Application of IDO1 non-enzymatic function inhibitor in preparation of medicine for treating cardiomyopathy

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of an IDO1 non-enzymatic function inhibitor in preparation of a medicine for treating cardiomyopathy. The cardiomyopathy is the cardiomyopathy induced by Ang II. The IDO1 non-enzymatic function inhibitor is Epacadstat, and the IDO1 non-enzymatic function inhibitor is Epacadstat. According to analysis, Ang II can induce IDO1 expression increase, under the high expression state of IDO1, the non-enzymatic function of the Ang II is in the dominant position along with expression increase of a core subunit gp91 phox, Epacadstat inhibits activation of ANGPTL4 / gp91 phox by antagonizing the non-enzymatic function of IDO1 and up-regulating Notch1 / HES1 signals, and then Ang II-induced myocardial oxidative damage is relieved, a theoretical basis is provided for application of Epacadstat to prevention and treatment of Ang II-related myocardial damage, and the application of Epacadstat to prevention and treatment of Ang II-related myocardial damage is developed. And a new thought and a new target are provided for prevention and treatment of oxidative stress related cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of an IDO1 non-enzymatic function inhibitor in the preparation of a drug for treating cardiomyopathy. Background Art

[0002] Angiotensin II (Ang II) in the local heart has been proven to cause oxidative stress by activating NADPH oxidase (NOXs) to produce excessive reactive oxygen species (ROS), thereby activating various signaling pathways such as inflammation, apoptosis, and fibrosis, and promoting the pathological processes of various heart diseases, such as acute myocardial infarction and diabetic cardiomyopathy. Oxidative stress is one of the core pathological mechanisms of myocardial injury. NOX2 containing the membrane catalytic subunit gp91 phox is the main source of ROS in the cardiovascular system, but the molecular mechanism of activating the core subunit gp91 phox is still unclear. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the metabolism of tryptophan to generate kynurenine and activates related pathways, participating in the regulation of functions such as redox, tumor resistance, and immunosuppression. IDO1 is not only a catalytic enzyme, but also can mediate downstream signal transduction through the phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the non-enzymatic domain; in the study of ischemic stroke, it was found that the expression of gp91 phox increased with the increase in the expression of IDO1, which was independent of the increase in the enzymatic activity of IDO1, but the specific mechanism was unknown.

[0003] Epacadostat is a specific inhibitor of IDO1, which exerts an anti-tumor effect by inhibiting the enzymatic reaction of IDO1 and the kynurenine pathway. However, there is no public information on its effect on the non-enzymatic function of IDO1 and whether it can reduce myocardial injury through the non-enzymatic function of IDO1.

[0004] The high expression of IDO1 may inhibit the Notch1 signal in intestinal epithelial tissues through a phosphorylation-mediated non-enzymatic pathway, and promoting Notch1 / HES1 signal transduction can improve oxidative damage and apoptosis of rat myocardium induced by ischemia-reperfusion. In addition, the Notch1 signal in endothelial cells promotes vascular lipolysis by inhibiting the expression of angiopoietin-like protein 4 (ANGPTL4), thereby improving the metabolism of cardiomyocytes and reducing myocardial hypertrophy; and ANGPTL4 induces the expression of gp91 phox by phosphorylating and activating focal adhesion kinase (FAK), promoting myocardial injury in diabetic mice.

[0005] In summary, antagonizing the non-enzymatic function of IDO1, promoting Notch / HES1 signaling, inhibiting the activation of ANGPTL4 / gp91 phox, can alleviate Ang II-induced myocardial oxidative damage, and provide new ideas for the preparation of drugs for treating cardiomyopathy. Summary of the Invention

[0006] The purpose of the embodiments of the present invention is to provide the application of an inhibitor of the non-enzymatic function of IDO1 in the preparation of drugs for treating cardiomyopathy, aiming to solve the problems proposed in the above background technology.

[0007] The embodiments of the present invention are implemented as follows: the application of an inhibitor of the non-enzymatic function of IDO1 in the preparation of drugs for treating cardiomyopathy.

[0008] Preferably, the cardiomyopathy is Ang II-induced cardiomyopathy.

[0009] Preferably, the inhibitor of the non-enzymatic function of IDO1 is Epacadostat.

[0010] Preferably, Epacadostat antagonizes the non-enzymatic function of IDO1, promotes Notch / HES1 signaling, inhibits the activation of ANGPTL4 / gp91 phox, and alleviates Ang II-induced myocardial oxidative damage.

[0011] Another purpose of the embodiments of the present invention is to provide a drug for treating cardiomyopathy, and the drug includes an inhibitor of the non-enzymatic function of IDO1.

[0012] Preferably, the cardiomyopathy is Ang II-induced cardiomyopathy.

[0013] Preferably, the inhibitor of the non-enzymatic function of IDO1 is Epacadostat.

[0014] The embodiments of the present invention analyzed that Ang II can induce an increase in IDO1 expression. In the state of high IDO1 expression, its non-enzymatic function is dominant, accompanied by an increase in the expression of the core subunit gp91 phox. Epacadostat can antagonize the non-enzymatic function of IDO1, up-regulate Notch1 / HES1 signaling, inhibit the activation of ANGPTL4 / gp91 phox, and thus relieve Ang II-induced myocardial oxidative damage, providing a theoretical basis for the application of Epacadostat in the prevention and treatment of Ang II-related myocardial injury, and also providing new ideas and new targets for the prevention and treatment of oxidative stress-related cardiomyopathy. Brief Description of the Drawings

[0015] Figure 1Results of Ang II-induced cardiac dysfunction and myocardial oxidative damage provided in Example 1 of the present invention (A, left ventricular end-diastolic diameter; B, left ventricular posterior wall thickness during diastole; C, ejection fraction; D, fractional shortening; E-F, protein expression and quantitative analysis of 4-HNE in mouse myocardial tissue detected by Western blot; G, MDA content in mouse myocardial tissue; H-I, protein expression and quantitative analysis of cleaved Caspase3 in mouse myocardial tissue detected by Western blot. *vs. Control, P <0.05; **vs. Control, P <0.01; ***vs. Control, P <0.001.). Figure 2 Results of Ang II-induced oxidative stress and apoptosis in AC16 cardiomyocytes provided in Example 1 of the present invention (A, AC16 cardiomyocytes were treated with 100 nM Ang II for 48 h, and the green fluorescence intensity was observed by fluorescence microscopy to reflect the content of intracellular ROS (Scale bar, 50 μm); B-C, the contents of ROS and MDA in AC16 cardiomyocytes were detected by fluorescence microplate reader; D-G, protein expression and quantitative analysis of 4-HNE and cleaved Caspase3 in AC16 cardiomyocytes were detected by Western blot. **vs. Control, P <0.01.). Figure 3 Results of Ang II-induced IDO1 expression in cardiomyocytes provided in Example 1 of the present invention (A-C, protein expression and quantitative analysis of gp91 phox and IDO1 in mouse myocardial tissue detected by Western blot; D, IDO1 mRNA level in mouse myocardial tissue detected by RT-qPCR; E-G, AC16 cardiomyocytes were treated with 100 nM Ang II for 48 h, and protein expression and quantitative analysis of gp91 phox and IDO1 in AC16 cardiomyocytes were detected by Western blot. *vs. Control, P <0.05; **vs. Control, P <0.01; ***vs. Control, P <0.001.). Figure 4Results of Epacadostat inhibiting IDO1 expression to alleviate Ang II-induced oxidative damage in cardiomyocytes provided in Example 1 of the present invention (A-B, AC16 cardiomyocytes were treated with different concentrations of Epacadostat for 48 h, and Western blot was used to detect the protein expression of IDO1 and quantitative analysis; C-H, AC16 cardiomyocytes were treated with 4 μM Epacadostat for 48 h, and Western blot was used to detect the protein expressions of IDO1, gp91 phox, cleaved Caspase3 and 4-HNE and quantitative analysis; I-J, a fluorescence microplate reader was used to detect the contents of ROS and MDA in AC16 cardiomyocytes; K, a fluorescence microscope was used to observe the green fluorescence intensity to reflect the content of intracellular ROS (Scale bar, 50 μm). *vs. Control, P <0.05; **vs. Control, P <0.01; ## vs. AngII, P <0.01; ### vs. Ang II, P <0.001.); Figure 5 Results of IDO1 promoting ANGPTL4 expression provided in Example 2 of the present invention (A-C, Western blot was used to detect the protein expressions of ANGPTL4, p-FAK and FAK in mouse myocardial tissue and quantitative analysis; D, RT-qPCR was used to detect the ANGPTL4 mRNA level in mouse myocardial tissue; E-G, AC16 cardiomyocytes were treated with 100 nM Ang II for 48 h, and Western blot was used to detect the protein expressions of ANGPTL4, p-FAK and FAK in AC16 cardiomyocytes and quantitative analysis; H-J, AC16 cardiomyocytes were treated with 100 nM Ang II combined with 4 μM Epacadostat for 48 h, and Western blot was used to detect the protein expressions of p-FAK, FAK and ANGPTL4 and quantitative analysis. *vs. Control, P <0.05; **vs. Control, P <0.01; *** vs. Control, P <0.001; ****vs. Control, P <0.0001; #### vs. Ang II, P <0.0001.); Figure 6Results of Ang II upregulating the expression of ANGPTL4 to induce oxidative damage in cardiomyocytes provided in Example 2 of the present invention (A-H, Western blot was used to detect the protein expression of ANGPTL4, IDO1, p-FAK, FAK, gp91 phox, cleaved Caspase3 and 4-HNE in AC16 cardiomyocytes and quantitative analysis; I-J, a fluorescence microplate reader was used to detect the contents of ROS and MDA in AC16 cardiomyocytes; K, a fluorescence microscope was used to observe the green fluorescence intensity to reflect the content of intracellular ROS (Scale bar, 50 μm). ** vs. Control, P <0.01; ***vs. Control, P <0.001; # vs. Ang II, P <0.05; ## vs. Ang II, P <0.01; ### vs. Ang II, P <0.001; ns, no statistical difference.); Figure 7 Results of Epacadostat antagonizing the non-enzymatic function of IDO1 to promote Notch1 / HES1 signaling provided in Example 3 of the present invention (A-B, Western blot was used to detect the protein expression of cleaved Notch1 in mouse myocardial tissue and quantitative analysis; C, RT-qPCR was used to detect the HES1 mRNA level in mouse myocardial tissue; D-E, AC16 cardiomyocytes were treated with 4 μM Epacadostat combined with 100 μM Kynurenine for 48 h, and Western blot was used to detect the protein expression of cleaved Notch1 and quantitative analysis; F, AC16 cardiomyocytes were treated with 4 μM Epacadostat for 48 h, and RT-qPCR was used to detect the HES1 mRNA level in AC16 cardiomyocytes; G, the tyrosine phosphorylation (p-TYR) level (IB) was detected after immunoprecipitation (IP) with an IDO1 antibody.*vs.Control, P <0.05; ** vs. Control, P <0.01; ***vs. Control, P <0.001; ## vs. AngII, P <0.01; ### vs. Ang II, P <0.001; ns, no statistical difference.); Figure 8Results provided by Example 3 of the present invention showing that HES1 is a transcriptional repressor of ANGPTL4 (A, Binding Motif map of HES1 extracted from the JASPAR database; B, ChIP-seq data extracted from the Cistrome Date Browser, showing that there is a binding peak of HES1 in the promoter region of the ANGPTL4 gene; C, Prediction of possible binding sites of the transcriptional repressor HES1 to the promoter region of the ANGPTL4 gene through the JASPAR database; D, Verification of the binding of HES1 to the promoter region of the ANGPTL4 gene by ChIP-PCR experiment; E-F, Analysis of the difference in the content of HES1 bound to the promoter region of the ANGPTL4 gene in AC16 cardiomyocytes of the control group and the Ang II group by ChIP-PCR and ChIP-qPCR. **** vs. Control, P <0.0001.). Detailed implementation mode

[0016] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] The following describes in detail the specific implementation of the present invention with reference to specific embodiments.

[0018] Example 1: Experimental analysis of Epacadostat alleviating Ang II-induced cardiac insufficiency and myocardial oxidative damage by inhibiting IDO1: After 8-week-old healthy male C57BL / 6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were adaptively fed for 1 week, a small dose of Ang II (0.5 mg / kg, Sigma-Aldrich) was subcutaneously injected every other day for 2 months. After the drug withdrawal, the mice were observed for 6 months. The control group was injected with the same dose of normal saline to establish a myocardial oxidative damage model. After 6 months, cardiac function was detected by echocardiography, and myocardial tissues were collected to extract proteins and RNA to detect oxidative stress indexes (4-HNE and MDA), apoptosis indexes (cleaved Caspase3), the expression of IDO1 and gp91 phox, and the results were as follows Figure 1As shown, it can be seen that after 6 months of chronic Ang II stimulation, the results of echocardiography showed that compared with the control group of mice, the left ventricular end-diastolic diameter (reflecting the degree of cardiac dilation) and the thickness of the left ventricular posterior wall in diastole (reflecting the degree of myocardial hypertrophy) in the Ang II group of mice increased, while the cardiac systolic function indexes ejection fraction and shortening fraction were significantly decreased; and in the myocardial tissue of the Ang II group of mice, the expression of the oxidative stress index 4-HNE protein was significantly increased, and the content of the lipid peroxidation product MDA and the protein expression of the apoptosis index cleaved Caspase3 were also significantly higher than those in the control group. These results indicate that low-dose, long-term Ang II stimulation can induce oxidative stress and apoptosis in the myocardial tissue of mice, and then lead to left ventricular dilation, myocardial hypertrophy and heart failure, that is, the occurrence of cardiomyopathy, which clarifies that Ang II induces myocardial oxidative damage and hypofunction in mice, and is accompanied by high expression of IDO1; Human AC16 cardiomyocytes were treated with 100 nM Ang II for 48 h to establish a cardiomyocyte oxidative damage model, and oxidative damage indexes such as 4-HNE, MDA, ROS and cleaved Caspase3 were detected. The results are as follows Figure 2 As shown, using the DCFH-DA fluorescent probe method to label intracellular ROS, it was found that in AC16 cardiomyocytes treated with 100 nM Ang II, the green fluorescence intensity was significantly higher than that in the control group, and the intracellular ROS and MDA contents, 4-HNE and the protein expression of the apoptosis index cleaved Caspase3 were also significantly higher than those in the control group, indicating that Ang II induces oxidative stress and apoptosis in cardiomyocytes in vitro; As follows Figure 3 As shown, in the myocardial tissue induced by Ang II and in the oxidative damage model group of AC16 cardiomyocytes in vitro, the transcriptional level and protein expression of IDO1 and the protein expression of gp91 phox were significantly higher than those in the control group, suggesting that IDO1 may be involved in Ang II-induced myocardial oxidative damage; AC16 cardiomyocytes were treated with different concentrations (0, 0.5, 2, 4, 8, 16 µM) of Epacadostat (purchased from Selleck) for 48 h respectively, and the protein expression level of IDO1 was detected. The results are as follows Figure 4As shown, it can be seen that when 4 μM Epacadostat (with the best inhibitory effect) acts on AC16 cardiomyocytes, the protein expression of IDO1 can be significantly inhibited. Compared with the control group, the expressions of gp91 phox and IDO1 in the Ang II group both increase. After treatment with Epacadostat, the increases in gp91 phox and IDO1 proteins induced by Ang II can be reversed, and the protein expressions of oxidative damage indicators 4-HNE and cleaved Caspase3, as well as the contents of ROS and MDA, also decrease significantly. The above results prove that Epacadostat reduces the expression of IDO1, inhibits the activation of the core subunit gp91 phox of NOX2, and alleviates the oxidative damage of cardiomyocytes induced by Ang II.

[0019] Example 2: Experimental analysis of Epacadostat targeting IDO1 to inhibit ANGPTL4 / gp91 phox and relieve myocardial oxidative damage induced by Ang II: The experimental procedure was referred to Example 1, and the expressions of ANGPTL4, p-FAK, and FAK in mouse myocardial tissues and AC16 cardiomyocytes were detected respectively, and the results are as Figure 5 shown. It can be seen that compared with the control group, the expression of ANGPTL4 in mouse myocardial tissues and the phosphorylation level of downstream FAK (i.e., the p-FAK / FAK ratio) in the Ang II group both increase significantly. The same changes were also observed in AC16 cardiomyocytes stimulated by Ang II in vitro. After inhibiting the high expression of IDO1 induced by Ang II with Epacadostat, the expression of ANGPTL4 and the phosphorylation level of FAK also decreased accordingly; The human AC16 cardiomyocyte cell line with low expression of ANGPTL4 was constructed by knocking down ANGPTL4, and IDO1 and the above oxidative damage indicators were detected to analyze the effect of knocking down ANGPTL4 on the oxidative damage of AC16 cardiomyocytes induced by Ang II, as Figure 6 shown. It can be seen that compared with the Ang II injury group, the expression of IDO1 in the ANGPTL4 knockdown group did not change, but the phosphorylation of FAK and the protein expression of gp91 phox both decreased, and the protein expressions of cleaved Caspase3 and 4-HNE, as well as the contents of ROS and MDA, also decreased accordingly, indicating that knocking down ANGPTL4 can reverse the oxidative damage of cardiomyocytes induced by Ang II, and Ang II plays a role in promoting the oxidative damage of cardiomyocytes by upregulating the expression of ANGPTL4.

[0020] Based on the above results, it is suggested that Epacadostat antagonizes the high expression of IDO1 induced by Ang II, inhibits the expression of ANGPTL4 and the activation of gp91 phox, and alleviates the oxidative damage of cardiomyocytes.

[0021] Example 3. Experimental analysis of Epacadostat antagonizing the non-enzymatic function of IDO1 to inhibit ANGPTL4 / gp91 phox: The experimental procedure was referred to Example 1, and then the expressions of cleaved Notch1 and HES1 in mouse myocardial tissues and AC16 cardiomyocytes were detected. The results were as Figure 7 shown. In in vivo experiments, it was found that compared with the control group, the protein expression of the activated form of Notch1 receptor - cleaved Notch1 and the mRNA level of downstream target gene HES1 in myocardial tissues of Ang II group mice were significantly decreased. Meanwhile, in AC16 cardiomyocytes induced by Ang II in vitro, the protein level of cleaved Notch1 and the mRNA level of HES1 were also significantly decreased. After using Epacadostat to inhibit the expression of IDO1, the inhibitory effect of Ang II on the expression of cleaved Notch1 and the transcription of HES1 could be reversed, that is, the inhibitory effect on the Notch1 / HES1 pathway; AC16 cardiomyocytes were treated with 4 μM Epacadostat combined with 100 μM Kynurenine (MedChemExpress) for 48 h, and the changes in the phosphorylation level of IDO1, the protein of the activated form of Notch1 - cleaved Notch1, and the mRNA expression of its downstream target gene HES1 were detected. As Figure 7 shown, exogenous supplementation of Kynurenine was used to restore the enzymatic function of IDO1, but the activation of Notch1 caused by Epacadostat inhibition of IDO1 was not restored. Therefore, it was considered that the effect of IDO1 on the Notch1 / HES1 signaling pathway might depend on its non-enzymatic function; further, through co-immunoprecipitation experiments, it was found that Epacadostat inhibited the phosphorylation of the tyrosine site (TYR) of IDO1, and this site was related to the non-enzymatic function of IDO1. Thus, it was speculated that IDO1 might inhibit the Notch1 / HES1 signaling pathway through non-enzymatic signals; To further clarify how the Nocth1 / HES1 signaling pathway regulates the expression of ANGPTL4, the promoter region of ANGPTL4 was identified through the NCBI database, that is, the DNA sequence from 2000 bp upstream to 100 bp downstream of the transcription starting site (TSS) of the ANGPTL4 gene. Then, the binding Motif map of HES1 was extracted through the JASPAR database, and the ChIP-seq data extracted from the Cistrome Date Browser, as Figure 8As shown, binding peaks of HES1 were found in the promoter region of the ANGPTL4 gene, predicting possible binding sites between the transcriptional repressor HES1 and the promoter region of the ANGPTL4 gene. Primer sequences were designed based on the three predicted binding sites respectively, and the binding of HES1 to the three potential sites in the promoter region of ANGPTL4 was verified by ChIP-PCR experiments. The results showed that the transcriptional repressor HES1 could directly bind to the first predicted site in the promoter region of the ANGPTL4 gene. The enrichment difference of HES1 in the promoter region of the ANGPTL4 gene before and after Ang II stimulation was analyzed by ChIP-PCR experiments and ChIP-qPCR. The results showed that the binding amount of HES1 to the promoter region of the ANGPTL4 gene decreased significantly after Ang II treatment, suggesting that the inhibitory effect of HES1 on ANGPTL4 weakened after Ang II treatment. The above results suggest that Ang II inhibits the Notch1 / HES1 signal through IDO1, reducing the inhibitory effect of HES1 on ANGPTL4 transcription, thereby leading to increased ANGPTL4 expression and promoting the occurrence of oxidative damage in cardiomyocytes.

[0022] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of an IDO1 non-enzymatic function inhibitor in the preparation of a drug for treating cardiomyopathy.

2. Use of the IDO1 non-enzymatic function inhibitor according to claim 1 in the preparation of a medicament for treating cardiomyopathy, characterized in that, The cardiomyopathy is Ang II-induced cardiomyopathy.

3. Use of the IDO1 non-enzymatic function inhibitor according to claim 1 in the preparation of a medicament for treating cardiomyopathy, characterized in that, The IDO1 non-enzymatic function inhibitor is Epacadostat.

4. Use of the IDO1 non-enzymatic function inhibitor according to claim 3 in the preparation of a medicament for treating cardiomyopathy, characterized in that, The Epacadostat antagonizes the non-enzymatic function of IDO1, promotes the Notch / HES1 signal, inhibits the activation of ANGPTL4 / gp91 phox, and reduces Ang II-induced myocardial oxidative damage.

5. A drug for treating cardiomyopathy, characterized in that, The drug comprises an IDO1 non-enzymatic function inhibitor.

6. The medicament for treating cardiomyopathy according to claim 5, wherein, The cardiomyopathy is Ang II-induced cardiomyopathy.

7. The drug for treating cardiomyopathy according to claim 5, wherein, The IDO1 non-enzymatic function inhibitor is Epacadostat.

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