Use of imidazole-4-acetic acid in preparation of drug or health care product for suppressing appetite, lowering blood glucose, reducing weight or promoting weight loss

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to the use of imidazole-4-acetic acid in the preparation of a drug or health care product for suppressing appetite, lowering blood glucose, reducing weight or promoting weight loss. Provided is the use of imidazole-4-acetic acid, and / or a pharmaceutically acceptable salt thereof, a solvate thereof or a precursor thereof in the preparation of a drug or health care product for suppressing appetite. As a natural metabolite, imidazole-4-acetic acid can alleviate toxic and adverse effects and is thus relatively safe. By means of providing the use of imidazole-4-acetic acid in the preparation of a drug or health care product for suppressing appetite, lowering blood glucose, reducing weight or promoting weight loss, it is expected to provide a new strategy for appetite control, blood glucose lowering and weight reduction in obesity.

Description

Use of imidazole-4-acetic acid in the preparation of a drug or health product for inhibiting appetite, reducing blood sugar, reducing weight or losing weight

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application CN202510106301.0, filed on January 23, 2025; the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure belongs to the technical field of biological medicine, and specifically relates to use of imidazole-4-acetic acid in the preparation of a drug or health product for inhibiting appetite, reducing blood sugar, reducing weight or losing weight. BACKGROUND

[0004] Obesity caused by appetite disorders is a predisposing factor for many metabolic diseases such as diabetes, fatty liver, cancer, etc. Therefore, it is necessary for obese people to reduce weight.

[0005] Inhibition of appetite is an effective and relatively safe weight loss method. Many appetite suppressants have been used in clinical practice. In recent years, appetite suppressants have developed rapidly, and the developed drugs mainly include: (1) catecholamines such as fenfluramine, amfepramone, etc., which can increase oxygen consumption by acting on beta receptors to promote energy release, thereby reducing appetite; common adverse reactions include insomnia, dry mouth, constipation, etc. (2) opioid receptor blockers such as naltrexone, etc., which can act on the central nervous system to suppress appetite; common adverse reactions include nausea, vomiting, headache, etc. (3) 5-hydroxytryptamine receptor agonists such as lorcaserin, fluoxetine, etc., which can selectively stimulate the hypothalamic satiety center to reduce food intake and promote satiety; common adverse reactions include headache, dizziness, dry mouth, fatigue, etc. (4) Other drugs can reduce appetite, reduce energy intake, inhibit fat synthesis, increase metabolic rate, increase energy consumption, and thus reduce fat accumulation through central and peripheral effects. The appetite suppressants on the market often cause many side effects, therefore, it is of great clinical significance to develop new safe appetite suppressants.

[0006] Imidazole-4-acetic acid (4-IAA) is one of the most important imidazole derivatives, and is a natural metabolite present in the brain. It has been reported that 4-IAA has multiple uses such as analgesic, sedative, hypnotic, treatment of retinal diseases, as a pharmaceutical intermediate, etc.

[0007] There is no relevant report on 4-IAA for inhibiting appetite, reducing blood sugar, reducing weight, losing weight, or for the preparation of a drug or health product for inhibiting appetite, reducing blood sugar, reducing weight, or losing weight. SUMMARY

[0008] The technical problem to be solved by the present disclosure is that the appetite suppressing, blood sugar reducing, weight reducing, and weight loss health care products or drugs on the market often cause many side effects, and new safe appetite suppressing, blood sugar reducing, weight reducing, and weight loss health care products or drugs are developed, and the use of imidazole-4-acetic acid in the preparation of appetite suppressing, blood sugar reducing, weight reducing, and weight loss drugs or health care products is provided.

[0009] To achieve the above technical purpose, the technical solution adopted by the present disclosure is:

[0010] In one aspect, the present disclosure provides the use of a compound and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a precursor thereof, which binds to the positions of D114, Y115, C118, Y374, F398, L401 and W402 of the histamine H3 receptor (HRH3) with the sequence of SEQ ID NO: 2, in the preparation of any one or more of the following drugs or health care products:

[0011] (1) appetite suppressing drugs or health care products;

[0012] (2) blood sugar reducing drugs or health care products;

[0013] (3) diabetes treating drugs or health care products;

[0014] (4) weight reducing drugs or health care products;

[0015] (5) weight loss drugs or health care products;

[0016] (6) fat reducing drugs or health care products;

[0017] (7) fatty liver treating drugs or health care products.

[0018] In another aspect, the present disclosure provides a method for preparing a drug or health care product, which comprises the step of preparing a compound and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a precursor thereof, which binds to the positions of D114, Y115, C118, Y374, F398, L401 and W402 of the histamine H3 receptor (HRH3) with the sequence of SEQ ID NO: 2, in the preparation of any one or more of the following drugs or health care products:

[0019] (1) appetite suppressing drugs or health care products;

[0020] (2) blood sugar reducing drugs or health care products;

[0021] (3) diabetes treating drugs or health care products;

[0022] (4) weight reducing drugs or health care products;

[0023] (5) a medicine or health food for weight loss;

[0024] (6) a medicine or health food for fat reduction;

[0025] (7) a medicine or health food for treating fatty liver.

[0026] In another aspect, the present disclosure provides a compound binding to the positions of D114, Y115, C118, Y374, F398, L401, and W402 of histamine H3 receptor (HRH3) having the sequence of SEQ ID NO: 2, and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a precursor thereof, for use in the manufacture of a medicine or health food for any one or more of the following:

[0027] (1) a medicine or health food for suppressing appetite;

[0028] (2) a medicine or health food for lowering blood sugar;

[0029] (3) a medicine or health food for treating diabetes;

[0030] (4) a medicine or health food for reducing body weight;

[0031] (5) a medicine or health food for weight loss;

[0032] (6) a medicine or health food for fat reduction;

[0033] (7) a medicine or health food for treating fatty liver.

[0034] In some preferred embodiments of the present disclosure, E206 has specificity for histamine binding.

[0035] In some preferred embodiments of the present disclosure, D114, Y115, C118, Y374, F398, L401, and W402 are residues necessary for HRH3 binding to 4-IAA.

[0036] In some preferred embodiments of the present disclosure, D114, Y115, C118, Y374, F398, L401, and W402 are sites in HRH3 that are required to be common to binding to 4-IAA and histamine.

[0037] In the present disclosure, D114 is aspartic acid at position 114. Y115 is tyrosine at position 115. C118 is cysteine at position 118. Y374 is tyrosine at position 374. F398 is phenylalanine at position 398. L401 is leucine at position 401. W402 is tryptophan at position 402.

[0038] While the disclosure gives some specific amino acid sequences or nucleotide sequences, such as those shown in the sequence listing, it is understood that a specific amino acid sequence or nucleotide sequence includes variants thereof with conservative sequence modifications, such as sequences with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% homology thereto, as long as the biological function or activity of the specific amino acid sequence or nucleotide sequence is not lost.

[0039] As used herein, the term "homology" has the art-recognized meaning and is a central concept in comparative biology. The basic meaning of homology is that two samples (e.g., a stretch of amino acid sequence or a stretch of nucleotide sequence) being compared share a common ancestor. In general, two traits (states) in two species can be said to be a pair of homologous traits if either of the following two conditions is met: 1. they are identical to a trait found in an ancestral group of these species; 2. they are different traits that have ancestor-descendant relationship. Amino acid sequence homology can be determined by methods known per se. For example, amino acid sequence homology (%) can be determined using programs commonly used in the art (e.g., BLAST, FASTA, etc.) according to initial settings. On the other hand, homology (%) can be determined using any algorithm known in the art, such as the algorithm of Needleman et al., Myers and Miller, etc. The algorithm of Needleman et al. is incorporated into the GAP program of the GCG software package (available through www.gcg.com), and homology (%) can be determined, for example, by using any of BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, 14, 12, 10, 8, 6 or 4 and length weight: 1, 2, 3, 4, 5 or 6. In addition, the algorithm of Myers and Miller is incorporated into the ALIGN program as part of the GCG sequence alignment software package. In the case of using the ALIGN program for comparing amino acid sequences, for example, PAM120 weight residue table, gap length penalty, gap penalty can be used.

[0040] In another aspect, the present disclosure provides a use of a compound binding to the positions D114, Y115, C118, Y374, F398, L401 and W402 of a histamine H3 receptor sequence of SEQ ID NO: 2 and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof in the preparation of a medicament or a health product for hypothalamic AgRP neuron-mediated inhibition of appetite, reduction of blood sugar, treatment of diabetes, weight loss, weight loss, fat reduction and / or treatment of fatty liver.

[0041] In another aspect, the present disclosure provides a method for hypothalamic AgRP neuron-mediated inhibition of appetite, reduction of blood sugar, treatment of diabetes, weight loss, weight loss, fat reduction and / or treatment of fatty liver, comprising administering to a patient in need a therapeutically effective amount of a compound binding to the positions D114, Y115, C118, Y374, F398, L401 and W402 of a histamine H3 receptor sequence of SEQ ID NO: 2 and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof.

[0042] In another aspect, the present disclosure provides a compound binding to the positions D114, Y115, C118, Y374, F398, L401 and W402 of a histamine H3 receptor sequence of SEQ ID NO: 2 and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof for use in therapy.

[0043] In another aspect, the present disclosure provides a compound binding to the positions D114, Y115, C118, Y374, F398, L401 and W402 of a histamine H3 receptor sequence of SEQ ID NO: 2 and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof for use in hypothalamic AgRP neuron-mediated inhibition of appetite, reduction of blood sugar, treatment of diabetes, weight loss, weight loss, fat reduction and / or treatment of fatty liver.

[0044] In some preferred embodiments of the present disclosure, the compound is imidazole-4-acetic acid and / or a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, imidazole-4-acetic acid has a structure as shown in formula (I):

[0045] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for one or more of the following uses: reducing body fat percentage, increasing muscle proportion, increasing glucose tolerance, increasing insulin sensitivity, inhibiting or reducing the weight of adipose tissue, reducing white adipose tissue or inhibiting the weight of white adipose tissue, reducing the size or area of white adipocyte cells, maintaining the browning of brown adipose tissue, reducing liver triglyceride levels, preventing or treating obesity, and / or for managing obesity, for an obese, overweight, or obesity-prone subject.

[0046] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for reducing body fat percentage, increasing muscle proportion, increasing glucose tolerance, increasing insulin sensitivity.

[0047] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for one or more of the following uses: inhibiting or reducing the weight of adipose tissue, reducing white adipose tissue or inhibiting the weight of white adipose tissue, reducing the size or area of white adipocyte cells, maintaining the browning of brown adipose tissue, reducing liver triglyceride levels.

[0048] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for one or more of the following uses: inhibiting or reducing the weight of adipose tissue, reducing white adipose tissue or inhibiting the weight of white adipose tissue, reducing the size or area of white adipocyte cells, and / or maintaining the browning of brown adipose tissue.

[0049] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for reducing liver triglyceride levels.

[0050] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for preventing or treating obesity, and / or for managing obesity.

[0051] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for increasing glucose tolerance.

[0052] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for increasing insulin sensitivity.

[0053] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for reducing body fat percentage and increasing muscle proportion.

[0054] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for an obese, overweight, or obesity-prone subject.

[0055] In some preferred embodiments of the disclosure, the pharmaceutical or nutraceutical product is used for inhibiting fatty liver.

[0056] In some preferred embodiments of the disclosure, the drug or nutraceutical is for reducing the pathology of fatty liver.

[0057] In some preferred embodiments of the disclosure, the drug or nutraceutical is for inhibiting or reducing the weight of adipose tissue.

[0058] In some preferred embodiments of the disclosure, the drug or nutraceutical is for reducing white adipose tissue or inhibiting the weight of white adipose tissue.

[0059] In some preferred embodiments of the disclosure, the drug or nutraceutical reduces the size or area of white adipocytes.

[0060] In some preferred embodiments of the disclosure, the drug or nutraceutical is for maintaining the browning of brown adipose tissue.

[0061] In some preferred embodiments of the disclosure, the drug or nutraceutical has at least one effect selected from the group consisting of:

[0062] does not affect the metabolic phenotype of a normal healthy organism;

[0063] does not affect exercise;

[0064] does not cause nausea;

[0065] does not affect preference;

[0066] does not affect learning cognition; and / or

[0067] does not cause anxiety depression.

[0068] In some preferred embodiments of the disclosure, the drug or nutraceutical has the effect of not affecting the metabolic phenotype of a normal healthy organism.

[0069] In some preferred embodiments of the disclosure, the drug or nutraceutical has the effect of not affecting exercise.

[0070] In some preferred embodiments of the disclosure, the drug or nutraceutical has the effect of not affecting total-activity.

[0071] In some preferred embodiments of the disclosure, the drug or nutraceutical has the effect of not causing nausea.

[0072] In some preferred embodiments of the disclosure, the drug or nutraceutical has the effect of not affecting preference.

[0073] In some preferred embodiments of the disclosure, the drug or nutraceutical has the effect of not affecting learning cognition.

[0074] In some preferred embodiments of this disclosure, the drug or health product has an effect that does not cause anxiety or depression.

[0075] In some preferred embodiments of this disclosure, the drug or health product has the effect of not causing nausea or affecting preferences.

[0076] In some preferred embodiments of this disclosure, the drug or health product has the effect of not affecting learning and cognition, and not causing anxiety or depression.

[0077] In some preferred embodiments of this disclosure, the drug or health product has the effect of not causing nausea, not affecting preferences, not affecting learning and cognition, and not causing anxiety or depression.

[0078] In some preferred embodiments of this disclosure, the drug or health product forms an appetite suppressant; the formulation uses the imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors as the sole active ingredient.

[0079] In this disclosure, the dosage form of the preparation is not limited and may be tablets, granules, capsules, pills, oral liquids, injections, etc.

[0080] In some preferred embodiments of this disclosure, the drug or health product forms an appetite-suppressing formulation; the formulation comprises the imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate thereof, or precursor thereof, and also comprises a pharmaceutically acceptable carrier. The type of pharmaceutically acceptable carrier is not limited.

[0081] In some preferred embodiments of this disclosure, the pharmaceutically acceptable carrier includes one or more of the following: solvent, solubilizer, co-solvent, emulsifier, flavoring agent, odorant, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, pH regulator, stabilizer, surfactant, and preservative. Fillers, also known as diluents, include, for example, wheat starch, tapioca starch, corn starch, potato starch, dextrin, microcrystalline cellulose, and lactose. Examples of flavoring agents include, but are not limited to, steviol glycosides, glycyrrhizin, mogrosides, acesulfame potassium, aspartame, sucralose, and isomaltulose. Examples of lubricants include, but are not limited to, magnesium stearate, talc, micronized silica gel, and magnesium lauryl sulfate.

[0082] In some preferred embodiments of this disclosure, the drug or health product forms an appetite-suppressing formulation; the formulation comprises imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, its solvates, its precursors, and one or more other appetite-suppressing active ingredients.

[0083] In some preferred embodiments of this disclosure, the drug or health product forms an appetite suppressant; the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor in a unit dosage for suppressing appetite is 5 to 100 mg, preferably 10 to 60 mg.

[0084] In some preferred embodiments of this disclosure, the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor in a unit formulation for combating diet-induced obesity is 20–300 mg, preferably 30–200 mg.

[0085] In some preferred embodiments of this disclosure, the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor in a unit formulation for weight loss is 50–1000 mg, preferably 100–500 mg.

[0086] In some preferred embodiments of this disclosure, the drug forms an appetite-suppressing formulation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors in a unit formulation for appetite suppression in mice is 75 mg / kg by a single intraperitoneal injection or 120 mg / kg by a single oral gavage.

[0087] In some preferred embodiments of this disclosure, the drug forms an appetite-suppressing formulation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, and precursors in a unit formulation for appetite suppression in mice is 75 mg / kg via a single intraperitoneal injection.

[0088] In some preferred embodiments of this disclosure, the drug forms an appetite-suppressing formulation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors in a unit formulation for appetite suppression in mice is 120 mg / kg via a single oral gavage.

[0089] In some preferred embodiments of this disclosure, the drug forms an appetite-suppressing formulation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, and prodrugs in a unit formulation for combating diet-induced obesity in mice is achieved by preparing the drug into a 3 mg / ml aqueous solution and allowing the mice free access to water. Based on the mice's daily water intake, the dosage for mice is approximately 3–25 mg, preferably 5–20 mg, and more preferably 8–15 mg.

[0090] In some preferred embodiments of this disclosure, the drug forms an appetite-suppressing formulation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, and precursors in a unit formulation for weight loss in obese mice is to prepare the drug into a 12 mg / ml aqueous solution, allowing the obese mice free access to water. Based on the mice's daily water intake, the dosage for mice is approximately 10–80 mg, preferably 30–60 mg, and more preferably 40–55 mg.

[0091] A unit dosage form refers to a dosage form that is prepared and used in a single unit, such as a tablet, a sachet of granules, a capsule, or a bottle of oral liquid. When the content of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, and precursors in a unit dosage form is within the above-mentioned range, it facilitates administration and also facilitates the suppression of appetite.

[0092] In some preferred embodiments of this disclosure, the compound exerts its effect by inhibiting the activity of hypothalamic AgRP neurons.

[0093] In some preferred embodiments of this disclosure, the drug or health product is administered via one or more of the following methods: oral, intravenous, intraperitoneal, intramuscular, rectal, or subcutaneous administration.

[0094] In some preferred embodiments of this disclosure, the drug is administered via one or more methods, including oral, intravenous, and intraperitoneal administration.

[0095] In some preferred embodiments of this disclosure, the formulation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form; preferably, the solid dosage form is a tablet, capsule, granule, or pill; the semi-solid dosage form is a gel, suppository, or ointment; and the liquid dosage form is an emulsion, mixture, suspension, or solution.

[0096] In some preferred embodiments of this disclosure, the drug or health product is a tablet, capsule, injection, powder, pellet, granule, syrup, chewable tablet, or patch.

[0097] In some preferred embodiments of this disclosure, the drug or health product is a tablet, capsule, injection, powder, pellet, granule, syrup, or chewable tablet.

[0098] This disclosure has the following advantages:

[0099] (1) This disclosure provides the use of imidazole-4-acetic acid in the preparation of medicaments or health products for suppressing appetite and medicament or health product compositions containing the imidazole-4-acetic acid. Since imidazole-4-acetic acid is a natural metabolite, it can reduce toxic side effects and is relatively safe.

[0100] (2) This disclosure provides the use of imidazole-4-acetic acid in the preparation of drugs or health products that suppress appetite. It can effectively alleviate metabolic symptoms such as weight gain, hyperglycemia, and insulin resistance caused by a high-fat diet by suppressing appetite, and is expected to provide a new strategy for the treatment of obesity-related appetite and weight loss by lowering blood sugar. Attached Figure Description

[0101] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The accompanying drawings are incorporated in and constitute a part of this specification, illustrating embodiments consistent with this specification, and are used together with the specification to explain the principles of this specification.

[0102] Figure 1 illustrates the identification and functional validation of the metabolite 4-IAA; values ​​are expressed as mean ± standard error, and statistical significance was analyzed using the Student's t-test. Figure 1A shows the daily food intake of mice trained on a restricted diet; Figure 1B shows the food intake of mice trained on a restricted diet at different time points; Figure 1C shows the identification of 4-IAA using non-targeted metabolomics, with the 4-IAA structural formula shown below; Figure 1D shows the plasma 4-IAA content of mice at different time points after feeding; Figure 1E shows the negative correlation between plasma 4-IAA levels and food intake. The correlation between food intake and plasma 4-IAA concentration in Figures 1B and 1D was evaluated using linear regression analysis, and statistical significance (P-value) and goodness of fit (R²) were calculated. Figure 1F shows the dynamic changes in plasma 4-IAA in mice on a free-feeding diet. Plasma was collected every 4 hours via tail vein puncture, and plasma samples were collected for 4-IAA determination (n=4); Figure 1H shows the food intake of mice at different time points after intraperitoneal injection of 4-IAA or 1-IAA, with the 1-IAA structural formula on the left; Figure 1G (left) shows the food intake of mice at different time points after intraperitoneal injection of 4-IAA; Figure 1G (middle) shows the food intake of mice at different time points after gavage administration of 4-IAA; Figure 1G (right) shows the food intake of mice at different time points after mixing 4-IAA into their drinking water; Figure 1I shows the concentration of 4-IAA in the blood of Chow (normal), HFD-fed, and ob / ob obese mice under starvation and feeding conditions, as analyzed by metabolomics; Figure 1J shows the changes in 4-IAA in human blood before and after feeding; Figure 1K shows the correlation between human BMI and 4-IAA in blood, where BMI refers to body mass index, also known as body weight index; R 2The coefficient of determination (R²) is an important concept in statistics used to measure the predictive power of a statistical model. It is primarily used in regression analysis and explains the proportion of variation in the response variable that is explained by the free variables. 2 The values ​​range from 0 to 1, with higher values ​​indicating that the model better explains the coefficient of variation; the p-values ​​were calculated by a two-tailed unpaired t-test without adjustment for multiple comparisons. Figure 1L shows the changes in blood 4-IAA before and after weight loss due to restricted feeding. Figure M shows mice in the Chow group and the HFD feeding group (n=5) that were fasted for 20 hours, then given different concentrations of 4-IAA in their drinking water, and then fed for 2 hours. Food intake was recorded and normalized to the total food intake over 2 hours. Values ​​represent mean ± standard error. The p-values ​​in (D and M) were calculated by a two-tailed unpaired t-test, while the p-values ​​in (I, J, and L) were calculated by a two-tailed paired t-test. The p-values ​​in (E and K) were calculated by one-way ANOVA without adjustment for multiple comparisons, while the p-value in (I) was generated using a linear regression model. Data are expressed as mean ± standard error (SEM). *, p≤0.05; **, p≤0.01; ***, p≤0.001. The EP tube project in Figure 1C was created using BioRender.com.

[0103] Figure 2 illustrates the pharmacological properties of 4-IAA; (A) Dynamic changes in plasma insulin in free-feeding mice. Blood was collected from the tail vein every 4 hours to determine insulin levels (n=4). (B and C) Pharmacokinetics of rats after oral administration of 4-IAA (30 mg / kg). Pharmacokinetic characteristics (B) and pharmacokinetic parameters (C) are shown. (D) In ​​(Figure 1G), plasma was collected from mice 30 minutes after 4-IAA administration. Plasma samples were collected to determine 4-IAA levels (n=6). P values ​​were calculated by one-way ANOVA without correction for multiple comparisons. Data are expressed as mean ± standard error. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

[0104] Figure 3 shows the improvement of abnormal metabolic indicators in HFD obese mice by 4-IAA; Figure 3A shows the food intake of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 3B shows the weight gain of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 3C shows the glucose tolerance of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 3D shows the insulin sensitivity of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 3E shows the weight of liver and adipose tissue in HFD obese mice under 4-IAA, 1-IAA, and water conditions, where iWAT refers to inguinal white blood cells. Figure 3F shows the morphology of adipose tissue and liver in HFD obese mice under 4-IAA, 1-IAA, and water conditions. Figure 3G shows the effects of HFD on liver and adipose tissue pathology. Figure 3H shows the effects of HFD on liver and adipose tissue triglyceride content in HFD obese mice under 4-IAA, 1-IAA, and water conditions. Figure 3H shows the effects of HFD on liver triglyceride content in HFD obese mice under 4-IAA, 1-IAA, and water conditions. Values ​​are expressed as mean ± standard error. P-values ​​were calculated using one-way ANOVA without multiple comparison correction. Data are expressed as mean ± standard error (SEM). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

[0105] Figure 4 illustrates the effect of 4-IAA on metabolic parameters in HFD obese mice using a metabolic cage analysis. Figure 4A shows the cumulative food intake, O2 consumption, CO2 exhalation, respiratory quotient, heat production, and total activity in HFD obese mice. Figure 4B shows the fat and muscle percentages in HFD obese mice under 4-IAA, 1-IAA, and water conditions, where fat percentage is the percentage of fat to body weight, and muscle percentage is the percentage of muscle to body weight. Figure 4C shows the quantitative analysis of white adipocyte area in HFD obese mice. Figure 4D shows the quantitative measurement of plasma 4-IAA levels after the experiment (n=9 per group). P-values ​​were calculated using one-way ANOVA without multiple comparison correction. Data are expressed as mean ± standard error (SEM). *, p≤0.01; ***, p≤0.001.

[0106] Figure 5 illustrates the improvement of multiple metabolic abnormalities in db / db obese mice by 4-IAA; Figure 5A shows the daily food intake of db / db mice (n=7 per group) fed with a diet treated with water or 4-IAA (3 mg / mL). (B) Body weight was recorded weekly. (C, D) Glucose tolerance test (C) and insulin tolerance test (D) were performed at weeks 8 and 9, respectively. (E) The effect of 4-IAA on metabolic parameters in db / db mice was analyzed using metabolic cages (n=4 per group). (F) Mice were dissected and tissue weights were measured at week 10. (GI) Photographs (G) and H&E staining (H) of iWAT, gWAT, BAT, and liver are shown. The areas of iWAT and gWAT are calculated in (I). (J) Liver triglyceride levels were measured. Scale bars are shown. (K) Experimental setup is shown in Figure 3 (n=8 per group). After 5 weeks of treatment, 4-IAA was withdrawn, and mouse body weight was monitored for another 3 weeks. P-values ​​were calculated using a two-tailed unpaired t-test without correction for multiple comparisons. Data are expressed as mean ± standard error. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

[0107] Figure 6 shows the effect of 4-IAA on abnormal metabolic indicators in ob / ob obese mice; Figure 6A shows the effect of 4-IAA on food intake in the ob / ob obese mouse model; Figure 6B shows the effect of 4-IAA on weight gain in the ob / ob obese mouse model; Figure 6C shows the effect of 4-IAA on glucose tolerance in the ob / ob obese mouse model; Figure 6D shows the effect of 4-IAA on insulin sensitivity in the ob / ob obese mouse model; Figure 6E shows the effect of 4-IAA on liver and adipose tissue weight in the ob / ob obese mouse model, where iWAT refers to inguinal white adipose tissue, gWAT refers to gonadal white adipose tissue (also known as visceral white adipose tissue), and BAT refers to brown adipose tissue; Figure 6F shows the effect of 4-IAA on the hepatic and hepatobiliary pathology of liver and adipose tissue in the ob / ob obese mouse model; Figure 6G shows the area of ​​white adipocytes; Figure 6H shows the effect of 4-IAA on liver triglyceride content in the ob / ob obese mouse model. Numerical values ​​are expressed as mean ± standard error. Statistical significance was analyzed using the Student's t-test. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

[0108] Figure 7 shows the effect of 4-IAA on the metabolic phenotype of mice with normal physiological conditions; Figure 7A shows the effect of 4-IAA on the food intake of normal healthy mice; Figure 7B shows the effect of 4-IAA on the weight gain of normal healthy mice; Figure 7C shows the effect of 4-IAA on the glucose tolerance of normal healthy mice; Figure 7D shows the effect of 4-IAA on the insulin sensitivity of normal healthy mice; Figure 7E shows the effect of 4-IAA on the body fat percentage of normal healthy mice; Figure 7F shows the effect of 4-IAA on the adipose tissue and liver weight of normal healthy mice; Figure 7G shows the effect of 4-IAA on the morphology of the liver and adipose tissue of normal healthy mice; Figure 7H shows the effect of 4-IAA on the pathology of hepatitis B surface antigen (HE) in normal healthy mice; Figure 7I shows the effect of 4-IAA on the area of ​​white adipocytes in normal healthy mice; Figure 7J shows the effect of 4-IAA on the triglyceride content in the liver of normal healthy mice. Numerical values ​​are expressed as mean ± standard error. Statistical significance was analyzed using the Student's t-test; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

[0109] Figure 8 illustrates the use of 4-IAA for weight loss; (A) C57BL / 6 mice were fed a high-fat diet (HFD) until they reached a weight of approximately 50 g. The mice were then divided into two groups (n=6 per group) and treated with 4-IAA (12 mg / mL) in their drinking water. (B) Weight was monitored weekly. (C and D) Glucose tolerance test (C) and insulin tolerance test (D) were performed at weeks 3 and 4, respectively. (E) Body composition was monitored at week 4. (F) Tissue weights of liver, gWAT (abdominal white adipose tissue), iWAT (intra-abdominal white adipose tissue), and BAT (brown adipose tissue). (G) Hematoxylin-eosin (H&E) analysis of liver and adipocyte tissue. Scale bars are shown. (H) Quantitative statistical analysis of adipocyte size in iWAT and gWAT. (I) Monitoring of liver triglyceride levels. (J) Quantitative determination of plasma 4-IAA levels. P-values ​​were calculated using a two-tailed unpaired t-test without correction for multiple comparisons. Data are expressed as mean ± standard error. *, p≤0.05; **, p≤0.01; ***, p≤0.001. Figure 3A was created by BioRender.com.

[0110] Figure 9 illustrates the behavioral effects of 4-IAA on mice. 4-IAA had no effect on movement, food preferences, or cognition in mice. (A) WTC57BL / 6 mice (n=8 per group) were fasted for 20 hours and then received intraperitoneal injection of 4-IAA (75 mg / kg) or saline. The effects of 4-IAA on food intake, water intake, and total activity were monitored using metabolic cages. (BH) Mice were administered 4-IAA by intraperitoneal injection (ip, 75 mg / kg), oral gavage (po, 120 mg / kg), or in-water addition (3 mg / mL) before the behavioral test. (B) Mice (n=5 per group) were fasted for 20 hours, administered 4-IAA, and then fed a normal diet (left) or kaolin (right). (C) The left figure is a schematic diagram of the conditioned taste avoidance test (n=6 per group). On day 11, 4-IAA was administered by intraperitoneal injection or oral gavage 5 minutes before the test. The right figure shows the effect of 4-IAA. LiCl was used as a positive control. (DH) For intraperitoneal injection or gavage, 4-IAA was administered 5 minutes before the test. For addition to drinking water, 4-IAA was administered 7 days before the test. (D) Effect of 4-IAA on learning ability in mice during the water maze test (n=9 per group). (E) Effect of 4-IAA on anxiety and depression in mice during the forced swimming test (n=9 per group). (F) Effect of 4-IAA on anxiety and depression during the tail suspension test (n=9 per group). (G and H) Anxiety levels were tested in the open field experiment (n=9 per group). P-values ​​were calculated using a two-tailed unpaired t-test without correction for multiple comparisons. Data are expressed as mean ± standard error. *, p≤0.05; ***, p≤0.001. Wherein, immobility is completely immobile; immobilitylow is low-frequency immobility.

[0111] Figure 10 shows the metabolic pathway of 4-IAA and the major metabolic enzyme AOC1; (B) mRNA levels of Hdc, Aoc1, and Hnmt in designated tissues. (C and D) mRNA (C) and protein (D) levels of AOC1 in different parts of the small intestine on day 0 and day 7 of TRF. (E) Expression analysis of Aoc1 in the BroadInstitute mouse single-cell sequencing database. (F) Jejunal fragments were collected from wild-type (WT) and Aoc1- / - mice. Frozen sections were prepared and immunostained with designated antibodies. Scale bars are shown. (G) Mice (n=3) were administered histamine (30 mg / kg) or histidine (30 mg / kg) by gavage. Plasma was collected 1 hour later to determine 4-IAA levels. (H) WT and Aoc1- / - mice (n=8 per group) were fasted for 20 hours and then refeeded for 2 hours. Plasma samples were collected to determine 4-IAA levels. (I) As shown in (G), WT and Aoc1- / - mice (n=6 per group) were administered histamine by gavage. (J) After a 20-hour fast, mice were intraperitoneally injected with leptin (5 mg / kg), liraglutide (0.1 mg / kg), or WAY161503 (7 mg / kg) one hour before refeeding. Food intake was recorded at specified time points. P-values ​​were calculated using a two-tailed unpaired t-test without correction for multiple comparisons. Data are presented as mean ± standard error (SEM). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

[0112] Figure 11 shows the identification of Aoc1 knockout mice; (A) Food intake of mice after intraperitoneal injection of saline, 4-IAA (75 mg / kg), or Me-IAA (75 mg / kg) (n = 6 per group). (B) Analysis of AOC1 mRNA expression from the DISCO human single-cell database. (C) Quantification of the Western blot bands in Figure E (n = 3). (D) Aoc1 expression in the small intestine of fasted and re-fed mice (n = 3). (E) Schematic design of Aoc1 knockout mice. (FH) Validation of Aoc1 knockout mice by genotyping (F), qRT-PCR (G), and Western blot (H). (I) Measurement of 4-IAA content in the tissues shown (n = 4). The p-values ​​in (A) were calculated by one-way ANOVA, and the p-values ​​in (C, G, and I) were calculated by two-tailed unpaired t-tests without multiple comparison correction. Data are presented as mean ± standard error. *, p≤0.05; **, p≤0.01; ***, p≤0.001.

[0113] Figure 12 shows the phenotypic analysis of Aoc1 knockout mice (Aoc1- / -). (A) Daily food intake of HFD-fed Aoc1+ / + (control group) and Aoc1- / - mice (n=8 per group). (B) Weekly body weight recording. (C and D) Glucose tolerance test (C) and insulin tolerance test (D) were performed at weeks 10 and 11, respectively. (E and F) Body composition of mice was monitored and tissues were dissected and weighed at week 12. (G) H&E of iWAT, gWAT, BAT, and liver are shown. (H) Liver triglyceride levels were measured. Scale bars are shown in the figure. (I) Aoc2 and Aoc3 mRNA levels were measured in designated tissues of C57BL / 6 mice (n=3). Cyclophilin was used as an internal control. (J) Aoc2 and Aoc3 mRNA levels in gonadal white adipose tissue of HFD-fed WT and Aoc1- / - mice (n=4 per group) were analyzed. (K) In vitro enzyme activity assays were performed on purified bacterial AOC1, AOC2, or AOC3 (30 ng) to assess their ability to synthesize 4-IAA, as described in the methods. P-values ​​were calculated using a two-tailed unpaired t-test without adjustment for multiple comparisons. Data are expressed as mean ± SEM. *, p ≤ 0.05; **, p ≤ 0.01.

[0114] Figure 13 illustrates the inhibition of AgRP neuron activity in the hypothalamic ARC region by 4-IAA. (A) Schematic diagram of whole-brain transparency and c-Fos staining. The lower left panel shows c-Fos staining in the appetite-controlling region. The lower right panel shows immunostaining of the brain alone using anti-c-Fos antibodies. (B and C) Mice were fasted for 20 hours and then injected into the third ventricle with 4-IAA (12 μg), 1-IAA (12 μg), or saline. After 90 minutes, the brain was dissected and immunostained with the specified antibody (B). Quantification of c-Fos activation in AgRP neurons was performed (C). (D and E) Expression of the Ca2+ sensor GCaMP in AgRP neurons using the AAV system. Mice were fasted for 20 hours and then injected intraperitoneally with saline or 4-IAA (75 mg / kg), and fiber photometric analysis was performed (n = 8 per group). (FH) Brain sections of AgRP-Cre;CAG-LSL-tdTomato mice were prepared and patch-clamped for analysis. Spontaneous action potential firing of AgRP neurons (n ​​= 10 cells from 4 mice) before and after administration of 4-IAA (1.5, 5, 15, or 50 μM) was recorded in F and G, and the inhibitory effect of 4-IAA was summarized in H. (I and J) AgRP-Cre;R26-LSL-hM3Dq mice (n = 9 per group) received intraperitoneal injections of CNO (0.5 mg / kg) and saline or 4-IAA (75 mg / kg) followed by feeding studies. P-values ​​in C were calculated using one-way ANOVA. P-values ​​in J were calculated using two-tailed unpaired t-tests without multiple comparison correction, and p-values ​​in G were calculated using two-tailed paired t-tests. Data are expressed as mean ± standard error. **, p ≤ 0.01; ***, p ≤ 0.001. Figure I was created by BioRender.com.

[0115] Figure 14 illustrates the whole-brain transparency assay for locating and validating 4-IAA target brain regions; (A) On days -9 and -7 of the experiment, mice (n=4 per group) were subcutaneously injected with either the control solvent or 50 mg / kg capsaicin. After one week of recovery, the mice were fasted overnight and intraperitoneally injected with either saline or 75 mg / kg 4-IAA before being refeeded. Food intake was recorded at specified times. (B) One week prior to the experiment, mice (n=6 per group) were cannulated in the third ventricle (ICV). The mice were fasted for 20 hours and infused with either 4-IAA (12 μg) or 1-IAA (12 μg) before being refeeded. Food intake was recorded at specified times. (C) As shown in Figure A, c-Fos staining analysis of the whole brain was performed. This showed areas of change between the saline and 4-IAA treatment groups. (D) The experiment was performed as shown in Figure B, but using anti-POMC antibody. (E and F) Overnight-fasted mice (n=3 per group) were intraperitoneally injected with saline, 4-IAA (75 mg / kg), or 1-IAA (75 mg / kg). After 90 minutes, the brains were dissected and immunostained using the designated antibody (E). Activation of c-Fos in AgRP neurons was quantified (F). (G and H) Brain sections were prepared from AgRP-Cre;CAG-LSL-tdTomato mice treated with PTX (5 μM), DNQX (20 μM), CNQX (20 μM), D-AP5 (50 μM), or a combination thereof. Spontaneous action potential firing of AgRP neurons (from 10 cells in 4 mice) was recorded using patch clamp. Scale bars are shown. P-values ​​in (A) were calculated using a two-tailed unpaired t-test. P-values ​​in (B and F) were calculated using one-way ANOVA without multiple comparison correction. P-values ​​in (H) were calculated using a two-tailed paired t-test. Data are expressed as mean ± standard error (SEM). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

[0116] Figure 15 illustrates 4-IAA targeting the AgRP-MC4R neuronal pathway to suppress appetite; (AC) 4-IAA reversed CNO-induced AgRP neuronal activation. The strategy of introducing hM3D(Gq) and mCherry into AgRP neurons via the AAV system is shown in Figure 15 (A). Brain slices from Agripp-Cre mice were treated with CNO (10 μM) followed by 4-IAA (50 μM). Spontaneous action potential firing of AgRP neurons was recorded using patch clamp (n = 10 cells from 4 mice) (B and C). (DF) Wild-type (WT) or Mc4r- / - mice (8 weeks old, n = 6 per group) were fed a high-fat diet (HFD) for 4 weeks, followed by treatment with water or 4-IAA (12 mg / mL) (D). Daily food intake of mice was monitored (E). Body weight was monitored every 2 days (F). On day 24, mice were dissected and tissues were weighed. The p-value in Figure (C) was calculated using a two-tailed paired t-test. The p-values ​​in Figure (DF) were calculated using a two-tailed unpaired t-test without correction for multiple comparisons. Data are expressed as mean ± standard error. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

[0117] Figure 16 shows the screening of receptors for the metabolite 4-IAA. (A) Isolated hypothalamus from wild-type (WT) mice were treated with forskolin (10 μM) for 15 min in the presence or absence of 4-IAA (1 nM to 10 μM), followed by measurement of cyclic adenosine monophosphate (cAMP) levels. Concentration curves of 4-IAA are plotted below. Each value represents the mean ± standard error (SEM) of 6 samples. (B) Candidate screening of 4-IAA receptors. On day 0, the 15 most abundant GPCRs expressed in ARC (GSE96627) were transfected into HEK293T cells expressing GloSensor. On day 2, cells were treated with forskolin (10 μM) in the presence of 100 pM, 1 nM, or 100 nM of 4-IAA or 1-IAA, and cAMP levels were monitored by GloSensor analysis. Each value represents the mean ± SEM of 3 samples. (C) Detailed concentration curves of 4-IAA inhibiting cAMP production by HRH3. Each value represents the mean ± SEM of 6 samples. (D) Forty-eight hours post-transfection, membrane components were isolated from HRH3-expressing HEK293 cells and incubated with 5 nM 3H-histamine in the presence of different concentrations of unlabeled histamine, iodophenpropit, or 4-IAA. Data were normalized to the binding values ​​of HRH3-overexpressing membranes to [3H]-histamine in the absence of unlabeled ligands. Representative binding curves for independent experiments are shown (histamine n = 6; iodophenpropit n = 5; 4-IAA n = 8). (E and F) Wild-type mouse hypothalamus was treated with forskolin (10 μM) for 15 min with or without specified combinations of histamine (10 nM and 100 nM), iodophenpropit (1 μM), and 4-IAA (100 nM), followed by measurement of cAMP levels. Each value represents the mean ± SEM of 3 samples. (G) HEK293T cells were transfected with HRH3, engineered Gβγ, and Gαi1, Gαi2, Gαi3, or Gαo, respectively. Cells were treated with different concentrations of 4-IAA and then subjected to BRET analysis. Each value represents the mean ± SEM of four samples. (H) Schematic diagram of the structure determination of the 4-IAA-HRH3-mGo complex. (I) Cryo-electron microscopy density map (PDB: 9K3J) and band representation of the 4-IAA-HRH3-mGo complex. The three-dimensional (3D) structural representation details the interaction between 4-IAA and HRH3 in the ligand-binding pocket. (J) Membrane components expressing specified HRH3 mutations were incubated with 5 nM3H-histamine and different concentrations of unlabeled 4-IAA. Values ​​represent the mean ± SEM of three independent experiments (n=3). NR, no response to the ligand. aND, activity was observed but not measured due to low signal. Comparison of the binding sites of (K)4-IAA and histamine with HRH3.The Ballesteros-Weinstein numbered residue positions of HRH3 are shown at the top of the diagram. Purple circles indicate HRH3 residues that interact with both 4-IAA and histamine. Orange circles indicate HRH3 residues that interact only with 4-IAA or histamine. Gray circles indicate HRH3 residues that do not interact with 4-IAA or histamine. Red lines indicate hydrogen bonds formed between acidic HRH3 residues and 4-IAA or histamine. *, p≤0.05; **, p≤0.01.

[0118] Figure 17 shows the structural screening of 4-IAA and HRH3 interaction; (A) saturation binding of [3H]-histamine to HRH3 or pcDNA3.1 overexpressing HEK293 cell membrane components. Specific binding curves were obtained by subtracting non-specific bindings (measured in the presence of 10 μM unlabeled histamine) from the total bindings. The data shown correspond to representative curves from three independent experiments (n=3). Histamine is a high-affinity endogenous ligand for HRH3. (B and C) Competition binding curves of 1-IAA (B) and 1-methyl-4-IAA (C) in HRH3 overexpressing HEK293 cell membranes. Membranes isolated from HRH3 overexpressing HEK293 cells were incubated with 5 nM [3H]-histamine and different concentrations of unlabeled 1-IAA and 1-methyl-4-IAA. Data were normalized to the binding values ​​of [3H]-histamine to HRH3 overexpressing cell membranes in the absence of unlabeled ligands. Representative binding curves from three independent experiments (n=3) are shown. (D) Expression of Hrh1-4 in AgRP neurons (GSE68177) of fasted and fed mice. (E) HEK293T cells expressing engineered G protein probes and HRH1 or HRH3 were treated with different concentrations of 4-IAA (1 pM to 1 mM) and BRET analysis was performed to assess the dissociation of Gαo-Gβγ. Values ​​represent the mean ± SEM of three independent experiments. (FI) Purification (F), 2D structural diagram (G), resolution analysis (H), and single transmembrane domain (I) of the 4-IAA-HRH3-mGo complex. Scale bars are shown. (JN) Comparison of 4-IAA-bound and unbound structures of HRH3. (O) Histamine-HRH3 complex (PDB: 8YUU). A magnified view shows the binding pocket of HRH3 with histamine. (P) Competition experiments were performed as shown in Figure 6J, but with different concentrations of histamine. Each value represents the mean ± standard error (SEM) of four samples. NR, no response to the ligand. aND, activity observed but not measured due to low signal. (Q) Detection of mutant expression levels. (R) Chemical structures of 4-IAA and several commercially available structural analogs. (S to V) Competitive binding curves of 1H-imidazole-4-carboxylicacid, 2-(pyridin-3-yl)aceticacid, 2-(pyridin-2-yl)aceticacid, or 2-(pyridin-4-yl)aceticacid were detected in membrane fractions prepared from HEK293 cells overexpressing the HRH3 receptor harvested 48 hours after transfection.Membrane components expressing the specified HRH3 wild-type (WT) were incubated with 5 nM 3H-histamine and different concentrations of unlabeled 1H-imidazolium-4-carboxylic acid, 2-(pyridin-3-yl)acetic acid, 2-(pyridin-2-yl)acetic acid, or 2-(pyridin-4-yl)acetic acid. Values ​​represent the mean ± standard error (SEM) of three independent experiments (n=3).

[0119] Figure 18 illustrates how 4-IAA inhibits appetite by acting on HRH3 receptors in AgRP neurons in the ARC region. (A and B) Brain slices from AgRP-Cre;CAG-LSL-tdTomato mice treated with 4-IAA (50 μM) or pretreated with the HRH3 antagonist ciproxifan (37.5 μM) or GSK189254 (20 μM). Spontaneous action potential firing of AgRP neurons (n ​​= 10 cells from 4 mice) was recorded by patch clamp. (C) C57BL / 6 mice (n = 5 per group) were fasted for 20 hours and then received intraventricular injection (ICV) of 4-IAA (12 μg) with or without ciproxifan (3 μg) before being refeeded. (D) HRH3 knockdown in AgRP neurons was achieved by unilateral injection of DIO-shHRH3-AAV into AgRP-Cre mice. Mice were fasted for 20 hours and then administered an intraperitoneal injection (IP) of 4-IAA (75 mg / kg). Immunostaining of c-Fos and AgRP was performed 90 minutes post-injection (n=8 per group). (EJ) HRH3 knockdown in AgRP neurons was achieved by bilateral injection of AAV encoding DIO-shHRH3 into Agrp-Cre mice (AgrpΔHRH3). (E) Mice were fasted for 20 hours and then administered an intraperitoneal injection of 4-IAA (75 mg / kg), followed by refeeding (n=5 per group). (FJ) AgrpΔHRH3 mice were fed a high-fat diet (HFD) (n=6 per group). (F) Daily food intake was recorded. (G) Body weight was monitored weekly. Glucose tolerance tests (H) and insulin tolerance tests (I) were performed at weeks 7 and 8, respectively. (J) At week 9, mice were dissected and tissue weights were measured. (K) Schematic diagram of the inhibitory effect of 4-IAA on food intake. The p-values ​​in (B) were calculated using a paired two-tailed t-test, while the p-values ​​in (CJ) were calculated using a non-paired two-tailed t-test without multiple comparison correction. Data are expressed as mean ± standard error (SEM). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Figure 7K is produced by BioRender.com.

[0120] Figure 19 shows the effect of 4-IAA on HRH3 receptors on AgRP neurons in the ARC region to suppress appetite. (A and B) Brain slices of AgRP-Cre;CAG-LSL-tdTomato mice treated with histamine (50 μM) or imepip (50 μM). Spontaneous action potential firing of AgRP neurons was recorded by patch clamp (n = 10 cells, from 4 mice). (C) Appetite suppression by the HRH3 agonist imepip. Mice were fasted for 20 hours, then received intraperitoneal injection of imepip (30 mg / kg), and then refeeded (n = 8 mice / group). (D) Knockdown efficiency of HRH3 shRNA. (E) Experiments were performed as shown in (Figure 7D). Immunostaining analysis results are shown. (FJ) Using the same mice as in (Figure 7E-7J). The figures show body composition (F), photographs (G), and H&E analysis (H) of liver and adipose tissue, as well as quantitative analysis of adipocyte size (I) and liver triglyceride levels (J) of intraperitoneal white adipose tissue (iWAT) and brown-white adipose tissue (gWAT). Scale bars are shown. P-values ​​in (B) were calculated using a two-tailed paired t-test. P-values ​​in (C) were calculated using one-way ANOVA without multiple comparison correction. P-values ​​in (F, I, and J) were calculated using a two-tailed unpaired t-test without multiple comparison correction. Data are expressed as mean ± standard error. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Detailed Implementation

[0121] Definitions and Explanations

[0122] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the cell and tissue culture, microbiology-related terms, and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this disclosure, definitions and explanations of relevant terms are provided below. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for describing specific embodiments only and is not intended to be limiting.

[0123] Unless otherwise expressly stated, the terms “a,” “an,” and “the” as used in this specification and the appended claims cover one or more types.

[0124] As used herein, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0125] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0126] In this disclosure, imidazole-4-acetic acid (4-IAA) is an important imidazole derivative. The molecular formula of imidazole-4-acetic acid is C5H6N2O2, and its molecular weight is 126.1. Imidazole-4-acetic acid is a known compound that can be prepared by chemical synthesis methods, such as the oxidation of α-hydroxy-β-imidazolium-4-propionic acid and the hydrolysis of 4-acrylonitrile-methylimidazolium. Imidazole-4-acetic acid can also be obtained by biosynthesis methods, for example, according to the method disclosed in patent number CN107177642B.

[0127] In this disclosure, pharmaceutically acceptable salts of imidazole-4-acetic acid refer to those that are non-toxic when administered in doses. Because imidazole-4-acetic acid in this disclosure contains a carboxyl group, it may be a base addition salt. Pharmaceutically acceptable base addition salts include metal salts and organic salts. More preferred metal salts include (but are not limited to) suitable alkali metal (Group Ia) salts, alkaline earth metal (Group IIa) salts, and other physiologically acceptable salts. Such salts can be prepared from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Preferred organic salts can be prepared from tertiary amines and quaternary ammonium salts including (partially) aminobutanetriol, diethylamine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine), and procaine.

[0128] Examples of solvates of imidazole-4-acetic acid in this disclosure include, for example, hydrates and alcohols.

[0129] In this disclosure, the precursor of imidazole-4-acetic acid refers to a compound that is inactive or has low activity in vitro, but releases imidazole-4-acetic acid through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect.

[0130] Imbalances in energy intake and expenditure lead to obesity, a major contributing factor to various chronic diseases such as diabetes, fatty liver, and cancer. The central nervous system regulates systemic metabolic activities, including food acquisition, energy intake, and energy expenditure, and therefore plays a crucial regulatory role in the development and progression of obesity.

[0131] The gut-brain axis plays a crucial role in the regulation of energy metabolism. The gut-brain axis refers to the interaction between the gut and the brain, involving complex interactions between the gut, nervous system, and immune system, and plays a vital role in human physical and mental health. The components of the gut-brain axis include: ① Gut microbiota: Microorganisms present in the gut, including bacteria, fungi, and viruses, which influence human psychological and emotional states through interactions with the immune and nervous systems. ② Immune system: The gut is an important component of the immune system, working closely with the gut microbiota to maintain gut immune homeostasis. Inflammatory responses and immunomodulatory substances of the immune system can affect brain function and emotional state. ③ Nervous system: The gut and brain have rich neurotransmission and chemical signaling pathways. The gut communicates with the brain through neurotransmitters and neuropeptides, influencing functions such as sleep, mood, cognition, and behavior.

[0132] Nutritional sensing and energy homeostasis signals from peripheral tissues project to the CNS via the nucleus of the solitary tract (NTS) in the brainstem through pathways such as hormones (e.g., leptin, insulin), cytokines (e.g., inflammatory factors), and neural impulses (e.g., mechanical tension). These signals are further processed, integrated, and released in the hypothalamic neuronal circuits. The main neurons regulating food acquisition and intake are located in the arcuate nucleus (ARC) of the hypothalamus. Within the ARC, there are two types of neurons closely related to appetite: appetite-stimulating neurons expressing neuropeptide Y (NPY) and agouti-related protein (AgRP); and appetite-suppressing neurons expressing proopiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript peptide (CART).

[0133] Gastrointestinal secretory factors such as ghrelin and glucagon-like peptide-1 (GLP-1) can project to the paraventricular nucleus (PVN) of the hypothalamus via the vagus nerve or directly across the blood-brain barrier through AgRP / NPY and POMC / CART neurons in the ARC, thereby regulating food intake and energy metabolism. Spinal afferents and vagal afferents in the gastrointestinal tract can directly sense changes in neurotransmitters, cytokines, and other signaling molecules and mechanotropic activity in the tissue, further regulating energy metabolism through the central nervous system. Gut microbiota can also regulate appetite through the brain-gut axis. For example, various metabolites produced by gut microbiota regulate food intake: short-chain fatty acids (SCFAs) regulate appetite by promoting the secretion of GLP-1 and gastrointestinal hormone peptides (PYY); neurotransmitters such as γ-aminobutyric acid (GABA) directly regulate appetite; and tryptophan, as a precursor for the synthesis of the neurotransmitter serotonin (5-HT), regulates appetite.

[0134] The blood-brain barrier in the ARC is relatively thin. Therefore, neurons in the ARC can sense peripheral metabolic information not only through neural pathways but also through humoral pathways, and regulate the body's energy intake via specific neural projection pathways. For example, recent studies have shown that metabolites can also directly regulate appetite across the blood-brain barrier of the ARC. For instance, bile acids suppress appetite by reducing AgRP / NPY secretion through TGR5 receptors on the surface of AgRP / NPY neurons.

[0135] AgRP (Agouti-related protein), also known as spiky-related peptide, is a neuropeptide produced by AgRP / NPY neurons in the brain. AgRP is synthesized only in cell bodies containing neuropeptide Y (NPY) located in the ventral part of the arcuate nucleus of the hypothalamus. AgRP is co-expressed with NPY and plays a role in increasing appetite, reducing metabolism, and decreasing energy expenditure. AgRP is one of the most effective and long-lasting appetite stimulants. In humans, spiky-related peptide is encoded by the AgRP gene. The relevant sequence of human AgRP can be found in the gene sequence information section.

[0136] The principle of c-fos staining is mainly based on antigen-antibody reactions. Specific antibodies bind to the c-fos protein, and then a staining agent stains the conjugate, thereby achieving the localization and quantification of the c-fos protein. Commonly used staining methods include immunohistochemical staining and Western blotting.

[0137] BMI (Body Mass Index) is an internationally used standard for measuring body fat and overall health. The formula is: BMI = weight ÷ height squared; where weight is measured in kilograms and height in meters.

[0138] Histamine, as a biogenic amine, plays an important role in various pathophysiological conditions. In peripheral tissues, histamine is mainly stored in mast cells and basophils. In allergic diseases, histamine is released from these cells, leading to several common symptoms in skin and respiratory allergic diseases. Histamine exerts its effects through at least four different receptor subtypes (histamine H1 receptor, histamine H2 receptor, histamine H3 receptor, and histamine H4 receptor). Molecular biological methods have shown that all histamine receptors belong to the large family of G protein-coupled receptors.

[0139] Histamine H3 receptor, or HRH3, is a member of the G protein-coupled receptor (GPCR) family. It is primarily expressed on histaminergic neurons in the central nervous system, and also functions in peripheral tissues such as immune cells. The mechanism of action of HRH3 mainly involves regulating the synthesis and release of neurotransmitters. Presynaptically, it acts as an autoreceptor to inhibit histamine synthesis and secretion, while postsynaptically, it acts as a heteroreceptor to regulate the activity of other neurotransmitters. The relevant sequence of the human histamine H3 receptor can be found in the gene sequence information section.

[0140] White adipose tissue is a type of adipose tissue in humans or animals, whose main function is to store excess fat in the body.

[0141] Brown adipose tissue refers to brown adipose tissue in the body of humans or animals.

[0142] Body fat percentage refers to the proportion of fat weight in the total body weight, also known as body fat percentage, which reflects the amount of fat in the body.

[0143] Muscle ratio, also known as muscle percentage, is the percentage of total body weight that is composed of muscle tissue. Generally speaking, the normal range for muscle ratio varies from person to person.

[0144] Glucose tolerance refers to the body's ability to absorb and utilize ingested glucose. When the body's glucose tolerance is reduced, insulin secretion is insufficient or cellular sensitivity to insulin decreases, leading to elevated blood glucose levels. To assess glucose tolerance, doctors usually recommend an oral glucose tolerance test (OGTT). In addition, fasting blood glucose testing is also a routine screening method.

[0145] D114 is aspartic acid at position 114.

[0146] Y115 is the tyrosine residue at position 115.

[0147] F398 is phenylalanine at position 398.

[0148] L401 is leucine at position 401.

[0149] W402 is the tryptophan at position 402.

[0150] C118 is the cysteine ​​residue at position 118.

[0151] Y374 is the tyrosine residue at position 374.

[0152] E206 is glutamic acid at position 206.

[0153] The ballesteros–weinstein number is a universal numbering scheme for identifying the transmembrane helical (TMH) region and binding site (BS) of G protein-coupled receptor (GPCR) proteins. Developed by Ballesteros and Weinstein, the scheme aims to associate the position of each amino acid in a GPCR sequence with a specific generalized number, thereby deriving the relative position of each amino acid and the specific amino acid information at that position.

[0154] Detailed description of the implementation method

[0155] Example

[0156] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. The following is merely a further description of this disclosure, and the protection scope of this disclosure is not limited thereto.

[0157] The experimental conditions and methods involved in Examples 1-9 are as follows:

[0158] (1) Population Sample

[0159] To investigate changes in plasma 4-IAA levels before and after meals, we recruited 17 healthy male student volunteers from Fudan University. Volunteers fasted for 14 hours prior to the experiment (8 PM to 10 AM) and drank no water for 12 hours prior to the experiment (10 PM to 10 AM). Volunteers began eating at 10 AM the following day. Blood samples were collected before and 2 hours after meals. This study was approved by the Fudan University Human Research Ethics Committee (B2022-553R).

[0160] To investigate the relationship between plasma 4-IAA levels and body mass index (BMI), we collected plasma samples from 42 healthy volunteers. Informed consent forms for blood collection from healthy volunteers were reviewed and approved by the Ethics Committee of Shanghai Xuhui Central Hospital (2011-37).

[0161] A randomized clinical trial (NCT04283942) conducted by Zhongshan Hospital affiliated with Fudan University compared the efficacy and safety of intermittent calorie restriction (ICR) versus continuous calorie restriction (CCR). All 10 participants were instructed to consume a prescribed daily calorie intake (25 kcal / kg x [height (cm) - 100] kg) of regular food, without time restrictions. During the 12-week intervention, the CCR group experienced a mean daily calorie expenditure reduction of 673.7 kcal from baseline (95% CI, -884.9 to -462.5 kcal) and a mean weight loss of 7.6%. Serum samples were collected to measure 4-IAA after a 10-hour fast before and after the 12-week dietary intervention. Written informed consent was obtained from all patients, and the study was approved by the Ethics Committee of Zhongshan Hospital affiliated with Fudan University (B2019-256).

[0162] (2) Mice

[0163] All mice were housed in group cages at 22°C with a 12-hour light / 12-hour dark cycle. The dark cycle began at 7 PM. All animal studies were approved by the Animal Care and Research Advisory Committees of Fudan University and Xiamen University. Mice were euthanized with isoflurane at appropriate times during the study, and tissue samples were collected for further experiments.

[0164] The normal diet (Xietong Organism, Nanjing, China) and the high-fat diet (HFD, Research Diet, D12492) contained 12% and 60% fat calories, respectively. C57BL / 6, db / db, Aoc1- / - (T027589), and CAG-LSL-tdTomato (T006163) mice were purchased from GemPharmatech Co., Ltd. AgRP-Cre (JAX: 012899) mice were purchased from Jackson Laboratory. Mc4r - / -(NM-KO-200357) mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd., and R26-LSL-hM3Dq-DREADD (JAX: 026220) mice were generously provided by Dr. Chen Ying from Xiamen University, China. AgRP-Cre and CAG-LSL-tdTomato mice were crossed to produce AgRP-Cre;CAG-LSL-tdTomato mice, used to specifically express tdTomato in AgRP neurons. AgRP-Cre and R26-LSL-hM3Dq-DREADD mice were crossed to produce AgRP-Cre;R26-LSL-hM3Dq mice, which specifically express hM3Dq in AgRP neurons. All transgenic mice used in this study had a C57BL / 6 background. Primer sequences are shown in Table 1.

[0165] Table 1. Primers used in the study (related to the STAR method) Primer sequences are available at: http: / / pga.mgh.harvard.edu / primerbank.

[0166] (3) Compounds

[0167] 4-Imidazole acetate hydrochloride (4-IAA, S64493), 2-(1-imidazolyl)acetic acid (1-IAA, S63507), 1-methyl-4-imidazolyl acetate hydrochloride (1-Me-4-IAA, Y35050), histamine (S20188) and L-histidine hydrochloride (S20127) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Ciproxifan maleate (HY-15289), GSK189254 (HY-14111), and clozapine-N-oxide (CNO) (HY-17366) were purchased from MCE (MedChemExpress). Mouse leptin was purchased from MedChemExpress (HY-P70704), Liraglutide from MedChemExpress (HY-P0014), WAY 161503 hydrochloride from Tocris Bioscience (Cat#1801), 1H-imidazole-4-carboxylic acid from Bide Pharmatech (BD40238), 2-(pyridin-3-yl)acetic acid from Bide Pharmatech (BD10526), ​​and 2-(pyridin-2-yl)acetic acid from [unclear - possibly a specific brand or product]. The acid was purchased from Macklin (P965236), the 2-(pyridin-4-yl)acetic acid was purchased from Macklin (P923984), and the 4-color Fluorescence Kit (enhanced) was purchased from AiFang Biological (Cat#AFIHC024).

[0168] (4) Plasmids and adeno-associated virus (AAV)

[0169] Mouse Hrh3 was cloned into the pcDNA 3.1-Flag vector. AAV2 / 9-hsyn-FLEX-GCaMP6s-WPRE-pA(S0226-9-H20) was purchased from Taitool. We modified 2543C to 2543T to create an AgeI cleavage site in PT-2788(rAAV-CMV-DIO-(mCherry-U6)-shRNA-WPRE-hGH, BrainVTA), which we call PT-2788*. HRH3 shRNA was cloned into PT-2788*. The preparation and isolation of AAV particles were as described in the reference "Wang, X. et al. Receptor-Mediated ER Export of Lipoproteins Controls Lipid Homeostasis in Mice and Humans. Cell Metab 33, 350-366e357 (2021)", and stored at -80°C before use. Primer sequences are shown in Table 1.

[0170] (5) Dietary training of mice

[0171] Mice that were previously kept in captivity were switched from free-feeding to time-restricted feeding (TRF), where they could eat between 1 p.m. and 5 p.m. After 6 days of training, food intake was measured within 4 hours. Plasma samples were collected at 0, 0.5, 1, 2, and 4 hours after feeding on days 0 and 6 of TRF for untargeted metabolomics studies.

[0172] (6) Quantitative analysis of plasma 4-IAA

[0173] To extract and quantify 4-IAA, 10 μL of plasma was mixed with 40 μL of MeOH:ACN (v / v, 1 / 1), and 62.5 nM L-phenyl-d5-alanine (BePure, MD-4580D5) was added as an internal standard. The mixture was vortexed for 30 seconds. The sample was sonicated in a water bath at 4 °C for 10 min. After incubation at -20 °C for 1 hour, the sample was centrifuged at 20000 g for 15 min at 4 °C. The supernatant was transferred to a new tube and dried using a nitrogen blower at 4 °C. The sample was reconstituted with 20 μL of MeOH:ACN:H2O (v / v / v, 2 / 2 / 1) and sonicated in a water bath at 4 °C for 10 min. After centrifugation at 20000 g for 10 min, the supernatant was collected and analyzed by AB SCIEX QTRAP 6500+ LC-MS / MS.

[0174] (7) Measurement of food intake in mice

[0175] Mice were housed alone for 3-5 days. They were then fasted for 20 hours (from 5 PM to 1 PM the following day), treated with the compound specified in the illustration, and then fed again. Food intake was recorded at the specified times.

[0176] To assess food intake in Aoc1- / - mice in an appetite suppression model, mice were fasted for 20 hours and then administered intraperitoneal injections of mouse leptin (5 mg / kg, HY-P70704, MedChemExpress), a GLP-1 agonist (liraglutide, 0.1 mg / kg, HY-P0014, MedChemExpress), and a 5HT receptor agonist (WAY 161503 hydrochloride, 7 mg / kg, Cat. No. 1801, Tocris Bioscience). One hour later, the mice were fed, and food intake was recorded at 0, 0.5, 1, and 2 hours.

[0177] (8) Mouse metabolic cage analysis

[0178] Metabolic cage analysis was performed in a 22°C system (Columbus, CLAMS-16M) with a 12-hour light / 12-hour dark cycle. Mice were housed alone for one week prior to the experiment and allowed to acclimatize to the metabolic cages for two days. Food intake, body weight, oxygen consumption, carbon dioxide production, and activity levels were monitored in the mice.

[0179] (9) c-Fos signaling staining of mouse whole brain

[0180] In summary, mice were perfused with 4% paraformaldehyde (PFA), and dissected brains were immersed in 4% PFA overnight at 4°C for further fixation. The brains were then immersed in a 10% hot gelatin solution and subsequently placed in ice-cold water for gelatin coagulation. The embedded samples were returned to 4% PFA for further fixation for 24 hours. Brains were sectioned into 300 μm thick sections using a Leica VT1200S Vibratome microtome. All portions of each brain were collected sequentially. Sections were washed with 0.5% Triton X-100 in PBS solution with gentle agitation at 37°C for 6 hours. Sections were immunostained with anti-Fos (#2250, CST, 1:1500) and anti-rabbit secondary antibody (#711-165-152, Jackson ImmunoResearch, 1:500). For ease of imaging, each brain section was mounted on a custom-made slide (100 mm x 100 mm). The sample slide was transferred to a refractive index-matched solution (RIMS) and left overnight. The slide was then imaged using the VISoR imaging system as described earlier. The image was reconstructed using Imaris 9.5.

[0181] (10) Histological and immunofluorescence analysis

[0182] For histological analysis, hematoxylin and eosin (H&E) staining were performed, followed by immunofluorescence. Briefly, after 5 hours of perfusion and fixation in mice using 4% PFA, the brain was transferred to 30% sucrose PBS solution for 24 hours and then embedded in OCT embedding medium (Sakura, 4583) at -80°C. Coronal sections of 16 μm were prepared serially using a cryostat. After drying at room temperature, the brain slices were stored at -80°C. For immunostaining, the brain slices were brought to room temperature (RT), placed in a 55°C oven for 30 minutes, and then transferred at low temperature to a boiling antigen retrieval solution (0.3782 g / L citric acid and 2.4116 g / L sodium citrate in MQ) for 10 minutes. Subsequently, the brain slices were washed three times with PBS and infiltrated with blocking solution (0.3% Triton X-100, 3% BSA in PBS) at room temperature for 0.5 hours. The sections were then incubated at room temperature for 24 hours with anti-Fos (sc-271243, Santa Cruz, 1:500) and anti-AgRP (A00272, BOSTER, 1:1200) or anti-POMC (BM5411, BOSDER, 1:250). After washing with PBS, the sections were incubated at room temperature for 6 hours with Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 594 goat anti-mouse IgG secondary antibody (CST, 1:50). The sections were then stained with DAPI and mounted. The slides were imaged under a Leica LSM880 confocal microscope.

[0183] Small intestine was collected using the Swiss roll method. Preparation of 8 μm frozen sections was performed using a similar method as before. For immunostaining, ABP1 (16338-1-AP, Proteintech, 1:400) and anti-EpCAM (A1107, Abclonal, 1:400) were stained using a 4-color Fluorescence Kit (enhanced).

[0184] In situ hybridization chain reaction (isHCR) combined with immunofluorescence:

[0185] AAV2 / 9-hSyn-DIO-EGFP (catalog number: S0746-9, Taitool, 2×10^12 V.G / ml, 300 nanoliters per side) was injected into Agrip-Cre mice via stereotactic injection. Brains were collected two weeks later. Four sets of splitting probes targeting HRH3 were synthesized (table), mixed, and diluted to a concentration of 2 μM. Slides were washed once with PBST, then treated with methanol for 10 min, and then washed twice with PBST. Subsequently, slides were in a humidified chamber at 37°C with hybridization buffer (5× saline sodium citrate solution (SSC), 10% dextran sulfate, 30% formamide, 0.1% Tween 20, 11 U / mL heparin sodium, Denhardt's solution, and distilled). Pre-hybridize for 5 minutes with water. Denature the HRH3 probe at 95°C for 5 minutes, then dilute it to a final concentration of 20 nM in hybridization solution. Add the solution to the slides, cover each slide with sealing film to ensure even spreading, and incubate overnight in a humidified room at 37°C. After hybridization, wash the slides three times with probe washing buffer (5×SSC, 30% formamide, and 0.1% Tween 20; 10 minutes each time), and wash once at 37°C with 5×SSCT (5×SSC and 0.1% Tween 20) for 10 minutes. Equilibrate the slides with amplification buffer (IPL-AB, Nepagene) for 5 minutes. Add fluorescent labeling. In situ hybridization (ISH) of short hairpin amplicon (ATTO550-S41, Nepagene) was performed by heating to 95°C for 1 minute, followed by gradual cooling to 65°C over 15 minutes and then to 25°C over 40 minutes to form hairpin structures. The hairpin DNA was diluted to a final concentration of 60 nM in amplification buffer and applied to slides. The slides were then incubated at 25°C for 2 hours for chain reaction color development. Next, the slides were washed with PBST and PBS for 5 minutes at room temperature, then co-incubated overnight at 4°C with anti-GFP antibody (rabbit-derived, ab290, Abcam, 1:200), and then incubated for 2 hours at room temperature with Alexa Fluor 488 goat anti-rabbit antibody (CST, 1:500). The slides were imaged under a Leica LSM880 7 confocal microscope. The percentage of GFP(AgRP) and HRH3 double-positive neurons in the arcuate nucleus (ARC) was analyzed relative to GFP(AgRP) positive cells.

[0186] Four sets of splitting probes were designed and synthesized for the HRH3 target as standard oligonucleotides, as shown in Table 2.

[0187] Table 2. Four sets of splitting probes designed and synthesized targeting HRH3.

[0188] (11) Gene expression analysis

[0189] Total RNA was isolated and measured by quantitative real-time PCR (qRT-PCR). Primers are listed in Table 1. All reactions were performed in triplicate. qRT-PCR was performed on an ABIQuantStudio 7Flex using Hieff qPCR SYBR Green Master Mix (High ROX Plus) (YEASEN, 11201ES08). The relative amount of each mRNA was calculated using the comparison threshold cycle (CT) method. Cyclophilin or 36B4 mRNA was used as a control.

[0190] In vitro 4-IAA synthesis

[0191] Individual reaction mixtures were prepared in 500 μL of 0.05 M phosphate buffer (pH 7.2) containing 30 ng of bacterially purified AOC1, AOC2, or AOC3 protein. These mixtures were incubated at 37 °C with continuous shaking (200 rpm) for 15 min in the presence of 4.5 μM histamine or an equal volume buffer control. After incubation, the reaction solutions were collected and analyzed by LC-MS to determine 4-IAA.

[0192] (12) Oral glucose tolerance test and insulin sensitivity test

[0193] Mice were fasted for 16 hours (from 5 p.m. to 9 a.m.) and orally administered D-glucose. The dosage was 2 mg / g body weight for mice with normal food intake, 1 mg / g body weight for mice fed HFD, and 0.1 mg / g body weight for db / db mice. Blood was collected from the tail vein at 0, 15, 30, 60, 90, and 120 minutes, and blood glucose was measured using a Bayer glucometer.

[0194] For the insulin sensitivity test, mice were fasted for 6 hours (from 8:00 AM to 2:00 PM). Mice that were fed normally were injected with insulin at 0.5 U / kg body weight, while mice fed HFD and db / db mice were injected intraperitoneally at 1 U / kg body weight. Blood was collected from the tail vein at 0, 15, 30, 60, 90, and 120 minutes, and blood glucose was measured using a Bayer glucometer.

[0195] (13) CNO chemical genetic activation experiment

[0196] After a 20-hour fast, AgRP-Cre;R26-LSL-hM3Dq mice were injected intraperitoneally with CNO (0.5 mg / kg), given either saline or 4-IAA (75 mg / kg), and fed. Food intake was recorded at 0, 0.5, 1, and 2 hours.

[0197] (14) Stereoscopic brain surgery

[0198] Mice were anesthetized with tribromoethanol (200 mg / kg). The skull was exposed and cleaned with cotton swabs soaked in 5% hydrogen peroxide. For third ventricle cannulation, the guiding cannula was inserted into the ventral third ventricle at a midline coordinate of 1.8 mm posterior to the anterior fontanelle and 5.45 mm below the surface of the skull. Drug delivery was performed using a microinfusion pump (R462, RWD Instruments).

[0199] For AAV delivery, 300-350 nL of AAV was stereotactically injected at a rate of 50 nL / min using a microinjector (R-480 Nanoliter Microinjection Pump, RWD Instruments) (anterior fontanelle: AP: -1.55 mm, ML: ±0.2 mm, DV: 5.95 mm). For fiber optic analysis experiments, a fiber optic cannula (400 μm, NA = 0.50) was implanted 0.1 mm above the injection site and fixed with dental cement. Experiments were performed at least 2 weeks post-operatively.

[0200] (15) Fiber optic recording and analysis of AgRP neurons

[0201] AAV2 / 9-hsyn-FLEX-GCaMP6-WPRE-pA was delivered to the target brain region of AgRP-Cre mice via stereotactic injection, followed by implantation of an optical fiber cannula. Fluorescence in AgRP neurons was recorded using an Inper multichannel fiber optic photometer with excitation channels set to wavelengths of 410 nm and 470 nm. The excitation power at the top of the fiber was adjusted to 20–30 mW, transmitted in 30 Hz pulses at 10 ms. To minimize autofluorescence, the recording fiber was pre-optically bleached using a high-power light source.

[0202] Photometric data were analyzed using Inper data analysis software with a deletion interval function (retention duration set to 3 seconds, no baseline correction) to eliminate redundancy in both channels. Background autofluorescence and baseline correction were handled using default settings (Polyflt, Poly Order=2). F0 was defined as the average fluorescence within the first 30 minutes of recording with saline or 4-IAA, and ΔF / F0 was calculated accordingly. 11 .

[0203] (16) Brain slice preparation and whole-cell patch-clamp recording

[0204] Mice were anesthetized with 2.5% tribromoethanol and decapitated. The brains were rapidly dissected and cut into 250 μm thick coronal sections using a Leica VT1200S Vibratome microtome (Leica, Germany) in a 95% O2 / 5% CO2-saturated, ice-cold N-methyl-D-glucosamine (NMDG, Sigma-Aldrich M2004) cutting solution. This solution contained 92 mM NMDG, 1.2 mM KCl, 30 mM NaHCO3, 1.2 mM KH2PO4, 25 mM D-glucose, 20 mM HEPES, 5 mM L-ascorbic acid, 3 mM sodium pyruvate, 2 mM thiourea, 10 mM MgSO4, and 0.5 mM CaCl2 (310 ± 5 mOsmol / L at pH 7.2 ± 0.1). Brain slices were incubated at 37°C in 95% O2 / 5% CO2-saturated artificial cerebrospinal fluid (ACSF) for at least 45 minutes, and then allowed to recover at room temperature for approximately 1 hour before recording. ACSF contained 125 mM NaCl, 25 mM NaHCO3, 1.25 mM KH2PO4, 25 mM D-glucose, 0.4 mM L-ascorbic acid, 2 mM sodium pyruvate, 2 mM CaCl2, and 1 mM MgCl2. After recovery, the slices were placed in the recording chamber and continuously perfused with ACSF at a rate of 1–2 mL / min.

[0205] Brain slices were visualized using an Olympus BX51WI microscope, infrared (IR) differential interference contrast (DIC), and charge-coupled device (CCD) camera (IR-2000; DAGE-MTI). Electrodes were drawn from borosilicate glass capillaries (outer diameter = 1.5 mm, inner diameter = 0.86 mm, Sutter Instrument) using a Model P-97 puller to achieve a final tip resistance of 4–7 MΩ. For recording action potentials (current clamps), pipettes were filled with a solution containing 26 mM potassium gluconate, 10 mM HEPE, 4 mM KCl, 4 mM magnesium adenosine 5′-triphosphate (ATP-Mg), 0.3 mM guanosine 5′-triphosphate sodium hydrate (5′-GTP-Na), and 10 mM creatine phosphate. All recordings were performed using a MultiClamp 700B amplifier (Axon instrumentation). Electrophysiological data were filtered at 2.9 kHz and sampled at 10 kHz using Clampex 11.1 and MultiClamp 700B software 8.

[0206] (17) Conditional Flavor Test

[0207] To test the effect of 4-IAA on flavor preference, a conditional flavor test was designed. The protocol involved an 11-day period, during which mice were introduced into a test area containing two water bottles for 90 minutes each day from 9:00 AM to 10:30 AM. From day 0, mice were allowed free access to food but were only allowed to drink water in the test area from 9:00 AM to 10:30 AM. From day 0 to day 6 (habituation day), mice were provided with two bottles of water. On day 7 (adjustment day), mice were given a cherry-flavored solution containing 0.2% saccharin (0.36 g / L Kool-Aid sweet cherry) and injected with saline at 10:30 AM. On day 8 (recovery day), mice were given only drinking water. On day 9, mice were randomly divided into three groups, receiving either a grape-flavored solution (0.36 g / L Kool-Aid sweet grape) or an intraperitoneal injection of saline, LiCl (200 mg / kg), or 4-IAA (75 mg / kg). Mice were then given drinking water again on day 8. On day 11 (test day), between 9:00 a.m. and 10:30 a.m., three groups of mice were provided with cherry and grape flavored solutions on the random side, and the consumption of each bottle was recorded.

[0208] (18) Kaolin Intake Test

[0209] To test the effect of 4-IAA on nausea, we conducted a kaolin ingestion experiment. For five days prior to the experiment, mice were housed alone and allowed free access to kaolin granules (Research Diets, K50001), food, and water. On the day of the experiment, mice were fasted for 20 hours, injected intraperitoneally with saline or 4-IAA (75 mg / kg), and then refeeded with food and kaolin. The intake of food or kaolin was recorded.

[0210] (19) Behavioral testing

[0211] After training in the Morris water maze test, mice were injected intraperitoneally (IP) with 4-IAA (75 mg / kg) and then tested. The CleverSys TopScanLite system was used for results analysis.

[0212] For the forced swimming test, mice were injected intraperitoneally (IP) with 4-IAA (75 mg / kg) or saline before the test. The cumulative duration of immobility was recorded during the last 4 minutes of the 6-minute test period. Immobility was defined as the mouse floating motionless in the water without struggling.

[0213] For the resting time in the tail suspension test, after a 30-minute tail suspension test, mice were injected with 4-IAA (75 mg / kg) via IP, followed by a 6-minute tail suspension test.

[0214] The open field test (OFT) was used to assess motor activity and anxiety-like behavior. In the test, mice were injected with 4-IAA (75 mg / kg) via intraperitoneal injection.

[0215] (20) cAMP assay

[0216] To measure cAMP levels in the arcuate nucleus (ARC) of the hypothalamus, freshly isolated hypothalamus was treated with 10 μM trichodin for 15 minutes, with or without 1 μM 4-IAA. The tissue was homogenized in cold 0.1 M HCl and centrifuged at 10,000 g for 10 minutes. The supernatant was neutralized with 1 M NaOH, and cAMP levels were then quantified using a commercially available ELISA kit (R&D Systems, KGE012B).

[0217] (21) GloSensor cAMP analysis

[0218] To screen for the 4-IAA receptor, a GloSensor cAMP assay was performed. On day 0, HEK293 cells were transiently co-transfected with the GloSensor cAMP probe, and each of the first 15 cells highly expressed the GPCR in the arcuate nucleus. On day 2, the transfected cells were divided into groups of 5 × 10⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells per well in 96-well plates. On day 3, cells were incubated for 2 hours at 37°C with serum-free DMEM containing 2% (v / v) GloSensor cAMP substrate stock solution (Progema). Different concentrations of 4-IAA or 1-IAA were added, and ligand-induced changes in luminescence intensity were recorded using an EnVision Multilabel microplate reader (Perkin-Elmer).

[0219] (22) Bioluminescence resonance energy transfer analysis (BRET)

[0220] To characterize G protein signaling, bioluminescent resonance energy transfer (BRET) analysis was performed. On day 0, HEK293 cells were transfected with GPCRs and G protein probes (Gαi1-3 or Gαo fused with Nluc, Gβ, Gγ fused with GFP2). On day 2, cells were divided into groups of 5 x 10⁻⁶ cells per well. 4 Cells were seeded at a density of 4-IAA into 96-well plates. On day 3, cells were treated with different concentrations of 4-IAA in the presence of the luciferase substrate coelentrin 400a (5 μM, Cayman), and the BRET signal between Nluc (440–480 nm) and GFP2 (510 nm) emission was measured using a Mithras LB 940 multimode microplate reader (Berthold Technologies).

[0221] (23) Purification of HRH3 and G protein complex

[0222] Fall armyworm (Spodoptera frugiperda) (Sf9) cells were cultured in insect ESF921 cell culture medium (Sino Biological). To express HRH3, miniGαo1, Gβ1, Gγ2, and scFv16, high-titer recombinant baculoviruses were generated using the Bac-To-Bac baculovirus system (Invitrogen) according to the previously described protocol. 19 Sf9 cells were transfected with a recombinant baculovirus carrying HRH3, miniGαo1, Gβ1, Gγ2, and scFv16 using FuGENE HD transfection reagent (Promega), generating high titers of recombinant virus. Sf9 cells were infected with this mixture of baculoviruses in a 1 / 1 / 1 / 1 ratio. After shaking at 27°C for 48 hours, cells were collected by centrifugation at 1000g for 20 minutes, and the particles were frozen in liquid nitrogen and stored at -80°C.

[0223] To prepare the 4-IAA-HRH3-mGo complex, frozen cell particles were thawed at room temperature and resuspended in lysis buffer (100 μM 4-IAA, 20 mM HEPES pH 7.5, 100 mM NaCl, 3 mM MgCl2, 5 mM CaCl2, 2.5 mg / mL leucine and 0.2 mg / mL benzidine) and rotated end-to-end at 50 rpm for 1.5 hours. The mixture was prepared by adding 0.5% (w / v) dodecyl maltose neopentyl glycol (LMNG, Anatrace) and 0.1% (w / v) benzoyl maltose neopentyl glycol (Anatrace) at 4°C. The mixture was then centrifuged at 100,000 g for 20 min. The supernatant containing the 4-IAA-HRH3-mGo complex was incubated with M1 anti-FLAG affinity resin at 4°C for 2 h. Flag-M1 beads were washed three times with a buffer containing 100 μM 4-IAA, 0.01% (w / v) LMNG, 0.002% (w / v) CHS, 0.2 mg / mL benzoamide, and 2.5 mg / mL leucine. The mixture was then incubated with M1 anti-FLAG affinity resin containing 100 μM 4-IAA, 20 mM HEPES pH 7.5, 100 mM NaCl, 0.01% (w / v) LMNG, 0.002% (w / v) CHS, 5 mM EGTA, and 0.2 mg / mL leucine. The FLAG peptide buffer was used to elute the 4-IAA-HRH3-mGo complex from M1 beads.

[0224] The eluted complex was concentrated using an Amicon ultracentrifuge filter with a molecular weight cutoff of 100 kDa and then injected into a Superose 6Increase 10 / 300GL column pre-equilibrated with a buffer containing 100 μM 4-IAA, 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w / v) LMNG, and 0.00025% (w / v) CHS for complex separation. Peak fractions containing the 4-IAA-HRH3-mGo complex were combined and concentrated to approximately 8 mg / mL for cryogenic grid preparation.

[0225] (24) Low-temperature mesh preparation and EM data acquisition

[0226] To prepare cryogenic EM meshes, 3 μL of purified 4-IAA-HRH3-mGo complex was applied to glow discharge porous carbon meshes. After rapid freezing using a FEI Vitrobot Mark IV (Thermo Fisher Scientific), the sample was placed in a storage chamber and stored in liquid nitrogen. The sample mesh was then mounted in a Titan Krios electron microscope (300 kV) equipped with a spherical aberration (Cs) corrector for data collection. Images of the 4-IAA-HRH3-mGo complex were captured using a Falcon 4 camera with a nominal magnification of 120,000x and a pixel size of [missing information]. SerialEM software with custom scripts is used to facilitate automated low-dose image acquisition. The defocus range of the image stack is set between -1.0 and -2.0 μm. The cumulative dose is set per... Each electron contains 32 frames in each photomicrograph.

[0227] (25) Data processing and 3D reconstruction

[0228] All cryogenic EM film stacks of 4-IAA-HRH3-mGo were processed with motion correction and dose weighting using MotionCor2. Contrast transfer function (CTF) parameters were then estimated using patch CTF estimation. The cryogenic EM data were then processed using CryoSPARC v4.4.1. A total of 8580 imaging records were manually examined to exclude low-quality images containing crystalline ice or other contaminants. To enhance particle picking, we used the Topaz method based on a traditional neural network in CryoSPARC. The dataset then underwent two rounds of no-reference 2D classification. The selected good 1367298 particle projections were used for de novo reconstruction to generate an initial reference map, followed by three rounds of non-uniform thinning in CryoSPARC. A high-quality subset containing 565614 particle projections was re-extracted and 3D classified in Relion 4.4.1 to further exclude particles belonging to undefined categories. This subset was further refined using uniform and non-uniform thinning to obtain a resolution of [resolution missing]. The final image is determined by the gold standard FSC standard. The overall density is refined using an EM-ready method through automatic local sharpening.

[0229] (26) Model building and optimization

[0230] The histamine-HRH3-Gi complex model (PDB: 8YUU) was used as the initial model for determining the HRH3 structure of the 4-IAA-HRH3-Go complex. Go heterotrimers (mGo, Gβ1, and Gγ2) and scFv16 were generated using the beclomethasone-GPR97-mGo complex model (7D76) as the initial model. These initial models were aligned to the EM density maps using UCSF Chimera. The models were then manually adjusted and reconstructed based on the EM density maps in COOT software, followed by refinement using Phenix tools. Residues or side chains that could not accurately adapt to explicit electron microscopy densities were excluded from the final models. The fit of the improved models was evaluated using model-mapped Fourier shell correlation (FSC). The structural diagrams were created using UCSF Chimera and UCSF ChimiraX. The 4-IAA model was generated using the eLBOW procedure in Phenix and validated using the MolProbity tool.

[0231] Molprobity is an online model analysis tool, available at http: / / molprobity.biochem.duke.edu / index.php. Currently, Molprobity has become a relatively comprehensive protein structure detection tool in structural biology.

[0232] (27) Quantitative and statistical analysis

[0233] All statistical analyses were performed using Student's paired two-tailed t-tests. Values ​​are expressed as mean ± SEM. Statistical details for all experiments, including the exact number of cell samples or mice, can be found in the legend. Ns represents no difference; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. No data were excluded for any experiment.

[0234] Example 1

[0235] Identification of 4-IAA, a substance that regulates food intake:

[0236] To identify metabolites related to appetite regulation, we established a time-restricted feeding model. Eight male mice (n=8) aged 8–10 weeks and weighing 22–25 g were given free access to food daily from 1 PM to 5 PM, while the mice were deprived of food at other times. The training period was 7 days. Mice that were not trained were defined as the untrained group (Day 0), and mice trained for 7 days were defined as the experimental group (Day 6). The normal daily total food intake for mice is approximately 4 g. We found that mice in the Day 0 group only consumed about 1 g during the restricted feeding period. For the experimental group mice, each day of training resulted in a higher food intake than the previous day. By Day 7, mice in the Day 6 group consumed a similar amount of food as normal mice during the restricted feeding period (Figure 1A). We found that the food intake of Day 0 and Day 6 mice differed by more than double after 1 hour of feeding, and the food intake of Day 0 mice stopped increasing after 1 hour, while Day 6 mice continued to eat (Figure 1B). Therefore, we trained 8-10 week old male mice and divided them into two groups, Day 0 and Day 6. We performed non-targeted metabolomics analysis on the plasma of the mice before feeding (0h) and 1h after feeding (n=3) to look for substances that control the mice's feeding behavior (Figure 1C). After screening, we identified the metabolite 4-IAA, which is related to feeding behavior (Figure 1C, 1D). Metabolite 4-IAA plays a role in inhibiting appetite.

[0237] To demonstrate that 4-IAA can suppress appetite, we significantly suppressed food intake in 8–10-week-old male mice via intraperitoneal injection (single dose 75 mg / kg, n=6), oral gavage (single dose 120 mg / kg, n=6), and drinking water supplementation with 4-IAA (the drug was prepared as a 3 mg / ml aqueous solution and allowed free access to water, n=6) (Figure 1E). These results indicate that the metabolite 4-IAA plays a significant role in suppressing appetite. A significant negative correlation was found between circulating 4-IAA concentration and food intake in the subsequent 0.5 hours (Figure 1E), suggesting that 4-IAA may play a role in appetite regulation. Furthermore, during free access to food, plasma 4-IAA peaked at midnight (0:00), 4 hours after the insulin peak (Figures 1F and 2A), further confirming the association between elevated 4-IAA and food intake.

[0238] 4-IAA is an oxidation product of histamine, and its structure is similar to that of γ-aminobutyric acid (GABA). Previous studies have identified the pharmacological effects of 4-IAA in sedation, analgesia, hypnosis, and hypotension. However, its role in appetite regulation has not been reported. We first characterized the pharmacokinetics of 4-IAA after oral administration in rats. Plasma concentration-time curves showed rapid absorption, reaching peak concentrations within 41.7 minutes after administration (Figure 2B). The terminal half-life (T1 / 2) was 53.3 minutes (Figure 2C). Subsequently, we investigated the effect of 4-IAA on food intake in mice. As shown in Figure 1G, both intraperitoneal injection and oral administration of 4-IAA showed a dose-dependent inhibition of fasting-induced food intake. Similarly, supplementation with 4-IAA in drinking water also reduced food consumption (Figure 1G). Plasma 4-IAA levels measured 30 minutes after administration increased in a dose-dependent manner and were negatively correlated with food intake (Figure 2D). At peak concentrations, plasma 4-IAA reached 2-5 μM, approximately 10 times the endogenous level (Figure 2D).

[0239] Furthermore, we found that the isomer 1-IAA of 4-IAA does not exert an appetite-suppressing effect, indicating that the appetite-suppressing effect of 4-IAA is structure-specific in 8- to 10-week-old male mice (n = 6) (Figure 1H).

[0240] In the correlation analysis, we found that the levels of the metabolite 4-IAA in the blood of three types of mice after feeding: normal diet (Chow, 12-14 week old mice, weighing about 26-28g), high-fat diet-induced obese mice (HFD, high-fat induced for 14 weeks, weighing 45g), and gene defect-induced obese model mice (ob / ob, mice around 10 weeks old, weighing about 55g) (Figure 1I).

[0241] Seventeen healthy male student volunteers were recruited. The volunteers were required to fast for 14 hours (8 PM to 10 AM) and abstain from water for 12 hours (10 PM to 10 AM). At 10 AM the following day, the volunteers began eating. Blood samples were collected from the volunteers before and 2 hours after eating. Changes in 4-IAA levels in their blood before and after eating were measured. We found that the level of 4-IAA in the blood was significantly increased after eating (Figure 1J).

[0242] To investigate the relationship between plasma 4-IAA levels and body mass index (BMI), researchers collected plasma samples from 42 healthy volunteers and calculated the correlation between BMI and blood 4-IAA levels. A strong positive correlation was found between plasma 4-IAA levels and BMI in the population (Figure 1K).

[0243] Ten adults were randomly selected from the continuous calorie restriction (CCR) group. All 10 participants were instructed to consume a prescribed daily calorie intake (25 kcal / kg x [height(cm)-100]kg) without time restrictions on regular food intake. During the 12-week intervention, the mean daily calorie expenditure in the CCR group decreased by 673.7 kcal from baseline (95% CI, -884.9 to -462.5 kcal), and the mean body weight decreased by 7.6%. Before and after the 12-week dietary intervention, patients fasted for 10 hours overnight, and serum samples were collected the following morning to measure changes in 4-IAA (Figure 1L). Obese patients showed a significant decrease in serum 4-IAA levels after weight loss. To assess the potential for obesity-related 4-IAA resistance, we administered two concentrations of 4-IAA (1 mg / mL and 3 mg / mL in drinking water) to lean mice fed a standard diet (chow-fed) and obese mice fed a high-fat diet (HFD-fed). Contrary to the expected development of resistance, obese mice fed a high-fat diet showed significantly greater sensitivity to 4-IAA treatment at both dose levels than lean mice (Figure 1M). These results suggest that 4-IAA can suppress food intake and modulate obesity.

[0244] Example 2

[0245] 4-IAA can combat obesity induced by a high-fat diet:

[0246] Since appetite regulation plays a crucial role in maintaining metabolic homeostasis, overeating can lead to metabolic imbalance, thereby inducing metabolic diseases such as obesity. Our study found that the metabolite 4-IAA has an inhibitory effect on appetite, suggesting that 4-IAA may inhibit the progression of obesity. Using a high-fat HFD-induced obese mouse model, we evaluated the role of 4-IAA in the development of obesity. Mice were induced with HFD at 4 weeks of age (starting at 20g body weight, n=9) and simultaneously subjected to water intake testing until week 12. Appetite was measured at week 3 of treatment; glucose tolerance was tested at week 10; insulin sensitivity was tested at week 11; and body composition analysis and dissection were performed at week 12.

[0247] We found that 4-IAA, compared to 1-IAA, specifically inhibited food intake in HFD obese mice (Fig. 3A), significantly reduced mouse body weight (Fig. 3B), and improved their glucose and insulin sensitivity (Fig. 3C, Fig. 3D).

[0248] Metabolic cage analysis showed that 4-IAA-treated mice had reduced food intake but no impact on kinetic activity (Fig. 4A). Although there may have been a consistent trend in oxygen consumption, carbon dioxide production, and thermogenesis in 4-IAA-treated mice, it did not reach statistical significance (Fig. 4A). After 12 weeks of HFD feeding, the fat mass of 4-IAA-treated mice was significantly lower than that of the control group and 1-IAA-treated mice (Fig. 4B). The weights of liver, inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), and brown adipose tissue (BAT), as well as liver triglyceride content, were significantly reduced in the 4-IAA-treated group (Figs. 2E-2H and 4C). Plasma 4-IAA concentration increased to 2.89 ± 0.32 μM (Fig. 4D). These results suggest that 4-IAA can prevent diet-induced obesity and related metabolic disorders.

[0249] Example 3

[0250] The role of 4-IAA in genetically obese mice (i.e., db / db mice lacking functional leptin receptors):

[0251] We also investigated the effects of 4-IAA in genetically obese mice (i.e., db / db mice lacking functional leptin receptors). Similarly, administration of 4-IAA significantly reduced daily food intake, prevented weight gain, accelerated glucose clearance, and improved insulin sensitivity in db / db mice (Fig. 5A-D). Metabolic cage analysis confirmed the reduced food intake in 4-IAA-treated db / db mice (Fig. 5E). No significant differences were observed in oxygen consumption, carbon dioxide production, thermogenesis, or kinetic activity (Fig. 5E). Consistent with the improved insulin sensitivity, 4-IAA significantly increased the respiratory exchange ratio in db / db mice (Fig. 5E). Adipose tissue was significantly reduced and hepatic triglyceride levels were decreased in 4-IAA-treated db / db mice (Fig. 5F-J). These results suggest that 4-IAA may be a candidate drug for treating patients with leptin or leptin receptor dysfunction.

[0252] Example 4

[0253] 4-IAA improves multiple metabolic abnormalities in obese mice with gene defects: ob / ob mice were subjected to a water intake test from 4 weeks of age (starting at 25g body weight, n=5) to 8 weeks. Appetite was measured at week 3 of treatment. Glucose tolerance was tested at week 6 of treatment, insulin sensitivity was tested at week 7, and body composition was analyzed and the mice were dissected at week 8.

[0254] To further demonstrate that 4-IAA can inhibit the development of obesity, we used a genetically defective obese mouse model (ob / ob) to evaluate the role of 4-IAA in ob / ob obese mice. We found that 4-IAA specifically inhibited food intake in ob / ob obese mice (Fig. 6A) and significantly reduced mouse body weight (Fig. 6B). 4-IAA did not significantly alter glucose tolerance in ob / ob obese mice (Fig. 6C), but significantly improved insulin sensitivity (Fig. 6D). 4-IAA significantly inhibited the weight of fatty liver and white adipose tissue in ob / ob obese mice (Fig. 6E), reduced the severity of fatty liver lesions (Fig. 6F), and reduced the size of white adipocytes (Fig. 6G). Simultaneously, 4-IAA significantly reduced triglyceride levels in the liver of ob / ob obese mice (Fig. 6H). These results indicate that 4-IAA can significantly improve multiple metabolic abnormalities in genetically defective obese mice.

[0255] Example 5

[0256] 4-IAA does not affect the metabolic phenotype of mice with normal physiological state: Mice were subjected to a drinking water test from 4 weeks of age (starting at 20g body weight, n=7) to 14 weeks of age. Appetite was measured at week 3 of treatment. Glucose tolerance was tested at week 11 of treatment, insulin sensitivity was tested at week 12, and body composition was analyzed and the mice were dissected at week 14.

[0257] We found that 4-IAA significantly improved metabolic abnormalities in obese mice induced by a high-fat diet and genetic defects, as well as in diabetic mice with genetic defects, suggesting that 4-IAA may significantly affect mice under pathological conditions. However, we did not know whether 4-IAA would affect healthy mice with normal physiological conditions. Therefore, we used a normal-diet mouse model (Chow Diet) to evaluate the effects of 4-IAA on healthy mice. We found that long-term administration of 4-IAA to drinking water did not change the food intake of healthy mice (Figure 7A), nor did it change their body weight (Figure 7B) or body fat percentage (Figure 7E). Administration of 4-IAA did not alter glucose tolerance in healthy mice (Figure 7C), but it did improve insulin sensitivity (Figure 7D). 4-IAA did not alter the weight, morphology, or pathology of the liver and adipose tissue in normal healthy mice (Fig. 7F, 7H), the size of white adipocytes in normal healthy mice (Fig. 7I), or the triglyceride content in the liver of normal healthy mice (Fig. 7J). These results indicate that 4-IAA does not affect the metabolic phenotype of normal healthy mice.

[0258] Example 6

[0259] Potential applications of 4-IAA in the treatment of obesity:

[0260] To explore the potential application of 4-IAA in the treatment of obesity, we fed C57BL6 mice a high-fat diet (HFD) to a weight of approximately 50 g and treated them by supplementing their drinking water with 4-IAA (Fig. 8A). As shown in Fig. 8B, 4-IAA treatment significantly reduced the body weight of obese mice. By week 4, the mice had lost 21.9% of their body weight. Similarly, 4-IAA treatment accelerated glucose clearance, improved insulin sensitivity, reduced fat mass, and decreased triglyceride levels in the liver (Fig. 8C-3I). The plasma concentration of 4-IAA increased to 4.98 ± 0.43 μM (Fig. 8J). Notably, after discontinuing 4-IAA, the mice regained their body weight within 3 weeks (Fig. 5K), indicating that continued administration of 4-IAA is necessary to maintain the metabolic benefits. These results highlight the potential application of 4-IAA in the treatment of obesity.

[0261] Example 7

[0262] 4-IAA does not affect muscular function in mice; 4-IAA does not cause nausea, irritability, learning, cognition, anxiety, or depression.

[0263] Male mice aged 8–10 weeks were intraperitoneally injected with 4-IAA (single dose 75 mg / kg, n=8). We analyzed the metabolic phenotype of mice before and after administration of 4-IAA using metabolic cages. We found that administration of 4-IAA inhibited feeding and significantly reduced oxygen uptake (VO2), carbon dioxide exhalation (VCO2), respiratory quotient (RER), and heat production. However, administration of 4-IAA did not affect the mice's total activity (Figure 9A), demonstrating that 4-IAA did not have an anesthetic or hypnotic effect to inhibit feeding in mice.

[0264] To demonstrate that the appetite-suppressing and ameliorative effects of 4-IAA on various metabolic indicators did not affect behavior or cognition, and to evaluate its mechanism of action and drug-likeness, we conducted a series of tests on 8-10 week old male mice after a 20-hour fast, including intraperitoneal injection (75 mg / kg, n=5), oral administration (120 mg / kg), or mixing the drug into drinking water (3 mg / mL). These tests showed that 4-IAA did not induce nausea in the mice (Figure 9B). A flavor preference test was also conducted on 8-10 week old male mice, using intraperitoneal injection (75 mg / kg, n=6) or oral administration (120 mg / kg) to confirm that 4-IAA did not induce any preference in the mice (Figure 9C). Finally, a water maze test was performed on 8-10 week old male mice, using intraperitoneal injection (75 mg / kg), oral administration (120 mg / kg), or mixing the drug into drinking water. In the test of 4-IAA (mixed in drinking water (3 mg / mL), n=7), 4-IAA did not alter the learning and cognitive abilities of mice (Fig. 9D). In the test of 4-IAA in male mice aged 8–10 weeks with forced swimming, 4-IAA did not induce anxiety or depression in mice. The test of 4-IAA in male mice aged 8–10 weeks with forced swimming, 4-IAA was administered intraperitoneally (75 mg / kg), or orally (120 mg / kg), or mixed in drinking water (3 mg / mL), n=13 (Fig. 6E). The test of 4-IAA in male mice aged 8–10 weeks with forced swimming, 4-IAA was administered intraperitoneally (75 mg / kg), orally (120 mg / kg), or mixed in drinking water (3 mg / mL), n=13 (Fig. 6F). The test of 4-IAA in male mice aged 8–10 weeks with open field, 4-IAA was administered intraperitoneally (75 mg / kg), orally (120 mg / kg), or mixed in drinking water (3 mg / mL), n=13 (Fig. 6G). Through these behavioral experiments, we have demonstrated that 4-IAA does not cause nausea or changes in preferences in mice, nor does it affect their learning and cognitive abilities, nor does it induce anxiety or depression.

[0265] Example 8

[0266] Analysis of the synthesis pathway of 4-IAA in the body: 4-IAA is synthesized via the intestinal pathway:

[0267] The mice used in this example were all 8 to 10 weeks old.

[0268] We further elucidated the synthesis pathway of 4-IAA. We found that 4-IAA is produced through a series of metabolic processes: L-histidine → histamine → tetraimidazole acetaldehyde → tetraimidazole acetic acid (4-IAA) (Figure 10A). The main enzyme catalyzing the conversion of L-histidine to histamine is HDC, which is expressed in low amounts in the gut. The main enzyme catalyzing the conversion of tetraimidazole acetaldehyde to 4-IAA is AOC1, which is highly expressed in the gut. Another histidine catalytic enzyme, HNMT, is also almost not expressed in the gut (Figure 10B). 4-IAA is a metabolite produced by histidine metabolism (Figure 10A). Histidine is first converted to histamine by histidine decarboxylase (HDC), an enzyme mainly expressed in the brain (Figure 10B). Histamine can then be converted to 4-IAA by copper-containing amine oxidase 1 (AOC1), or to 1-methyl-4-imidazolium acetic acid (1-methyl-4-IAA) by histamine N-methyltransferase (HNMT1). Although 1-methyl-4-IAA is similar to 4-IAA, it has no effect on food intake (Fig. 11A). Notably, the enzymatic expression patterns of AOC1 and HNMT1 are mutually exclusive. AOC1 is primarily expressed in the gut, with the highest expression level in the jejunum, while HNMT1 is expressed in other tissues besides the gut (Fig. 10B). Surprisingly, on day 7, the expression levels of AOC1 in the duodenum and jejunum were significantly reduced compared to day 0 (Fig. 10C, 4D, and 11C), consistent with the difference in 4-IAA plasma levels (Fig. 1D). In contrast, feeding / fasting after acute fasting did not alter AOC1 expression (Fig. 11D). Reanalysis of single-cell sequencing databases showed that AOC1 mRNA is primarily expressed in intestinal epithelial cells of the mouse and human small intestine (Fig. 10E and 11B). Immunostaining further confirmed the expression of AOC1 in intestinal epithelial cells (Fig. 10F). These results indicate that in the intestinal epithelial cells of the small intestine, 4-IAA is mainly derived from histamine, rather than directly from histidine.

[0269] To demonstrate this, we treated mice with either histamine or histidine and measured plasma 4-IAA levels. Figure 10G shows that histamine (but not histidine) resulted in elevated plasma 4-IAA levels. To confirm the role of AOC1, we subsequently bred Aoc1- / - mice (Figures 11E-H). Indeed, plasma 4-IAA levels in Aoc1- / - mice were significantly lower than in wild-type (WT) mice under both fasting and fasting-refeeding conditions (Figure 10H). In Aoc1- / - mice, 4-IAA levels in the jejunum, duodenum, liver, hypothalamus, and brain (without hypothalamus) were significantly reduced (Figure 11I). Furthermore, in Aoc1- / - mice, histamine gavage significantly reduced 4-IAA production (Figure 10I).

[0270] We also characterized Aoc1- / - mice by feeding them a high-fat diet (HFD). As shown in Figure 12A, knocking out Aoc1 did not increase daily food intake. After 12 weeks of high-fat diet, Aoc1- / - mice showed a slight increase in body weight and exhibited mildly impaired glucose clearance (Figures 12B-D). Aoc1- / - mice also showed a slight increase in fat mass and elevated liver triglyceride levels (Figures 12E-H). Although the observed weight gain was consistent with our hypothesis, the overall phenotypic effect was less severe than expected.

[0271] To further investigate the physiological role of 4-IAA in appetite regulation, we designed a functional challenge experiment. We hypothesized that if the AOC1 / 4-IAA pathway acts as an endogenous brake in the appetite regulation network, its necessity would become apparent when the system is challenged. Therefore, we pretreated wild-type (WT) and Aoc1- / - mice with three established anorexics: leptin, liraglutide (a GLP-1 analog), and WAY-161503 (a 5-HT receptor agonist). Under basal fasting conditions, the food intake of mice of both genotypes was comparable, but after being challenged with any of the anorexics, the anorexic response in Aoc1- / - mice was significantly reduced, and their food intake was greater than that of the WT control group (Figure 10). This result indicates that the AOC1 / 4-IAA pathway is physiologically essential, providing direct in vivo evidence for its endogenous role. These results suggest that 4-IAA is derived from histamine by AOC1 in the small intestinal epithelial cells.

[0272] These results also suggest the possible existence of a compensatory mechanism in Aoc1- / - mice. Given that plasma 4-IAA levels were reduced by approximately 40% in Aoc1- / - mice (Fig. 10H), we hypothesized the existence of other enzymatic pathways for 4-IAA production. We noted that AOC1 has two homologs in mammals, namely AOC2 and AOC3. Aoc2 and AOC3 are primarily expressed in adipose tissue (Fig. 12I). Notably, in the gonadal white adipose tissue of Aoc1- / - mice, the mRNA expression of Aoc3 was upregulated by 2.5-fold (Fig. 12J), indicating compensatory regulation. In vitro enzymatic assays confirmed that recombinant AOC2 and AOC3 possessed comparable activity to AOC1 in the conversion of histamine to 4-IAA (Fig. 12K). In summary, these findings suggest that 4-IAA production regulates appetite through both intestinal (AOC1-mediated) and adipose tissue (AOC2 / AOC3-mediated) pathways. The above experiments demonstrate that AOC1 catalyzes the synthesis of tetraimidazole acetic acid (4-IAA) from histamine, thereby regulating appetite and metabolism in the body.

[0273] Example 9

[0274] 4-IAA exerts its appetite-suppressing effect by inhibiting the activity of AgRP neurons in the hypothalamus.

[0275] Hypothalamic nuclei play a crucial role in appetite regulation. To investigate how 4-IAA functions, we first examined whether its effect depends on vagal signaling. Capsaicin-mediated vagal blockade did not alter the inhibitory effect of 4-IAA on food intake (Fig. 14A), ruling out the vagus nerve as the primary agent. Since 4-IAA has been previously found in the brain, we tested whether it might act directly on central circuits. Third ventricle (ICV) injection of 4-IAA significantly reduced food intake (Fig. 14B), supporting the hypothesis that 4-IAA targets the brain to mediate its anorectic effects.

[0276] To identify brain regions responsive to 4-IAA, we performed whole-brain decongestion combined with c-Fos immunostaining, a marker of neuronal activation. Notably, 4-IAA treatment significantly reduced c-Fos+ neurons in the arcuate nucleus (ARC) and produced downstream effects in connective tissue (Fig. 13A and 14C). Further characterization revealed that these c-Fos+ neurons were AgRP neurons, not POMC neurons (Fig. 13B and 14D). Quantitative statistics showed that 4-IAA reduced the proportion of active (c-Fos+) AgRP neurons from 60% to 35% (Fig. 13B and 13C). This inhibitory effect was also observed when 4-IAA was administered via intraperitoneal injection (Fig. 14E and 14F). These results indicate that 4-IAA inhibits the activity of AgRP neurons.

[0277] To validate our results, we first expressed the calcium sensor GCaMP in AgRP neurons using adeno-associated virus (AAV) and monitored neuronal activity using fiber photometric analysis (Fig. 13D). Fig. 13E shows that administration of 4-IAA significantly reduced GCaMP signaling, indicating decreased neuronal activity. We then confirmed these results using patch-clamp analysis. Consistently, 4-IAA dose-dependently reduced the peak firing rate of AgRP neurons, with an IC50 of 7.825 μM (Figs. 13F-13H).

[0278] To determine whether the inhibitory effect of 4-IAA on AgRP neurons is cell-autonomous, we pretreated brain slices with several synaptic blockers, including PTX (a GABA-A antagonist), DNQX (an AMPA antagonist), CNQX (a fumaryl antagonist), and D-AP5 (an NMDA antagonist). Notably, these blockers, whether used alone or in combination, attenuated the inhibitory effect of 4-IAA on AgRP neurons (Figures 14G and 14H). This finding suggests that 4-IAA acts directly on AgRP neurons, rather than through synaptic regulation, supporting a cell-autonomous mechanism of action.

[0279] Furthermore, we validated the effect of 4-IAA on AgRP neurons using a designed receptor (hM3Dq) activated solely by the designed drug (DREADD) system. Clozapine N-oxide (CNO) stimulation of food intake activates AgRP neurons; however, 4-IAA significantly inhibits this effect (Figs. 13I and 13J). Consistently, electrophysiological analyses showed that 4-IAA reversed CNO activation of AgRP neurons (Figs. 15A-C).

[0280] To further confirm the downstream pathway, we investigated the effects of 4-IAA on Mc4r- / - mice, since Mc4r neurons are the main downstream neurons of AgRP. Unlike wild-type mice, 4-IAA showed little reduction in food intake, weight gain, or tissue weight of adipose tissue and liver (Fig. 15D-F), indicating that the anorectic effect of 4-IAA depends on the AgRP-MC4R pathway.

[0281] These experiments collectively demonstrate that 4-IAA exerts its appetite-suppressing effect by inhibiting the activity of AgRP neurons in the ARC region of the hypothalamus.

[0282] Example 10

[0283] Receptor screening revealed that HRH3 is the active receptor for 4-IAA:

[0284] We went on to identify the receptor for 4-IAA. To this end, we first investigated the downstream signaling of 4-IAA. Since cAMP is known to stimulate c-Fos expression, we tested the effect of 4-IAA on cAMP production. Figure 16A shows that 4-IAA dose-dependently reduced forskolin-induced cAMP production, with an EC50 of 28.3 ± 6.6 nM.

[0285] As shown in Figures 1H and 11A, neither of the two structural analogs of 4-IAA, 1-IAA and 1-methyl-4-IAA, exhibited any inhibitory effect on food intake. This finding strongly suggests that the molecular target of 4-IAA has strict stereochemical requirements for ligand recognition and activation. We inferred that this receptor might be a G protein-coupled receptor (GPCR), as they tend to be highly selective for ligands. We then established a GPCR screening system by expressing each of the top 15 highly expressed GPCRs in the ARC of HEK293 cells, treating them with either 4-IAA or 1-IAA, and monitoring cAMP levels using the GloSensor system 41 (Figure 16B). Among the candidates, the histamine H3 receptor (HRH3) specifically responded to 4-IAA, with an EC50 of 10.7 ± 2.01 nM (Figures 16B and 16C).

[0286] To biochemically verify the 4-IAA-HRH3 interaction, we used 3 Competitive radioligand binding analysis was performed on H-histamine (Figure 17A). Our results indicate that 4-IAA is dose-dependently replaced from HRH3. 3H histamine produces a binding affinity of 75 ± 12 nM (Ki) (Fig. 16D). For validation, we included two established HRH3 ligands as controls: unlabeled histamine (agonist Ki, 84 ± 13 nM) and iodophenpropit (antagonist Ki) (19 ± 2.6 nM) (Fig. 16D). Consistent with the finding that 1-IAA and 1-methyl-4-IAA have no effect on food intake (Fig. 1H and Fig. 11A), they failed to displace HRH3. 3 H-histamine (Figs. 17B and 17C). Furthermore, histamine effectively inhibited forskolin-induced hypothalamic cAMP production (Fig. 16E). In contrast, iodophenpropit reversed the inhibitory effect of 4-IAA on cAMP production (Fig. 16F). Moreover, considering that 4-IAA is a histamine metabolite, and that knockout of Hrh3 increases appetite and leads to obesity in mice, we hypothesize that Hrh3 may be the functional receptor for 4-IAA's appetite-inhibiting effect.

[0287] We continued to identify downstream Gα proteins of the 4-IAA-HRH3 complex. Since 4-IAA triggers inhibitory signaling, we co-expressed each inhibitory Gα protein, including Gαi1, Gαi2, Gαi3, and Gαo, with HRH3 and performed bioluminescent resonance energy transfer (BRET) analysis. Figure 16G shows that the 4-IAA-HRH3 complex triggers inhibitory signaling via Gαo, with an EC50 of 27.4 ± 7.1 nM.

[0288] Previous studies have shown that HRH3 is expressed on the presynaptic membrane of histaminergic neurons and functions as an inhibitory autoreceptor for histamine. However, its expression and role in AgRP neurons remain unexplored. To investigate this, we analyzed published databases. As shown in Figure 17D, HRH3 is indeed expressed in AgRP neurons at levels far higher than the other three members of the histamine receptor family. Since HRH1 is also expressed in AgRP neurons, albeit at much lower levels, we tested its binding to 4-IAA using BRET analysis and found that 4-IAA, up to 1 mM, cannot trigger Gαo signaling via HRH1 (Figure 17E).

[0289] To further confirm the direct binding of 4-IAA to HRH3, we expressed HRH3, miniGαo, Gβ1, Gγ2, and scFv16 in insect cells, purified the 4-IAA-HRH3-mGo complex, and resolved its structure using cryo-electron microscopy (cryo-EM) (Figs. 16H, 16I, and 17F). After a series of classifications and purifications, 138,771 complex particles were obtained, with a resolution of [missing information - likely a resolution value]. (Figs. 16H, 17G, and 17H). The binding pocket for 4-IAA is located in transmembrane 3 (TM3) and TM6-TM7 of HRH3 (Figs. 16I and 17I). The imidazole and carboxyl groups of 4-IAA are in the same plane (Fig. 16I). Through structural analysis, mutagenesis, and radioligand competition analysis, we identified D114, Y115, C118, Y374, F398, L401, and W402 as the residues required for binding to 4-IAA in HRH3 (Figs. 16I-6K and 17Q).

[0290] We compared the 4-IAA-bound HRH3 Go structure with the previously reported inactive HRH3 structure (PDB: 7F61) to investigate the 4-IAA binding-induced HRH3 activation mechanism. Upon 4-IAA binding, the extracellular region of TM5 in HRH3 shifted inward by approximately 25°, while the intracellular region of TM6 shifted outward by approximately 28° (Fig. 17J). 4-IAA binding also triggered a rearrangement of residues within the ortho-pocket of HRH3, which was then passed on to conformational changes at W371 and F367 (Figs. 17K and 17L). On the cytoplasmic side, D131, R132, and Y222 residues also rearranged, and the salt bridge formed by R132 and D131 was disrupted (Fig. 17M). Furthermore, N408, P409, and Y412 residues in the NPxxY motif flipped down, resulting in a conformational rearrangement (Fig. 17N).

[0291] We also compared the structures of the 4-IAA-HRH3 and histamine-HRH3 complexes. The binding pockets of 4-IAA and histamine are both similar and different (Fig. 17O). D114, Y115, C118, Y374, F398, L401, and W402 are essential for binding to both 4-IAA and histamine (Figs. 17P and 17Q), but C118 and Y374 form hydrogen bonds with 4-IAA but not with histamine. Conversely, E206 is specific for histamine (Fig. 16K).

[0292] To determine the structural determinants of binding, we evaluated several analogues. Removing the methylene linker (imidazolium-4-carboxylic acid) or replacing the imidazolium ring with a pyridine ring eliminated activity (Figure 17R-V). These structure-activity relationship data indicate that the carboxylate, methylene linker, and imidazolium ring are all essential for HRH3 binding, which explains why 1-IAA and 1-methyl-4-IAA are inactive.

[0293] Example 11

[0294] 4-IAA acts on HRH3 receptors, thereby inhibiting the activity of AgRP neurons to regulate appetite.

[0295] We further validated that HRH3 is a functional receptor for 4-IAA. To investigate the effect of the 4-IAA-HRH3 complex on AgRP neuronal activity, we performed patch-clamp analyses. First, histamine and immepip, two agonists of HRH3, also effectively inhibited AgRP neuronal activity (Fig. 19A and 19B). Like 4-IAA, immepip also inhibited fasting-induced food intake (Fig. 19C). In contrast, the two HRH3 antagonists, ciproxifan or GSK189254, largely reversed the inhibitory effect of 4-IAA on the peak firing rate of AgRP neurons (Fig. 18A and 18B). Sustained, third ventricle ICV infusion of ciproxifan eliminated the inhibitory effect of 4-IAA on food intake (Fig. 18C). Furthermore, adeno-associated virus (AAV) knockout of HRH3 in AgRP neurons largely eliminated the inhibitory effect of 4-IAA on c-Fos+ AgRP neurons (Fig. 18D, 19D, and 19E). Bilateral knockout of HRH3 in AgRP neurons (AgRPΔHhr3) eliminated the inhibitory effect of 4-IAA on food intake (Figure 18E). These results indicate that HRH3 on AgRP neurons is a functional receptor for 4-IAA.

[0296] To further elucidate the physiological role of HRH3 in AgRP neurons, we fed AgRPΔHhr3 mice with high-frequency dispensing (HFD). Compared with control mice, AgRPΔHhr3 mice showed a significantly increased daily food intake (Fig. 18F). AgRPΔHhr3 mice had significantly higher body weight than control mice (Fig. 18G), impaired glucose clearance, and insulin resistance (Fig. 18H and 18I). These mice exhibited increased fat mass, decreased lean body mass (Fig. 19F), and significantly larger adipose tissue than control mice (Fig. 18J and 19G-19I). Hepatic triglyceride levels were significantly elevated in AgRPΔHhr3 mice (Fig. 19J). Overall, the AgRPΔHhr3 mouse phenotype reproduced the obesity-related phenotype of systemic Hrh3 knockout mice, indicating that Hrh3 plays a role in controlling appetite and body weight in AgRP neurons.

[0297] To summarize our findings, we propose a comprehensive metabolic neural circuit in which 4-IAA acts as a key anorexia mediator (Figure 18K). This metabolite is synthesized from histamine via enzymatic conversion of AOC1 in the small intestine and its homologs (AOC2 / 3) in adipose tissue. Upon release into circulation, 4-IAA crosses the blood-brain barrier and acts directly on AgRP neurons in the arcuate nucleus of the hypothalamus. Through high affinity binding to HRH3, 4-IAA activates a Gαo-coupled inhibitory signaling cascade, inhibiting AgRP neuronal activity and ultimately leading to decreased appetite and weight control. These results demonstrate that in the absence of the HRH3 receptor, 4-IAA fails to exert its significant therapeutic effect on metabolic abnormalities in obese mice, loses its ability to improve abnormal metabolic indicators, and contributes to obesity and abnormal metabolism in mice.

[0298] This disclosure proposes a novel appetite-suppressing metabolite, 4-IAA, identifies its synthetic origin, and clarifies its specific mechanism of action. 4-IAA acts on HRH3 receptors on AgRP neurons in the ARC region, thereby inhibiting appetite and regulating metabolism. The regulatory pattern is summarized in Figure 14. We are the first to discover that 4-IAA exerts its effects on appetite regulation and blood sugar reduction / weight loss through neuro-metabolic modulation. Furthermore, it provides a promising preclinical target receptor, HRH3, for obesity and other metabolic diseases. This has significant theoretical and applied value for the research and development of novel drugs targeting appetite suppression and blood sugar reduction / weight loss.

[0299] Gene sequence information:

[0300] Human HRH3 sequence: (from https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=GENEID&DATA=11255&BUILDS=CURRENTBUILDS) HRH3 histamine receptor H3 [Homo sapiens(human)]

[0301] Gene ID: 11255

[0302] SEQ ID NO: 1: Nucleotide sequence of human HRH3 (1338nt):

[0303] SEQ ID NO: 2: Translation of human HRH3 (445 amino acids):

[0304] Human AgRP sequence: (from https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=CCDS&GO=MainBrowse&DA=CCDS10839.1) AGRP agouti-related neuropeptide [Homo sapiens (human)]

[0305] Gene ID: 181

[0306] SEQ ID NO: 3: Nucleotide sequence of human AgRP (399nt):

[0307] SEQ ID NO: 4: Translation of human AgRP (132 amino acids):

[0308] Mouse HRH3 sequence: (from https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=GV&DATA=603534&BUILDS=CURRENTBUILDS) Hrh3 histamine receptor H3 [Mus musculus (house mouse)]

[0309] Gene ID: 99296.

[0310] SEQ ID NO: 5: Nucleotide sequence of mouse HRH3 (1338nt):

[0311] SEQ ID NO: 6: Translation of mouse HRH3 (445 amino acids):

[0312] Mouse AgRP sequence: (from https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=GV&DATA=613553&BUILDS=CURRENTBUILDS) AgRP agouti-related neuropeptide [Mus musculus (house mouse)]

[0313] Gene ID: 11604.

[0314] SEQ ID NO: 7: Nucleotide sequence of mouse AgRP (396nt):

[0315] SEQ ID NO: 8: Translation of mouse AgRP (131 amino acids):

[0316] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of this disclosure, as well as various different choices and variations. The scope of this disclosure is intended to be defined by the claims and their equivalents.

Claims

1. The use of compounds and / or pharmaceutically acceptable salts thereof, solvates thereof, or precursors thereof binding to the D114, Y115, C118, Y374, F398, and L401 positions of the histamine H3 receptor (HRH3) in the preparation of a pharmaceutical or health product, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2, and the pharmaceutical or health product is any one or more of the following: (1) Appetite suppressants or health products; (2) Medications or health products that lower blood sugar; (3) Medications or health products for treating diabetes; (4) Weight loss drugs or health products; (5) Weight loss drugs or health products; (6) Weight loss drugs or health products; (7) Medications or health products for treating fatty liver; Alternatively, a method for preparing a drug or health product includes the steps of preparing a compound and / or a pharmaceutically acceptable salt thereof, a solvate thereof, or a precursor thereof that binds to the D114, Y115, C118, Y374, F398, and L401 positions of a histamine H3 receptor (HRH3), wherein the histamine H3 receptor sequence is SEQ ID NO: 2, and the drug or health product is any one or more of the following: (1) Appetite suppressants or health products; (2) Medications or health products that lower blood sugar; (3) Medications or health products for treating diabetes; (4) Weight loss drugs or health products; (5) Weight loss drugs or health products; (6) Weight loss drugs or health products; (7) Medications or health products for treating fatty liver; Alternatively, compounds and / or their pharmaceutically acceptable salts, solvates, or precursors that bind to the D114, Y115, C118, Y374, F398, and L401 positions of the histamine H3 receptor (HRH3), for use in the preparation of pharmaceuticals or health products, wherein the histamine H3 receptor sequence is SEQ ID NO: 2, and the pharmaceutical or health product is any one or more of the following: (1) Appetite suppressants or health products; (2) Medications or health products that lower blood sugar; (3) Medications or health products for treating diabetes; (4) Weight loss drugs or health products; (5) Weight loss drugs or health products; (6) Weight loss drugs or health products; (7) Medications or health products for treating fatty liver.

2. The use of compounds and / or pharmaceutically acceptable salts of the histamine H3 receptor at positions D114, Y115, C118, Y374, F398, and L401, solvates thereof, or precursors thereof, in the preparation of medicaments or health products for the treatment of hypothalamic AgRP neuron-mediated appetite suppression, blood glucose reduction, diabetes treatment, weight loss, fat reduction, and / or treatment of fatty liver, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2; Alternatively, a method for suppressing appetite, lowering blood glucose, treating diabetes, reducing weight, losing weight, reducing fat, and / or treating fatty liver mediated by hypothalamic AgRP neurons, comprising administering to a patient in need a therapeutically effective amount of a compound and / or its pharmaceutically acceptable salts, solvates, or precursors that bind to histamine H3 receptors at positions D114, Y115, C118, Y374, F398, and L401, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2; Alternatively, compounds and / or their pharmaceutically acceptable salts, solvates, or precursors that bind to the D114, Y115, C118, Y374, F398, and L401 positions of the histamine H3 receptor, for therapeutic purposes, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2; Alternatively, compounds and / or their pharmaceutically acceptable salts, solvates, or precursors that bind to the D114, Y115, C118, Y374, F398, and L401 positions of the histamine H3 receptor, for the purpose of suppressing appetite, lowering blood glucose, treating diabetes, reducing weight, losing weight, reducing fat, and / or treating fatty liver mediated by hypothalamic AgRP neurons, wherein the histamine H3 receptor sequence is: SEQ ID NO:

2.

3. The use or method according to claim 1 or 2, characterized in that, The compound is imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor, wherein imidazole-4-acetic acid has the structure shown in formula (I):

4. The use or method according to any one of claims 1 to 3, characterized in that, The medicine or health product is used for one or more of the following purposes: reducing body fat percentage, increasing muscle mass, improving glucose tolerance, improving insulin sensitivity, inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, maintaining the browning of brown fat, reducing liver triglyceride levels, preventing and treating obesity, and / or for regulating obesity, for treatment of obese, overweight or obese patients. Preferably, the drug or health product is used to reduce body fat percentage, increase muscle mass, improve glucose tolerance, and improve insulin sensitivity. Preferably, the drug or health product is used for one or more of the following purposes: inhibiting or reducing the weight of adipose tissue, reducing white adipose tissue or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, maintaining the browning of brown adipose tissue, and reducing liver triglyceride levels. Preferably, the drug or health product is used for one or more of the following purposes: inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, and / or maintaining the browning of brown fat; Preferably, the drug or health product is used to lower liver triglyceride levels; Preferably, the drug or health product is used to improve insulin sensitivity; Preferably, the drug or health product is used to reduce body fat percentage and increase muscle mass. Preferably, the drug or health product is used for patients who are obese, overweight, or have a predisposition to obesity.

5. The use or method according to any one of claims 1 to 4, characterized in that, The drug or health product has at least one effect selected from the following: It does not affect the metabolic phenotype of a normal healthy organism; It does not affect exercise; It will not cause nausea; It does not affect preferences; It does not affect learning or cognition; and / or It will not cause anxiety or depression.

6. The use or method according to any one of claims 1 to 5, characterized in that, The drug or health product forms an appetite suppressant; the formulation uses the imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors as the sole active ingredient.

7. The use or method according to any one of claims 1 to 6, characterized in that, The drug or health product forms an appetite suppressant; the formulation comprises the imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, its solvate, its precursor, and also comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes one or more of the following: solvent, solubilizer, cosolvent, emulsifier, flavoring agent, odorant, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, pH regulator, stabilizer, surfactant, and preservative.

8. The use or method according to any one of claims 1 to 7, characterized in that, The drug or health product forms an appetite suppressant formulation; the formulation comprises imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, its solvate, its precursor, and one or more other appetite suppressant active ingredients.

9. The use or method according to any one of claims 1 to 8, characterized in that, The drug or health product forms an appetite suppressant; the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor in a unit formulation for suppressing appetite is 5 to 100 mg, preferably 10 to 60 mg. The dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, and precursors in a unit formulation for combating diet-induced obesity is 20–300 mg, preferably 30–200 mg. The dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors in a unit formulation for weight loss is 50–1000 mg, preferably 100–500 mg.

10. The use or method according to any one of claims 1 to 9, characterized in that, The compound works by inhibiting the activity of AgRP neurons in the hypothalamus; Preferably, the drug or health product is administered via one or more of the following methods: oral, intravenous, intraperitoneal, intramuscular, rectal, or subcutaneous administration; preferably, the drug is administered via one or more of the following methods: oral, intravenous, or intraperitoneal administration. Preferably, the formulation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form; preferably, the solid dosage form is a tablet, capsule, granule, or pill; the semi-solid dosage form is a gel, suppository, or ointment; and the liquid dosage form is an emulsion, mixture, suspension, or solution. Preferably, the drug or health product is a tablet, capsule, injection, powder, drop, granule, syrup, chewable tablet, or patch.

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