Use of a small molecule compound in the manufacture of a medicament for inhibiting MRGPRX4 and for preventing and / or treating pruritus

Abstract

The application provides use of a small molecule compound in preparation of a MRGPRX4 inhibitor and a medicine for preventing and / or treating itching, and belongs to the field of chemical medicines. The application finds that the small molecule compound has inhibitory effect on MRGPRX4 and can significantly reduce itching, in particular, biliary stasis-induced itching. The effect of compound 6'-O-malonyldaidzin is optimal. The compound found by the application can be used in preparation of a MRGPRX4 inhibitor and a medicine for preventing and treating itching. The application lays a foundation for development of a new MRGPRX4 inhibitor and a medicine for preventing and treating itching, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceuticals, specifically relating to the use of a small molecule compound in the preparation of MRGPRX4 inhibitors and in drugs for the prevention and / or treatment of pruritus. Background Technology

[0002] Chronic pruritus is a common and debilitating condition that severely impairs patients' quality of life and constitutes a significant unmet medical need. Unlike acute pruritus, which is a protective response, chronic pruritus can persist for weeks or months without external stimulation and is often resistant to conventional antihistamines or corticosteroids. It typically occurs in the context of systemic diseases, including cholestatic liver disease, renal dysfunction, and neurological disorders, but its molecular mechanisms remain unclear. Cholestatic pruritus, in particular, is common in patients with primary biliary cholangitis (PBC) and intrahepatic cholestasis of pregnancy (ICP). Existing treatments such as ursodeoxycholic acid, macibatide, and rifampin have limited efficacy, highlighting the urgent need for new targeted interventions. This type of pruritus severely impacts patients' sleep, mental health, and treatment adherence, becoming a critical clinical problem that urgently needs to be addressed.

[0003] Recent research indicates that MRGPRX4 (Mas-related G-protein coupled receptor member X4) is primarily expressed in human sensory neurons and is a key mediator of cholestatic pruritus. MRGPRX4 belongs to the G protein-coupled receptor (GPCR) family and is an orphan receptor expressed in nociceptive sensory neurons. Studies have found that bile acids, especially 3-hydroxy-1-sulfated derivatives accumulated under cholestatic conditions, can directly activate MRGPRX4, driving pruritus signal transduction. Furthermore, certain phosphate-containing drugs (such as creatine phosphate) also induce pruritus through MRGPRX4. Unlike the classic histaminergic pathway, MRGPRX4 represents a unique mechanism linking hepatobiliary dysfunction and pruritus, providing a novel target for the precision treatment of cholestatic pruritus.

[0004] However, targeted therapy for MRGPRX4 faces significant challenges. Although G protein-coupled receptors are among the most drug-targetable protein families, the development of specific small-molecule inhibitors for MRGPRX4 has not yet been successful. Currently, there are no approved MRGPRX4 antagonists or inverse agonists, and existing drugs cannot effectively block the pruritus signaling mediated by this receptor. Therefore, the development of highly effective and selective MRGPRX4 inhibitors has significant clinical value and urgent market demand for the treatment of cholestatic pruritus and related chronic pruritus. Summary of the Invention

[0005] The purpose of this invention is to provide the use of a small molecule compound in the preparation of MRGPRX4 inhibitors and medicaments for the prevention and / or treatment of pruritus.

[0006] This invention provides the use of a compound or a pharmaceutically acceptable salt thereof in the preparation of an MRGPRX4 inhibitor, said compound being selected from one of the following compounds: .

[0007] Furthermore, the compound is selected from... .

[0008] Furthermore, the MRGPRX4 inhibitor is a pharmaceutical preparation made by using one or more of the aforementioned compounds as active ingredients, plus pharmaceutically acceptable excipients.

[0009] Furthermore, the pharmaceutical preparation is a topical preparation or an oral preparation.

[0010] The present invention also provides the use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of pruritus, said compound being selected from one of the following compounds: .

[0011] Furthermore, the compound is selected from... .

[0012] Furthermore, the itching is chronic itching.

[0013] Furthermore, the pruritus is cholestatic pruritus.

[0014] Furthermore, the drug is a pharmaceutical preparation made by using one or more of the aforementioned compounds as active ingredients, plus pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutical preparation is a topical preparation or an oral preparation.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention identified MRGPRX4-targeting molecules through virtual screening of 13,144 compounds. Further research revealed small molecule compounds that inhibit MRGPRX4 and significantly alleviate itching, particularly cholestatic pruritus. Compound 6'-O-malonyldaidzin showed the best efficacy. The compounds discovered in this invention can be used to prepare MRGPRX4 inhibitors and drugs for the prevention and treatment of pruritus. This invention lays the foundation for the development of novel MRGPRX4 inhibitors and drugs for the prevention and treatment of pruritus, and has promising application prospects.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 Figure 1 shows the calcium flux detection results for the compounds: A is a schematic diagram of screening MRGPRX4 inhibitors; B is the calcium flux after adding 10 μM DCA for 20 seconds. 2+ The response represents the trajectory plot; C is the Ca after adding 10 μM UDCA for 20 s. 2+ The response represents the trajectory plot; D represents the result of adding 10 μM DCA at 20 s and pre-incubating cells with the U73122 inhibitor for 30 min before detection. 2+ The response represents the trajectory plot; E represents the result of adding 10 μM UDCA at 20 s and pre-incubating cells with the U73122 inhibitor for 30 min before detection. 2+ Response trajectory diagram; F represents the Ca2+ of different concentrations of DCA and UDCA. 2+ Response curves; G represents the inhibition rate of compounds Hit 1~Hit 5 on MRGPRX4 at a concentration of 50 μM; H represents the dose-effect relationship of compound Hit 1 on calcium flow response in MRGPRX4-HEK293 cells.

[0020] Figure 2 Figure 1 shows the effect of compounds Hit 1-Hit 5 on scratching behavior in DCA-5S-induced mice: A is the experimental flowchart for evaluating the antipruritic effect of compounds Hit 1-Hit 5; B is the result of scratching behavior in mice before and after injection of the pruritin. ±s; n=6; * P<0.05 vs Model group; ** P<0.005 vs Model group; *** P<0.001 vs Model group; ns, not significant. Detailed Implementation

[0021] Unless otherwise specified, the chemical reagents used in the specific embodiments are all conventional commercially available reagents, and the technical means used are all conventional means well known to those skilled in the art.

[0022] This invention employs virtual screening based on the MRGPRX4 receptor structure to select five compounds from 13,144 compounds for further study. The specific screening procedure utilizes conventional techniques in the field (molecular docking screening using Schrödinger Maestro software). These five compounds are all commercially available and can be purchased; they are 6'-O-malonyldaidzin (Hit 1), 4-Caffeoylquinic acid (Hit 2), Folinic acid (Hit 3), Ganoderic acid C6 (Hit 4), and Adenosine triphosphate (Hit 5), and their structures are shown below: .

[0023] Example 1: Detection of calcium flux in compounds 1. Experimental Methods HEK293 cells stably expressing MRGPRX4 (constructed by Sangon Biotech) were placed in DMEM containing 10% fetal bovine serum (FBS), 1 μg / ml puromycin, and 1% penicillin-streptomycin. The cell suspension was then adjusted to a density of 2 × 10⁻⁶ cells / mL. 5 100 μL / well was seeded into 96-well black-walled transparent-bottom culture plates coated with poly-D-lysine solution. Cells were incubated overnight at 37°C in a 5% CO2 incubator. After approximately 24 hours, the culture medium in the wells was discarded, and the cells were seeded with Flexbuffer (containing Ca2+). 2+ Mg 2+ Na +Gently wash cells twice with Flex buffer (pH 7.4), aspirate the Flex buffer, and then add 50 µL of Fluo-4 buffer (containing 4 μM Fluo-4, pH 7.4). Incubate at 37°C in the dark for 60 minutes. Afterward, discard the dye working solution, gently wash cells twice with Flex buffer, and then perform the following experimental procedures: (1) In a 96-well black-walled transparent plate containing cells, add 50 µL of Flex buffer and 20 μM U73122 inhibitor dissolved in Flex buffer to each well, and incubate at room temperature for 30 minutes. Then, place 200-300 μL of agonist DCA (20 μM) and UDCA (20 μM) into a regular 96-well plate, respectively, in Flex buffer. The baseline of the 96-well black-walled transparent plate was detected by a Flex Station 3 multi-microplate reader. After 20 seconds of baseline measurement, 50 μL of agonist DCA buffer and 50 μL of agonist UDCA buffer were automatically added (to make the final concentrations of DCA and UDCA 10 μM respectively), and the fluorescence value was recorded. The measurement was repeated for 90 seconds.

[0024] (2) In each well of a 96-well black-walled transparent plate containing cells, add 50 µL of Flex buffer and incubate at room temperature for 30 minutes. Then, place different concentrations of agonist DCA (i.e., concentrations of 1, 2, 10, 20, 60, 100 μM) and UDCA (i.e., concentrations of 1, 2, 10, 20, 60, 100 μM) in a regular 96-well plate, 200-300 μL per well, in Flex buffer. The baseline of the 96-well black-walled transparent plate was detected by a Flex Station 3 multi-microplate reader. After 20 seconds of baseline measurement, 50 μL of agonist DCA buffer and 50 μL of agonist UDCA buffer were automatically added (to make the final concentrations of DCA and UDCA 0.5, 1, 5, 10, 30, 50 μM, respectively), and the fluorescence value was recorded. The measurement was repeated for 90 seconds.

[0025] (3) In each well of a 96-well black-walled transparent plate containing cells, add 50 µL of a compound at twice the concentration (i.e., 100 μM) dissolved in Flex buffer or a U73122 inhibitor (20 μM) dissolved in Flex buffer, and incubate at room temperature for 30 minutes. Next, place 200-300 μL of agonist DCA buffer (containing 20 μM DCA) in a standard 96-well plate, using Flex buffer as the solvent. Detect the baseline of the 96-well black-walled transparent plate using a Flex Station 3 multi-mode microplate reader. After 20 seconds of baseline measurement, automatically add 50 μL of agonist DCA buffer and record the change in fluorescence value. Continuous measurement is performed for 90 seconds.

[0026] (4) In each well of a 96-well black-walled transparent plate containing cells, add 50 µL of Hit1 compound at twice the concentration dissolved in Flex buffer (i.e., concentrations of 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, and 400 μM) and incubate at room temperature for 30 minutes. Next, place 200-300 μL of agonist DCA buffer (containing 20 μM DCA) in a standard 96-well plate using Flex buffer. Detect the baseline of the 96-well black-walled transparent plate using a Flex Station 3 multi-microplate reader. After 20 seconds of baseline measurement, automatically add 50 μL of agonist DCA buffer and record the fluorescence value change. Repeat this measurement for 90 seconds.

[0027] 2. Experimental Results In this embodiment, deoxycholic acid (DCA) (an effective endogenous MRGPRX4 agonist) is used to induce Ca2+ via the Gq pathway. 2+ In the presence of MRGPRX4 inhibitors, DCA-induced Ca 2+ The reduced influx is reflected in the decreased fluorescence intensity in the Flex Station3 assay (see schematic diagram of screening MRGPRX4 inhibitors). Figure 1 (As shown in A). In the test, both DCA and UDCA stimulated the release of related Ca in HEK293 cells that stably express MRGPRX4. 2+ In addition, the inhibitor U73122 was verified to inhibit Ca. 2+ Signals (such as) Figure 1 (as shown in BE), and the EC50 of DCA and UDCA are approximately 4.8 μM and 6.8 μM, respectively (as shown in BE). Figure 1(As shown in F in the diagram). Next, HEK293 cells stably expressing MRGPRX4 were stimulated with 10 μM DCA. Compounds Hit1-5 (all 50 μM) were screened to identify MRGPRX4 inhibitors. The results showed that compounds Hit1, Hit2, Hit3, and Hit5 inhibited MRGPRX4, with inhibition rates of 91%, 68%, 19%, and 45%, respectively. Compound Hit4 had no inhibitory effect on MRGPRX4, and compound Hit1 showed the best inhibitory effect (as shown in F in the diagram). Figure 1 (as shown in G).

[0028] To further investigate the inhibitory effect of the initially screened active molecule Hit 1, the inhibitory effect of compound Hit 1 on MRGPRX4 was examined at final concentration gradients of 0.39 μM, 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100, and 200 μM to determine whether the inhibitory effect of compound Hit 1 on MRGPRX4 was concentration-dependent. Figure 1 As shown in H, the fluorescence value gradually increases with the increase of Hit 1 concentration, and its IC50 is 2.66 μM.

[0029] Example 2: Study on the inhibitory effect of the compound on itching 1. Experimental Methods To evaluate the antipruritic efficacy of compounds Hit 1–Hit 5, scratching behavior in mice was assessed after DCA-3S-induced pruritus. Adult male C57BL / 6J mice, weighing 20–22 g, were housed in an SPF laboratory. For three consecutive days prior to the behavioral test, they were allowed to acclimatize in individual recording boxes for 30 minutes each day to minimize stress-induced variability. On the day of the experiment, animals were randomly assigned to treatment groups. Specifically, 2.5 hours before injection of the pruritogen, compounds Hit 1–Hit 5 (5 mg / kg) were administered via gavage (using CMC-Na aqueous solution at a concentration of 0.5%). 2.5 hours later, 50 μL of DCA-3S solution (1 mg / mL physiological saline) was injected intradermally into the neck and back of the mice. Scratching behavior in mice was recorded 30 minutes before and 30 minutes after injection using a high-definition camera. Scratching was defined as raising the hind paw to the neck area, followed by a series of rapid scratching motions, and then placing the paw back on the ground. The number of scratches was manually counted by trained observers unaware of the treatment. The experiment used model mice as the Control group (model group), which were administered an equal volume of solvent via gavage.

[0030] 2. Experimental Results DCA-3S (disodium deoxycholic acid-3-sulfate)-induced pruritus is a typical model of cholestatic pruritus. This invention established a mouse model to evaluate the therapeutic potential of compounds Hit 1–Hit 5 for cholestatic pruritus. Compounds Hit 1–Hit 5 were orally administered at doses of 5 mg / kg and 10 mg / kg for 2.5 hours, followed by intradermal injection of DCA-3S to induce pruritus. The number of scratches was quantified before and after DCA-3S administration as behavioral readings of pruritus severity (experimental flowchart shown in Figure 2A). The results showed (e.g.) Figure 2 (As shown in B): Before injection of the pruritin, there was no statistically significant difference in the number of scratches among the groups of mice. After injection of the pruritin, the number of scratches in the model group (Control) mice increased significantly, reaching more than 10 times. Compared with the Control, compound Hit 4 not only did not inhibit the number of scratches, but actually increased the number of scratches, indicating that compound Hit 4 not only failed to inhibit pruritus, but also promoted it. Compared with the Control, compounds Hit 1, Hit 2, Hit 3, and Hit 5 all reduced the number of scratches. Among them, compounds Hit 1 and high-dose compound Hit 2 showed significant differences compared with the Control, indicating a significant reduction in DCA-3S-induced scratching and a significant inhibitory effect on pruritus. Compound Hit 1 showed the best antipruritic effect, significantly better than other compounds. The above findings make compound Hit 1 (6'-O-malonyldaidzin) a promising candidate drug for the treatment of cholestatic pruritus.

[0031] In summary, this invention identified MRGPRX4-targeting molecules through virtual screening of 13,144 compounds. Further research revealed small molecule compounds that inhibit MRGPRX4 and significantly alleviate itching, particularly cholestatic pruritus. Among these, compound 6'-O-malonyldaidzin showed the best efficacy. The compounds discovered in this invention can be used to prepare MRGPRX4 inhibitors and drugs for the prevention and treatment of pruritus. This invention lays the foundation for the development of novel MRGPRX4 inhibitors and drugs for the prevention and treatment of pruritus, and has promising application prospects.

Claims

1. The use of a compound or a pharmaceutically acceptable salt thereof in the preparation of an MRGPRX4 inhibitor, characterized in that: The compound is selected from one of the following compounds: 。 2. The use according to claim 1, characterized in that: The compound is selected from .

3. The use according to claim 1 or 2, characterized in that: The MRGPRX4 inhibitor is a pharmaceutical preparation made by using one or more of the compounds of claim 1 or 2 as active ingredients, plus pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The pharmaceutical preparation is either a topical or oral preparation.

5. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of pruritus, characterized in that: The compound is selected from one of the following compounds: 。 6. The use according to claim 5, characterized in that: The compound is selected from .

7. The use according to claim 5 or 6, characterized in that: The itching is chronic itching.

8. The use according to claim 5 or 6, characterized in that: The itching is cholestatic pruritus.

9. The use according to claim 5 or 6, characterized in that: The drug is a pharmaceutical preparation made by using one or more of the compounds of claim 5 or 6 as active ingredients, plus pharmaceutically acceptable excipients.

10. The use according to claim 9, characterized in that: The pharmaceutical preparation is either a topical or oral preparation.

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