CN113244237B - Application of BI8622 in preparation of medicine for relieving acute kidney injury induced by cisplatin - Google Patents

Abstract

The invention discloses a novel application of BI8622, namely an application of BI8622 in preparation of a medicine for relieving acute kidney injury induced by cisplatin. The application finds that BI8622 can be an effective clinical medicine for preventing and treating AKI by inhibiting the activity of E3 ubiquitinase HUWE1 and up-regulating the protein level of MUTYH to improve DNA damage and further relieve cisplatin-related acute kidney damage.

Description

Application of BI8622 in preparation of medicine for relieving acute kidney injury induced by cisplatin

Technical Field

The invention relates to a novel medicinal application of BI8622, in particular to an application of BI8622 in preparing a medicament for relieving acute kidney injury caused by cisplatin serving as a chemotherapeutic medicament.

Background

In the 70 s of the 20 th century, cisplatin began to be widely used clinically for treating various malignant tumors, including bladder cancer, cervical cancer, head and neck malignant tumors, small cell or non-small cell lung pain and the like, and is one of the most effective and commonly used chemotherapeutic drugs for treating solid tumors. However, cisplatin can cause serious adverse reactions such as damage to normal tissues and organs while killing tumor cells, thereby greatly limiting the clinical application of cisplatin. The adverse reactions of cisplatin mainly include a series of systemic organ injury reactions such as nephrotoxicity. Of these, nephrotoxicity is most common, and after cisplatin treatment, renal dysfunction has occurred in approximately 1/3 patients leading to acute renal failure, and its dose-related nephrotoxicity has greatly limited cisplatin clinical use. The mechanism of normal cell damage caused by cisplatin has not been completely understood for a long time, and numerous studies have found that DNA damage, inflammatory mediators, necrosis, iron death, apoptosis, oxidative stress, autophagy, etc. may be the cause of normal tissue organ damage caused by cisplatin (Ozkok A, Edelstein CL. Pathophysiology of pain-induced access kit in J.BioMed research international 2014; 2014: 967826).

Although the platinum chemotherapeutic drugs have been gradually replaced by new chemotherapeutic drugs in recent years, the traditional platinum drugs and derivatives thereof are still widely applied to clinical antitumor chemotherapeutic drugs due to the effective effect of the platinum drugs and derivatives thereof in antitumor. However, the toxic and side effects of the kidney injury and the like caused by the platinum drugs greatly limit the clinical application of the drugs, and at present, an effective drug for treating the acute kidney injury induced by the cisplatin is still lacking clinically, so that the development of a new effective drug capable of improving the acute kidney injury caused by the cisplatin has important clinical significance.

HUWE1 inhibitor BI8622 is a class of small molecule compounds having the following structural formula:

BI8622 is obtained by screening of Stefanie Peter et al, can specifically inhibit ubiquitin ligase HUWE1 through ubiquitination modification function, and finds that the inhibitor has a certain inhibiting effect on tumorigenesis and development (Peter S, Bumultick J, Myant K, Jaenicke LA, Walz S, Muller J, et al. Tumor cell-specific inhibition of MYC functional using small molecule inhibitors of the HUWE1ubiquitin Ligase. EMBO Mol. Med. 2014; 6: 5. 1541.), CN111358793A discloses that HUWE1 inhibitor BI8622 can be used for inhibiting activation of the inflammasome, but no published literature exists yet that BI8622 can reduce acute kidney injury induced by cisplatin.

Disclosure of Invention

The application unexpectedly finds that the level of the MUTYH protein in the kidney tissue of the mouse with acute kidney injury induced by cisplatin is remarkably reduced, and the E3 ubiquitination ligase HUWE 1-mediated MUTYH ubiquitination degradation is probably a molecular mechanism, which suggests that the inhibition of HUWE1 function by using a related inhibitor may improve the DNA injury induced by cisplatin so as to improve the acute injury, and further the experiment verifies the speculation.

Specifically, the invention provides an application of a ubiquitin ligase HUWE1 protein inhibitor BI8622 in preparation of an acute kidney injury medicament, and the application of the inhibitor in preparation of a medicament for relieving cisplatin-induced acute kidney injury related symptoms solves the technical problem that no suitable medicament is available in the prior art for treating cisplatin-induced kidney structural and functional damage.

Specifically, the application proposes that BI8622 improves DNA damage by inhibiting the activity of E3 ubiquitinase HUWE1, up-regulating MUTYH protein levels, and thereby reduces cisplatin-related acute kidney injury.

The invention uses BI8622 on a mouse model and an in vitro cell model of acute kidney injury induced by cisplatin, and discusses the application of BI8622 as an inhibitor of ubiquitin-mediated enzyme HUWE1 in medicaments for relieving acute kidney injury related diseases induced by cisplatin. The results show that intervention treatment by using BI8622 on a cisplatin mouse model and in vitro cells can obviously improve the acute kidney injury kidney structure and function, DNA injury, renal tubular cell apoptosis and the like; our findings will most likely provide effective clinical drugs for the prevention and treatment of AKI.

Drawings

Fig. 1 shows that BI8622 significantly improved kidney injury and kidney function in cisplatin-induced acute kidney injury;

FIG. 2 shows that BI8622 down-regulates NGAL and KIM-l protein levels in renal tissue in a cisplatin-induced acute kidney injury model;

fig. 3 shows that BI8622 significantly improved cisplatin-induced DNA damage;

fig. 4 shows that BI8622 significantly inhibits MUTYH ubiquitination and up-regulates its protein levels;

fig. 5 shows that BI8622 significantly reduced cisplatin-induced tubular epithelial apoptosis in an in vitro tubular epithelial cell model.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which will help understanding the present invention, but the scope of the present invention is not limited to the following examples.

The materials and methods used in the following examples are as follows:

materials and reagents:

BI8622 available from MCE, USA; NGAL, MUTYH antibody was purchased from Abcam; KIM-1, antibody, available from R & D Systems; clean caspase3, gamma H2AX, purchased from CST; beta-actin antibodies were all purchased from proteintech; fluorescent secondary antibodies were purchased from Invitrogen; apoptosis detection kits were purchased from B & D.

(II) cell culture and treatment:

mouse tubular epithelial cells (mPTCs) were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 0.5% penicillin and streptomycin at 37 ℃ in 5% carbon dioxide and 95% air. To investigate the role of BI8622 in cisplatin-induced injury to tubular epithelial cells, mPTC was grown to 70% density and replaced with DMEM/F12 medium without fetal bovine serum, and BI8622 (dissolved in DMSO) was added for 1h or treated identically with DMSO. After the cells are incubated in an incubator for 24h, the cells are finally collected to carry out tests such as Western Blot detection, flow cytometry analysis of apoptosis and the like.

(III) cisplatin-induced acute renal injury mouse model and experimental grouping:

mice were randomized into four groups of 6 mice, each group consisting of a placebo group (vehicle), a BI8622 group, a cisplatin injection model group (cissplatin), and a cissplatin + BI8622 group (cissplatin + BI 8622). BI8622 was dissolved in PEG300+ TWEEN80+ DMSO and administered by intraperitoneal injection at a dose of 5 mg/kg. Mice in the control group or BI 8622-treated group were given either vehicle or BI8622, respectively, by intraperitoneal injection once a day starting 12h before cisplatin injection. Cisplatin model mice were injected with 20mg/kg of cisplatin at a time intraperitoneally, and mice were sacrificed and harvested 72h after cisplatin injection. Mice in the control group received the same dose of saline injection. After 72 hours, all groups of mice were euthanized and kidney and blood samples were collected. All animal experiments followed the Chinese regulations for the management and use of experimental animals.

(IV) renal function detection:

blood serum components are collected after a mouse blood specimen is centrifuged, and serum creatinine and serum urea nitrogen indexes are detected on a full-automatic biochemical instrument of children hospital in Nanjing.

(V) renal tubular injury score:

the degree of tubular injury was observed in the pathological staining of PAS in the kidney and evaluated according to a semi-quantitative injury score with 0 score for normal tubular tissue, 1 score for 30% of tubular injury, 2 scores for 30% -60% of tubular injury and 3 scores for greater than 60%.

(VI) histological and immunofluorescence staining:

kidney tissues are fixed by paraformaldehyde, wrapped by paraffin, and subjected to histology and immunofluorescence staining after being sliced.

Primary antibody concentrations of γ H2AX, etc. were all 1:100, fluorescent secondary antibody concentrations were 1:400, DAPI staining was performed as indicated.

(seventh) western immunoblotting:

the kidney tissue extracts proteins and operates according to literature procedures. Results of Western immunoblotting (Western blot) were subjected to grayscale analysis using ImageJ software.

(eight) flow cytometric detection of apoptosis:

culturing mouse renal tubular epithelial cells, pretreating with BI8622 for 1h, adding cisplatin for 24h, analyzing apoptosis level of the cells by a flow cytometer, and repeating cell experiments for three times and counting.

(nine) statistical analysis:

data are represented using the mean SD. Multiple comparisons were performed using one-way analysis of variance (ANOVA) and two data comparisons were performed using T-test. P <0.05 is statistically significant.

Example 2BI8622 improves kidney injury and kidney function in a cisplatin-induced acute kidney injury model.

In order to evaluate and detect the effect of BI8622 in protection of cisplatin-induced acute kidney injury, related biochemical indexes of kidney in mouse serum are detected, after 72 hours of intraperitoneal cisplatin modeling, indexes of serum muscle and urea nitrogen are obviously increased, and pathological injury manifestations of kidney such as renal tubular dilatation necrosis are also serious, while after BI8622 treatment, corresponding kidney injury and kidney function indexes are obviously reduced (fig. 1A, 1B and 1C). In addition, the renal tubular injury score also suggests that trehalose treatment may ameliorate cisplatin-induced pathological renal injury. Thus, BI8622 may not only improve kidney function, but also reduce pathological damage to the kidney. These results indicate that BI8622 can protect cisplatin-induced acute kidney injury and has no significant toxic side effects by itself.

To further demonstrate the role of BI8622 in protecting cisplatin-induced acute kidney injury, we examined specific biomarkers, NGAL and KIM-1, early in acute kidney injury. As shown in fig. 2, the western blot detection results show that KIM-1 and NGAL are highly expressed in the kidney of cisplatin-induced acute kidney injury mice, and the expression level thereof is significantly reduced after BI8622 treatment, which indicates that BI8622 can improve cisplatin-induced acute kidney injury.

Example 3 in the cisplatin-induced acute kidney injury model, BI8622 significantly improved cisplatin-induced DNA damage.

The main principle that cisplatin acts on cells is that cisplatin enters cell nucleus and is combined with DNA to form a DNA-platinum complex, so that DNA damage is caused, therefore, the DNA damage level in mouse kidney tissues after BI8622 treatment is further detected, comet-event electrophoresis can detect the DNA breakage level, and the result shows that BI8622 remarkably improves cisplatin-induced DNA double strand breakage (fig. 3A); further, γ H2AX protein levels could indicate DNA fragmentation, and γ H2AX immunofluorescent staining showed that cisplatin-induced upregulation of γ H2AX levels was significantly down-regulated following BI8622 treatment (fig. 3B). The above results suggest that BI8622 can significantly improve cisplatin-induced DNA damage.

Example 4 BI8622 inhibits cisplatin-induced ubiquitination of the MUTYH protein and up-regulates its protein levels in a cisplatin-induced acute kidney injury model.

We further explored the possible mechanism of action of BI8622, which could act as an inhibitor of E3 ubiquitin ligase HUWE1 by regulating ubiquitination of substrate proteins. One of the substrate proteins of HUWE1, MUTYH, is a glycosylase which is widely expressed in mammalian cells and participates in the repair of base mismatch excision, and plays an important role in the repair of DNA oxidative damage. Our experimental results show that treatment with BI8622 can significantly down-regulate cisplatin-induced levels of MUTYH ubiquitination (fig. 4A); thereby significantly improving cisplatin-induced down-regulation of MUTYH protein levels (figure 4B).

Example 5 in an in vitro cisplatin model of tubular epithelial cells, BI8622 treatment improved cisplatin-induced apoptosis.

Further, using tubular epithelial cells cultured in vitro, pre-treating for 1h with BI8622(1 μ M), then adding 5g/ml cisplatin to continue treating for 24h, collecting cells and detecting the apoptosis level by a flow cytometer, and the result shows that BI8622 can significantly improve cisplatin-induced apoptosis (fig. 5A); further, western blot results showed that apoptosis-related protein clear caspase3 was significantly down-regulated after BI8622 treatment, while cisplatin-induced down-regulation of MUTYH protein levels was significantly improved (fig. 5B).

In conclusion, the invention provides an application of HUWE1 ubiquitinase inhibitor BI8622 in preparation of a medicine for relieving cisplatin-induced acute kidney injury related symptoms, the medicine is administered by intraperitoneal injection, the mouse dose is 5mg/kg, and the MUTYH protein level is up-regulated by inhibiting MUTYH ubiquitination degradation, so that DNA injury is improved, and acute kidney injury is improved.

Claims (2)

1. Use of BI8622 in the manufacture of a medicament for the reduction of cisplatin-induced acute kidney injury.
2. 2. The use of claim 1, wherein BI8622 ameliorates DNA damage by inhibiting the activity of E3 ubiquitinase HUWE1, up-regulating MUTYH protein levels, thereby reducing cisplatin-related acute kidney injury.

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