Use of the TRPML1-specific small molecule agonist ML-SA1 in pharmaceuticals

Abstract

The present invention provides the use of a TRPML1-specific small molecule agonist ML-SA1 in the pharmaceutical industry. Through single and multiple uranium exposures to simulate acute and chronic uranium-exposed mouse models, it was confirmed that delayed administration of the small molecule compound ML-SA1 can significantly promote the urinary uranium excretion of acute and chronic uranium-exposed mice, reduce renal uranium accumulation, alleviate the pathological damage and apoptosis of uranium-induced renal proximal tubular epithelial cells, protect renal function, and is closely related to the significant improvement of lysosomal exocytosis; through the uranium-exposed human renal proximal tubular epithelial HK-2 cell model, it was confirmed that delayed administration of the small molecule compound ML-SA1 significantly promotes the excretion of uranium accumulated in HK-2 cells by promoting lysosomal exocytosis and accompanying compensatory lysosomal biogenesis, and significantly reduces uranium-induced cell death. Since the small molecule compound ML-SA1 excretes uranium deposited in lysosomes out of the cell through lysosomal exocytosis, thereby effectively reducing the intracellular uranium content, it can also be used for the enhanced excretion and detoxification of other heavy metal poisonings deposited in lysosomes.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an application of a small molecule compound ML-SA1. Background Art

[0002] Uranium (U) has been produced and used on a large scale worldwide as an important nuclear fuel in the nuclear industry. Its byproduct, depleted uranium (DU), is also widely used in military and industrial fields. 238 U. 235 U and 234 U composition) for nuclear fuel 235 The production, processing and post-processing of U and the use of depleted uranium weapons may cause a certain degree of pollution to the surrounding environment or cause accidents, and then enter the human body through the respiratory tract, digestive tract or skin wounds. The resulting health hazards have attracted much attention from countries around the world.

[0003] Uranium has chemical and radiotoxic effects on the human body. Natural uranium and DU are mainly composed of 238 U( 238 U content is 99.28% and >99.8% respectively), and its radioactivity is very low, mainly chemical toxicity. The kidney is the main target organ of U toxicity, among which the renal proximal tubules, especially the S3 segment renal proximal tubule epithelial cells, are the main target cells of U toxicity. Both acute and chronic U exposure can cause varying degrees of renal damage, and severe cases can lead to renal failure or even death. The use of chelators to accelerate the excretion of U in the body is its main treatment measure, but at present, there is only one drug, sodium bicarbonate, for clinical treatment of acute U poisoning at home and abroad, and the efficacy of promoting excretion is still uncertain. It also has the side effect of causing mild alkalosis and electrolyte imbalance in the body, and has no obvious therapeutic effect on chronic U poisoning; furthermore, the biggest defect of effective U excretion chelators such as catechols and hydroxypyridone chelators in the research and development stage at home and abroad in recent years is that they are ineffective in delayed administration of acute U poisoning and chronic U poisoning treatment. The fundamental reason is that the U accumulated in the cells cannot be excreted, which has become an important problem facing the current U excretion and detoxification treatment.

[0004] Previous studies have found that needle-like U-phosphate crystals can be observed deposited in lysosomes in vitro cultured renal proximal tubular epithelial cells (porcine LLC-PK1 cells and rat NRK-52E cells) exposed to soluble U. This phenomenon has also been observed in vitro cultured rat ROS 17 / 2.8 osteosarcoma cells and UMR-106 osteosarcoma cells exposed to soluble U. Moreover, the more U-phosphate deposited intracellularly, the greater the toxicity to the cells. U-phosphate crystal precipitation has also been observed extracellularly, presumably as a result of cell death and disintegration or lysosomal exocytosis, but no direct experimental evidence has been obtained to support this. In addition, in vitro cultured human renal HEK-293 cells, human hepatoma HepG2 cells, and human neuroblastoma IMR-32 cells exposed to DU, it has also been observed that the higher the DU exposure concentration or the longer the exposure time, the earlier the intracellular DU deposition forms and the greater the deposition amount. Transmission electron microscopy has observed that the DU crystals in lysosomes develop from single needle-like structures to clustered distributions with the prolongation of DU exposure time. There are also studies reporting that mineral metal elements such as cerium and niobium are deposited in the lysosomes of renal tubular epithelial cells of rats exposed to cerium and niobium. The above studies have shown that after U enters the cell, it is deposited in lysosomes in the form of U-phosphate crystals. As the amount of U entering the cell increases and the U-phosphate crystals in lysosomes continue to grow, the lysosomal membrane ruptures and the acidic hydrolases therein are released into the cytoplasm, ultimately leading to cell death and disintegration. Therefore, promoting lysosomal exocytosis before the lysosomal membrane containing U-phosphate crystals is damaged, thereby promoting the excretion of intracellular U, may be the most promising way to solve the bottleneck problem of "unable to excrete the U accumulated in cells", and may also be an effective way for the enhanced excretion and detoxification treatment of other harmful metal elements deposited in lysosomes.

[0005] Lysosomes are vesicular organelles widely present in eukaryotic cells and enclosed by a single membrane, with dual functions of digestion and transportation: their lumen contains more than 60 acidic hydrolases, which can degrade exogenous particles from endocytosis / phagocytosis and intracellular components from autophagy in cells to defend against the invasion of foreign pathogens and recycle the "waste" in cells after degradation to maintain the normal metabolic activities of cells. They are the main "digestive" organelles in cells. Lysosomal exocytosis is an important export pathway of lysosomes, which can release lysosomal contents such as degradation products to the outside of the cell and plays an important role in multiple cellular physiological processes such as secretion, neurotransmitter release, and plasma membrane repair. Lysosomal exocytosis requires two consecutive steps: the first step is Ca 2+ independent, after being stimulated, lysosomes move along microtubules from the perinuclear region to near the plasma membrane; the second step is Ca 2+ dependent, local Ca 2+The increase causes the lysosomes pre-anchored to the plasma membrane to fuse with the plasma membrane, releasing the contents extracellularly. Transient receptor potential cation channel mucolipin subfamily-2 (TRPML) protein subfamily member TRPML1 is an important Ca 2+ channel mainly distributed on the lysosomal membrane. When it is activated, it mediates the release of Ca 2+ from the lysosomal lumen into the cytoplasm, triggering the fusion of lysosomes with the plasma membrane. Transcription factor EB (TFEB) is a key regulator mainly controlling lysosome biogenesis and autophagy. Once activated, it translocates from the cytoplasm to the nucleus, inducing the transcriptional activation of coordinated lysosome expression and regulation (CLEAR) genes such as TRPML1, lysosome-associated membrane protein 1 (LAMP1), and autophagy genes. While TRPML1 is transcriptionally regulated as a TFEB target gene, it can also activate TFEB through TRPML1-mediated Ca 2+ release, and TRPML1 is also the main effector molecule for TFEB activation. Studies have found that overexpression of TFEB effectively reduces the accumulation of stored substances in lysosomes in the lysosomal storage disease (LSD) cell model through lysosomal exocytosis mediated by the activation of TRPML1. Thus, it can be seen that the Ca 2+ channel protein TRPML1 on the lysosomal membrane is a key regulator of lysosomal exocytosis. ML-SA1 is a specific small molecule agonist of TRPML1 and has been used in the study of the regulatory mechanism of lysosomal exocytosis. Moreover, ML-SA1 effectively promotes the clearance of cholesterol in lysosomes by triggering lysosomal exocytosis through the activation of TRPML1 in LSD cells cultured in vitro, but whether it can be used for the promotion of heavy metal excretion and detoxification has not been reported yet.

[0006] In recent years, studies have shown that the translocation of lysosomal molecular markers LAMP1 / 2 or TRPML1 to the plasma membrane is a marker of lysosomal exocytosis (Reddy, A., Caler, E.V. & Andrews, N.W. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell 106, 157-169 (2001). Xu, H.X. & Ren, D.J. Lysosomal Physiology. Annu Rev Physiol 77, 57-80 (2015).)

[0007] The asymmetric distribution of LAMP1 / 2 on the plasma membrane is a hallmark of lysosomal polarized exocytosis (Toops KA, Lakkaraju A. Let’s play a game of chutes and ladders: Lysosome fusion with the epithelial plasma membrane. Commun Integr Biol 6, e24474 (2013)), and lysosomes translocate from the perinuclear region to the vicinity of the plasma membrane before exocytosis (Xu, H. X. & Ren, D. J. Lysosomal Physiology. Annu Rev Physiol 77, 57 - 80 (2015).), indicating that the distribution of LAMP1 / 2 and TRPML1 on the apical membrane of the proximal renal tubule marks the exocytosis of lysosomes from the proximal renal tubular epithelial cells into the renal tubule lumen. Summary of the Invention

[0008] The present invention intends to provide a new drug for promoting the excretion and detoxification of acute / chronic uranium poisoning / internal contamination targeting a novel target to meet the needs of nuclear accident emergency treatment and chronic uranium poisoning / internal contamination, and specifically relates to the application of a pharmaceutically acceptable small molecule compound ML - SA1 and its pharmaceutical composition in the treatment of acute / chronic uranium poisoning / internal contamination.

[0009] The object of the present invention is to overcome the problems of delayed administration of existing uranium excretion and detoxification drugs for acute uranium poisoning / internal contamination and no therapeutic effect on chronic uranium poisoning / internal contamination, and to develop a therapeutic drug targeting the promotion of lysosomal exocytosis and new pathways of lysosome biogenesis.

[0010] The present invention provides the application of a small molecule compound ML - SA1 in the preparation of drugs for promoting the excretion and detoxification of heavy metal poisoning / internal contamination. Preferably, the heavy metal is uranium.

[0011] Furthermore, the uranium poisoning / internal contamination is acute uranium poisoning / internal contamination, and / or chronic uranium poisoning / internal contamination.

[0012] Furthermore, the small molecule compound ML - SA1 is effective for delayed administration after acute uranium poisoning / internal contamination.

[0013] Furthermore, the small molecule compound ML - SA1 is used after chronic uranium poisoning / internal contamination.

[0014] Furthermore, the small molecule compound ML - SA1 promotes the excretion of heavy metals accumulated in the proximal renal tubular epithelial cells by promoting lysosomal exocytosis and lysosome biogenesis.

[0015] Furthermore, the heavy metal excretion and detoxification drug includes active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient includes the small molecule compound ML-SA1.

[0016] The present invention also discloses a pharmaceutical composition, characterized in that it comprises an effective therapeutic amount of the small molecule compound ML-SA1 and pharmaceutically acceptable excipients.

[0017] Furthermore, the pharmaceutical composition is in the form of injection, tablet, capsule, granule, aerosol or oral liquid.

[0018] Furthermore, the pharmaceutical composition is used to treat acute / chronic heavy metal poisoning / internal pollution. Preferably, the heavy metal is uranium.

[0019] The small molecule compound ML-SA1 (CAS: 332382-54-4) of the present invention is a TRPML1 channel specific agonist, and its chemical structure is as follows:

[0020]

[0021] Beneficial effects of the present invention:

[0022] The small molecule compound ML-SA1 of the present invention is a TRPML1 channel-specific agonist because its mechanism of action is to activate the uranium-accumulating renal proximal tubular epithelial cell lysosomal membrane channel protein TRPML1 to release Ca 2+ , triggering the fusion of lysosomes and the plasma membrane to promote lysosomal exocytosis, and after TRPML1 activation, it then promotes TFEB nuclear translocation to increase lysosomal biogenesis, synergistically promoting the excretion of uranium accumulated in cells, rather than forming chelates with uranium to promote its excretion. Therefore, in theory, the delayed administration of the small molecule compound ML-SA1 after acute uranium poisoning / internal pollution and the treatment of chronic uranium poisoning / internal pollution are effective. This speculation has been confirmed by the present invention, and the present invention further confirms from the cellular level that ML-SA1 has the effect of promoting excretion and reducing uranium-induced cytotoxicity in renal proximal tubular epithelial cells with uranium accumulation caused by short-term or long-term uranium poisoning; because the small molecule compound ML-SA1 expels uranium deposited in lysosomes out of cells through lysosomal exocytosis, thereby effectively reducing the role of intracellular uranium, it can also be used for the promotion and detoxification of other heavy metal poisoning deposited in lysosomes. In addition, the CCK-8 method shows that the small molecule compound ML-SA1 has no obvious toxicity to HK-2 cells when it is 36 times higher than its uranium promotion and detoxification concentration and the action time is extended by 48 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the effect of the small molecule compound ML-SA1 of the present invention on promoting excretion and alleviating kidney damage in mice exposed to acute uranium poisoning;

[0024] For a - g, after mice were intramuscularly injected (im) with 0.4 mg / kg and 2 mg / kg uranium and then intraperitoneally injected (ip) with 400 μg / kg and 800 μg / kg ML - SA1 24 h later, relevant indexes were detected 24 h after administration. Among them, a is the result of detecting the 24 - h urinary uranium excretion by ICP - MS; b is the result of detecting the renal uranium accumulation by ICP - MS; c is the detection result of serum creatinine and urea nitrogen; d and e are the pathological image of kidney tissue and the injury score results of epithelial cells in S1, S2 and S3 segments of renal proximal tubules respectively; f and g are the image and data processing results of detecting apoptosis of epithelial cells in S3 segment of renal proximal tubules in kidney tissue sections by TUNEL method.

[0025] Figure 2 It is the effect diagram of the small - molecule compound ML - SA1 in promoting uranium excretion and reducing kidney injury in chronically uranium - poisoned mice in the present invention;

[0026] For a - h, mice were intramuscularly injected (im) with 80 μg / kg uranium every day for 5 consecutive days, and then intraperitoneally injected (ip) with 400 μg / kg ML - SA1 24 h after the last uranium poisoning. Relevant indexes were detected 24 h after administration. Among them, a is the result of detecting the 24 - h urinary uranium excretion by ICP - MS; b is the result of detecting the renal uranium accumulation by ICP - MS; c and d are the image of detecting the expression of KIM - 1 in epithelial cells of S3 segment of renal proximal tubules by immunohistochemistry and the data processing results of the expression levels of KIM - 1 in epithelial cells of S1, S2 and S3 segments of renal proximal tubules respectively; e and f are the pathological image of kidney tissue and the injury score results of epithelial cells in S1, S2 and S3 segments of renal proximal tubules respectively; g and h are the image and data processing results of detecting apoptosis of epithelial cells in S3 segment of proximal tubules in kidney tissue sections by TUNEL method.

[0027] Figure 3 It is the effect diagram of the small - molecule compound ML - SA1 on the lysosomal exocytosis of epithelial cells in renal proximal tubules of acutely and chronically uranium - poisoned mice in the present invention;

[0028] Among them, a and b are respectively the images of immunohistochemical detection of the expression of LAMP1 and TRPML1 proteins in the epithelial cells of the S3 segment of the renal proximal tubule and the data processing graphs of the expression in the epithelial cells of the S1, S2, and S3 segments of the renal proximal tubule after 24 hours of intraperitoneal injection (ip) of 400 μg / kg ML-SA1 24 hours after intramuscular injection (im) of 0.4 mg / kg uranium in mice; c and d are respectively the images of immunohistochemical detection of the expression of LAMP1 and TRPML1 proteins in the epithelial cells of the S3 segment of the renal proximal tubule and the data processing graphs of the expression in the epithelial cells of the S1, S2, and S3 segments of the renal proximal tubule after 24 hours of intraperitoneal injection (ip) of 400 μg / kg ML-SA1 24 hours after the last uranium exposure in mice that were intramuscularly injected (im) with 80 μg / kg uranium daily for 5 consecutive days.

[0029] Figure 4 It is the effect diagram of the small molecule compound ML-SA1 in the present invention promoting the excretion of intracellular uranium and reducing uranium-induced cell death by activating lysosomal exocytosis mediated by TRPML1 in uranium-accumulated HK-2 cells;

[0030] Among them, a is that HK-2 cells were exposed to different concentrations of uranyl acetate from 0 to 1200 μM for 24 hours, and a lactate dehydrogenase (LDH) kit was used to detect the LDH content in the cell culture medium to evaluate the cytotoxicity of uranium at different concentrations; b is that HK-2 cells were exposed to 0 and 600 μM uranyl acetate for 24 hours, and after treatment with 10 μM of ML-SA1 for 30 minutes, the Ca 2+ release was detected by the Fura-2 AM (calcium ion fluorescence probe) method; c-d is that HK-2 cells were exposed to 0, 50, 100, and 600 μM uranyl acetate for 24 hours, and were treated with 10 μM ML-SA1 alone, or 2 μM Vacuolin-1, or both 10 μM ML-SA1 and 2 μM Vacuolin-1 for 30 minutes. Among them, c is to detect the β-hex content in the cell culture medium using a β-hexosaminidase (β-hex) kit, and d is to detect the uranium content in the cells and the uranium content in the cell culture medium using ICP-MS; e is that HK-2 cells were exposed to 0, 100, and 600 μM uranyl acetate solutions for 24 hours, and were treated with 10 μM ML-SA1 alone, or 2 μM Vacuolin-1, or both 10 μM ML-SA1 and 2 μM Vacuolin-1 for 30 minutes, and the death of uranium-accumulated HK-2 cells was detected by calcein / propidium iodide staining.

[0031] Figure 5 It is the effect diagram of the small molecule compound ML-SA1 in the present invention activating the TFEB activity of uranium-accumulated HK-2 cells to promote lysosome biogenesis;

[0032] Among them, for HK-2 cells exposed to 0 and 600 μM uranyl acetate for 24 h and then treated with 10 μM ML-SA1 for 30 min, relevant indicators were detected. Among them, a and b are the images and data processing diagrams of detecting TFEB nuclear translocation by immunofluorescence method; c and d are the images and gray value analysis data diagrams of detecting the protein expressions of LAMP1 and TRPML1 by Western blot method.

[0033] Figure 6 It is the effect diagram of the small molecule compound ML-SA1 in the present invention promoting lysosomal exocytosis by activating the TRPML1 and TFEB targets of uranium-accumulated HK-2 cells, thereby increasing the uranium excretion in cells and reducing uranium-induced cell death.

[0034] Among them, a and b are the images and gray value analysis data diagrams of detecting the TRPML1 protein expression 48 h after HK-2 cells are transfected with TRPML1 shRNA plasmid by Western blot method; c and d are the images and gray value analysis data diagrams of detecting the TFEB protein expression 48 h after HK-2 cells are transfected with TFEB shRNA plasmid by Western blot method; e, f, and i are for HK-2 cells with transient knockdown of TRPML1 expression and empty vector HK-2 cells exposed to 0, 50 and / or 100, 600 μM uranyl acetate for 24 h and then treated with 10 μM ML-SA1 for 30 min. Among them, e is to detect the β-hex content in the cell culture medium by β-hex activity detection kit, f is to detect the uranium content in cells and the uranium content in the cell culture medium by ICP-MS, and i is to detect cell death by calcein / PI staining; g, h, and j are for HK-2 cells with transient knockdown of TFEB expression and empty vector HK-2 cells exposed to 0, 50 and / or 100, 600 μM uranyl acetate for 24 h and then treated with 10 μM ML-SA1 for 30 min. Among them, g is to detect the β-hex content in the cell culture medium by β-hex activity detection kit, h is to detect the uranium content in cells and the uranium content in the cell culture medium by ICP-MS, and j is to detect cell death by calcein / PI staining.

[0035] Figure 7 It is the effect diagram of the small molecule compound ML-SA1 in the present invention on the excretion of accumulated uranium in HK-2 cells exposed to uranium for a long time.

[0036] Among them, for HK-2 cells exposed to 1 μM uranyl acetate for 10, 20, and 30 days, 10 μM ML-SA1 was given for 30 min, and the intracellular uranium content was detected by ICP-MS; b was for HK-2 cells exposed to 5 μM uranyl acetate for 10, 20, and 30 days, 10 μM ML-SA1 was given for 30 min, and the intracellular uranium content was detected by ICP-MS; c was for HK-2 cells exposed to 10 μM uranyl acetate for 10, 20, and 30 days, 10 μM ML-SA1 was given for 30 min, and the intracellular uranium content was detected by ICP-MS.

[0037] Figure 8 It is the effect diagram of the cytotoxicity of the small molecule ML-SA1 on HK-2 cells in the present invention. Specific embodiments

[0038] The following further describes the specific embodiments of the present invention in detail in conjunction with the examples and the drawings. The following examples and drawings are used to illustrate the present invention, but not to limit the scope of the present invention.

[0039] Example 1:

[0040] The uranium excretion-promoting effect of the small molecule compound ML-SA1 on acutely uranium-exposed mice and its role in reducing uranium-induced proximal renal tubular injury

[0041] SPF-grade male Balb / c mice with a body weight of 20 ± 2 g were randomly grouped by body weight stratification, with 5 - 6 mice in each group. They were intramuscularly injected (im) with 0.4 and 2 mg / kg of uranium to simulate acute internal uranium contamination / uranium poisoning. At 24 h after uranium exposure, they were intraperitoneally injected (ip) with 400 and 800 μg / kg of ML-SA1. Urine was collected 24 h after administration. The mice were anesthetized 24 h after administration, blood was taken by eye enucleation, and then tissue perfusion was performed with 4°C phosphate buffer (PBS), and bilateral kidneys were taken. After 24 h urine and unilateral kidneys were digested with nitric acid / perchloric acid, the urinary uranium excretion and renal uranium accumulation were measured by ICP-MS; the other kidney was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and the pathological morphological changes of the S1, S2, and S3 segments of the proximal renal tubules were observed under a microscope, and the apoptosis of the S3 segment of the proximal renal tubules was detected by the TUNEL method.

[0042] As can be seen from Figure 1 a and 1b, the delayed administration of 400 μg / kg ML-SA1 for 24 h can significantly increase the 24 h urinary uranium excretion in mice with acute uranium exposure of 0.4 mg / kg and 2 mg / kg, which are increased by about 86% and 105% respectively compared with the group with uranium exposure alone ( Figure 1 a), and the renal uranium accumulation is significantly reduced, which are reduced by about 58% and 52% respectively compared with the group with uranium exposure alone ( Figure 1b); Similarly, administration of 800 μg / kg ML-SA1 with a 24-hour delay can significantly increase the 24-hour urinary uranium excretion in mice acutely exposed to 0.4 mg / kg and 2 mg / kg uranium, which are approximately 113% and 106% higher than those in the group with uranium exposure alone ( Figure 1 a), and the renal uranium accumulation is significantly reduced, approximately 43% and 57% lower than those in the group with uranium exposure alone ( Figure 1 b), but there is no significant difference compared with the effect of delayed administration of 400 μg / kg ML-SA1 ( Figure 1 a,1b).

[0043] The results of the effects of different doses of uranium exposure on renal injury in mice showed that there was no significant difference in the levels of serum creatinine and urea nitrogen, the renal function indicators, in mice exposed to 0.4 mg / kg uranium for 48 hours compared with the blank control group ( Figure 1 c), indicating that renal function injury had not occurred yet, but the pathological scores of necrosis and exfoliation of epithelial cells in the S1, S2, and S3 segments of the proximal renal tubules and the apoptosis detected by TUNEL were significantly higher than those in the mice of the blank control group ( Figure 1 d,1e,1f,1g); the levels of serum creatinine and urea nitrogen in mice exposed to 2 mg / kg uranium for 48 hours were significantly higher than those in the blank control group ( Figure 1 c), indicating that renal function injury had been induced, and the pathological injury of necrosis and exfoliation of epithelial cells in the S1, S2, and S3 segments of the proximal renal tubules and the apoptosis detected by TUNEL were significantly higher than those in the blank control group ( Figure 1 d,1e,1f,1g), and were significantly higher than those in mice exposed to 0.4 mg / kg uranium ( Figure 1 d,1e,1f), indicating that the renal injury in mice exposed to 2 mg / kg uranium was significantly more severe than that in mice exposed to 0.4 mg / kg uranium. Delayed administration of 400 μg / kg ML-SA1 for 24 hours can not only significantly reduce the pathological injury of necrosis and exfoliation of epithelial cells in the S1, S2, and S3 segments of the proximal renal tubules and the apoptosis detected by TUNEL in mice exposed to 0.4 mg / kg and 2 mg / kg uranium ( Figure 1 d,1e,1f,1g), but also significantly reduce the levels of serum creatinine and urea nitrogen in mice exposed to 2 mg / kg uranium ( Figure 1 c), showing an obvious effect of reducing uranium-induced renal injury.

[0044] The above results indicate that delayed administration of ML-SA1 can not only significantly promote the urinary uranium excretion in mice with mild renal injury caused by low-dose uranium exposure, significantly reduce the renal uranium accumulation, and alleviate uranium-induced injury of proximal renal tubular epithelial cells, but also has a significant effect of promoting uranium excretion in mice with renal function injury caused by high-dose uranium exposure, and can also significantly alleviate uranium-induced injury of proximal renal tubular epithelial cells and protect renal function.

[0045] Example 2:

[0046] Promoting effect of small molecule compound ML-SA1 on uranium excretion in chronically uranium-exposed mice and its role in alleviating uranium-induced renal proximal tubular injury

[0047] SPF-grade male Balb / c mice weighing 20±2 g were randomly divided into groups according to body weight in a stratified manner, with 4 mice in each group. They were injected intramuscularly (im) with 80 μg / kg uranium once a day for 5 consecutive days to simulate chronic uranium exposure. At 24 h after the last uranium exposure, 400 μg / kg ML-SA1 was injected intraperitoneally (ip). Urine was collected 24 h after drug administration. After anesthetizing the mice 24 h after drug administration and performing tissue perfusion with 4°C PBS, bilateral kidneys were taken. After digesting 24 h urine and a single kidney with nitric acid / perchloric acid, inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the urinary uranium excretion and renal uranium accumulation. The other kidney was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and immunohistochemistry was used to detect the protein expression of kidney injury molecule-1 (KIM-1), an early renal injury molecular marker in the S1, S2, and S3 segments of the renal proximal tubules. After hematoxylin-eosin (HE) staining of renal tissue sections, the pathological morphological changes in the S1, S2, and S3 segments of the renal proximal tubules were observed under a microscope, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptosis in the S3 segment of the renal proximal tubules.

[0048] As shown in Figure 2 a and 2b, administration of 400 μg / kg ML-SA1 significantly increased the 24 h urinary uranium excretion in chronically uranium-exposed mice, which was approximately 117% higher than that in the group with uranium exposure alone ( Figure 2 a), and significantly decreased the renal uranium accumulation, which was approximately 33% lower than that in the group with uranium exposure alone ( Figure 2 b); similarly, administration of 800 μg / kg ML-SA1 also significantly increased the 24 h urinary uranium excretion in chronically uranium-exposed mice, which was approximately 118% higher than that in the group with uranium exposure alone ( Figure 2 a), and significantly decreased the renal uranium accumulation, which was approximately 35% lower than that in the group with uranium exposure alone ( Figure 2 b), but there was no significant difference in the effect compared with administration of 400 μg / kg ML-SA1 ( Figure 2 a, 2b).

[0049] The results of the effect of delayed administration of 400 μg / kg ML-SA1 on uranium-induced renal injury showed that although there were no abnormalities in the serum creatinine and blood urea nitrogen levels in chronically uranium-exposed mice at 80 μg / kg / d * 5 d (data not shown), the early renal injury markers KIM-1 in the epithelial cells of the S1, S2, and S3 segments of the renal proximal tubules, the pathological scores of cell necrosis and exfoliation, and the apoptosis detected by TUNEL were all significantly higher than those in the blank control group of mice. Figure 2c, 2d, 2e, 2f, 2g, 2h). Administration of 400 μg / kg ML-SA1 significantly decreased the protein level of KIM-1 in the epithelial cells of the S1, S2, and S3 segments of the proximal renal tubules, the pathological score of cell necrosis and exfoliation, and the apoptosis detected by TUNEL in chronically uranium-exposed mice Figure 2 c, 2d, 2e, 2f, 2g, 2h).

[0050] The above results indicate that administration of ML-SA1 can significantly increase the urinary uranium excretion in chronically uranium-exposed mice, significantly reduce the renal uranium accumulation, and alleviate the damage of uranium-induced proximal renal tubular epithelial cells.

[0051] Example 3:

[0052] Effect of small molecule compound ML-SA1 on promoting lysosomal exocytosis in acutely and chronically uranium-exposed mice

[0053] SPF-grade male Balb / c mice weighing 20 ± 2 g were randomly divided into groups according to body weight in a stratified manner, with 4 - 6 mice in each group. They were intramuscularly injected (im) with 0.4 mg / kg uranium to simulate acute uranium exposure and intramuscularly injected (im) with 80 μg / kg uranium once a day for 5 consecutive days to simulate chronic uranium exposure. At 24 h after the last uranium exposure, 400 μg / kg ML-SA1 was intraperitoneally injected (ip). At 24 h after administration, the mice were anesthetized and perfused with 4°C PBS. One kidney was taken, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and the expression of lysosomal molecular markers lysosome-associated membrane protein 1 (LAMP1) and lysosomal non-selective cation channel 1, namely TRPML1, in the epithelial cells of the S1, S2, and S3 segments of the proximal renal tubules was detected by immunohistochemistry.

[0054] In recent years, studies have shown that the translocation of lysosomal molecular markers LAMP1 / 2 or TRPML1 to the plasma membrane is a sign of lysosomal exocytosis, and the asymmetric distribution of LAMP1 / 2 on the plasma membrane is a sign of lysosomal polarized exocytosis. Moreover, lysosomes will shift from the perinuclear region to near the plasma membrane before exocytosis, indicating that the distribution of LAMP1 / 2 and TRPML1 on the apical membrane of the proximal renal tubules marks the occurrence of lysosomal exocytosis towards the renal tubular lumen in the epithelial cells of the proximal renal tubules. By Figure 3As shown in a, 3b, 3c and 3d, administration of ML-SA1 (400 μg / kg), a specific agonist of TRPML1, significantly increased the distribution of LAMP1 and TRPML1 on the apical membrane of epithelial cells in the S1, S2 and S3 segments of the proximal renal tubules in mice, indicating that ML-SA1 promoted lysosomal exocytosis in the epithelial cells of the S1, S2 and S3 segments of the proximal renal tubules; whether it was acute uranium exposure or chronic uranium exposure, compared with the blank control group, simple uranium exposure alone could also significantly increase the distribution of LAMP1 and TRPML1 on the apical membrane of epithelial cells in the S1, S2 and S3 segments of the proximal renal tubules, but it was significantly lower than the effect of ML-SA1; importantly, administration of ML-SA1 could further increase the distribution of LAMP1 and TRPML1 on the apical membrane of epithelial cells in the S1, S2 and S3 segments of the proximal renal tubules in mice with acute and chronic uranium exposure.

[0055] The above results indicate that ML-SA1 can trigger lysosomal exocytosis in the epithelial cells of the S1, S2 and S3 segments of the proximal renal tubules in mice with acute and chronic uranium exposure, and its effect is significantly stronger than that of simple uranium exposure alone.

[0056] Example 4:

[0057] The small molecule compound ML-SA1 promotes the excretion of intracellularly accumulated uranium and reduces uranium-induced cell death by activating TRPML1-mediated lysosomal exocytosis in HK-2 cells with uranium accumulation caused by short-term uranium exposure

[0058] Human renal proximal tubular epithelial HK-2 cells were exposed to 0, 50, 100, 600, 900 and 1200 μM uranyl acetate solution for 24 h, and a lactate dehydrogenase cytotoxicity detection kit was used to detect the death of uranium-induced HK-2 cells. HK-2 cells were exposed to 0, 600 μM uranyl acetate solution for 24 h, and 10 μM of ML-SA1 was given for 30 min. The Fura-2AM fluorescence probe method was used to detect the Ca 2+ release. HK-2 cells were exposed to 0, 50, 100 and 600 μM uranyl acetate solution for 24 h. 10 μM of ML-SA1 was given alone, 2 μM of the lysosomal exocytosis inhibitor Vacuolin-1 was given alone, and 10 μM of ML-SA1 and 2 μM of Vacuolin-1 were given simultaneously for 30 min. A β-hexosaminidase activity detection kit was used to detect the content of β-hex in the cell culture medium. ICP-MS was used to detect the uranium content in HK-2 cells and the cell culture medium, and calcein / propidium iodide staining was used to detect cell death.

[0059] By Figure 4a It can be seen that the relative percentage of LDH release (i.e., cell mortality) of HK-2 cells induced by uranium exposure at different concentrations for 24 h increased significantly with the increase in uranium exposure concentration. Among them, uranium exposure at 100 μM induced obvious cytotoxicity, and uranium exposure at 600 μM led to nearly 50% cell death. From Figure 4 b It can be seen that the treatment of HK-2 cells with 10 μM of ML-SA1 for 30 min could significantly increase the release of Ca 2+ in lysosomes mediated by TRPML1 (lysosomal exocytosis is Ca 2+ -dependent), which was significantly higher than that of the vehicle control group, indicating that ML-SA1 activated the lysosomal exocytosis triggered by the release of Ca 2+ mediated by TRPML1 in HK-2 cells; importantly, ML-SA1 could also significantly increase the release of Ca 2+ mediated by TRPML1 in HK-2 cells with uranium accumulation induced by uranium exposure at cytotoxic doses, which was significantly higher than that of the group with only uranium exposure, indicating that ML-SA1 could effectively activate the lysosomal exocytosis triggered by the release of Ca 2+ mediated by TRPML1 in HK-2 cells with uranium accumulation induced by uranium exposure at cytotoxic doses. From Figure 4 c It can be seen that the treatment of HK-2 cells with 10 μM of ML-SA1 alone for 30 min could make the content of β-hex in the cell culture medium of HK-2 cells significantly higher than that of the vehicle control group, indicating that ML-SA1 triggered the lysosomal exocytosis of HK-2 cells; the treatment of HK-2 cells with 2 μM of the lysosomal exocytosis inhibitor Vacuolin-1 alone for 30 min could make the content of β-hex in the cell culture medium of HK-2 cells significantly lower than that of the vehicle control group, indicating that Vacuolin-1 inhibited the lysosomal exocytosis of HK-2 cells; while the simultaneous treatment of HK-2 cells with 10 μM of ML-SA1 and 2 μM of Vacuolin-1 for 30 min could significantly reduce the effect of ML-SA1 in increasing the content of β-hex in the cell culture medium of HK-2 cells, further indicating that ML-SA1 played a role in promoting lysosomal exocytosis. Importantly, the treatment of HK-2 cells with 10 μM of ML-SA1 for 30 min could make the content of β-hex in the cell culture medium of HK-2 cells with uranium accumulation induced by uranyl acetate at 50, 100, and 600 μM significantly higher than that of the vehicle control group, the treatment of HK-2 cells with 2 μM of the lysosomal exocytosis inhibitor Vacuolin-1 for 30 min could make the content of β-hex in the cell culture medium of HK-2 cells with uranium accumulation induced by uranyl acetate at 50, 100, and 600 μM significantly lower than that of the vehicle control group, and the simultaneous treatment of HK-2 cells with 10 μM of ML-SA1 and 2 μM of Vacuolin-1 for 30 min could significantly reduce the effect of ML-SA1 in increasing the content of β-hex in the cell culture medium of HK-2 cells with uranium accumulation induced by uranyl acetate at 50, 100, and 600 μM ( Figure 4c), indicating that ML-SA1 can significantly promote lysosomal exocytosis in HK-2 cells with uranium accumulation induced by uranium exposure at non-cytotoxic and cytotoxic doses. Meanwhile, more importantly, treatment with 10 μM ML-SA1 for 30 min can significantly reduce the intracellular uranium content and increase the extracellular uranium content in HK-2 cells with uranium accumulation induced by 50, 100, and 600 μM uranyl acetate exposure compared with the vehicle control group; treatment with 2 μM lysosomal exocytosis inhibitor Vacuolin-1 for 30 min can significantly increase the intracellular uranium content and decrease the extracellular uranium content in HK-2 cells with uranium accumulation induced by 50, 100, and 600 μM uranyl acetate exposure compared with the vehicle control group; while simultaneous treatment with 10 μM ML-SA1 and 2 μM Vacuolin-1 for 30 min can significantly increase the intracellular uranium content and decrease the extracellular uranium content in HK-2 cells with uranium accumulation induced by 50, 100, and 600 μM uranyl acetate exposure compared with the vehicle control group ( Figure 4 d), indicating that ML-SA1 promotes the excretion of intracellular uranium by activating lysosomal exocytosis. As Figure 4 shown in e, there were no significant changes in the cell death rate of cells treated with 10 μM ML-SA1 alone, 2 μM lysosomal exocytosis inhibitor Vacuolin-1 alone, or simultaneous treatment with 10 μM ML-SA1 and 2 μM Vacuolin-1 for 30 min compared with the vehicle control group, indicating that ML-SA1 and Vacuolin-1 alone and their simultaneous action have no cytotoxicity to HK-2 cells; treatment with 10 μM ML-SA1 for 30 min can significantly reduce the death rate of HK-2 cells exposed to 100 and 600 μM uranyl acetate, treatment with 2 μM lysosomal exocytosis inhibitor Vacuolin-1 for 30 min can significantly increase the death rate of HK-2 cells exposed to 100 and 600 μM uranyl acetate, while simultaneous treatment with 10 μM ML-SA1 and 2 μM Vacuolin-1 for 30 min can significantly increase the death rate of HK-2 cells exposed to 100 and 600 μM uranyl acetate, indicating that ML-SA1 significantly reduces uranium-induced HK-2 cell death by triggering lysosomal exocytosis.

[0060] The above results indicate that ML-SA1 can promote the excretion of intracellular uranium by promoting lysosomal exocytosis in HK-2 cells with uranium accumulation induced by uranium exposure at non-cytotoxic and cytotoxic doses, and significantly reduce uranium-induced cell death.

[0061] Example 5:

[0062] The small molecule compound ML-SA1 promotes lysosome biogenesis by activating TFEB in HK-2 cells with uranium accumulation induced by short-term uranium exposure

[0063] Human kidney proximal tubular epithelial HK-2 cells were used and exposed to 0 and 600 μM uranyl acetate solution for 24 h, then treated with 10 μM ML-SA1 for 30 min. Immunofluorescence was used to detect the nuclear translocation of the transcription factor TFEB that regulates lysosome biogenesis, and Western blot was used to detect the protein expression of the lysosomal molecular markers LAMP1 and TRPML1, so as to observe the activation effect of ML-SA1 on TFEB and the influence on lysosome biogenesis in HK-2 cells with uranium accumulation caused by cytotoxic dose of uranium exposure.

[0064] As shown in Figure 5 a, 5b, 5c and 5d, treatment with 10 μM ML-SA1 for 30 min could significantly increase the positive cell rate of TFEB in the nucleus of HK-2 cells, which was significantly higher than that of the vehicle control group. The protein expression of LAMP1 and TRPML1, the downstream target genes of TFEB and related to lysosome biogenesis, was also significantly increased compared with the vehicle control group, indicating that ML-SA1 activated the function of TFEB in HK-2 cells and enhanced lysosome biogenesis. Importantly, ML-SA1 could also significantly increase the positive cell rate of TFEB in the nucleus of HK-2 cells with uranium accumulation caused by cytotoxic dose of uranium exposure, which was significantly higher than that of the group with uranium exposure alone. The protein expression of the downstream target genes LAMP1 and TRPML1 of TFEB was also significantly increased compared with the group with uranium exposure alone, indicating that ML-SA1 could effectively activate the function of TFEB in HK-2 cells with uranium accumulation caused by cytotoxic dose of uranium exposure and enhance lysosome biogenesis.

[0065] The above results indicate that ML-SA1 can promote lysosome biogenesis by activating TFEB in HK-2 cells with uranium accumulation caused by cytotoxic dose of uranium exposure.

[0066] Example 6:

[0067] The effect of the small molecule compound ML-SA1 on promoting the exocytosis of lysosomes to excrete intracellularly accumulated uranium and reducing uranium-induced cell death in HK-2 cells with uranium accumulation caused by short-term uranium exposure depends on TRPML1 and TFEB

[0068] TRPML1 shRNA (Origene, USA, catalog number: TR303307) and TFEB shRNA (Miaoling Biotechnology, Wuhan, catalog number: P31675) were used to knockdown the expression of TRPML1 and TFEB genes in human renal proximal tubular epithelial HK-2 cells, respectively. Western blot was used to detect the protein expression of TRPML1 and TFEB, respectively. HK-2 cells with transient knockdown expression of TRPML1 and TFEB were established, and HK-2 cells transfected with the corresponding empty vectors (Origene, USA, catalog number: TR30012; Miaoling Biotechnology, Wuhan, catalog number: P0684) were established. The HK-2 cells with transient knockdown expression of TRPML1 and TFEB and the corresponding empty vector cells were exposed to 0, 50, 100, and 600 μM uranyl acetate solution for 24 h, or treated with 10 μM ML-SA1 for 30 min after uranium exposure for 24 h. The content of β-hex in the cell culture medium was detected using a β-hexosaminidase activity detection kit, the uranium content in HK-2 cells and the cell culture medium was detected using ICP-MS, and cell death was detected using calcein / propidium iodide staining.

[0069] As shown in Figure 6 a, 6b, 6c, and 6d, HK-2 cells with transient knockdown expression of TRPML1 and TFEB and the corresponding empty vector HK-2 cells were established by transfection with TRPML1 shRNA, TFEB shRNA, and the corresponding empty vector plasmids, respectively, to observe the roles of TRPML1 and TFEB in the promotion of intracellular uranium excretion mediated by lysosomal exocytosis and the reduction of uranium-induced cell death by ML-SA1. As shown in Figure 6 e, 6g, knockdown of TRPML1 or TFEB expression not only significantly inhibited the content of β-hex in the culture medium of blank control cells and the content of β-hex in the culture medium of blank control cells treated with ML-SA1, but also significantly inhibited the content of β-hex in the culture medium of HK-2 cells exposed to 50, 100, and 600 μM uranyl acetate for 24 h and the content of β-hex in the culture medium of HK-2 cells exposed to 50, 100, and 600 μM uranyl acetate for 24 h treated with ML-SA1, indicating that the effect of ML-SA1 in promoting lysosomal exocytosis in uranium-accumulated HK-2 cells depends on TRPML1 and TFEB; at the same time, knockdown of TRPML1 or TFEB expression not only significantly increased the uranium content in HK-2 cells exposed to 50, 100, and 600 μM uranyl acetate for 24 h and decreased the uranium content in the cell culture medium ( Figure 6 f, 6h), but also significantly increased the uranium content in HK-2 cells exposed to 50, 100, and 600 μM uranyl acetate for 24 h treated with ML-SA1 and decreased the uranium content in the cell culture medium ( Figure 6f, 6h), indicating that the effect of ML-SA1 on promoting the intracellular uranium efflux mediated by lysosomal exocytosis in uranium-accumulating HK-2 cells depends on TRPML1 and TFEB. As Figure 6 shown in i, 6j, knockdown of TRPML1 or TFEB not only significantly increased the cell death rate of HK-2 cells exposed to 100 and 600 μM uranyl acetate for 24 h, but also significantly increased the cell death rate of HK-2 cells exposed to 100 and 600 μM uranyl acetate for 24 h treated with ML-SA1, indicating that the effect of ML-SA1 on reducing the death of uranium-accumulating HK-2 cells depends on TRPML1 and TFEB.

[0070] The above results indicate that the effect of ML-SA1 on promoting uranium efflux and reducing uranium-induced cell death in HK-2 cells by triggering lysosomal exocytosis depends on TRPML1 and TFEB.

[0071] Example 7:

[0072] Effect of small molecule compound ML-SA1 on uranium efflux in HK-2 cells with uranium accumulation caused by long-term uranium exposure.

[0073] Human renal proximal tubular epithelial HK-2 cells were used and exposed to 1, 5, 10 μM uranyl acetate solutions for 10, 20, and 30 days respectively. ML-SA1 at 10 μM was administered for 30 min 30 min before the termination of uranium exposure, or cells were transiently transfected with TRPML1 shRNA (Origene, USA, catalog number: TR303307) 2 days before the termination of uranium exposure to knockdown the expression of TRPML1, and a group treated with transient knockdown of TRPML1 expression combined with 10 μM ML-SA1 for 30 min was set up. The treated cells were collected by trypsin digestion, lysed with alkaline lysis buffer, and the intracellular uranium content was detected by ICP-MS.

[0074] As Figure 7 shown in a, 7b and 7c, treatment with 10 μM ML-SA1 for 30 min could significantly reduce the intracellular uranium content of HK-2 cells exposed to 1, 5, 10 μM uranyl acetate solutions for 10, 20, and 30 days respectively, while transient knockdown of TRPML1 expression significantly increased the intracellular uranium content of the above long-term uranium-exposed cells and significantly reduced the effect of ML-SA1.

[0075] The above results indicate that ML-SA1 can significantly reduce the intracellular uranium content of HK-2 cells with chronic uranium exposure, and its effect depends on TRPML1.

[0076] Example 8:

[0077] Toxic effect of small molecule compound ML-SA1 on HK-2 cells

[0078] Human renal proximal tubular epithelial HK-2 cells were used and treated with different concentrations (0, 10, 20, 40, 80, 120, 160, 200, 240, 280, 320, 360 μM) of ML-SA1 for 0.5 and 24 h respectively. The cell viability was detected by the CCK-8 method.

[0079] It can be seen that when the small molecule compound ML-SA1 acts on HK-2 cells for 0.5 and 24 h respectively in the concentration range of 10 - 360 μM, it has no obvious effect on the cell viability of HK-2 cells. Figure 8

[0080] The above results indicate that when the small molecule compound ML-SA1 is 36 times higher than its concentration for promoting excretion and detoxification (10 μM) and the action time is 48 times its excretion and detoxification time, it has no obvious cytotoxic effect on HK-2 cells.

[0081] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.​

Claims

1. Use of a small molecule compound ML-SA1 in the preparation of a drug for promoting the excretion and detoxification of renal toxicity caused by heavy metal poisoning / internal contamination; The heavy metal is uranium.

2. The use according to claim 1, characterized in that: Heavy metal uranium poisoning / internal contamination is acute uranium poisoning / internal contamination, and / or chronic uranium poisoning / internal contamination.

3. The use according to claim 1, characterized in that: The small molecule compound ML-SA1 is effective when administered at a delayed time after acute uranium poisoning / internal contamination.

4. The use according to claim 1, characterized in that: The small molecule compound ML-SA1 is used after chronic uranium poisoning / internal contamination.

5. The use according to claim 1, characterized in that: The small molecule compound ML-SA1 promotes the excretion of heavy metals accumulated in renal proximal tubular epithelial cells by promoting lysosomal exocytosis and lysosomal biogenesis.

6. The use according to claim 1, characterized in that: The heavy metal-induced renal toxicity-promoting and detoxifying drug comprises an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the small molecule compound ML-SA1.

7. The use according to claim 6, characterized in that: The dosage form of the drug is injection, tablet, capsule, granule, aerosol or oral liquid.

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