G protein biased apelin-13 polypeptide mutant and application

Abstract

The invention discloses a G protein biased apelin-13 polypeptide mutant and an application of the G protein biased apelin-13 polypeptide mutant. The invention is based on polypeptide design with a reasonable structure. Two amino acids M11 and F13 which are combined on'twin hotspots' on a locked polypeptide ligand are mutated or modified to regulate a receptor to adopt a G protein biased conformation. Functional verification is carried out on part of the designed polypeptides, the result shows that the polypeptides designed based on the structure conform to expectation and can block or retain weak beta-arrest activation capacity, and the verification result of part of the polypeptides can also be popularized to other polypeptides.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a G protein-biased apelin-13 polypeptide mutant and application thereof. Background Art

[0002] Cardiovascular disease is one of the major diseases that threaten human physical and mental health. Its prevalence rate is increasing year by year. Countries are facing major challenges in the prevention and treatment of cardiovascular diseases, which has caused a major social and economic burden. Scientists have long been committed to the discovery and development of cardiovascular disease drug targets. However, under the existing treatment strategies, the clinical control of cardiovascular diseases is still not ideal. Although some GPCR antagonists, including antagonists for β-adrenergic receptors, angiotensin II receptors and aldosterone receptors, have been widely used in the treatment of cardiovascular diseases, they often cause adverse side effects or insufficient efficacy. Therefore, there is an urgent need for the discovery of new drug targets and the development of drugs targeting new targets.

[0003] Apelin receptor (APJ) is a typical class A GPCR with important functions in the cardiovascular system, including promoting cardiac contraction, enhancing left ventricular stroke volume, vasodilation, diuresis, and lowering systemic blood pressure. In addition, the Apelin / APJ system also plays an important role in the pathophysiology of cardiovascular diseases. Animal experiments and clinical data have shown that APJ and its endogenous peptide ligand apelin can be used to intervene in a variety of cardiovascular diseases such as heart failure, pulmonary hypertension, atherosclerosis, coronary heart disease, and ischemia-reperfusion lesions. It is a very promising new therapeutic target in clinical applications to improve cardiovascular diseases. Although drug development targeting APJ has a long history, most of the initial patents focused on the development of small molecule agonists and apelin peptide analogs that improve metabolic stability for the treatment of various acute and chronic cardiovascular diseases and diabetes, and all clinical applications ended in failure. Subsequently, some new peptide analogs and small molecules were developed to reduce adverse reactions. In 2014, CMF-019 was developed as an effective APJ receptor Gαi biased agonist that can effectively promote cardiac contraction and vasodilation (patent application number: WO2014044738). MM07 is also a G protein biased agonist. It is an apelin analog formed by replacing the first and sixth amino acids of apelin-13 with cysteine. It can increase the half-life of rat plasma by 8 times, delay receptor internalization, and thus prolong cardiovascular effects (Brame, AL, et al., Design, characterization, and first-in-human study of the vascular actions of a novel biased apelin receptor agonist. Hypertension, 2015. 65(4): p.834-40.). Bpa and (L-α-Me)Phe analogs are G protein biased agonists formed by replacing the C-terminal residue of apelin-13, showing good cardiovascular protective effects. Although this biased design strategy has shown great advantages in in vivo validation, these agonists still retain strong activity in β-arrestin recruitment, which may be the main reason why they have not been clinically tested or terminated clinical trials early. Therefore, the development of fully G protein-biased agonists targeting APJ has great potential as drugs for the treatment of cardiovascular diseases.

[0004] The rapid degradation of apelin peptides in the body and their potential side effects limit their clinical application. At present, many apelin-based peptides and small molecule agonists have been developed to solve the problem of its short half-life, and some biased agonists can significantly improve the cardioprotective effect. However, the existing agonists cannot completely block the β-arrestin signaling pathway, thereby failing to avoid the side effects caused by APJ receptor activation of adverse signals, which poses a great safety hazard and limits its further evaluation in patients with heart failure. Therefore, a fully G protein-biased agonist of APJ is a necessary condition to ensure its safe use. Summary of the invention

[0005] The present invention aims to solve the problem of side effects associated with APJ agonists in the treatment of heart failure and pulmonary hypertension, develop a complete G protein-biased agonist, and avoid adverse reactions caused by the β-arrestin signaling pathway.

[0006] A G protein-biased apelin-13 polypeptide mutant is obtained by further mutation based on the G protein-biased agonist MM07, wherein the amino acid sequence of MM07 is CRPRLCHKGPMPF, wherein a disulfide bond is formed between the cysteine ​​residues between positions 1 and 6, and the mutant is any one of the following:

[0007] (1) Mutate M11 to D-amino acid based on MM07;

[0008] (2) Mutate M11 to any of the following based on MM07: A, G, Nal, V, P;

[0009] (3) Insert A or G between P10 and M11 based on MM07;

[0010] (4) Based on MM07, P3 was mutated to W, L5 was mutated to F, and M11 was mutated to Nal;

[0011] (5) Based on MM07, P3 was mutated to W, L5 was mutated to F, and G was inserted between P10 and M11.

[0012] Preferably, the G protein-biased apelin-13 polypeptide mutant is obtained by further mutation of mutant MM07, and the mutant is obtained by mutation of M11 to D-type amino acid on the basis of MM07, specifically any one of the following: D-type M, D-type Nal.

[0013] More preferably, the G protein-biased apelin-13 polypeptide mutant is obtained by further mutation of mutant MM07, wherein M11 is mutated to a D-type amino acid on the basis of MM07, and an amino acid is inserted between the amino acids at positions 11 and 12, and the inserted amino acid is specifically any one of the following: G, A.

[0014] Further preferably, the G protein-biased apelin-13 polypeptide mutant is obtained by further mutation based on the mutant MM07, and the mutant is any one of the following:

[0015] (1) WN561: Based on MM07, M11 was mutated to D-type M, and G was inserted between D-type M11 and P12. The sequence is: CRPRLCHKGPM D GPF, where M D Indicates D-type M, a disulfide bond is formed between the cysteine ​​residues at positions 1 and 6;

[0016] (2) Based on MM07, M11 was mutated to D-type M, and A was inserted between D-type M11 and P12.

[0017] Further preferably, the G protein-biased apelin-13 polypeptide mutant is obtained by further mutation based on the mutant WN561, and the mutant is any one of the following:

[0018] (1) Mutation of R2 to A, S, K, C, or T based on WN561;

[0019] (2) P3 was mutated to Y, S, T, or C based on WN561;

[0020] (3) Mutation of R4 to A or K based on WN561;

[0021] (4) L5 was mutated to A, I, or V based on WN561;

[0022] (5) Mutation of H7 to A, S, R, K, T, C, or G based on WN561;

[0023] (6) Mutation of K8 to A or R based on WN561;

[0024] (7) Mutation of G9 to S, C, or T based on WN561;

[0025] (8) Mutation of G12 to F, S, W, C, or Y based on WN561;

[0026] (9) Mutate F14 to L, I, P, V or W based on WN561.

[0027] The present invention further provides the use of the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing a disease or condition responsive to APJ receptor agonism.

[0028] Preferably, the disease or condition is selected from: acute decompensated heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, cancer, Alzheimer's disease, Parkinson's disease, stroke, neurodegenerative disease.

[0029] The present invention also provides a drug for treating or preventing diseases or conditions that respond to APJ receptor stimuli, wherein the active substance is the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof.

[0030] The present invention explains the detailed interaction mechanism between endogenous ligands and G protein-biased agonists and APJ, which is the basis for agonist design. We analyzed the structures of APJ-G protein complex activated by endogenous balanced agonist [Pyr1]-apelin-13 and reported G protein-biased agonist MM07, revealing the receptor-ligand interaction mechanism.

[0031] The present invention clarifies the APJ receptor biased signal transduction mechanism. We combined structural and functional experiments to analyze the APJ biased signal transduction mechanism, indicating that two important binding sites "twin hotspots" on the receptor are the key to affecting APJ biased signal transduction, and that the G protein-biased ligand is closer to TM6 / 7.

[0032] The present invention is based on the design of a structurally rational peptide. We target two amino acids M11 and F13 on the peptide ligand that bind to the "twin hotspots", and mutate or modify these two amino acids to regulate the receptor to adopt a G protein-biased conformation.

[0033] Functional verification of some of the designed peptides showed that the peptides we designed based on the structure were in line with expectations and could block or retain weak β-arrestin activation ability. The verification results of some peptides can also be extended to other peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 apelin-APJ-G i1 The structure of the complex and the results of the interaction analysis between the ligand and the receptor, where A: apelin-APJ-Gi1 A: Cryo-EM density map and molecular model of the complex, B: Cryo-EM density map and molecular model of the polypeptide ligands apelin and MM07, C: Dose-dependence curves of apelin and MM07 inducing Gi protein dissociation (left) and β-arrestin recruitment (right).

[0035] Figure 2 The results of the analysis of the APJ bias signal transduction mechanism include: A: density map of the interaction between apelin and the key amino acid residues affecting bias in APJ, B: density map of the interaction between MM07 and the key amino acid residues affecting bias in APJ, CF: G protein activation and β-arrestin recruitment detection of key amino acid mutations interacting with M11 and F13, G: interaction pattern of "twin hotspots" on the receptor with peptide agonists M11 and F13.

[0036] Figure 3 MM07 is a positive control and the designed peptides WN370, WN353, and WN123 induce G i Dose-dependency curves of protein dissociation and β-arrestin recruitment.

[0037] Figure 4 For structural analysis, the peptides with M11 site mutations to A, Nal, V, P, G, and P3 mutations to W and L5 mutations to F based on Nal interact with the receptor.

[0038] Figure 5 The positive control MM07 and the designed peptides WN680, WN386, and WN648 induced G i Dose-dependency curves of protein dissociation and β-arrestin recruitment.

[0039] Figure 6 For structural analysis, G and A were inserted between P10 and M11, and the peptide with mutation of P3 to W and L5 to F based on the insertion of G between P10 and M11 interacted with the receptor.

[0040] Figure 7 MM07 is a positive control and WN561 is a designed peptide that induces G i Dose-dependency curves of protein dissociation and β-arrestin recruitment.

[0041] Figure 8 For structural analysis, peptides in which M11 was mutated to DMet and G or A was inserted between DMet at position 11 and P at position 12 interacted with the receptor.

[0042] Fig. 9MM07 is a positive control, and WN561, WN562, and WN563 induce G i Dose-dependency curves of protein dissociation and β-arrestin recruitment.

[0043] Fig.10 The interaction with the receptor is determined by mutating R2 to A, S, K, C, or T based on WN561.

[0044] Fig.11 Induced G in WN561, WN564, WN565 and MM07 i Dose-dependency curves of protein dissociation and recruitment of β-arrestin.

[0045] Fig.12 The interaction between P3 and the receptor was determined by mutation of P3 to Y, S, T, or C based on WN561.

[0046] Fig.13 Induced G in WN561, WN566 and MM07 i Dose-dependency curves of protein dissociation and recruitment of β-arrestin.

[0047] Fig.14 The interaction between R4 and receptor was determined by mutation of R4 to A and K based on WN561.

[0048] Fig.15 Induced G in WN561, WN567 and MM07 i Dose-dependency curves of protein dissociation and recruitment of p-arrestin.

[0049] Fig.16 The interaction between L5 and the receptor was determined by mutation of L5 to A, I, or V based on WN561.

[0050] Fig.17 Induced G in WN561, WN571, WN572 and MM07 i Dose-dependency curves of protein dissociation and recruitment of β-arrestin.

[0051] Fig.18 The interaction between H7 and the receptor was determined by mutation of H7 to A, S, R, K, G, T, and C based on WN561.

[0052] Fig.19 Induced G in WN561, WN573, WN574 and MM07 i Dose-dependency curves of protein dissociation and recruitment of β-arrestin.

[0053] Fig. 20 The mutation of K8 to A and R on the basis of WN561 interacts with the receptor.

[0054] Fig.21 Induced G in WN561, WN575 and MM07 i Dose-dependency curves of protein dissociation and recruitment of β-arrestin.

[0055] Fig. 22 The interaction between G9 and the receptor was determined by mutation of G9 to S, C, or T based on WN561.

[0056] Fig.23 Dose-dependency curves of WN561, WN580, WN581, WN582 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0057] Fig.24 The interaction between G12 mutated to F, S, W, C, Y and the receptor based on WN561.

[0058] Fig.25 Induced G in WN561, WN584 and MM07 i Dose-dependency curves of protein dissociation and recruitment of β-arrestin.

[0059] Fig.26 The interaction between F14 and the receptor was determined by mutation of F14 to L, I, P, V, and W based on WN561. DETAILED DESCRIPTION

[0060] The amino acid sequence of the wild-type apelin-13 polypeptide is Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe (represented by single letters as QRPRLSHKGPMPF).

[0061] The amino acid sequence of MM07 is Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Met-Pro-Phe (disulfide bridge 1-6, i.e., the Cys between positions 1 and 6 forms a disulfide bond) (expressed in single letters as CRPRLCHKGPMPF (disulfide bridge 1-6)).

[0062] The remaining mutant polypeptide sequences used in this application are shown in Table 1.

[0063] Table 1

[0064]

[0065]

[0066] 1.Nanobit experiment:

[0067] Experimental principle: Binary Technology (NanoBiT) is a reporter gene technology used to detect structural complementation in protein interaction studies. The system consists of two subunits, Large BiT (LgBiT; 18kDa) and Small BiT (SmBiT; 11 amino acids), which can be fused with target proteins. The LgBiT and SmBiT subunits have been optimized to have good stability and minimized self-association. When the fusion target protein with these two subunits interacts, the two subunits will bind to each other to form an active enzyme, which will produce a bright luminescent signal. We connected the Gα protein to LgBiT and Gγ to SmBiT, and cloned the receptor genes into the pcDNA3.1 vector. The dissociation of Gα and Gγ was detected to reflect the ability of ligands to activate receptor-coupled G proteins.

[0068] Experimental method: The above plasmids were co-transfected into a 6-well plate in which HEK293T cells were cultured for expression. After 24 hours of cell transfection, the culture medium was replaced with a complete culture medium containing 10% FBS overnight. After 24h-48h of cell transfection, the transfected 6-well plate was removed, washed with PBS, and 1ml of trypsin was added for 1min, and the cells were resuspended with 3ml of DEMEM complete culture medium. The resuspended cells were inoculated into a 96-well plate for overnight culture. The next day, the 96-well plate was removed, washed with D-PBS, and the coelenterazine 400a substrate prepared in HBSS was added and incubated for 50min. The basal signal was read, and then different concentrations of ligands were added and mixed, and the signal after ligand stimulation was read. The ligand-induced signal was normalized to the baseline signal to show the dissociation reaction of the G protein.

[0069] 2.BRET experiment:

[0070] Experimental principle: BRET experiment is to study the interaction between proteins by labeling bioluminescent donors such as Renilla luciferase (Rluc) or fluorescent receptors such as enhanced green or yellow fluorescent protein (EGFP or EYFP) fusion protein. When the interacting parts are co-expressed in cells, in the presence of substrate coelenterazine, the distance between the proteins is close enough (in Energy transfer from Rluc to EYFP will occur if the donor and acceptor molecules are within a certain distance. This makes BRET an ideal tool for studying receptor-protein interactions involved in all GPCR functions and regulation by agonists and antagonists.

[0071] Experimental methods:

[0072] The BRET method was used to detect the recruitment of β-arrestin. The APJ receptor C-terminus was fused with the Rluc8 fragment, and the Venus fragment was inserted into the N-terminus of β-arrestin2 and cloned into the pBiT1.1 plasmid. The above plasmids were co-transfected into a 6-well plate in which HEK293T cells had been cultured for expression. 24 hours after cell transfection, the culture medium was replaced with a complete culture medium containing 10% FBS overnight. After 24h-48h of cell transfection, the transfected 6-well plate was removed, washed with PBS, digested with 1ml of trypsin for 1min, and resuspended with 3ml of DMEM complete culture medium. The resuspended cells were inoculated into a 96-well plate for overnight culture. The next day, the 96-well plate was removed, washed with D-PBS, and 40μl HBSS was added, and then the transfected cells were stimulated with different concentrations of ligand (5μl) for 40min. Then, the luciferase substrate luminal luciferin was added for 400 h, and the BRET signals were detected: Venus (520-560 nm), RLuc8 (460-485 nm).

[0073] 3. Protein Purification

[0074] Preparation of APJ-G i1 Combined with apelin-, MM07-complex cryo-EM samples. We combined human wild-type (WT) APJ, G il -Gβγ, scfv16 genes were cloned into pFastBac1 vector (Invitrogen) and expressed in Sf9 insect cells at a ratio of 1:1:1. i1Complex, the cell pellet was suspended in a buffer containing 20mM HEPES pH 7.5, 2mMMgCl2, 100mM NaCl and a protease inhibitor cocktail (Bimake), and then 1.2mg scFv16 antibody, 12mU / ml apyrase (Sigma) and agonists (50μM apelin, 50μM MM07) were added. The mixture was incubated at room temperature for 1 hour to form a complex. Then 0.5% (w / v) dodecyl maltose neopentyl glycol and 0.1% (w / v) hemisuccinate cholesterol were added and incubated at 4°C for 2h. After incubation with MBP (NEB), the protein was eluted with 20mM maltose. Then TEV protease was added for 1 hour to remove the MBP protein. The eluate was concentrated with a 100 kDa concentrator, and the sample was injected into a Superose 6increase 10 / 300GL column (GE Healthcare). The running buffer contained 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% dodecyl glutamic acid neopentyl glycol, 0.0002% (w / v) hemiacetyl cholesterol and 0.00025% (w / v) glycosapogenin, agonist (50 μM apelin, 50 μM MM07). The peak fractions were collected, concentrated to 10-15 mg / ml, and subjected to electron microscopy.

[0075] 4. Data collection and structural analysis

[0076] For APJ-G i1 Complex, cryo-EM data acquisition was performed on a Titan Krios at 300 kV accelerating voltage. Micrographs were recorded in super-resolution mode using a Gatan K2 detector using SerialEM software (Schorb, Haberbosch et al. 2019). The pixel size of the apelin complex is At about 8.0 electrons / angstrom 2 Image stacks were acquired at a dose rate of 1.5 Å / s with defocus values ​​ranging from -1.0 to -2.5 μm. The total exposure time was 8 s and 40 frames were recorded for each micrograph. A total of 2520 images of apelin-bound complexes were collected. A total of 4454 micrographs were collected, and the micrographs were recorded at a dose rate of 21.3 electrons / angstroms / second, with a defocus value range of -0.8 to -1.8 microns. The total exposure time was 2.35 seconds, and 36 frames were recorded for each micrograph. The collected data were processed using RELION 3.1 and CryoSPARCv3.1. The model was docked with the cryo-EM density map using UCSF Chimera (Pettersen, Goddard et al. 2004). The final model was refined using the "Comprehensive Validation (cryo-EM)" module in Phenix. The structure was drawn using UCSF Chimera and UCSF Chimera X software.

[0077] Example 1

[0078] Structural analysis of the APJ-G protein complex.

[0079] The amino acid sequence of human APJ is shown in SEQ ID No. 1 (NCBI sequence number: NP_005152.1), and the coding gene sequence is shown in SEQ ID No. 2 (NCBI sequence number: NM_005161.6). i1 (G i1 G i The amino acid sequence is shown in SEQ ID No. 3 (NCBI sequence number: NP_001027619.1), and the coding gene sequence is shown in SEQ ID No. 4 (NCBI sequence number: NM_001032447.1).

[0080] To investigate the ligand recognition mechanism of APJ, we resolved the cryo-EM structures of APJ-Gi complex activated by the balanced agonist apelin and the G protein-biased agonist MM07 (CAS No. 1876450-21-3) at resolutions of and

[0081] First, apelin-13-APJ-G was purified by baculovirus in an insect expression system and stabilized by scFv16 antibody (laboratory expression and purification, amino acid sequence shown in SEQ ID No. 5) to obtain a stable apelin-13-APJ-G i1 (apelin-13 and APJ, G i1 complex) and MM07-APJ-G i1 (MM07 and APJ, G i1The ternary complex was then analyzed by cryo-electron microscopy at 300KV. RELION software was used for data processing and three-dimensional reconstruction, and WINCOOT and PHENIX software were used for modeling and structural refinement to obtain a high-resolution ternary complex structure.

[0082] The experimental results are as follows Figure 1 The experimental results show that we have resolved the high-resolution apelin-APJ--G i1 Cryo-EM structure of the complex ( Figure 1 A and B), we can analyze the interaction between ligand and receptor very well. We overexpressed APJ receptor in HEK293 cells, and used nanobit assay to detect the ability of apelin and MM07 to activate APJ to recruit G protein, and bret assay to detect the ability of ligand to activate arrestin. The experimental results are shown in Figure 1 As shown in C, the EC50 of apelin activating G protein signaling is 9.793×10 -10 M, the EC50 of recruiting β-arrestin is 8.688×10 -9 The EC50 of MM07 activating G protein signaling was 1.377×10 -8 M, the EC50 of recruiting β-arrestin is 5.495×10 -6 Compared with apelin, MM07's ability to activate arrestin was nearly 1000-fold weaker, while its G protein activity was about 10-fold weaker than that of apelin. These results suggest that MM07 is a G protein-biased agonist but still retains considerable arrestin activity.

[0083] Example 2

[0084] Analysis of APJ-biased signal transduction mechanisms.

[0085] By comparing the differences in the two ligand-activated APJ binding pockets, the specific amino acid sites where different agonists bind to APJ were identified, and the recognition and activation mechanisms of the endogenous peptide apelin and the G protein-biased agonist MM07 were explained. The key amino acid sites involved in ligand recognition in APJ were mutated, and the ligand-activated downstream G protein and β-arrestin activities were detected by nanobit and BRET experiments, respectively, to determine the effects of point mutations on the ability of APJ to bind to downstream G proteins and β-arrestin under the action of different ligands, and to explain the biased signal transduction mechanism.

[0086] The results are as follows Figure 2As shown. The residues of the ligand binding pocket of the APJ structure bound to apelin (A) and MM07 (B) are displayed, and the density of the residues M11 and F13 of the peptide ligand and the receptor that interact with these two amino acids are displayed in a surface map. The functional experimental results show that the mutation of key amino acids located in the "twin hotspots" will affect the activity of G protein or β-arrestin ( Figure 2 The amino acid mutations shown in C and E in the figure show that the activity of β-arrestin is reduced more than that of G protein, and the receptor prefers to bind to G protein after mutation; Figure 2 The amino acid mutations shown in D and F in the figure show that the G protein activity is reduced more than that of β-arrestin, and the receptor prefers to bind to β-arrestin after mutation, indicating that the two pockets "twin hotspots" on the receptor are the key to affecting the selectivity of APJ to downstream signaling molecules. M11 and F13 of apelin and MM07 are inserted into the two pockets, respectively, and interact with the key amino acid residues of the "twin hotspots". Therefore, changing the 11-position Met and 13-position Phe that interact with the receptor is helpful to design G protein-biased peptide agonists.

[0087] Example 3

[0088] (1) Based on the above structural analysis, we conducted mutation studies on MM07, mutated M11 to A (WN123), Nal (WN353), and DNal (WN370) to synthesize three peptides, and conducted functional experiments. The results are as follows: Figure 3 As shown, functional experiments showed that the three polypeptides of WN123, WN353 and WN370 almost lost the ability to recruit β-arrestin, but retained the ability to activate G protein.

[0089] We performed structural analysis of the corresponding amino acid mutations in WinCoot, e.g. Figure 4As shown in the figure, it was found that mutation to a small residue such as A greatly weakened the interaction with the first hotspot, while mutation to F (large residue) did not affect its insertion into the hotspot pocket. Other small residue mutations were simulated in WinCoot, and it was found that mutations to V (WN148), P (WN149), and G (WN150) were similar to the M11A mutation, all of which were non-polar amino acids, and all weakened the interaction with the first hotspot, thus also leading to the loss of the receptor's β-arrestin activity. We solved the structure of M11 after it mutated to Nal (WN353) and found that Nal was completely deflected in the opposite direction of M11, and the interaction with Y35 and T295 in the first hotspot was enhanced, while the interaction with I109, F110, and Y264 was weakened, which was consistent with the characteristics of G protein bias. Although the structure of M11 mutated to DNal (WN370) has not been solved, our functional experimental data show that mutation to DNal will also lead to the loss of the receptor's ability to recruit β-arrestin without affecting G protein activity. Based on Nal11, P3 was mutated to W and L5 to F (WN386). Functional experiments showed that it was also a complete G protein-biased agonist.

[0090] (2) Figure 5 As shown, we also designed a peptide (WN680) with Gly inserted between P10 and M11 and found that the first hotspot could not be inserted into G11, thus greatly weakening the interaction with the key residues and weakening the activity of β-arrestin, which was consistent with the functional experiments.

[0091] like Figure 6 As shown, structural analysis showed that inserting a small residue A (WN682) between P10 and M11 would also lead to a weakened interaction with the first hotspot, thus achieving the same effect. Based on WN386, the third and fifth amino acid residues were mutated to W and F to form WN648. Functional experimental results showed that WN648 could not recruit β-arrestin either, but retained G protein activity.

[0092] (3) In addition, when M11 was mutated to DMet and G was inserted between DMet at position 11 and P at position 12 (WN561), functional data showed that β-arrestin activity was completely lost. Figure 7 As shown. Structural data analysis showed that it mainly affected the interaction between the ligand and the two hotspots. This indicates that weakening the interaction between the 13th amino acid and the second hotspot will weaken the β-arrestin activity. Therefore, inserting A into DMet and 12 P can also have the same effect (DMet CA) ( Figure 8 ).

[0093] Example 4

[0094] (1) Based on WN561, we mutated R2: WN562 (R2A), WN563 (R2S). The functional experimental results are as follows Fig. 9 As shown in Figure 3, β-arrestin activity is lost, while G protein activity is retained, indicating that the mutation of R2 to small residues A and S is also consistent.

[0095] According to the structural analysis, K and R are polar positively charged amino acids, which can form electrostatic interactions with the surrounding E; C, T and S are polar uncharged amino acids, which can form polar interactions with the surrounding E. Structural analysis shows that mutations R2K (WN586), R2C (WN587), and R2T (WN588) can also lead to loss of β-arrestin activity. Structural data such as Fig.10 shown.

[0096] (2) Based on WN561, mutations were performed on P3: WN564 (P3Y), WN565 (P3S). The functional experimental results are shown in Fig.11 As shown in the figure, β-arrestin activity is lost, while G protein activity is retained, indicating that P3 mutation to Y and S is also consistent.

[0097] According to the structural analysis, P3Y can form polar interactions with the surrounding Y, P3S can form polar interactions with the surrounding E, and C, T and S are polar uncharged amino acids, and can also form polar interactions with the surrounding E. Therefore, the structural analysis shows that the mutations P3T (WN589) and P3C (WN590) will also lead to the loss of β-arrestin activity. Structural data such as Fig.12 shown.

[0098] (3) Based on WN561, a mutation was made on R4: WN566 (R4A). The functional experimental results are as follows Fig.13 As shown in Figure 3, β-arrestin activity is lost, while G protein activity is retained, indicating that the mutation of R4 to A is also consistent.

[0099] According to structural analysis, K and R are both polar positively charged amino acids, which can form polar interactions with the surrounding Y. Therefore, structural analysis shows that R4K (WN591) mutation will also lead to loss of β-arrestin activity. Structural data such as Fig.14 shown.

[0100] (4) Based on WN561, a mutation was made on L5: WN567 (L5A). ​​The functional experimental results are as follows Fig.15 As shown in Figure 2, β-arrestin activity is lost, while G protein activity is retained, indicating that the mutation of L5 to A is also consistent.

[0101] According to structural analysis, V and I are non-polar amino acids like L, and can form non-polar interactions with the surrounding F and Y. Therefore, structural analysis shows that mutations L5I (WN592) and L5V (WN593) will also lead to loss of β-arrestin activity. Structural data such as Fig.16 shown.

[0102] (5) Based on WN561, mutations were performed on H7: WN571 (H7A), WN572 (H7S). The functional experimental results are as follows Fig.17 As shown in the figure, β-arrestin activity is lost, while G protein activity is retained, indicating that H7 mutation to A and S is also consistent.

[0103] According to the structural analysis, R, K, like H, are polar positively charged amino acids, which can form polar interactions with the surrounding N and Y. Therefore, the structural analysis shows that the mutations H7R (WN594) and H7K (WN595) will also lead to the loss of β-arrestin activity. The mutation of H7A to a small side chain residue is also consistent, so the structural analysis shows that H7G (WN606) will also lead to the loss of β-arrestin activity. C, T, like S, are polar uncharged amino acids, which can form polar interactions with the surrounding Y. Therefore, the structural analysis shows that the mutations H7T (WN596) and H7C (WN597) will also lead to the loss of β-arrestin activity. Structural data such as Fig.18 shown.

[0104] (6) Based on WN561, mutations were performed on K8: WN573 (K8A), WN574 (K8R). The functional experimental results are shown in Fig.19 As shown in the figure, β-arrestin activity is lost, while G protein activity is retained, indicating that the mutation of K8 to A and R is also consistent.

[0105] According to the structural analysis, the mutation of H7A to a small side chain residue is also consistent. R, like K, is a polar positively charged amino acid that can form electrostatic interactions with the surrounding E and polar interactions with the surrounding Y. Therefore, the structural analysis shows that the K8R mutation will also lead to the loss of β-arrestin activity. Structural data such as Fig. 20 shown.

[0106] (7) Based on WN561, a mutation was made on G9: WN575 (G9S). The functional experimental results are as follows Fig.21 As shown in Figure 3, β-arrestin activity is lost, while G protein activity is retained, indicating that the mutation of G9 to S is also consistent.

[0107] According to structural analysis, C and T are polar uncharged amino acids like S, and can form polar interactions with the surrounding Y, S, and T. Therefore, structural analysis shows that G9C (WN598) and G9T (WN599) mutations will also lead to loss of β-arrestin activity. Structural data such as Fig. 22 shown.

[0108] (8) Based on WN561, mutations were performed on G12: WN580 (G12F), WN581 (G12S), and WN582 (G12W). The functional experimental results are shown in Fig.23 As shown in the figure, β-arrestin activity is lost, while G protein activity is retained, indicating that the mutation of G12 to S and the F and W of the large side chain are also consistent.

[0109] According to structural analysis, mutations to large side chain amino acids such as F and W will enhance the non-polar interaction with M183 on the receptor, making the receptor more biased towards the G protein. Y is also a large side chain amino acid like F and W, so structural analysis shows that the G12Y (WN601) mutation will also lead to the loss of β-arrestin activity. C, like S, is a polar uncharged amino acid that can form polar interactions with the surrounding S, so structural analysis shows that the G12C (WN600) mutation will also lead to the loss of β-arrestin activity. Structural data such as Fig.24 shown.

[0110] (9) Based on WN561, a mutation was performed on F14 (because WN561 has an insertion mutation, the original F13 position became F14 position): WN584 (F14L). The functional experimental results are as follows Fig.25 The β-arrestin activity was lost, while the G protein activity was retained, indicating that the mutation of F14 to the non-polar amino acid L was also consistent.

[0111] According to the structural analysis, the 14th amino acid is close to the phospholipid bilayer. I, P, V, W are non-polar amino acids like F and L, which can increase the stability of the receptor and form non-polar interactions with the surrounding V. Therefore, the structural analysis shows that F14I (WN602), F14P (WN603), F14V (WN604), and F14W (WN605) mutations will also lead to the loss of β-arrestin activity. Structural data such as Fig.26 shown.

Claims

1. A G protein-biased apelin-13 polypeptide mutant, It is characterized in that The amino acid sequence of MM07 is CRPRLCHKGPMPF, which is a G protein-biased agonist. The cysteine ​​residues at positions 1 and 6 form a disulfide bond. The mutant is any of the following: (1) Mutate M11 to D-amino acid based on MM07; (2) Mutate M11 to any of the following based on MM07: A, G, Nal, V, P; (3) Insert A or G between P10 and M11 based on MM07; (4) Based on MM07, P3 was mutated to W, L5 was mutated to F, and M11 was mutated to Nal; (5) Based on MM07, P3 was mutated to W, L5 was mutated to F, and G was inserted between P10 and M11.

2. The G protein-biased apelin-13 polypeptide mutant according to claim 1, It is characterized in that The mutant is obtained by further mutation on the basis of mutant MM07, when M11 is mutated to D-type amino acid on the basis of MM07, specifically any one of the following: D-type M, D-type Nal.

3. The G protein-biased apelin-13 polypeptide mutant according to claim 2, It is characterized in that The mutant is obtained by further mutation based on mutant MM07, in which M11 is mutated to D-type amino acid based on MM07, and an amino acid is inserted between amino acids at positions 11 and 12, and the inserted amino acid is specifically any one of the following: G, A.

4. The G protein-biased apelin-13 polypeptide mutant according to claim 3, It is characterized in that Obtained by further mutation based on mutant MM07, the mutant is any of the following: (1) WN561: Based on MM07, M11 was mutated to D-type M, and G was inserted between D-type M11 and P12. The sequence is: CRPRLCHKGPM D GPF, where M D Indicates D-type M, a disulfide bond is formed between the cysteine ​​residues at positions 1 and 6; (2) Based on MM07, M11 was mutated to D-type M, and A was inserted between D-type M11 and P12.

5. The G protein-biased apelin-13 polypeptide mutant according to claim 4, It is characterized in that The mutant WN561 is further mutated and is any of the following: (1) Mutation of R2 to A, S, K, C, or T based on WN561; (2) P3 was mutated to Y, S, T, or C based on WN561; (3) Mutation of R4 to A or K based on WN561; (4) L5 was mutated to A, I, or V based on WN561; (5) Mutation of H7 to A, S, R, K, T, C, or G based on WN561; (6) Mutation of K8 to A or R based on WN561; (7) Mutation of G9 to S, C, or T based on WN561; (8) Mutation of G12 to F, S, W, C, or Y based on WN561; (9) Mutate F14 to L, I, P, V or W based on WN561.

6. Use of the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of a medicament for treating or preventing a disease or condition responsive to APJ receptor agonism.

7. The use according to claim 6, It is characterized in that The disease or condition is selected from the group consisting of acute decompensated heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, cancer, Alzheimer's disease, Parkinson's disease, stroke, and neurodegenerative diseases.

8. A medicament for treating or preventing a disease or condition responsive to agonism of an APJ receptor, It is characterized in that The active substance is the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5.

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