Application of PARP1 PROTAC A19 in preparation of medicine for preventing and treating acute kidney injury

Abstract

The invention discloses an application of PARP1PROTACA19 in preparation of a medicine for preventing and treating acute kidney injury, and relates to the technical field of medicines, and the key points of the technical scheme are as follows: PARP1PROTAC A19 can reduce the expression level of PARP1 in HK2 cells and mice, reduce the levels of acute kidney injury markers KIM-1 and inflammatory factors of the HK2 cells and the mice, improve pathological changes of kidney tissues, and inhibit the occurrence of acute kidney injury. The traditional Chinese medicine composition has obvious prevention and treatment effects on acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more specifically, to the use of PARP1 PROTAC A19 in preparing drugs for preventing and treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a common clinical syndrome characterized by a rapid deterioration in renal function, and is associated with significant short-term and long-term morbidity and mortality, particularly in critically ill patients. Numerous factors, including ischemia-reperfusion injury, nephrotoxic drugs, and sepsis, can trigger AKI, yet the pathogenesis of AKI remains unclear. The primary treatments include treating underlying diseases, removing risk factors (such as diabetes and hypertension), maintaining acid-base balance, and maintaining water and electrolyte balance, and ultimately, employing renal replacement therapy. Currently, there is a lack of effective treatments to limit renal damage or promote recovery and survival. Therefore, identifying regulatory factors that play an important role in AKI and developing targeted therapeutic strategies are key goals for developing effective treatments for AKI.

[0003] Poly(ADP-ribose) polymerase 1 (PARP1) is the founding member of the PARP family. It binds to damaged DNA and is activated by nicotinamide adenine dinucleotide (NAD + ) as a substrate to add poly (ADP-ribose) chains (poly (ADP-ribose), PAR) to itself and other proteins. PARP1 plays a role in a variety of basic cellular processes, such as DNA repair, maintenance of genome stability, chromatin remodeling, gene expression, cell differentiation, and cell survival and death, and is also a target for clinical cancer treatment inhibitors. In recent years, the study of PARP1 in kidney disease has become one of the hot topics and has made some progress. Currently, there are five PARP inhibitors (olaparib, rucaparib, niraparib, tarazopanib and pamiparib) clinically approved for the treatment of ovarian cancer, pancreatic cancer, prostate cancer and breast cancer. Although their initial treatment is effective and the survival rate is high, most cancers will eventually develop drug resistance.

[0004] Proteolysis-targeting chimera (PROTAC) is an emerging therapeutic entity designed to hijack the ubiquitin-proteasome system (UPS) and degrade target proteins, which are often associated with various diseases. PROTAC does not require tight or persistent binding like small molecule inhibitors to work. It can enhance the effect and minimize drug resistance by regulating related signaling pathways. Therefore, the development of a new PARP1 PROTAC drug for the prevention or treatment of acute kidney injury has broad clinical application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of PARP1 PROTACA19 in the preparation of a medicament for preventing and treating acute kidney injury, so as to solve the problems existing in the above-mentioned prior art.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions:

[0007] The first aspect of the present invention provides PARP1 PROTAC A19, the structural formula of which is as follows:

[0008]

[0009] The present invention further provides a synthetic route for PARP1 PROTAC A19:

[0010]

[0011] The present invention further provides the use of PARP1 PROTAC A19 in the preparation of drugs for preventing and treating acute kidney injury and reducing the cell injury marker KIM-1 and inflammatory factors.

[0012] The present invention is further configured as follows: the drug includes a pharmaceutically acceptable carrier or excipient.

[0013] The present invention is further configured such that the drug is an oral administration preparation, a transdermal administration preparation or an injectable administration preparation.

[0014] The present invention is further configured as follows: the dosage form of the drug is tablets, capsules, granules, oral liquid, patch, ointment, gel or injection.

[0015] In summary, the present invention has the following beneficial effects: the PARP1 PROTAC A19 synthesized herein can reduce the expression level of PARP1 in HK2 cells and mice, reduce the levels of KIM-1, an acute kidney injury marker, and inflammatory factors in HK2 cells and mice, and improve renal tissue pathological changes, showing significant preventive and therapeutic effects on acute kidney injury. This is conducive to the further development of new drugs for the prevention and treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A is the synthetic route of PARP1 PROTAC A19 in Example 1 of the present invention; B is the computer simulation of the molecular docking of PARP1 and A19;

[0017] Figure 2 The results of MTT assay for the effect of A19 on HK2 cell viability in Example 2 of the present invention are as follows;

[0018] Figure 3 The results of Western blot detection of PARP1 protein levels in HK2 cells of each experimental group in Example 2 of the present invention are as follows;

[0019] Figure 4 The results of Western blot and immunofluorescence (IF) detection of KIM-1 expression levels and NF-κB pathway activation in HK2 cells of each experimental group in Example 2 of the present invention are as follows;

[0020] Figure 5 These are the results of Real-time PCR detection of changes in inflammatory factor levels in HK2 cells of each test group in Example 2 of the present invention;

[0021] Figure 6 Schematic diagram of the dosing regimen for establishing a mouse acute kidney injury model induced by cisplatin (CIS) in Example 3 of the present invention;

[0022] Figure 7 The results of Western blot detection of PARP1 protein levels in mice of each experimental group in Example 3 of the present invention are as follows;

[0023] Figure 8 This is a graph showing the serum creatinine and urea nitrogen levels of mice in each experimental group in Example 3 of the present invention;

[0024] Figure 9 The results of Western blot detection of KIM-1 expression levels and NF-κB pathway activation in mice of each experimental group in Example 3 of the present invention are as follows;

[0025] Figure 10This is a graph showing the results of mouse kidney tissue staining using immunohistochemistry and glycogen (PAS) staining in Example 3 of the present invention;

[0026] Figure 11 The results of Real-time PCR detection of changes in inflammatory factor levels in mice in each experimental group in Example 3 of the present invention are as follows;

[0027] Figure 12 This is a diagram showing the staining results of mouse heart, liver, spleen and lung using hematoxylin-eosin (HE) staining in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] The following is combined with Figure 1-12 The present invention is described in further detail.

[0029] Example 1: Synthesis of PARP1 PROTAC A19

[0030] The structural formula of PARP1 PROTAC A19 is as follows:

[0031]

[0032] The synthetic route of PARP1 PROTAC A19 is as follows:

[0033]

[0034] Thalidomide derivative 3 is synthesized by heating a mixture of commercially available fluorinated phthalic anhydride derivative 1 and starting material 2. Following this step, a nucleophilic substitution reaction involving an N-heterocycloalkane derivative 4 in the presence of N,N-diisopropylethylamine (DIPEA) produces compound 5. Intermediate 5 is then converted to the corresponding aldehyde derivative 6 via an oxidation process using Dess-Martin periodinane (DMP). A tert-butyloxycarbonyl (Boc)-piperidine fragment 8 is reductively aminized with commercially available niraparib 7 to produce compound 9. Intermediate 9 is deprotected to produce an imine fragment, which is then reductively aminized with a tert-butyloxycarbonyl-piperidine fragment to produce intermediate 10. After further deprotection, the imine fragment of intermediate 10 undergoes reductive amination with intermediate 6 to produce product A19. The efficient degradation of PARP1 by PROTAC A19 relies on the formation of a ternary complex. The PARP1-A19-CRBN ternary complex modeling structure supports the beneficial role of A19 in PARP1 degradation ( Figure 1 (Figure B).

[0035] Example 2: Effect of A19 in CIS-induced HK2 cell model

[0036] The MTT method was used to evaluate the effect of A19 on the activity of HK2 cells. Take a 96-well plate, add 200 μL PBS buffer to each of the 36 wells on the outermost circle, then seed HK2 cells in the plate, and start corresponding treatment when the cells grow to 60-70%. Treat HK2 cells with A19 at a dose gradient of 0.0625-8 μM for 24 hours. Then add MTT solution to the wells, and then put the 96-well plate back into the incubator and incubate for 5 hours. Aspirate the supernatant, add DMSO, and use a multifunctional microplate reader to detect the absorbance after 8 minutes. Figure 2 As shown in Figure A, when the concentration of A19 exceeded 8 μM, cell viability was significantly affected. The effect of A19 on CIS-induced cell damage was then determined. Figure 2 As shown in Figure B, HK2 cells were treated with A19 at a dose gradient of 0.125-16 μM and stimulated with 20 μM CIS for 24 hours. The results showed that A19 at concentrations of 0.5 μM, 1 μM, and 2 μM could restore the viability of HK2 cells treated with CIS in a dose-dependent manner.

[0037] HK2 cells in the logarithmic growth phase were seeded in 6-well plates and divided into seven groups: control, 2μM A19, model, model + 0.5μM A19 low-concentration, model + 1μM A19 medium-concentration, model + 2μM A19 high-concentration, and model + 2μM PJ34. Twelve hours after drug administration, HK2 cells were stimulated with 20μM CIS, and samples were collected 24 hours later.

[0038] Western blot: extract tissue proteins and perform immunoblotting experiments. Figure 3 As shown, A19 can effectively degrade PARP1 in a concentration-dependent manner. PJ34 is a highly selective PARP1 inhibitor. At the same dose, A19 has a more significant effect in reducing PARP1 expression levels. KIM-1 is a biomarker for acute kidney injury, and p-P65 is one of the key molecules in the NF-κB signaling pathway. Under CIS stimulation, the expression of KIM-1 and p-P65 is significantly upregulated, and A19 can inhibit the levels of KIM-1 and p-P65 in a concentration-dependent manner ( Figure 4 Compared with the positive drug PJ34, A19 showed a better damage alleviation effect.

[0039] Immunofluorescence: Wash three times with PBS, fix with polymethanol for 15 minutes, seal the slide with a histochemical pen, block with 10% BSA (prepared in PBS), add KIM-1 primary antibody and incubate overnight at 4°C. After returning to room temperature the next day, wash three times with PBS, incubate with secondary antibody for 1.5 hours, wash three times with PBS, incubate with DAPI for 10 minutes, wash three times with PBS, and seal with anti-fluorescence quencher. The results of immunofluorescence experiments are as follows Figure 4As shown in middle B, consistent with the Western blot results, A19 effectively reduced the expression level of KIM-1.

[0040] Real-time PCR: Extract tissue RNA, reverse transcribe, and amplify. Real-time PCR results are as follows: Figure 5 As shown in the results, A19 significantly reduced the levels of inflammatory factors TNF-α, MCP-1 and IL-1β in the CIS-induced cell model, and A19 showed a better injury-alleviating effect than PJ34.

[0041] Example 3: Effect of A19 in CIS-induced acute kidney injury model in mice

[0042] Healthy male C57-BL / 6J mice were acclimated for one week. Mice meeting the required weight were randomly divided into seven groups: a control group, a 10 mg / kg A19 group, a model group, a model group plus a low-dose 2.5 mg / kg A19 group, a model group plus a medium-dose 5 mg / kg A19 group, a model group plus a high-dose 10 mg / kg A19 group, and a model group plus a 10 mg / kg PJ34 group. Each group had 6-8 mice.

[0043] like Figure 6 As shown, a cisplatin-induced AKI model was established with a single intraperitoneal injection of 20 mg / kg cisplatin. A19 or PJ34 was administered intraperitoneally 12 hours before cisplatin administration and daily thereafter. A control group received normal saline. After model establishment, mice were anesthetized and placed in a sealed chamber filled with 5% isoflurane. Blood samples were then obtained by cardiac exsanguination. After sacrifice, kidney tissue was obtained for subsequent pathological and molecular analysis.

[0044] Western blot: extract tissue proteins and perform immunoblotting experiments. Figure 7 As shown in the results, A19 can also reduce the expression level of PARP1 in a dose-dependent manner in vivo, and its effect is better than that of the positive drug PJ34.

[0045] Serum creatinine and urea nitrogen: After the mice were modeled and administered, the blood was drawn and allowed to stand for 40-45 minutes. The blood samples were then centrifuged at 4°C and 3600 rpm for 20 minutes. After centrifugation, the upper serum was collected and the experiment was performed according to the instructions. Figure 8 As shown in Figures 8A and 8B, A19 could significantly reduce the serum creatinine and urea nitrogen levels in mice induced by CIS.

[0046] Western blot: extract tissue proteins and perform immunoblotting experiments. Figure 9 As shown, A19 significantly reduced the increase in KIM-1 and p-P65 levels caused by CIS in vivo, and its therapeutic effect was significantly stronger than that of the positive drug PJ34.

[0047] Immunohistochemistry: Wash three times with PBS, seal the slides with a histochemical pen, add endogenous peroxidase, and repair. After blocking, add KIM-1 primary antibody and incubate overnight at 4°C. Return to room temperature the next day, wash three times with PBS, incubate with secondary antibody for 30 minutes, wash three times with PBS, use DAB for color development, rinse with running water and stain the nucleus with hematoxylin, rinse clean, differentiate with acid differentiation solution for 10s, then rinse with running water for anti-blue, then dehydrate, air dry, and seal. Figure 10 As shown in middle A, A19 significantly reduced KIM-1 expression in mice and alleviated renal tubular tissue damage.

[0048] PAS staining: PAS staining kit was used for staining, and the results were as follows: Figure 10 As shown in Figure B, A19 significantly reduced glycogen deposition and improved renal pathology compared with the model group.

[0049] Real-time PCR: Extract tissue RNA, reverse transcribe, and amplify. Real-time PCR results are as follows: Figure 11 As shown in the figure, the levels of inflammatory factors TNF-α, MCP-1 and IL-1β were also significantly reduced after using A19.

[0050] Example 4: Effects of A19 on other organs of mice

[0051] A19 (10 mg / kg) was administered to mice, and a group receiving physiological saline was set up as a control group.

[0052] HE staining: Wash three times with PBS, seal the slide with a histochemical pen, add hematoxylin solution to stain the cell nucleus, rinse with running water, add acid differentiation solution, rinse with running water, add eosin solution, rinse with running water, then dehydrate, air dry, and seal the slide. The results showed that the heart, liver, spleen, and lungs of the A19-treated mice did not show any pathological abnormalities compared with the control mice ( Figure 12 ), which showed that A19 was safe in mice at a therapeutic dose (10 mg / kg) and no obvious toxic effects were observed.

[0053] The above experimental results show that A19 can inhibit CIS-induced HK2 cell damage and inflammation, alleviate acute kidney injury in mice, improve renal tissue pathology, and has significant preventive and therapeutic effects on acute kidney injury.

[0054] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. PARP1 PROTAC A19, characterized by: The structural formula of the PARP1 PROTAC A19 is as follows:

2. The synthesis of PARP1 PROTACA19 according to claim 1, characterized in that: The synthetic route is as follows:

3. Use of the PARP1 PROTACA19 according to claim 1 in the preparation of a drug for preventing and treating acute kidney injury and reducing the cell injury marker KIM-1 and inflammatory factors.

4. The use according to claim 3, characterized in that: The medicine includes a pharmaceutically acceptable carrier or excipient.

5. The use according to claim 3, characterized in that: The medicine is an oral administration preparation, a transdermal administration preparation or an injection preparation.

6. The use according to claim 3, characterized in that: The dosage form of the drug is tablet, capsule, granule, oral solution, patch, ointment, gel or injection.

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