Biased polypeptide ligands for protease-activated receptor 2 and uses thereof

Abstract

The application discloses a biased polypeptide ligand of protease-activated receptor 2 and application thereof, and belongs to the technical field of proteins and polypeptides. The application aims to provide a biased polypeptide ligand of protease-activated receptor 2, which only activates the Gq signal pathway of PAR2. The application provides a biased polypeptide ligand of protease-activated receptor 2, which is obtained by any one or both of mutation and modification of amino acids of SLIGRL-NH2 as a starting sequence. The biased PAR2 polypeptide ligand designed in the application has unique advantages in treating diseases caused by nerve injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein polypeptide technology, and particularly relates to a biased polypeptide ligand of protease-activated receptor 2 and application thereof. BACKGROUND

[0002] Protease-activated receptor 2 (PAR2) is a member of the protease-activated receptors (PARs) family, which belongs to the G protein-coupled receptor (GPCR) family. PAR2 has a wide range of biological effects and is involved in the occurrence and formation of various diseases, such as pain, arthritis, asthma, inflammation, itching and neurological diseases. PAR2 is distributed in many tissues and cells, including the central nervous system, skin, respiratory tract, bone joints and pancreas. PAR2 is an important target for the treatment of many diseases, including arthritis, pain, neurological diseases and cancer. PAR2 is cleaved at the N-terminal of its receptor by interstitial proteases such as trypsin, producing a specific peptide segment (tethered peptide segment). This peptide segment is then inserted into the receptor ligand binding pocket to activate the receptor and trigger downstream signaling pathways, including Gq, G13, Gs and β-Arrestin pathways. In addition to proteases, synthetic peptides and small molecules can also bind and activate PAR2. Generally speaking, these polypeptides or small molecules can simultaneously activate Gq, G13, Gs and β-Arrestin signaling pathways, and these ligands are called balanced ligands.

[0003] In animal models, activation of PAR2, particularly in brain astrocytes, has a protective effect on the central nervous system of animals. When PAR2 is activated in these cells, it induces the secretion of glutamate, which plays an important role in excitatory transmission, synaptic plasticity, neural development and cognitive function, and has a protective effect on nerves. Glutamate secretion caused by PAR2 activation is achieved through the activation of ERK and p38 MAPK signals of its Gq pathway. The most commonly used PAR2 tethered polypeptides currently include SLIGRL-NH2 (mouse origin) and SLIGKV-NH2 (human origin), which are balanced ligands of PAR2 and can simultaneously activate Gq, G13, Gs and other signaling pathways. Therefore, when used in the treatment of brain neuron damage, they can have strong side effects, including inducing strong central nervous system inflammatory reactions.

[0004] Ligands that only activate a single signaling pathway are called biased ligands, and biased ligands are the hot spot and trend of modern G protein-coupled receptor drug design and development, because only activating a single targeted pathway and avoiding other pathways can maximize the efficiency of reducing side effects caused by activating other pathways. Therefore, when treating diseases related to G protein-coupled receptors such as PAR2, biased ligands are preferred, and therefore it is urgent to find available biased polypeptide ligands. SUMMARY

[0005] The purpose of the present application is to provide a biased polypeptide ligand of protease-activated receptor 2 that only activates the Gq signaling pathway of PAR2.

[0006] The present application provides a biased polypeptide ligand of protease-activated receptor 2, which is obtained by any one or both of mutation and modification of amino acids from SLIGRL-NH2.

[0007] Further limited as follows:

[0008] 1) SLVGRL-NH2;

[0009] 2) SLVGRL-OH;

[0010] 3) SLVGKV-NH2;

[0011] 4) 2-furoyl-LVGRL;

[0012] 5) 2-furoyl-LVGRL-NH2;

[0013] 6) 2-furoyl-LVAAAI-NH2;

[0014] 7) 5-isoxazolyl-LVGRL;

[0015] 8) 5-isoxazolyl-LVGRL-NH2;

[0016] 9) 5-isoxazolyl-Cha-VGRL-NH2;

[0017] 10) 5-isoxazolyl-Cha-V-NH2;

[0018] 11) 5-isoxazolyl-Cha-VAR-NH2;

[0019] 12) S-pFPhe-VGRL;

[0020] 13) S-pFPhe-VGRL-NH2;

[0021] 14) S-pFPhe-Cha-VRK;

[0022] 15) S-pFPhe-Cha-VRK-NH2;

[0023] 16) SLVGRL;

[0024] 17) SLVGKV;

[0025] 18) 2-furoyl-LVGRLO-amide;

[0026] 19) 2-furoyl-LVGKV-OH.

[0027] The present application provides a nucleic acid molecule of the above-mentioned biased polypeptide ligand of protease-activated receptor 2.

[0028] The present application provides a recombinant vector containing the above-mentioned nucleic acid molecule.

[0029] Further limited, the starting vector of the recombinant vector is any one of pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series.

[0030] The present application provides a recombinant microbial cell carrying the above-mentioned biased polypeptide ligand of protease-activated receptor 2 or expressing the above-mentioned nucleic acid molecule.

[0031] Further limited, the recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0032] The present application provides the use of the above-mentioned biased polypeptide ligand of protease-activated receptor 2 in the preparation of a drug for treating inflammation or neuropathic pain.

[0033] Beneficial effects: SLIGRL-NH2 is modified to change some amino acid residues, so as to selectively activate one of the β-arrestin signaling pathway or the G protein-mediated signaling pathway, rather than simultaneously activating multiple pathways like traditional activation. These biased polypeptide ligands help researchers to better understand the functions of different signaling pathways of PAR2, and provide more accurate tools for the development of drugs targeting PAR2. The study of biased polypeptide ligands of PAR2 is an active field, and has important significance for understanding the function of PAR2 and developing related therapeutic drugs.

[0034] The application designs a biased PAR2 peptide segment, which only activates the Gq signal pathway of PAR2 and has little activity on other pathways, including G13, and thus the biased PAR2 ligand has a unique advantage in treating diseases caused by nerve injury. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Figure 1 is a whole structure result diagram of PAR2 / G protein complex; A is a whole structure result diagram of PAR2 / Gq complex, and B is a whole structure result diagram of PAR2 / G13 complex;

[0036] Figure 2 Figure 2 is a whole structure result diagram of the binding of the tethered peptide ligand TL to PAR2;

[0037] Figure 3 Figure 3 is a biased result diagram of PAR2 binding to TL; a is a comparison result diagram of TL / receptor binding between PAR2 / Gq and PAR2 / G13 complexes; and b is a result diagram of G protein coupling activity of the D62A mutant on the receptor side detected by different function experiments;

[0038] Figure 4 Figure 4 is a result diagram of G protein coupling activity of the I39 mutant of the third amino acid of the TL part of the receptor; A is a signal response result diagram of the I39(V) mutant, and B is a signal response result diagram of the I39(L,D,A,F) mutant;

[0039] Figure 5 Figure 5 is a result diagram of molecular dynamics simulation analysis of PAR2; a is a result diagram of the interaction between the N-terminal region of TL and WT by molecular dynamics simulation analysis, b is a result diagram of the interaction between the N-terminal region of TL and D62A by molecular dynamics simulation analysis, and c is a result diagram of the interaction between the N-terminal region of TL and I39V by molecular dynamics simulation analysis;

[0040] Figure 6 Figure 6 is a result diagram of G protein coupling activity of the synthesized PAR2 AP detected by different detection methods. DETAILED DESCRIPTION

[0041] furoyl: furan carboxyl; isoxazolyl: isoxazolyl; pFPhe = L-(4-fuorophenyl)alanine, L-(4-furan phenyl)alanine; Cha, L-cyclohexylalanine, propionyl alanine cyclohexyl ester; -OH, tail end is hydroxyl; -NH2, tail end is amino.

[0042] Original polypeptide sequence (PAR2): SLIGRL-NH2, also called PAR2 balanced ligand.

[0043] Example 1. Construction of biased polypeptide ligands for protease-activated receptor 2

[0044] I. 1) SLVGRL-NH2;

[0045] 2) SLVGRL-OH;

[0046] 3) SLVGKV-NH2;

[0047] 4) 2-furoyl-LVGRL;

[0048] 5) 2-furoyl-LVGRL-NH2;

[0049] 6) 2-furoyl-LVAAAI-NH2;

[0050] 7) 5-isoxazolyl-LVGRL;

[0051] 8) 5-isoxazolyl-LVGRL-NH2;

[0052] 9) 5-isoxazolyl-Cha-VGRL-NH2;

[0053] 10) 5-isoxazolyl-Cha-V-NH2;

[0054] 11) 5-isoxazolyl-Cha-VAR-NH2;

[0055] 12) S-pFPhe-VGRL;

[0056] 13) S-pFPhe-VGRL-NH2;

[0057] 14) S-pFPhe-Cha-VRK;

[0058] 15) S-pFPhe-Cha-VRK-NH2;

[0059] 16) SLVGRL;

[0060] 17) SLVGKV;

[0061] 18) 2-furoyl-LVGRLO-amide;

[0062] 19) 2-furoyl-LVGKV-OH;

[0063] The above compounds can also be synthesized directly by the company.

[0064] II. Basic steps for polypeptide synthesis:

[0065] Method: Solid phase FMOC synthesis, hand operation on solid phase synthesis tube, using nitrogen blowing agitation reaction.

[0066] Resin selection: C-terminal with the requirement of amide closed modification, can choose Rink amide-AM Resin or Rinkamide-MBHA Resin;

[0067] Required raw materials and reagents: amino acids: Fmoc-Leu-OH, FMOC-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, FMOC-Ser(tbu)-OH;

[0068] Condensing agent: DIEA HBTU HOBT;

[0069] Solvent: DMF, DCM, MeOH;

[0070] Detection of (-NH2) coupling reaction process completeness: 5% indantrione solution, 30% phenol ethanol solution, pyridine.

[0071] 1. Resin pretreatment

[0072] Weigh the appropriate amount (1.5-2 times the theoretical amount) of Rink amide-MBHA Resin, add DCM to swell for 30 minutes. Wash with DMF, discard the solvent, and add the first amino acid.

[0073] 2. Coupling of the first amino acid

[0074] ① Add Fmoc-Leu-OH (2-3 times the theoretical amount) in turn, DMF, DIEA (DIEA usage 0.5-1 ml / g resin), react for 30 min-1 h, add MeOH to close the unreacted site.

[0075] ② Remove Fmoc of Fmoc-Leu-OH, add piperidine (20% hexahydro-piperidine in DMF) to immerse the resin, twice, 7-10 min each time. Wash with DMF, MeOH alternately, and detect the removal of Fmoc. Blue color

[0076] 3. Coupling of the next amino acid

[0077] ① Add FMOC-Arg(pbf)-OH, HBTU, DMF, DIEA in turn, react for 15-20 min.

[0078] ② Monitor the reaction degree

[0079] Wash with DMF, MeOH for 3-5 times, take 20 or so resin detection, add 30% phenol ethanol solution 2d, 5% ninhydrin solution 2d, 1d pure pyridine solution, placed in 100 ℃ or so heating 3-5 minutes, observe the resin color.

[0080] If colorless, the coupling is complete. Then proceed to deprotection (FMOC): add piperidine (20% hexahydro-piperidine in DMF) over the resin, twice, 7-10 min each. Wash with DMF, MeOH for 6-10 times alternately, check the de-Fmoc condition, which should be blue. Continue the coupling of the next amino acid, cycle until all the sequence coupling is completed.

[0081] The experimental effect is verified by the following experiments:

[0082] Firstly, the activated state PAR2 and Gq complex (PAR2 / Gq) and the complex of PAR2 and G13 (PAR2 / G13) structure were analyzed by cryo-EM, with resolutions of and , Figure 1 .

[0083] These structures show that the tethered ligand (TL) of the PAR2 receptor is almost vertically inserted into the ligand binding pocket of the receptor, and forms a beta sheet with the β3 strand of the extracellular loop 2 (ECL2) above the orthosteric pocket to activate the receptor. In addition, the inverse parallel beta sheet formed by the β1 and β2 strands of the ECL2 of the receptor also helps the correct folding of the entire ECL2 to maintain the correct insertion of the TL Figure 2 .

[0084] By comparing the binding of the tethered peptide ligand TL in the PAR2 / Gq complex and the PAR2 / G13 complex Figure 3 a), it is found that there are slight differences in the orientation of TL in the PAR2 / Gq complex and the PAR2 / G13 complex: 1) the carboxyl group of the 62th aspartic acid (D62) of the transmembrane helix part of the receptor is oriented towards TL in the PAR2 / G13 complex, but turns in the PAR2 / Gq complex; 2) the side chain of the third amino acid isoleucine (I39) of the TL part of the receptor is oriented towards D62 in the PAR2 / G13 complex, but changes its orientation in the PAR2 / Gq complex. Combined with site-directed mutagenesis experiments and multiple functional experimental data, it is found that the mutation of the receptor side aspartic acid D62 to alanine A can selectively activate the activity of the Gq pathway, while almost completely eliminating the activity of the G13 pathway Figure 3b). And in the receptor TL part, the third amino acid I39 which can interact with D62 was mutated and the intrinsic activity of PAR2 (37-397) was detected by functional experiment, it was found that the Gq and G13 activity of I39V mutant had obvious difference, showing strong Gq pathway selectivity, while other mutants did not show obvious signal bias( Figure 4 ).

[0085] With the aid of molecular dynamics simulation, it was found that there was a persistent hydrogen bond interaction between D62 and I39, neither D62A nor I39V mutant could form hydrogen bond interaction between them( Figure 5 ), indicating that the interaction mediated by D62 / I39 may play a very important role in stabilizing the extracellular conformation of transmembrane helix 1 (TM1) of the receptor.

[0086] Based on the above cell function experiment data, it is inferred that the hydrogen bond interaction between D62 and I39 is very important for G13 signal pathway, while Gq signal pathway is not sensitive to this interaction.

[0087] According to the findings of the above structural study, a third residue with valine mutation polypeptide (PAR2 AP (SLVGRL-NH2)) was designed and synthesized, and its pathway selectivity was detected by different cell function experiments, the results showed that the synthetic polypeptide (SLVGRL-NH2) retained most of the Gq activity of wild type TL, but lost most of the G13 activity, showing strong Gq activity bias( Figure 6 ).

[0088] Other biased polypeptide ligands of protease-activated receptor 2 obtained also underwent the above structural study, all of which retained most of the Gq activity of wild type TL, but lost most of the G13 activity, showing strong Gq activity bias.

[0089] Example 2. Application of biased polypeptide ligand of protease-activated receptor 2

[0090] (1) Protease-activated receptor 2 has a protective effect on neurons in the central nervous system, promotes the secretion of glutamate in astrocytes in the brain, and plays an important role in neural development and cognitive function, so the biased polypeptide ligand of protease-activated receptor 2 can be used for the recovery of diseases caused by nerve damage, including epilepsy, concussion, etc.

[0091] (2) Protease-activated receptor 2 plays an important role in inflammation, especially in the occurrence, development and deterioration of arthritis, so the biased polypeptide ligand of protease-activated receptor 2 has potential value in the treatment of inflammation including arthritis.

[0092] (3) Protease-activated receptor 2 plays an important role in the generation of pain, especially long-term neuropathic pain, so biased polypeptide ligands of protease-activated receptor 2 have potential applications in the treatment of long-term neuropathic pain.

[0093] (4) Protease-activated receptor 2 also plays an important role in the occurrence and migration of cancer, especially in the process of tumor angiogenesis, so biased polypeptide ligands of protease-activated receptor 2 have potential value in the treatment of tumors.

Claims

1. A biased polypeptide ligand for protease-activated receptor 2, characterized in that, The biased polypeptide ligand for the protease-activated receptor 2 is SLVGRL-NH2.

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