CN112791178A - GLP-1R/GCGR double-agonist polypeptide derivative for preventing or treating renal fibrosis - Google Patents

GLP-1R/GCGR double-agonist polypeptide derivative for preventing or treating renal fibrosis Download PDF

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CN112791178A
CN112791178A CN202110094194.6A CN202110094194A CN112791178A CN 112791178 A CN112791178 A CN 112791178A CN 202110094194 A CN202110094194 A CN 202110094194A CN 112791178 A CN112791178 A CN 112791178A
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gly
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lys
asp
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刘琦
张斌智
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Shenzhen Turier Biotech Co ltd
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Shenzhen Turier Biotech Co ltd
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Abstract

The invention discloses application of a GLP-1R/GCGR double-agonist polypeptide derivative in preventing or treating renal fibrosis. The GLP-1R/GCGR double agonist polypeptide can inhibit epithelial-mesenchymal transition of renal tubular cells in vivo and in vitro, can improve mitochondrial morphology, and can inhibit PERK-mediated endoplasmic reticulum stress pathway in the TGF-beta induced EMT process, thereby treating or improving kidney fibrosis and inflammation caused by kidney fibrosis.

Description

GLP-1R/GCGR double-agonist polypeptide derivative for preventing or treating renal fibrosis
Technical Field
The invention relates to a GLP-1R/GCGR double-agonist polypeptide derivative, in particular to application of the GLP-1R/GCGR double-agonist polypeptide derivative in preventing or treating renal fibrosis, and especially relates to application of the GLP-1R/GCGR double-agonist polypeptide derivative in improving renal fibrosis by regulating PERK-mediated endoplasmic reticulum stress pathway.
Background
Chronic renal Disease (CKD) remains a major Burden on the healthcare system, with a Global prevalence of 9.1% and is associated with increased morbidity and mortality from cardiovascular Disease (collagen GBDCKD. Global, regional, and national Burden of cardiovascular Disease,1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.Lancet. 2020; 395(10225):709-33.Said S, Hernandez GT. the link between cardiovascular Disease and cardiovascular Disease. J Nephropathol. 2014; 3(3): 99-104). CKD may develop into end-stage renal disease (ESRD) requiring dialysis or renal replacement therapy. 120 million people are reported to die globally in 2017 from CKD (Collaboration GBDCKD. Global, regional, and national Burden of respiratory Disease, 1990-) 2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet.2020; 395(10225): 709-33.). Many factors, such as diabetes, hypertension, obesity, infectious infections and autoimmune diseases, contribute to the development of CKD (Hall ME, do Carmo JM, da Silva AA, Juncos LA, Wang Z, Hall je. obesite, hypertension, and respiratory kidney disease. int J Nephrol Renovasc disp. 2014; 7: 75-88). Regardless of the initial etiology, renal fibrosis is the ultimate histological hallmark of CKD, characterized by excessive pathological extracellular matrix protein production and deposition in the interstitium, leading to structural damage, impaired renal function, and ultimately ESRD (Zeisberg M, Neilson eg. mechanisms of tuberculosis. j Am Soc nephrol. 2010; 21(11): 1819-34). Despite advances in the study of CKD, few FDA-approved drugs are available for the treatment of renal fibrosis, and challenges remain in the study of renal fibrosis therapies.
Similar to the wound healing response, renal fibrosis is also a dynamic and complex process. Key events of renal interstitial fibrosis include peritubular inflammatory cell infiltration, which may secrete a variety of pro-fibrotic stimulators including TGF- β and IL-4, tubular apoptosis, epithelial-to-mesenchymal transition (EMT), and activation and expansion of myofibroblasts (Liu y. new insights into epithelial-mesenchymal transition in kidney fibrosis. j Am Soc nephrol.2010; 21(2): 212-22.).
The Endoplasmic Reticulum (ER) is critical for many cellular functions, including protein synthesis, folding, modification and trafficking. Disruption of these functions by extracellular or intercellular stimuli can affect proper folding of the protein and lead to endoplasmic reticulum stress. ER is controlled by three ER stress sensors, including PERK, IRE1 α and ATF-6. The PERK pathway is activated by autophosphorylation, then phosphorylates eIF2 α and promotes expression of pro-apoptotic proteins including C/EBP homologous protein (CHOP). At present, it is unclear whether the endoplasmic reticulum stress pathway plays a role in the renal protective role of GLP-1R/GCGR agonists.
The medicines for treating renal fibrosis in the prior art have little effect, and actually, a new medicine for effectively treating renal fibrosis is needed to be provided.
Disclosure of Invention
In order to solve the problems, the invention provides novel biological activity of a GLP-1R/GCGR dual-agonist polypeptide derivative and application thereof in treating or preventing renal fibrosis, wherein the GLP-1R/GCGR dual-agonist polypeptide derivative improves the renal fibrosis by regulating PERK-mediated endoplasmic reticulum stress pathway.
A large number of experimental studies prove that the GLP-1R/GCGR double-agonist polypeptide derivative can obviously improve Unilateral Ureteral Obstruction (UUO) -induced renal fibrosis. Proves that the GLP-1R/GCGR double-target agonist polypeptide derivative has obvious therapeutic effect on renal fibrosis.
The invention aims to provide a new application of the GLP-1R/GCGR dual agonist polypeptide derivative in treating indications. The GLP-1R/GCGR double-agonist polypeptide derivative is expected to be used as a new generation of medicaments for preventing or treating renal fibrosis.
The GLP-1R/GCGR double agonist polypeptide derivative has a parent peptide represented by the following amino acid sequence:
His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-COR1
wherein R is1＝-NH2；
Xaa2 ═ Aib or D-Ser;
xaa10 ═ Lys or Tyr;
xaa13 ═ Lys or Tyr;
xaa16 ═ Ser, Aib, Lys, or Glu;
xaa17 ═ Lys or Arg;
xaa18 ═ Arg or Ala;
xaa20 ═ His, Gln, or Lys;
xaa21 ═ Asp or Glu;
Xaa23＝Ile，Val；
xaa24 ═ Glu or Gln;
Xaa27＝Met，Leu，Nle；
xaa28 ═ Asn, Asp, Arg, Ser or deleted;
xaa29 ═ Gly, Thr or absent;
xaa30 ═ Gly or absent;
xaa31 ═ Gly or absent;
xaa32 — Pro or absent;
xaa33 ═ Ser, Val, or absent;
xaa34 or absent;
xaa35 ═ Gly or absent;
xaa36 ═ Ala or absent;
xaa37 — Pro or absent;
xaa38 — Pro or absent;
xaa39 — Pro or absent;
xaa40 or absent;
at least one of Xaa10, position 12, Xaa16, Xaa17 or Xaa20 is Lys and the side chain of said at least one Lys is linked to a lipophilic substituent in such a way that the lipophilic substituent forms an amide bond with the amino group of a bridging group, the carboxyl group of an amino acid residue of said bridging group forms an amide bond with the N-terminal residue of the Lys of the parent peptide to which said parent peptide is linked, said bridging group being Glu, Asp and/or (PEG)mWherein m is an integer of 2 to 10; the lipophilic substituent is selected from CH3(CH2)nCO-or HOOC (CH)2)nAn acyl group of CO-, wherein n is an integer of 10 to 24. A preferred bridging group is Glu- (PEG)mOr Asp- (PEG)mOr (PEG)mIs connected toIn the manner shown in figure 1.
The compounds of the invention stabilize the helical structure of the molecule based on theoretical intramolecular bridges, thereby improving potency and/or selectivity against GLP-1R or GCGR. The compounds of the invention carry one or more intramolecular bridges in the sequence. Such bridges are formed between the side chains of two amino acid residues, which are usually separated by three amino acids in a linear sequence. For example, the bridge may be formed between the side chains of residue pairs 12 and 16, 16 and 20, 17 and 21, or 20 and 24. The two side chains may be linked to each other by ionic interaction or by covalent bonds. Thus, these residue pairs may contain oppositely charged side chains, forming salt bridges through ionic interactions. For example, one residue may be Glu or Asp and the other may be Lys or Arg, the Lys and Glu pair and the Lys and Asp pair, respectively, also being capable of reacting to form a lactam ring.
When the amino acid sequence is Lys at position 10, 12, 16, 17 or 20, the Lys side chain is attached to the lipophilic substituent and the bridging group in one of the following structures:
2 2 2 14 3Lys(PEG-PEG-CO(CH)CH)：
Figure BDA0002911582310000041
2 2 2 14 2Lys(PEG-PEG-CO(CH)COH)：
Figure BDA0002911582310000051
2 2 2 16 2Lys(PEG-PEG-CO(CH)COH)：
Figure BDA0002911582310000052
2 2 2 16 3Lys(PEG-PEG-CO(CH)CH)：
Figure BDA0002911582310000061
the amino acid sequence of the parent peptide is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48 amino acid sequence of the parent peptide.
The invention also aims to provide a pharmaceutical composition containing the GLP-1R/GCGR dual-agonist polypeptide derivative, wherein the GLP-1R/GCGR dual-agonist polypeptide derivative is used as an active ingredient and is added with a pharmaceutically acceptable carrier and/or an auxiliary material to prepare the pharmaceutical composition.
The GLP-1R/GCGR double-agonist polypeptide derivative has the effects of improving and treating renal fibrosis. The GLP-1R/GCGR dual agonist polypeptide derivatives can be used for directly or indirectly treating diseases caused by or characterized by renal fibrosis.
It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention are suitable for various modes of administration, such as oral, transdermal, intravenous, intramuscular, topical, nasal, and the like. Depending on the mode of administration used, the polypeptide pharmaceutical composition of the present invention may be formulated into various suitable dosage forms comprising at least one effective amount of the polypeptide of the present invention and at least one pharmaceutically acceptable carrier.
Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions for injection.
Pharmaceutical compositions containing the polypeptides of the invention may be formulated as solutions or lyophilized powders for parenteral administration, the powders being reconstituted with a suitable solvent or other pharmaceutically acceptable carrier prior to use, the liquid formulations typically being buffers, isotonic solutions and aqueous solutions.
The amount of the polypeptide of the present invention in the pharmaceutical composition may vary within wide limits and can be readily determined by one skilled in the art based on objective factors such as the type of disease, the severity of the condition, the weight of the patient, the dosage form, the route of administration, and the like.
The invention has the advantages that:
1) the GLP-1R/GCGR double agonist polypeptide can inhibit epithelial-mesenchymal transition of renal tubular cells in vivo and in vitro, can improve mitochondrial morphology, and can inhibit PERK-mediated endoplasmic reticulum stress pathway in a TGF-beta induced EMT process by a polypeptide compound, so that kidney fibrosis and inflammation caused by kidney fibrosis are treated or improved.
2) Has good biological activity;
3) the stability is shown in the pharmaceutical experiment of the medicine, the stability is good, the scale-up production is easy, and the cost is low;
4) compared with small molecular compounds, the compound has lower toxicity, larger safety window and smaller dosage.
In particular embodiments, the following GLP-1R/GCGR dual agonist polypeptides are contemplated having the following amino acid sequence:
compound 1 (related to SEQ ID NO: 1):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYS-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 2 (related to SEQ ID NO: 2):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYS-K(PEG2-PEG2-CO(CH2)16CO2H)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 3 (related to SEQ ID NO: 3):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-CO(CH2)16CH3)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYS-K(PEG2-PEG2-CO(CH2)16CH3)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 4 (related to SEQ ID NO:4)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS
Compound 5 (related to SEQ ID NO: 5):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 6 (related to SEQ ID NO: 6):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 7 (related to SEQ ID NO: 7):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-Aib-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-RAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 8 (related to SEQ ID NO: 8):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 9 (related to SEQ ID NO: 9):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-CO(CH2)14CH3)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYS-K(PEG2-PEG2-CO(CH2)14CH3)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 10 (related to SEQ ID NO: 10):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-Aib-K(PEG2-PEG2-CO(CH2)16CO2H)-RAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 11 (related to SEQ ID NO: 11):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)14CO2H)-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 12 (related to SEQ ID NO: 12):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 13 (related to SEQ ID NO: 13):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)14CO2H)-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 14 (related to SEQ ID NO: 14):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 15 (related to SEQ ID NO: 15):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 16 (related to SEQ ID NO: 16):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 17 (related to SEQ ID NO: 17):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-NH2
H-(d-S)-QGTFTSDYSKYLDS-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-AAHDFVEWLLRA-NH2
compound 18 (related to SEQ ID NO: 18):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Arg-Ala-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-EFIEWLLRA-NH2
compound 19 (related to SEQ ID NO: 19):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-Aib-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH2
compound 20 (related to SEQ ID NO: 20):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2H-(d-S)-QGTFTSDYS-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 21 (related to SEQ ID NO: 21):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLDS-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-AAHDFVEWLLNGGPSSGAPPPS-NH2
compound 22 (related to SEQ ID NO: 22):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLDS-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-AAHDFVEWLLNGGPSSGAPPPS-NH2
compound 23 (related to SEQ ID NO: 23):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Arg-Ala-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-EFIEWLLRA-NH2
compound 24 (related to SEQ ID NO: 24):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Arg-Ala-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-EFIEWLLRA-NH2
compound 25 (referring to SEQ ID NO: 25):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Arg-Ala-NH2
H-(d-S)-QGTFTSDYSKYLDS-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-AAHDFVEWLLRA-NH2
compound 26 (related to SEQ ID NO:26)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-EFIEWLLNGGPSSGAPPPS-NH2
Compound 27 (related to SEQ ID NO:27)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)14CO2H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)14CO2H)-EFIEWLLNGGPSSGAPPPS-NH2
Compound 28 (related to SEQ ID NO:28)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-EFIEWLLNGGPSSGAPPPS-NH2
Compound 29 (related to SEQ ID NO:29)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLDEKAA-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-EFIEWLLNGGPSSGAPPPS-NH2
Compound 30 (related to SEQ ID NO:30)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-CO(CH2)16CO2H)-SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
Compound 31 (related to SEQ ID NO:31):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-CO(CH2)14CH3)-SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
Compound 32 (related to SEQ ID NO:32)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)16CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-CO(CH2)16CH3)-SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
Compound 33 (referring to SEQ ID NO: 33):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-Aib-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-RAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 34 (related to)SEQ ID NO:34)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
Compound 35 (referring to SEQ ID NO: 35):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-CO(CH2)16CO2H)-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 36 (referring to SEQ ID NO: 36):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)16CH3)-RRAQDFVQWLMNTGGPSSGAPPPS-NH2
compound 37 (related to SEQ ID NO: 37):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH2
compound 38 (related to SEQ ID NO: 38):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH2
compound 39 (referring to SEQ ID NO: 39):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-RRAQDFVQWLLNTGGPSSGAPPPS-NH2
compound 40 (related to SEQ ID NO: 40):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-RRAQDFVQWLLNTGGPSSGAPPPS-NH2
compound 41 (referring to SEQ ID NO: 41):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-CO(CH2)16CO2H)-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH2
compound 42 (related to SEQ ID NO: 42):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-CO(CH2)14CH3)-RRAQDFVQWLLNTGGPSSGAPPPS-NH2
compound 43 (related to SEQ ID NO: 43):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)14CH3)-SKYLD-Aib-RRAQDFVQWLLNTGGPSSGAPPPS
compound 44 (related to SEQ ID NO: 44):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-CO(CH2)14CH3)-SKYLD-Aib-RRAQDFVQWLLNTGGPSSGAPPPS
compound 45 (related to SEQ ID NO: 45):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)14CH3)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-CO(CH2)14CH3)-SKYLD-Aib-RRAQDFVQWL-Nle-NTGGPSSGAPPPS
compound 46 (related to SEQ ID NO: 46):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-CO(CH2)16CO2H)-SKYLD-Aib-RRAQDFVQWL-Nle-NTGGPSSGAPPPS
compound 47 (referring to SEQ ID NO: 47):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-SKYLD-Aib-RRAQDFVQWLLNTGGPSSGAPPPS
compound 48 (related to SEQ ID NO: 48):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
H-(d-S)-QGTFTSD-K(PEG2-PEG2-γGlu-CO(CH2)16CO2H)-SKYLD-Aib-RRAQDFVQWL-Nle-NTGGPSSGAPPPS
in the amino acid sequence of the parent peptide of the invention, Lys of Xaa10, position 12, Xaa16, Xaa17 or Xaa20 linked to the lipophilic substituent is replaced by HomoLys, Orn, Dap or Dab.
Figure BDA0002911582310000161
Abbreviations used in the present invention have the following specific meanings:
boc is t-butyloxycarbonyl, Fmoc is fluorenylmethyloxycarbonyl, t-Bu is t-butyl, ivDDe is the removal and lipophilic substituent of 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methyl-butyl, resin, TFA is trifluoroacetic acid, EDT is 1, 2-ethanedithiol, Phenol is Phenol, FBS is fetal bovine serum, BSA is bovine serum albumin, HPLC is high performance liquid phase, GLP-1R is glucagon-like peptide 1 receptor, GCGR is glucagon receptor, GLP-1 is glucagon-like peptide, mPEG is monomethoxypolyethylene glycol, OXM is oxyntomodulin, His is histidine, Ser, D-Ser is D-serine, Gln is glutamine, Gly is glycine, Glu is glutamic acid, Ala is alanine, Thr is threonine, lys is lysine, Arg is arginine, Tyr is tyrosine, Asp is aspartic acid, Trp is tryptophan, Phe is phenylalanine, Ile is isoleucine, Leu is leucine, Cys is cysteine, Pro is proline, Val is valine, Met is methionine, Asn is asparagine, HomoLys is homolysine, Orn is ornithine, Dap is diaminopimelic acid, Dab is 2, 4-diaminobutyric acid, Nle is norleucine, Aib is 2-aminoisobutyric acid, AEEA is [2- [2- (amino) ethoxy ] acetic acid.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: is a schematic representation of the attachment of the bridging group to the peptide chain in the compounds of the invention.
FIG. 2: section images of mouse kidney tissue after H & E staining were used.
FIG. 3: section images of mouse kidney tissue after Masson staining were used.
FIG. 4: the section after CD68 staining of mouse kidney tissue is shown.
FIG. 5: is a graph of gene expression of type I collagen alpha 1 chain in mouse kidney tissue.
FIG. 6: an IHC image of mouse kidney tissue stained with E-cad and alpha-SMA during EMT in vivo.
FIG. 7: is a graph of the percentage of E-cad cells in mouse kidney tissue in FIG. 6.
FIG. 8: is a graph of the percent alpha-SMA cells in mouse kidney tissue in FIG. 6.
FIG. 9: IF images of mouse renal tubular epithelial cells stained with alpha-SMA during in vitro EMT.
FIG. 10: is a graph of the percent alpha-SMA cells in the renal tubular epithelial cells of the mice in FIG. 9.
FIG. 11: FIG. 9 is a graph showing the gene expression of type I collagen alpha 1 chain in renal tubular epithelial cells of a mouse.
FIG. 12: RNAseq analysis of mouse tubular cells.
FIG. 13: is a KEGG pathway enrichment analysis diagram of the differentially expressed genes in FIG. 12.
FIG. 14: the expression condition of the mouse renal tubular cell PERK gene is shown.
FIG. 15: western blot analysis for mouse tubular cells PERK and p-PERK and CHOP.
FIG. 16: sections were stained for PERK-mediated endoplasmic reticulum stress PERK and p-eIF2 α IHC during in vitro EMT.
FIG. 17: the percentage of PERK positive cells in figure 16 is plotted.
FIG. 18: is a graph of the percentage of p-eIF2 alpha positive cells in FIG. 16.
FIG. 19: is a graph of mitochondrial staining in mouse tubular cells.
FIG. 20: a histogram of mitochondrial strength in mouse tubular cells is presented.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, reagents or equipment used are commercially available.
Example 1 Synthesis of Dual agonist Polypeptides
Materials:
all amino acids were purchased from NovaBiochem. All other reagents were analytical grade, purchased from Sigma, unless otherwise specified. Protein Technologies PRELUDE 6 channel polypeptide synthesizer was used. Phenomenex Luna C18 preparative columns (46 mm. times.250 mm) were used to purify the polypeptides. The high performance liquid chromatograph is a product of Waters company. Mass spectrometry was performed using an Agilent mass spectrometer.
The synthesis method of the polypeptide compound of the present invention is illustrated by polypeptide compound 6:
the structural sequence is as follows:
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
a) assembling a main peptide chain:
the following polypeptides were synthesized on a 0.25mmol scale on a CS336X polypeptide synthesizer (CS Bio, USA) according to the Fmoc/t-Bu strategy:
Boc-His (Boc) -D-Ser (t-Bu) -Gln (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (tBu) -Asp (OtBu) -Tyr (t-Bu) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Asp (OtBu) -Lys (ivDde) -Arg (Pbf) -Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Gln (Trt) -Trp (Boc) -Leu-Met-Asn-Trt) -Thr (t-Bu) -Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin.
(1) The first step is as follows: 0.75 g Rink amide MBHA-LL resin (Novabiochem, 0.34mmol/g loading) was swollen in Dichloromethane (DCM) for one hour, and the resin was washed thoroughly 3 times with N, N-Dimethylformamide (DMF);
(2) the second step is that: taking Rink amide resin as a carrier, taking a coupling agent comprising 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea Hexafluorophosphate (HCTU) and organic base N, N-Diisopropylethylamine (DIEPA) as solvents according to the mass ratio of 1:1, carrying out a programmed reaction, and sequentially carrying out a condensation reaction to connect
Fmoc-Ser (t-Bu) -OH, Fmoc-Pro-OH (3x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (t-Bu) -OH (2x), Fmoc-Pro-OH, Fmoc-Gly-OH (2x), Fmoc-Thr (t-Bu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gln (t) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH (2x), Fmoc-Lys (ivDde) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (t-Bu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ser (t-Bu) -OH, Fmoc-Tyr (t-Bu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (t-Bu) -OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Phe-OH, Thr (t-Bu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-D-Ser (t-Bu) -OH, Boc-His (Boc) -OH:
Boc-His (Boc) -D-Ser (t-Bu) -Gln (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (tBu) -Asp (OtBu) -Tyr (t-Bu) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Asp (OtBu) -Lys (ivDde) -Arg (Pbf) -Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Gln (Trt) -Trp (Boc) -Leu-Met-Asn-Trt) -Thr (t-Bu) -Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-B u) -rink amide resin. The resin was then washed thoroughly 3 times with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), Methanol (Methanol), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
In the reaction, 1) the mass ratio of the dosage of the first amino acid Fmoc-Ser (t-Bu) -OH to the dosage of the resin is 1: 1-6: 1; 2) in each subsequent condensation reaction, the dosage of Fmoc protected amino acid, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea Hexafluorophosphate (HCTU) and organic base N, N-Diisopropylethylamine (DIEPA) is excessive by 2-8 times, and the reaction time is 1-5 hours.
b) Removal of 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methyl-butyl (ivDde) and introduction of lipophilic substituents:
the resin was washed twice with a solution of N, N-Dimethylformamide (DMF)/Dichloromethane (DCM) at 1:1 (volume ratio), a freshly prepared 3.0% solution of hydrazine hydrate in N, N-Dimethylformamide (DMF) was added, and the reaction mixture was subjected to a trap treatment step with shaking at room temperature for 10-30 minutes, and then filtered. The hydrazine treatment step was repeated 5 times to give:
Boc-His (Boc) -D-Ser (t-Bu) -Gln (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (tBu) -Asp (OtBu) -Tyr (t-Bu) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Asp (OtBu) -Lys-Arg (Pbf) -Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Gln (Trt) -Trp (Boc) -Leu-Met-Asn Trt- (t-Bu) -Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Burin) -k amide resin. The resin was then washed thoroughly 3 times with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), Methanol (Methanol), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
Adding FmocNH-PEG2-OH (Quanta BioDesign), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), Diisopropylethylamine (DIEPA) in N, N-Dimethylformamide (DMF) mixed coupling solution (both in 5-fold excess), after shaking for 2 hours, filtered. After this time the resin was washed thoroughly 3 times with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), Methanol (Methanol), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in sequence to give:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(Fmoc-PEG2) -arg (pbf) -Ala-gln (trt) -asp (otbu) -Phe-Val-gln (trt) -trp (boc) -Leu-Met-asn (trt) -Thr (t-Bu) -Gly-Pro-Ser (t-Bu) -Gly-Ala-Pro-Ser (t-Bu) -rink amide resin. After this time the resin was washed thoroughly with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), Methanol (Methanol), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in that order3 times each.
Removal of Fmoc group in 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) (30 min, repeat removal twice) and addition of Fmoc-PEG2Mixing (5 times excess) N, N-Dimethylformamide (DMF) of-OH, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) and Diisopropylethylamine (DIEPA), and performing coupling reaction to obtain
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(Fmoc-PEG2-PEG2) -arg (pbf) -Ala-gln (trt) -asp (otbu) -Phe-Val-gln (trt) -trp (boc) -Leu-Met-asn (trt) -Thr (t-Bu) -Gly-Pro-Ser (t-Bu) -Gly-Ala-Pro-Ser (t-Bu) -rink amide resin. The resin was then washed thoroughly 3 times with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), Methanol (Methanol), Dichloromethane (DCM), N, N-Dimethylformamide (DMF) in succession.
Removing Fmoc group from 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) (30 min, repeated twice), coupling Fmoc-gamma Glu-OtBu in sequence according to conventional conditions, and adding palmitic acid (palmitic acid) to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(PEG2-PEG2-C16) -Arg (Pbf) -Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Gln (Trt) -Trp (Boc) -Leu-Met-Asn (Trt) -Thr (t-Bu) -Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin. After this time the resin was washed thoroughly 3 times with N, N-Dimethylformamide (DMF), Dichloromethane (DCM), Methanol (Methanol), Dichloromethane (DCM) and dried in vacuo.
c) And (3) removing full protection of polypeptide:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(PEG2-PEG2-C16)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn (Trt) -Thr (t-Bu) -Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin is added with cutting fluid TFA/Phenol/thioanisole/EDT/H2In O (82.5:5:5:2.5:5, volume ratio), the temperature is raised, the temperature of the lysate is controlled at 25 ℃, and the reaction lasts for 2.5 hours. Filtering, washing the filter cake with a small amount of lysate for 3 times, and combining the filtrates. The filtrate was slowly poured into ice-cold ether with stirring. Standing for more than 2 hours until the precipitate is complete, centrifuging, washing the precipitate with glacial ethyl ether for 3 times to obtain a crude compound:
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2)14CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
d) and (3) refining and purifying the polypeptide compound:
dissolving the crude compound in Acetonitrile (ACN)/H2In a solution of O1: 2 (vol/vol), preparative HPLC purification was performed on a 46mm x 250mm column packed with 5.0mm reverse phase C18. With 30% acetonitrile (containing 0.05% trifluoroacetic acid)/H2Starting with O (containing 0.05% trifluoroacetic acid), the column was eluted with a gradient (increasing acetonitrile ratio at a rate of 1.33%/min) at a flow rate of 15mL/min for 30 minutes, and the fractions containing the peptide were collected, lyophilized to give a pure product with an HPLC purity of greater than 95%. The isolated product was analyzed by LC-MS.
Based on the above synthetic procedures, the following polypeptide compounds of the present invention were synthesized (table 1):
table 1, structures of the polypeptide compounds synthesized in the examples of the present invention:
Figure BDA0002911582310000211
Figure BDA0002911582310000221
Figure BDA0002911582310000231
Figure BDA0002911582310000241
Figure BDA0002911582310000251
Figure BDA0002911582310000261
Figure BDA0002911582310000271
example 2 amelioration and therapeutic Effect of GLP-1R/GCGR Dual agonist Polypeptides on UUO-induced renal fibrosis
The mouse renal fibrosis model is established by a Unilateral Ureteral Obstruction (UUO) method, and polypeptide compounds 1,3, 11, 21, 28, 40 and 41 are randomly selected for testing. Adult B6 mice were used for a mouse model of Unilateral Ureteral Obstruction (UUO), with sham operated mice as a control group. Mice were dosed daily after surgery and sacrificed on day 14. Tissue samples were collected for immunohistochemistry and kidney mRNA gene expression analysis, and mouse tubular cells (mTECs) stimulated with TGF- β were used as an in vitro model of fibrosis. The specific operation method comprises the following steps:
mice were treated:
80 male B6(H2B) mice were selected, 6-8 weeks old, and were all manufactured by Wintolite laboratory animals of Beijing. Mice were divided into 10 groups of 8 mice each, of which 9 groups were subjected to Unilateral Ureteral Obstruction (UUO) surgical treatment to establish a mouse renal fibrosis model, and 1 group was subjected to pseudo-surgical treatment as a control group. For mice subjected to UUO surgical treatment, the treatment method comprises the following steps: the abdominal skin of the mouse was incised using surgical scissors, the small intestine was moved to the right side of the mouse using a cotton swab, the left ureter was ligated using 8-0 suture, and the abdominal incision was closed. For the mice subjected to the pseudo-surgical treatment, the treatment method comprises the following steps: the abdomen of the mice was opened, the small intestine was removed and replaced, and the abdomen was closed.
After the surgery, 10 groups of mice were treated as follows:
1) a sham operation control group, without any treatment, n is 8;
2) saline group, mice treated with UUO were injected intraperitoneally with 200uL of saline daily, n-8;
3) somalufu group, UUO treated mice were injected intraperitoneally daily with somalufu (80ug/kg), n ═ 8;
4) polypeptide compound 1 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 1(80ug/kg), n-8;
5) polypeptide compound 3 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 3(80ug/kg), n-8;
6) polypeptide compound 11 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 11(80ug/kg), n-8;
7) polypeptide compound 21 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 21(80ug/kg), n-8;
8) polypeptide compound 28 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 28(80ug/kg), n-8;
9) polypeptide compound 40 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 40(80ug/kg), n-8;
10) polypeptide compound 41 group, UUO-treated mice were injected intraperitoneally daily with polypeptide compound 41(80ug/kg), n-8.
Mice were sacrificed on day 14, kidney tissues were collected and sectioned at a thickness of 4um, and stained with hematoxylin-eosin (H & E) and Masson (Masson), the results of which are shown in fig. 2 and 3.
The efficacy of the polypeptide compounds of the present invention in treating and improving renal fibrosis was determined using a mouse Unilateral Ureteral Obstruction (UUO) model. After UUO occlusion for 14 days in 8-12 week old mice, it can be seen from the results of fig. 2 and 3 that the renal tubules of mice treated with Unilateral Ureteral Occlusion (UUO) are significantly damaged and have an area of infiltrating cells, and that after 14 days of administration, Masson stained sections show diffuse collagen deposition in the mouse kidneys after UUO treatment; among them, the treatment with the polypeptide compound of the present invention can significantly improve the tissue morphology of the kidney and reduce the deposition of collagen. In addition, the stained sections of fig. 2 and 3 show that the mouse kidney tissue treated with the polypeptide compounds 1,3, 11, 21, 28, 40, 41 of the present invention shows less macrophage infiltration, indicating that the mouse kidney tissue has less inflammation and the effect is better than the thaumatin.
Therefore, the polypeptide compound can obviously reduce the inflammation caused by the renal fibrosis, and has better treatment effect.
Example 3 study of GLP-1R/GCGR dual agonist Polypeptides to improve and treat the mechanism of action of renal fibrosis
To further understand the mechanism of action of the polypeptide compounds of the present invention in improving and treating renal fibrosis, UUO mice treated with the polypeptide compound 40 of example 2, sham-operated control group and saline group were randomly selected for study.
Mice were sacrificed on day 14, mouse kidney tissue was collected and sectioned at a thickness of 4um, Immunohistochemical (IHC) staining of mouse kidney tissue sections was performed using CD68 (including antibody E-cad, anti- α -SMA, antibody PERK, and antibody p-eIF2 α), followed by observation of stained mouse kidney tissue sections for mouse kidney morphology and fibrosis, the results of which are shown in fig. 4. Tissue samples were collected for immunohistochemistry and kidney mRNA gene expression analysis, and mouse tubular cells (mTECs) stimulated with TGF- β were used as an in vitro model of fibrosis.
In vitro cell culture:
tubular epithelial cells (mTECs) were isolated from mouse kidney. mTECs were then grown in kidney-supplemented epithelial cell growth medium at 37 ℃ and 5% CO2Culturing in medium. mTECs were seeded overnight in 6-or 24-well plates and then exposed to 10ng/mL TGF-. beta.1 and/or 10umol/L of a polypeptide compound of the invention for 48 hours.
Protein extraction and western blotting:
mtecs 48 hours after TGF- β 1 and/or the polypeptide compound of the present invention were collected, the mtecs 48 hours after treatment were lysed on ice using a lysis buffer containing a protease inhibitor and a phosphatase inhibitor, and proteins were extracted. Protein concentration was quantified by BCA assay, and then the protein was mixed with loading buffer and boiled for western blot experiments. Proteins were separated on 10% SDS-PAGE in running buffer, then transferred to polyvinylidene fluoride (PVDF) membranes, blocked with BSA buffer, and incubated with anti-PERK, anti-p-PERK, anti-CHOP and anti- β -actin overnight at 4 ℃, washed 3 times with PBST, signal detected by chemiluminescence, and visualized by the chemi scope 3300Mini Imaging System.
Real-time polymerase chain reaction:
total RNA was extracted from mouse kidneys using Trizol reagent according to the manufacturer's instructions () from Thermo Fisher Scientific. Reverse transcriptase reactions were performed using the qScript cDNA Synthesis kit (Quanta Biosciences, Gaithersburg, Md.), 1. mu.g RNA, real-time quantitative PCR was run on a Bio-Rad IQ2PCR machine, each PCR mixture containing 40ng of cDNA template and 10. mu.nM primer in 15. mu.L SYBR green reaction mix (Bio-Rad), and changes in gene expression levels were calculated using the 2-. DELTA.Ct method.
RNAseq analysis:
mTEC treated with or without TGF-. beta.1 (10ng/mL) were collected 48 hours post-treatment. RNA from these samples was extracted (n-3/group) and subjected to RNAseq analysis. RNA integrity was assessed using RNA Nano 6000 assay kit from Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The total amount of each sample was 1ug of RNA used as input material for RNA sample preparation. Use for
Figure BDA0002911582310000301
Is/are as follows
Figure BDA0002911582310000302
UltraTM RNA library preparation kit (NEB, USA) generates sequencing library and adds index code toAttribute sequence for each sample. Index-coded samples were clustered on the cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on the Illumina Novaseq platform and paired-end reads of 150bp were generated. Differential Expression Gene (DEG) analysis was performed using the edgeR software package and the cutoff CPM was set to greater than 0.4 and the FDR to less than 5%. Statistical enrichment of differentially expressed genes in the KEGG pathway was tested using the clusterProfiler R software package.
Data analysis was performed using the mapping tool GraphPad Prism (GraphPad Software, inc., san diego, california). Statistical significance between groups was determined by Wilcoxon nonparametric test or paired or unpaired t-test. Data representing more than two groups were analyzed using one-way analysis of variance. P <0.05 was considered to represent a statistically significant difference. Error bars represent standard error of the mean (SEM) throughout.
And (3) analyzing test results:
(1) the polypeptide compound of the invention can inhibit the expression of type I collagen gene ColI.
After 14 days of administration, IHC stained sections using CD68 in fig. 4 showed less macrophage infiltration in kidney tissue of mice treated with the polypeptide compound of the present invention, indicating less inflammation in kidney tissue of mice, and thus it could be demonstrated that inflammation caused by renal fibrosis can be reduced using the polypeptide compound of the present invention. Figure 5 is a graph of the gene expression of collagen type I α 1 chain (ColI α 1) in mouse kidney tissue, # p <0.05, # p <0.01, # p <0.0001, one-way anova; the type I collagen α 1 chain gene was used to encode type I collagen, and the expression of the type I collagen α 1 chain (coliα 1) gene was analyzed by analyzing mouse kidney tissue using the qPCR method, and the results are shown in fig. 5. The results shown in FIG. 5 show that the mRNA of the type I collagen gene ColI α was also decreased in the treated group using the polypeptide compound of the present invention, as compared with the untreated group, and thus it can be confirmed that the polypeptide compound of the present invention can inhibit the expression of the type I collagen gene.
(2) The polypeptide compounds of the present invention inhibit tubular cell epithelial-mesenchymal transition (EMT) in vivo and in vitro.
FIG. 6 is an IHC image of mouse kidney tissue stained with E-cad and α -SMA during EMT in vivo. Fig. 7 is a graph of the percent of E-cad cells of the mouse kidney tissue in fig. 6, wherein p <0.05, p <0.01, p <0.0001, one-way anova. Fig. 8 is a graph of the percentage of α -SMA cells in the rat kidney tissue of fig. 6, wherein p <0.05, p <0.01, p <0.0001, one-way anova. Epithelial-to-mesenchymal transition of renal tubule cells is a key process leading to renal fibrosis. To determine whether the polypeptide compound of the present invention plays a role in altering EMT, the control group, the saline group, and the kidney tissue of mice treated with the polypeptide compound of the present invention were subjected to IHC staining for E cadherin (E-cad) and α -actin (α -SMA), the results of which are shown in fig. 6. The percentage of E cadherin (E-cad) cells and α -SMA cells in FIG. 6 were calculated using Image J software, the results of which are shown in FIGS. 7 and 8. The results in FIGS. 6 to 8 show that most of the normal renal tubules in the control group were positive for E-cad, while the expression of E-cad was significantly reduced in the kidney of UUO-treated mice. However, the group treated with the polypeptide compound of the present invention had significantly more normal tubules than the saline group. alpha-SMA is a sensitive marker of fibroblast activation, and is mainly located in blood vessels in the kidney of the mouse in the control group, while alpha-SMA in the tubules and the tubulointerstitial organs is obviously up-regulated in the kidney of the mouse treated by UUO, but the alpha-SMA in the tubules and the tubulointerstitial organs is obviously reduced by the mouse treated by the polypeptide compound. It can be thus demonstrated that the polypeptide compound of the present invention can inhibit epithelial-mesenchymal transition (EMT) of renal tubular cells during renal fibrosis in vivo.
FIG. 9 is an IF image of mouse tubular epithelial cells stained with α -SMA during in vitro EMT. Fig. 10 is a graph of the percentage of α -SMA cells in the mouse tubular epithelial cells in fig. 9, wherein p <0.05, p <0.01, p <0.0001, one-way anova. Fig. 11 is a graph of gene expression of type I collagen alpha 1 chain (ColI alpha 1) in mouse tubular epithelial cells in fig. 9, { p > <0.05, { p > <0.01, { p > <0.0001, { single-way variance analysis, }; the type I collagen alpha 1 chain gene is used for coding type I collagen, and the qPCR method is used for analyzing mouse kidney tissues to analyze the gene expression condition of the type I collagen alpha 1 chain (ColI alpha 1). To determine whether the polypeptide compounds of the present invention play a role in altering the EMT process in vitro, mouse renal tubular epithelial cells were cultured in vitro and the EMT process was induced using TGF- β, the results of which are shown in fig. 9 to 11. As shown in FIGS. 9 to 11, the addition of the polypeptide compounds of the present invention to the culture system significantly inhibited TGF-. beta.induced fibroblast activation, as demonstrated by the α -SMA staining and the ColI α 1 gene expression.
(3) The polypeptide compounds of the invention inhibit the PERK-mediated endoplasmic reticulum stress pathway in TGF-beta induced EMT processes.
To determine the transcriptome profile of renal tubule cells stimulated by TGF-. beta.we performed an RNAseq analysis of mTEC with or without TGF-. beta.1 (10ng/mL) stimulation at 48 hours, the results of which are shown in FIG. 12. The results according to fig. 12 show that over 8000 genes are differentially regulated. The results of the KEGG pathway enrichment analysis of these differentially expressed genes are shown in fig. 12. The results according to fig. 12 indicate that the signaling pathway regulating glucose metabolism and Endoplasmic Reticulum (ER) stress is the major pathway of tubular cell alteration following TGF- β stimulation.
The endoplasmic reticulum stress pathway plays a key role in kidney injury and renal fibrosis, and endoplasmic reticulum kinase (PERK) and C/EBP homologous protein (CHOP) are key regulators of ER stress regulators, leading to renal tubular epithelial cell injury and fibroblast activation. To determine whether ER stress is regulated by GLP1R/GCGR, ER stress genes (including XBP1s, PERK and ATF-6) were analyzed by qPCR, the results of which are shown in FIG. 14. The results shown in FIG. 14 indicate that the expression of the PERK gene in renal tubular cells is significantly upregulated under the induction of TGF-. beta.and that the expression of the PERK gene is significantly inhibited by the addition of the polypeptide compound of the present invention. FIG. 15 shows Western blot results demonstrating that the polypeptide compounds of the present invention reduce protein expression of PERK, p-PERK and CHOP during TGF- β induced EMT.
To further determine whether the polypeptide compounds of the invention function in an in vitro procedure, the activity of the TGF- β peptide was determined in the control group (blank control, no TGF- β treatment), the TGF- β group (treated with 10ng/ml of TGF- β); TGF-. beta. + polypeptide compound 40 of the invention (treated with 10ng/ml of GF-beta. and 10. mu. mol/L of polypeptide compound 40 of the invention) resulted in PERK and p-eIF 2. alpha. IHC staining in the obstructive kidney, as shown in FIG. 16. Fig. 17 and 18 are the percentage of PERK and p-eIF2 α positive cells, where p <0.05, p <0.001, p <0.0001, one-way anova, respectively. The results in fig. 16-18 show that PERK and p-eIF2 α are predominantly located in damaged tubular cells. The obstructive kidney showed a significant increase in PERK and p-eIF2 alpha positive tubular cells compared to the normal kidney of the control group. However, treatment with the polypeptide compounds of the present invention significantly reduced PERK and p-eIF2 α expression in the obstructive kidney. Therefore, the polypeptide compound can inhibit and inhibit the PERK-mediated endoplasmic reticulum stress pathway in the EMT process.
(4) The polypeptide compound of the present invention can improve mitochondrial morphology.
The ER stress pathway is closely related to mitochondrial function, and mitochondrial dysfunction secondary to endoplasmic reticulum stress is associated with a variety of renal diseases. To further determine whether the polypeptide compounds of the present invention have an effect on the mitochondrial morphology of tubular cells, a control group (blank control, no TGF-. beta.treatment), a TGF-. beta.group (treated with 10ng/ml TGF-. beta.) were tested in an in vitro cell system; mitochondria from the TGF-. beta. + polypeptide compound group (treated with 10ng/ml of GF-. beta.and 10. mu. mol/L of the polypeptide compound of the present invention) were stained; mitochondria were stained with green stain and nuclei were stained with Hoechst, and the results are shown in fig. 19; the mitochondrial intensities were detected, and the results are shown in fig. 20. From the results of FIGS. 19 and 20, it was shown that TGF-. beta.induction decreased the mitochondrial mass of cultured tubular cells, and that this effect could be reversed by the addition of the polypeptide compounds of the present invention. It can be thus demonstrated that the polypeptide compounds of the present invention can play a role in mitochondrial metabolism.
In conclusion, the polypeptide compound can inhibit epithelial-mesenchymal transition of renal tubular cells in vivo and in vitro, can improve mitochondrial morphology, simultaneously determines that PERK-mediated ER stress pathway participates in the process of renal fibrosis, and can inhibit PERK-mediated endoplasmic reticulum stress pathway in the TGF-beta-induced EMT process, thereby treating or improving renal fibrosis and inflammation caused by renal fibrosis.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and modifications as fall within the true spirit and scope of the invention as defined by the appended claims be interpreted in accordance with the breadth to which they are fairly, if not explicitly recited herein.
Sequence listing
<110> Shenzhen City diagram micro-Anchu science and technology development Limited
<120> GLP-1R/GCGR dual agonist polypeptide derivatives for preventing or treating renal fibrosis
<130> GD2300-20P125135
<160> 48
<170> PatentIn version 3.5
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His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Xaa
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Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
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Ser Ser Gly Ala Pro Pro Pro Ser
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1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 9
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)14CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 9
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 10
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CO2H
<400> 10
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Lys Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 11
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 11
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 12
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 12
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 13
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys- (PEG2-PEG 2-gamma Glu-CO (CH2)14CO2H
<400> 13
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 14
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<400> 14
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 15
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 15
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 16
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 16
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 17
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 17
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Lys Ala Ala His Asp Phe Val Glu Trp Leu Leu Arg Ala
20 25
<210> 18
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 18
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Arg Ala
20 25
<210> 19
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 19
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 20
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 20
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 21
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 21
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Lys Ala Ala His Asp Phe Val Glu Trp Leu Leu Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 22
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<400> 22
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Lys Ala Ala His Asp Phe Val Glu Trp Leu Leu Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 23
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 23
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Arg Ala
20 25
<210> 24
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<400> 24
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Arg Ala
20 25
<210> 25
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<400> 25
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Lys Ala Ala His Asp Phe Val Glu Trp Leu Leu Arg Ala
20 25
<210> 26
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 26
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 27
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CO2H
<400> 27
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 28
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<400> 28
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 29
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 29
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Leu Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 30
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 30
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 31
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)14CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 31
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 32
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 32
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 33
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 33
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Lys Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 34
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 34
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 35
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CO2H
<400> 35
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 36
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CH3
<400> 36
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 37
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<220>
<221> MISC_FEATURE
<222> (27)..(27)
<223> Xaa=Nle
<400> 37
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 38
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (27)..(27)
<223> Xaa=Nle
<400> 38
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 39
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<400> 39
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 40
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 40
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 41
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (27)..(27)
<223> Xaa=Nle
<400> 41
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 42
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)14CH3
<400> 42
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 43
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 43
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 44
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)14CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 44
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 45
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)14CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (27)..(27)
<223> Xaa=Nle
<400> 45
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 46
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (27)..(27)
<223> Xaa=Nle
<400> 46
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 47
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<400> 47
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 48
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> Xaa=Aib
<220>
<221> MISC_FEATURE
<222> (27)..(27)
<223> Xaa=Nle
<400> 48
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Xaa Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40

Claims (10)

  1. The application of GLP-1R/GCGR double-agonist polypeptide derivatives in preventing or treating renal fibrosis.
  2. 2. The use according to claim 1, wherein the GLP-1R/GCGR dual agonist polypeptide derivative has the parent peptide represented by the amino acid sequence:
    His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-COR1
    wherein R is1＝-NH2；
    Xaa2 ═ Aib or D-Ser;
    xaa10 ═ Lys or Tyr;
    xaa13 ═ Lys or Tyr;
    xaa16 ═ Ser, Aib, Glu, or Lys;
    xaa17 ═ Lys or Arg;
    xaa18 ═ Arg or Ala;
    xaa20 ═ His, Gln, or Lys;
    xaa21 ═ Asp or Glu;
    Xaa23＝Ile，Val；
    xaa24 ═ Glu or Gln;
    xaa27 ═ Met, Leu, or Nle;
    xaa28 ═ Asn, Asp, Arg, Ser or deleted;
    xaa29 ═ Gly, Thr or absent;
    xaa30 ═ Gly or absent;
    xaa31 ═ Gly or absent;
    xaa32 — Pro or absent;
    xaa33 ═ Ser, Val, or absent;
    xaa34 or absent;
    xaa35 ═ Gly or absent;
    xaa36 ═ Ala or absent;
    xaa37 — Pro or absent;
    xaa38 — Pro or absent;
    xaa39 — Pro or absent;
    xaa40 is Ser or absent.
  3. 3. The use according to claim 2, wherein at least one of Xaa10, position 12, Xaa16, Xaa17 or Xaa20 is Lys and the side chain of said at least one Ly is linked to a lipophilic substituent in such a way that the lipophilic substituent forms an amide bond with the amino group of a bridging group whose carboxyl group forms an amide bond with the N-terminal residue of Lys of the parent peptide to which the bridging group is Glu, Asp and/or (PEG)mWherein m is an integer of 2 to 10; the lipophilic substituent is selected from CH3(CH2)nCO-or HOOC (CH)2)nAn acyl group of CO-, wherein n is an integer of 10 to 24.
  4. 4. Use according to claim 2, wherein the bridging group is Glu- (PEG)mOr Asp- (PEG)mOr (PEG)m。
  5. 5. Use according to claim 2, wherein the bridging group forms a molecular bridge between the side chains of residue pairs 12 and 16, 16 and 20, 17 and 21 or 20 and 24 of the amino acid sequence.
  6. 6. Use according to claim 2, characterized in that Lys linked to the lipophilic substituent is replaced by HomoLys, Orn, Dap or Dab.
  7. 7. The use according to any one of claims 2 to 5, wherein when position 10, 12, 16, 17 or 20 of the amino acid sequence is Lys, the Lys side chain is attached to the lipophilic substituent and the bridging group in one of the following structures:
    Figure FDA0002911582300000031
    Figure FDA0002911582300000041
  8. 8. the use according to claim 2, wherein the amino acid sequence of the parent peptide is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47 and 48 amino acid sequences of the parent peptides.
  9. 9. A composition comprising a GLP-1R/GCGR dual agonist polypeptide derivative of any one of claims 1-8 and at least one pharmaceutically acceptable pharmaceutical carrier and/or adjuvant.
  10. 10. The composition of claim 9, wherein the composition is in the form of at least one of a tablet, a capsule, a sugar-coated tablet, a granule, an oral solution, a syrup, an ointment and patch for skin surface, an aerosol, a nasal spray, and a sterile solution for injection.
CN202110094194.6A 2021-01-22 2021-01-22 GLP-1R/GCGR double-agonist polypeptide derivative for preventing or treating renal fibrosis Pending CN112791178A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790236A (en) * 2022-03-09 2022-07-26 深圳市图微安创科技开发有限公司 Synthetic method of GCGR/GLP-1R double-target-point agonist polypeptide
WO2024032523A1 (en) * 2022-08-10 2024-02-15 成都奥达生物科技有限公司 Long-acting dual-agonist compound

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110257092A1 (en) * 2008-06-17 2011-10-20 Indiana University Research And Technology Corp. Glucagon/glp-1 receptor co-agonists
CN106046145A (en) * 2016-04-22 2016-10-26 深圳市图微安创科技开发有限公司 Application of GLP-1R/GCGR double-target agonist polypeptide to treatment of fatty liver diseases, hyperlipidemia and arteriosclerosis
CN111278853A (en) * 2017-11-06 2020-06-12 深圳市图微安创科技开发有限公司 Treatment of biliary cirrhosis based on oxyntomodulin analog GLP-1R/GCGR dual-target agonist polypeptide
CN111372945A (en) * 2017-11-06 2020-07-03 中山大学 Treatment of idiopathic pulmonary interstitial fibrosis based on oxyntomodulin analog GLP-1R/GCGR dual-target agonist polypeptide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110257092A1 (en) * 2008-06-17 2011-10-20 Indiana University Research And Technology Corp. Glucagon/glp-1 receptor co-agonists
CN106046145A (en) * 2016-04-22 2016-10-26 深圳市图微安创科技开发有限公司 Application of GLP-1R/GCGR double-target agonist polypeptide to treatment of fatty liver diseases, hyperlipidemia and arteriosclerosis
CN111278853A (en) * 2017-11-06 2020-06-12 深圳市图微安创科技开发有限公司 Treatment of biliary cirrhosis based on oxyntomodulin analog GLP-1R/GCGR dual-target agonist polypeptide
CN111372945A (en) * 2017-11-06 2020-07-03 中山大学 Treatment of idiopathic pulmonary interstitial fibrosis based on oxyntomodulin analog GLP-1R/GCGR dual-target agonist polypeptide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
安春妹: "胃泌酸调节素（OXM）类似物改善慢性肾病肾脏纤维化的作用研究", 中国优秀硕士学位论文全文数据库医药卫生科技辑, no. 1, 15 January 2021 (2021-01-15) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790236A (en) * 2022-03-09 2022-07-26 深圳市图微安创科技开发有限公司 Synthetic method of GCGR/GLP-1R double-target-point agonist polypeptide
WO2024032523A1 (en) * 2022-08-10 2024-02-15 成都奥达生物科技有限公司 Long-acting dual-agonist compound

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