Use of zedoarone in preparation of olfactory receptor olfr43 specific agonist drugs

Abstract

This invention provides the application of cyperene in the preparation of olfactory receptor Olfr43 specific agonist drugs, relating to the field of small molecule pharmaceutical technology of traditional Chinese medicine. This invention constructs Hana3A... Olfr43‑FLuc‑RLuc A heterologous OR dual-luciferase screening platform revealed that cyperene can activate the downstream pathway of the olfactory receptor Olfr43, significantly increasing intracellular cAMP levels. Cellular experiments further verified that cyperene stimulation significantly increased the mNRA and protein expression levels of Olfr43 in cells, raising downstream cAMP levels. This, in turn, inhibited the expression of lipid synthesis-related genes by phosphorylating CREB, promoting HES-1, and inhibiting DGAT2 protein expression, ultimately reducing TC and TG in the HepG2 hyperlipidemia model. This invention is the first to propose that cyperene can act as a specific agonist targeting the olfactory receptor Olfr43 to regulate downstream cAMP changes. This invention provides a new option for the development of highly effective agonists specifically targeting Olfr43 / OR1A1.

Description

Technical Field

[0001] This invention relates to the field of small molecule pharmaceutical technology of traditional Chinese medicine, specifically to the application of cyperone in the preparation of olfactory receptor Olfr43 specific agonist drugs. Background Technology

[0002] The olfactory system is one of the chemical sensing systems essential for the survival of many organisms. Olfactory receptors (ORs) are mainly distributed on olfactory sensory neurons. When they bind to specific odor ligands, they activate traditional G protein signaling pathways, promoting adenylate cyclase (AC) to induce a large secretion of intracellular cyclic adenosine monophosphate (cAMP). This triggers olfactory-specific cyclic nucleic acid-gated cation channels to increase the influx of Na+ and Ca2+ ions, causing changes in membrane potential and converting the chemical information of the odor into an electrical signal that is transmitted to the brain, thus producing the sense of smell. Initially, it was thought that ORs were only expressed in olfactory neurons. However, since the first OR gene transcript outside the nasal mucosa was discovered in mammalian germ cells in 1992, a large number of ORs have been found to be expressed in multiple non-olfactory tissues such as the testes, sperm, skin, adipose tissue, and intestines, and this expression has been described as "ectopic" OR expression. In recent years, studies have shown that ectopic ORs have a variety of biological functions, including influencing sperm chemotaxis, wound healing, glucose and lipid metabolism, and the regulation of intestinal secretion. They also participate in the regulation of cell proliferation, apoptosis, invasion, and metastasis of malignant tumors.

[0003] Numerous studies have demonstrated that olfactory receptors (ORs) can regulate glucose and lipid metabolism. Olfr43 expression in the liver is second only to nasal tissue, followed by the kidneys, heart, spleen, colon, and muscle. Its homolog, OR1A1, is also highly expressed in the liver. Reports indicate that Olfr43 / OR1A1 activates HES-1 and inhibits PPAR-γ expression through the cAMP / PKA / CREB pathway, thereby affecting the expression of two key enzymes, DGAT and GPAM, regulating triglyceride synthesis, and thus regulating lipid metabolism. L cells, intestinal endocrine cells, secrete glucagon-like peptide-1 (GLP-1). Activation of OR1A1 and OR1G1 can induce GLP-1 secretion from L cells, which then regulates pancreatic β-cell function through multiple pathways, including AC / cAMP, stimulating insulin secretion and regulating glucose metabolism. These studies suggest that the olfactory receptor Olfr43 / OR1A1 has a significant regulatory effect on glucose and lipid metabolism and could serve as an effective therapeutic target for metabolic diseases such as obesity and diabetes.

[0004] Olfactory receptors (ORs) belong to the G protein-coupled receptor (GPCR) family. Currently, approximately 40% of approved targeted drugs on the market target GPCRs. As the largest gene family within GPCRs, ORs play a crucial regulatory role in glucose and lipid metabolism. These olfactory receptors have the potential to become novel drug targets for the treatment of metabolic diseases. Given the important role of the olfactory receptor Olfr43 / OR1A1 in lipid and glucose metabolism, developing highly effective agonists that specifically target Olfr43 / OR1A1 is of great significance. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides the application of cyperene ketone in the preparation of olfactory receptor Olfr43 specific agonist drugs.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides the application of cyperene ketone in the preparation of olfactory receptor Olfr43 specific agonist drugs.

[0010] This invention constructs Hana3A Olfr43-FLuc-RLuc A heterologous OR dual-luciferase screening platform was developed. Stimulation with different concentration gradients of cyperone and detection using a luciferase reporter assay revealed a significant increase in intracellular cAMP levels in cells overexpressing the olfactory receptor Olfr43. Cyperone can activate downstream tracts of Olfr43. Furthermore, cyperone did not exhibit activating effects on other odor receptors such as Olfr544, Olfr734, and Olfr16. This indicates that cyperone can serve as a specific targeting agonist of the olfactory receptor Olfr43.

[0011] This invention also utilizes cell experiments to further verify the role of Cyp as a specific targeting agonist of the olfactory receptor Olfr43. Under the stimulation and activation of Cyperene, it can significantly increase the mNRA and protein expression levels of Olfr43 in cells, thereby increasing the downstream cAMP level. By phosphorylating CREB, promoting HES-1 and inhibiting the expression of DGAT2 protein, it can inhibit the expression of lipid synthesis-related genes, ultimately achieving the effect of reducing TC and TG in the HepG2 high-fat model.

[0012] The chemical formula of the cyperone described in this invention is shown in Formula I:

[0013] Formula I.

[0014] Furthermore, the drug is composed of an effective dose of cyperone and a pharmaceutically acceptable carrier or excipient.

[0015] (III) Beneficial Effects

[0016] This invention is the first to discover that cyperene can act as a specific targeting agonist of the olfactory receptor Olfr43 to regulate downstream cAMP changes. This invention provides a new option for the development of highly effective agonists specifically targeting Olfr43 / OR1A1. Attached Figure Description

[0017] Figure 1 Establishment of a dual-luciferase detection platform for Olfr43 agonists in Hana3AOlfr43-FLuc-RLuc cells (A); relative fluorescence intensity triggered by different concentrations of Cyp in Hana3AOlfr43-FLuc-RLuc cells expressing Olfr43, Olfr544, Olfr734, and Olfr16, respectively (B).

[0018] Figure 2 The effects of Cyp on lipid secretion in the HepG2 hyperlipidemic cell model of this invention are as follows: (A) Cyp cytotoxicity assay; (BC) Amounts of TC and TG after Cyp treatment; (D) Time-dependent cAMP production by Olfr43 cells activated by Cyp; (E) Relative gene expression (n=6); (F) Western blot images and semi-quantitative data of Olfr43 and downstream factors after Cyp intervention; Data are expressed as mean ± SEMs (n=8). *p<0.05, **p<0.01; ns P>0.05.

[0019] Figure 3 This invention relates to the effect of Cyp on lipid secretion in the Olfr43 knockdown hyperlipidemic cell model HepG2; (A) Olfr43 knockdown in HepG2 cells using shRNA; (B) cAMP time-dependent effects of Cyp intervention on Olfr43 knockdown in HepG2 cells; (CD) TC and TG levels in Olfr43 knockdown HepG2 cells after Cyp treatment. *p<0.05, **p<0.01, ***p<0.001; ns P>0.05. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1

[0022] Cyperone specifically binds to Olfr43 and activates Olfr43 downstream signaling pathways.

[0023] 1. Experimental cells.

[0024] HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in DMEM high-glucose medium containing 10% FBS at 37°C and 5% CO2.

[0025] Hana3A cells, originally from Hiroaki Matsunami's (Duke University) laboratory, accession number: CVCL_RW32; were cultured under standard conditions (37°C, 5% CO2) in minimum necessary medium (MEM) supplemented with 10% fetal bovine serum.

[0026] 2 Experimental drugs

[0027] Cyperone (abbreviated as Cyp), CAS No.: 3466-15-7, obtained by purchasing pure product, with a purity of over 99%, refrigerated for later use.

[0028] Free fatty acids (FFA): Accurately weigh fatty acid-free bovine serum albumin (BSA) powder (1g / 2g) into a 15mL centrifuge tube, add deionized water, vortex to mix, centrifuge at 4000rpm for 5min, and bring the volume to 5mL to prepare 20% and 40% BSA solutions, respectively. Weigh 0.06g NaOH and dissolve it in 10mL of deionized water to prepare a 0.15mM solution. After equalizing the solutions, add 63.474μL of oleic acid (OA) or 0.025672g of palmitic acid (PA) to each solution, and react in a 75℃ water bath for 30min to obtain 40mM OA and 20mM PA stock solutions. Mix the OA stock solution with 40% BSA and the PA stock solution with 20% BSA in the specified proportions, adjust the pH to a similar value to the culture medium with hydrochloric acid, and centrifuge again to remove foam. Filter sterilize using a 0.22pm microporous membrane and store at 4℃ for later use. Before the experiment, mix equal volumes of OA and PA stock solutions (final concentration 15mM, OA:PA = 2:1), dilute to the target concentration with 10% FBS medium, and filter again with a 0.22μm filter membrane before use.

[0029] SQ22536: Dissolved in a complete culture medium containing 0.1% DMSO and 10% FBS using sonication to prepare a stock solution of 5 mg / mL. Filtered through a 0.22 μm sterile filter membrane and stored at 4°C in the dark.

[0030] Sylibin: Prepare a stock solution of 1 mM / mL using sterile pure water. Dilute with complete culture medium containing 10% FBS to the required concentration during the experiment. Filter through a 0.22 μm sterile filter membrane and store at 4°C in the dark.

[0031] 3 Experimental Methods

[0032] 3.1 Hana3A Olfr43-FLuc-RLuc Construction and fluorescence detection of a heterologous OR dual-luciferase screening platform

[0033] This system includes transfection of all constructs using Lipofectamine 2000 (ThermoFisher). For each 96-well plate, a total of 12 μg of plasmid DNA containing the Olfr43 receptor expression gene, 2.4 μg of CRE-Luc, 2.4 μg of mouse RTP1S, and 2.4 μg of pRL-SV40 were introduced into the cells. 24 hours post-transfection, messenger RNAs containing Olfr544 (NM_020289.2), Olfr734 (NM_146664.2), Olfr16 (NM_008763.2), and Olfr43 (NM_207622.1) amplified from a mouse liver cDNA library were cloned into pCI vectors. Details of platform setup are as follows... Figure 1 A.

[0034] Luciferase detection: The luminescence signal was quantified using a dual-luciferase reporter assay system (Dual-Lumi, product number RG088S, Beyotime) and a microplate reader. Twenty-four hours post-transfection, the culture medium was replaced with 100 µL of Cyp solution at different concentrations (diluted in Opti-MEM to 0.00001, 0.0001, 0.001, 0.01, 0.1, and 1 mM, with each concentration repeated three times), and then incubated at 37°C and 5% CO2 for 24 hours. The activity of luciferase in fireflies and sea cucumbers was detected using a dual-luciferase reporter assay. The OR activity was normalized and calculated as FLuc / RLuc.

[0035] 3.2 Cell viability test

[0036] HepG2 cells were fed at a rate of 1×10 4 Seeds were placed in each well of a 96-well plate and cultured for 12 hours until cells were fully adherent. The culture medium was discarded, and the cells were washed twice with PBS. The following protocol was followed.

[0037] Blank control group: Cell-free, with 100 μL DMEM complete culture medium added to each well;

[0038] Normal control group: cells were present, and 100 μL of DMEM complete culture medium was added to each well;

[0039] Experimental control group: containing cells, 100 μL of Cyp solution of different concentrations (0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL and 256 μg / mL, diluted from the stock solution with DMEM complete medium) was added to each well in sequence.

[0040] Each group was set up with 6 replicates. After culturing for 36 hours, the culture medium was discarded, and 100 μL of prepared CCK-8 solution (CCK-8 stock solution: complete culture medium = 1:9) was added. The mixture was then incubated in an incubator for 40 minutes. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Each experiment was repeated three times independently.

[0041] 3.3 HepG2 cell culture and grouping

[0042] HepG2 cells were loaded at a rate of 2 × 10 5 Cells were seeded per well in 6-well plates and cultured for 12 hours until fully adherent. The culture medium was discarded, and the cells were washed twice with PBS. A NAFLD cell model was constructed and appropriate drug interventions were administered as follows, with 6 replicates per group.

[0043] Normal cell control group (NC): After culturing in DMEM complete culture medium for 12 h, the cells were replaced with fresh DMEM complete culture medium and cultured for another 36 h.

[0044] NAFLD cell model control group (free fatty acids, FFA): After culturing in DMEM complete culture medium for 12 h, the cells were replaced with FFA solution (final concentration 1 mM) and cultured for another 36 h.

[0045] Positive control group (silymarin): After culturing in DMEM complete medium for 12 h, the culture was continued for 36 h with a mixture of FFA solution (final concentration 1 mM) and Sylibin (final concentration 10 μM).

[0046] Cyperone high-dose group (Cyp-H): After culturing in DMEM complete culture medium for 12 h, the culture was continued for 36 h in a mixed solution of FFA solution (final concentration of 1 mM) and Cyp (final concentration of 10 μg / mL);

[0047] Cyperone low-dose group (Cyp-L): After culturing in DMEM complete culture medium for 12 h, the culture was continued for 36 h in a mixed solution of FFA solution (final concentration of 1 mM) and Cyp (final concentration of 5 μg / mL);

[0048] SQ22536 inhibitor group (SQ22536): After culturing in DMEM complete medium for 12 h, the culture was continued for 36 h in a mixed solution of FFA solution (final concentration 1 mM), Cyp (final concentration 10 μg / mL) and SQ22536 (final concentration 100 μmol / L).

[0049] 3.4 RNA knockdown in Olfr43 cells

[0050] To suppress Olfr43 gene expression in HepG2 cells, shRNA-Olfr43 was inserted into the shRNA insertion site of the pLKO.1-EGFP-puro vector. psPAX2 and pMD2.G vectors were co-transfected into 293T cells to generate lentiviruses expressing shRNA and the aforementioned recombinant vector. Forty-eight hours after transfection, the supernatant containing the lentivirus was collected and filtered through a 0.45 μm filter. HepG2 cells were infected with the lentivirus for 48 hours and selected using puromycin (2.5 μg / mL). The constructed Olfr43 RNA-knockdown HepG2 cells were grouped and treated according to 3.3 as NC, FFA, and Cyp-H cells. Another negative control group was set up, consisting of HepG2 cells without Olfr43 knockout (constructed in a similar manner to HepG2 cells with Olfr43 RNA knockdown, except that shRNA-Olfr43 was replaced with shRNA-NC). The cells were grouped and treated according to the conditions in 3.3 for NC, FFA, and Cyp-H.

[0051] 4 detection indicators

[0052] 4.1 Measurement of TC, TG, cAMP, etc. in cell supernatant

[0053] Cells were analyzed using triglyceride and total cholesterol assay kits, and were sonicated after collection. The remaining steps were performed strictly according to the kit instructions, and cellular protein concentrations were measured using BCA reagent. Intracellular TG levels were measured, and TC was determined using a similar method to TG.

[0054] The steps for detecting cAMP content in cell supernatant using a cAMP kit are as follows: Collect cell supernatant, centrifuge at 3000 rpm for 10 minutes to remove impurities; add the supernatant to a microplate pre-coated with antibody, incubate, and then add enzyme-labeled secondary antibody; wash, add substrate TMB for color development, and terminate the reaction; measure absorbance at 450 nm using a microplate reader, and calculate the cAMP concentration using a standard curve. Avoid repeated freeze-thaw cycles and ensure reagents are equilibrated to room temperature.

[0055] 4.2 Expression levels of lipid metabolism-related genes and Western blot analysis

[0056] Total RNA was extracted and reverse transcribed into cDNA using a reverse transcription kit. The cDNA was then used as a template for quantitative real-time PCR amplification. GAPDH was used as an internal control. -ΔΔCT The mNRA expression of related genes was calculated using a method.

[0057] For Western blot (WB), the electrophoresis buffer, transfer buffer, and blocking buffer are prepared first. The protein sample is mixed with a denaturing agent and injected into a gel plate for electrophoresis. After separation, it is transferred to a PVDF membrane. After blocking, the primary and secondary antibodies for the corresponding proteins are added sequentially. After color development, the grayscale values ​​are analyzed using ImageJ to calculate the relative expression levels.

[0058] Primer sequences are as follows

[0059]

[0060] Note: Olfr43: Ectopic olfactory receptor 43; PPAR-γ: Peroxisome proliferator-activated receptor gama; FAS: Fatty acid synthase; SCD-1: Stearoyl-CoA desaturase-1; SREBP-1C: Key gene sterol regulatory element binding protein 1c; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

[0061] 5 Statistical Methods

[0062] The quantitative data were processed and statistically analyzed using Prism software, with mean ± standard error. For the four-parameter fitted curves, GraphPad Prism 9.0 was used for normalization to generate four-parameter agonist dose-response curves. Other parameters were analyzed using one-way ANOVA for inter-group significance. P < 0.05 was considered statistically significant, and P < 0.01 indicated a statistically significant difference. For quantitative experiments, unless otherwise specified, three replicates were performed, and the results were averaged.

[0063] 6 Results

[0064] 6.1 Screening of Hana3A with Olfr43 agonists Olfr43-FLuc-RLuc The establishment of the platform and the verification of Cyp

[0065] The principle behind the dual-luciferase screening platform is as follows: Activation of the G protein-coupled receptor (Olfr43) triggers an increase in cAMP, ultimately driving luciferase gene expression. When odor molecules bind to the G protein-coupled receptor on the cell membrane, a conformational change in the receptor is triggered. The activated receptor binds to the G protein, which activates adenylate cyclase, converting ATP into cyclic adenosine monophosphate (cAMP). cAMP acts as a second messenger, activating protein kinase A (PKA), which then phosphorylates transcription factors (such as CREB), promoting the transcription and expression of the firefly luciferase gene in the CRE-Luc plasmid. Subsequently, firefly luciferase catalyzes substrate luminescence. The activation level of the olfactory receptor can be quantified by detecting the fluorescence intensity. This mechanism is highly consistent with the classical pathway of olfactory signal transduction. Based on the above, this invention constructs the Olfr43 agonist screening platform Hana3A.Olfr43 -FLuc-RLuc The platform construction diagram is as follows: Figure 1 As shown in Figure A.

[0066] like Figure 1 Results B show that Cyp can activate the olfactory receptor Olfr43, leading to an increase in intracellular cAMP. Statistical analysis showed that the half-maximal effective concentration (EC50) of Cyp for Olfr43 activation was 97.56 μM. Furthermore, we investigated the activation effects of Cyp on other ectopic olfactory receptors in the liver, such as Olfr544, Olfr734, and Olfr16. The results indicate that Cyp did not show any activation effect on Olfr544, Olfr734, or Olfr16. In conclusion, Cyp can serve as a specific agonist of the olfactory receptor Olfr43.

[0067] 6.2 Preliminary study on the effects and mechanisms of Cyp on lipid metabolism indicators in the FFA-induced HepG2 high-fat model in this invention

[0068] Cell viability assay results showed that Cyp was non-toxic to cells in the range of 0-32 μg / mL. Figure 2 A). Furthermore, the cAMP level in the cell supernatant of the high-dose Cyp group was detected. Figure 2 Results showed that high-dose Cyp significantly increased cAMP levels in the hyperlipidemic HepG2 cell model after 30-45 min of treatment (P<0.01). Compared with the NC group, the levels of TC and TG in the cell supernatant of the FFA group were significantly increased (P<0.01); after drug treatment, both high and low doses of Cyp reduced the levels of TC and TG in the hyperlipidemic HepG2 cell model (P<0.01, P<0.05). Figure 2 CD).

[0069] Figure 2 E showed that high-dose Cyp increased mNRA and protein expression in Olfr43 cells in a significantly hyperlipidemic HepG2 cell model, while decreasing the expression of genes positively related to lipid synthesis, such as PPARγ, SREBP-1C, FAS, and SCD-1, with significant differences (P<0.05, P<0.01). Figure 2 F-tests showed that Cyp significantly increased the levels of PKA, CREB, phosphorylated CREB, and HES-1, while decreasing the levels of DGAT2 protein (P<0.05). This indicates that Cyp's main functional pathway is achieved through the activation of Olfr43.

[0070] 6.3 Effects of Cyp on lipid metabolism parameters in HepG2 hyperlipidemic model cells with knocked-down Olfr43 RNA in this invention

[0071] To further verify that Cyp is a specific agonist of the olfactory receptor Olfr43, we designed shRAN-Olfr43 using RNA silencing technology and constructed HepG2 cells with Olfr43 RNA knocked down. Figure 3 As shown in Figure A), a HepG2 high-fat cell model with Olfr43 RNA knockdown (referred to as Olfr43 shRNA) was further constructed and treated with Cyp. The results are as follows. Figure 3 As shown in Figure B, in the high-lipid model cells with un-knocked Olfr43HepG2 (Scr shRNA), the Cyp-H group showed a significant increase in cAMP levels compared to the FFA group (P<0.001); in Olfr43 shRNA, the Cyp-H group showed no significant change in cAMP levels compared to the FFA group (P>0.05). These results further verify that Cyp increases cAMP levels by specifically activating the olfactory receptor Olfr43. Figure 3 CD results showed that Cyp in Olfr43 shRNA could not reduce TC and TG in HepG2 hyperlipidemic model cells (P>0.05).

[0072] The above results indicate that Cyp, as a specific agonist of the olfactory receptor Olfr43, can significantly increase the mNRA and protein expression levels of Olfr43 in cells, enhance cellular cAMP levels, and inhibit the expression of lipid synthesis-related genes by phosphorylating CREB, promoting HES-1, and inhibiting DGAT2 protein expression, ultimately reducing TC and TG in the HepG2 hyperlipidemia model. This invention provides a new option for the development of highly effective agonists specifically targeting Olfr43 / OR1A1.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Cyperene in the preparation of olfactory receptor Olfr43 specific targeted agonist drugs.

2. The use of cyperone as described in claim 1 in the preparation of olfactory receptor Olfr43 specific agonist drugs, characterized in that, Cyperone regulates the level of downstream cyclic adenosine monophosphate (cAMP) by activating the olfactory receptor Olfr43.

3. The use of cyperone as described in claim 1 in the preparation of olfactory receptor Olfr43 specific agonist drugs, characterized in that, The chemical formula of the cyperone is shown in Formula I: Formula I.

4. The use of cyperone as described in claim 1 in the preparation of an olfactory receptor Olfr43 specific agonist drug, characterized in that, The drug is composed of an effective dose of cyperone and a pharmaceutically acceptable carrier or excipient.

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