Use of yap / taz in preparation of drugs for treating ed

Abstract

The application discloses application of YAP / TAZ as an action target and a YAP / TAZ agonist in preparation of an ED treatment drug, and simultaneously provides an ED treatment drug, and belongs to the field of erectile dysfunction medicine. The application proves that YAP / TAZ is related to erectile dysfunction by establishing an ED model caused by BCNI, an ED model caused by X-rays and an ED model caused by a YAP / TAZ inhibitor, and proving that the expression amount of YAP / TAZ is improved by a PY-60 agonist, and then YAP / TAZ is taken as a related target for research, so that the problem that a PDE5 inhibitor is not sensitive is solved, and a new treatment direction is provided for treatment of erectile dysfunction.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for erectile dysfunction, and more specifically to the application of YAP / TAZ in the preparation of drugs for treating ED. Background Technology

[0002] Erectile dysfunction (ED), also known as impotence, is characterized by the inability of the penis to maintain an erection sufficient for sexual intercourse. Many factors can contribute to ED, including psychological factors, obesity, cardiovascular disease, and age. Furthermore, some medical procedures can cause serious complications of ED, significantly impacting the quality of life for many patients. For example, two common treatments for prostate cancer—radical prostatectomy and prostate radiotherapy—can both cause severe ED complications.

[0003] Oral PDE5 inhibitors are currently considered first-line drugs for treating erectile dysfunction (ED). These drugs promote erection by inhibiting the PDE5 enzyme, which specifically degrades cGMP in the smooth muscle of the corpus cavernosum. This inhibition leads to prolonged cGMP activity, which further reduces intracellular calcium concentration, maintains smooth muscle relaxation, and thus prolongs the erection. Although PDE5 inhibitors are a good first-line treatment, up to 35% of patients with ED may not respond to this treatment, especially since PDE5 inhibitors have no significant effect on ED complications caused by prostate cancer treatment. Therefore, finding new therapeutic targets for ED is an urgent issue.

[0004] When individuals receive sexual stimulation, neuronal nitric oxide synthase (nNOS) in non-adrenergic, non-cholinergic nerve fibers and endothelial nitric oxide synthase (eNOS) in vascular endothelial cells synthesize and release nitric oxide (NO). The generated nitric oxide diffuses across the smooth muscle cell membrane, activating soluble guanylate cyclase (GC), which further activates guanylate cyclase (cGMP) in smooth muscle cells. Increased cGMP in the cytoplasm activates protein kinase G (PKG). PKG phosphorylates some related intracellular proteins and ion channels, causing cell hyperpolarization and reducing intracellular calcium levels. 2+ At rest, the smooth muscle cells relax, the cavernous arteries are no longer compressed, and venous return from the surrounding area is blocked, allowing blood to fill the cavities within the corpus cavernosum, resulting in penile erection. In the resting state, G protein-coupled receptors (GPCRs) on the smooth muscle cells of the corpus cavernosum bind to ligands such as norepinephrine and angiotensin II, initiating the transition of the penis from an erect to a relaxed state. The GPCRs amplify the upstream signal cascade, increasing intracellular calcium levels. 2+ As concentration increases, Ca 2+After binding to calmodulin, it then binds to and activates myosin light chain kinase (MLCK). Activated MLCK phosphorylates myosin light chain (MLC), causing myosin to bind to actin, resulting in smooth muscle contraction, compressing the arteries supplying the corpus cavernosum, and simultaneously relieving pressure on the surrounding venous plexus, thus relaxing the penis.

[0005] YAP / TAZ was initially identified as an effector molecule of the Hippo pathway, controlling organ size during development. Research on YAP / TAZ regulation has primarily focused on the Hippo pathway. When the Hippo pathway is activated, LATS1 / 2 kinases phosphorylate YAP / TAZ; YAP / TAZ is thus retained in the cytoplasm or degraded, losing its transcriptional activity by failing to bind to its transcriptional platform TEAD. The movement of YAP / TAZ between the cytoplasm and nucleus is widely considered an important indicator of YAP / TAZ activity. Nuclear accumulation of YAP / TAZ is a key determinant of its function. Phosphorylation of LATS is the main controller of YAP / TAZ subcellular localization, leading to its degradation in the cytoplasm. Subsequent studies have found that proper regulation and normal expression of YAP / TAZ are crucial for organismal development and regeneration / repair. For example, it regulates homeostasis after tissue damage repair, determines cell fate, senses cell-cell contact inhibition, and regulates cell proliferation.

[0006] YAP overexpression significantly enhances intestinal cell proliferation without affecting overall organ size. Interestingly, intranuclear YAP is confined to intestinal stem cells at the base of intestinal crypts, and this cell population can extend to the villous apex after YAP overexpression. Following DSS treatment, YAP deficiency impairs epithelial and crypt proliferation, leading to rapid cell death, while combined YAP / TAZ knockout inhibits crypt growth in vitro, ultimately resulting in the death of mutant crypts. Furthermore, combined YAP and TAZ knockout rescues intestinal hyperplasia induced by acute activation of the Wnt pathway following APC depletion. YAP / TAZ is involved in the restoration of intestinal crypt homeostasis following radiation-induced intestinal injury and GSI-induced enteritis.

[0007] Overexpression of YAP in the liver of transgenic animals was sufficient to induce a fourfold increase in liver mass due to the proliferation of mature hepatocytes, resulting in hepatocytes acquiring cholangiocarcinoma progenitor characteristics. This overgrowth phenotype depends on a TEAD-mediated gene response. Liver overgrowth was also observed when MST1 / 2 or NF2 / merlin was deleted from the liver using gene-editing models. In these cases, it was primarily due to enhanced proliferation of oval cells, which are progenitor cells of both hepatocytes and bile duct epithelial cells after liver injury, although recent lineage-tracing data suggest that the additional oval cells in Hippo mutant mice were actually acquired through hepatocyte dedifferentiation. The enlarged liver can develop into a tumor with mixed features, resembling both hepatocellular carcinoma and cholangiocarcinoma, which is associated with abnormal migration of progenitor cells such as oval cells. Furthermore, in the Sav mutant, oval cells rapidly expand after liver injury. Bile duct hamartomas also occur in Nf2-deficient livers, with onset far earlier than signs of liver overgrowth. Importantly, the phenotype resulting from NF2 inactivation could be rescued by combined knockout of a YAP allele, establishing NF2 as a true upstream regulator of YAP in mammals. Liver-specific YAP knockout led to decreased hepatocyte proliferation and increased apoptosis, but it also induced bile duct morphology defects due to cholangiocellular dysfunction, resulting in mild hepatomegaly, steatosis, and progressive fibrosis in mice. Following bile duct ligation (a cholestatic injury model), YAP-knockout mice showed reduced ductal cell proliferation and enhanced parenchymal damage, suggesting a positive role for YAP in liver regeneration. Similar to the need for YAP, the NF2 knockout liver phenotype could also be rescued by AMOT knockout. These results are consistent with the positive effects of AMOT on YAP / TAZ function in vivo. Mice with severe bile acid homeostasis defects exhibited enlarged livers, progenitor cell proliferation, and YAP activation, and spontaneous liver tumor development.

[0008] YAP / TAZ plays a crucial role in maintaining the homeostasis and function of various organs. Numerous studies have demonstrated the importance of YAP / TAZ in maintaining the function of organs including the intestines, liver, pancreas, and heart. However, the role of YAP / TAZ in penile erectile function remains unexplored.

[0009] Therefore, how to apply YAP / TAZ protein to drugs for the treatment of erectile dysfunction is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0010] In view of this, the present invention aims to prepare a drug for treating ED by targeting the enhancement of YAP / TAZ protein activity, thus finding a new treatment strategy for ED patients who are insensitive to PDE5 inhibitors.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] Application of YAP / TAZ as a target in the preparation of drugs for treating erectile dysfunction.

[0013] As with the same inventive concept as the above-described technical solution, the present invention also claims protection for the use of YAP / TAZ agonists in the preparation of drugs for treating erectile dysfunction, wherein the YAP / TAZ agonists include PY-60.

[0014] As with the same inventive concept as the above-described technical solution, the present invention also claims protection for a drug for treating ED, the drug comprising: a substance capable of inhibiting the degradation of YAP / TAZ.

[0015] As a preferred embodiment of the above technical solution, the substance capable of inhibiting the degradation of YAP / TAZ is PY-60.

[0016] As a preferred technical solution to the above technical solutions, ED includes ED that is insensitive to PDE5 inhibitors.

[0017] As a preferred embodiment of the above technical solution, the ED that is insensitive to PDE5 inhibitors includes ED caused by BNCI.

[0018] As a preferred embodiment of the above technical solution, the ED that is insensitive to PDE5 inhibitors includes ED caused by bilateral prostate X-ray irradiation.

[0019] As demonstrated by the above technical solution, compared with existing technologies, this study has discovered a novel mechanism for erectile dysfunction (ED) beyond the classic PDE5-related pathway: YAP / TAZ. Studies have found that YAP / TAZ exhibits low levels and low activity in clinical ED patients and various ED models. Furthermore, blocking YAP / TAZ activity in mice immediately induces ED symptoms. In ED mice after radical prostatectomy who have not responded to or have had low response to PDE5 inhibitor treatment, inducing YAP / TAZ activity effectively treats ED, indicating that YAP / TAZ may indeed serve as a novel target for ED treatment and holds significant promise for drug target research. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1The attached figure shows the erectile function of mice after knocking out YAP, knocking out TAZ, and knocking out both YAP and TAZ simultaneously.

[0022] Figure 2 The attached figure shows the erectile function of mice after knocking out YAP, knocking out TAZ, and knocking out both YAP and TAZ simultaneously.

[0023] Figure 3 The attached figure shows the contractile capacity of smooth muscle cells after simultaneous knockout of YAP and TAZ.

[0024] Figure 4 The attached figure shows the contractile capacity of smooth muscle cells after simultaneous knockout of YAP and TAZ.

[0025] Figure 5 The attached figure shows a graph of erectile function in rats after BCNI.

[0026] Figure 6 The attached figure shows the YAP / TAZ immunofluorescence detection results at corresponding time points after BCNI in rats;

[0027] Figure 7 The attached figure shows the results of real-time PCR detection of the YAP / TAZ target gene Ctgf at the corresponding time points after rat BCNI. *, p<0.05; **, p<0.01;

[0028] Figure 8 The attached figure shows the results of erectile function testing in mice after treatment with the YAP / TAZ agonist PY-60 following BCNI surgery to induce erectile dysfunction.

[0029] Figure 9 The attached figure shows an image of erectile function in a rat model of erectile dysfunction induced by bilateral prostate local X-ray irradiation simulating prostate cancer treatment.

[0030] Figure 10 The attached figure shows the YAP / TAZ immunofluorescence detection results at corresponding time points after a model of erectile dysfunction (ED) induced by X-ray radiation simulating prostate cancer treatment.

[0031] Figure 11 The attached figure shows the results of quantitative real-time PCR detection of the YAP / TAZ target gene Ctgf at corresponding time points after a rat model of erectile dysfunction induced by bilateral prostate local X-ray irradiation simulating prostate cancer treatment. *, p<0.05; **, p<0.01;

[0032] Figure 12 The attached figure shows the results of erectile function testing in mice after treatment with the YAP / TAZ inhibitor Verteporfin.

[0033] Figure 13The attached figure shows the results of detecting erectile function in mice after knocking out YAP / TAZ in mouse smooth muscle cells. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] This invention provides the relationship between YAP / TAZ and related genes and erectile dysfunction (ED), and verifies this relationship by establishing different ED models. The detection methods and approaches used in the examples are as follows:

[0036] Immunofluorescence: Frozen tissue sections were fixed with 4% paraformaldehyde for 10 minutes. They were washed three times with PBS for 5 minutes each time, then perforated in PBS containing 0.5% Triton X-100 for 10 minutes, washed three times with PBS for 5 minutes each time, blocked in PBS containing 10% BSA and 0.1% Triton X-100 for 1 hour, incubated overnight with primary antibody at 4°C, and washed three times with PBS for 5 minutes each time. Secondary antibody was incubated at room temperature for 1.5 hours. They were washed three times with PBS for 5 minutes each time. Phalloidin was incubated at room temperature for 15 minutes, and finally ProLong-DAPI was added.

[0037] Intraperitoneal pressure (ICP) testing in rats and mice: Rats or mice were anesthetized by intraperitoneal injection of 40 mg / kg sodium pentobarbital. Penile tissue was exposed, and the corpus cavernosum was punctured using a 23-gauge needle connected to a catheter. Simultaneously, the right carotid artery was cannulated using a polyethylene cannula containing heparin and PBS to continuously monitor mean arterial pressure (MAP). Both were connected to a pressure sensor (Biopac System Inc., Goleta, CA, USA). The cavernous nerve was stimulated using a bipolar electrode connected to a stimulator. The stimulator was set to 5 volts 1.5 mA for 1 minute, with a minimum interval of 5 minutes between stimulations. All data were recorded using an MP100 data acquisition system and analyzed using Acqknowledge software (Biopac System Inc., Goleta, CA, USA). All ICP tests were performed by a single person to ensure minimal intragroup variability.

[0038] Quantitative Real-Time PCR: Collect rat and mouse penile tissue flash-frozen in liquid nitrogen, grind it using a grinder, and add 1 ml of Trizol. Incubate on ice for 10 min, add 200 μl of chloroform, vortex for 30 s, and centrifuge at 12000 rpm for 15 min at 4°C. Extract the supernatant RNA into a new EP tube, add 500 μl of isopropanol, vortex to mix, incubate at room temperature for 20 min, centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant. Add 1 ml of 75% ice-cold ethanol, vortex to mix, centrifuge at 7500 rpm for 5 min, discard the supernatant, and repeat 3 times. Dry at room temperature for 10 min, add an appropriate amount of DEPC water, and incubate at 55-60°C for 10-15 min. Detect the concentration; store the sample at -70°C. Take 1 μg of RNA, and perform reverse transcription under the following conditions: 37°C for 15 min, 85°C for 5 s. The reaction mixture was prepared according to the SYBR Green kit: 7.5 μl SYBR Green, 5 μl cDNA, 0.4 μl each of forward and reverse primers, and 1.7 μl DEPC water. The reaction conditions were: 95℃ for 10 min, 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 30 s, for 40 cycles. The experimental results were analyzed using the relative quantification method ΔΔCT, and the 22 was calculated. -ΔΔ CT represents the ratio of expression levels in the experimental group to those in the control group, with GAPDH used as the internal reference.

[0039] Example 1

[0040] Gene-edited mouse breeding: Myh11Cre ERT2 (019079) and YAP fl / fl ;TAZ fl / fl The (030532) genotype mouse was introduced from the Jackson laboratory. These two mouse types were crossbred to expand the genotype, and the offspring genotypes were identified, resulting in the Myh11Cre mouse. ERT2 YAP fl / + ;TAZ fl / fl (TAZ ρSM Myh11Cre ERT2 YAP fl / fl ;TAZ fl / + (YAP ρSM ) and Myh11Cre ERT2 YAP fl / fl ;TAZ fl / fl (Y / T ρSM Mice were given tamoxifen for 7 consecutive days, after which YAP and TAZ were knocked out. ICP values ​​were measured to evaluate erectile function. (See below) Figures 1-2 .

[0041] Depend on Figure 1 and Figure 2It can be seen that simply knocking out YAP or TAZ can reduce erectile function in mice. When YAP / TAZ is completely knocked out, the erectile function of mice is basically lost. This suggests that YAP / TAZ can be used as a target to treat erectile dysfunction.

[0042] Example 2

[0043] Gene-edited mouse breeding and smooth muscle cell extraction: Myh11Cre ERT2 (019079) and YAP fl / fl ;TAZ fl / fl The (030532) genotype mouse was introduced from the Jackson laboratory. These two mouse types were crossbred to expand the genotype, and the offspring genotypes were identified, resulting in the Myh11Cre mouse. ERT2 YAP fl / fl ;TAZ fl / fl (Y / T ρSM Mice. Y / T ρSM The corpus cavernosum of the penis of mice was minced and then digested with collagenase I, II, and IV at 37°C. After 4 hours, the cells were removed and cultured in primary corpus cavernosum smooth muscle culture medium. The smooth muscle cells were then knocked out by YAP / TAZ (Y / T KO).

[0044] The two groups of cells underwent a contraction-relaxation assay as follows: Cells from each group were digested into a cell suspension, mixed with rat tail collagen, and the mixture was added to agar plates. After solidification, the appropriate culture medium was added, and the gel was released from the bottom of the plate after 24 hours. After another 24 hours, photographs were taken to measure the contraction-relaxation area of ​​each group of gels.

[0045] The results are as follows Figure 3 and Figure 4 As shown, compared with the control group, Y / T KO significantly improved the contractile ability of smooth muscle cells. This suggests that YAP / TAZ knockout can lead to enhanced smooth muscle cell contractile ability. Smooth muscle cell contraction results in insufficient blood flow during penile erection, weakened erectile function or inability to achieve erection, leading to ED.

[0046] Example 3: Establishment of a rat model of erectile dysfunction induced by BCNI

[0047] Male SD rats (approximately 250g, ordered from the Animal Center of Soochow University) were anesthetized with sodium pentobarbital at 40mg / kg. The animals were placed in a sterile environment constructed with sterile drapes, and all instruments were autoclaved to ensure sterility. A 1-2 cm incision was made along the midline of the animal's abdomen. The cavernous nerve, located posterolateral to the prostate, was located using a sterile cotton swab. The cavernous nerve was isolated as much as possible and clamped with hemostatic forceps for 4 minutes on each side. After clamping, the wound was sutured and disinfected. In the sham surgery group, the cavernous nerve was not clamped; after confirming the prostate and cavernous nerve, the wound was sutured and disinfected. All BCNI and sham surgeries were performed by the same person to minimize intra-group variability. Erectile function testing of rats after BCNI surgery is shown in [link to BCNI procedure]. Figure 5 ,Depend on Figure 5 It can be seen that erectile dysfunction was observed in rats after BCNI, indicating successful modeling.

[0048] Then, mice that successfully modeled erectile dysfunction were selected for immunofluorescence detection of YAP / TAZ and quantitative real-time PCR detection of the YAP / TAZ target gene Ctgf to clarify the role of YAP / TAZ in erectile dysfunction. Results are shown below. Figures 6-7 This indicates that in mice with erectile dysfunction, the expression levels of YAP / TAZ protein and the target gene Ctgf are both reduced, suggesting that YAP / TAZ protein and Ctgf are associated with ED.

[0049] Example 4

[0050] In the BCNI mouse model established in Example 1, mice were treated with the YAP / TAZ agonist PY-60 at a concentration of 2.5 mg / kg in DMSO.

[0051] PY-60 was ordered from MCE, product number HY-141644, administered every two days for 20 days. Erectile function in mice was then assessed. (See below) Figure 8 .

[0052] Example 5: Rats with erectile dysfunction induced by bilateral prostate X-ray irradiation

[0053] Male SD rats weighing approximately 250g were anesthetized with 40mg / kg sodium pentobarbital and placed in a prone position on an X-ray small animal therapy device (X-RAD SmART, Precision X-Ray Inc., USA). Whole-body CT scans were performed to obtain 3D images, followed by detailed CT scans of the abdominal and pelvic cavities to obtain more detailed 3D images. The location of the prostate was determined by the position of the bladder, and the posterolateral portion of the prostate was selected as the irradiation area, irradiated with a single 20Gy X-ray. All irradiation site selection was performed by a single person to ensure minimal intragroup variation. The role of YAP / TAZ in erectile dysfunction was clarified through erectile function testing, immunofluorescence, and quantitative real-time PCR detection of the YAP / TAZ target gene Ctgf (Ctgf is only used to demonstrate the function of YAP / TAZ; since YAP / TAZ needs to enter the cell nucleus to participate in transcriptional regulation and does not function in the cytoplasm, and the protein level of YAP is relatively stable, directly detecting the protein and RNA levels of YAP cannot reflect its activity. YAP / TAZ activity is determined by detecting its transcriptional activity, i.e., its regulation of the downstream target gene Ctgf). See [link to quantitative real-time PCR]. Figures 9-11 This indicates that YAP / TAZ is related to ED.

[0054] Example 6: Mouse YAP / TAZ Inhibitor Model

[0055] Mice were administered Verteporfin 100 mg / kg via intraperitoneal injection (0.2 ml / 10 g) while protected from light. After injection, the needle was rotated and withdrawn, and pressure was applied for two minutes to prevent leakage. This was repeated three times a week for two weeks. The posterolateral ganglion of the prostate was electrically stimulated, and ICP values ​​were measured to assess erectile function. (See below) Figure 12 .

[0056] Example 7

[0057] Gene-edited mouse breeding: Myh11Cre ERT2 (019079) and YAP fl / fl ;TAZ fl / fl The (030532) genotype mouse was introduced from the Jackson laboratory. These two mouse types were crossbred to expand the genotype, and the offspring genotypes were identified, resulting in the Myh11Cre mouse. ERT2 YAP fl / fl ;TAZ fl / fl (Y / TSM) mice were administered tamoxifen for 7 consecutive days, after which YAP and TAZ were knocked out. ICP values ​​were measured to evaluate erectile function. Figure 13 .

[0058] Depend on Figure 13It is known that inhibiting or knocking out YAP / TAZ expression can induce erectile dysfunction in mice, indicating that YAP / TAZ can be used as a target for the treatment of erectile dysfunction.

[0059] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0060] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of YAP / TAZ agonists in the preparation of drugs for treating erectile dysfunction (ED), wherein the YAP / TAZ agonist is PY-60.

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