A polypeptide and its use as an olfactory receptor antagonist

Abstract

The application belongs to the technical field of diabetes treatment drugs, and particularly relates to a polypeptide and application thereof as an olfactory receptor antagonist. In the homologous sequences of insB:9-23 of different species, three homologous sequences Hgy, Gal and Ssth of insB:9-23 of other species are found, which can be used as antagonists of the targeted olfactory receptor Olfr109 / OR12D3, antagonize the agonistic effect of the self insB:9-23 of the mouse on the Olfr109 / OR12D3, improve the sugar metabolism of the diabetes model mouse, and can be used as a new type of diabetes clinical treatment drug for further research.

Description

[0001] This application is a divisional application of the patent entitled "A polypeptide and its application as an olfactory receptor antagonist", filed on April 12, 2023, application number: 2023103863219. Technical Field

[0002] This invention belongs to the field of diabetes treatment drug technology, specifically relating to a polypeptide and its application as an antagonist of the olfactory receptor Olfr109 / OR12D3. Background Technology

[0003] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0004] Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia and disordered glucose metabolism. Absolute insulin deficiency and insulin resistance are its core causes. Diabetes mellitus is mainly divided into type 1 and type 2.

[0005] Non-natural amino acids, also known as synthetic amino acids, are a class of amino acids that can be manufactured using synthetic methods. Their structures differ significantly from those of natural amino acids, but they possess the same functions. Due to their diverse effects, including enhancing physiological activity, metabolism, solubility, and stability, they have attracted considerable attention from drug researchers.

[0006] In our previous studies, we discovered that S9L (dextrorotatory) and L11F can serve as antagonists for the diabetes treatment target Olfr109 / OR12D3, but they have problems such as short half-life and poor efficacy. Therefore, it is necessary to develop antagonists with stronger antagonistic ability and better effect on improving glucose metabolism. Summary of the Invention

[0007] Based on the aforementioned research background, Olfr109 / OR12D3 can serve as a novel target for diabetes treatment. Its agonist, murine insB:9-23, can act on the Gαi signaling pathway of Olfr109, leading to a decrease in intracellular cAMP levels and inhibiting insulin secretion. Antagonists modified based on the amino acid sequence of murine insB:9-23 can effectively alleviate the inhibition of insulin secretion by murine insB:9-23, while simultaneously improving pancreatic inflammation and oxidative stress, effectively maintaining pancreatic homeostasis. In our studies of homologous sequences of insB:9-23 from different species, we discovered three other species' insB:9-23 homologous sequences, Hgy, Gal, and Ssth, which can act as antagonists targeting the olfactory receptor Olfr109 / OR12D3. These sequences antagonize the agonistic effect of insB:9-23 on Olfr109 / OR12D3 in mice, improving glucose metabolism in diabetic model mice. Furthermore, through substitution of non-natural amino acids, we obtained the antagonists Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X, which to some extent prolonged the half-life and enhanced the therapeutic effect. These sequences could be further investigated as novel clinical treatments for diabetes.

[0008] In a first aspect, the present invention provides a polypeptide, the sequence of which is shown below: Hgy: SQLVDTLYSVCRHRG, as shown in SEQ ID NO: 1; Gal: PALVEAIEFVCGERG, as shown in SEQ ID NO: 2; Ssth: PQLVDALVLVCGDRG, as shown in SEQ ID NO: 3; Gal-S9L (right-handed): LALVEAIEFVCGERG, where the first L is right-handed, as shown in SEQ ID NO: 4; Gal-2X: PXLVEAIEFVCGERG, where X is a 2-aminoisobutyric acid residue, as shown in SEQ ID NO: 5; Gal-S9L(dextral)-2X:LXLVEAIEFVCGERG, where the first L is dextrorotatory and X is a 2-aminoisobutyric acid residue, as shown in SEQ ID NO:6.

[0009] In a second aspect, the present invention provides a gene sequence, said gene sequence: (1) Used to encode the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; (2) An amino acid sequence used to encode the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, obtained by modifying or substituting one or more amino acids with non-natural amino acids; Preferably, the modification includes amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation, or glycosylation.

[0010] In a third aspect, the present invention provides the use of the polypeptide described in the first aspect as an Olfr109 and / or OR12D3 antagonist.

[0011] The OR12D3 mentioned is the OR12D3 gene in human pancreatic islet tissue.

[0012] Olfr109 is the homolog of OR12D3 in mice.

[0013] Furthermore, the peptide is used to improve glucose metabolism in diabetic model mice.

[0014] Furthermore, the use of the polypeptide as a treatment for diabetes. The diabetes can be type 1 diabetes and / or type 2 diabetes.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides peptides that antagonize the olfactory receptor Olfr109 / OR12D3 through screening. Based on the comparison of amino acid sequences of insB:9-23 from different species, insB:9-23 with significant sequence differences was selected for synthesis. Through biochemical screening, an antagonist targeting the olfactory receptor Olfr109 / OR12D3 was obtained, which can antagonize the agonistic effect of Olfr109 on Olfr109 by the mouse's own insB:9-23. Based on the important regulatory role of Olfr109 in diabetic metabolic diseases such as insulin secretion, pancreatic islet inflammation, and dedifferentiation, this antagonist can specifically act on the Olfr109 / OR12D3 to regulate downstream signaling pathways, and can be further studied as a novel clinical treatment drug for diabetes. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The binding ability of 13 different species of synthesized insB:9-23 to Olfr109 and its activation ability to its downstream Gi signaling pathway were investigated. Figure 2Schematic diagram of the effect of insB:9-23 antagonizing the downstream Gi signaling pathway of olfactory receptor Olfr109 in different species (Hgy, Gal and Ssth); Figure 3 Schematic diagram of the effect of insB:9-23 antagonizing the Gi signaling pathway downstream of olfactory receptor OR12D3 in different species (Hgy, Gal and Ssth); Figure 4 The pharmacokinetic effects of Gal, Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X in rats are shown in the figure. Figure 5 Diagram showing the effect of Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X on antagonizing the Gi signaling pathway downstream of the olfactory receptor OR12D3; Figure 6 The antagonistic effects of five antagonists (Hgy, Gal, Ssth, Gal-S9L (dextral), Gal-S9L (dextral)-2X) on the inhibition of insulin secretion from mouse pancreatic islets by agonist murine insB:9-23; Figure 7 The effects of five antagonists (Hgy, Gal, Ssth, Gal-S9L (dextral), Gal-S9L (dextral)-2X) on the improvement of type 1 diabetes model mice; Figure 8 The effects of five antagonists (Hgy, Gal, Ssth, Gal-S9L (dextral), Gal-S9L (dextral)-2X) on the amelioration of type 2 diabetes model mice were investigated. Detailed Implementation

[0018] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0020] As described in the background section, existing research indicates that the olfactory receptor Olfr109 is a key factor in maintaining blood glucose homeostasis and is closely related to insulin secretion. This invention proposes an antagonist targeting the olfactory receptor Olfr109 / OR12D3.

[0021] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0022] Example 1: Specific antagonistic effect of peptides on olfactory receptor Olfr109 / OR12D3 Experimental steps: 1. Synthesize the following peptides (represented by single-letter amino acids) using a 431A peptide synthesizer (Perkin Elmer): Mus:9-23:SHLVEALYLVCGERG Hoplomys gymnurus (Hgy): SQLVDTLYSVCRHRG (amino acid sequence as shown in SEQ ID NO:1) Gonatodes albogularis (GAL): PALVEAIEFVCGERG (amino acid sequence as shown in SEQ ID NO:2) Stenodactylus sthenodactylus(Ssth): PQLVDALVLVCGDRG (amino acid sequence as shown in SEQ ID NO:3) Cyrtodactylus quadrivirgatus(Cqu): RQLVEALALICGDQG Chondrodactylus turneri(Ctu): KQLVDALILICGDRG Gekko vittatus(Gvi): RQLVDALILVCGDRG Gekko hokouensis(Gho): RQLVDSLNLICGDRG Myocastor coypus(Mco): SQLVDTLYSVCKHRG Cavia porcellus(Cpoc): SNLVETLYSVCQDDG Myotis brandtii(Myb): EDLVDTLTMVCGDRG Abrocoma bennettii(Abe): SNLVDALFLLCERNG Galea musteloides(Gmu): SNLVDALYVVCKDKG Microcavia niata(Mni):SNLVETLYEVCRDKG 2-1. Detection of the activation ability of different species of insB:9-23 synthesized in step 1 on the Gi signaling pathway of Olfr109. Using molecular biology methods, HEK293 cells overexpressing Olfr109 were employed. The Glosensor method was used to detect the second messenger cAMP. Cells were stimulated to produce cAMP by Forskolin, an agonist of adenylate cyclase, while simultaneously stimulated with mouse insB:9-23, an agonist of Olfr109. The degree of decrease in intracellular cAMP concentration was used to characterize the activation of the downstream Gi signaling pathway of Olfr109. The test peptide synthesized in step 1 was prepared into a working concentration of 10 μL using PBS. -12 M, 10 -11 M, 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M、、10 -6 In HEK293 cells overexpressing Olfr109 and Glosensor, after stimulation with 10 μM Forskolin, different concentrations (10 μM Forskolin, 10 μM Forskolin, 10 μM Forskolin, Glosensor ... -12 -10 -6 The test peptide synthesized in step 1 of experiment M) was used for stimulation, with HEK293 cells expressing empty vector pcDNA3.1 and Glosensor as the negative control. Fluorescence values ​​were recorded in real time using a multi-functional microplate reader, and the results were statistically analyzed. The results are shown in [Figure 1]. Figure 1 .

[0023] 2-2. Detection of the binding ability of insB:9-23 with receptors in different species HEK293 cells overexpressing Olfr109 and Glosensor were seeded into 96-well plates. The culture medium was aspirated from each well, and 40 μL was added to each well using a pipette. FITC-insB 9-23 (original concentration 2 mM) was diluted 200-fold with PBS to a final concentration of 10 μM. Then, 5 μL of the synthesized test peptide (different species insB: 9-23) from step 3 (original concentration 5 mM) was added to each well using a pipette. This diluted solution was further serially diluted to 1 μM, 100 nM, 10 nM, 1 nM, 100 pM, and 10 pM, and then added to the corresponding wells (5 μL per well). The plates were incubated at 37°C for 45 minutes. Detection was performed using a Mithras LB 940, and the results were statistically analyzed. The results are shown in the table below. Figure 1 .

[0024] exist Figure 1 Based on the experimental results, the binding ability of insB:9-23 to the receptor remained unchanged or improved in different species, and EC50 or Bmax decreased in the G protein activation experiment, indicating that it may act as an antagonist of the olfactory receptor Olfr109 / OR12D3.

[0025] 3. Detect the antagonistic effect of insB:9-23 of different species of Hgy / Gal / Ssth on the downstream Gi signaling pathway of olfactory receptor Olfr109 / OR12D3. Using molecular biology methods, HEK293 cells overexpressing Olfr109 / OR12D3 were used. The Glosensor method was employed to detect the second messenger cAMP. Cells were stimulated to produce cAMP by the adenylate cyclase agonist Forsoklin, while simultaneously stimulated with the Olfr109 / OR12D3 agonist, murine insB:9-23. The degree of intracellular cAMP concentration reduction was used to characterize the activation of the downstream Gi signaling pathway of Olfr109 / OR12D3. Furthermore, the species antagonists Hgy / Gal / Ssth insB:9-23 were administered, and the degree of intracellular cAMP concentration recovery was used to characterize the agonistic effect of the peptides on the Olfr109 / OR12D3 downstream Gi signaling pathway. The insB:9-23 antagonists of the test peptides were prepared with PBS to a working concentration of 10. -11 M, 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, in HEK293 cells overexpressing Olfr109 / OR12D3 and Glosensor, after stimulation with 10 μM Forskolin, different concentrations (10 -12 -10 -6 Different species of Hgy / Gal / Ssth cells (M) were incubated with insB:9-23 and stimulated with 100 nM insB:9-23 ligand. HEK293 cells expressing empty vector pcDNA3.1 and Glosensor served as negative controls. Fluorescence values ​​were recorded in real-time using a multi-functional microplate reader, and the results were statistically analyzed. The results are shown in [Figure 1]. Figure 2 , Figure 3 .

[0026] Figure 2 and Figure 3 The results show that the insB:9-23 of species Hgy, Gal, and Ssth are associated with the olfactory receptor Olfr109 ( Figure 2 ) and OR12D3 ( Figure 3 Downstream Gi signaling pathways all have a certain degree of antagonistic effect.

[0027] 4. Detection of the pharmacokinetics of Gal and its modified peptides in rats. Twenty male SD rats were randomly divided into four groups of five each. They were fasted for 12 hours prior to drug administration but had free access to water. Gal, Gal-S9L (dextrorotatory), Gal-2X, and Gal-S9L (dextrorotatory)-2X were administered via tail vein injection at a dose of 5 mg / kg. Approximately 0.2 mL of blood was collected via the clavicular sinus at 2 min, 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, and 8 h post-injection. 25 μL of internal standard working solution was added to a 1.5 mL EP tube, followed by 50 μL of biological samples (standard curve, serum sample, and blank sample), and then 300 μL of methanol as precipitation reagent. The mixture was vortexed and centrifuged at 13000 rpm for 5 min. The supernatant was then transferred to a clean mass spectrometry vial. The concentrations of Gal, Gal-S9L (dextrorotatory), Gal-2X, and Gal-S9L (dextrorotatory)-2X in plasma samples were determined using LC-MS. The non-compartmental model of the DAS 2.0 pharmacokinetic program was used to fit and analyze the measured plasma drug-time data, calculating key pharmacokinetic parameters such as Cmax, AUC, Tmax, T1 / 2, MRT, and CL, and plotting the mean plasma drug concentration-time curve.

[0028] Depend on Figure 4 It can be seen that the half-life of Gal in rats is about 0.1 h. The drug metabolism was improved to 0.2 h by introducing dextrorotatory amino acid substitution to Gal-S9L (dextral). The half-life can be improved to 1.06 h by substitution with non-natural amino acid X. Finally, the half-life can be effectively extended to 1.46 h after being modified into Gal-S9L (dextral)-2X.

[0029] 5. The antagonistic effects of different species of Hgy / Gal / Ssth insB:9-23 antagonists, as well as Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X, on the inhibition of insulin secretion by mouse insB:9-23 agonists in mice. The experimental principle is that high glucose stimulates the pancreas to secrete more insulin. The agonist murine insB:9-23 acts on Olfr109 to activate the downstream Gi signaling pathway, reducing intracellular cAMP levels and thus decreasing insulin secretion. On this basis, a peptide antagonist is given to restore the decrease in cAMP levels caused by the action of murine insB:9-23 on the receptor, thereby characterizing the antagonistic effect of the peptide on the downstream Gi signaling pathway of Olfr109 and alleviating the reduction in insulin secretion caused by the action of the ligand.

[0030] Pancreatic islets were randomly divided into 6 groups of 10 islets each. The islets were starved with mKRBB buffer for 30 minutes. The control group received 2.8 mM glucose mKRBB. The experimental groups received 20 mM glucose mKRBB, 20 mM glucose mKRBB stimulated with the agonist mouse insB:9-23, and 20 mM glucose mKRBB pre-incubated with different species of Hgy / Gal / Ssth insB:9-23, Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X before stimulation with the agonist mouse insB:9-23 for 20 minutes. The supernatant was collected, diluted 5-fold, and insulin secretion was detected using an ELISA kit. The luminescence value was measured using a multi-functional microplate reader. Statistical analysis of the results was performed. (See attached graph). Figure 6 .

[0031] Depend on Figure 6 It can be seen that stimulation with agonist mouse insB:9-23 can significantly reduce insulin secretion. However, the reduction in insulin secretion after pre-incubation with insB:9-23 and Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X from different species of Hgy / Gal / Ssth followed by stimulation with agonist mouse insB:9-23 was less than that after stimulation with agonist mouse insB:9-23 alone. This confirms that insB:9-23 and Gal-S9L (dextral), Gal-2X, and Gal-S9L (dextral)-2X from different species of Hgy / Gal / Ssth can act as Olfr109 / OR12D3 antagonists to increase insulin secretion.

[0032] 6. Detection of glucose tolerance and insulin tolerance in HFD mice after administration of different species of Hgy / Gal / Ssth insB:9-23 antagonists, as well as Gal-S9L (dextrorotatory), Gal-2X, and Gal-S9L (dextrorotatory)-2X. Glucose tolerance: HFD mice (control / Hgy / Gal / Ssth / Gal-S9L (dextral) / Gal-2X / Gal-S9L (dextral)-2X) were subjected to glucose tolerance tests in different drug groups after being starved for 16 hours. Plasma glucose levels in mice were recorded at 0 or 15, 30, 60, and 120 minutes before intraperitoneal injection of 2 mg / g body weight glucose using a FreeStyle LiteGlucose Meter (Roche). To investigate the effects of different antagonist stimulation on glucose tolerance, HFD mice were intraperitoneally injected with either the control solvent or different antagonists (1 μg / g, ip) every other day for two weeks.

[0033] Depend on Figure 7 and Figure 8 It can be seen that after administration of antagonists to Akita (type 1 diabetes model) and HFD mice (type 2 diabetes model), the species antagonists Hgy / Gal / Ssth can all improve glucose tolerance to varying degrees, with Gal showing the strongest improvement. The pharmacokineticly optimized peptides Gal-S9L(dextral) / Gal-2X / Gal-S9L(dextral)-2X based on Gal can also improve glucose tolerance in Akita and HFD mice, with no significant difference in improvement compared to Gal.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, the polypeptide sequence of which is shown below: SQLVDTLYSVCRHRG, as shown in SEQ ID NO: 1; PALVEAIEFVCGERG, as shown in SEQ ID NO: 2; PQLVDALVLVCGDRG, as shown in SEQ ID NO:

3.

2. A gene sequence, said gene sequence: (1) Used to encode the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; (2) An amino acid sequence used to encode the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, which is obtained by modifying or substituting one or more amino acids with non-natural amino acids.

3. The gene sequence as described in claim 2, characterized in that, The modifications include amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation, or glycosylation.

4. The use of the polypeptide as described in claim 1 as an Olfr109 and / or OR12D3 antagonist in pharmaceutical preparation.

5. The application of the polypeptide as described in claim 1 in improving glucose metabolism in diabetic model mice.

6. The use of the polypeptide as described in claim 1 as a treatment for diabetes.

7. The application as described in claim 6, characterized in that, The diabetes can be type 1 diabetes and / or type 2 diabetes.

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