Preparation method of Drp1 protein degradation targeting chimera compound

Abstract

The invention relates to the technical field of protein targeted degradation, in particular to a preparation method of a Drp1 protein degradation targeted chimera compound with a structure of # imgabs0 #, and provides and synthesizes a protein degradation targeted chimera for targeted degradation of Drp1 for recovering IHD and DCM myocardial cell mitochondrial division dysfunction for the first time. The invention provides a complete synthesis route of the MLTAC, and the degradation efficiency of the Drp1 protein is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein targeted degradation, and in particular to a method for preparing a Drp1 protein degradation targeted chimera compound. Background Art

[0002] Ischemic heart disease (IHD) and diabetic cardiomyopathy (DCM) can both lead to irreversible myocardial lesions and ultimately develop into heart failure (HF). During the course of IHD and DCM, the mitochondrial fusion / fission kinetics is imbalanced, and excessive fission of a large number of mitochondria causes mitochondrial fragmentation, leading to pathological fatty acid oxidation, necrotic apoptosis of cardiomyocytes and an increase in the level of myocardial fibrosis. Abnormal mitochondrial function has a great impact on IHD and DCM. Dynamin-related protein 1 (Drp1) is a key protein regulating mitochondrial fission. Studies have found that reducing the level of Drp1 is beneficial to maintaining the stability of mitochondrial fusion / fission kinetics and improving mitochondrial function. However, current drugs are mostly small molecule drugs or polypeptides that reduce the activity of Drp1. After being metabolized and inactivated, the inhibitory effect on Drp1 will disappear, and a long-term therapeutic effect cannot be achieved. Protein degradation targeted chimera is a newly emerging small molecule drug technology used to selectively target and degrade specific proteins in order to achieve long-term clearance of Drp1 and reduce the continuous damage of cardiomyocytes caused by excessive fission of cardiomyocyte mitochondria in IHD and DCM. However, there is currently no protein degradation targeted chimera for Drp1 protein.

[0003] Currently, drugs targeting Drp1 protein are small molecule drugs or polypeptide drugs. The inhibition of Drp1 protein by small molecule Drp1 protein inhibitors represented by Mdivi-1 is not long-lasting, and the inhibitory effect on Drp1 protein will gradually disappear as Mdivi-1 is metabolized. The high development and manufacturing costs of polypeptide drugs and the great influence of storage and transportation on the environment have greatly restricted the research and application of such drugs. On the other hand, polymers and polypeptide mitochondrial directed clearance organelle targeted chimeras developed based on protein degradation targeted chimera technology have the problem of non-selectively clearing mitochondria, and this problem cannot be solved at present. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing a Drp1 protein degradation-targeting chimeric compound, aiming to use a polyethylene glycol fragment as a linker chain, Mdivi-1 as the Drp1 recognition and binding end, and guanosine monophosphate as the induced protein clearance end. The MLTAC recognizes and binds to the Drp1 protein in the cytoplasm. Mdivi-1 first inhibits the Drp1 protein and tags the Drp1 protein with guanosine monophosphate, and then is polyubiquitinated via the K63 protein to form an autophagosome, and the Drp1 protein is cleared through the ubiquitin-proteasome system (UPS). The effect of long-term clearance of the Drp1 protein and reduction of excessive mitochondrial fission in IHD and DCM cardiomyocytes is achieved.

[0005] To achieve the above object, in the first aspect, the present invention provides a Drp1 protein degradation-targeting chimeric compound, the structure of which is shown as follows:

[0006]

[0007] In the second aspect, the present invention provides a method for preparing a Drp1 protein degradation-targeting chimeric compound, including: adding N-tert-butoxycarbonyl-diglycolic acid to tetrahydrofuran, stirring until completely dissolved, then adding succinic anhydride, fully stirring and adding triethylamine to completely react N-tert-butoxycarbonyl-pentaglycolic acid, adjusting the pH with hydrochloric acid, and separating and purifying by water extraction, washing and drying to obtain a first intermediate;

[0008] Dissolving the first intermediate in N,N-dimethylformamide, stirring until completely dissolved, then adding N,N'-dicyclohexylcarbodiimide, stirring and adding thalidomide-4-hydroxy and 4-dimethylaminopyridine, monitoring by thin-layer chromatography until the reaction is completed, extracting with water, washing and drying, and obtaining a second intermediate after separation;

[0009] Dissolving the second intermediate in dichloromethane, adding an excess of trifluoroacetic acid and stirring until completely reacted, and obtaining a de-Boc protected product, a third intermediate, after extraction, washing and drying;

[0010] Dissolving the third intermediate in THF, adding dicyclohexylcarbodiimide, stirring and adding Mdivi-1 and adding 4-dimethylaminopyridine, filtering and vacuum drying after extraction, washing and drying after the reaction is completed, and obtaining the Drp1 protein degradation-targeting chimeric compound after separation and purification.

[0011] Wherein, the hydrochloric acid adjusts the pH of the solution to be less than 4.

[0012] In the third aspect, the present invention provides an application of the Drp1 protein degradation-targeting chimeric compound in restoring the abnormal mitochondrial fission function of IHD cardiomyocytes.

[0013] Fourthly, the present invention provides an application of a Drp1 protein degradation-targeting chimeric compound in restoring abnormal mitochondrial fission function in DCM cardiomyocytes.

[0014] Fifthly, the present invention provides a pharmaceutical composition comprising the Drp1 protein degradation-targeting chimeric compound described above.

[0015] The preparation method of a Drp1 protein degradation-targeting chimeric compound of the present invention uses a polyethylene glycol fragment as a linker chain, Mdivi-1 as the Drp1 recognition and binding end, and guanosine monophosphate as the induced protein clearance end. MLTAC recognizes and binds to the Drp1 protein in the cytoplasm. Mdivi-1 first inhibits the Drp1 protein and tags the Drp1 protein with guanosine monophosphate, and then forms an autophagosome through K63 protein and polyubiquitination, and the Drp1 protein is cleared through the ubiquitin-proteasome system (UPS). The effect of long-term clearance of the Drp1 protein and reduction of excessive mitochondrial fission in IHD and DCM cardiomyocytes is achieved, breaking through the problem of non-selective degradation existing in directly promoting mitophagy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 is a flowchart of the preparation method of a Drp1 protein degradation-targeting chimeric compound of the present invention.

[0018] Figure 2 is the nuclear magnetic resonance spectrum of the first intermediate in the first embodiment of the present invention.

[0019] Figure 3 is the nuclear magnetic resonance spectrum of the first intermediate in the second embodiment of the present invention.

[0020] Figure 4 is the nuclear magnetic resonance spectrum of the second intermediate in the first embodiment of the present invention.

[0021] Figure 5 is the nuclear magnetic resonance spectrum of the second intermediate in the second embodiment of the present invention.

[0022] Figure 6 is the nuclear magnetic resonance spectrum of Mdivi-1 of the present invention.

[0023] Figure 7 is the nuclear magnetic resonance spectrum of the finished product of the first embodiment of the present invention.

[0024] Figure 8 is the nuclear magnetic resonance spectrum of the finished product of the second embodiment of the present invention.

[0025] Figure 9 is the effect diagram of MLTAC of the present invention on the expression and accumulation of Drp1 protein.

[0026] Figure 10 is the effect diagram of MLTAC of the present invention on mitochondrial fission and morphology. Detailed implementation manners

[0027] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0028] First embodiment

[0029] Please refer to Figure 1 , the present invention provides a preparation method of a Drp1 protein degradation-targeted chimeric compound, including:

[0030] S101 Add 500 mg of N-tert-butoxycarbonyl-diglycolic acid to 50 mL of tetrahydrofuran, stir until completely dissolved, then add 3 equivalents of excessive succinic anhydride, stir well and add triethylamine. N-tert-butoxycarbonyl-pentaglycolic acid reacts completely, and the pH is adjusted to less than 4 with hydrochloric acid. After water extraction, washing and drying, the first intermediate is separated and purified;

[0031] S102 Dissolve 30 mg of intermediate 1 in 50 mL of N,N-dimethylformamide, stir until completely dissolved, then add 1.5 equivalents of N,N'-dicyclohexylcarbodiimide (DCC), stir for 10 min and then add 1 equivalent of thalidomide-4-hydroxy and 1.5 equivalents of 4-dimethylaminopyridine. Monitor the reaction by thin-layer chromatography until it ends. Add water for extraction, washing and drying, and obtain the second intermediate after separation (calculated based on the first intermediate in terms of equivalent).

[0032] S103 Dissolve the second intermediate in dichloromethane, add excessive trifluoroacetic acid and stir until the reaction is complete. After extraction, washing and drying, the de-Boc protected product, the third intermediate, is obtained;

[0033] Dissolve 20 mg of the third intermediate in 40 mL of THF, add 1.5 equivalents of dicyclohexylcarbodiimide, stir for 20 minutes, then add 1 equivalent of Mdivi-1 and 1.5 equivalents of 4-dimethylaminopyridine. After the reaction is completed, filter and vacuum dry after extraction, washing, and drying. After separation and purification, MLTAC is obtained (the equivalents are all calculated based on intermediate 3). Here, MLTAC is the English abbreviation of the Drp1 protein degradation-targeting chimeric compound.

[0034] The synthetic route is shown in the following figure, and "n" in the synthetic route can be any integer value from 2 to 5:

[0035]

[0036] Second Embodiment

[0037] The present invention provides a method for preparing a Drp1 protein degradation-targeting chimeric compound, including:

[0038] S201 Add 550 mg of N-Boc-diglycolic acid to 56 mL of tetrahydrofuran, stir until completely dissolved, then add 3 equivalents of excessive succinic anhydride, stir well and add triethylamine. After N-Boc-pentaglycolic acid completely reacts, adjust the pH to less than 4 with hydrochloric acid. After water extraction, washing, and drying, separate and purify to obtain the first intermediate;

[0039] S202 Dissolve 35 mg of intermediate 1 in 52 mL of N,N-dimethylformamide, stir until completely dissolved, then add 2 equivalents of N,N'-dicyclohexylcarbodiimide (DCC), stir for 10 min, then add 2 equivalents of thalidomide-4-hydroxy and 2 equivalents of 4-dimethylaminopyridine, and monitor the reaction by thin-layer chromatography until it ends. Add water for extraction, washing, and drying, and obtain the second intermediate after separation (the equivalents are all calculated based on the first intermediate).

[0040] S203 Dissolve the second intermediate in dichloromethane, add excessive trifluoroacetic acid and stir until completely reacted, and obtain the de-Boc protected product, the third intermediate, after extraction, washing, and drying;

[0041] S204 Dissolve 30 mg of the third intermediate in 45 mL of THF, add 2 equivalents of dicyclohexylcarbodiimide, stir for 20 minutes, then add 1.5 equivalents of Mdivi-1 and 1.5 equivalents of 4-dimethylaminopyridine. After the reaction is completed, filter and vacuum dry after extraction, washing, and drying. After separation and purification, MLTAC is obtained (the equivalents are all calculated based on intermediate 3).

[0042] According to 1 the HNMR results, compare Figure 2 andFigure 3 As a result, Figure 3 a distinct carboxyl-terminal methylene peak appeared at 2.39 ppm, indicating the successful synthesis of the first intermediate. Subsequently, we performed 1 1H NMR detection on the second intermediates of the first and second embodiments, and the results are as Figure 4 , Figure 5 shown. Distinct triplets appeared at 7.32 ppm, 7.51 ppm, and 7.92 ppm, which are characteristic peaks of the benzene ring region, indicating the successful grafting of the first intermediate and guanylic acid. Combining with the 1 1H NMR spectrum of Mdivi-1 ( Figure 6 ) and the 1 1H NMR spectra of the final products of the first and second embodiments ( Figure 7 , Figure 8 ), in Figure 7 , Figure 8 , the peak of Mdivi-1 at 0.75 ppm disappeared significantly. Compared with Figure 4 , Figure 5 , Figure 7 , Figure 8 a peak at 0.91 ppm increased. At the same time, in the benzene ring region of Figure 7 , Figure 8 , namely at 7.24 ppm, 7.52 ppm, and 7.88 ppm, the peak areas increased significantly, indicating the successful grafting of the second intermediate and Mdivi-1 and the successful preparation of MLTAC, obtaining the finished product of the first embodiment (MLTAC1) and the finished product of the second embodiment (MLTAC2).

[0043] Subsequently, the Drp1 protein clearance effect of MLTAC was evaluated. The Western Blot results showed ( Figure 9 A - B) that Mdivi-1 could reduce the level of Drp1 and was significantly different from the control group (P < 0.05). In contrast, both MLTAC1 and MLTAC2 could significantly reduce the expression level of Drp1 protein in cells (P < 0.0001), and the effect was much stronger than that of Mdivi-1. The Elisa detection results were consistent with those of Western Blot ( Figure 9 C). It was shown that both MLTAC1 and MLTAC2 could effectively clear Drp1 protein and the effect was much stronger than that of the Drp1 protein inhibitor Mdivi-1.

[0044] Transmission electron microscope photos of mitochondrial structures also showed that a decrease in the level of Drp1 protein was beneficial to improving the problem of excessive mitochondrial fragmentation ( Figure 10) Compared with the severe mitochondrial size heterogeneity and fragmentation in the hypoxic control group, the mitochondrial morphology in the Mdivi-1 group showed obvious improvement, but there were still some fragmented mitochondria. The mitochondrial morphology in the MLTAC1 and MLTAC2 groups showed obvious improvement, with the mitochondrial morphology being more complete and almost no mitochondrial fragments appeared, indicating that the problem of excessive mitochondrial fragmentation was significantly improved and the effect was better than directly using an inhibitor against Drp1

[0045] Second Embodiment

[0046] The present invention provides a pharmaceutical composition comprising the Drp1 proteolysis-targeting chimera compound described above.

[0047] Drp1 (Dynamin-related protein 1) is a protein crucial for the fission of mitochondria and peroxisomes during cell division and plays an important role in regulating various biological processes such as apoptosis, metabolism, and stress response. However, under certain disease states, such as neurodegenerative diseases and cardiovascular diseases, the dysfunction of Drp1 may lead to pathological changes.

[0048] The Drp1 proteolysis-targeting chimera compound in this invention aims to regulate or inhibit the abnormal activity of Drp1 through a novel method, thus providing a new approach for the treatment of related diseases. This chimera compound can specifically bind to the Drp1 protein and promote the selective degradation of the Drp1 protein using the ubiquitin-proteasome system or lysosomal pathway within the cell. Therefore, it not only improves the targeting of treatment but also may reduce the common side effect problems in traditional drug therapies.

[0049] In addition, the pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients, or adjuvants according to actual needs to facilitate the diversification of dosage forms, such as tablets, capsules, injections, etc., to adapt to different administration routes and clinical application requirements. By optimizing the formulation composition, the stability, bioavailability, and patient compliance of the drug can also be improved.

[0050] What is disclosed above is only a preferred embodiment of the present invention. Of course, it cannot be used to limit the scope of the rights of the present invention. Those of ordinary skill in the art can understand the entire or partial processes of implementing the above embodiments, and the equivalent changes made according to the claims of the present invention still fall within the scope covered by the invention.

Claims

1. A Drp1 protein degradation-targeting chimeric compound, characterized in that the structure is as follows:

2. A method for preparing a Drp1 protein degradation-targeting chimeric compound for preparing the Drp1 protein degradation-targeting chimeric compound according to claim 1, characterized in that including: Add N-tert-butoxycarbonyl-diglycolic acid to tetrahydrofuran, stir until completely dissolved, then add succinic anhydride, stir well and add triethylamine to completely react N-tert-butoxycarbonyl-pentaglycolic acid. After adjusting the pH with hydrochloric acid, it is extracted with water, washed and dried, and then separated and purified to obtain the first intermediate; Dissolve the first intermediate in N,N-dimethylformamide, stir until completely dissolved, then add N,N'-dicyclohexylcarbodiimide, stir and then add thalidomide-4-hydroxy and 4-dimethylaminopyridine. Monitor the reaction by thin-layer chromatography until it ends, extract with water, wash and dry, and obtain the second intermediate after separation; Dissolve the second intermediate in dichloromethane, add an excess of trifluoroacetic acid and stir until completely reacted. After extraction, washing and drying, obtain the de-Boc protected product, the third intermediate; Dissolve the third intermediate in THF, add dicyclohexylcarbodiimide, stir and then add Mdivi-1 and add 4-dimethylaminopyridine. After the reaction is completed, it is extracted, washed, dried, filtered and vacuum dried, and the Drp1 protein degradation-targeting chimeric compound is obtained after separation and purification.

3. The method for preparing a Drp1 protein degradation-targeting chimeric compound according to claim 2, characterized in that the hydrochloric acid adjusts the pH to make the solution pH less than 4.

4. The application of the Drp1 protein degradation-targeting chimeric compound according to claim 1 in restoring the abnormal mitochondrial fission function of IHD cardiomyocytes.

5. The application of the Drp1 protein degradation-targeting chimeric compound according to claim 1 in restoring the abnormal mitochondrial fission function of DCM cardiomyocytes.

6. A pharmaceutical composition, characterized in that, It includes the Drp1 protein degradation-targeting chimeric compound according to claim 1.

Images