Polypeptides targeting apj and uses thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a polypeptide targeting APJ and application thereof. The polypeptide contains an amino acid sequence as shown in formula 1. The polypeptide provided in the present application has high affinity to APJ, thereby being capable of preventing and / or treating diseases related to the signal pathway thereof.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a polypeptide targeting APJ and its applications. Background Technology

[0002] APJ (apelin receptor) is a typical class A G protein-coupled receptor, consisting of a complete seven-span membrane domain composed of 308 amino acid residues. It is widely and specifically expressed in the core functional regions of human cardiovascular endothelial tissue, cardiomyocytes, central nervous system vascular regulatory units, renal microvascular epithelium, and pulmonary circulation thin-walled tissue. It is a core functional membrane receptor for endogenous active signal transduction in the body. At the physiological mechanism level, APJ can specifically couple to multiple downstream Gαi and Gαq signaling pathways, precisely regulating core physiological and pathological processes such as vascular endothelial homeostasis remodeling, cardiomyocyte energy metabolism compensation, systemic blood pressure rhythm balance, sustained release of inflammatory factor cascades, and organ ischemic injury repair. It is deeply involved in the entire process of the occurrence, progression, and prognosis of heart failure, refractory hypertension, acute myocardial ischemia injury, pulmonary vascular remodeling syndrome, ischemic stroke, and microcirculatory disorders. It is a recognized high-potential precision drug target in the fields of cardiovascular metabolism, vascular targeted diagnosis and treatment, and critical organ protection. The drugability of the target has been fully validated, and its clinical translation adaptability is extremely strong, making it a valuable target for the development and commercialization of new drugs.

[0003] Currently, the mainstream intervention agents targeting APJ pathway regulation in clinical practice are mainly developed based on two core technology routes: artificial replication and modification of endogenous natural ligands and small molecule targeted agonists / antagonists. While natural endogenous ligands such as apelin and eLABELA can specifically bind to APJ and activate downstream protective signaling pathways, the natural peptides themselves have inherent structural limitations, resulting in extremely poor clinical suitability: First, they have insufficient tolerance to the plasma environment and are easily hydrolyzed and degraded by broad-spectrum aminopeptidases and carboxylpeptidases in vivo, with a circulating half-life of only a few minutes, requiring high-frequency continuous intravenous infusion, which significantly increases the cost of clinical administration and the physical and mental burden on patients; Second, they have weak targeting enrichment capabilities, lacking tissue-specific target recognition domains, and are widely and non-specifically distributed throughout the body, making it difficult to accurately enrich them in ischemic areas of myocardial infarction, damaged vascular endothelium, and the core areas of microcirculatory lesions in target organs, resulting in a low effective target occupancy rate, and high-dose administration can easily cause off-target adverse reactions such as abnormal vasodilation, transient heart rate fluctuations, and peripheral microcirculatory disturbances; Third, they have insufficient physicochemical suitability, with poor physicochemical stability of natural ligands, requiring stringent storage conditions for formulations, high costs for cold chain transportation control, and significant difficulties in large-scale industrial-scale production.

[0004] While existing small-molecule APJ-targeting compounds circumvent the shortcomings of short half-lives and cumbersome formulation storage and transportation of natural ligands, their clinical application bottlenecks are becoming increasingly prominent, with core defects proving difficult to overcome: First, there is an imbalance in target binding specificity. Small-molecule compounds have a single molecular spatial conformation, only able to bind to a single extracellular binding site of APJ, making them prone to cross-binding with other subtypes of the homologous G protein-coupled receptor family in vivo. This interferes with normal neural and endocrine homeostasis regulation, and long-term administration can easily induce metabolic accumulation toxicity in liver and kidney function, persistent gastrointestinal adverse reactions, and abnormal stress damage to vascular endothelium; Second, pathway regulation efficacy is limited. Small molecules cannot precisely match the activation conformation of the natural APJ receptor, exhibiting mostly incomplete agonist / weak antagonist activity. This makes it difficult to fully activate the entire protective signaling network of myocardial protection, vascular repair, anti-inflammatory, and anti-remodeling. The effective response rate of monotherapy in high-risk critically ill patients is less than 60%, and combination therapy carries additional risks of adverse reactions. Thirdly, the applicable scenarios for critical care are narrow. The transmembrane penetration efficiency of small molecules is uneven, and their penetration ability into damaged target tissues under ischemic and hypoxic pathological microenvironments is significantly reduced. This makes them unsuitable for the urgent clinical need for rapid targeted intervention in acute myocardial infarction, severe heart failure, and fulminant pulmonary vascular injury, significantly limiting their use in emergency situations. Furthermore, existing targeted agents do not address the five core clinical requirements of precise lesion enrichment, long-term circulation retention, low toxicity and safety, room-temperature storage and transportation, and universal administration across multiple scenarios. No mature long-acting targeted APJ peptide formulation has yet been approved for clinical application, and the gap in clinical targeted intervention for critically ill patients continues to widen.

[0005] Compared to small molecule compounds and natural endogenous ligands, artificially customized targeted peptide drugs possess inherent core R&D advantages: biocompatibility aligns with the human endogenous metabolic system, with no cumulative toxic side effects and low immunogenicity; molecular structure can be precisely and directionally optimized, allowing for the addition of targeted enrichment groups and anti-enzymatic modification sites, simultaneously enhancing target affinity and long-term in vivo stability; controllable molecular weight adapts to the penetration requirements of vascular microcirculation, resulting in lesion-targeted enrichment efficiency far exceeding that of conventional small molecules; mature and controllable synthesis processes enable low-cost industrial-scale mass production; and formulations are suitable for intravenous injection, subcutaneous administration, and nebulization, catering to multiple clinical scenarios, perfectly meeting the core upgrade demands of current APJ targeted therapy. However, existing targeted APJ peptide sequences generally suffer from technical deficiencies such as weak binding activity, unreasonable design of anti-enzymatic modification sites, missing targeted enrichment modules, and insufficient tolerance to the pathological microenvironment. They have not yet formed a dedicated peptide drug system with integrated optimization of high affinity, high stability, and high lesion targeting, failing to address the core shortcomings of existing clinical intervention technologies.

[0006] In summary, the current clinical practice lacks a dedicated APJ-targeted peptide formulation that is highly specific, stable in vivo, has controllable toxicity and side effects, is suitable for the diagnosis and treatment of multiple diseases, and is convenient for industrial mass production. The industry pain points of existing intervention drugs, such as insufficient target precision, poor drug safety, weak suitability for severe diseases, and low clinical compliance, have long remained unresolved.

[0007] Based on this, the present invention specifically develops a new type of peptide drug that targets APJ, which efficiently and specifically activates the target protective pathway by occupying the site, and has multiple core advantages such as long-lasting circulation, precise accumulation in lesions, low toxicity and safety, and easy dosage production. It effectively solves the shortcomings of existing pharmaceutical technologies and meets the rigid clinical needs for precise targeted therapy for severe cases. Summary of the Invention

[0008] The purpose of this invention is to provide a drug polypeptide that targets APJ, which has a high affinity for APJ.

[0009] To achieve the above objectives, a first aspect of the present invention provides a polypeptide targeting APJ or a pharmaceutically acceptable salt thereof, the polypeptide containing an amino acid sequence as shown in Formula 1:

[0010] X1RRPX2X3X4KFX5X6X7X8X9F Formula 1

[0011] Among them, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each an arbitrary amino acid.

[0012] A second aspect of the present invention provides a gene whose nucleotide sequence is capable of encoding the polypeptide targeting APJ described in the first aspect above.

[0013] A third aspect of the present invention provides a vector containing the gene described in the second aspect above.

[0014] A fourth aspect of the present invention provides a host cell containing the vector described in the third aspect above.

[0015] A fifth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned first aspect of a polypeptide targeting APJ or a pharmaceutically acceptable salt thereof.

[0016] The sixth aspect of the present invention provides the use of at least one of the following: the APJ-targeting polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect; the gene described in the second aspect; the vector described in the third aspect; the host cell described in the fourth aspect; and the pharmaceutical composition described in the fifth aspect, in the preparation of a medicament for the prevention and / or treatment of diseases related to the APJ signaling pathway; wherein the diseases related to the APJ signaling pathway are selected from at least one of cardiovascular diseases, nervous system diseases, metabolic diseases, and tumors; wherein the cardiovascular diseases are selected from at least one of heart failure, pulmonary hypertension, hypertension, myocardial hypertrophy, and ischemic heart disease; wherein the nervous system diseases are selected from at least one of ischemic stroke, depression, and epilepsy; wherein the metabolic diseases are selected from at least one of type 2 diabetes and obesity; and wherein the tumors are selected from at least one of glioblastoma, lung cancer, ovarian cancer, esophageal cancer, and lymphoma.

[0017] Other features and advantages of the present invention will be described in detail in the following detailed description section. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] It should be noted that, unless otherwise defined herein, the scientific and technical terms used in this invention shall have the meanings commonly understood by those skilled in the art.

[0020] As previously stated, a first aspect of the present invention provides a polypeptide targeting APJ or a pharmaceutically acceptable salt thereof, the polypeptide containing an amino acid sequence as shown in Formula 1:

[0021] X1RRPX2X3X4KFX5X6X7X8X9F Formula 1

[0022] Among them, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each an arbitrary amino acid.

[0023] Preferably, in formula 1,

[0024] X1 is selected from R, hArg, or does not exist;

[0025] X2 is selected from G and Y;

[0026] X3 is selected from L and W;

[0027] X4 is selected from N and R;

[0028] X5 is selected from L and G;

[0029] X6 is selected from N, P, W, Pip, Hyp, and 4-MePro;

[0030] X7 is selected from D-Met or does not exist;

[0031] X8 is selected from W, G, 1-Nal, 2-Nal, Nle, 2-MeTrp, or does not exist;

[0032] X9 is selected from P, Hyp, Pip, Aze, and 4-MePro.

[0033] More preferably, in Equation 1,

[0034] X2 is G;

[0035] X3 is L;

[0036] X4 is N.

[0037] Particularly preferably, the polypeptide contains an amino acid sequence as shown in any one of SEQ ID NO: 1-26.

[0038] In a preferred embodiment, the amino terminus and / or carboxyl terminus of the amino acid sequence of the polypeptide are modified.

[0039] Preferably, the modification is selected from at least one of the following: amylation, hydroxylation, carboxylation, carbonylation, amidation, alkylation, phosphorylation, glycosylation, cyclization, biotinylation, acetylation, esterification, fluorescent group modification, polyethylene glycol modification, and immobilization modification.

[0040] Particularly preferred is that the modification is an amidation modification.

[0041] It should be noted that the present invention does not impose any particular limitation on the method of synthesizing the polypeptide, and those skilled in the art can choose from the techniques known in the art. Exemplarily, a preferred polypeptide synthesis method is provided below, and those skilled in the art should not construe it as a limitation of the present invention.

[0042] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0043] In this invention, the amino acid sequences shown in SEQ ID NO: 1-26 are listed in Table 1.

[0044] Table 1

[0045]

[0046] As previously stated, a second aspect of the present invention provides a gene whose nucleotide sequence is capable of encoding the polypeptide targeting APJ described in the first aspect.

[0047] As previously stated, a third aspect of the present invention provides a vector containing the gene described in the second aspect.

[0048] As previously stated, a fourth aspect of the present invention provides a host cell containing the vector described in the third aspect.

[0049] As previously described, a fifth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide targeting APJ described in the first aspect thereof or a pharmaceutically acceptable salt thereof.

[0050] Preferably, the composition further contains excipients.

[0051] It should be noted that the present invention does not impose any particular restrictions on the type of excipients. Those skilled in the art can select them according to known techniques in the field, as long as the polypeptides provided in the present invention can target APJ.

[0052] As previously stated, the sixth aspect of the present invention provides the use of at least one of the following: the APJ-targeting polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect; the gene described in the second aspect; the vector described in the third aspect; the host cell described in the fourth aspect; and the pharmaceutical composition described in the fifth aspect, in the preparation of a medicament for the prevention and / or treatment of diseases related to the APJ signaling pathway; wherein the diseases related to the APJ signaling pathway are selected from at least one of cardiovascular diseases, nervous system diseases, metabolic diseases, and tumors; wherein the cardiovascular diseases are selected from at least one of heart failure, pulmonary hypertension, hypertension, myocardial hypertrophy, and ischemic heart disease; wherein the nervous system diseases are selected from at least one of ischemic stroke, depression, and epilepsy; wherein the metabolic diseases are selected from at least one of type 2 diabetes and obesity; and wherein the tumors are selected from at least one of glioblastoma, lung cancer, ovarian cancer, esophageal cancer, and lymphoma.

[0053] The present invention will be described in detail below through examples.

[0054] In the following examples, unless otherwise specified, all raw materials used were commercially purchased.

[0055] In the following examples, unless otherwise specified, room temperature refers to 25±5℃.

[0056] In the following examples:

[0057] HEK293 cell line overexpressing human AGTRL1, abbreviated as HEK293 / APJ-EGFP(A5 2nd-A5B1) cells;

[0058] The CHO-K1 / Gα15 cell line overexpressing human AGTRL1 is abbreviated as CHO-K1 / AGTRL1 / Gα15 cells;

[0059] Apelin13: Apelin-13, an endogenous natural APJ polypeptide ligand, a balanced full agonist, CAS number 217082-58-1;

[0060] MM07: A synthetic cyclic polypeptide, an APJ G protein-biased agonist, CAS number 1876450-21-3;

[0061] Example 1: Synthesis of Polypeptides

[0062] The present invention provides a method for synthesizing linear precursors of polypeptide compounds and their derivatives using solid-phase synthesis, followed by cleavage to obtain crude target polypeptides. The solid-phase support is 2-Chlotrityl Resin resin. During synthesis, the 2-Chlotrityl Resin resin is first fully swollen in N,N-dimethylformamide (DMF). Then, the swollen solid-phase support is repeatedly subjected to condensation with activated amino acid derivatives, followed by washing, deprotection of the Fmoc layer, washing, and another round of amino acid condensation to achieve the desired polypeptide chain length. Finally, the polypeptide is cleaved from the solid-phase support by reacting a mixed solution of trifluoroacetic acid:water:triisopropylsilane:aniline sulfide (v:v:v:v = 90:2.5:2.5:5) with the solid-phase resin. The cleavage is then carried out by precipitation with frozen methyl tert-butyl ether to obtain the crude target polypeptide. The crude target polypeptide is purified and separated by C18 reversed-phase preparative chromatography in a 0.1% trifluoroacetic acid / acetonitrile / water system to obtain pure polypeptides and their derivatives. The following examples provide specific methods for synthesizing several polypeptides; the specific methods for synthesizing other polypeptides are similar.

[0063] 1.1: Synthesis of a polypeptide compound with the amino acid sequence shown in SEQ ID NO: 7

[0064] Step 1: Couple the first amino acid Fmoc-Phe-OH

[0065] 0.2 mmol of 2-Chlorotrityl chloride resin was fully swollen in dichloromethane (DCM) for 1 h. 0.16 mmol of Fmoc-Phe-OH and 0.64 mmol of diisopropylethylamine (DIEA) were dissolved in 5 mL of DCM, and the swollen resin was added. The reaction was carried out at room temperature for 2 h. After the reaction was complete, 10 mL of blocking solution (DCM:methanol:DIEA (v:v:v = 85:10:5) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and then 5 times with N,N-dimethylformamide (DMF) before use.

[0066] Step 2: Synthesis of linear precursor peptide chains

[0067] The linear precursor peptide chain of the polypeptide compound with the amino acid sequence shown in SEQ ID NO: 7 is: RRPGLNKFGP-Nle-PF.

[0068] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second P-position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0069] (1) The swollen resin was deprotected twice with Fmoc using a mixture of 10 mL piperidine:DMF (v:v=20:80), each time for 8 min;

[0070] (2) Wash the resin with DMF 6-8 times until the liquid pH is neutral;

[0071] (3) Dissolve 0.5 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, then add the washed resin and react at room temperature for 1 h;

[0072] (4) Wash the resin with DMF 4-6 times before coupling the next amino acid;

[0073] After the linear precursor peptide chain is synthesized, the resin is washed 5 times with DMF and then 5 times with DCM. The washed resin is then dried under vacuum for later use.

[0074] Step 3: Cleavage of the linear precursor peptide chain

[0075] 10 mL of freshly prepared cutting solution (hexafluoroisopropanol:DCM (v:v:=30:70)) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 h. After the reaction was complete, the reaction solution was filtered, and the crude peptide was precipitated with 4 volumes of frozen methyl tert-butyl ether (MTBE). The crude peptide was washed three times with MTBE and then dried under vacuum for later use.

[0076] Step 4: Remove the protective substrate

[0077] 10 mL of the prepared protecting group removal solution (trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5, v:v:v)) was added to the crude peptide obtained in step 3, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was completed, 4 volumes of cold MTBE were added to precipitate the crude peptide, which had the protecting group removed. The crude peptide was washed three times with MTBE and dried under vacuum.

[0078] Step 5: Preparation of Peptides

[0079] The crude peptide obtained in step 4 was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were phase A (0.1% trifluoroacetic acid aqueous solution) and phase B (0.1% trifluoroacetic acid acetonitrile solution). A BR-C18 (Saifen Technology) reversed-phase column was used. During purification, the chromatograph detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was phase B changing from 20% to 50% within 0-40 min. The relevant fractions of the product were collected, and after HPLC purity identification, the fractions >95% were combined and lyophilized to obtain the pure peptide.

[0080] 1.2: Synthesis of a polypeptide compound with the amino acid sequence shown in SEQ ID NO: 17

[0081] Step 1: Couple the first amino acid Fmoc-Phe-OH

[0082] 0.2 mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. 0.16 mmol of Fmoc-Phe-OH and 0.64 mmol of DIEA were weighed and dissolved in 5 mL of DCM, and the swollen resin was added. The reaction was carried out at room temperature for 2 h. After the reaction was complete, 10 mL of blocking solution (DCM:methanol:DIEA (v:v:v = 85:10:5) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and then 5 times with DMF before use.

[0083] Step 2: Synthesis of linear precursor peptide chains

[0084] The linear precursor peptide chain of the polypeptide compound with the amino acid sequence shown in SEQ ID NO: 17 is: RRRPGLNKFGP-{1-Nal}-Aze-F.

[0085] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second Aze position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0086] (1) The swollen resin was deprotected twice with Fmoc using a mixture of 10 mL piperidine:DMF (v:v=20:80), each time for 8 min;

[0087] (2) Wash the resin with DMF 6-8 times until the liquid pH is neutral;

[0088] (3) Dissolve 0.5 mmol of Fmoc-AA, 1.0 mmol of HCTU and 2 mmol of NMM in DMF, then add the washed resin and react at room temperature for 1 h;

[0089] (4) Wash the resin with DMF 4-6 times before coupling the next amino acid;

[0090] After the linear precursor peptide chain is synthesized, the resin is washed 5 times with DMF and then 5 times with DCM. The washed resin is then dried under vacuum for later use.

[0091] Step 3: Cleavage of the linear precursor peptide chain

[0092] 10 mL of freshly prepared cutting solution (hexafluoroisopropanol:DCM (v:v:=30:70)) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 h. After the reaction was complete, the reaction solution was filtered, and the crude peptide was precipitated with 4 times its volume of frozen MTBE. The crude peptide was washed three times with MTBE and then dried under vacuum for later use.

[0093] Step 4: Remove the protective substrate

[0094] 10 mL of the prepared protecting group removal solution (trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5, v:v:v)) was added to the white solid obtained in step 3, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was completed, 4 volumes of cold MTBE were added to precipitate the solid, which had the protecting group removed. The crude product was washed three times with MTBE and dried under vacuum.

[0095] Step 5: Preparation of Peptides

[0096] The crude peptide obtained in step 4 was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were phase A (0.1% trifluoroacetic acid aqueous solution) and phase B (0.1% trifluoroacetic acid acetonitrile solution). A BR-C18 (Saifen Technology) reversed-phase column was used. During purification, the chromatograph detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was phase B changing from 20% to 50% within 0-40 min. The relevant fractions of the product were collected, and after HPLC purity identification, the fractions >95% were combined and lyophilized to obtain the pure peptide.

[0097] 1.3: Synthesis of a polypeptide compound with the amino acid sequence shown in SEQ ID NO: 20

[0098] Step 1: Couple the first amino acid Fmoc-Phe-OH

[0099] 0.2 mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. 0.16 mmol of Fmoc-Phe-OH and 0.64 mmol of DIEA were weighed and dissolved in 5 mL of DCM, and the swollen resin was added. The reaction was carried out at room temperature for 2 h. After the reaction was complete, 10 mL of blocking solution (DCM:methanol:DIEA (v:v:v = 85:10:5) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and then 5 times with DMF before use.

[0100] Step 2: Synthesis of linear precursor peptide chains

[0101] The linear precursor peptide chain of the polypeptide compound with the amino acid sequence shown in SEQ ID NO: 20 is: RRRPGLNKFGP-{1-Nal}-Hyp-F.

[0102] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second Hyp at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0103] (1) The swollen resin was deprotected twice with Fmoc using a mixture of 10 mL piperidine:DMF (v:v=20:80), each time for 8 min;

[0104] (2) Wash the resin with DMF 6-8 times until the liquid pH is neutral;

[0105] (3) Dissolve 0.5 mmol of Fmoc-AA, 1.0 mmol of HCTU and 2 mmol of NMM in DMF, then add the washed resin and react at room temperature for 1 h;

[0106] (4) Wash the resin with DMF 4-6 times before coupling the next amino acid;

[0107] After the linear precursor peptide chain is synthesized, the resin is washed 5 times with DMF and then 5 times with DCM. The washed resin is then dried under vacuum for later use.

[0108] Step 3: Cleavage of the linear precursor peptide chain

[0109] 10 mL of freshly prepared cutting solution (hexafluoroisopropanol:DCM (v:v:=30:70)) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 h. After the reaction was complete, the reaction solution was filtered, and the crude peptide was precipitated with 4 times its volume of frozen MTBE. The crude peptide was washed three times with MTBE and then dried under vacuum for later use.

[0110] Step 4: Remove the protective substrate

[0111] 10 mL of the prepared protecting group removal solution (trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5, v:v:v)) was added to the white solid obtained in step 3, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was completed, 4 volumes of cold MTBE were added to precipitate the solid, which had the protecting group removed. The crude product was washed three times with MTBE and dried under vacuum.

[0112] Step 5: Preparation of Peptides

[0113] The crude peptide obtained in step 4 was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were phase A (0.1% trifluoroacetic acid aqueous solution) and phase B (0.1% trifluoroacetic acid acetonitrile solution). A BR-C18 (Saifen Technology) reversed-phase column was used. During purification, the chromatograph detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was phase B changing from 20% to 50% within 0-40 min. The relevant fractions of the product were collected, and after HPLC purity identification, the fractions >95% were combined and lyophilized to obtain the pure peptide.

[0114] 1.4: Synthesis of a polypeptide compound with the amino acid sequence shown in SEQ ID NO: 21

[0115] Step 1: Couple the first amino acid Fmoc-Phe-OH

[0116] 0.2 mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. 0.16 mmol of Fmoc-Phe-OH and 0.64 mmol of DIEA were weighed and dissolved in 5 mL of DCM, and the swollen resin was added. The reaction was carried out at room temperature for 2 h. After the reaction was complete, 10 mL of blocking solution (DCM:methanol:DIEA (v:v:v = 85:10:5) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and then 5 times with DMF before use.

[0117] Step 2: Synthesis of linear precursor peptide chains

[0118] The linear precursor peptide chain of the polypeptide compound with the amino acid sequence shown in SEQ ID NO: 21 is: RRRPGLNKFG-{4-MePro}-{1-Nal}-PF.

[0119] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the linear precursor sequence from the second 1-Nal at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0120] (1) The swollen resin was deprotected twice with Fmoc using a mixture of 10 mL piperidine:DMF (v:v=20:80), each time for 8 min;

[0121] (2) Wash the resin with DMF 6-8 times until the liquid pH is neutral;

[0122] (3) Dissolve 0.5 mmol of Fmoc-AA, 1.0 mmol of HCTU and 2 mmol of NMM in DMF, then add the washed resin and react at room temperature for 1 h;

[0123] (4) Wash the resin with DMF 4-6 times before coupling the next amino acid;

[0124] After the linear precursor peptide chain is synthesized, the resin is washed 5 times with DMF and then 5 times with DCM. The washed resin is then dried under vacuum for later use.

[0125] Step 3: Cleavage of the linear precursor peptide chain

[0126] 10 mL of freshly prepared cutting solution (hexafluoroisopropanol:DCM (v:v:=30:70)) was added to the resin obtained in step 2, and the mixture was shaken and reacted at room temperature for 2 h. After the reaction was complete, the reaction solution was filtered, and the crude peptide was precipitated with 4 times its volume of frozen MTBE. The crude peptide was washed three times with MTBE and then dried under vacuum for later use.

[0127] Step 4: Remove the protective substrate

[0128] 10 mL of the prepared protecting group removal solution (trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5, v:v:v)) was added to the white solid obtained in step 3, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was completed, 4 volumes of cold MTBE were added to precipitate the solid, which had the protecting group removed. The crude product was washed three times with MTBE and dried under vacuum.

[0129] Step 5: Preparation of Peptides

[0130] The crude peptide obtained in step 4 was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were phase A (0.1% trifluoroacetic acid aqueous solution) and phase B (0.1% trifluoroacetic acid acetonitrile solution). A BR-C18 (Saifen Technology) reversed-phase column was used. During purification, the chromatograph detection wavelength was set to 230 nm, the flow rate was 15 mL / min, and the gradient was phase B changing from 20% to 50% within 0-40 min. The relevant fractions of the product were collected, and after HPLC purity identification, the fractions >95% were combined and lyophilized to obtain the pure peptide.

[0131] Test Example 1: Calcium flow EC of peptides 50 test

[0132] 24 hours before the test, CHO-K1 / AGTRL1 / Gα15 cells were digested with 0.25% trypsin (containing 0.5mM EDTA) and suspended in cell culture medium. 8000 cells were added to each well of a 384-well black transparent plate and cultured overnight at 37°C with 5% CO2 (25 μL per well). The polypeptide sample was diluted 8-12 times with loading buffer containing 0.1 wt% BSA to obtain the polypeptide sample solution. Component A from the Fluo-4 Direct™ Calcium Content Detection Kit was dissolved in 1L of calcium dye buffer (Loading buffer, 1×HBSS, 20mM HEPES, 1g glucose) to obtain the calcium staining solution. This calcium staining solution was mixed with the loading buffer at a v:v = 1:3 ratio, and a stock solution of 500mM probenecid was added to prepare a calcium dye solution containing 2.5mM probenecid.

[0133] After aspirating the cell culture supernatant from the 384-well plate, 50 μL of calcium dye solution was added to each well. The plate was incubated at 37°C for 30 min, then transferred to room temperature and incubated for another 30 min. The cell culture plate and the sample plate containing the polypeptide compound were then placed into a FLIPR Tetra instrument for detection. Apelin-13 was used as a positive control for the agonist. The detected fluorescence signal values ​​were subjected to a four-parameter nonlinear fitting to calculate the EC50. 50 The values ​​are shown in Table 2.

[0134] Table 2: Test results of some peptides

[0135]

[0136] As can be seen from Table 2, the peptides of the present invention all have good agonistic activity against APJ, with some of them approaching the level of the endogenous ligand Apelin-13.

[0137] Test Example 2: cAMP EC of peptides 50 test

[0138] 24 hours before the test, HEK-293 / APJR-EGFP (A5 2nd-A5B1) cells were digested with 0.25% trypsin (containing 0.5mM EDTA) and suspended in cell culture medium. The cells were added to 384-well plates with an opaque white bottom using a pipette at a density of 5000 cells per well. 25 μL per well was incubated overnight at 37°C with 5% CO2.

[0139] After gently patting the cell culture supernatant in the 384-well plate to dry, add 5 μL of the peptide compound test solution diluted with IBMX-containing stimulation buffer and 5 μL of 1.4 μM Forskolin solution to each well. Centrifuge and incubate at 37°C for 1 h. Then add cAMP-A2 reagent diluted with Lysis Detection Buffer and Anti-cAMP-Eu reagent diluted with Lysis Detection Buffer, and incubate at room temperature in the dark for 1 h. Read the FRET signal at 665 / 620 nm using a Cytation 5 microplate reader. Perform a four-parameter nonlinear fitting on the detected FRET signal values ​​to calculate EC. 50 The values ​​are shown in Table 3.

[0140] Table 3: Test results of some peptides

[0141]

[0142] As can be seen from Table 3, the peptides of the present invention have good Gαi pathway agonist activity, and the levels of some peptides are comparable to Apelin13 and superior to MM07.

[0143] Test Example 3: Endocytosis EC of Peptides 50 test

[0144] 16-24 hours before the experiment, HEK293 / APJ-EGFP (A5 2nd-A5B1) cells were digested, centrifuged, and the cell pellet was collected. The volume of the suspension was adjusted to achieve a suitable cell concentration using cell growth medium. Cells were seeded at 100 μL, 7000 cells / well, into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator (the 96-well plates were pre-coated with Poly-D-lysine).

[0145] Polypeptide samples were dissolved in sterile ultrapure water to a concentration of 2 mM or higher. During the experiment, FBS-free medium was used to dilute the peptide samples to the required concentration (0-30 μM). After overnight inoculation, the cell plate was removed, the old medium was aspirated from the wells, and the diluted peptide sample was added at 100 μL / well. The plate was then incubated at 37°C for 30 min in a 5% CO2 incubator. After incubation, the cells were washed three times with DPBS, then fixed with 4% paraformaldehyde fixative. Cells were washed again with DPBS, and then Hoechst 33342 (diluted to 2 μg / mL, 50 μL per well) was added for nuclei staining. Cells were washed with DPBS after each incubation. The final DPBS was retained in the cell wells. Fluorescence imaging was performed using a confocal microplate imaging system, and the endocytic fluorescence was statistically analyzed. Four-parameter nonlinear fitting was then performed to calculate the EC50. 50 The values ​​are shown in Table 4.

[0146] Table 4: Test results of some peptides

[0147]

[0148] As can be seen from the results in Table 4, the peptide of the present invention can effectively activate the Gαi signaling pathway. Its ability to induce receptor endocytosis is weaker than that of Apelin13, but stronger than that of MM07 (>10~100 times). Combined with the results in Table 3, the peptide of the present invention has the potential to become a G protein biased agonist.

[0149] Test Example 4: β-arrestin EC of peptides 50 test

[0150] 24 hours before the test, HEK293 / hAPLNR-LgBit / SmBiT-hARRB2_1st-C2 cells were digested with 0.25% trypsin (containing 0.5mM EDTA) and suspended in cell culture medium. The cells were added to white opaque bottom 96-well plates at a density of 8000 cells per well using a pipette, 100 μL per well, and cultured overnight at 37°C with 5% CO2.

[0151] After gently patting the cell culture supernatant in the 96-well plate to dry, add 100 μL of Opti-MEM medium containing 5% FBS to each well and pre-treat at 37°C for 10 min. Add the luminescent working solution (Nano-Glo substrate and dilution buffer prepared at a ratio of v:v = 1:19) and the peptide compound test solution diluted with Opti-MEM medium containing 5% FBS, mix well, and incubate at 37°C for 10 min. Measure the fluorescence value using a microplate reader. Perform a four-parameter nonlinear fitting on the detected fluorescence signal values ​​to calculate the EC50. 50 The values ​​are shown in Table 5.

[0152] Table 5: Test results of some peptides

[0153]

[0154] As can be seen from the results in Table 5, the peptide of the present invention has a weaker ability to recruit β-arrestin than Apelin13, but a stronger ability than MM07 (>50 times). Combining the results in Tables 3 and 4, the peptide of the present invention has the potential to become a G protein-biased agonist.

[0155] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A polypeptide targeting APJ or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 7, 17, 20, 21.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the polypeptide targeting APJ as described in claim 1 or a pharmaceutically acceptable salt thereof.

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