A high-throughput method for rapid detection and assessment of skeletal muscle toxicity of compounds

Abstract

The present invention discloses a method for rapid high-throughput detection and evaluation of the skeletal muscle toxicity of a compound. The method takes the skeletal muscle-specific calcium ion channel RYR1 as a target, combines a cell line stably expressing RYR1 with a calcium-sensitive fluorescent probe, screens and identifies compounds that can induce activation of the calcium ion channel RYR1 and lead to an increase in calcium ions in cells stably expressing RYR1, and then detects and evaluates the skeletal muscle toxicity of the compound. Compared with existing muscle toxicity detection methods, the method has high sensitivity, short time consumption and low cost, provides a rapid screening method for muscle toxicity adverse reactions in drug development and compound muscle toxicity monitoring, and has good development prospects.

Description

Technical Field

[0001] The present invention relates to a method for detecting and evaluating the skeletal muscle toxicity of a compound, and in particular to a method for high-throughput rapid detection and evaluation of the skeletal muscle toxicity of a compound. Background Art

[0002] Skeletal muscle is the main organ for body movement and an important organ for energy metabolism. Many toxic and harmful pollutants such as botulinum toxin target skeletal muscle for damage. Skeletal muscle toxicity is also a common adverse reaction of many drugs such as anesthetics, lipid-lowering drugs statins, and immunotherapy agents PD-1 inhibitors. Therefore, identifying and evaluating skeletal muscle toxicity is of great significance to protecting life and health and reducing drug development costs.

[0003] There is currently no recognized method for detecting and evaluating skeletal muscle toxicity, nor is there a mature animal model for muscle toxicity. In muscle toxicity studies, muscle injury modeling is generally performed by intramuscular injection of glycerol, but this method simulates mechanical muscle damage and cannot reflect chemical muscle damage after compound exposure. In addition, this method has the inherent defects of animal experiments, such as high cost, long cycle, and inability to quantify and batch test. Moreover, there is currently no in vitro test method for muscle toxicity. This lack of mature screening methods has seriously hindered the quantification and standardization of muscle toxicity assessment and subsequent toxicity control work. It is urgent to establish a high-throughput rapid quantitative assessment system for muscle toxicity.

[0004] The main physiological function of skeletal muscle is muscle relaxation and contraction, and the excitation-contraction coupling of skeletal muscle is the molecular basis of relaxation and contraction. In skeletal muscle, the calcium channel RYR1 (Ryanodine receptor 1) is directly involved in the excitation-contraction coupling of skeletal muscle cells. Excitation is transmitted to the skeletal muscle membrane and transmitted to the deep part of the cell through the T-tubule, triggering the activation of the terminal pool L-type calcium channel, which in turn activates the sarcoplasmic reticulum RYR1 to release Ca 2+ Entering the cytoplasm, it produces excitation-contraction coupling (Du et al., 2002, PNAS, 99:26). RYR1 abnormalities affect the release and uptake of calcium ions in muscle cells, causing skeletal muscle contractile dysfunction. Studies have confirmed that the structural and functional abnormalities of RYR1 are closely related to skeletal muscle dysfunction-related diseases such as myasthenia gravis, central axis cavitation, malignant hyperthermia, and rhabdomyolysis (Brennan et al., 2019, Human Molecular Genetics, 28:18; Voermans et al., 2016, Revue Neurologique, 172:10). RYR1 is a biomarker for diagnosing skeletal muscle damage. Therefore, the state of the RYR1 channel can indicate whether skeletal muscle is damaged or not.

[0005] Currently, the method for monitoring RYR1 is generally [ 3 H]ryanodine isotope binding experiments and patch clamp-based calcium flux experiments. 3 H]ryanodine isotope binding assay is based on the characteristics that low concentration ryanodine binds to RYR1 with high affinity without changing the function of RYR1 channel. 3 The binding level of H]ryanodine to RYR1 reflects the functional state of RYR1. This method has been widely used in the functional study of RYR1, but the experiment involves isotopes, which poses a health hazard to the experimenters, and the experiment needs to be done overnight, which is time-consuming. Patch clamp technology is the "gold standard" for studying ion channels. It reflects the molecular activity of a single or multiple ion channels on the cell membrane by measuring the ionic current of ion channels. Patch clamp technology has high requirements for instrument and personnel operation, is expensive, and cannot be screened on a large scale. Moreover, both of the above techniques need to be performed on isolated and purified RYR1 or sarcoplasmic reticulum, which makes the experiment difficult. Therefore, it is of great significance to develop high-throughput, rapid, simple, and low-cost technologies for the discovery and identification of skeletal muscle toxins. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a method for high-throughput rapid detection and evaluation of the skeletal muscle toxicity of a compound using the calcium ion channel RYR1 as a biomarker to simulate the physiological conditions of skeletal muscle motor function in vivo to the greatest extent.

[0007] Technical solution: The present invention provides a high-throughput rapid detection and evaluation method for the skeletal muscle toxicity of a compound, which uses the skeletal muscle-specific calcium channel RYR1 as a biomarker, and uses a cell line stably expressing RYR1 and a calcium-sensitive fluorescent probe to screen and identify compounds that can induce activation of the calcium channel RYR1 and lead to an increase in calcium ions in cells stably expressing RYR1, thereby detecting and evaluating the skeletal muscle toxicity of the compound.

[0008] Among them, the method is direct excitation or secondary excitation: the direct excitation is to inoculate and plate a cell line stably expressing RYR1, add a calcium ion dye for incubation, add the test compound and positive control, solvent control and negative control, and detect real-time fluorescence; the secondary excitation is to inoculate and plate a cell line stably expressing RYR1, add a calcium ion dye for incubation, add the test compound for pre-stimulation, add the test compound again and detect real-time fluorescence.

[0009] Wherein, the real-time fluorescence is detected, the fluorescence signal excitation wavelength is 470-495 nm, the measurement wavelength is 515-575 nm, the fluorescence signal is detected 600-800 times at intervals of 1-10 seconds.

[0010] Wherein, in the direct stimulation, the time of adding the test compound is 20 to 60 seconds from the start of detection, with 1 second as the detection interval.

[0011] Wherein, the secondary excitation has a detection interval of 1 s when the test compound is added for pre-stimulation, and a detection interval of 1 to 10 s when the test compound is added again after 300 to 350 s of pre-stimulation.

[0012] The cell line stably expressing RYR1 is a cell line overexpressing RYR1, including tool cells stably expressing RYR1 obtained by plasmid transfection or lentivirus infection.

[0013] Wherein, the cell line stably expressing RYR1 is a HEK293 cell line stably expressing RYR1.

[0014] The positive control is caffeine, the negative control is Hank's Balanced Salt Solution (HBSS), the solvent control is HBSS containing the solvent of the test compound, and the solvate includes one or more of dimethyl sulfoxide, HBSS, physiological saline, and phosphate buffer saline (PBS).

[0015] Principle of the invention: The present invention is a fluorescence rapid detection method based on the principle of real-time monitoring of calcium ion concentration. Based on HEK293 cells that stably express RYR1 ion channel protein, calcium ion fluorescent dyes are used to optimize experimental conditions to establish a fluorescence screening technology for real-time monitoring of calcium ion channel RYR1. By monitoring the functional status of RYR1, toxins with skeletal muscle toxicity can be screened, agonists and inhibitors can be quickly screened, and agonism / inhibition curves can be obtained, providing a strategy for low-cost rapid screening and evaluation of skeletal muscle toxins.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: Compared with the existing muscle toxicity detection methods, the method for screening muscle toxins through RYR1 described in the present invention has high sensitivity, short time consumption and low cost, and provides a rapid screening method for muscle toxicity adverse reactions in drug development and compound muscle toxicity monitoring, and has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of RYR1 activation by caffeine and inhibition by 10 μM Yanodine;

[0018] Figure 2 This is a schematic diagram of activating RYR1 by simvastatin at different concentrations in Example 3 of the present invention;

[0019] Figure 3This is a schematic diagram of different concentrations of atorvastatin activating RYR1 in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with embodiments and drawings.

[0021] Among them, the FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices) instrument is a high-throughput kinetic screening platform;

[0022] HEK293 cell line stably expressing RYR1, i.e., RYR1-HEK293 (gene information: human RYR1, NM_000540.3, CDS size is 15117 bp). The cell culture medium is DMEM medium containing 10% FBS, 1% penicillin / streptomycin mixture (100×) and 1 mg / mL G418. Cell culture flasks (T25, T75) were coated with 0.1 mg / mL poly-D-lysine (PDL) aqueous solution for 1 to 2 h before use and cultured in a humidified incubator (5% CO2, 37°C) for routine culture.

[0023] Example 1

[0024] A high-throughput rapid method for detecting and evaluating caffeine skeletal muscle toxicity:

[0025] Direct stimulation method: When the cells are in the logarithmic growth phase, RYR1-HEK293 cells are inoculated into a 96-well black transparent flat-bottom cell culture plate, with 2×10 cells per well. 4 Cells were plated with PDL in advance in 96-well plates. After about 18 hours of plating, the cells grew to about 80% and could be tested.

[0026] The culture medium in the wells was discarded, and calcium ion dye was added for incubation for 45 minutes. The cell culture plate was placed in the FLIPR instrument for detection, and RYR1 agonist caffeine (final concentration of 300 μM) was added 20 seconds after the start of the detection. The fluorescence signal was detected by the FLIPR instrument with an excitation wavelength of 470-495 nm, a measurement wavelength of 515-575 nm, a detection interval of 1 second, and a detection time of 1000-1500 seconds. The experimental data were analyzed and processed using GraphPad (Version 8.0) software.

[0027] like Figure 1As shown, 300 μM caffeine can induce the opening of RYR1 channels, and the intracellular calcium ion concentration increases rapidly. 10 μM Ryanodine was added at 80 s to inhibit RYR1, and the channels were closed. Compared with the caffeine group, the intracellular calcium ion concentration was significantly reduced, confirming that the method of the present invention can indeed monitor the state of RYR1 channels in real time.

[0028] Example 2

[0029] A high-throughput rapid method for detecting and evaluating skeletal muscle toxicity of simvastatin:

[0030] The secondary stimulation method was used: when the cells were in the logarithmic growth phase, RYR1-HEK293 were inoculated into a 96-well black transparent flat-bottom cell culture plate, with 2×10 cells per well. 4 Cells were plated with PDL in advance in 96-well plates. After about 18 hours of plating, the cells grew to about 80% and could be tested.

[0031] The culture medium in the wells was discarded, and calcium ion dye was added and incubated for 45 minutes. When measuring, the test compound simvastatin (diluted stepwise into 3 concentrations) was first added to each well at 20 seconds after the start of the test, and the fluorescence signal was detected in real time at intervals of 1 second. At 300 seconds, simvastatin of the corresponding concentration was added to each well again, and the fluorescence signal was detected 200 times at intervals of 1 second, and then 300 times at intervals of 10 seconds. The fluorescence signal was detected by the FLIPR instrument with an excitation wavelength of 470 to 495 nm and a measurement wavelength of 515 to 575 nm. The experimental data were analyzed and processed using GraphPad (Version 8.0) software.

[0032] Example 3

[0033] A high-throughput rapid assay for evaluating atorvastatin skeletal muscle toxicity:

[0034] The secondary stimulation method was used: when the cells were in the logarithmic growth phase, RYR1-HEK293 were inoculated into a 96-well black transparent flat-bottom cell culture plate, with 2×10 cells per well. 4 The 96-well plate was coated with 0.1 mg / mL poly-D-lysine aqueous solution in advance. After about 18 hours of plating, the cells grew to about 80% and could be tested.

[0035] The culture medium in the wells was discarded, and calcium ion dye was added and incubated for 45 minutes. When measuring, the test compound atorvastatin (diluted stepwise into 3 concentrations) was first added to each well at 20 seconds after the start of the test, and the fluorescence signal was detected in real time at intervals of 1 second. At 300 seconds, the corresponding concentration of atorvastatin was added to each well again, and the fluorescence signal was detected 200 times at intervals of 1 second, and then 300 times at intervals of 10 seconds. The fluorescence signal was detected by the FLIPR instrument with an excitation wavelength of 470 to 495 nm and a measurement wavelength of 515 to 575 nm. The experimental data were analyzed and processed using GraphPad (Version 8.0) software.

[0036] like Figure 2 and Figure 3 As shown in the figure, the agonist caffeine induced the opening of RYR1 channels at the first administration, and the intracellular calcium ion concentration increased significantly. In the secondary agonist mode, both simvastatin and atorvastatin showed obvious agonist effects at 50μM and 100μM. Among them, simvastatin had a stronger agonist effect. The solvent control DMSO and the negative control HBSS had no reaction, indicating that the detection system is accurate and reliable.

[0037] As can be seen from the above examples, the present invention uses RYR1 as a biomarker for screening skeletal muscle toxins and establishes a monitoring system for the RYR1 channel with the help of high-throughput real-time fluorescence screening technology. Real-time monitoring of RYR1 stable transfected cells shows significant calcium ion influx after exposure to skeletal muscle toxins simvastatin and atorvastatin, indicating that skeletal muscle toxins can be screened and identified by the method described in the present invention. Compared with the reported animal modeling detection method (Matsubara et al., 2018, Toxicology Letters, 291; Watanabe et al., 2019, Toxicology Letters, 307), the detection time of the method described in the present invention is only 1 hour, which is much shorter than the 3-14 days of the animal modeling method, and is also much shorter than the conventional ion channel detection method—[ 3 H] ryanodine isotope binding experiment (Zhang Yukun et al., 2010, Journal of East China Normal University (Natural Science Edition), 2: 92). In addition, the method described in the present invention can monitor channel activity in real time, identify different calcium oscillation patterns, and detect agonist effects at the μM level. Therefore, the RYR1 monitoring method described in the present invention can become an ideal method for screening skeletal muscle toxins.

Claims

1. A method for high-throughput rapid detection and evaluation of skeletal muscle toxicity of a compound, characterized in that: The method uses the skeletal muscle-specific calcium channel RYR1 as a target, and uses a cell line stably expressing RYR1 and a calcium-sensitive fluorescent probe to screen and identify compounds that can induce activation of the calcium channel RYR1 and increase calcium ions in cells stably expressing RYR1, and then detect and evaluate the skeletal muscle toxicity of the compound; the method is direct stimulation or secondary stimulation; the cell line stably expressing RYR1 is a cell line overexpressing RYR1, including a tool cell stably expressing RYR1 obtained by plasmid transfection or lentivirus infection; The direct excitation comprises inoculating and plating a cell line stably expressing RYR1, adding a calcium ion dye for incubation, adding a test compound and a positive control, a solvent control and a negative control, and detecting real-time fluorescence; the secondary excitation comprises inoculating and plating a cell line stably expressing RYR1, adding a calcium ion dye for incubation, adding a test compound for pre-stimulation, adding the test compound again and detecting real-time fluorescence, the secondary excitation comprises adding the test compound for pre-stimulation for 300 to 350 seconds, and then adding the test compound again, with a detection interval of 1 to 10 seconds, and the cell line stably expressing RYR1 is a HEK293 cell line stably expressing RYR1.

2. The method according to claim 1, characterized in that The real-time fluorescence detection has an excitation wavelength of 470-495 nm, a measurement wavelength of 515-575 nm, and the fluorescence signal is detected 600-800 times.

3. The method according to claim 1, characterized in that For the direct stimulation, the time of adding the test compound is 20 to 60 seconds from the start of the test.

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