Application of fuloacetone in preparation of TRPV3 ion channel inhibitor

Abstract

The invention discloses application of fuloacetone in preparation of a TRPV3 ion channel inhibitor, and belongs to the field of biology. According to the present invention, the TRPV3 can be adopted as the target spot, and the activity of the TRPV3 ion channel can be inhibited by acting on the E501 or / and the I505 on the TRPV3S2 spiral. As a selective inhibitor of the TRPV3 channel, the fuloacetone not only provides a valuable molecular tool for understanding TRPV3 channel pharmacology, but also has reutilization potential in the aspect of inhibiting skin inflammation (such as associated dermatitis and pruritus).

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to the application of floxacin in preparing TRPV3 ion channel inhibitors. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Atopic dermatitis (AD) is a common chronic inflammatory skin disease that is estimated to affect 230 million people worldwide. It is characterized by recurrent eczematous skin lesions accompanied by severe itching, which seriously affects the patient's physical and mental health. In recent years, the incidence of atopic dermatitis has continued to rise, and it has become a huge economic burden on families and society. The pathogenesis of atopic dermatitis is complex and has not yet been fully clarified. It is related to genetic susceptibility, skin barrier dysfunction, skin microbial abnormalities, and immune disorders. There is no effective cure for atopic dermatitis, and its related symptoms can only be alleviated or the disease can be avoided from recurring. The main therapeutic drugs currently are glucocorticoids, antihistamines, antibiotics, immunosuppressants, etc., but these drugs are accompanied by some serious adverse reactions.

[0004] Transient Receptor Potential Channels (TRP channels) are a superfamily of non-selective cation channels that can sense a variety of stimuli such as temperature, pain, taste, and osmotic pressure. Research results show that a variety of transient receptor potential (TRP) channels play an important role in the regulatory function of the skin, especially transient receptor potential vanilloid type 3 (TRPV3). As a member of the TRPV subfamily, TRPV3 is abundantly expressed in the epidermis and keratinocytes of the skin.

[0005] The cryo-electron microscopy structure shows that the TRVP3 channel protein is composed of a tetramer, and a single subunit mainly includes 6 transmembrane α-helices, the N-terminus and C-terminus located in the cell, and the "inverted tent"-shaped selectivity filter formed by S5 and S6. At present, it is known that there are three identified domains in the TRPV3 channel that play an important role in mediating the binding of channel inhibitors. The first site is residue Y564 located in the S4 helix, which is also the same binding site for the agonist 2-APB; the second is residue F666 located in the S6 helix, which is important for channel opening and interacts with dyclonine, isochlorogenic acid A, isochlorogenic acid B and baicalein; third, trpvicin binds and stabilizes the channel in a closed state involving two residues, A556 located in S4 and A560 located in S5. These observations indicate that known TRPV3 modulators interact with multiple sites within the channel.

[0006] The multimodal receptor TRPV3 channel is closely related to the pathological process of atopic dermatitis. More and more research results show that TRPV3 is a potential drug target for the treatment of skin diseases such as dermatitis and chronic pruritus. TRPV3 gain-of-function mutations can cause skin dysfunctions such as human Olmsted syndrome and focal palmoplantar keratoderma. Pharmacological inhibition of TRPV3 or genetic silencing of TRPV3 can relieve itching and inflammation. Therefore, it is necessary to find efficient and specific inhibitors targeting TRPV3. It is worth noting that the currently available TRPV3 inhibitors either lack selectivity or lack sufficient evaluation of other members of the TRP subfamily. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an application of spasmolytic agent floxacin as a TRPV3 inhibitor. The present invention constructs a cell line expressing TRPV3 channel protein, chemically activates TRPV3, puts the TRPV3 channel protein in a functionally enhanced state, and then uses a compound to act on cells expressing TRPV3 channel protein, and screens out TRPV3 blockers by detecting the TRPV3 channel function of the cells after the action, thereby providing floxacin that relieves skin inflammation by selectively inhibiting TRPV3.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] The first aspect of the present invention provides the use of floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof in the preparation of TRPV3 ion channel inhibitors;

[0010] The structural formula of the floxacin is shown in Formula I;

[0011]

[0012] Preferably, the floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof target TRPV3 and inhibit the activity of the TRPV3 ion channel by acting on E501 and / or I505 on the TRPV3 S2 helix.

[0013] Preferably, the TRPV3 ion channel inhibitor includes drugs and experimental reagents.

[0014] Preferably, the experimental reagents are used for basic research.

[0015] The second aspect of the present invention provides the use of floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof in the preparation of drugs for TRPV3 ion channel-related diseases.

[0016] Preferably, the floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof have TRPV3 ion channels as targets, and selectively inhibit the activity of TRPV3 ion channels to prevent and reduce / reduce tissue pathological changes caused by enhanced TRPV3 activity.

[0017] Preferably, the TRPV3 ion channel-related diseases include skin inflammation, damage, or enhanced TRPV3 activity caused by UVB irradiation, resulting in pathological changes in the tissue.

[0018] The third aspect of the present invention provides a drug for inhibiting TRPV3 ion channels, wherein the active ingredient of the drug is floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof.

[0019] Further, the drug also contains one or more pharmaceutically or food-acceptable excipients. The excipients used may be solid or liquid. Solid-state preparations include powders, tablets, dispersed granules, capsules, pills and suppositories. Powders and tablets may contain about 0.1% to about 99.9% of active ingredients. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talcum powder, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid-state preparations include solutions, suspensions and emulsions, or oral solutions with added sweeteners and contrast agents. In addition, it can also be made into small water injections for injection, freeze-dried powder injections for injection, large infusions or small infusions.

[0020] The fourth aspect of the present invention provides the use of floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof in the preparation of a drug for treating or preventing atopic dermatitis.

[0021] The beneficial effects of the present invention are:

[0022] Temperature-sensitive Ca 2+ The permeability TRPV3 ion channel is strongly expressed in skin keratinocytes, and its gain-of-function mutation is associated with the pathology of skin lesions. Here, the present invention constructs a cell line expressing TRPV3 channel protein, chemically activates TRPV3 to put the TRPV3 channel protein in a functionally enhanced state, and then uses a compound to act on cells expressing TRPV3 channel protein, and screens out TRPV3 blockers by detecting the TRPV3 channel function of the cells after the action, thereby providing a spasmolytic agent, floxacin, that relieves skin inflammation by selectively inhibiting TRPV3.

[0023] In whole-cell patch clamp recordings, floxacin selectively inhibited macroscopic TRPV3 currents in a concentration-dependent manner with an IC50 value of 17.8±3.5 μM. At the single-channel level, floxacin inhibited the probability of TRPV3 channel opening without changing its unit conductance. In an in vivo mouse model of skin inflammation induced by the skin sensitizer DNFB, floxacin also reduced back skin damage and ear skin swelling. Further molecular docking combined with site-directed mutagenesis revealed that two residues E501 and I505 in the channel S2 helix are critical for floxacin-mediated inhibition of TRPV3. The present invention also discloses the molecular mechanism of the interaction between floxacin and TRPV3 channel protein. As a selective inhibitor of TRPV3 channels, floxacin not only provides a valuable molecular tool for understanding TRPV3 channel pharmacology, but also has the potential for re-use in inhibiting skin inflammation (such as associated dermatitis and pruritus). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 The FlexStation 3 method was used to evaluate the effect of floxacin on TRPV3 channels. 200 μM floxacin (green) inhibited the calcium fluorescence signal generated by 200 μM 2-APB activating TRPV3. 0.2% DMSO was the negative control (black), and 200 μM 2-APB was the positive control (red).

[0026] Figure 2 A shows the effect of 50 μM floxacin on TRPV3 currents induced by 2-APB; Figure 2 B: 1μM, 10μM, 30μM, 100μM and 300μM floxacin inhibited the TRPV3 current activated by 50μM 2-APB in a concentration-dependent manner; Figure 2C is the IC50 of floxacin dose-dependent inhibition of TRPV3 channels fitted by the Hill equation, which is 17.80±3.48μM; Figure 2 D is a representative TRPV3 single-channel current trace and full-point amplitude histogram of single-channel current during 2-APB activation; Figure 2 E is a representative TRPV3 single-channel current trace and full-point amplitude histogram of single-channel current during floxacin inhibition; Figure 2 F is a bar chart showing the probability of TRPV3 channel opening when activated by 30 μM 2-APB or inhibited by 50 μM floxacin; Figure 2 G is the bar chart of the unit conductance of TRPV3 channels when activated by 30 μM 2-APB or inhibited by 50 μM floxacin;

[0027] Figure 3 A shows that 100 μM floxacin had no inhibitory effect on TRPA1 currents induced by 300 μM allyl isothiocyanate; Figure 3 B: 100 μM floxacin had no inhibitory effect on TRPV1 current induced by 1 μM capsaicin; Figure 3 C: 100 μM floxacin had no inhibitory effect on TRPV4 current induced by 0.1 μM GSK101; Figure 3 D: 100 μM floxacin had no inhibitory effect on the TRPM8 current induced by 500 μM menthol; Figure 3 E: 100 μM floxacin had a moderate inhibitory effect on TRPV2 current induced by 2 mM 2-APB; Figure 3 F is the comparison of the average current inhibition effect of 100 μM floxacin on TRPV3, TRPA1, TRPV1, TRPV2, TRPV4, and TRPM8 channels;

[0028] Figure 4 A shows the experimental flow chart of the generation of a mouse back skin specific dermatitis model by topical application of the sensitizer DNFB and treatment with floxacin; Figure 4 B shows the phenotypic characteristics of the back skin of mice in the blank group, DNFB model group, 0.1 mM floxacin group, 1 mM floxacin group, and 10 mM floxacin group for 8 consecutive days; Figure 4 C is the change curve of dermatitis scores of mice in each group for 8 consecutive days; Figure 4 D is the statistical analysis of the thickness of the back skin tissue sections of mice in different groups; Figure 4 E is H&E staining of skin tissues of mice in each group.

[0029] Figure 5 A shows the flow chart of floxacin treatment of DNFB-induced ear swelling in mice; Figure 5B shows the ear swelling of mice in the blank group, DNFB group, 0.1 mM floxacin group, 1 mM floxacin group and 10 mM floxacin group on the 9th day of the experiment; Figure 5 C is the statistical change of ear thickness in mice with DNFB-induced ear swelling treated with floxacin;

[0030] Figure 6 A shows the top view of the binding of floxacin to TRPV3 protein inferred by molecular docking; Figure 6 B is a side view of the binding of floxacin to TRPV3 protein inferred by molecular docking (left) and an expanded view of the binding pocket of floxacin (red) to TRPV3 channel (right); Figure 6 C is a representative whole-cell current trace of mutant E501 (E501A) in response to 50 μM 2-APB and 100 μM floxacin (left) and a representative single-channel current trace when 30 μM 2-APB (middle) or 50 μM floxacin was administered (right); Figure 6 D is a representative whole-cell current trace of mutant F666 (F666A) in response to 50 μM 2-APB and 100 μM floxacin (left) and a representative single-channel current trace when 30 μM 2-APB (middle) or 50 μM floxacin was administered (right); Figure 6 E is a representative whole-cell current graph of wild-type TRPV3 expressed in HEK293 cells in response to 50 μM 2-APB and 100 μM floxacin; Figure 6 F is a representative whole-cell current graph of 50 μM 2-APB and 100 μM floxacin when mutant I505 (I505A) was expressed in HEK293 cells; Figure 6 G is a representative whole-cell current graph of 50 μM 2-APB and 100 μM floxacin when mutant R509 (R509A) was expressed in HEK293 cells; Figure 6 H is a representative whole-cell current graph of 50 μM 2-APB and 100 μM floxacin when mutant A697 (A697D) was expressed in HEK293 cells; Figure 6 I is the statistic of 100 μM floxacin inhibiting the current of wild-type TRPV3 or mutant channels. DETAILED DESCRIPTION

[0031] Flocacetone, a derivative of acylphloroglucinol isolated from Tribus Inulea, Compositeae is a perennial medicinal plant widely used in traditional medicine to treat various diseases, including skin diseases, wounds, hypertension, diabetes, cancer, bronchitis and gastroduodenal diseases. Flocacetone was also previously known to act by inhibiting catechol-O-methyltransferase (COMT) and antagonizing the serotonin receptor 5-HT1A.

[0032] The present invention began with a Schrödinger virtual screening of a library of 813 FDA-approved drugs against the cryo-EM structure of TRPV3. Among the identified compounds, the inventors identified 50 drugs with docking scores higher than -4, compared to known TRPV3 inhibitors used as controls. To evaluate the effects of these drugs on hTRPV3 channels expressed in HEK293 cells, the inventors performed whole-cell patch clamp recordings on the cells. Perfusion of floxacin at a concentration of 100 μmol / L before washout inhibited TRPV3 currents activated by the agonist 2-APB at 50 μmol / L by approximately 84%. In addition, a dose-dependent inhibition of TRPV3 currents by floxacin was determined, with an IC50 value of 17.8±3.5 μM ( Figure 2 A- Figure 2 C). Further experimental results showed that floxacin directly acts on individual TRPV3 channels by reducing the probability of channel opening, rather than by reducing the conductivity of the channel ( Figure 2 D- Figure 2 G).

[0033] To determine the selective inhibition of TRPV3 currents by floxacin, the inventors conducted further tests on other subtypes of hot TRP channels such as TRPV1, TRPV2, TRPV4, TRPM8, and TRPA1 channels transiently expressed in HEK293 cells. Compared with the positive control of 1 μM agonist capsaicin that activates TRPV1, 100 μM floxacin had no effect on TRPV1. 100 μM floxacin had no effect on TRPV4, TRPM8, and TRPA1 induced by its agonists GSK1016970A (0.1 μM), menthol (500 μM), and AITC (300 μM), respectively. Only floxacin (100 μM) was found to have an effect on TRPV2 ion channels, and inhibition of TRPV2 currents activated by 2 mM2-APB was observed by about 60%. Further docking of floxacin with the three-dimensional structure of TRPV2 protein (PDB ID code: 7XEV) also confirmed the putative floxacin binding pocket formed by residues Y442 and L443 of one subunit S2 and E665 of the TRP helix. These results indicate that floxacin is a relatively selective inhibitor of TRPV3 compared to TRPV1, TRPV4, TRPM8 and TRPA1 channel subtypes, and has a moderate inhibition on TRPV2 (TRPV2 is not expressed in skin tissue).

[0034] To identify the key residues for floxacin inhibition of TRPV3, the inventors utilized the cryo-EM structure of human TRPV3 bound to 2-APB (PDB: 6DVY) and used Fluoropropionic acid was docked into the structure. The docking results showed that fluopropionic acid had four binding pockets, with the highest docking score of -5.5 kcal / mol. Further analysis of the binding pose and energy decomposition showed that floxiproline bound to the S2 helix through an obvious hydrogen bond, and residue E501 contributed a binding energy of -5.7 kcal / mol, which was the largest contribution compared to other related amino acid residues. In addition, residues I497 and I505 can also assist in ligand binding. In order to confirm that residue E501 is essential for floxiproline binding, the inventors performed site-directed mutagenesis and tested the effects of floxiproline on these channel mutants. Figure 6 As shown, whole-cell or single-channel recordings showed that mutating residue E501 (E501A) significantly reduced the inhibitory effect of floxacin on TRPV3. Similarly, mutating residue I505 also partially reduced the inhibitory effect of floxacin on TRPV3. In contrast, mutation F666 (F666A) had no effect on floxacin-mediated TRPV3 inhibition. In addition, the inventors introduced two mutations, R509A in pocket 3 and A697D in pocket 4, which still maintained sensitivity to floxacin inhibition, similar to the wild-type channel. These results indicate that residues E501 and I505 in TRPV3 channel S2 are critical for interacting with floxacin, unlike other known TRPV3 inhibitors.

[0035] It is known that inhibiting overactive TRPV3 channel function by gene silencing or using channel inhibitors can reduce skin damage. Therefore, the inventors established a mouse model of back and ear skin inflammation induced by topical application of the skin sensitizer DNFB to further confirm the anti-inflammatory effect of floxacin. Topical application of different concentrations (0.1 to 10 mM) of floxacin reduced skin inflammation in a concentration-dependent manner and significantly reduced the dermatitis score (95% confidence interval [CI], 2.0) compared with the group using DNFB alone. Figure 4 , Figure 5 ) and confirmed by histopathological examination.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0037] Example 1

[0038] Evaluation of the inhibitory effect of floxacin on transient receptor potential vanilloid receptor 3 channel by calcium fluorescence method

[0039] HEK293 cells were cultured and passaged using DMEM complete medium containing 10% fetal bovine serum. When the HEK293 cells in a 10 cm diameter culture dish grew to 90-100% density, 2 mL of 0.25% trypsin was added for digestion, and then 4 mL of culture medium was added to terminate the digestion. After centrifugation, 2 mL of cell suspension was prepared, 150 μL of cell suspension was added to each well of a 6-well plate, and 2 mL of complete culture medium was added to each well. After shaking, the cells were placed in a constant temperature incubator at 37°C and 5% CO2 for overnight culture. When the cells in the 6-well plate grew to 80-90% density, the human transient receptor potential vanilloid receptor 3 plasmid was transfected using the liposome transfection reagent Lipofectamine 2000. When performing transient transfection, add 600μL Opti-MEM medium and 19.2μL liposomes to microcentrifuge tube No. 1, add 600μL Opti-MEM medium and 9600ng human transient receptor potential vanilloid receptor 3 plasmid to microcentrifuge tube No. 2, mix them separately, place them at room temperature for 5 minutes, add the solution in microcentrifuge tube No. 1 to microcentrifuge tube No. 2, and mix them. After placing them at room temperature for 20 minutes, add the mixture to a 6-well plate, 200μL per well, and place them in an incubator for culture. After 4 hours, discard the solution in the well, add 160μL 0.25% trypsin to each well for digestion, then add 1.6mL complete medium, slowly blow and mix, transfer to a black-walled transparent bottom 96-well plate, 100μL per well, transfer one well of the 6-well plate to two rows of the 96-well plate, and place the incubator at 37°C and 5% CO2 for culture. Cells were cultured overnight until they grew to 80-90% density and tested using the calcium indicator Cal-520TM PBX calcium assay kit. Before use, 200 μL DMSO was added to component A, mixed evenly, and stored at -20°C. 60 μL calcium dye was added to each well of a 96-well plate to prepare 6 mL of dye: 5.4 mL Hank's balanced salt solution was taken to the preparation tank, 600 μL component B (dark purple) and 12 μL component A (colorless) were added, and mixed evenly. 40 μL of culture medium was discarded from each well of cells, 60 μL calcium dye was added, and the cells were incubated at 37°C and 5% CO2 for 1 to 1.5 hours. The compounds to be tested were prepared using a transparent 96-well plate, the final concentration of floxacin test was 200 μM, and the volume per well was 200 μL. When testing on the machine, the cell plate was placed on the bottom layer of the FlexStation 3 multi-function microplate reader, the compound plate was placed in the middle layer, and the special gun tip was placed on the top layer. The fast kinetic fluorescence analysis module was used, with an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The reading was taken every 1.6 seconds. 30 μL of the test substance was added at 17 seconds, and 30 μL of 2-aminoethylbiphenyl borate (2-APB, 200 μM), a transient receptor potential vanilloid receptor 3 channel agonist, was added at 100 seconds. The data was collected until 180 seconds. The results showed that floxacin could inhibit the calcium influx caused by 2-APB, a transient receptor potential vanilloid receptor 3 channel agonist.

[0040] Example 2

[0041] Patch clamp technique to determine the inhibitory effect of floxacin on transient receptor potential vanilloid receptor 3 channels and its dose-effect relationship

[0042] After culturing HEK293 cells transiently expressing human transient receptor potential vanilloid receptor 3 channels for 24 hours, whole-cell patch clamp recordings were performed using the EPC10 amplifier and Patchmaster software of HEKA, Germany, and single-channel recordings were performed using the MultiClamp 700B patch clamp amplifier and Clampex 11.0.3 software of AXON, USA. The glass electrode was pulled using a DMZDMZ electrode puller, and the water resistance after polishing was 3-8MΩ. The external solution and compound solution used a gravity perfusion device. In the whole-cell patch clamp recording, the cell membrane potential was maintained at 0mV, and the current was recorded for 500ms with a voltage ramp from -100 to +100mV, and the current analysis was performed at ±80mV. The results showed that 100 μM floxacin inhibited the transient receptor potential vanilloid receptor 3 channel current induced by 2-aminoethyl biphenyl borate (2-APB, 50 μM) with an inhibition rate of 84.0±2.4%. The half inhibitory concentration IC of floxacin on transient receptor potential vanilloid receptor 3 channel was 50 The single-channel recording mode was adopted, and the voltage of -60mV was continuously applied when recording the current, and the samples were sampled at 20kHz and filtered at 2kHz. The results showed that floxacin directly acts on single TRPV3 channels by reducing the probability of channel opening without changing the channel unit conductance.

[0043] Note: Measurement data were expressed as mean ± standard deviation (mean ± SD). Paired t-test was used for analysis. In the analysis results, ** indicates P < 0.01, and ns indicates no significant difference.

[0044] Example 3

[0045] Inhibitory effects of floxacin on transient receptor potential ankyrin receptor 1 channel, transient receptor potential vanilloid receptor 1 channel, transient receptor potential vanilloid receptor 2 channel, transient receptor potential vanilloid receptor 4 channel, and cold-stimulated transient potential receptor subtype 8 channel

[0046] HEK293 cells transiently transfected with human transient receptor potential ankyrin receptor 1 channel (hTRPA1), human transient receptor potential vanilloid receptor 1 channel (hTRPV1), human transient receptor potential vanilloid receptor 4 channel (hTRPV4), human transient receptor for cold stimulation subtype 8 (hTRPM8), and mouse transient receptor potential vanilloid receptor 2 channel (mTRPV2) were tested by patch clamp technique. The results showed that 100 μM floxacin had an inhibitory effect on the transient receptor potential ankyrin receptor 1 channel current induced by 300 μM allyl isothiocyanate. The inhibition rate was 0.57±5.25%, the inhibition rate of 100μM floxacin on the transient receptor potential vanilloid receptor 1 channel current induced by 1μM capsaicin was 9.32±13.65%, the inhibition rate of 100μM floxacin on the transient receptor potential vanilloid receptor 4 channel current induced by 0.1μM GSK101 was -2.70±6.71%, the inhibition rate of 100μM floxacin on the cold stimulus transient potential receptor subtype 8 channel current induced by 500μM menthol was 13.51±10.02%, and the inhibition rate of 100μM floxacin on the transient receptor potential vanilloid receptor 2 channel current induced by 2mM 2-APB was 59.94±13.69%, which were significantly weaker than the inhibitory effect on the transient receptor potential vanilloid receptor 3 channel. Floxacin is a specific inhibitor of the transient receptor potential vanilloid receptor 3 channel.

[0047] Note: Measurement data were expressed as mean ± SD and tested using one-way ANOVA. In the analysis results, ** indicates **P < 0.01, ****P < 0.0001.

[0048] Example 4

[0049] This example is used to illustrate the application of the TRPV3 inhibitor floxacin provided by the present invention in the prevention or treatment of atopic dermatitis

[0050] C57BL / 6 male mice aged 6-8 weeks and weighing about 20g were purchased from Beijing Weitong Lihua Company. The mice were allowed to eat and drink freely. The breeding environment was clean, the temperature was 22±2℃, and the lighting cycle was 12 hours. The experiment was conducted after one week of adaptive breeding. The mice were randomly divided into a blank control group, a DNFB model group, a 0.1mM floxacin treatment group, a 1mM floxacin treatment group, and a 10mM floxacin treatment group, with 5-10 mice in each group. Before using the DNFB-sensitized mouse back skin, shave the hair on the back of the mouse with a razor, and then use a depilatory cream for depilatory. After applying the depilatory cream for 8 minutes, wipe off the depilatory cream with a wet wipe soaked in saline. Figure 4As shown in A, the experiment officially started 24 hours after hair removal. 100 μL of 0.5% DNFB was applied to the hair removal area on the back of the mouse. No treatment was performed from the second to the fourth day. On the fifth day, 50 μL of 0.2% DNFB solution was applied to the same area. On the sixth day, 50 μL of 0.2% DNFB was repeatedly applied for re-stimulation. The blank group only used DNFB solvent. During the experiment, isoflurane was added to the gas anesthesia device to assist hair removal. According to the selected concentration, the 100mM floxacin stock solution was diluted 10 times, 100 times and 1000 times with normal saline to obtain 10mM, 1mM and 0.1mM floxacin solutions. The blank group and DNFB model group were treated with 10% DMSO normal saline solution as a control. On the first day of treatment, 100 μL of different concentrations of floxacin were used to treat the back skin at 30 minutes, 24.5 hours, 48.5 hours, and 72.5 hours after 0.2% DNFB restimulation.

[0051] The back skin of the mice was photographed and recorded every day, and the degree of inflammation was scored. The content and criteria of the mouse specific dermatitis-like dermatitis scoring include erythema / hemorrhage, scarring / dryness, edema and scratching / erosion. The score of each symptom ranged from 0 to 3 points (none, 0 points; mild, 1 point; moderate, 2 points; severe, 3 points). The dermatitis score was defined as the sum of the individual scores, ranging from 0 to 12 points. The results are shown in Figure 4 B and Figure 4 As shown in C, topical application of DNFB resulted in a time-dependent development of inflammation in the back skin of mice compared with the blank control group. In contrast, topical application of different concentrations of floxacin reduced skin inflammation in a concentration-dependent manner compared with the DNFB group.

[0052] Note: Measurement data were expressed as mean ± standard deviation (mean ± SD) and tested using two-way ANOVA. In the analysis results, ****P<0.0001.

[0053] 24 hours after the last treatment with floxacin, the mice were killed by cervical dislocation. The skin tissue on the back of the mice was removed with scissors and forceps, fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced ​​with a thickness of 5-8 μm, and stained with H&E. The Eclipse Ti inverted microscope (Nikon Corporation, Tokyo, Japan) was used for observation and photography, and the tissue was photographed and recorded using NIS-element software (Nikon Corporation, Tokyo, Japan). The results are shown in Figure 4 D and Figure 4 (E) Compared with the DNFB group, topical application of floxacin resulted in a dose-dependent decrease in epidermal thickness and reduced inflammatory cell infiltration in the dermis of mice.

[0054] Note: Measurement data were expressed as mean ± SD and tested using one-way ANOVA. In the analysis results, *** indicates P < 0.001, ****P < 0.0001, and ns indicates no significant difference.

[0055] Example 5

[0056] This example is used to further illustrate the application of the TRPV3 inhibitor floxacin provided by the present invention in the prevention or treatment of atopic dermatitis

[0057] C57BL / 6 male mice aged 6-8 weeks and weighing about 20g were purchased from Beijing Weitong Lihua Company. The mice were allowed to eat and drink freely. The breeding environment was clean, the temperature was 22±2℃, and the lighting cycle was 12 hours. The experiment was conducted after one week of adaptive breeding. The mice were randomly divided into a blank control group, a DNFB model group, a 0.1mM floxacin treatment group, a 1mM floxacin treatment group, and a 10mM floxacin treatment group, with 5-10 mice in each group. Except that no hair removal was required in advance, the induction and treatment process of the ear model was consistent with the back-specific dermatitis described above and will not be repeated. On the 9th day, the characteristics of the right ear of the mice were recorded by photographing. Compared with the DNFB group, the swelling of the right ear of the mice in the floxacin treatment group was significantly reduced. The thickness of the mouse ears was measured for the first time 30 minutes after the last application of 0.2% DNFB solution using a vernier caliper. The thickness of the mouse ears was measured with a vernier caliper at 24h, 48h, 72h, and 96h after the first measurement. The results are as follows Figure 5 As shown in C, the floxacin-treated group significantly reduced ear thickness in a dose-dependent manner on measurement days 4 and 5. These results suggest that inhibition of TRPV3 by floxacin can attenuate the development of DNFB-induced ear inflammation.

[0058] Note: Measurement data were expressed as mean ± SD and tested using two-way ANOVA. In the analysis results, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0059] Example 6

[0060] Molecular docking combined with point mutation electrophysiology to identify the key amino acid residues for the binding of floxacin to transient receptor potential vanilloid receptor 3 channel

[0061] The structure of floxacin was obtained from the listed compound library (TargetMol). The cryo-EM structure of TRPV3 (PDB ID: 6DVY) was downloaded from the Protein Data Bank (PDB). The default parameters of Glid were used to dock floxacin with TRPV3. The compound and protein were optimized by energy minimization and semi-flexible docking using the built-in program Ligprep. The docking results showed that floxacin bound to the transmembrane α-helix S2 of the TRPV3 channel protein, and the hydroxyl group of floxacin interacted with the 501st amino acid of TRPV3 through hydrogen bonds.

[0062] The patch clamp recording was completed using the MultiClamp 700B patch clamp amplifier and Clampex11.0.3 software from AXON, USA. The glass electrode was made using the DMZDMZ electrode puller, and the water resistance after polishing was 3-6MΩ. The extracellular solution and compound solution were perfused using a gravity perfusion device. In the whole-cell patch clamp recording, the cell membrane potential was kept at 0mV, and the voltage ramp from -100 to +100mV lasted for 500ms to record the current, and the current analysis was performed at ±80mV. In single-channel recording, the inner membrane-out single-channel recording mode was used, and a voltage of -60mV was continuously applied during the current recording to observe the opening of the single channel. The results showed that the current of HEK293 cells expressing TRPV3-E501A did not show inhibition after perfusion of 100μM floxacin, but the channel current of HEK293 cells expressing TRPV3-F666A showed inhibition immediately after perfusion of 100μM floxacin. In addition, HEK293 cells expressing TRPV3-I505A also showed partial inhibition of current after perfusion of 100 μM floxacin. HEK293 cells expressing TRPV3-R509A and TRPV3-A697D residues still maintained sensitivity to floxacin inhibition, similar to wild-type channels. This suggests that the two amino acid residues E501 and I505 are essential for floxacin inhibition of TRPV3.

[0063] Note: Measurement data were expressed as mean ± SD and tested using one-way ANOVA. In the analysis results, **** means P < 0.0001.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof in the preparation of TRPV3 ion channel inhibitors; The structural formula of the floxacin is shown in Formula I; 2. The use according to claim 1, characterized in that The TRPV3 ion channel inhibitors include drugs and experimental reagents.

3. The use according to claim 1, characterized in that The experimental reagents are for basic research use.

4. The use according to claim 1, characterized in that The floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof take TRPV3 as a target, and inhibit the activity of the TRPV3 ion channel by acting on E501 and / or I505 on the TRPV3S2 helix.

5. Use of floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof in the preparation of drugs for TRPV3 ion channel-related diseases; Preferably, the floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof have TRPV3 ion channels as targets, and selectively inhibit the activity of TRPV3 ion channels to prevent and reduce / reduce tissue pathological changes caused by enhanced TRPV3 activity.

6. The use according to claim 5, characterized in that The TRPV3 ion channel-related diseases include skin inflammation, damage, or enhanced TRPV3 activity caused by UVB irradiation, resulting in pathological changes in the tissues.

7. A drug for inhibiting TRPV3 ion channels, characterized in that: The active ingredient in the drug is floxacin and its stereoisomers, and its pharmaceutically acceptable salts and / or solvates; Preferably, the drug further comprises one or more pharmaceutically or food-acceptable excipients.

8. The drug according to claim 7, characterized in that The excipients used may be solid or liquid; Preferably, the solid excipient is magnesium carbonate, magnesium stearate, talc, sugar or lactose; Liquid form preparations include solutions, suspensions and emulsions.

9. The drug according to claim 8, characterized in that: Solid form preparations include powders, tablets, dispersible granules, capsules, pills and suppositories; The powders and tablets may be comprised of from about 0.1% to about 99.9% active ingredient.

10. Use of floxacin and its stereoisomers, and pharmaceutically acceptable salts and / or solvates thereof in the preparation of a medicament for treating or preventing atopic dermatitis.

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