Use of small molecule inhibitors in the treatment of mucus secretion related diseases

Abstract

The present application relates to the field of biological medicine, and in particular to the use of small molecule inhibitors in the treatment of mucus secretion related diseases. The present application provides the use of a small molecule inhibitor in the preparation of a medicament for treating and / or preventing diseases related to excessive mucus secretion, characterized in that the disease is a respiratory disease, the small molecule inhibitor includes one or more of casimycin, cyanidin-3-O-glucoside, ellagic acid or a pharmaceutically acceptable salt thereof, and the medicament is used to inhibit the secretion of the mucus. The small molecule inhibitor of the present application can effectively inhibit the secretion of mucus and is suitable for various types of respiratory mucus obstruction diseases (such as asthma, chronic bronchitis, cystic fibrosis and chronic obstructive pulmonary disease).

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of small molecule inhibitors in the treatment of mucin secretion-related diseases. Background Technology

[0002] The respiratory tract can be divided into two regions: the upper respiratory tract and the lower respiratory tract. Within the respiratory tract, airway mucus secretion is typically the first line of defense and an important component of innate immunity. Mucus plays a crucial role in protecting the respiratory tract from harmful chemical, microbial, and physical environmental factors. However, excessive mucin secretion can cause airway obstruction, leading to obstructive pulmonary disease (OPD). OPD often causes breathing difficulties and can even be life-threatening.

[0003] Common respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) are characterized by airway obstruction as their main pathological feature. Conventional treatments for asthma and COPD primarily involve bronchodilators and anti-inflammatory drugs, which have limited effectiveness in treating excessive airway mucus secretion. Because airway epithelial cells have multiple pathways that activate mucin synthesis, drugs currently under development or investigation that target specific points to inhibit mucin synthesis (such as EGFR tyrosine kinase inhibitors) not only treat specific respiratory diseases but also have limited efficacy and even significant side effects (such as adverse reactions like papulopustular eruptions) and poor patient tolerance.

[0004] In summary, although many drugs and treatments aimed at directly or indirectly inhibiting excessive mucus secretion are available or under development, the vast majority of them are drugs developed to relieve respiratory smooth muscle contraction or reduce epithelial cell mucin synthesis, and their therapeutic effect on excessive mucin secretion is not ideal. Summary of the Invention

[0005] This invention provides the use of a small molecule inhibitor in the preparation of a medicament for treating and / or preventing diseases associated with excessive mucin secretion, characterized in that the diseases include respiratory diseases, and the small molecule inhibitor comprises one or more of cathinone, cyanidin-3-O-glucoside, ellagic acid, or pharmaceutically acceptable salts thereof, the medicament being used to inhibit the secretion of mucin.

[0006] In some embodiments, the respiratory disease includes obstructive pulmonary disease.

[0007] In some embodiments, the obstructive lung disease includes one or more of asthma, chronic bronchitis, cystic fibrosis, and chronic obstructive pulmonary disease.

[0008] In some embodiments, the secretion includes stimulative secretion.

[0009] In some embodiments, the secretion is mediated by SNARE / SYT2.

[0010] In some embodiments, the symptoms of the disease include one or more of excessive mucus secretion, mucus buildup, airway obstruction, difficulty breathing, infection, and inflammation.

[0011] In some embodiments, the mucin includes the MUC5AC protein.

[0012] In some embodiments, the drug is used to inhibit the fusion of intracellular vesicles with the cell membrane.

[0013] In some embodiments, the drug is administered orally or intranasally.

[0014] In some embodiments, the medicament further includes a pharmaceutically acceptable carrier.

[0015] Beneficial effects

[0016] Excessive mucin secretion can cause airway obstruction, leading to diseases associated with excessive mucin secretion. Diseases characterized by excessive mucin secretion and mucus obstruction (such as obstructive pulmonary disease) often cause breathing difficulties and can even be life-threatening. Current technologies typically aim to indirectly control mucin in the airways by improving mucociliary clearance, accelerating mucus transport, and reducing mucus viscoelasticity, but the therapeutic effects are very limited. Furthermore, because airway epithelial cells have multiple pathways that activate mucin synthesis, currently developed or under investigation drugs that inhibit mucin synthesis (such as EGFR tyrosine kinase inhibitors) are disease-specific, treating only specific respiratory diseases. They also suffer from limited efficacy and even significant side effects (such as adverse reactions like papulopustular eruptions) and poor patient tolerance.

[0017] Through various experiments (such as lipid fusion experiments, GST-Pull-down experiments, and IL-13-induced mucin experiments), this invention has creatively discovered that the three small molecule compounds 11-F-5, 10-B-9, and 11-B-6 can directly control mucin in the airways by inhibiting mucin secretion. Specifically, these three small molecule compounds can act as small molecule inhibitors, effectively inhibiting SNARE / SYT2-mediated mucin excitation, thereby alleviating, to some extent, various symptoms caused by airway mucus accumulation (such as airway obstruction, dyspnea, infection, and inflammation) from the source of airway mucus obstruction (i.e., mucin secretion). In addition, since the pathway of mucin-induced secretion is unique, the small molecule inhibitor of the present invention can effectively inhibit airway obstruction caused by excessive mucin secretion by blocking mucin-induced secretion, and is applicable to various types of respiratory mucoid obstructive diseases (such as asthma, chronic bronchitis, cystic fibrosis and chronic obstructive pulmonary disease).

[0018] Furthermore, the small molecule inhibitor (<1kDa) provided by this invention has good stability, low production cost, convenient storage and transportation, good cell permeability, and can be administered through various routes of administration (e.g., oral, intranasal), making it suitable for clinical applications.

[0019] As used herein, “pharmaceutically acceptable” (e.g., pharmaceutically acceptable carriers, pharmaceutically acceptable salts, etc.) means pharmacologically acceptable and substantially non-toxic to the individual to which the specific compound is administered.

[0020] As used herein, a "pharmaceutically acceptable salt" refers to a salt of a specific compound (in this invention, including one or more of cathinone, cyanidin-3-O-glucoside, and ellagic acid) that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and can be acid addition salts and base addition salts. Exemplary acid addition salts include those derived from inorganic acids (such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid) and those derived from organic acids (such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, etc.). Exemplary base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides (e.g., tetramethylamine hydroxide).

[0021] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to a mammalian individual unless otherwise stated. The small molecule inhibitors of the present invention can be designed to be administered to individuals via a variety of different routes of administration, such as oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal (e.g., inhalation, nebulization), oral, topical, or transdermal routes.

[0022] As used herein, “diseases associated with excessive mucin secretion” refers to a class of diseases characterized by excessive mucin secretion and airway obstruction. Exemplary “diseases associated with excessive mucin secretion” include obstructive lung diseases such as asthma, chronic bronchitis, cystic fibrosis, and chronic obstructive pulmonary disease.

[0023] As used in this article, “reduce,” “reduc,” “mitigate,” and “inhibit” generally mean a reduction in a statistically significant amount, such as any amount between approximately 10% and approximately 100% relative to a reference level.

[0024] As used in this article, “increase,” “enhance,” “boost,” “activate,” “upregulate,” and “induce” generally mean an increase in a statistically significant amount, such as any amount between about 10% and about 100% relative to the reference level, or an increase of about two times or more relative to the reference level. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0026] Figure 1 This invention relates to a plasmid expression map of the plasmid.

[0027] Figure 2 The image shows the purification results of the SNARE protein;

[0028] Figure 3 The image shows the purification results of the SYT2 protein.

[0029] Figure 4 This represents the interaction interface between the C2B protein and the SNARE complex.

[0030] Figure 5 The purification results are for the C2B protein and its mutant proteins.

[0031] Figure 6 The purification results of SNARE truncated protein;

[0032] Figure 7 Figure showing the verification results of in vitro recombination of secretory granules and cell membrane vesicles;

[0033] Figure 8 This is a schematic diagram of lipid fusion;

[0034] Figure 9 The graph shows the results of vesicle fusion efficiency;

[0035] Figure 10A The structural formula of the small molecule compound 11-F-5 (kaxamycin);

[0036] Figure 10B The structural formula of the small molecule compound 10-B-9 (cyanidin-3-O-glucoside);

[0037] Figure 10C The structural formula of the small molecule compound 11-B-6 (ellagic acid);

[0038] Figure 11 The graph shows the inhibition rate of the small molecule compound 11-B-6.

[0039] Figure 12A The graph shows the statistical data of the decrease in fusion efficiency at 30 min compared to the fusion efficiency of the control group (the concentration of the small molecule compound was 100 μM, and the control group was the same volume of DMSO added; error bars are shown as ±SEM, t test, **P<0.01, ***P<0.001).

[0040] Figure 12B The ΔFRET signal values ​​of different small molecule compounds at concentrations of 100 μM (red), 10 μM (blue), and 1 μM (green) (the control group was the addition of an equal volume of DMSO (black));

[0041] Figure 12C Statistical analysis of the inhibition rates of different small molecule compounds (i.e. Figure 12B Data analysis graph of the ratio of the decrease in fusion efficiency of small molecule compounds at different concentrations in the medium to the fusion efficiency of the control group at 30 min (error bars are shown as ±SEM, t test, *P<0.1, ns indicates no significant difference));

[0042] Figure 13 Validation of the Soluble SNARE complex by molecular sieve (left) and SDS-PAGE (right, lane 1);

[0043] Figure 14 The figure shows the experimental results of the pull-down experiment to verify the binding of small molecule compounds with C2B-WT;

[0044] Figure 15 The figure shows the experimental results of the pull-down experiment to verify the binding of small molecule compounds with C2B-QM;

[0045] Figure 16 The figure shows the experimental results of the pull-down experiment to verify the binding of small molecule compounds with C2B-QQQ;

[0046] Figure 17 The figure shows the experimental results of the toxicity of small molecule compounds to cells;

[0047] Figure 18 A schematic diagram of ALI culture;

[0048] Figure 19 This was done to verify, using a Dot Blot experiment, that the small molecule compound could inhibit the excitatory secretion of mucin.

[0049] Figure 20 The presence of mucin (MUC5AC protein) residues within cells suggests that SNARE / SYT2 can mediate the secretion of stimulant mucin;

[0050] Figure 21 The presence of mucin (MUC5AC protein) residues within cells demonstrates that small molecules can inhibit the stimulative secretion of mucin. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0052] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0053] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0054] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0055] As used in this specification, the term "about" typically means + / - 5% of the value, more typically + / - 4%, more typically + / - 3%, more typically + / - 2%, even more typically + / - 1%, even more typically + / - 0.5% of the value.

[0056] In this specification, some embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as specifically disclosing all possible subranges and the individual numerical values ​​within that range. For example, a description of the range 1–6 should be considered as specifically disclosing subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0057] Detailed description of the attached figures

[0058] Figure 1 A. Expression map of pGEX-6p-1-C2B plasmid. B. Expression map of pGEX-6p-1-C2B-QM plasmid. C. Expression map of pGEX-6p-1-C2B-QQQ plasmid.

[0059] Figure 2 A. Purification results of STX3 protein. Anion exchange column (left) and SDS-PAGE (right, lane 1) were used to verify the purity of STX3 protein. B. Purification results of VAMP8 protein. Molecular sieve (left) and SDS-PAGE (right, lane 2) were used to verify the purity of VAMP8 protein. C. Purification results of SNAP23 protein. Anion exchange column (left) and SDS-PAGE (right, lane 1) were used to verify the purity of SNAP23 protein.

[0060] Figure 3 Cation exchange column (left) and SDS-PAGE (right, lane 2) were used to verify the purity of SYT2 protein.

[0061] Figure 4 A. Hydrophobic interaction region. B. Electrostatic interaction region. C. Sequence alignment of C2B protein and C2B mutant protein.

[0062] Figure 5A. Purification results of C2B protein. Cation exchange column (left), molecular sieve (middle), and SDS-PAGE (right, lane 2) were used to verify the purity of C2B protein. B. Purification results of C2B-QM protein. Cation exchange column (left), molecular sieve (middle), and SDS-PAGE (right, lane 2) were used to verify the purity of C2B-QQQ protein. C. Purification results of C2B-QQQ protein. Cation exchange column (left), molecular sieve (middle), and SDS-PAGE (right, lane 2) were used to verify the purity of C2B-QQQ protein.

[0063] Figure 6 A. Purification results of STX3(1-263) protein. Anion exchange column (left) and SDS-PAGE (right, lane 1) were used to verify the purity of STX3(1-263) protein. B. Purification results of VAMP8(1-76)-GST protein. Anion exchange column (left) and SDS-PAGE (right, lane 1) were used to verify the purity of VAMP8(1-76)-GST protein.

[0064] Figure 9 A. Average ΔFRET signal value, differences in lipid fusion under different conditions. ΔFRET values ​​for vesicle fusion in the presence of SNARE only (grey line), in the presence of SNARE and SYT2 (blue line), and in the presence of SNARE and SYT2 with the addition of 100 μM CaCl2 (red line). BA plot shows the statistical data of fusion percentage at 30 min (error bars are shown as ±SEM, t-test, **P<0.01, ***P<0.001).

[0065] Figure 11 A. The ΔFRET signal value, taking 11-B-6 as an example, represents the vesicle fusion efficiency. F DMSO : Add DMSO's ΔFRET value, F 小分子 : The ΔFRET value of the small molecule compound; the inhibition rate is the ratio of the decrease in fusion efficiency at 30 min to the fusion efficiency of the control group, which is the addition of an equal volume of DMSO. B. Inhibition rate of the small molecule compound.

[0066] Figure 14A. SDS-PAGE was used to verify the effect of small molecule compounds on the binding of C2B-WT to the SNARE complex. Lane 1 is the protein marker, lane 2 is VAMP8(1-76)-GST, lane 3 is STX3(1-263), lane 4 is SNAP23, lane 5 is the Soluble SNARE complex, lane 6 is C2B-WT, lane 7 is DMSO, lane 11 is 11-F-5, lane 13 is 10-B-9, and lane 14 is 11-B-6. Quantitative analysis of C2B-WT protein is shown in Figure BA (error bars are shown as ±SEM, t-test, *P<0.1, **P<0.01).

[0067] Figure 15 A. SDS-PAGE was used to verify the effect of small molecule compounds on the binding of C2B-QM to the SNARE complex. Lane 1 is the protein marker, lane 2 is VAMP8(1-76)-GST, lane 3 is STX3(1-263), lane 4 is SNAP23, lane 5 is the Soluble SNARE complex, lane 6 is C2B-QM, lane 7 is DMSO, lane 11 is 11-F-5, lane 13 is 10-B-9, and lane 14 is 11-B-6. Quantitative analysis of C2B-QM protein is shown in Figure BA (error bars are shown as ±SEM, t-test, ns indicates no significant difference).

[0068] Figure 16 A. SDS-PAGE was used to verify the effect of small molecule compounds on the binding of C2B-QQQ to the SNARE complex. Lane 1 is the protein marker, lane 2 is VAMP8(1-76)-GST, lane 3 is STX3(1-263), lane 4 is SNAP23, lane 5 is the Soluble SNARE complex, lane 6 is C2B-QQQ, lane 7 is DMSO, lane 11 is 11-F-5, lane 13 is 10-B-9, and lane 14 is 11-B-6. Quantitative analysis of C2B-QQQ protein is shown in Figure BA (error bars are shown as ±SEM, t-test, *P<0.1, **P<0.01, ***P<0.001).

[0069] Figure 17 A. Toxicity results for 11-F-5: No toxicity. B. Toxicity results for 10-B-9: No toxicity. C. Toxicity results for 11-B-6: No toxicity.

[0070] Figure 19A. Immunoblotting of MUC5AC protein: the left side shows baseline secreted mucin, the right side shows stimulated secreted mucin, and the control group is DMSO. Quantitative analysis of MUC5AC protein in Figure BA (error bars are shown as ±SEM, t-test, ****P<0.0001, ns indicates no significant difference).

[0071] Figure 20 A. Confocal immunofluorescence staining. APC: MUC5AC protein (red), FITC: cell membrane (green), DAPI: cell nucleus (blue). B. Statistical graph of intracellular MUC5AC protein residues (error bars are shown as ±SEM, t-test, ***P<0.001).

[0072] Figure 21 A. Confocal immunofluorescence staining. APC: MUC5AC protein (red), FITC: cell membrane (green), DAPI: cell nucleus (blue). B. Statistical graph of intracellular MUC5AC protein residue after adding small molecules (error bars are shown as ±SEM, t-test, *P<0.1, ****P<0.0001, ns indicates no significant difference).

[0073] Example 1

[0074] 1.1 Construction of protein expression vector

[0075] SNARE proteins from the respiratory tract (including STX3, SNAP23, and VAMP8), SYT2, SYT2 C2B and their mutant proteins, as well as SNARE-related truncated proteins, were expressed in E. coli. The optimized coding sequences, based on the prokaryotic expression system, were cloned into the pET-28a or pGEX-6p-1 vector, and an N-terminal 6His or GST tag was added to aid in subsequent protein purification. Figure 1 As shown, the relevant protein expression cloning vector was successfully constructed for subsequent protein purification.

[0076] 1.2 Protein purification

[0077] 1.2.1 Purification of SNARE protein

[0078] Purification of STX3 protein

[0079] After cell lysis and centrifugation, the protein supernatant was collected. The supernatant was then ultracentrifuged to obtain the cell membrane. The protein was dissolved from the membrane using 2% DDM (w / v), and the supernatant was collected after centrifugation. Preliminary purification was performed using a Ni-NTA nickel affinity chromatography column, followed by further purification using ion exchange chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 2 A). Figure 2The middle horizontal axis represents the volume of the eluted protein, the left vertical axis represents the ultraviolet absorbance of the protein at a wavelength of 280 nm, and the right vertical axis represents the conductivity. Figure 2 Two peaks were observed in sample A. The protein at the first peak was eluted at a conductivity of 25.6 mS / cm, consistent with the theoretical value. SDS-PAGE analysis of the protein eluent from both peaks confirmed that the protein at the first peak was the target protein. Micro-spectrophotometry determined the protein concentration to be 200 μM, with a purity exceeding 90%. Finally, the protein was aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0080] Purification of VAMP8 protein

[0081] After cell lysis and centrifugation, the protein supernatant was collected. The supernatant was then ultracentrifuged to obtain the cell membrane. The protein was dissolved from the membrane using 2% DDM (w / v), and the supernatant was collected after centrifugation. Preliminary purification was performed using a GST column, followed by further purification using molecular sieve chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 2 B). Figure 2 Two peaks were observed in sample B. The protein at the second peak was eluted at approximately 16 mL, consistent with the theoretical value. SDS-PAGE confirmed that the protein at the second peak was the target protein. Micro-spectrophotometry determined the protein concentration to be 200 μM, with a purity exceeding 90%. Finally, the protein was aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0082] Purification of SNAP23 protein

[0083] After cell lysis and centrifugation, the protein supernatant was collected and then initially purified using a Ni-NTA nickel affinity chromatography column. The eluted protein was then further purified by ion-exchange chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 2 C). Figure 2 The main peak in C was single but broad, and the protein at the peak was eluted at a conductivity of 23 mS / cm, consistent with the theoretical value. SDS-PAGE verification confirmed that the peak represented the target protein. Micro-spectrophotometry determined the protein concentration to be 330 μM, with a purity exceeding 85%. The protein was aliquoted into 60 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use. Due to the instrument's limited peak detection capacity (maximum peak value of 3000 mAU), a concave absorption peak appeared when the protein concentration exceeded this maximum.

[0084] 1.2.2 Purification of SYT2 protein

[0085] After cell lysis and centrifugation, the protein supernatant was collected. The supernatant was then ultracentrifuged to obtain the cell membrane. The protein was dissolved from the membrane using 2% DDM (w / v), and the supernatant was collected after centrifugation. Preliminary purification was performed using a GST column, followed by further purification of the eluted protein using ion-exchange chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 3 ). Figure 3 The horizontal axis represents the volume of eluted protein, the vertical axis represents the UV absorbance of the protein at 280 nm, and the vertical axis represents conductivity. The main peak in the graph is single and narrow, indicating that the protein at the peak was eluted at a conductivity of 53 mS / cm, consistent with theoretical values. SDS-PAGE analysis confirmed that the peak represents the target protein. Micro-spectrophotometry determined the protein concentration to be 50 μM, with a purity exceeding 85%. Finally, the protein was aliquoted into 90 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0086] 1.2.3 Purification of SYT2 C2B protein and its mutant protein

[0087] The SYT2 C2B protein is a soluble protein composed of amino acids 272-422 of the SYT2 protein, hereinafter referred to as the C2B protein. The C2B protein is one of the main functional regions of the SYT2 protein, and its structural domain has two main regions that interact with the SNARE complex: one is through hydrophobic interactions to form an α-helix, and this interaction region is called the primary interface. Figure 4 A); Another method involves the electrostatic interaction of the C2B multibase region with the t-SNARE and SNARE complex. This interaction region is called the polybasic region. Figure 4B). Subsequent experiments of this invention mainly focused on the binding site of the C2B-SNARE complex. Therefore, mutant C2B proteins were constructed targeting these two interaction regions: one type mutated the binding site on the C2B protein primary interface that interacts with the SNARE complex, by mutating R282, E296, R399, and R400 to alanine and Y338 to tryptophan, and named C2B-QM; the other type mutated the polybasic region on the C2B protein that interacts with the SNARE protein, by mutating amino acids at positions K327, K328, and K332 to glutamine, and named C2B-QQQ. The selection of these mutation sites is based on the research on the interaction between SYT1 and SNARE by Zhou et al. (Zhou Q, Lai Y, Bacaj T, et al. Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis. Nature. 2015; 525(7567):62-67. doi:10.1038 / nature14975) and Lai et al. (Lai Y, Lou X, Diao J, Shin YK. Molecular origins of synaptotagmin1 activities on vesicle docking and fusion pore opening. Sci Rep. 2015; 5:38-40. doi:10.1038 / srep09267). The sequence alignment of C2B protein and C2B mutant protein is as follows: Figure 4 As shown in C. Then, the C2B protein and its mutant protein were expressed and purified.

[0088] Purification of C2B protein

[0089] After cell lysis and centrifugation, the protein supernatant was collected and initially purified using a GST column. The eluted protein was then further purified by ion-exchange chromatography, followed by further purification using molecular sieve chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 5 A). Figure 5The middle horizontal axis represents the volume of eluted protein, the left vertical axis represents the UV absorbance of the protein at 280 nm, and the right vertical axis represents conductivity. The ion exchange column shows a single peak, and the protein at the peak is eluted at approximately 58 mS / cm, consistent with theoretical values. The molecular sieve diagram also shows a single peak, eluted at 16 mL, consistent with theoretical values. SDS-PAGE verification confirmed that the peak represents the target protein. Micro-spectrophotometry determined the protein concentration to be 160 μM, with a purity exceeding 95%. Finally, the protein was aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0090] Purification of C2B-QM protein

[0091] After cell lysis and centrifugation, the protein supernatant was collected and initially purified using a GST column. The eluted protein was then further purified by ion-exchange chromatography, followed by further purification using molecular sieve chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 5 B). The ion exchange column graph shows two peaks; the protein at the second peak was eluted at approximately 53 mS / cm, consistent with the theoretical value. The molecular sieve graph shows three peaks; the first peak indicates elution at 16 mL, also consistent with the theoretical value. SDS-PAGE verification confirmed that the first peak represents the target protein. Micro-spectrophotometry determined the protein concentration to be 80 μM, with a purity exceeding 95%. Finally, the protein was aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0092] Purification of C2B-QQQ protein

[0093] After cell lysis and centrifugation, the protein supernatant was collected and initially purified using a GST column. The eluted protein was then further purified by ion-exchange chromatography, followed by further purification using molecular sieve chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 5 C). The ion exchange column graph shows a single peak, indicating that the protein at the peak was eluted at approximately 29 mS / cm, consistent with theoretical values. The molecular sieve graph also shows a single peak, indicating elution at 16 mL, consistent with theoretical values. SDS-PAGE verification confirmed that the peak represents the target protein. Micro-spectrophotometry determined the protein concentration to be 80 μM, with a purity exceeding 95%. Finally, the protein was aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0094] 1.2.4 Purification of SNARE truncated protein

[0095] Purification of STX3(1-263) protein

[0096] After cell lysis and centrifugation, the protein supernatant was collected and then initially purified using a Ni-NTA nickel affinity chromatography column. The eluted protein was then further purified by ion-exchange chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 6 A). Figure 6 The middle horizontal axis represents the volume of the eluted protein, the left vertical axis represents the ultraviolet absorbance of the protein at a wavelength of 280 nm, and the right vertical axis represents the conductivity. Figure 6 Three peaks were observed in sample A. The protein at the second peak was eluted at approximately 21 mS / cm, consistent with the theoretical value. SDS-PAGE confirmed that the protein at the second peak was the target protein. Micro-spectrophotometry determined the protein concentration to be 110 μM, with a purity exceeding 90%. Finally, the protein was aliquoted into 500 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0097] Purification of VAMP8(1-76)-GST protein

[0098] After cell lysis and centrifugation, the protein supernatant was collected and then preliminarily purified using a GST affinity chromatography column. The eluted protein was then further purified by ion-exchange chromatography. Finally, the protein purity was determined by SDS-PAGE. Figure 6 B). Figure 6 In sample B, a single peak was observed, and the protein at the peak was eluted at approximately 32 mS / cm, consistent with theoretical values. SDS-PAGE analysis confirmed that the peak represented the target protein. Micro-spectrophotometry determined the protein concentration to be 500 μM, with a purity exceeding 75%. Finally, the protein was aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0099] Example 2

[0100] 2.1 Reconstruction of vesicles mimicking secretory granules and cell membranes in vitro

[0101] This embodiment uses a direct recombination method to generate in vitro recombinant vesicles. The in vitro recombination experiment was conducted according to the following steps:

[0102] Step 1 - Fat blowing: Mix the fat in a glass test tube according to the proportions in Table 1 and Table 2, blow it dry with argon gas, wrap the glass test tube with aluminum foil and make a hole at the opening of the glass tube with a needle to allow chloroform to evaporate, and then put it in a vacuum pump to evacuate overnight.

[0103] Step 2 - Resuspension: Using a vortex apparatus, slowly add STX3 protein solution (containing 3% OG (isopropyl-β-D-thiogalactoside)) to the PM-vesicles glass tube, and slowly add a mixed protein solution of VAMP8 and SYT2 (containing 3% OG) to the SG-vesicles glass tube. Incubate at room temperature for 30 minutes. The molar ratio of SNARE protein to lipids is 1:200, and the mass ratio of SYT2 to lipids is 1:800.

[0104] Step 3 - Dilution: Slowly add SNAP23 protein solution dropwise to the PM-vesicles glass tube using a vortex mixer and incubate at room temperature for 30 minutes; slowly add Vesicle Buffer (25 mM HEPES pH 7.4, 100 mM NaCl) dropwise to the SG-vesicles glass tube. The volume of the added dilution solution should be 3.5 times the volume of the protein solution in Step 2, thereby reducing the concentration of OG below the CMC (critical micelle concentration).

[0105] Step 4 - Column chromatography: Separate proteins, lipids, detergents and Vesicles by passing the sample solution from Step 3 through a CL4B column, and elute with Vesicle Buffer to collect the recombinant Vesicles.

[0106] Step 5 - Dialysis: Add the collected vesicles to a 10 kDa dialysis bag, secure with a dialysis clamp, and dialyze overnight in Vesicle Buffer at 4°C. The dialysis process must be protected from light. After overnight dialysis, transfer the vesicles to 1.5 mL centrifuge tubes and centrifuge at 12000 rpm for 90 seconds at 4°C. Collect the supernatant. Analyze the protein recombination efficiency in the vesicles using SDS-PAGE gel electrophoresis.

[0107] Table 1. Proportion of PM-vesicles

[0108] Table 2. Proportion of SG-vesicles

[0109] In this embodiment, STX3 and SNAP23, purified from prokaryotic expression, are mixed with phospholipids. The detergent is then removed via CL4B chromatography and dialysis, allowing STX3 and SNAP23 to be recombined onto vesicles, resulting in cell membrane-mimicking vesicles, i.e., PM-vesicles. Simultaneously, VAMP8 and calcium ion-sensing protein SYT2 are recombined onto another group of vesicles to obtain secretory granule-mimicking vesicles, i.e., SG-vesicles. The phospholipid composition of the vesicles is a mixture of major phospholipid molecules such as POPC, DOPS, POPE, PIP2, and Cholesterol, in proportions corresponding to the various phospholipid molecules found on the cell membrane. DID and DiI fluorescent molecules are then labeled onto the vesicle membranes as needed for the experiment.

[0110] The recombination efficiency and activity of vesicles were assessed using SDS-PAGE and lipid molecule fusion experiments. The results of SDS-PAGE showed (…). Figure 7 The PM-vesicles had two lanes, and based on the molecular weights of STX3 (33kDa) and SNAP23 (23kDa), the PM-vesicles recombination was successful. Similarly, the SG-vesicles had two lanes, and based on the molecular weights of VAMP8 (11kDa) and SYT2 (47kDa), the SG-vesicles recombination was also successful. Therefore, this embodiment successfully reconstituted vesicles mimicking secretory granules and cell membranes in vitro.

[0111] 2.2 SNARE / SYT2 regulates vesicle membrane fusion, and calcium ions promote fusion.

[0112] This experiment utilizes fluorescence energy resonance transfer (FRET) technology to detect lipid fusion, specifically vesicle fusion efficiency. FRET is an energy transfer phenomenon that occurs when two fluorescent molecules are very close to each other. One fluorescent molecule acts as a donor fluorophore, and the other as an acceptor fluorophore. The emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore. When the distance between them is less than 10 nm, non-radioactive energy transfer occurs, also known as the FRET phenomenon. At this point, the fluorescence intensity of the donor fluorophore is lower than its intensity when it exists alone, while the fluorescence emission intensity of the acceptor fluorophore increases significantly. The experimental samples were added to cuvettes according to Table 3 and placed in a fluorescence spectrophotometer for testing. The vesicle fusion efficiency was detected through a lipid fusion experiment.

[0113] Table 3. Vesicle testing system (50 μL)

[0114] Cell membrane vesicles containing STX3 and SNAP23 were mixed with secretory granule vesicles containing VAMP8 and SYT2 at a 1:1 molar ratio and added to a quartz cuvette. Fluorescence changes were observed and recorded using a fluorescence spectrophotometer. The cell membrane vesicles contained fluorescein DiI, and the secretory granule vesicles contained fluorescein DiD. DiI was excited with a 530nm laser at 37°C, and the fluorescence emission of the DiD dye was detected using the FRET phenomenon. This was used to assess the degree and efficiency of lipid molecule fusion, ultimately determining the vesicle activity. A schematic diagram of lipid fusion is shown below. Figure 8 As shown.

[0115] When lipid fusion occurs in vesicles, the fluorescence signal intensity of the donor's emitted light decreases, while the fluorescence signal intensity of the acceptor's emitted light increases. The efficiency of vesicle lipid fusion is ultimately calculated as (acceptor emission intensity / (donor emission intensity + acceptor emission intensity)). In this experiment, the difference in lipid fusion efficiency under different conditions was used to determine the activity of both vesicles and proteins. Figure 9 As can be seen, the ΔFRET value is 0.15 in the presence of SYT2, which is higher than the ΔFRET value of vesicle fusion with only SNARE protein, indicating that SYT2 can promote vesicle fusion. Adding 100 μM CaCl2 to the presence of SYT2 significantly increases the ΔFRET value, indicating that calcium ions can further promote vesicle fusion. Lipid fusion experiments show that the reconstituted vesicles in this embodiment exhibit good activity in vitro and show good calcium ion dependence, indicating that the purified protein has good activity and can be used for further experimental research. This also demonstrates that calcium ions can promote SNARE / SYT2-mediated vesicle membrane fusion.

[0116] Example 3: Inhibitory effect of small molecule compounds on in vitro recombinant vesicle fusion

[0117] First, fluorescence energy resonance transfer technology was used to analyze small molecule compounds (i.e., 11-F-5 (cathinone)). Figure 10A ), 10-B-9 (cyanidin-3-O-glucoside) Figure 10B ) and 11-B-6 (ellagic acid) Figure 10C The effect of the small molecule compound on vesicle fusion was verified. Experimental samples were added to cuvettes according to Table 4 and placed in a fluorescence spectrophotometer for testing. The effect of the small molecule compound on fusion was detected through a lipid fusion experiment. An equal volume of DMSO was added to the control group.

[0118] Table 4. Vesicle testing system (50 μL)

[0119] Cell membrane vesicles containing STX3 and SNAP23 were mixed with secretory granule vesicles containing VAMP8 and SYT2 at a 1:1 molar ratio. Calcium ions were then added to bring the final concentration to 100 μM. A small molecule compound was then added to bring the concentration to 100 μM. Fluorescence changes were observed and recorded using a fluorescence spectrophotometer. DiI dye was excited with a 530 nm laser at 37 °C, and the fluorescence emission of the DiD dye was detected using the FRET phenomenon to assess the vesicle fusion efficiency after the addition of the small molecule compound. This determined whether the small molecule compound inhibited vesicle fusion (e.g., [missing information]). Figure 11 ).

[0120] The inhibitory effects of the above-mentioned small molecule compounds were evaluated multiple times using lipid fusion assays. The results showed that all of the above small molecule compounds had a certain inhibitory effect on vesicle fusion, with 11-B-6 showing a more significant inhibitory effect, demonstrating a good gradient dependence. Figure 12A , Figure 12B , Figure 12C ).

[0121] This embodiment further verifies the interaction between small molecule compounds and C2B in a pull-down experiment. The pull-down experiment is an in vitro affinity purification technique. Protein pull-down technology can provide a bait protein to specifically recognize a ligand protein, thereby achieving the enrichment, separation, and purification of this protein ligand. This is a powerful tool for studying protein-protein interactions in vitro. The basic principle of this method is to first immobilize a protein on a matrix, and then pass another protein solution through the matrix. If the protein can interact with the immobilized protein, it will be adsorbed, while the unadsorbed "impurities" will flow out with the eluent. The adsorbed protein can be recovered by changing the eluent or elution conditions. We can determine the interactions between proteins using the pull-down technique. This experiment uses the GST-Pulldown technique to verify the interaction between small molecule compounds and SNARE protein and SYT2 C2B protein. The main experimental steps are as follows:

[0122] Constructing the Soluble SNARE complex. 1) First, truncated VAMP8(1-76) and STX3(1-263) proteins were expressed and purified in vitro, with VAMP8(1-76) tagged with GST. Then, VAMP8(1-76)-GST, STX3(1-263), and SNAP23 were mixed in a 1:1:1 ratio, and an appropriate amount of GST Sepharose was added to the protein mixture. The mixture was incubated at 4°C for 2 hours. 2) After incubation, GST Sepharose was washed with washing buffer (25 mM HEPES, pH 7.4, 150 mM NaCl) until the protein reagent no longer changed color. 3) GST Sepharose was washed with elution buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 20 mM GSH) until the protein reagent no longer changed color, and the eluted protein solution was collected. Size exclusion chromatography column purification: Concentrate the eluent collected in step 3) to 2 mL, load the sample through a molecular sieve, and elute with elution buffer (25 mM HEPES, pH 7.4, 150 mM NaCl) for one column volume, then collect the eluent. 5) SDS-PAGE detection: Take samples of proteins near the peak and run them on an SDS-PAGE gel. Determine whether the Soluble SNARE complex has formed by observing the protein bands on the SDS-PAGE gel.

[0123] The Soluble SNARE complex, SYT2 C2B protein, small molecule compound and GSTSepharose were mixed in a certain proportion and incubated at room temperature for 2 hours.

[0124] GSTSepharose was washed with washing buffer (25 mM HEPES, pH 7.4, 150 mM NaCl) by centrifugation. The washing was repeated four times at 2000 rpm for 150 s at room temperature.

[0125] The differences in GST Sepharose protein bands among different groups were compared using SDS-PAGE, and the interaction between the proteins was analyzed.

[0126] This embodiment uses a GST-Pull-down experiment to further verify the effect of small molecule compounds interacting with SNARE on the binding of C2B protein. In the entire system, VAMP8 (1-76) carries a GST tag, thus it can bind to the GST Sepharose on the SNARE complex formed by SNAP23 and STX3 (1-263). With 5 μM of C2B protein in the system, 100 μM of different small molecule compounds were added, and their effects on the binding of C2B to the SNARE complex were observed.

[0127] After co-incubating VAMP8(1-76)-GST, STX3(1-263), SNAP23, and GST Sepharose, the resulting Soluble SNARE complex was purified by molecular sieve analysis and used for subsequent pull-down experiments of small molecule compounds. The results showed ( Figure 13 (Left) Two peaks appear in the molecular sieve plot; the first peak is at 14 mL, consistent with the theoretical value. This was verified by SDS-PAGE. Figure 13 (Right) The Soluble SNARE complex was successfully obtained.

[0128] After co-incubating the Soluble SNARE complex, C2B-WT protein, and small molecule compounds with GST Sepharose, the effect of the small molecule compounds on the binding of C2B-WT to the SNARE complex was observed by SDS-PAGE. The results showed that ( Figure 14 ), 11-F-5, 10-B-9 and 11-B-6 inhibit the binding of C2B-WT to the SNARE complex.

[0129] Secondly, after co-incubating the Soluble SNARE complex, C2B-QM protein, and small molecule compounds with GSTSepharose, the effect of the small molecule compounds on the binding of C2B-QM to the SNARE complex was observed by SDS-PAGE. The results showed that ( Figure 15 ), 11-F-5, 10-B-9 and 11-B-6 do not affect the binding of C2B-QM to the SNARE complex.

[0130] Then, the Soluble SNARE complex, C2B-QQQ protein, and small molecule compound were co-incubated with GSTSepharose, and the effect of the small molecule compound on the binding of C2B-QQQ to the SNARE complex was observed by SDS-PAGE. The results showed ( Figure 16 ), 11-F-5, 10-B-9 and 11-B-6 inhibit the binding of C2B-QQQ to the SNARE complex.

[0131] The above experimental results indicate that the interaction sites of the three small molecules 10-B-9, 11-F-5, and 11-B-6 may be on the primary interface of the C2B-SNARE complex interaction site, and therefore further experiments were conducted on them.

[0132] Example 4: Effect of small molecule compounds on mucin release

[0133] 4.1 Cytotoxicity of Small Molecule Compounds

[0134] The cytotoxicity of 11-F-5, 10-B-9, and 11-B-6 to HEK293T cells was verified using the CCK-8 assay. The specific methods included: Cell plating: 4000 cells per well (100 μL) were plated in 96-well plates. An equal volume of cell-free DMEM medium was added to the outermost ring of wells. The 96-well plates were incubated in a cell culture incubator (37°C, 5% CO2). When the cells reached 80%-90% confluence, 100 μL of a small molecule compound was added per well. The concentration of the small molecule compound was started at 100 μM and then sequentially diluted 4-fold, resulting in nine different concentrations. An equal volume of cell-free DMEM medium was added to the control group. The 96-well plates were incubated in a cell culture incubator (37°C, 5% CO2). After 48 hours of culture, 10% CCK-8 was added, and the cells were incubated in the dark for another 4 hours. The entire process required protection from light. Cell viability was detected by measuring the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0135] The results show ( Figure 17 The three small molecules 11-F-5, 10-B-9, and 11-B-6 are non-toxic to cells. Therefore, this invention uses these three small molecules for subsequent experimental verification.

[0136] 4.2 Validation of small molecule inhibition of mucin release by Dot Blot experiment

[0137] It has been reported that respiratory epithelial cells mainly secrete two mucins, MUC5B and MUC5AC. MUC5B is a baseline-secreted mucin that primarily mediates the baseline barrier and clearance function of the airway; while MUC5AC is mainly a stimulated-secreted mucin that promotes mucosal clearance. Excessive mucin secretion in the airways can easily lead to airway obstruction, thereby causing various obstructive lung diseases, such as asthma, COPD, and CF.

[0138] Dot blotting is a simple and rapid method for protein quantification that quantifies target proteins through the binding of antigens to antibodies or receptor ligands. It is used in ALI culture (…). Figure 18 Calu-3 cells were used, and their mucin metaplasia was induced with IL-13, followed by ATP stimulation to induce mucin secretion. Finally, MUC5AC protein secreted by cells with and without ATP was collected during a 30-minute experimental period (i.e., the upper layer of culture medium in the Transwell chamber was collected), and a Dot Blot experiment was performed to quantify the MUC5AC protein. Specific methods included:

[0139] Collagen was used to coat Transwell chambers. A 4 mL solution of 3 mg / mL collagen was prepared, and 0.5 mL of 10×PBS was added. The pH was adjusted to 6-7, and the pH was tested using pH paper. The solution was then filtered in a biosafety cabinet. In the biosafety cabinet, 160 μL of the collagen mixture was added to each Transwell chamber, and the chamber was gently agitated for 10 seconds to ensure even distribution of the collagen mixture on the semi-permeable membrane. The chamber was capped, and the collagen was allowed to polymerize for 10 seconds. After 10 seconds, the plate was tilted, and excess collagen mixture was aspirated using a pipette. The chamber was then left uncovered and placed in the biosafety cabinet to dry for 1 hour. After 1 hour, the chamber was capped again and left to dry.

[0140] Cell differentiation culture. Preheat the growth medium in a 37°C water bath for 10 minutes. Resuspend the digested cells in growth medium and count them, then dilute the cells to 1.75 × 10⁻⁶. 5 Cells / mL, add 200 μL of cells to each Transwell chamber. Add cells to the top and 600 μL of growth medium to the bottom. Change the cell culture every other day until the cells reach 100% confluence. After confluence, begin ALI culture (Day 0). Remove the culture medium from the top and add 600 μL of differentiation medium to the bottom. Change the cell culture every other day. Starting Day 14, wash with 1×PBS for 30 min every 3 days.

[0141] Small molecule compound treatment. After complete cell differentiation, small molecule compound treatment was performed. 24 hours before mucin stimulation, small molecule compound treatment was administered by adding 20 μL of DMEM medium (containing 100 μM small molecule compound) to the top of the Transwell chamber. The next day, the cells were washed with 100 μL of DMEM medium for 1 hour, for a total of 5 washes.

[0142] Baseline secretion: Add 100 μL of DMEM medium (containing 100 μM small molecule compound) and incubate for 30 min. Take the supernatant for later use and store at -80℃.

[0143] Stimulating secretion: Add 100 μL of DMEM medium (containing 100 μM ATP) and incubate for 30 min. Take a sample of the supernatant for later use. It can be frozen and stored at -80℃.

[0144] Baseline and stimulated secretion samples were analyzed using Dot Blot experiments to observe mucin secretion. The experimental steps are as follows:

[0145] First, activate the PVDF membrane with methanol for 15 seconds. Rinse the PVDF membrane three times with 1×PBS. Add 10 μL of sample to the PVDF membrane using a pipette and allow it to dry in a vacuum pump. Place the PVDF membrane in an incubation chamber and incubate it on a shaker with 5% skim milk blocking buffer for 1 hour. Incubate overnight at 4°C with primary antibody in blocking buffer at a ratio of 1:1000. Place the PVDF membrane in an incubation chamber and wash four times with 1×PBST for 10 minutes each time. Incubate on a shaker at 37°C with secondary antibody in blocking buffer at a ratio of 1:5000 for 90 minutes. Place the PVDF membrane in an incubation chamber and wash four times with 1×PBST for 10 minutes each time. After washing, place the PVDF membrane in developing solution and let it stand for 15 seconds before exposing it using an automatic exposure machine. Save the images and perform image analysis.

[0146] The results of the Dot Blot experiment showed that ( Figure 19 Upon ATP stimulation, the amount of MUC5AC protein secreted voluntarily increased compared to the baseline secretion. However, the addition of 100 μM of the small molecules 11-F-5, 10-B-9, and 11-B-6 significantly reduced the amount of MUC5AC protein secreted voluntarily, without significantly affecting the baseline secretion. This indicates that these three small molecules can inhibit voluntarily secreted mucin and may serve as potential drugs for treating obstructive pulmonary disease.

[0147] 4.3 Validation of intracellular residual mucin by immunofluorescence staining

[0148] Immunofluorescence is a powerful detection technique for detecting and identifying the subcellular distribution and migration of proteins and other molecules in cells and tissues. Its main principle is the antigen-antibody reaction. First, a known antigen or antibody is labeled with fluorescein to become a fluorescent antibody. This fluorescent antibody or antigen is then used as a probe to detect the corresponding antigen or antibody within tissues or cells. Consequently, an antigen-antibody complex containing the labeled fluorescein is formed within the tissue or cells. Finally, the sample is observed using a fluorescence microscope. When irradiated with external excitation light, the fluorescein emits bright fluorescence, allowing observation of the tissue or cells where the fluorescence is located. This enables determination of the nature and location of the antigen or antibody, and allows for quantitative determination of its concentration. The experimental steps of this method are as follows:

[0149] Transfer the baseline secretion and post-secretion chambers of cells to a new 24-well plate and fix with 2 mL of 4% paraformaldehyde solution for 15 min. Wash 3 times with 2 mL of 1×PBS solution for 5 min each. Incubate overnight at 4°C with 1 mL of primary antibody (prepared with 1×PBS) at a dilution of 1:500; or at room temperature for at least 1 h. Wash 3 times with 2 mL of 1×PBS solution for 5 min each. Incubate 3 times at room temperature with 1 mL of secondary antibody containing fluorescein (prepared with 1×PBS) at a dilution of 1:400. From this step onwards, all subsequent steps require protection from light. Wash 3 times with 2 mL of 1×PBS solution for 5 min each. Incubate 20 min at room temperature with 1 mL of cell membrane dye at a dilution of 1:200. Wash 3 times with 2 mL of 1×PBS solution for 5 min each. Stain the cell nuclei with 1 mL of DAPI and incubate at room temperature for 3–5 min. Wash 3 times with 2 mL of 1×PBS solution for 5 min each.

[0150] Sample preparation: Place a drop of antiquenching agent on a glass slide, then gently scrape off the membrane of the cell chamber along the edge with a clean blade, invert the side with cells, immerse it in the antiquenching agent, fix it with nail polish, and dry at room temperature.

[0151] Detection: The residual mucin in the cells was observed and detected using a fluorescence microscope.

[0152] By using various fluorescent antibodies, target proteins can be observed under a microscope and quantitatively analyzed. This embodiment uses immunofluorescence staining to observe the secretion of mucin in Calu-3 cells. Simultaneously, the residual mucin in the cells after treatment with small molecule compounds is observed, further verifying the inhibitory effect of small molecule compounds on induced secretion.

[0153] SNARE / SYT2 can mediate the secretion of stimulating mucin.

[0154] First, the secretion of mucin was observed after ATP stimulation without the addition of small molecule compounds. The results showed ( Figure 20 Upon ATP stimulation, a large amount of mucin was released, and the amount of mucin residue in the cell was significantly reduced. This indicates that SNARE / SYT2 can mediate the secretion of stimulant mucin.

[0155] Inhibitory effect of small molecule compounds on mucin-induced secretion

[0156] In this embodiment, a small molecule compound was added to the culture medium of cultured cells, and the effect of the small molecule compound on the stimulated secretion of mucin was observed. The results showed that ( Figure 21In cells treated with 100 μM 11-F-5, 10-B-9, and 11-B-6, ATP stimulation significantly increased mucin residue. Baseline secretion showed no significant difference in cells treated with 10-B-9 and 11-B-6, but a slight increase in baseline secretion was observed in cells treated with 11-F-5. These results further suggest that these three small molecules, 11-F-5, 10-B-9, and 11-B-6, can act as small molecule inhibitors to suppress stimulative mucin secretion, potentially facilitating their application in respiratory diseases associated with excessive mucin secretion (e.g., obstructive pulmonary disease).

[0157] Summarize

[0158] This invention expresses and purifies SNARE proteins (STX3, VAMP8, and SNAP23, with STX3 and SNAP23 highly expressed in airway epithelial cells and VAMP8 associated with mucin granules in goblet cells of the airway epithelium) and SYT2 protein (a calcium ion sensor that regulates calcium ion-triggered membrane fusion) from the respiratory system in vitro. Then, by reconstructing vesicles in vitro to simulate the fusion of secretory granules with the plasma membrane, it was found that SYT2 acts as a calcium ion sensor in the respiratory system to regulate SNARE-mediated membrane fusion. In the presence of both SYT2 and calcium ions, compared with SNARE complex alone, calcium ions significantly promote membrane fusion in reconstructed vesicles in vitro. Furthermore, this invention demonstrates that SNARE / SYT2-mediated vesicle membrane fusion regulates mucin-induced secretion. In the presence of calcium ions, compared with the presence of SNARE complex and SYT2 alone, calcium ions significantly promote vesicle membrane fusion, and in cell experiments, ATP stimulation significantly increases induced mucin secretion.

[0159] Based on the SNARE / SYT2 regulation of mucin induced secretion, this invention focuses on the primary interface binding site on the SYT2 C2B domain. Through various experiments (e.g., lipid fusion experiments of recombinant vesicles in vitro, GST-Pull-down experiments, etc.), it was verified that small molecule compounds 11-F-5, 10-B-9, and 11-B-6 can act as small molecule inhibitors to suppress mucin secretion. Cytotoxicity experiments confirmed that 11-F-5, 10-B-9, and 11-B-6 are non-cytotoxic. To further demonstrate that small molecule compounds 11-F-5, 10-B-9, and 11-B-6 can inhibit mucin induced secretion, this invention uses an IL-13-induced cell model. IL-13 was used to induce mucin production, and then Calu-3 cells were stimulated with ATP to observe mucin secretion and intracellular residues, verifying the inhibitory effect of small molecule compounds on mucin induced secretion. This invention, through Dot Blot and immunofluorescence staining experiments, revealed that treatment with the three small molecule compounds 11-F-5, 10-B-9, and 11-B-6 reduced the release of mucin while significantly increasing intracellular residual mucin. In other words, these three small molecules can effectively inhibit the stimulative secretion of mucin. Therefore, these three small molecule compounds can serve as potential small molecule inhibitors for the prevention and / or treatment of respiratory diseases (e.g., obstructive pulmonary disease) associated with excessive mucin secretion.

[0160] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. The use of cathinone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting excessive mucus secretion in respiratory diseases, characterized in that, The respiratory disease is selected from one or more of asthma, chronic bronchitis, and chronic obstructive pulmonary disease, and the drug is used to inhibit the secretion of mucin; the secretion is mediated by SNARE / SYT2; the secretion includes stimulative secretion, and the mucin includes the MUC5AC protein.

2. The use as described in claim 1, characterized in that, The symptoms of the disease include one or more of the following: excessive mucus secretion, mucus buildup, airway obstruction, difficulty breathing, infection, and inflammation.

3. The use as described in claim 1, characterized in that, The drug is used to inhibit the fusion of intracellular vesicles with the cell membrane.

4. The use as described in claim 1, characterized in that, The drug is administered orally or intranasally.

5. The use as described in claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

Images