Selective Kv1.3 channel inhibitor

Abstract

The invention discloses a selective Kv1.3 channel inhibitor, isoimperatorin capable of effectively inhibiting the concentration of a Kv1.3 channel is used, the isoimperatorin has the following chemical structure: # imgabs0 #, the selective Kv1.3 channel inhibitor has the advantages that the isoimperatorin is used as a Kv1.3 selective inhibitor for the first time, and an electrophysiological experiment proves that the isoimperatorin can specifically inhibit the current of the Kv1.3 channel, so that the selectivity of the Kv1.3 channel is improved. Compared with the prior art, the compound disclosed by the invention has the advantages that the structure is simple, the activity of inhibiting Kv1.1, Kv1.2 and Kv1.5 channels is almost avoided, the compound has extremely outstanding selectivity, a new lead molecule is provided for developing autoimmune disease drugs, and the compound has important value for developing and preparing pharmaceutical compositions for treating and / or preventing autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to a Kv1.3 channel inhibitor, in particular to a selective Kv1.3 channel inhibitor. Background Art

[0002] Kv1.3 channels are widely distributed in multiple tissues of the human body and participate in many physiological activities, which have important physiological significance. In the immune system and nervous system, Kv1.3 channels play a key role, especially on the surface of T cells. It is a key factor in the sustained activation of effector T cells and plays an important role in the occurrence and development of autoimmune diseases.

[0003] Autoimmune diseases refer to pathological immune responses to self-antigens, leading to abnormal local or systemic inflammatory responses. Common autoimmune diseases include multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, ulcerative colitis, and Crohn's disease. According to statistics from 2019 to 2020, the global prevalence of autoimmune diseases is approximately 7.6% to 9.4%, which has become the third largest chronic disease after cardiovascular disease and cancer, seriously endangering human health.

[0004] In the process of autoimmune diseases, calcium release activated calcium channels (CRAC channels) and voltage-gated potassium channels Kv1.3 on effector memory T cells (TEM cells) are the molecular basis for the activation, proliferation and differentiation of TEM cells, and are considered to be targets for the development of new therapeutic drugs for autoimmune diseases. Therefore, selective Kv1.3 channel inhibitors can target self-reactive TEM cells, inhibit their functional activation, while retaining other lymphocyte subsets to maintain normal immune function, and are potential drug lead molecules for the treatment of autoimmune diseases.

[0005] If Kv1.3 inhibitors also have inhibitory effects on other Kv family members, it will cause a series of side effects. First, other channels in the Kv1.x family also have important functions in the heart. For example, Kv1.5 channels are highly expressed in atrial myocytes. Inhibition of them may lead to delayed repolarization of atrial myocytes and increase the risk of atrial fibrillation. In addition, Kv1.1 channels and Kv1.2 channels are widely present in the nervous system and are involved in the excitability and signal transduction of neurons. Inhibition of these channels may cause excessive neuronal excitation, triggering epileptic seizures or motor coordination disorders. In terms of the immune system, Kv1.1 channels are also expressed in T cells. Inhibition of them may affect the activation and proliferation of T cells, thereby affecting the body's immune response. In addition, Kv1.4 channels are involved in regulating the balance of potassium ions inside and outside cells in liver and kidney cells. Inhibition of them may lead to liver metabolic dysfunction and abnormal renal potassium excretion. Therefore, in order to reduce these potential side effects, it is necessary to improve the selectivity of Kv1.3 inhibitors when developing them to minimize the inhibitory effects on other Kv family members.

[0006] There are currently no selective inhibitors that are completely specific to a single Kv1.3 channel. Although some inhibitors such as Psora-4 and PAP-1 show high selectivity for Kv1.3, they also have a high degree of inhibitory effect on other channels. Therefore, the current Kv1.3 inhibitors still need to be further optimized in clinical applications to improve their selectivity and safety. Future research should be devoted to improving the selectivity of these inhibitors through structural optimization and drug design in order to more accurately target Kv1.3 channels, thereby reducing adverse reactions and improving efficacy. Currently available inhibitors still affect other ion channels to varying degrees. Therefore, the current Kv1.3 inhibitors still need to be further improved in clinical applications to achieve selective blocking of Kv1.3 channels. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a selective Kv1.3 channel inhibitor which can significantly reduce the current of the Kv1.3 channel, thereby inhibiting the activation and proliferation of effector memory T cells (TEM cells) and has almost no inhibitory activity on Kv1.1, Kv1.2 and Kv1.5 channels.

[0008] The first technical solution adopted by the present invention to solve the above technical problems is: a selective Kv1.3 channel inhibitor, using isoimperatorin that effectively inhibits the concentration of Kv1.3 channels, and the isoimperatorin has the following chemical structure: .

[0009] The second technical solution is: use of isoimperatorin in the preparation of a selective Kv1.3 channel inhibitor, wherein the isoimperatorin has the following chemical structure: .

[0010] The third technical solution is: isoimperatorin is used as a selective Kv1.3 channel inhibitor, and the isoimperatorin has the following chemical structure: .

[0011] The fourth technical solution is: a pharmaceutical composition for treating and / or preventing autoimmune diseases, wherein the pharmaceutical composition contains the selective Kv1.3 channel inhibitor according to claim 1 or 2.

[0012] The fifth technical solution is: a pharmaceutical composition for treating and / or preventing autoimmune diseases, characterized in that the pharmaceutical composition contains isoimperatorin, and the isoimperatorin has the following chemical structure: .

[0013] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0014] The autoimmune diseases are multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, ulcerative colitis and Crohn's disease.

[0015] The sixth technical solution is: use of the above-mentioned selective Kv1.3 channel inhibitor in the preparation of a pharmaceutical composition for treating and / or preventing autoimmune diseases.

[0016] The seventh technical solution is: use of isoimperatorin in preparing a pharmaceutical composition for treating and / or preventing autoimmune diseases, wherein the isoimperatorin has the following chemical structure: .

[0017] Compared with the prior art, the advantage of the present invention is that isoimperatorin is used for the first time as a Kv1.3 selective inhibitor. Electrophysiological experiments have confirmed that isoimperatorin can specifically inhibit the current of Kv1.3 channels and has almost no inhibitory activity on Kv1.1, Kv1.2 and Kv1.5 channels, which provides a new lead molecule for the development of drugs for autoimmune diseases and is of great value for the development and preparation of pharmaceutical compositions for the treatment and / or prevention of autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a scatter plot of the blocking effect of isoimperatorin on Kv1.1 current in the examples of the present invention; Figure 2 It is a scatter plot of the blocking effect of isoimperatorin on Kv1.2 current in the examples of the present invention; Figure 3 It is a scatter plot of the blocking effect of isoimperatorin on Kv1.5 current in the examples of the present invention; Figure 4 It is a scatter plot of the blocking effect of isoimperatorin on Kv1.3 current in the examples of the present invention; Figure 5 It is a scatter plot of the blocking effect of the existing inhibitor PAP-1 on Kv1.1 current; Figure 6 It is a scatter plot of the blocking effect of the existing inhibitor PAP-1 on Kv1.2 current; Figure 7 It is a scatter plot of the blocking effect of the existing inhibitor PAP-1 on Kv1.5 current; Figure 8 It is a scatter plot of the blocking effect of the existing inhibitor PAP-1 on Kv1.3 current. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to the accompanying drawings.

[0020] Example 1: Sample preparation: 1. Procurement of the compound Isoimperatorin The isoimperatorin used in the present invention is purchased through commercial channels, and the sample in this example is purchased from Targetmol.

[0021] 2. Quality Control of Isoproteroside The purchased isoimperatorin needs to be quality controlled, and the purity of isoimperatorin must be tested by high performance liquid chromatography (HPLC), mass spectrometry (MS) and other analytical methods to ensure that its purity and activity meet the experimental requirements.

[0022] Example 2: Test of the selective inhibitory activity of isoimperatorin: 1. Experimental materials: DMSO (dimethyl sulfoxide) (purchased from Sigma, catalog number: D5879-500mL), hKv1.1-CHO cell line (Chinese hamster ovary cells stably expressing hKv1.1 channel), hKv1.2-CHO cell line (Chinese hamster ovary cells stably expressing hKv1.2 channel), hKv1.5-CHO cell line (Chinese hamster ovary cells stably expressing hKv1.5 channel).

[0023] 2. Experimental principle: Kv1.1, Kv1.2, Kv1.3 and Kv1.5 ion channels are stably expressed in CHO cells. After the current is stabilized, the current size before and after the application of different compound concentrations can be compared to obtain the effect of the compound on the ion channel.

[0024] 3. Experimental instruments: patch clamp amplifier (Multiclamp 700B, Axon, USA), digital-to-analog converter (DigiData 1440A, Axon, USA), inverted microscope (IX71, Olympus, Japan), rapid drug delivery system (RSC-200, Bio-Logic, France), micromanipulator (MX7600R, Syskiyou, USA), electrode pulling instrument (P-97, Sutter, USA), glass electrode (BF150-86-10, Sutter, USA), anti-vibration table and shielding net (63-534, TMC, USA), data acquisition and analysis software (pClamp 10, Axon, USA), carbon dioxide incubator (HERAcell 150i, Thermo, USA), biological safety cabinet (MODEL 1384, Thermo, USA), water purifier (Milli Q, Millipore, USA).

[0025] 4. Drug preparation: All the compounds required for the test, except NaOH and KOH for acid-base titration, were purchased from Sigma (St. Louis, MO). The final concentration of isoimperatorin was prepared on the same day and then dissolved in the extracellular fluid. Extracellular fluid formula (mM): 140 NaCl, 5 KCl, 1 CaCl 2 , 1.25 MgCl 2 , 10 HEPES (4-hydroxyethylpiperazineethanesulfonic acid) and 10 Glucose (glucose), pH adjusted to 7.4 with NaOH. Intracellular solution formula (mM): 140 KCl, 1 MgCl 2 ,1 CaCl 2 , 10 EGTA and 10 HEPES, pH adjusted to 7.2 with KOH.

[0026] 5. Experimental methods: Electrophysiological techniques, such as manual patch clamp technique, were used to measure the effects of isoimperatorin on Kv1.3, Kv1.1, Kv1.2, and Kv1.5 channel currents. The experimental results showed that isoimperatorin had a significant inhibitory effect on Kv1.3 channels, but had almost no inhibitory activity on Kv1.1, Kv1.2, and Kv1.5 channels, confirming its selective inhibitory properties.

[0027] ①Cell culture and treatment CHO cells stably expressing hKv1.x were cultured in a 35 mm diameter cell culture dish in an incubator at 37°C and 5% CO2. The culture medium was 90% DMEM / F12, 10% fetal bovine serum, and 200 g / mL hygromycin B. On the day of the experiment, the cell culture medium was aspirated, the cells were rinsed with extracellular fluid, and then 0.25% trypsin-ethylenediaminetetraacetic acid solution (Trypsin-EDTA solution) was added and digested at room temperature for 3-5 minutes. The Trypsin-EDTA solution was aspirated, the cells were resuspended with extracellular fluid, and then transferred to the experimental dish for electrophysiological recording for later use.

[0028] ② Compound preparation The compound was prepared into a stock solution with dimethyl sulfoxide (DMSO). On the day of the test, the stock solution was serially diluted with DMSO to obtain an intermediate concentration, and then diluted with extracellular fluid to obtain the final concentration to be tested. The DMSO content in the final test concentration did not exceed 0.2%.

[0029] ③Electrophysiological recording process CHO cells stably expressing hKv1.x potassium channels were used to record hKv1.x potassium channel currents using the whole-cell voltage clamp technique at room temperature. Glass microelectrodes were pulled from glass electrode blanks (BF150-86-10, Sutter) using a puller. The tip resistance after perfusion of the electrode solution was about 2-5 MΩ. The glass microelectrode was connected to the patch clamp amplifier by inserting it into the amplifier headstage. The clamping voltage and data recording were controlled and recorded by pClamp software through a computer, with a sampling frequency of 10 kHz and a filter frequency of 2 kHz. After obtaining whole-cell recordings, the cells were clamped at -80 mV, and the hKv1.x potassium current (I hKv1.x) was induced by a 300 ms depolarizing step voltage of 0 mV or +40 mV. This step voltage stimulus was given every 5 s, and the drug administration process was started after the current was confirmed to be stable (at least 1 minute). Each test concentration of the compound was given for at least 1 minute until the steady state of action or a maximum of 3 minutes, and at least 3 cells (n≥3) were tested for each concentration.

[0030] ④Data processing Data analysis and processing were performed using pClamp 10, GraphPad Prism 8 and Excel software. The degree of inhibition of hKv1.x potassium current (steady-state current amplitude induced at 0 mV or +40 mV) by different compound concentrations was calculated using the following formula: Inhibition % = [1 – (I / Io)]×100% Wherein, Inhibition % represents the inhibition percentage of the compound on hKv1.x potassium current, and I and Io represent the amplitude of hKv1.x potassium current before and after drug addition, respectively.

[0031] Compound IC 50 The results were calculated using GraphPad Prism 8 software by fitting the following equation: Y=Bottom + (Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)) Among them, X is the Log value of the test sample concentration, Y is the inhibition percentage at the corresponding concentration, Bottom and Top are the minimum and maximum inhibition percentages, respectively. HillSlope represents the Hill coefficient (Hill Slope), which is used to describe the steepness of the receptor-ligand binding curve and reflects the synergistic effect between the ligand (inhibitor) and the target (such as Kv1.3 channel).

[0032] Experimental results: Relevant experimental data are shown in Tables 1-8.

[0033] Table 1: Blocking effect of isoimperatorin on Kv1.3 current

[0034] Table 2: Blocking effect of isoimperatorin on Kv1.1 current

[0035] Table 3: Blocking effect of isoimperatorin on Kv1.2 current

[0036] Table 4: Blocking effect of isoimperatorin on Kv1.5 current

[0037] Table 5: Blocking effect of positive control (PAP-1) on Kv1.1 current

[0038] Table 6: Blocking effect of positive control (PAP-1) on Kv1.2 current

[0039] Table 7: Blocking effect of positive control (PAP-1) on Kv1.3 current

[0040] Table 8: Blocking effect of positive control (PAP-1) on Kv1.5 current

[0041] In Tables 1 to 8, 001, 002, and 003 represent the inhibition rate results of three parallel experiments. Figure 1-Figure 8 The scatter plot of isoimperatorin and PAP-1 on Kv1.1, Kv1.2, Kv1.3 and Kv1.5. The horizontal axis of the scatter plot is the concentration, and the vertical axis is the inhibition rate. It is used for the IC values ​​of isoimperatorin on the four ion channels. 50 Value display.

[0042] from Figure 1 , Figure 2 and Figure 3 It can be seen that even when the concentration of isoimperatorin was increased to 100 μM, the inhibition rate of Kv1.1, Kv1.2 and Kv1.5 channels still did not reach 50%, indicating that its inhibitory effect on these channels was weak. Figure 4 It can be seen that in the Kv1.3 channel, when the concentration reaches 4 μM, the inhibition rate has exceeded 50%, indicating that isoimperatorin has a strong inhibitory activity on Kv1.3 and may have a certain selectivity. Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, the positive inhibitor PAP-1 has a broad-spectrum inhibitory effect on Kv1.1, Kv1.2, Kv1.5 and Kv1.3 channels, and the inhibition rate of all channels can exceed 50% at a low concentration of 0.37 μM, indicating that its inhibitory efficacy is much higher than that of isoimperatorin, but its selectivity is poor.

[0043] In summary, compared with the positive control PAP-1, although PAP-1 showed high inhibitory activity on the four Kv subtypes, isoimperatorin showed excellent selectivity, mainly targeting the Kv1.3 channel, and its inhibitory effect on Kv1.1, Kv1.2 and Kv1.5 was much lower than that of PAP-1. This shows that isoimperatorin has significant advantages in selectively inhibiting the Kv1.3 potassium ion channel and has the potential to become a candidate compound for the treatment of related immune diseases. When comparing the selectivity of PAP-1 and isoimperatorin on Kv1.3, we found that isoimperatorin had significantly higher selectivity. Specifically, the IC of PAP-1 on Kv1.1, Kv1.2 and Kv1.5 50 The values ​​of isoimperatorin on Kv1.1, Kv1.2 and Kv1.5 were 2.59, 3.46 and 2.96 times those of Kv1.3, respectively, indicating that although its inhibitory activity on Kv1.3 is strong, the difference from the inhibition of other potassium ion channels is small, so its selectivity is relatively limited. 50The values ​​are 843.51, 79.58 and 267.53 times of Kv1.3, respectively. Especially, the IC 50 Significantly higher than Kv1.3, showing extremely outstanding selectivity. 50 The inhibitory effect of PAP-1 on Kv1.3 was 1.54 μM, which was higher than that of PAP-1 at 0.081 μM, but its inhibitory effect on other channels was much weaker than that on Kv1.3, indicating that isoimperatorin has a particularly prominent advantage in selectivity. Therefore, although PAP-1 has a strong inhibitory effect on Kv1.3, isoimperatorin is more selective on Kv1.3 because its inhibitory effect on other channels is significantly weaker, showing higher selectivity.

[0044] Further analysis of the experimental data showed that isoimperatorin's blocking effect on Kv1.3 ion channels showed an IC 50 IC 50 The value reflects the inhibitory potency of the drug. The lower the value, the stronger the inhibitory ability of the drug. In contrast, the IC values ​​of isoimperatorin for blocking Kv1.1, Kv1.2 and Kv1.5 are 50 All are greater than 100 μM, indicating that its effect on these channels is relatively weak. Kv1.3 channels play a key role in the immune system, especially on the surface of T cells. They are important factors for the sustained activation of effector T cells and play an important role in the occurrence and development of autoimmune diseases. Isoimperatorin in the present invention is very likely to become a new leading drug for the treatment of autoimmune diseases in the future due to its strong specific inhibitory effect on Kv1.3 channels and its very weak blocking effect on Kv1.1, Kv1.2, and Kv1.5 ion channels.

[0045] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A selective Kv1.3 channel inhibitor, characterized in that An isoimperatorin at a concentration effective in inhibiting Kv1.3 channels is used, wherein the isoimperatorin has the following chemical structure: .

2. Use of isoimperatorin in the preparation of a selective Kv1.3 channel inhibitor, wherein the isoimperatorin has the following chemical structure: .

3. Isoimperatorin is used as a selective Kv1.3 channel inhibitor, and the isoimperatorin has the following chemical structure: .

4. A pharmaceutical composition for treating and / or preventing autoimmune diseases, characterized in that The pharmaceutical composition contains the selective Kv1.3 channel inhibitor according to claim 1 or 2.

5. A pharmaceutical composition for treating and / or preventing autoimmune diseases, characterized in that The pharmaceutical composition contains isoimperatorin, and the isoimperatorin has the following chemical structure: .

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that The autoimmune diseases are multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, ulcerative colitis and Crohn's disease.

8. Use of the selective Kv1.3 channel inhibitor according to claim 1 or 2 in the preparation of a pharmaceutical composition for treating and / or preventing autoimmune diseases.

9. Use of isoimperatorin in preparing a pharmaceutical composition for treating and / or preventing autoimmune diseases, wherein the isoimperatorin has the following chemical structure: .

Images