal β-chains to form a functional receptor kinase. The inventors co-expressed TRAF6 with Flag-labeled INSRα and INSRβ in HEK293 cells to test whether TRAF6 led to their degradation. Co-expression of TRAF6 had little effect on Flag-INSRα, but led to a significant increase in Flag-INSRβ. Figure 8 Therefore, the likelihood of TRAF6 inducing INSRβ degradation is small. The inventors co-expressed Flag-tagged TRAF6 and pro-INSR fused to the C-terminus with an HA tag in HEK293 cells. Flag-TRAF6 co-expression moderately increased the protein level of HA-pro-INSR (approximately 250 kDa glycosylated form and approximately 180 kDa unglycosylated form). Interestingly, Flag-TRAF6 co-expression significantly reduced the INSRβ protein derived from HA-pro-INSR (approximately 105 kDa). Figure 9 a). The TRAF6-induced decrease in INSRβ is unlikely to be due to protein degradation by the proteasome or lysosome, because treatment with the lysosomal inhibitor bafilomycin-A1 (Baf-A1) or the proteasome inhibitor MG-132 did not reverse the effect of TRAF6 on INSRβ. Figure 9 b). Conversely, bafimycin-A1 or MG-132 led to the accumulation of LC3 and ubiquitinated proteins, respectively. Figure 9b). However, the TRAF6-induced decrease in INSRβ depends on its E3 ligase activity. Compared to WT TRAF6, TRAF6... I72A Co-expression of the mutant with HA-pro-INSR did not lead to a decrease in INSRβ. Figure 9 a) To further confirm that the E3 ligase activity of TRAF6 is the cause of the decrease in INSRβ, the inventors added a critical residue Cys on TRAF6. 70 The mutation to alanine inactivates its E3 ligase. (This is related to TRAF6.) I72A Similar mutations occur when TRAF6 C70A When the mutant is co-expressed with HA-pro-INSR, TRAF6 C70A The mutation prevented TRAF6-induced reduction in INSRβ. Figure 9 a).

[0327] These data led the inventors to hypothesize that TRAF6 might inhibit the processing of pro-INSR into mature INSRβ. The inventors used a labeling assay to monitor the conversion of nascent pro-INSR to INSRβ. The results showed that within 90 minutes of labeling, in cells expressing only pro-INSR-Flag, nascent pro-INSR could produce a large amount of INSRβ. Figure 9 Conversely, co-expression of HA-TRAF6 overexpression significantly inhibited the production of INSRβ from its precursor pro-INSR-Flag (cd). Figure 9 (cd). The inventors then used this labeling assay to monitor the processing of endogenous pro-INSRβ in mouse primary hepatocytes. Endogenous INSRβ exists in two forms in mouse hepatocytes: a major form (approximately 100 kDa) and a minor form (approximately 95 kDa). The nature of these two forms of INSRβ is currently unknown. However, compared to WT cells during the experiment, TRAF6... I72A The amount of newly generated INSRβ (approximately 95 kDa) in hepatocytes increased significantly, while the amount of newly synthesized INSRβ (approximately 100 kDa) increased moderately. Figure 8 c, Figure 9 ef).

[0328] These data suggest that TRAF6 may regulate INSR maturation by inhibiting the processing of pro-INSR into mature INSRβ.

[0329] Example 5: TRAF6 promotes the processing of prelysosomal protein enzymes, such as FURIN.

[0330] FURIN is an important proprotein convertase that mediates the conversion of pro-INSR to mature INSR. Interestingly, TRAF6... I72AThe protein level of FURIN was significantly increased in the skeletal muscle and liver of mice, while its mRNA level remained unchanged. Figure 10 ab, Fig. 11a). In TRAF6 I72A FUIRN activity was significantly increased in both skeletal muscle and liver of mice. Figure 10 c). Similarly, FURIN protein levels were also elevated in TRAF6-KD L6 myocytes and TRAF6-KD primary hepatocytes. Figure 11 bc). To investigate whether the TRAF6 inactivation-induced increase in INSRβ was attributable to increased FURIN protein levels and activity, the inventors also silenced FURIN in primary TRAF6-KD mouse hepatocytes using siRNA. Notably, in hepatocytes, the TRAF6 knockout-induced increase in INSRβ protein and pro-INSR processing (INSRβ / pro-INSR as a substitute) could be prevented by knocking out FURIN. Figure 10 According to reports, INSR protein cleavage in the liver of FURIN knockout mice was not altered, possibly due to compensation from other PCSKs. Compared to the negative control group, FURIN knockout did not lead to a further reduction in INSRβ protein and pro-INSR processing. Figure 10 (de). FURIN on the cell surface can detach and be released into the circulation, inactivating mature PCSK9, a key regulator of cholesterol metabolism. In TRAF6... I72A In mouse serum, FURIN, considered a high-molecular-weight dimer (approximately 180 kDa), was significantly increased, accompanied by more extensively cleaved inactive PCSK9 and less mature active PCSK9. Figure 11 (de). It is generally agreed that TRAF6 I72A The mice had lower serum total cholesterol (TC) levels than their WT littermates, while their liver TC levels were higher than the control group. Figure 11 fg). Then, the inventors tested from WT and TRAF6. I72A The stability of FURIN in primary mouse hepatocytes, in which protein synthesis is inhibited by cycloheximine (CHX). After the addition of cycloheximine, TRAF6... I72A FURIN turnover in hepatocytes is slower than in WT cells. Figure 10 fg) indicates that TRAF6 inactivation can stabilize FURIN.

[0331] To investigate how TRAF6 regulates FURIN stability, the inventors co-expressed WT or mutant Flag-TRAF6 with HA-FURIN in HEK293 cells. Notably, co-expression of WT Flag-TRAF6 reduced HA-FURIN protein levels in HEK293 cells, while co-expression of mutant Flag-TRAF6... I72A No( Figure 10 h). This reduction in TRAF6-dependent FURIN is likely due to lysosomal degradation, rather than proteosome-mediated degradation. Figure 10 i) Treatment with the lysosomal inhibitor bafimycin-A1 prevented TRAF6-induced decrease in FURIN, while simultaneously increasing LC3. In contrast, the proteasome inhibitor MG-132 did not prevent the decrease in FURIN induced by TRAF6 co-expression, although, as expected, it led to the accumulation of ubiquitinated proteins.

[0332] In summary, these data indicate that TRAF6 promotes the lysosomal degradation of the pro-INSR processing enzyme FURIN.

[0333] Example 6: TRAF6 interacts with FURIN to promote its lysosomal degradation.

[0334] Flag-TRAF6 was immunoprecipitated from cell lysate, and HA-FURIN was found in the immunoprecipitate. Figure 10 j) suggests that the two proteins may interact. A reciprocal immunoprecipitation experiment showed that GFP-TRAF6 was present in the immunoprecipitate of Flag-FURIN, further confirming their interaction. Figure 10 k). Furthermore, endogenous TRAF6 and FURIN in HEK293 cells ( Figure 10 l) and mouse liver ( Figure 10 Interactions are observed in the m) region. Mature FURIN is transported from the endoplasmic reticulum to the Golgi apparatus for processing pre-proteins, and its Golgi localization was confirmed by subcellular isolation from the mouse liver. Figure 10 Interestingly, TRAF6 is present in the Golgi apparatus fraction, but almost entirely absent in the ER fraction of mouse liver (n). Figure 10 Consistent with this, when expressed in primary hepatocytes, a portion of GFP6-TRAF6 co-localizes with the Golgi marker RCAS1. Figure 11 h).

[0335] To gain a deeper understanding of the interaction between TRAF6 and FURIN, the inventors mapped the interaction domain of FURIN on TRAF6. TRAF6 contains a loop domain with E3 ligase activity at its N-terminus and a TRAF-C / MATH domain at its C-terminus, mediating its binding to various receptors. Figure 12 a). TRAF6 fragmented surface, containing the C-terminal portion (Asn) of the TRAF-C / MATH domain. 358 The terminus is the N-terminal portion (Met) containing the RING, Zn fingers, and TRAF-N domains. 1 -Cys 357 ), interacting with HA-FURIN in the immunoprecipitation assay ( Figure 12 b). PA treatment had no effect on the interaction between TRAF6-MATH and FURIN. Figure 13 a).

[0336] Intracellular FURIN is a transmembrane protein that has the ability to derive from His... 736 To the cytoplasmic tail at the end ( Figure 12 c). Similar to full-length FURIN, in immunoprecipitation experiments using Flag-TRAF6 as a decoy, the cytoplasmic domain of FURIN can interact with Flag-TRAF6. Figure 12 d). In the cross-immunoprecipitation assay, Flag-TRAF6 was present in the immunoprecipitate of GFP-labeled FURIN and its cytoplasmic domains, but not in the immunoprecipitate of free GFP. Figure 13 b). PA treatment does not affect TRAF6 and FURIN ( Figure 13 c). Four of the nine PCSKs, namely FURIN, PCSK5B, PCSK7, and PCSK8 / SKI-1, contain transmembrane regions with cytoplasmic domains. Does TRAF6 also interact with the other three PCSKs containing cytoplasmic domains? In fact, co-immunoprecipitation experiments showed that TRAF6 also interacts with PCSK5B, PCSK7, and PCSK8 / SKI-1. Figure 13 PCSK5A and PCSK5B are two isoforms encoded by two splice variants from the same gene. PCSK5A is a soluble protein that lacks the transmembrane and cytoplasmic domains present in PCSK5B. Compared to PCSK5B, PCSK5A shows co-immunization in precipitation assays and does not interact with TRAF6. Figure 13 g). These data indicate that the cytoplasmic domains of PCSKs are crucial for their interaction with TRAF6.

[0337] In summary, these data suggest that the interaction between TRAF6 and FURIN promotes its subsequent degradation.

[0338] Example 7: TRAF6 ubiquitination of FURIN for lysosomal degradation

[0339] When co-expressed with TRAF6, the mCherry or GFP-labeled FURIN cytoplasmic domain showed multiple bands on Western blotting, with a molecular weight larger than expected. Figure 12 d, Figure 13 b) indicates that TRAF6 may ubiquitinate FURIN through its cytoplasmic domain. TRAF6 co-expression increased FURIN ubiquitination, while E3 ligase-inactivated C70A and I72A mutations attenuated FURIN ubiquitination. Figure 12 e). PA treatment enhanced TRAF6-mediated FURIN ubiquitination ( Figure 12 f). The inventors then established an experiment to investigate whether TRAF6 could ubiquitinate FURIN on the Golgi apparatus, in which FURIN-GFP, Myc-TRAF6, and Flag-Ub were co-expressed in HEK293 cells expressing the 3×HA-labeled Golgi localizer TMEM115. TMEM115-3×HA was isolated from the Golgi apparatus using immunoprecipitation. Figure 13 As expected, most FURIN-GFP is localized to the Golgi apparatus, while a portion of Myc-TRAF6 is localized to the Golgi apparatus (h). Figure 13 h). Then, the inventors performed immunoprecipitation on the Golgi apparatus to detect localized FURIN ubiquitination, finding that overexpression of TRAF6 enhanced FURIN ubiquitination in the Golgi apparatus (h). Figure 12 g).

[0340] When co-expressed in cells, TRAF6 can ubiquitinate the cytoplasmic domains of FURIN. Figure 12 h), while the loss of the cytoplasmic domain can prevent TRAF6-mediated ubiquitination of FURIN ( Figure 12 i). To determine whether the cytoplasmic domain might mediate TRAF6-regulated FURIN degradation, the inventors examined WT FURIN and a FURIN-deficient cytoplasmic domain in HEK293 cells co-transfected with Flag-TRAF6. △CD The protein stability of FURIN. Notably, when protein synthesis is inhibited by cyclohexylimide, FURIN... △CD The mutant degrades more slowly than WT FURIN. Figure 12 (jk). Compared to wild-type FURIN, when co-expressed with INSR-Flag in HEK293 cells, FURIN... △CD Overexpression of the mutant significantly reduced mature INSRβ without affecting the expression of Insr mRNA. Figure 13 ik), indicating FURIN △CD The mutant may have a dominant negative effect on endogenous FURIN. Using INSRβ / pro-INSR as an alternative, FURIN... △CD Overexpression of the mutant alleviated the inhibitory effect of TRAF6 on pro-INSR processing. Figure 13 ik).

[0341] Traf6i-13 treatment blocked TRAF6-mediated FURN ubiquitination in HEK293 cells. Figure 12 In contrast, Traf6i-13 cannot inhibit TRAF2 ubiquitination of p62 (l). Figure 14 a). Eleven potential Traf6i-13 binding sites predicted by molecular docking were subjected to alanine mutations to identify key binding sites. Some mutations eliminated the E3 ligase activity of TRAF6 against FURIN, while others did not affect the inhibitory effect of Traf6i-13 on FURIN. Figure 14 bd). The K67A and H87A mutations maintained the E3 ligase activity of TRAF6 and were not inhibited by Traf6i-13. Figure 14 (cd). Subsequently, the inventors used K67A as the research object to further verify its resistance to Traf6i-13 (cd). Figure 14 e). Surface plasmon resonance experiments confirmed that Traf6i-13 cannot interact with purified recombinant TRAF6. K67A mutant protein binding ( Figure 14 f).

[0342] In summary, these data indicate that TRAF6 is degraded via its cytoplasmic domain ubiquitination of FURIN.

[0343] Example 8: TRAF6 inactivation can alleviate fatty acid-induced reduction in INSRβ.

[0344] Primary mouse hepatocytes and L6 myocytes were used as cell models. Treatment of primary mouse hepatocytes with fatty acids (PA) reduced the levels of FURIN and INSRβ proteins and inhibited pro-INSR processing (INSRβ / pro-INSR as a substitute). Figure 15 ab, d). PA treatment inhibited phosphorylation of PKB in primary mouse hepatocytes (ab, d). Figure 15 c). Similarly, PA treatment reduced INSRβ protein and INSRβ / pro-INSR, and inhibited PKB phosphorylation in L6 myotubules. Figure 15Notably, TRAF6 gene deficiency prevented the reduction in PA-induced phosphorylation of INSRβ protein, INSRβ / pro-INSR, and PKB in L6 muscle cells. Figure 15 The inventors also reexpressed TRAF6 in TRAF6-KD L6 muscle cells. WT and TRAF6 K67A And it was processed with PA and Traf6i-13 ( Figure 15 g). In TRAF6-KD / TRAF6 WT In muscle cells, PA treatment reduced FURIN and INSRβ protein levels, while Traf6i-13 prevented these PA-induced reductions. PA treatment also led to a decrease in TRAF6-KD / TRAF6 levels. K67A The levels of FURIN and INSRβ proteins in muscle cells were decreased. Importantly, Traf6i-13 could no longer prevent PA-induced TRAF6-KD / TRAF6 K67A The decrease in FURIN and INSRβ in muscle cells indicates that the effects of Traf6i-13 on FURIN and INSRβ are mediated by TRAF6. These data suggest that the TRAF6-INSR axis is involved in the development of PA-induced insulin resistance, and that TRAF6 inactivation may alleviate insulin resistance in muscle cells.

[0345] The TRAF6-INSR pathway was examined in DIO mice and obese human patients. DIO mice showed significantly reduced INSRβ in the liver, accompanied by a significant reduction in FURIN. Figure 16 ab). The ratio of INSRβ to pro-INSR in the liver of DIO mice was decreased, suggesting impaired pro-INSR processing. Figure 16 c). HFD had similar effects on the processing of FURIN protein and pro-INSR in the skeletal muscle of DIO mice. Figure 15 In the human liver, both INSRβ and FURIN proteins are negatively correlated with body mass index (BMI). Figure 16 The ratio of INSRβ to pro-INSR, as a proxy for pro-INSR treatment, is also negatively correlated with BMI. Figure 16 f). In contrast, PKB phosphorylation and TRAF6 expression were independent of BMI ( Figure 15 To further determine the role of the TRAF6-INSR axis in the development of HFD-induced insulin resistance, the inventors studied TRAF6... I72A Mice and their WT litters were fed HFD. Compared with WT litters that were obese on HFD, TRAF6 I72A Mice are resistant to HFD-induced obesity. Figure 16 g). Mice fed a similar diet ( Figure 6 b) HFD group TRAF6 I72A The mice had a lower percentage of body fat than their littermates at WT, but a higher percentage of body muscle than the WT control group. Figure 16 h). HFD's TRAF6 I72A Mice that were orally administered large amounts of glucose still had low blood glucose levels and cleared glucose faster than their WT littermates. Figure 16 As expected, HFD reduced the protein levels of INSRβ and FURIN in the liver of WT mice, as well as the INSRβ / pro-INSR ratio. Figure 16 (kn). However, it is worth noting that HFD-induced TRAF6 I72A The decrease in INSRβ and FURIN protein expression and the decrease in the INSRβ / pro-INSR ratio in mouse liver were prevented. Figure 16 kn). In TRAF6 I72A Similar trends were observed in the skeletal muscle of mice. Figure 15 np).

[0346] Although INSRβ and FURIN proteins and INSRβ / pro-INSR were decreased, TRAF6 protein was not altered in PA-treated L6 muscle cells. Figure 17 Furthermore, HFD did not cause any change in TRAF6 protein levels in the livers of DIO mice. Figure 16 ab). Protein palmitoylation can regulate the membrane binding or distribution of target proteins. Interestingly, TRAF6 has been predicted as a palmitoylated protein (https: / / gpspalm.biocuckoo.cn). This application confirms that Flag-TRAF6 is indeed palmitoylated when expressed in cells. Figure 17 d). In addition, the N-terminal portion containing RING (Met) 1 -Cys 357 ) and the C-terminal portion (Asn) containing the TRAF-C / MATH domain. 358 Both ends are palmitoylated, although the degree of palmitoylation is more severe in the N-terminal portion than in the C-terminal portion. Figure 17 d). However, HFD did not alter the Golgi binding of TRAF6 in the liver of DIO mice ( Figure 17 ef).

[0347] In summary, these data suggest that the TRAF6-INSR axis is involved in the development of PA / HFD-induced insulin resistance, and that TRAF6 inactivation may alleviate insulin resistance in muscle and hepatocytes.

[0348] This application demonstrates that TRAF6 is a key regulator of INSR maturation, dependent on its E3 ligase activity. The data in this application are consistent with a model of TRAF6 ubiquitination of pro-INSR converting enzyme FURIN for lysosomal degradation, inhibiting the processing of pro-INSR into mature INSR, thereby leading to insulin resistance. Figure 18 ) Targeting the E3 ligase activity of TRAF6 can increase mature INSR and alleviate PA / HFD-induced insulin resistance.

[0349] The development of obesity-induced insulin resistance involves multiple mechanisms at both the receptor and post-receptor levels. At the receptor level, glypican-4, released from adipose tissue, enhances insulin signaling through direct interaction with the insulin receptor agonist (INSR), and its circulating levels are reduced in obesity. In contrast, macrophage-derived galectin-3 directly binds to and inhibits the INSR in various insulin-sensitive cells such as hepatocytes, adipocytes, and myocytes, contributing to obesity-induced inflammation-induced insulin resistance. Furthermore, INSR has been documented to be downregulated in obese mouse models. For example, the reduced INSR in the heart of obese mice is likely due to decreased FURIN caused by sarcoplasmic reticulum calcium depletion. Data from this application suggest that obesity-induced downregulation of INSRβ in the liver and skeletal muscle is likely due to TRAF6-mediated inhibition of pro-INSR processing via FURIN. TRAF6 ubiquitinates the cytoplasmic domain of FURIN, leading to degradation by protonopeptidase via lysosomes. Calcium ions in the endoplasmic reticulum (ER) and Golgi apparatus enhance the activity of FURIN while inhibiting its lysosomal degradation. This application demonstrates that the TRAF6-FURIN axis plays a crucial role in the development of insulin resistance in obesity and can target and improve insulin sensitivity.

[0350] Data from this application indicate that Golgi-associated TRAF6 promotes FURIN degradation in response to PA / HFD treatment. TRAF6 expression was not altered in the liver of DIO mice or in PA-treated myotubes. Studies have shown that palmitoylation of the cyclic E3 ligase Gp78 on its cyclic finger cysteine ​​residues regulates its distribution to the peripheral ER. TRAF6 is also palmitoylated on its cyclic N-terminal portion and the C-terminal portion of the TRAF-C / MATH domain. However, in the liver of DIO mice, the relationship between TRAF6 and the Golgi apparatus was not altered. PA / HFD treatment may increase the palmitoylation of TRAF6, thereby enhancing its E3 ligase activity. Another possibility is that PA / HFD treatment may promote the binding of the ZDHHC family of S-acyltransferases that catalyze the palmitoylation of proteins to TRAF6, thereby enhancing their E3 ligase activity. Consistent with this possibility, the interaction of ZDHHC11 with TRAF6 may enhance its E3 ligase activity. The enhancement of TRAF6 activity by ZDHHC11 is independent of ZDHHC11 palmitoylation activity. The TRAF-C / MATH domain of TRAF6 is involved in its interaction with FURIN. Therefore, palmitoylation of the TRAF-C / MATH domain may affect its interaction with FURIN. TRAF6 is a key component of TLR4 signaling, which is activated in PA / HFD treatment. TLR4 is essential in fatty acid-induced muscle insulin resistance but not in fatty acid-induced hepatic insulin resistance. These data suggest that TLR4 may not be involved in the regulation of the hepatic TRAF6-FURIN-INSR axis by HFD.

[0351] Reports indicate that TRAF6 can directly ubiquitinate PKB, enhancing its membrane localization and activation in mouse embryonic fibroblasts under growth factor stimulation. This application demonstrates that the E3 ligase activity of TRAF6 is crucial for controlling insulin sensitivity, potentially involving multiple mechanisms, including inflammatory responses, serine phosphorylation of IRS1 / 2, and pro-INSR processing. In addition to these possible mechanisms, leanness may also contribute to improving TRAF6 levels. I72A Insulin sensitivity in mice. Contrary to predictions of TRAF6-mediated ubiquitination-dependent PKB activation in response to insulin-like growth factor-1 (IGF-1) stimulation, inhibition of the TRAF6 E3 ligase improved insulin sensitivity. One possibility for this discrepancy is that TRAF6 regulation of PKB signaling is environmentally dependent, with cell type and stimulation likely being important factors. Although both insulin and IGF-1 activate PKB signaling, these two stimuli are transmitted through structurally related but functionally different receptors that are differentially expressed in different cells / tissues.

[0352] FURIN is a proprotein processing enzyme belonging to the proprotein convertase subtilisin / kexin (PCSK) family of serine proteases. The PCSK family consists of nine members with proteolytic activity against hundreds of substrates, ranging from hormones / growth factors to receptors and adhesion molecules. In addition to mammalian substrates, FURIN can cleave and activate bacterial substrates (such as Shiga / Shiga-like toxins) and viral substrates (including the SARS-CoV-2 spike protein) for viral or bacterial infection. TRAF6-mediated FURIN degradation may link endotoxin sensing with Shiga / Shiga-like toxin-dependent activation of FURIN in the Golgi apparatus as a means of combating bacterial invasion, potentially complementing the LPS-induced antimicrobial innate immune response. Four of the nine PCSKs (FURIN, PCSK5B, PCSK7, and PCSK8 / SKI-1) contain transmembrane regions with cytoplasmic domains and interact with TRAF6. Conversely, PCSK5A, lacking a cytoplasmic domain, does not interact with TRAF6. Therefore, the cytoplasmic domains of PCSKs may be crucial to their regulation. Consistent with this view, the cytoplasmic domains of PCSKs are regulated by post-translational modifications. For example, the cytoplasmic domain of PCSK7 is located in Cys... 699 and Cys 704 Palmitoylation of the upper surface increases its stability without altering its subcellular localization. In contrast, the EXEXXXL domain of the PCSK7 cytoplasmic structure... 725 The motif is crucial for intracellular transport by interacting with adaptor protein 2 (AP-2).

[0353] The results of this application indicate that TRAF6 is a key regulator of INSR maturation by ubiquitinizing the pro-INSR processing enzyme FURIN in muscle and hepatocytes. The experiments in this application demonstrate that targeting the E3 ligase activity of TRAF6 can prevent PA / HFD-induced INSR reduction, thus representing a potential drug for combating T2D.

[0354] Example 9: Traf6i-13 reduces the relative proliferation rate of tumor cell lines.

[0355] Empty vector (control), Traf6i-13, and HepG2, Huh7, and HCT116 cell lines were co-cultured, respectively. Figure 19 As shown in the results, the experimental results indicate that Traf6i-13 can inhibit the growth of liver cancer cells and colon cancer cells. Further research showed that Traf6i-13 can inhibit TRAF6 activity in HepG2, Huh7, and HCT116 cell lines.

[0356] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. The use of Traf6i-n compounds or salts thereof in the preparation of medicaments for treating diseases caused by abnormal TRAF6 expression; wherein the Traf6i-n is selected from Traf6i-1 to 14, and the Traf6i-1 to 14 have the following structural formulas: Traf6i-1: Traf6i-2: Traf6i-3: Traf6i-4: Traf6i-5: Traf6i-6: Traf6i-7: Traf6i-8: Traf6i-9: Traf6i-10: Traf6i-11: Traf6i-12: Traf6i-13: And Traf6i-14:

2. In the application according to claim 1, the abnormal TRAF6 expression is an increase in the expression level of TRAF6 protein.

3. According to the application described in claim 1, the diseases caused by abnormal TRAF6 expression include, but are not limited to, diabetes, abnormal glucose metabolism, tumors, rheumatoid arthritis, inflammatory bowel disease, breast cancer bone metastasis, neurodegenerative diseases, and infectious diseases.

4. In the application according to claim 1, the Traf6i-n can inhibit the activity of TRAF6.

5. In the application according to claim 1, the Traf6i-n can regulate protease activity.

6. The application according to claim 5, wherein protease activity is regulated by ubiquitination, phosphorylation or methylation; preferably, protease activity is regulated by ubiquitination of the cytoplasmic domain of the protease.

7. The application according to claim 5, wherein the protease is a PCSK family protein; preferably, the protease is a PCSK family protein having a transmembrane domain.

8. The application according to claim 7, wherein the PCSK family protein comprises: FURIN, PCSK5B, PCSK7, PCSK8, PCSK9.

9. The application according to claim 3, wherein the abnormal glucose metabolism is caused by decreased insulin sensitivity in the subject, decreased glucose uptake in the subject's muscles, or decreased hepatic gluconeogenesis in the subject.

10. The application according to claim 3, wherein the abnormal glucose metabolism is caused by a high-fat diet, inhibition of FURIN activity, decreased INSRβ / pro-INSR, abnormal muscle glucose uptake, or abnormal hepatic gluconeogenesis.

11. The application according to claim 3, wherein, The diabetes mentioned is type 2 diabetes.

12. The use of any Traf6i-n compound or its salt in the preparation of an inhibitor of intracellular TRAF6 activity.

13. A composition comprising any Traf6i-n compound or a salt thereof, said composition being effective in treating diseases caused by abnormal TRAF6 expression.

14. A TRAF6 inhibitor comprising any Traf6i-n compound or a salt thereof.

15. A drug or drug composition for enhancing glucose metabolism in a subject, comprising one or more of an active substance that inhibits TRAF6, FURIN, and INSRβ.

16. The medicament or pharmaceutical composition according to claim 15, wherein the active substance is a small molecule compound; preferably, the small molecule compound is a Traf6i-n compound or a salt thereof.

17. The medicament or pharmaceutical composition according to claim 15, wherein the active substance is a siRNA or shRNA that silences TRAF6; preferably, the sequence of the siRNA is shown in SEQ ID NO. 7 or SEQ ID NO. 8; and the sequence of the shRNA is shown in SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO.

13.

18. The medicament or pharmaceutical composition according to claim 15, wherein the active substance is a carrier comprising site-directed mutagenesis of TRAF6, wherein the site-directed mutagenesis does not include TRAF6. K67 and / or TRAF6 H87 Preferably, the site-directed mutation of TRAF6 is a mutation of TRAF6. Q54 TRAF6 S66 TRAF6 C70 TRAF6 P71 TRAF6 I72 TRAF6 L74 TRAF6 L77 TRAF6 G86 TRAF6 R88 and / or TRAF6 C93 Site-directed mutation.

19. Use of a drug or pharmaceutical composition in the preparation of a pharmaceutical composition that enhances glucose metabolism in a subject; wherein the drug or pharmaceutical composition comprises one or more of an active substance that inhibits TRAF6, FURIN, and INSRβ.

20. The application according to claim 19, wherein the pharmaceutical composition alters the C-terminal portion (Asn) of the TRAF-C / MATH domain. 358 (Terminal) activity.

21. The application according to claim 19, wherein the pharmaceutical composition alters the activity of the cytoplasmic domain portion of FURIN.

Images