A selective positive allosteric modulator of the proton channel otop1 and methods of making and using the same

Abstract

The application belongs to the field of ion channel pharmacology, and particularly relates to a selective positive allosteric modulator of proton channel OTOP1 and a preparation method and application thereof. The small-molecule compound can significantly enhance acid-activated current of the proton channel OTOP1, does not affect base-activated current and ammonia-activated current of the OTOP1 channel, and has no effect on other two subtypes OTOP2 and OTOP3 channels of the OTOP family. After being combined with the channel, the small-molecule compound does not change the characteristics of the OTOP1 channel itself. In addition, an arginine residue on an extracellular loop of the OTOP1 channel can eliminate the enhancement effect of the small-molecule compound on the acid-activated current of the OTOP1 channel.

Description

Technical Field

[0001] This invention belongs to the field of ion channel pharmacology, specifically relating to a selective positive allosteric modulator of proton channel OTOP1, its preparation method, and its application. Background Technology

[0002] The taste system analyzes soluble chemicals in food before ingestion to maximize nutrient intake and avoid toxins and other harmful substances. Most mammalian species can perceive five basic tastes—bitter, sweet, sour, salty, and umami—primarily detected by specialized taste receptor cells (TRCs) on the tongue and palate. These specialized epithelial cells possess receptors that respond to these five basic tastes. Bitter, sweet, and umami are detected by G protein-coupled receptors. Sourness typically triggers an instinctive aversion response, ensuring that spoiled or rotten food is not ingested. Modern humans also utilize microorganisms to ferment foods that are not inherently sour, making sourness seem particularly mysterious compared to other tastes.

[0003] The first step in acid transduction is generally considered to be H. + The influx of protons into type III TRCs leads to a decrease in intracellular pH. This decrease in intracellular pH is thought to block the resting potassium channel, Kir2.1, thereby triggering membrane depolarization. In its protonated form, weak acids such as acetic acid can cross the cell membrane and acidify the cytoplasm, but strong acids such as hydrochloric acid require the assistance of specific membrane proteins for transport. Recordings from isolated type III TRCs show that protons penetrate the plasma membrane through a zinc-sensitive membrane protein that is specific to type III TRCs compared to other types. Differential RNA sequencing (RNA-seq) screening of the membrane protein encoding genes expressed in type III TRCs confirmed that the OTOP1 protein forms a proton channel and functions as a sour taste receptor in mammals. The gene encoding the OTOP1 channel is highly conserved in vertebrates and flying vertebrates. Due to the unique structure and gating properties of the OTOP1 channel, its gating mechanism remains unknown, and molecular tools for investigating this mechanism are lacking. Therefore, seeking OTOP1 channel modulators to elucidate the channel gating mechanism is crucial. Summary of the Invention

[0004] To address the above problems, this invention provides a 5-methyl-N-naphthol-2-furan-2-carboxamide with the following structural formula:

[0005] The present invention also provides a method for preparing the above-mentioned compound, comprising the following steps:

[0006] (1) Add naphthol-2-amine, HATU and DIEA to a tetrahydrofuran (THF) solution of 5-methyl-2-carboxylic acid furan, and mix well to obtain a mixture;

[0007] (2) The mixture was concentrated and purified to obtain the target compound 5-methyl-N-naphthol-2-furan-2-carboxamide.

[0008] Furthermore, the mixing step in step (1) specifically involves stirring at 70°C for 2 hours.

[0009] Furthermore, the concentration and purification steps in step (2) are specifically performed by preparative high-performance liquid chromatography.

[0010] On the other hand, the present invention also provides the application of the above-mentioned compounds in the preparation of selective positive allosteric modulators of proton channel OTOP1.

[0011] The present invention has the following beneficial effects:

[0012] The small molecule compound provided by this invention can significantly enhance the acid (proton) activation current of the OTOP1 proton channel without affecting the base activation current and ammonia activation current of the OTOP1 channel. It has no effect on the other two subtypes of the OTOP family, OTOP2 and OTOP3 channels, and does not alter the characteristics of the OTOP1 channel itself after binding to it. Furthermore, adding an arginine residue to the extracellular ring of the OTOP1 channel can eliminate the enhancing effect of the small molecule compound on the acid activation current of the OTOP1 channel. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 : Figure 1 A is the structural formula of a small molecule MFaN; Figure 1 B represents the pH value of the extracellular fluid before and after the addition of 100 μM MFaN.

[0015] Figure 2 : Figure 2 A shows representative current diagrams of acid-activated and base-activated currents of the OTOP1 channel (top) and representative current diagrams after adding 10μMMFaN (bottom); Figure 2 B represents a 10μM MFaN with increased channel current ratio; Figure 2C represents the concentration-efficiency relationship curves of MFaN acting on the channel at pH 7.4, pH 6.0, and pH 5.5; Figure 2 D is a representative current diagram of the ammonia current applied by 50μM MFaN to OTOP1; Figure 2 E is a representative current diagram of the effect of MFaN on channels OTOP2 and OTOP3.

[0016] Figure 3 : Figure 3 A is a representative current diagram of the channel before and after zinc ions bind to MFaN; Figure 3 B is the concentration-effect curve of zinc ions acting on the channel before and after MFaN binding; Figure 3 C is the current activation time constant of the OTOP1 channel at pH 5.5; Figure 3 D represents the channel current activation time constant at pH 5.5 after the OTOP1 channel binds to MFaN; Figure 3 E is a statistical graph of the current activation time constant of the channel before and after MFaN binding at pH 5.5; Figure 3 F represents the switching potential of the OTOP1 channel before and after the addition of MFaN at pH 5.5; Figure 3 G is the main cation in the external liquid, which is NMDG and Na. + K + and Cs + A statistical chart of the flip potential (Vrev) of the OTOP1 channel before and after combining MFaN. Detailed Implementation

[0017] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Example 1 Material Synthesis

[0023] 1. Experimental apparatus:

[0024] The whole-cell patch-clamp recording was performed using an EPC10 USB Amplifier (HEKA, Germany), and the data recording and control software was PatchMaster.

[0025] (HEKA, Germany). Other experimental instruments included a PC-10 electrode pulling apparatus (NARISHIGE, Japan), an IX70 inverted microscope (Olympus, Japan), a clean bench, and a carbon dioxide incubator.

[0026] 2. Experimental materials:

[0027] Plasmids: mOTOP1-pcDNA3.1-3×Flag-C were purchased from UBO Biotechnology, and mOTOP2-pcDNA3.1 and mOTOP3-pcDNA3.1 were kindly provided by Professor Emily Liman of the University of Southern California.

[0028] Cell line: HEK293T (human embryonic kidney cells);

[0029] 3. Experimental reagents:

[0030] (1) The DMEM high glucose medium, fetal bovine serum, penicillin and streptomycin and Opti-MEM used for cell culture were all purchased from Thermo Fisher Scientific. Lipfectamine 2000 used for transient cell transfection was purchased from Thermo Fisher Scientific.

[0031] (2) The composition of the cell bath solution and electrode internal solution is shown in Table 1:

[0032] Table 1 Composition of cell bath solution and electrode internal fluid

[0033]

[0034] After the above solutions are prepared, their osmotic pressure needs to be adjusted to a suitable range using sucrose. Generally, the osmotic pressure range of extracellular fluid is between 300-320 mOsm, and that of the electrode solution is between 290-310 mOsm. After adjusting the osmotic pressure, the solutions are filtered through a 0.22 μM filter. The extracellular fluid is stored at 4°C, and the electrode solution is stored at -20°C.

[0035] 4. Experimental Methods:

[0036] 4.1 Synthesis of compound MFaN

[0037] To a solution of 5-methyl-2-carboxylic acid furan (500 mg, 3.96 mM) in tetrahydrofuran (THF) (5 mL), naphthol-2-amine (567.71 mg, 3.96 mM), HATU (1.81 g, 4.76 mM), and DIEA (768.65 mg, 5.95 mM) were added. The mixture was stirred at 70 °C for 2 hours. Then, it was concentrated and purified by preparative high-performance liquid chromatography (prep-HPLC) to obtain the target compound 5-methyl-N-naphthol-2-furan-2-carboxamide, i.e., compound MFaN (400 mg, yield 40.15%), as a yellow solid.

[0038] 4.1 Cell transfection:

[0039] (1) Culture HEK-293T cells. When the cell density reaches 80-90%, discard the culture medium, wash once with 1×PBS, and add 2mL of serum-free culture medium (Opti-MEM).

[0040] (2) Prepare solution A: 250 μL serum-free culture medium + 3 μg DNA, mix gently, and let stand at room temperature for 5 min.

[0041] (3) Prepare solution B: 250 μL serum-free culture medium + 6 μL Lipofectamine 2000, mix gently, and let stand at room temperature for 5 min.

[0042] (4) Gently mix solution A and solution B and let stand at room temperature for 20 minutes.

[0043] (5) After mixing the DNA-liposomes, gently add them dropwise into the cells.

[0044] (6) After 4-6 hours, replace with a full culture medium containing serum.

[0045] 4.2 Activity detection:

[0046] Before the experiment, remove the extracellular fluid from the refrigerator and allow it to reach room temperature. Replace the culture medium in the culture dish with cell culture medium prepared in advance. Handle the solution gently when changing the medium to prevent cells from detaching from the bottom of the dish. Select cells with relatively smooth membranes and homogeneous cytoplasm under an inverted microscope and perform patch-clamp experiments at room temperature (20–25°C). Use borosilicate glass capillaries as the glass electrode material. The glass electrodes are fabricated in two steps using a PC-10 stretching apparatus. After fabrication, fill the glass electrode with intracellular fluid. The water resistance of the glass electrode is 1.5–2.5 MΩ. After a high-impedance king ohm (GΩ) seal is formed between the electrode and the cell membrane, compensate for the electrode's fast capacitance, switch to whole-cell recording mode, apply a short but strong negative pressure to the cell to quickly break the cell membrane clamped in the electrode, and then compensate for the cell's slow capacitance. Clamp the cell at the appropriate test voltage, and begin recording the current after the cell has stabilized for 4–6 minutes. The system resistance (Rs) remained within the range of 5 to 10 MΩ throughout the experiment, essentially unchanged, with the system series resistance (Rseries compensation) being 80% compensated.

[0047] Results and Analysis of Example 2

[0048] 1. Small molecule MFaN enhances the acid activation current of mOTOP1 channel but does not affect the base activation current and ammonia activation current.

[0049] Using the OTOP1 channel as the screening target, a diverse library of parent nuclei (containing approximately 4000 small molecules) was screened using whole-cell patch-clamp recording. A novel small molecule positive allosteric modulator, MFaN (e.g., ...), was identified. Figure 1 As shown in Figure A), at pH 5.5, 10 μM MFaN significantly enhanced the acid activation current of the OTOP1 channel, with an increase of approximately 50%-70%, and the effect was reversible. Subsequent work focused on exploring the interaction mechanism between MFaN and the OTOP1 channel. First, to confirm that the characteristic of MFaN enhancing the OTOP1 channel activation current was not due to the change in pH of the external solution after the addition of MFaN, the real-time pH changes of external solutions with different pH values ​​before and after the addition of 100 μM MFaN were measured and compared using a pH meter. The results of three repeated experiments showed that the addition of MFaN did not change the pH value of the corresponding solution (e.g., ...). Figure 1(As shown in B). Subsequently, 10 μM MFaN was acutely perfused into the same cells, and its enhancement of the acid activation current of the mOTOP1 channel was detected at external solution pH of 7.4, 6.0, 5.5, 5.0, 4.5, 4.0, and 3.5 (internal electrode solution pH of 7.35) (e.g., ...). Figure 2 As shown in Figure A), the experimental results show that MFaN can enhance the acid activation current of the channel at all external pH values ​​except for a pH of 7.4, and the proportion of current increase from MFaN decreases as the pH of the external solution decreases (e.g., ...). Figure 2 B). When the pH of the internal electrolyte solution was 7.35, the external electrolyte solution at pH 7.4 could not activate the OTOP1 channel due to the low proton concentration and lack of a proton concentration gradient. Even after adding MFaN, the OTOP1 channel remained inactive, indicating that MFaN may only function when an external condition exists that allows the OTOP1 channel to be activated; it cannot independently activate the OTOP1 channel to generate current. The concentration-efficiency relationship of MFaN on the mOTOP1 channel was examined at external electrolyte solutions of pH 7.4, pH 6.0, and pH 5.5. At pH 7.4, none of the MFaN concentrations activated the channel, while at pH 6.0 and pH 5.5, the EC50 concentration was significantly lower. 50 The concentrations were 9.21 ± 0.63 μM and 20 ± 1.02 μM, respectively (e.g., ...). Figure 2 (as shown in C).

[0050] Recent studies have found that OTOP1 channels can be activated by alkali and respond to extracellular acid and alkali stimuli. This study indicates that acid and alkali activation of OTOP1 channels may be independent gating processes. Further investigation into the physiological function of alkali activation revealed that, under physiological conditions, taste receptor cells (TRCs) generate inward currents in response to extracellular alkaline stimuli, possessing the potential to induce action potentials, suggesting that TRCs may sense environmental alkaline stimuli. Similarly, two dosing methods were used to perfuse 10 μM MFaN, and its effect on the alkali activation current of mOTOP1 channels was examined at external solution pH of 8.5, 9.0, and 9.5 (internal electrode solution pH of 7.35) (e.g., [missing information]). Figure 2 As shown in Figure A), the experimental results show that MFaN has no effect on the base activation current of the mOTOP1 channel (e.g., Figure 2 (As shown in B and 2C). Small molecule MFaN can significantly enhance the acid-activated current of the mOTOP1 channel while having no effect on the base-activated current, which indirectly confirms that acid and base activation of the OTOP1 channel may be two independent gating processes. Regarding the ammonia-activated current of the OTOP1 channel, experimental results show that small molecule MFaN has no effect on the ammonia-activated channel current (e.g., ...). Figure 2 (As shown in D). Furthermore, the small molecule MFaN has no effect on the currents of the mOTOP2 and mOTOP3 channels (e.g., ...). Figure 2 (As shown in E). The increase in acid activation current of the mOTOP1 channel by 2 small molecule MFaN did not alter the channel's intrinsic properties.

[0051] Small molecule MFaN can significantly increase the acid-activated current of the OTOP1 channel. Therefore, this part of the data aims to investigate whether the increase in channel current after MFaN binding has the same characteristics as the current before binding, and whether it affects the channel properties. First, the effect of MFaN binding on Zn... 2+ Sensitivity and current activation time constant. For Zn in the mOTOP1 channel. 2+ In terms of sensitivity, by detecting Zn 2+ The concentration-effect relationship between the activation current at pH 5.5 before and after the addition of MFaN was investigated, and the results showed that Zn 2+ The inhibition effect of adding 10 μM MFaN on the mOTOP1 channel activation current was not significantly different from that of the DMSO control group: MFaN: IC 50 =72.8±7.6μM; DMSO:IC 50 =58.34±1.5μM (e.g.) Figure 3 (As shown in A and 3B), the results indicate that MFaN binding to the channel does not change the Zn channel. 2+ Sensitivity. The activation time constant (τ) of the mOTOP1 channel activated by the external solution at pH 5.5 before and after drug addition was obtained by fitting a single exponential equation. on A t-test analysis was performed, and the results showed that there was no significant difference in the channel current activation time constant between the 10 μM MFaN group and the DMSO group: MFaN: τ on =457.5±35.4ms; DMSO: τ on =407.2±53.5ms (e.g.) Figure 3 C Figure 3 D and Figure 3 As shown in E.

[0052] The OTOP1 channel exhibits extremely high proton selectivity, the mechanism of which is currently unclear. However, based on studies of other ion channels, the pore diameter of the OTOP1 channel and certain amino acids in the selective filter region of the pore area likely play a role in the selective passage of protons. MFaN binding to the OTOP1 channel may alter the pore diameter and / or amino acid interactions at the selective filter through direct or allosteric mechanisms. Therefore, further investigation was conducted on the channel's flip potential before and after MFaN interaction, as well as the Na+... + K + Cs + Ca 2+ The plasma selectivity was tested, and the results showed that MFaN did not change the channel switching potential or ion selectivity (e.g., Figure 3 (as shown in F and 3G).

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of 5-methyl-N-naphthol-2-furan-2-carboxamide in the preparation of a selective positive allosteric modulator of the proton channel OTOP1, characterized in that, The structural formula of the 5-methyl-N-naphthol-2-furan-2-carboxamide is as follows: .

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