A specific palmitoyl transferase inhibitor and uses thereof

Abstract

The application discloses a specific palmitoyl transferase inhibitor and application thereof, and an active ingredient of the palmitoyl transferase inhibitor is 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol or a salt thereof. Current palmitoylation inhibitors, 2-bromohexadecanoic acid (2-BP), cerulenin and tunicamycin can all inhibit protein palmitoylation in cells, but all are not selective. And the existing palmitoylation inhibitors are similar to endogenous fatty acids, and inevitably affect lipid metabolism in the body, which limits the clinical application. The application discloses, for the first time, the application of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol as a specific palmitoyl transferase inhibitor, and the palmitoyl transferase inhibitor is better in specificity and reversible in inhibition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a specific palmitoyl transferase inhibitor and application thereof. BACKGROUND

[0002] Palmitoyl transferase modifies other proteins by linking lipid or fatty acid chains of different lengths together. This modification, known as palmitoylation, can change many properties of the target protein, such as its structure, function and location in the cell. Current palmitoylation inhibitors, 2-bromohexadecanoic acid, cerulenin and tunicamycin, can all inhibit protein palmitoylation in cells, but are not selective; and the existing palmitoylation inhibitors are similar to endogenous fatty acids, and inevitably affect lipid metabolism in the body, limiting their clinical application. Therefore, it is necessary to find a palmitoyl transferase inhibitor with better specificity and reversible inhibition. SUMMARY

[0003] The present application discloses a specific palmitoyl transferase inhibitor and application thereof, and particularly relates to a palmitoyl transferase DHHC5 inhibitor.

[0004] The present application adopts the following technical scheme:

[0005] The active ingredient of the specific palmitoyl transferase inhibitor is 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol or a salt thereof.

[0006] The present application discloses the application of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol as a palmitoyl transferase inhibitor, preferably as a specific palmitoyl transferase inhibitor.

[0007] The present application discloses the application of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol in the preparation of a palmitoyl transferase inhibitor; preferably in the preparation of a specific palmitoyl transferase inhibitor.

[0008] In the present application, the active ingredient of the palmitoyl transferase inhibitor is 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol or a salt thereof, and can also include pharmaceutical excipients, the selection of specific pharmaceutical excipients being a conventional technology, and the palmitoyl transferase inhibitor can also not include pharmaceutical excipients; the dosage form of the palmitoyl transferase inhibitor can be a conventional dosage form such as solution, suppository, tablet, powder and ointment.

[0009] In the present application, the pharmaceutically acceptable salt is a salt formed by metal ions, pharmaceutically acceptable amines, ammonium ions or choline, wherein the metal includes sodium, potassium, calcium and other ions; the pharmaceutically acceptable amines include ethylenediamine, tromethamine and the like.

[0010] The auxiliary materials of the present application can be changed according to the dosage form, administration form, etc. The auxiliary materials include excipients, binders, disintegrants, lubricants, flavoring agents, odorants, colorants, and sweeteners, etc. The administration route of the drug can be oral, sublingual, transdermal, intramuscular or subcutaneous, skin mucosa, or intravenous, etc. The drug can be in the form of a conventional pharmaceutical preparation such as a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, cream, ointment, suppository, or patch, etc.

[0011] The current palmitoylation inhibitors, 2-bromohexadecanoic acid (2-BP), cerulenin and tunicamycin, can all inhibit the intracellular protein palmitoylation, but all are not selective. The structure of 2-BP is similar to that of endogenous fatty acids, which inevitably affects the lipid metabolism in vivo. Cerulenin can also inhibit fatty acid synthesis, thereby affecting the lipid metabolism of cells, and the mechanism of action may be to act on the active pocket that determines the length of the lipid chain, and to irreversibly inhibit the activity of palmitoyl transferase. The structure of tunicamycin is similar to that of 2-BP and cerulenin, and it can also inhibit the lipid metabolism, and the inhibition mechanism may be the same as 2-BP. When the active pocket of 2-BP is selected for drug design, the lipid metabolism is inevitably affected, which limits its clinical application.

[0012] The application discloses a specific palmitoyl transferase inhibitor and application thereof, in particular to a palmitoyl transferase DHHC5 inhibitor, and the active ingredient is 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol or a salt thereof. 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol has a powerful blocking dopamine receptor effect, in addition to blocking D1 and D2 receptors, it can also inhibit D2 autoreceptors, so that the inter-synaptic dopamine concentration is increased; and has no sedative effect. The application discloses, for the first time, the application of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol as a specific palmitoyl transferase inhibitor, and the palmitoyl transferase inhibitor has better specificity and reversible inhibition effect. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a chemical structural formula of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol.

[0014] Figure 2 It is a 2D schematic diagram of molecular docking of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol and Human DHHC5 protein.

[0015] Figure 3 Molecular docking 3D diagram of 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1-piperazine ethanol and Human DHHC5 protein.

[0016] Figure 4 Diagram of the effect of 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1-piperazine ethanol on palmitoylation transferase DHHC5, the expression of DHHC5 was detected by Western blotting, and β-actin was used as an internal reference.

[0017] Figure 5 Diagram of the experimental results of the comparison with the existing palmitoylation inhibitor 2-bromohexadecanoic acid and the clinically used stroke drug edaravone dexkanol.

[0018] Figure 6 Diagram of the change of palmitoylation transferase DHHC5 at different time points of ischemic stroke.

[0019] Figure 7 Effect of 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1-piperazine ethanol on the volume of cerebral infarction in the acute stage of cerebral ischemia. DETAILED DESCRIPTION

[0020] The application discloses a palmitoyl transferase inhibitor and application thereof, in particular to a palmitoyl transferase DHHC5 inhibitor, and an active ingredient of the palmitoyl transferase DHHC5 inhibitor is 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1-piperazine ethanol or a salt thereof. Figure 1 .

[0021] Western blotting method.

[0022] 1. Preparation of protein sample: the mouse cerebral cortex tissue is added with RIPA lysis solution (strong) (main components are 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100), the cells are ultrasonically broken, centrifuged at 12000 g at 4 DEG C for 15 min, and the supernatant is transferred to a new centrifuge tube.

[0023] 2. Protein concentration determination: after the protein is placed in a 37 DEG C incubator for 30 min, the absorbance is detected by using an enzyme marker at 570 nm.

[0024] 3. Electrophoresis: according to the molecular weight of the protein run, prepare the corresponding concentration of gel, add the prepared brain tissue or human astrocyte sample. Run the gel at 90V, after the gel is run, transfer the membrane, transfer the membrane at 200 mA for 90 min.

[0025] 4. Immunoreaction: After the transfer is completed, move the PVDF membrane to a dish with blocking solution (5% skim milk), and block it on a shaker at room temperature for 1 h. Dilute the primary antibody with 1% BSA and add it to the hybridization bag with the membrane, remove the air bubbles, and incubate at room temperature for 3 h or overnight on a shaker at 4°C. Wash the membrane with TBST for 3 times, 10 min each time, add the fluorescent secondary antibody, and incubate at room temperature for 1 h. Wash the membrane with TBST for 3 times, 10 min each time. Develop with the Odyssey infrared fluorescence scanning imaging system.

[0026] Example One

[0027] 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol as a specific DHHC5 palmitoyl transferase inhibitor.

[0028] The proportion of fragments with a score of more than 70 in the AlphaFold-predicted three-dimensional structure of Human DHHC5 is high, indicating that the reliability of the AlphaFold-predicted structure is good (AlphaFold number: AF-Q9C0B5-F1). At the same time, Uniprot shows that C134 is the active site of Human DHHC5 (UniprotKB: Q9C0B5). In addition, the fragment of Human DHHC5 where C134 is located is scored as 97.98 by AlphaFold, and the reliability is extremely high. Therefore, the present application selects C134 as the binding site for verification in the AlphaFold-predicted Human DHHC5 protein structure.

[0029] Figure 2 is a 2D schematic diagram of the molecular docking of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol and Human DHHC5 protein; Figure 3Molecular docking 3D diagram of 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9- ylidene)propyl)-1-piperazinethanol and Human DHHC5 protein. Human DHHC5 protein C skeleton is shown in green, N atom is shown in blue, O atom is shown in dark red, H atom is shown in white, 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1- piperazinethanol is shown in orange stick. Hydrogen bond length is shown as a red dotted line, the longer the bond length, the weaker the hydrogen bond. 4-(3-(2-(trifluoromethyl)-9H- thioxanthen-9-ylidene)propyl)-1-piperazinethanol can form 1 hydrogen bond, 2 π-π interactions and hydrophobic interactions with Human DHHC5 protein: one nitrogen atom of the piperidine ring acts as a hydrogen bond acceptor to form 1 hydrogen bond with TRP136, with a distance of 2.2 Å; two benzene rings can form 2 π-π interactions with TRP136, PHE196; in addition, HY-15856B forms hydrophobic interactions with ALA20, PHE23, PHE152, LEU153, TRP136, etc. amino acid residues.

[0030] Figure 4 Schematic diagram and quantitative analysis statistics of the effect of 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1-piperazinethanol on palmitoyl transferase DHHC5. The expression of DHHC5 was detected by Western blotting, and β-actin was used as an internal reference. As can be seen from the figure, 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1- piperazinethanol inhibits palmitoyl transferase DHHC5 in a dose-dependent manner. Specific implementation method: human astrocyte cell line, randomly divided into control group (normal saline treatment) and drug group (different concentrations of trifluorothioxanthene, diluted with normal saline). Cells were plated at 10 5 cells / well, and after 24 hours of pre-culture, drugs were added, and after 24 hours of action, cells were collected for Western blotting detection.

[0031] As can be seen from the above, 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1- piperazinethanol can specifically inhibit palmitoyl transferase DHHC5.

[0032] Example Two

[0033] Animal experiments comply with relevant requirements of Suzhou University, and the specific operation is a routine technique.

[0034] Male C57 mice were randomly divided into model group (I / R), drug group (I / R+2-bromohexadecanoic acid 20 mg / kg, I / R+edaravone dextro alcohol 10 mg / kg, I / R+4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol 10 mg / kg), 10 in each group. The mouse transient middle cerebral artery occlusion model (tMCAO) was made by using a thread plug method. After ischemia for 60 min, reperfusion was performed, and drugs were injected intraperitoneally immediately after reperfusion. Single dose was given. After 24 hours, the changes of DHHC5 in the brain were observed, see Figure 5 For comparison with existing 2-bromohexadecanoic acid, edaravone dextro alcohol. As can be seen from the figure, compared with existing 2-bromohexadecanoic acid, edaravone dextro alcohol, 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol has the best inhibitory effect on DHHC5.

[0035] Male C57 mice were randomly divided into control group (sham), model group (I / R), and the mouse transient middle cerebral artery occlusion model (tMCAO) was made by using a thread plug method. After ischemia for 60 min, reperfusion was performed, and mice were randomly selected for 3 days, 7 days, and 14 days after reperfusion. The changes of DHHC5 in the brain were observed, see Figure 6 For the changes of palmitoyl transferase DHHC5 at different time points after ischemia-reperfusion in mice. As can be seen from the figure, the expression of DHHC5 significantly increased with the extension of reperfusion time.

[0036] Male C57 mice were randomly divided into control group (sham), model group (I / R), and drug group (I / R+4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol), 10 in each group. The mouse transient middle cerebral artery occlusion model (tMCAO) was made by using a thread plug method. After ischemia for 60 min, reperfusion was performed, and 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol (20 mg / kg, dissolved in normal saline) was injected intraperitoneally immediately after reperfusion. Single dose was given. After 24 hours, the mice were decapitated to take the brain and sliced for TTC staining to observe the effect of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol on the volume of cerebral infarction in acute phase of cerebral ischemia, see Figure 7 For the effect of trifluorothioxanthene on reducing the volume of cerebral infarction in acute phase of cerebral ischemia in mice. As can be seen from the figure, trifluorothioxanthene can reduce the volume of cerebral infarction in acute phase of cerebral ischemia in mice p <0.0001, with extremely significant difference).

[0037] The present application firstly proves that 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol can obviously inhibit DHHC5 palmitoyl transferase, and 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol can be used as a specific DHHC5 palmitoyl transferase inhibitor. The prior art considers that various cancers and nervous system diseases can be treated by intervening palmitoyl transferase. Protein palmitoylation and depalmitoylation modification enzymes are potential drug targets for diseases such as tumors, Alzheimer's disease, epilepsy and Huntington's disease.

Claims

1. Use of 4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)-1-piperazineethanol or its salt as an active ingredient of a palmitoyltransferase inhibitor in the preparation of a drug for treating acute cerebral ischemia and cerebral infarction, characterized in that: The palmitoyltransferase is DHHC5.

2. The use according to claim 1, characterized in that The palmitoyltransferase inhibitor may further include pharmaceutical excipients.

3. The use according to claim 2, characterized in that The auxiliary material is selected from excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, colorants or sweeteners.

4. The use according to claim 1, characterized in that The dosage form of the palmitoyltransferase inhibitor is selected from the group consisting of solution, suppository, tablet, powder, and ointment.

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