Fto protein degradation targeting chimera, and preparation method and application thereof

Abstract

The application discloses an FTO protein degradation targeting chimera as well as a preparation method and application thereof, and belongs to the technical field of biotechnology and biological medicine. The FTO protein degradation targeting chimera has a protein targeting chimera (PROTAC) molecular structure, and a general structural formula thereof is shown as formula I or formula II. E is an E3 ligase ligand with ubiquitination function. L is a connecting group, and the connecting group is one of an alkylene group or an alkoxy group. The FTO protein degradation targeting chimera provided by the application can effectively degrade FTO protein as an FTO protein degradation agent, thereby improving obesity and fatty liver, can be developed as a new medicine for preventing and / or treating obesity, fatty liver and diabetes, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and biomedicine, and particularly relates to an FTO protein degradation targeting chimera, its preparation method and application. Background Technology

[0002] FTO is a key gene associated with obesity and playing a regulatory role in adipogenesis, identified through genome-wide association analysis. The protein it encodes belongs to the AlkB family and is an Fe-dependent protein. 2+ FTO, along with α-ketoglutarate oxygenase, catalyzes RNA demethylation. Recent studies have found that abnormal activity of hepatic FTO is associated with metabolic damage such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease. It regulates hepatic glucose and lipid metabolism by altering the methylation status of genes involved in fatty acid oxidation, lipolysis, and lipogenesis, as well as the activity of transcription factors. Disruption of these regulatory mechanisms is closely related to the pathogenesis of obesity and metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease, and restoration of these damage may be beneficial in reversing metabolic abnormalities. Therefore, inhibiting FTO expression or activity may be a treatment approach for obesity, diabetes, and non-alcoholic fatty liver disease. However, current small molecules that inhibit FTO lack specificity and cannot address compensatory mechanisms in other signaling pathways. Furthermore, CRISPR-CAS9 and siRNA / shRNA technologies acting at the DNA or RNA level face drawbacks such as irreversible editing and off-target effects.

[0003] PROTAC, a targeted degradation chimera, has emerged as an innovative protein degradation technology in recent years, attracting widespread attention from scholars and pharmaceutical companies. As a bifunctional small molecule compound, the PROTAC consists of three parts: a ligand that binds to the target protein at one end, a ligand that binds to E3 ubiquitin ligase at the other end, and a linker chain connecting the two ligands. In the cell, the PROTAC molecule recognizes and selectively binds to the target protein and recruits a specific E3 ubiquitin ligase, shortening the spatial distance between the E3 ubiquitin ligase and the target protein to form a "target protein-PROTAC-E3 ubiquitin ligase" ternary complex. Then, through synergy with E2 ubiquitin conjugation enzyme, it promotes the ubiquitination of the target protein, allowing it to enter the ubiquitin-proteasome pathway, thereby achieving the goal of degrading the target protein. Unlike traditional small molecules that block protein function by directly binding to the active site of the target protein, PROTAC is an "event-driven" type. It does not need to directly inhibit the functional activity of the target protein and does not require high affinity for the target protein ligand. It can exert protein degradation function with only a low concentration, thus achieving a more lasting effect and effectively overcoming the drug resistance problem of existing drugs. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an FTO protein degradation targeting chimera, its preparation method and application. The FTO protein degradation targeting chimera provided by this invention, as an FTO protein degrading agent, can effectively degrade FTO protein, thereby improving obesity and fatty liver. It can be developed and applied as a new drug for the prevention and / or treatment of obesity, fatty liver and diabetes.

[0005] The objective of this invention is achieved by the following technical solution:

[0006] An FTO protein degradation targeting chimera, which is a protein degradation targeting chimera with FTO degradation activity, has the general structural formula shown in Formula I or Formula II:

[0007]

[0008] Wherein, E is an E3 ligase ligand with ubiquitination function;

[0009] L is a linking group, which is either an alkylene group or an alkoxy group.

[0010] As a preferred embodiment, the E3 ligase ligand is a VHL (von Hippel-Lindau) protein ligand or a CRBN (Cereblon) protein ligand (i.e., a small molecule ligand of the protease encoded by the CRBN gene).

[0011] Furthermore, the CRBN protein ligand is pomalidomide (structural formula: ) and its derivatives, lenalidomide (structural formula is ) and its derivatives, thalidomide (structural formula is ) and its derivatives, amide compounds or phthalimide compounds;

[0012] The VHL-type protein ligand is a VHL protein ligand (structural formula: ) or methylated VHL protein ligands (structural formula is One or more of the following.

[0013] In a preferred embodiment, the alkylene or alkoxy group is -(CH2). n -、-(CH2) n CO-, -NR1(CH2) n CO-, -NR1(CH2)n-, -NR1CO-, -(CH2CH2O) n -、-(OCH2CH2O) n -、-(OCH2CH2OCH2) n -、-(CH2CH2OCH2)n -、-(CH2CH2OCH2CH2) n - One or more combinations of alkenyl, alkyneyl, cycloalkylene, or heteroaryl hydroxyl groups (combinations include straight-chain or branched combinations), where n is a natural number from 1 to 20, and R1 is H or C. 1-10 alkyl.

[0014] In a preferred embodiment, the FTO protein degradation-targeting chimera specifically includes one or more of the compounds listed in Table 1 below:

[0015] Table 1

[0016]

[0017]

[0018]

[0019]

[0020] Furthermore, the FTO protein degradation targeting chimera is preferably a compound represented by Formula IV.

[0021] As a preferred embodiment, an FTO protein degradation-targeting chimera comprises a chimeric compound that is pharmacologically or physiologically acceptable salt.

[0022] As a preferred embodiment, the above-mentioned FTO protein degradation targeting chimera is used in the preparation of FTO protein degrading agents; these FTO protein degrading agents are used in research and treatment related to obesity, fatty liver, and diabetes.

[0023] As a preferred embodiment, the above-mentioned FTO protein degradation targeting chimera or its pharmacologically or physiologically acceptable salt is used in the preparation of drugs for the prevention and / or treatment of obesity, fatty liver or diabetes. The prevention and / or treatment of obesity, fatty liver or diabetes includes prevention and treatment of reducing fat deposition in the body, anti-obesity, and alleviating fatty liver and diabetes.

[0024] In a preferred embodiment, the drug for the prevention and / or treatment of obesity, fatty liver and diabetes, in addition to containing an FTO protein degradation-targeting chimera or a pharmacologically or physiologically acceptable salt thereof, also contains a pharmaceutically acceptable carrier or excipient.

[0025] The preparation method of the above-mentioned FTO protein degradation targeting chimera specifically includes the following steps:

[0026] 1) The linker donor undergoes a substitution reaction with the E3 ligase ligand to obtain the linker-substituted E3 ligase ligand;

[0027] 2) The E3 ligase ligand with the linker group substituted with the FTO ligand undergoes a condensation reaction to obtain the FTO protein degradation targeting chimera;

[0028] Wherein, the linking group donor is a donor compound in which the linking group (alkylene or alkoxy) is not linked;

[0029] The FTO ligand is one of formula XIII or XIV;

[0030]

[0031] Furthermore, the FTO protein degradation targeting chimera is a compound of formula I, where L is an alkoxy chain of n = 1-20 and E is pomalidomide; this invention provides a method for preparing an FTO protein degradation targeting chimera (as shown in formula XIX), comprising the following steps:

[0032] The E3 ligand compound substituted with tert-butyl propionate polyethylene glycol chain undergoes a condensation reaction with FTO ligand to obtain an FTO protein degrading agent.

[0033] The E3 ligand compound substituted with the tert-butyl propionate polyethylene glycol chain is shown in Formula XV:

[0034]

[0035] The FTO ligands shown are as shown in Formula XIII.

[0036] Furthermore, the preparation method of the tert-butyl propionate polyethylene glycol chain-substituted E3 ligand compound (as shown in Formula XV) includes the following steps:

[0037] (1) A polyethylene glycol compound with the structure shown in Formula XVI is subjected to a substitution reaction with a compound with the structure shown in Formula XVII to obtain a polyethylene glycol chain heterocyclic compound with the structure shown in Formula XVIII.

[0038]

[0039] (2) Compounds having the structure shown in Formula XVIII are subjected to substitution reactions to obtain E3 ligand compounds with the structure shown in Formula XV, which are substituted with polyethylene glycol chains of tert-butyl propionate.

[0040] (3) A compound having the structure shown in Formula XV is subjected to a condensation reaction with an FTO ligand having the structure shown in Formula XIII to obtain an FTO protein degradation targeting chimeric compound having the structure shown in Formula XIX;

[0041]

[0042] This invention successfully prepared a PROTAC molecule targeting FTO, which can effectively target the target protein and reduce the content of FTO protein in cells, thereby reducing intracellular demethylation and inhibiting intracellular lipid deposition. Cellular and in vivo activity evaluations show that the FTO protein degradation targeting chimera provided by this invention has good FTO protein degrading activity. At nanomolar levels, this degrader can rapidly and efficiently induce ubiquitination and degradation of FTO protein in HepG2 cells. Based on the efficacy verification of the FTO protein degradation targeting chimera, the FTO protein degradation targeting chimera provided by this invention, as an FTO protein degrader, can effectively degrade FTO protein, thereby improving obesity and fatty liver. It can be developed as a new anti-obesity, fatty liver relief, and diabetes treatment drug, and has broad application prospects. Attached Figure Description

[0043] Figure 1 The images show the LC-MS, HP-LC, and HNMR spectra of the protein degradation chimera in Example 6 of this invention; where: A is the LC-MS spectra, B is the HP-LC spectra, and C is the HNMR spectra.

[0044] Figure 2 The following are the degradation detection results of the FTO protein degradation chimera in Example 6 of the present invention: A is the detection result of intracellular FTO protein content in HepG2 cells after treatment with different concentrations of degradation agent for 24 h; B is the detection result of FTO protein content after adding 0.33 nM degradation agent for different time periods; C is the detection result of intracellular FTO protein content after washing with degradation agent and culturing for different time periods.

[0045] Figure 3 The above figures show the Western Blot results of FTO protein degradation by the FTO protein degradation chimera according to an embodiment of the present invention; wherein, A is the result of the competition test between the FTO inhibitor meclofenamic acid and the CRBN inhibitor pomalidomide on the degradation of FTO by the degradation agent; B is the result of the test on the degradation mechanism verification test of the proteasome inhibitor borzotimibe.

[0046] Figure 4 The figures show the experimental results of the study on the inhibition of lipid deposition and methylation level of HepG2 cells by a protein degradation chimera according to an embodiment of the present invention; wherein, A is the detection result of the effect of different concentrations of degradation agent on the triglyceride content of HepG2 cells, B is the detection result of Oil Red O staining of HepG2 cells, C is the image analysis result of Oil Red O staining using ImageJ software, and D is the detection result of the N6-methyladenosine RNA modification level of mRNA after the addition of degradation agent.

[0047] Figure 5The image shows the results of detecting the effects of a protein degradation chimera according to an embodiment of the present invention on FTO protein degradation in the liver, mouse body weight, and fat percentage in mice on a high-fat diet; wherein: A is the result of detecting FTO protein degradation in the liver, B is the result of detecting the trend of mouse body weight, C is the result of detecting the trend of mouse fat percentage, and D is a computed tomography scan of the mouse.

[0048] Figure 6 The figure shows the effect of a protein degradation chimera on glucose metabolism in mice according to an embodiment of the present invention; wherein, A is the result of glucose tolerance test in mice; B is the statistical result of the area under the curve of drug-time in figure A; C is the result of insulin tolerance test; and D is the statistical result of the area under the curve of drug-time in figure C.

[0049] Figure 7 The image shows the effect of a protein degradation chimera according to an embodiment of the present invention on lipid deposition in mouse liver; wherein, A is a liver photograph, B is a liver H&E staining result, C is a liver Oil Red O staining result, D is a liver triglyceride content detection result, and E is a liver cholesterol content detection result.

[0050] Figure 8 The graph shows the results of detecting the effect of a protein degradation chimera on serum biochemical indicators in mice according to an embodiment of the present invention; wherein, A is the result of detecting serum triglyceride content, B is the result of detecting serum cholesterol content, C is the result of detecting serum aspartate aminotransferase content, and D is the result of detecting serum alanine aminotransferase content. Detailed Implementation

[0051] 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 a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0052] Furthermore, the technical solutions of the various embodiments can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0053] Currently, there is a lack of effective methods to combat obesity and alleviate fatty liver disease.

[0054] In order to solve the above-mentioned technical problems, this invention proposes an FTO protein degradation targeting chimera, its preparation method and application.

[0055] Example 1

[0056]

[0057] A mixture of compound b (900 mg, 3.46 mmol, 1.00 eq), compound a (731 mg, 4.15 mmol, 1.20 eq), Pd2(dba)3 (316 mg, 346 μmol, 0.10 eq), BINAP (215 mg, 346 μmol, 0.10 eq), and Cs2CO3 (2.26 g, 6.92 mmol, 2.00 eq) was weighed into toluene (20 mL) and washed three times with nitrogen. The mixture was then stirred for 12 hours. The reaction mixture was diluted with 50 mL of ethyl acetate and filtered through a silica gel pad (20.0 g). The filtrate was concentrated under reduced pressure to obtain a residue. The crude product was purified by reversed-phase high-performance liquid chromatography (0.1% FA conditions) and freeze-dried to give compound c (750 mg, 2.11 mmol, 61.01% yield), which was a yellow oil and confirmed by ¹H NMR.

[0058] 1 ¹H NMR: Compound c (400MHz, DMSO-d6)

[0059] δ8.11(s,1H),7.52(d,J=8.3Hz,1H),7.47-7.34(m,3H),6.51(dd,J=0.9,8.3Hz,1H),3.83(s,3H),2.38(s,3H).

[0060] Example 2

[0061]

[0062] LiOH·H2O (3M, 3.64mL) was added to compound c (700mg, 1.97mmol, 1.00eq) dissolved in THF (7mL); the mixture was stirred at 55°C for 8 hours; the reaction mixture was separated, the organic layer was dried on Na2SO4, filtered and concentrated under reduced pressure to give yellow oily compound d (600mg, 1.76mmol, 89.2% yield).

[0063] Example 3

[0064]

[0065] Under nitrogen atmosphere, Pt / V / C (956 mg, 73.2 μmol, 2% purity, 0.05 eq) was added to a solution of compound d (500 mg, 1.47 mmol, 1.00 eq) in THF (10 mL); the mixture was stirred for 6 hours under hydrogen (15 Psi) at 25 °C; the reaction mixture was filtered through a diatomaceous earth mat, and the filtrate was concentrated under vacuum to give compound e (450 mg, 1.45 mmol, 98.6% yield) as a pale yellow solid, which was confirmed by ¹H NMR.

[0066] 1 ¹H NMR: Compound e (400MHz, DMSO-d6)

[0067] δ12.29-11.65(m,1H),7.37(d,J=8.2Hz,1H),7.15(d,J=8.2Hz,1H),6.57( t,J=8.0Hz,1H),5.89(d,J=7.9Hz,1H),5.18(d,J=7.9Hz,1H),2.33(s,3H).

[0068] Example 4

[0069]

[0070] DIEA (2.46 g, 19.0 mmol, 3.31 mL, 1.50 eq) was added to a solution of compound f (3.50 g, 12.66 mmol, 1.00 eq) in dioxane (70.0 mL) to initiate the reaction. The reaction mixture was poured into water (250 mL) and extracted with ethyl acetate (300 mL × 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was purified by reversed-phase high-performance liquid chromatography (0.1% FA conditions) and lyophilized to give compound h (2.70 g, 5.06 mmol, 39.9% yield), which was a yellow oil, as confirmed by ¹H NMR.

[0071] 1 ¹H NMR: Compound h (400MHz, DMSO-d6)

[0072] δ11.33-10.85(m,1H),7.58(dd,J=7.3,8.4Hz,1H),7.15(d,J=8.6Hz,1H),7.04(d,J=7.0Hz,1H),6.60(t,J=5.7Hz,1H),5.05(dd,J= 5.4,12.8Hz,1H),3.64-3.44(m,14H),2.95-2.82(m,1H),2.69-2.55(m,2H),2.39(t,J=6.2Hz,2H),2.06-1.97(m,1H),1.38(s,9H).

[0073] Example 5

[0074]

[0075] HCl / dioxane (2M, 46.0 mL, 21.3 eq) was added to a solution of compound h (2.30 g, 4.31 mmol, 1.00 eq), and the reaction was concentrated under reduced pressure to give compound i (1.70 g, 3.56 mmol, 82.6% yield), which was a dark brown oil, as confirmed by ¹H NMR.

[0076] 1 ¹H NMR: Compound i (400MHz, DMSO-d6)

[0077] δ11.09(s,1H),7.59(dd,J=7.2,8.5Hz,1H),7.16(d,J=8.6Hz,1H),7.05(d,J=7.0Hz,1H),6.61(br s,1H),5.06(dd,J=5.4,12.8Hz,1H),3.64-3.59(m,4H),3.56-3.47(m,10H),2.94-2.83 (m,1H),2.63-2.53(m,2H),2.43(t,J=6.3Hz,2H),2.08-2.00(m,1H),1.78-1.75(m,1H).

[0078] Example 6

[0079]

[0080] DIEA (243 mg, 1.88 mmol, 328 μL, 3.00 eq) and HATU (238.75 mg, 627.5 μmol, 1 eq) were added to 4 mL of DMF containing dissolved compound i (300 mg, 628 μmol, 1.00 eq), and the mixture was reacted for 0.5 h. Then, compound e (195 mg, 628 μmol, 1.00 eq) was added, and the resulting reaction mixture was stirred for 1 h. The reaction mixture was purified by reversed-phase high-performance liquid chromatography (0.1% FA conditions) and lyophilized to give compound f (120.04 mg, 149 μmol, 23.8% yield, 96.2% purity) as a yellow solid, which was further purified by LC-MS (…). Figure 1 A) HPLC Figure 1 B) HNMR ( Figure 1 C) Confirm.

[0081] 1 H NMR (400MHz, DMSO-d6)

[0082] δ11.10(s,1H),10.58(br d,J=2.1Hz,1H),9.11-8.68(m,1H),7.56(t,J=7.8Hz,1H),7.48(d,J=8.3Hz,1H),7.43(d ,J=8.0Hz,1H),7.31(d,J=8.4Hz,1H),7.21-7.09(m,2H),7.03(d,J=7.0Hz,1H),6.59(br s,1H),5.91(d,J=8.3Hz,1H),5.05(dd,J=5.3,12.8Hz,1H),3.68(t,J=6.1Hz,2H),3.61-3.57(m,2H),3.52(d,J=2.4Hz,8H),3.44(br d,J=4.8Hz,3H),2.94-2.81(m,1H),2.60(br s,1H),2.54(br t,J=6.3Hz,3H),2.36(s,3H),2.06-1.97(m,1H).

[0083] The following biological tests were performed on the FTO protein degradation-targeting chimera (compound f) prepared in Example 6 above:

[0084] Example 1: Degrading agent induces FTO protein degradation in HepG2 cells

[0085] Cells were seeded in 24-well plates. After 24 hours, different concentration gradients of FTO degrading agent (compound f) were added (0 nM, 0.1 nM, 0.33 nM, 1 nM, 3.33 nM, 10 nM) and cultured for 24 hours. Cells were then collected, and protein was collected by adding cell lysis buffer containing protease inhibitors for Western blotting. Alternatively, the same concentration (0.33 nM) of FTO degrading agent was added, and cells were cultured for different times (0 h, 2 h, 4 h, 8 h, 12 h, 24 h). Cells were then collected, and protein was collected by adding lysis buffer for Western blotting. Additionally, cells were cultured for different times after adding the same concentration of FTO degrading agent and changing the culture medium, and then collected for Western blotting. The Western blotting assays were used to detect the FTO protein degradation ability of HepG2 human hepatocellular carcinoma cells involving the synthesized degrading agent molecules. Degrading agents with polyethylene glycol (PEG) structures as linkers were also tested. The degradation results of the FTO protein degrading chimera and their concentration- and time-dependent results are shown below. Figure 2 As shown, Figure 2 A represents the intracellular FTO protein content of HepG2 cells after treatment with different concentrations of degradation agent for 24 hours; Figure 2 B represents the FTO protein content after adding 0.33 nM degrading agent for different time periods; Figure 2 C represents the intracellular FTO protein content after culturing for different time periods following washing with the degradation agent. The results indicate that significant degradation occurred with the addition of 0.33 to 1 nM of the degradation agent, with significant degradation occurring within 2 hours after the addition of 0.33 nM, and the degradation effect persisting up to 36 hours after washing.

[0086] Example 2: The degrading agent degrades FTO protein via the ubiquitin-proteasome pathway.

[0087] This invention uses the FTO inhibitor meclofenamic acid, the CRBN inhibitor pomalidomide, and the proteasome inhibitor borzotimibe to incubate cells for 2 hours before adding an FTO degrading agent. Finally, the changes in FTO protein bands are detected by Western blotting. The results are as follows: Figure 3 As shown, Figure 3 A is a competitive test of the degradation of FTO by the FTO inhibitor meclofenamic acid and the CRBN inhibitor pomalidomide; Figure 3 B represents the assay used to verify the degradation mechanism of the proteasome inhibitor borzotimibe. Figure 3 As shown in Figure A, the degradation of FTO protein by the degrading agents was competitively inhibited after the addition of meclofenamic acid and pomalidomide. From... Figure 3 As shown in B, the degradation effect of the degrading agent on FTO protein can be significantly blocked by borzotimibe, indicating that the degradation of FTO protein induced by the degrading agent is achieved through the ubiquitin-proteasome pathway.

[0088] Example 3: The degrading agent reduced OA / PA-induced lipid deposition and m6A levels in HepG2 cells.

[0089] (1) The degrading agent reduces OA / PA-induced lipid deposition in HepG2 cells.

[0090] HepG2 cells were seeded in 24-well plates and cultured in growth medium for 24 h. Then, 0.33 nM degradation agent and fresh OA / PA medium were added. A control group without the agent was used. After 24 h of culture, the medium in each well was aspirated, and each well was washed with PBS. Then, 200 μL of lysis buffer was added to each well, and the cells were lysed on a shaker at 4 °C for 30 min. Adherent cells were scraped off using a pipette tip, and the lysis buffer was transferred to a 1.5 mL centrifuge tube. The cells were centrifuged at 12,000 rpm for 10 min at room temperature. A portion of the supernatant was used for protein content detection. The remaining portion was heated in a 70 °C incubator for 10 min, then centrifuged at 12,000 rpm for 10 min. 64 μL of sample and 200 μL of working solution were added to each well of a 96-well microplate, and the plates were incubated at 37 °C for 10 min. The absorbance at 510 nm was measured using a microplate reader. Triglyceride content was determined according to the following formula. In addition, the cells cultured according to the above protocol were stained with Oil Red O, and the number and size of lipid droplets were analyzed using ImageJ software. The results are as follows: Figure 4 As shown, Figure 4 A represents the effect of different concentrations of the degrading agent on the triglyceride content of HepG2 cells. Figure 4 B shows the Oil Red O staining results for HepG2 cells. Figure 4 C represents the results of ImageJ software analysis using Oil Red O staining. After the addition of the degrading agent, the intracellular triglyceride content decreased, indicating that the degrading agent can reduce lipid accumulation in HepG2 cells, increase the proportion of small lipid droplets, and reduce the proportion of large lipid droplets.

[0091] formula:

[0092] (2) The degrading agent reduces the m6A level in HepG2 cells.

[0093] HepG2 cells were seeded in 24-well plates and cultured in growth medium for 24 h. Then, 0.33 nM degradation agent and fresh OA / PA medium were added, with the untreated group serving as a control. After 24 h of culture, the medium in each well was aspirated, and each well was washed with PBS. RNA was then extracted using the Trizol method and diluted to 20 ng / µl with RNA-free water. The membrane was heated at 95°C for 3 minutes to disrupt secondary structures, followed immediately by cooling on ice to prevent secondary structure recovery. 2 µl of mRNA was directly added to a nitrocellulose membrane and cross-linked using a UV lamp. The membrane was then washed with 10 mL of PBST in a clean washing dish at room temperature for 5 minutes to remove unbound mRNA. Subsequent experiments were performed according to the dot blot assay method. Results are as follows. Figure 4 As shown in Figure D, overexpression of FTO decreased the N6-methyladenosine RNA modification level of mRNA, while the addition of the degrading agent increased the N6-methyladenosine RNA modification level of mRNA. This indicates that the degrading agent can increase the N6-methyladenosine RNA modification level in HepG2 cells by degrading FTO.

[0094] Example 4: Effects of the degrading agent on high-fat diet-induced fatty liver in mice

[0095] This invention establishes a mouse model of hepatic steatosis using a high-fat diet to evaluate the in vivo pharmacodynamic effects of FTO degrading agents.

[0096] (1) Mice were intraperitoneally injected with degradation agents (0.04 mg / kg and 0.2 mg / kg) and a blank solvent, respectively, every four days. After 14 weeks of feeding, the mice were anesthetized and sacrificed. Liver tissue was collected, and proteins were extracted for Western blotting. The results are as follows: Figure 5 As shown in Figure A, FTO protein degradation was observed in the liver. Furthermore, we compared the effects of multiple administrations on fat deposition in mice by weighing them weekly during the feeding period, measuring fat percentage every two weeks, and performing computed tomography scans before sacrifice. Results are as follows: Figure 5 As shown, Figure 5 B represents the trend of mouse body weight change, and convex 5C represents the trend of mouse body fat percentage change. Figure 5 D is a computed tomography (CT) image of a mouse. From Figure 5 As can be seen, the body weight and fat percentage of mice in the high-fat model group increased significantly, and both ratios decreased after drug administration, while the fat percentage also showed a decreasing trend. These results indicate that the degrading agent still has a significant FTO protein degradation effect on the liver in in vivo experiments and can effectively inhibit the weight gain and fat percentage increase induced by a high-fat diet.

[0097] (2) The degrading agent can improve glucose tolerance and insulin tolerance in mice.

[0098] In this invention, glucose tolerance and insulin tolerance tests were conducted at weeks 12 and 13 after a high-fat diet. Mice were intraperitoneally injected with glucose solution at a dose of 2 g / kg and intraperitoneally injected with insulin at a dose of 0.5 U / kg. Blood glucose levels were measured by tail sampling at 0, 15, 30, 60, 90, and 120 minutes. The results are as follows: Figure 6 As shown, Figure 6 In the diagram, AB represents mouse glucose tolerance, and CD represents insulin tolerance. From... Figure 6 The results show that a high-fat diet leads to decreased glucose tolerance and insulin tolerance in mice, while the group treated with the degradation agent was able to improve the decreased glucose and insulin tolerance caused by a high-fat diet.

[0099] (3) Effects of degradation agents on lipid deposition in mouse liver

[0100] In this invention, mice were anesthetized and sacrificed at week 14 after being fed a high-fat diet. The liver was separated, photographed, and a portion was placed in tissue fixative for H&E and Oil Red O staining. The remaining portion was placed in a 1.5 ml centrifuge tube, 1 ml of lysis buffer and a steel ball were added, and the tissue was lysed using a tissue homogenizer to extract triglycerides and proteins. After extraction, the tissue was centrifuged multiple times at 12000 rpm, and the supernatant was collected. 2.5 μL and 5 μL were respectively used to detect triglycerides and cholesterol in a 96-well microplate, and 10 μL was used for protein content detection from the remaining portion. Results are as follows: Figure 7 As shown, Figure 7 Image A shows a photograph of the liver and its weight; image B shows liver H&E staining; image C shows liver Oil Red O staining; image D shows liver triglyceride content; and image E shows liver cholesterol content. From... Figure 7 The results showed that the livers of mice in the high-fat diet model group were larger and paler. H&E staining revealed unstained lipids in the livers of the high-fat diet model group, while the amount of unstained lipids in the drug-treated group was reduced. Oil Red O staining further confirmed that, compared with the control group, neutral lipids accumulated more extensively in the livers of the model group, indicating that the drug-treated group could reduce lipid accumulation in the liver to some extent. Furthermore, triglyceride and cholesterol levels in the livers of the model group were significantly elevated, while the injection of the degradation agent reversed this phenomenon in a dose-dependent manner, reducing triglyceride levels to control levels. These results indicate that the degradation agent can effectively alleviate hepatic lipid accumulation caused by a high-fat diet and mitigate the occurrence of fatty liver disease.

[0101] (4) Effects of degradation agents on blood biochemical indicators

[0102] In this invention, mice were anesthetized at week 14 after being fed a high-fat diet. Blood was collected via orbital sampling, and serum was collected by centrifugation at 3000 rpm for the detection of physiological and biochemical indicators. The results are as follows: Figure 8 As shown, Figure 8 In the figure, A represents serum triglyceride level, B represents serum cholesterol level, C represents serum aspartate aminotransferase (AST) level, and D represents serum alanine aminotransferase (ALT) level. As can be seen from the figure, the serum triglyceride, cholesterol, AST, and ALT levels in the high-fat diet model group were all significantly increased, while the injected degradation agent reduced the increase in these indicators in a dose-dependent manner. These results indicate that the degradation agent can effectively reduce the increased serum lipid transport induced by a high-fat diet and reduce liver damage.

[0103] This invention successfully prepared a PROTAC molecule targeting FTO, which can effectively target the target protein and reduce the content of FTO protein in cells, thereby reducing intracellular demethylation and inhibiting intracellular lipid deposition. Cellular and in vivo activity evaluations show that the FTO protein degradation targeting chimera provided by this invention has good FTO protein degrading activity. At nanomolar levels, this degrader can rapidly and efficiently induce ubiquitination and degradation of FTO protein in HepG2 cells. Based on the efficacy verification of the FTO protein degradation targeting chimera, the FTO protein degradation targeting chimera provided by this invention, as an FTO protein degrader, can effectively degrade FTO protein, thereby improving obesity and fatty liver. It can be developed as a new anti-obesity and fatty liver relief drug with broad application prospects.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An FTO protein degradation-targeting chimera, characterized in that: Its general structural formula is shown in equation f: 。 2. The FTO protein degradation targeting chimera according to claim 1, characterized in that: It contains a chimeric compound that is pharmacologically or physiologically acceptable salt.

3. The use of the FTO protein degradation targeting chimera according to any one of claims 1-2 in the preparation of FTO protein degrading agents.

4. The use of the FTO protein degradation targeting chimera according to any one of claims 1-2 in the preparation of drugs for the prevention and / or treatment of obesity, fatty liver or diabetes.

5. The use of the FTO protein degradation-targeting chimera according to claim 4 in the preparation of drugs for the prevention and / or treatment of obesity, fatty liver and diabetes, characterized in that: It also contains pharmaceutically acceptable carriers or excipients.

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