Use of a compound as an agonist of receptor GPR101

Abstract

The present invention provides the use of four compounds as agonists of receptor GPR101. The compounds of the present invention can activate the activity of GPR101 in vitro, bind to GPR101, target the allosteric site of GPR101, significantly enhance the GH / IGF-I axis function of aged mice, significantly improve the blood glucose metabolism function of mice, and reduce the body fat rate. It shows a very good anti-aging effect. It can be used to prepare drugs for treating aging.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-metabolic disease drugs, and specifically relates to the application of various compounds as orphan receptor GPR101 agonists. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins encoded by the human genome and couple extracellular stimuli such as light, ions, neurotransmitters, and hormones to intracellular signals (Wacker et al., 2017). In addition to being activated by ligand stimulation, an increasing number of GPCRs with high intrinsic constitutive activity play important regulatory roles in physiological processes such as neural development, brain development, metabolic balance, and reproductive function. Many of these GPCRs with high intrinsic activity are still orphan receptors, and there is a lack of effective strategies to regulate their activity, which poses a huge obstacle to the development of therapeutics involving these disease-related GPCRs.

[0004] GPR101 is an orphan GPCR of the class A family with constitutive Gs and Gq activities but no known endogenous ligands. GPR101 is highly expressed in the nucleus accumbens and hypothalamus, and GPR101 gene duplication in humans causes X-linked hypertrophy (X-LAG), which is characterized by infantile-onset overgrowth and hypertrophy due to excessive secretion of growth hormone (GH). Transgenic mice with pituitary-specific overexpression of GPR101 show enhanced skeletal growth and elevated GH and prolactin (PRL) secretion. Notably, GPR101 mutations have been found in patients with different endocrine diseases, such as Cushing's disease and acromegaly. Together, these emerging evidences suggest that manipulating the constitutive activity of GPR101 has important therapeutic potential. Summary of the invention

[0005] The present invention provides the use of four compounds as agonists of receptor GPR101. The compounds of the present invention can activate the activity of GPR101 in vitro, bind to GPR101, target the allosteric site of GPR101, significantly enhance the GH / IGF-I axis function of aged mice, significantly improve the blood glucose metabolism function of mice, and reduce the body fat rate. It shows a very good anti-aging effect. It can be used to prepare drugs for treating aging.

[0006] In the first aspect of the present invention, four compounds are provided for use as agonists of the receptor GPR101. The compounds of formula AA-14, formula AA-41, formula AA-63 and formula AA-88 are shown in the following table:

[0007] Table 1 Structures of compounds of the present invention

[0008]

[0009] The second aspect of the present invention provides use of the compound described in the first aspect in the preparation of a medicament for preventing and / or treating aging-related diseases.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The compound of the present invention is the first to be found to have agonist activity of GPR101, and is the first small molecule agonist of GPR101, and is also the first small molecule compound that targets GPR101 to exert anti-aging effects. Moreover, the compound of the present invention can significantly enhance the GH / IGF-I axis function of aged mice, promote the synthesis and secretion of multiple hormones such as growth hormone and prolactin, significantly improve the blood sugar metabolism function of mice, improve physical fitness, increase memory cognition, and play an anti-aging effect.

[0012] This work has unique value in the field of aging research. It opens a new chapter in intervening in aging by targeting GPCRs. Through structure-based design and screening of small molecule ligands, it provides a potential anti-aging strategy. The related results are of great significance to GPCR pharmacology research and anti-aging drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 : Effects of compounds on GPR101 activity;

[0015] Figure 2 : Effects of compound AA-14 on GH, PRL and IGF-1 hormone levels in mice;

[0016] Figure 3 : Effects of compound AA-14 on the expression of aging markers in mice;

[0017] Figure 4 : Compound AA-14 monitors the food intake, body weight changes, and whole body energy expenditure of mice;

[0018] Figure 5: Compound AA-14 in glucose tolerance test in mice;

[0019] Figure 6 :Results of mouse rotarod test;

[0020] Figure 7 : Results of Morris water maze experiment;

[0021] Figure 8 : 1 H NMR Spectrum of AA-14in CDCl 3 ;

[0022] Fig. 9 : 13 C NMR Spectrum of AA-14in CDCl 3 ;

[0023] Fig.10 (+)-HR-ESIMS spectrum of AA-14. DETAILED DESCRIPTION

[0024] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0025] Example 1 Effects of Compounds on GPR101 Activity

[0026] Experimental method: The compounds described in the present invention were purchased from suppliers in the SPECS compound database (https: / / www.specs.net / ). Then, Gs, Gq dissociation assays and β-arrestin2 recruitment assays were used to detect the activation of Gs, Gq and β-arrestin proteins downstream of GPR101 under stimulation by different compounds. HEK293 cells were transiently co-transfected with GPR101 and G protein BRET probes and β-arrestin2 recruitment probes. After transfection for 24 hours, 5×10 4The cells were distributed into 96-well microplates at a density of 10 cells and incubated at 37°C for 24 hours. The cells were washed twice with Tyrode's buffer (140mM NaCl, 2.7mM KCl, 1mM CaCl2, 12mM NaHCO3, 5.6mM D-glucose, 0.5mM MgCl2, 0.37mM NaH2PO4 and 25mM HEPES, pH7.4) and stimulated with the compounds of the present invention at different concentrations. After the addition of coelenterazine (final concentration of 5μM), the BRET signal was measured using a Mithras LB940 microplate reader equipped with a BRET filter set. The BRET signal was calculated as the ratio of the emitted light at 510nm / 400nm (G protein dissociation experiment) and 540nm / 460nm (β-arrestin2 recruitment experiment).

[0027] Experimental results: The small molecules obtained by screening were further tested for their activity, such as Figure 1 As shown, it was confirmed that four small molecules including AA-14, AA-41, AA-63 and AA-88 showed strong Gs, Gq and βarr2 agonism on GPR101, and these small molecules showed preferential activation of different pathways of GPR101. The above results suggest that we can develop preferential ligands for constitutively activated orphan GPCRs, thereby selectively regulating their functions.

[0028] Example 2 Effect of Compound AA-14 on GH, PRL and IGF-1 Hormone Levels in Mice: Experimental Method: GPR101fl / fl mice were treated with AA-14 (0.1 mg / kg / d) or solvent (corn oil) for 2 months. The plasma of mice was separated at different time points (0, 1, 2, 3 and 4 hours) after the last administration. Plasma GH, IGF-1 and PRL concentrations were determined using rat / mouse GH ELISA kit (Merck Millipore, EZRMGH-45K), mouse IGF-1 ELISA kit (Abcam, ab100695), and mouse PRL ELISA kit (Invitrogen, EMPRL).

[0029] Experimental results: Figure 2 As shown, the plasma levels of GH, IGF-1, and PRL were significantly increased after AA-14 administration to 8-week-old and 18-month-old WT male mice.

[0030] Example 3 Effect of compound AA-14 on the expression of aging markers in mice:

[0031] Experimental methods: Total RNA was extracted from the skin of 8-week-old and 18-month-old mice treated with AA-14 or control solvent using TRIzol reagent (Invitrogen). Reverse transcription into cDNA was performed using a qRT-PCR kit (Toyobo, FSQ-101). The relative mRNA levels of selected cellular senescence biomarker genes (Glb1, p21, Il6, Il1α) were calculated using the 2-ΔΔCt method.

[0032] Experimental results: Figure 3 As shown, biomarkers of cellular senescence, including β-galactosidase, P21, IL6, and IL1α, were decreased in AA-14-treated 18-month-old mice.

[0033] Example 4 Energy consumption and body fat measurement in mice:

[0034] Experimental methods: Food intake, body weight change, and whole body energy expenditure of male mice aged 8 weeks and 18 months were monitored using a laboratory animal comprehensive monitoring system (CLAMS-16, Columbus Instruments, Columbus, OH, USA). Body fat percentage of mice was measured by a body composition analyzer (Minispec LF90, Bruker Co., Billerica, MA, USA).

[0035] Experimental results: Figure 4 As shown, after treatment with AA-14, the food intake and body weight changes of male mice aged 8 weeks and 18 months showed obvious anti-aging effects.

[0036] Example 5 Glucose tolerance test:

[0037] Experimental methods: Male mice aged 8 weeks and 18 months were treated with AA-14 or control solvent for 2 months and fasted for 16 hours before glucose tolerance test. Plasma glucose levels were measured before (baseline) and 15, 30, 60, 90, and 120 minutes after intraperitoneal injection of 2 mg / g glucose using FreeStyle Lite blood glucose meter (Roche). Figure 5 As shown, compound AA-14 exhibits better anti-aging effect.

[0038] Example 6 Rotarod experiment:

[0039] Experimental methods: Male mice aged 8 weeks and 18 months were treated with AA-14 or control solvent for 2 months and placed on a motorized rotating rod (Med Associates, St Albans, VT). The latency of the mice to fall from the rotating rod within 300 seconds was measured. During one measurement day, each mouse was tested three times with an interval of 30 minutes between trials. The average latency of the mice to fall was calculated for analysis.

[0040] Experimental results: Figure 6 As shown, after drug treatment, the time mice stayed on the rotating rod increased, indicating that the physical fitness of mice was significantly improved after drug treatment.

[0041] Example 7 Morris water maze experiment:

[0042] Experimental methods: Male mice aged 8 weeks and 18 months were treated with AA-14 or control solvent for 2 months and then placed in a cylindrical water tank with a diameter of 1.2 meters for water maze training. The water tank contained a platform. The training time was five consecutive days, and each mouse received four training sessions per day to learn to use visual cues to find the platform and generate memory. The trial interval was 15 minutes. On the test day, the support platform in the water was removed, and the time spent by the mouse in the target area where the platform was previously placed was recorded using ANYmaze software. The time was 60 seconds.

[0043] Experimental results: Figure 7 As shown, after drug treatment, the mice's patrol time in the target area increased, indicating that the mice's cognitive memory had been significantly improved.

[0044] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a compound in the preparation of an anti-aging drug, the compound is shown in the following table: 。

Images