IL-11 long-acting protein antagonist compound and application thereof

Abstract

The invention discloses an IL-11 long-acting protein antagonist compound and application thereof, according to the sequence characteristics (SEQ ID NO.1) and receptor binding characteristics of human IL11 mutein, Cys mutation is introduced at a specific site by using site-specific mutagenesis, and an activity maintenance mutant is screened by means of cell activity. By utilizing a chemical reaction, fatty acids with different lengths of side chains C16-C22 are introduced into a Cys side chain for modification, and a long-acting product with kept activity is screened by virtue of cell activity again.

Description

Technical Field

[0001] This invention relates to compounds and their applications, particularly long-acting IL-11 protein antagonist compounds and their applications. Background Technology

[0002] Research has found that interleukin-11 (IL-11), a downstream signaling factor of TGF-β1, is a novel target for the treatment of fibrosis and cancer. In some cancers, fibrosis, and related cells (such as mesenchymal cells), IL-11 and its receptor IL-11Rα (interleukin-11 receptor subunit alpha) are abnormally overexpressed, leading to extracellular matrix (ECM) deposition and myofibroblast activation. Therefore, inhibiting the activity of IL-11R can prevent the occurrence of organ fibrosis and cancer-related diseases, such as renal fibrosis, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, and cancers such as kidney cancer, liver cancer, lung cancer, breast cancer, and uterine cancer. Therefore, the search for IL-11 antagonists for the treatment of fibrosis and cancer has attracted widespread interest.

[0003] Neutralizing antibodies, peptides, siRNAs, and protein antagonists for IL-11 have been discovered. Antibodies X203 and X209 against IL-11 and IL-11Rα have shown some efficacy in treating fibrosis, but are still in early clinical trials. Furthermore, difficulties in antibody acquisition and immunogenicity limit their development. This year, IL-11 peptide antagonists and siRNAs have been reported, exhibiting some inhibitory activity, but further activity evaluation, toxicology, and pharmacokinetic studies are needed. Previously, IL-11 mutants were discovered. 58 PAIDY 62 -W147A (IL-11 mutein) can block the formation of the IL-11_IL11RA_gp130 hexamer complex, thereby inhibiting signal transduction and alleviating the occurrence of disease. However, IL-11 mutein has poor plasma stability and is easily hydrolyzed by proteases, resulting in a short half-life in vivo, which makes it unsuitable for direct clinical application. Therefore, improving its pharmacokinetic properties through modification is a relatively simple, rapid and convenient way to obtain lead compounds. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to: (1) provide a class of activity-preserving IL-11 mutein cysteine ​​mutants for fatty acid modification at specific sites; (2) provide a method for preparing a class of fatty acid side chains; (3) provide a class of long-acting IL-11 antagonist compounds; (4) provide pharmacokinetic parameters of a compound; (5) provide a method for preparing the compound; (6) provide a modification means comprising the compound; and (7) provide a pharmaceutical application of the compound and its pharmaceutical composition.

[0005] First, a class of cysteine ​​(Cys) mutants of IL-11mutein were provided to offer chemically reactive side chains, along with fatty acid derivatives of varying side chain lengths for specific modification. Cys mutant proteins maintaining activity were screened using in vitro activity assay cell lines, followed by screening for fatty acid types. This method is simple, readily available, and produces homogeneous products. Next, the pharmacokinetic properties of the compound were examined in mice to verify that its half-life was indeed improved. Finally, the therapeutic effect of this compound on a specific disease was investigated.

[0006] Technical solution: The IL-11 mutein cysteine ​​mutant of the present invention replaces any amino acid from position 1 to 178 in the IL-11 mutein sequence as shown in SEQ ID NO.1 with cysteine.

[0007] The cysteine ​​mutant has amino acids at positions 19, 39, 40, 43, 45, 46, 108, 109, 112, 118, and 121 replaced with cysteine.

[0008] The method for preparing the cysteine ​​mutant, wherein the method for constructing the mutant gene includes: performing overlap PCR using pET28a-smt3-hIL-11 mutein as a template, extracting the plasmid and sequencing it to obtain the Cys mutant plasmid.

[0009] The long-acting IL-11 antagonist compound is an IL-11 mutein cysteine ​​mutant that undergoes nucleophilic substitution between the thiol group of the cysteine ​​side chain and the bromoacetyl group of the fatty acid, resulting in the departure of the bromide ion and the formation of a thioether.

[0010] The long-acting IL-11 antagonist compound has a fatty acid structure in which a monoacid or diacid with 16-22 carbon atoms is linked to a linker, including PEG, γE and hexane.

[0011] The long-acting IL-11 antagonist compound is prepared by the following steps: ultrafiltration and concentration of IL-11mutein cysteine ​​mutant protein to a final volume of 1-2 ml, addition of TCEP, mixing at room temperature for 10-30 min; addition of fatty acid side chain, reaction at room temperature for about 8-15 h; purification of the product by gel filtration chromatography, analysis of the target peak by SDS-PAGE, and concentration of the liquid in the collection tube corresponding to the target protein.

[0012] The structure of the fatty acid in the aforementioned long-acting IL-11 antagonist compound is shown below: .

[0013] The method for preparing the long-acting IL-11 antagonist compound includes the following steps: The IL-11 mutein cysteine ​​mutant protein was concentrated by ultrafiltration to a final volume of 1-2 ml, TCEP was added, and the mixture was stirred at room temperature for 10-30 min; then fatty acid side chains were added, and the reaction was carried out at room temperature for about 8-15 h. The product was purified by gel filtration chromatography, and the target peak was analyzed by SDS-PAGE. The liquid in the collection tube corresponding to the target protein was collected and concentrated.

[0014] The application of the long-acting IL-11 antagonist compound in the preparation of drugs for treating organ fibrosis.

[0015] The aforementioned applications include organ fibrosis, such as renal fibrosis, pulmonary fibrosis, cardiac fibrosis, and liver fibrosis.

[0016] Beneficial Effects: This invention has the following advantages over existing technologies: Based on the sequence characteristics (SEQ ID NO.1) and receptor binding properties of human IL11 mutein, this invention introduces Cys mutations at specific sites using site-directed mutagenesis, and then uses cell activity screening to select for activity-maintaining mutants. Using a chemical reaction, fatty acids of varying C16-C22 lengths are introduced into the Cys side chain for modification, and again, cell activity screening is used to select for long-acting products with maintained activity. Attached Figure Description

[0017] Figure 1 This is an SDS-PAGE result of the expression purification of SEQ ID NO.1; Figure 2This is the C-2 mass spectrum of compound C; Figure 3 This is a molecular sieve result diagram of the reaction product of D46C and C-13; Figure 4 These are the reaction products of D46C and C-13 and their corresponding SDS-PAGE images after passing through a molecular sieve; Figure 5 This is the mass spectrum of the reaction products of D46C and C-13; Figure 6 Compound FA-13 has an anti-fibrotic effect in UUO mice; Figure 7 It is a mass spectrum of C-13; Figure 8 It is a mass spectrum of C-1; Figure 9 It is a C-2 MS; Figure 10 It is a C-4 MS; Figure 11 It's a C-6 MS; Figure 12 It's a C-9 MS; Figure 13 It's a C-10 MS; Figure 14 It's a C-11 MS; Figure 15 It is a C-12 MS; Figure 16 This is the mass spectrum of the reaction product of D19C and C-1; Figure 17 This is the mass spectrum of the reaction product of L108C and C-1; Figure 18 This is the mass spectrum of the reaction product of Q109 and C-1; Figure 19 This is the mass spectrum of the reaction product of A45C- and C-1; Figure 20 This is the mass spectrum of the reaction products of D19C and C-11; Figure 21 This is the mass spectrum of the reaction products of D46C and C-4; Figure 22 This is the mass spectrum of the reaction products of A45C and C-12; Figure 23 This is the mass spectrum of the reaction product of L112C and C-12. Detailed Implementation

[0018] Example 1 SEQ ID NO.1 (IL11 mutein) PGPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLAAQLRDKFPADGDHNLDSLPTLPAIDYALGALQLPGVLTRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRLQLLMSRLALPQPPPDPPAPPLAPPSSAAGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL Obtaining high-purity SEQ ID NO.1 protein: The DNA fragment corresponding to SEQ ID NO.1, as shown in SEQ ID NO.2, was constructed into the pET28a-smt3 vector using seamless cloning technology. A SUMO tag was fused to its N-terminus to facilitate soluble expression. Soluble expression of IL-11 mutein was then performed using the E. coli expression system BL21(DE3): The pET28a-smt3-IL-11 mutein plasmid was chemically transformed into BL21(DE3). Single clones were picked and cultured overnight in a sterile LB medium at 37°C and 180 rpm. The next day, the culture was amplified by inoculation into 500 ml of LB medium. OD was then calculated. 600 When the concentration reached 0.8-1.0, 0.2 mM of the inducer isopropyl-β-D-thiogalactoside (IPTG) was added. After overnight culture at 25°C, the bacterial culture was collected, resuspended in 1xPBS (pH 7.4), sonicated to lyse the bacteria, and the supernatant was collected by high-speed centrifugation. The supernatant was then purified by nickel affinity chromatography. The elution was performed with PBS containing 300 mM imidazole. The eluent was dialyzed to remove imidazole and digested with SUMO enzyme. The free SUMO tag was removed again by nickel affinity chromatography to obtain the target protein with high purity. Finally, the purity was verified by SDS-PAGE.

[0019] SEQ ID NO.2 CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAAT TTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCCGGGATCTGCTGAGCTATCTGCGC CATGTGCAGTGGCTGCGCCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCT GCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC Introducing the Cys-mutated IL-11mutein sequence: SEQ ID NO.3 (its corresponding amino acid sequence has a G mutation at position 2, G2C) SEQ ID NO.4 (The corresponding amino acid sequence has a D mutation at position 19, which is D19C) SEQ ID NO.5 (its corresponding amino acid sequence has a mutation at position 39, where L is changed to C, L39C) SEQ ID NO.6 (its corresponding amino acid sequence has a mutation at position 40, where R is changed to C, R40C) SEQ ID NO.7 (its corresponding amino acid sequence has an F mutation at position 43, which is C, F43C) SEQ ID NO.8 (its corresponding amino acid sequence has a mutation at position 45, where A is changed to C, A45C) SEQ ID NO.9 (its corresponding amino acid sequence has a D mutation at position 46, which is changed to C, D146C) SEQ ID NO.10 (its corresponding amino acid sequence has an L mutation at position 108, which is C, L108C) SEQ ID NO.11 (its corresponding amino acid sequence has a Q mutation at position 109, Q109C) SEQ ID NO.12 (its corresponding amino acid sequence has an L mutation at position 112, which is C, L112C) SEQ ID NO.13 (its corresponding amino acid sequence has an R-to-C mutation at position 118, R118C) SEQ ID NO.14 (its corresponding amino acid sequence has an L mutation at position 121, which is C, L121C) DNA sequence: Mutation locations are underlined. SEQ ID NO.3 (its corresponding amino acid sequence has a G mutation at position 2, G2C) CCG TGC CCACCGCCGGGCCCGCCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCG GCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGGATCTGCTGAGCTATCTGCGCCAT GTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTG CCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.4 (its corresponding amino acid sequence has a D mutation at position 19, which is D19C) CCGGGCCCACCGCCGGGCCCGCCGCGTGAGCCCGGACCCGCGCGCGGAACTG TGCAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.5 (L at position 39 of the corresponding amino acid sequence is mutated to C, L39C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAG TGCCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.6 (R at position 40 of its corresponding amino acid sequence is mutated to C, R40C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTG TGC GATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.7 (F at position 43 of its corresponding amino acid sequence is mutated to C, F43C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAA TGC CCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.8 (A at position 45 of its corresponding amino acid sequence is mutated to C, A45C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCG TGCGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.9 (where D at the 46th position of its corresponding amino acid sequence is mutated to C, D46C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCG TGC GGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.10 (where the 108th L in its corresponding amino acid sequence is mutated to C, L108C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACC TGC CAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.11 (where the 109th Q in its corresponding amino acid sequence is mutated to C, Q109C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTG TGCGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.12 (where the 112th L in its corresponding amino acid sequence is mutated to C, L112C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGC TGC GATCGTCTGCTGCGCCGCCTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.13 (where the 118th R in its corresponding amino acid sequence is mutated to C, R118C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGC TGC CTGCAATTACTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC SEQ ID NO.14 (where the 121st L in its corresponding amino acid sequence is mutated to C, L121C) CCGGGCCCACCGCCGGGCCCGCCGCGCGTGAGCCCGGACCCGCGCGCGGAACTGGATAGCACCGTGCTGCTGACCCGCAGCCTGCTGGCGGATACCCGTCAGCTGGCGGCGCAGCTGCGCGATAAATTTCCGGCGGATGGCGATCATAATCTGGACAGCCTGCCGACCCTGCCGGCGATTGATTATGCCCTGGGCGCGCTGCAACTGCCGGGTGTGCTGACCCGCCTGCGCGCGGATCTGCTGAGCTATCTGCGCCATGTGCAGTGGCTGCGCCGCGCGGGCGGCAGCAGCCTGAAAACCCTGGAACCGGAACTGGGCACCCTGCAAGCGCGCCTGGATCGTCTGCTGCGCCGCCTGCAA TGCCTGATGAGCCGTCTGGCGCTGCCGCAGCCGCCGCCGGACCCGCCGGCGCCGCCACTGGCCCCGCCAAGCAGCGCGGCGGGCGGCATTCGCGCGGCGCATGCGATTCTGGGCGGCCTGCATCTGACCCTGGATTGGGCGGTGCGCGGCC 1. Experimental Method:

[0020] (a) Introduction of cysteine ​​mutation. Using a site-directed mutagenesis kit and designed site-directed mutagenesis primers, PCR was performed using the KOD-plus enzyme system (TOYOBO, Japan) with pET28a-smt3-hIL-11 mutein as a template. The PCR products were verified by agarose gel electrophoresis, and transformed after DnpI digestion. Single clones were picked and amplified, plasmids were extracted and sequenced to obtain the Cys mutant plasmid.

[0021] (b) Obtaining the Cys mutant protein. The plasmids corresponding to the constructed SEQ ID NO.3-NO.14 sequences were transformed into the expression strain BL21(DE3). Single clones were picked and cultured overnight in 5 ml sterile LB medium at 37°C and 180 rpm. The next day, the culture was transferred to 500 ml LB medium for amplification. OD was then calculated. 600 When the concentration reached 0.8-1.0, 0.2 mM of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) was added. After overnight culture at 25°C, the bacterial culture was collected, resuspended in 1x PBS (pH 7.4), sonicated to lyse the bacteria, and the supernatant was collected by high-speed centrifugation. The supernatant was then purified by nickel affinity chromatography. The elution was performed with PBS containing 300 mM imidazole. The eluent was dialyzed to remove imidazole and digested with SUMO enzyme. The free SUMO tag was removed again by nickel affinity chromatography to obtain the Cys mutant protein with high purity. Finally, the protein was verified by SDS-PAGE.

[0022] 2. Experimental Results Taking the expression and purification of SEQ ID NO.1 as an example, the SDS-PAGE results are shown below. Figure 1 .

[0023] Example 2: Fatty acid modification Fatty acid structure:

[0024] Experimental Method 1: Taking C-2 as an example, the synthesis of fatty acid chains Reagents and materials used in the embodiments of this invention: The chemical reagents used in the preparation of the compounds were obtained from Shanghai Bide Pharmaceutical Technology Co., Ltd. and Shanghai Haohong Biomedical Technology Co., Ltd. The instrument used to confirm the structure of compounds in this embodiment of the invention is a G6125B single quadrupole mass spectrometer.

[0025] Synthesize compound C-2:

[0026] Add 0.1 mmol Rink Amide Resin to a peptide synthesis tube, add 5 ml DCM, bubble with N2 for 30 min, then filter and discard the filtrate. Add 5 ml DMF to the synthesis tube, bubble with N2 for 1 min, then filter. Repeat three times. Add 5 ml 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq Fmoc-Lys(Alloc)-OH, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent) to the synthesis tube, and bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq Fmoc-8-amino-3,6-dioxaoctanoic acid, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to a synthesis tube. Bubble with N2 for 30 min, then remove the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and remove the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq Fmoc-8-amino-3,6-dioxaoctanoic acid, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to a synthesis tube. Bubble with N2 for 30 min, then remove the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq Fmoc-L-Glutamic acid 1-tert-butyl ester, 5 eq HBTU, 5 eq HOBT, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, and bubble with N2 for 120 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml of 20% Piperidine, bubble with N2 for 5 min, then filter and discard the filtrate. Repeat twice, then wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times.Add 5 eq 20-(tert-Butoxy)-20-oxoicosanoic acid, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to a synthesis tube. Bubble with N2 for 60 min, repeat twice. Wash the solid resin with DMF, bubble with N2 for 1 min, repeat three times. Add DCM for washing, bubble with N2 for 1 min, then dry under vacuum. Repeat 4 times. Add 15 eq phenylsilane, bubble with N2 for 5 min. Add 0.25 eq Pd(PPh3)4, bubble with N2 for 40 min. Add DCM for washing, bubble with N2 for 1 min, then dry under vacuum. Repeat 4 times. Wash the solid-phase resin with DMF, add 5 eq Fmoc-8-amino-3,6-dioxaoctanoic acid, 5 eq HATU, and 10 eq DIPEA (using DMF as solvent), add to the synthesis tube, bubble with N2 for 30 min, then remove the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat 3 times. Add 5 ml 20% Piperidine, bubble with N2 for 5 min, then filter and remove the filtrate. Repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, repeat 4 times. Add 5 eq Fmoc-8-amino-3,6-dioxaoctanoic acid, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min, remove the filtrate, and repeat twice. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add 5 ml 20% Piperidine, bubble with N2 for 5 min, and filter to remove the filtrate. Repeat twice, wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat four times. Add 5 eq Bromoacetyl bromide, 5 eq HATU, and 10 eq DIPEA (DMF as solvent) to the synthesis tube, bubble with N2 for 30 min. Wash the solid-phase resin with DMF, bubble with N2 for 1 min, and repeat three times. Add DCM for washing, bubble with N2 for 1 min, and then dry under vacuum. Repeat four times, pour off and air dry. The solid product was obtained and poured into a 25 ml flask. 5 ml of 95% TFA (TFA:Tips:H2O = 95%:2.5%:2.5%) was added. After stirring at room temperature for 2 h, the mixture was filtered. The filtrate was poured into cold diethyl ether, dried under vacuum, and methanol was added to dissolve the sample. The mixture was filtered through a microporous membrane and purified by HPLC to obtain the product. After mass spectrometry analysis, the product was lyophilized to obtain compound C-2.

[0027] Experimental results: C-2 mass spectrometry analysis is shown in [the table below]. Figure 2Predicted molecular weight: 1300.3, Detected molecular weight: 1301.2 ([M]) +2 1301.2).

[0028] Depending on the different fatty acids, PEG linker, and specific amino acids in the compounds, the corresponding raw materials were replaced, and the following compounds were prepared using a procedure similar to that in Example 1. The compounds were then confirmed by mass spectrometry analysis:

[0029] The mass spectra of C-13 are shown below. Figure 7 The mass spectra of C-1 are shown below. Figure 8 The mass spectra of C-2 are shown below. Figure 9 The mass spectra of C-4 are shown below. Figure 10 The mass spectra of C-6 are shown below. Figure 11 The mass spectra of C-9 can be found in [link to mass spectra]. Figure 12 The mass spectra of C-10 can be found here. Figure 13 The mass spectra of C-11 can be found here. Figure 14 The mass spectra of C-12 can be found in [link to mass spectra]. Figure 15 .

[0030] Experimental Method 2: Taking the reaction of D46C mutant with C13 as an example, fatty acid modification of IL-11 Cys mutant: D46C protein was ultrafiltered and concentrated to a final volume of 1-2 ml, the concentration was measured with nanodrop, and the molar concentration was calculated. TCEP was added at 5 molar equivalents and mixed at room temperature for 20 min; then 5 molar equivalents of fatty acid side chains were added, and the reaction was carried out at room temperature for approximately 12 h. The product was purified by gel filtration chromatography, and the target peak was analyzed by SDS-PAGE. The liquid in the collection tube corresponding to the target protein was collected and concentrated, and the purity was detected by mass spectrometry.

[0031] Molecular sieve results: The molecular sieve results for the reaction products of D46C and C-13 are shown in [the table below]. Figure 3 .

[0032] Figure 4 The reaction products of D46C and C13 and their corresponding SDS-PAGE chromatograms after passing through a molecular sieve are shown below. The mass spectrometry identification results of the reaction products are as follows: Figure 5 As shown in the mass spectrometry results, the product has high purity.

[0033] The mass spectrum of the product of the reaction between D19C and C1 is shown below. Figure 16 The mass spectrum of the reaction product of L108C and C1 is shown below. Figure 17 The mass spectrum of the product from the reaction of Q109 and C1 is shown below. Figure 18 The mass spectrum of the product from the reaction of A45C- with C1 is shown below. Figure 19 The mass spectrum of the reaction products of D19C and C11 is shown below. Figure 20 The mass spectrum of the reaction products of D46C and C4 is shown below. Figure 21The mass spectrum of the reaction products of A45C and C-12 is shown below. Figure 22 The mass spectrum of the reaction products of L112C and C-12 is shown below. Figure 23 .

[0034] Cysteine ​​mutants undergo nucleophilic substitution between the thiol group of the cysteine ​​side chain and the bromoacetyl group of the fatty acid, resulting in the departure of the bromide ion and the formation of thioethers.

[0035] Example 3: Inhibitory effect of the compound on IL-11 (IC50) 50 ) detection 1. Experimental Methods: hIL-11RA stable cell line was used for in vitro viability (IC50) screening. Cells were cultured in complete medium (DMEM + 10% FBS + 1% P.S + 4 μg / mL Blasticidin + 400 μg / mL G418 + 125 μg / mL Hygromycin). After trypsin digestion, cells were cultured at a density of 1.5*102 4 Cells were seeded in 96-well plates and cultured overnight before compound activity assays were performed. Compound solutions of varying concentrations were prepared using assay buffer (DMEM + 1% FBS + 1% PS), with the highest concentration set at 1 mg / ml. Other concentrations were prepared using a 10-fold dilution method, with three replicates per well. Cell supernatant was discarded, and 50 μL of the compound was added to each well. After incubation for 1 h, 50 μL of assay buffer containing 1 ng / ml hIL-11 was added to each well. Cells were cultured for another 6 h, and chemiluminescence was detected using a luciferase assay kit. IC50 was calculated using GraphPad 9.5. 50 .

[0036] 2. Experimental Results

[0037] As shown in Table 2, for compounds 1-49 with C mutations, D19C, R40C, A45C, D46C, L108C, Q109C, and L112C exhibited good activity. Fatty acid modification was performed using the exposed outer components D46C, A45C, and G3C. Among these, the product of D46C and C-13 (compound FA-13) showed the best activity, with an IC50 value of [missing value]. 50 The value is 263 nM.

[0038] Example 5: Evaluation of the anti-renal fibrosis activity of the compound in a mouse model 1. Experimental Methods: A mouse model of renal fibrosis was established using the unilateral ureteroligation (UUO) method. An incision was made on the left side of the mouse abdomen, and the left ureter was double-ligated using 3-0 silk sutures to establish the UUO model. UUO mice were divided into three groups: a solvent group, a low-dose group, and a high-dose group. The treatment groups received a subcutaneous injection of compound FA-13 at 50 ug / kg or 200 ug / kg on the first day of modeling, with injections every two days for three consecutive days. The sham-operated group and the solvent group received the same volume of physiological saline. Urine was collected on day 6, and serum was collected on day 7. The mice were then euthanized, and kidney tissue was harvested for data analysis and evaluation.

[0039] 2. Experimental Results: like Figure 6 As shown, compound FA-13 has a significant effect in the UUO-induced mouse renal fibrosis model, and can significantly inhibit the expression of fibrotic factors FN1 and α-SMA.

Claims

1. An IL-11 mutein cysteine mutant, characterized in that, The amino acid sequence of the IL-11 mutein sequence shown as SEQ ID NO. 1 is replaced by any amino acid from 1 to 178 with cysteine.

2. The cysteine mutant according to claim 1, characterized in that, The amino acid at position 19, 39, 40, 43, 45, 46, 108, 109, 112, 118, 121 is replaced by cysteine.

3. A method for preparing the cysteine mutant of claim 1, characterized by, The method for constructing the mutant gene comprises: performing overlap PCR with pET28a-smt3-hIL-11 mutein as a template, and performing sequencing after plasmid extraction to obtain a Cys mutant plasmid.

4. A long-acting IL-11 antagonist compound, characterized in that, The IL-11 mutein cysteine mutant of claim 1 is subjected to nucleophilic substitution of the thiol group of the cysteine side chain with the bromoacetyl group of the fatty acid, resulting in the removal of bromide ion and the formation of a thioether.

5. The long-acting IL-1 1 antagonist compound according to claim 4, wherein The fatty acid is a structure of a monoacid or a diacid with a linker, and the linker comprises PEG, γE and hexaalanine.

6. The long-acting IL-1 1 antagonist compound according to claim 4, wherein The IL-11 mutein cysteine mutant protein is prepared by the following method, comprising the steps of: ultrafiltration and concentration to a final volume of 1-2 ml, addition of TCEP, mixing at room temperature for 10-30 min, addition of a fatty acid side chain, reaction at room temperature for about 8-15 h, purification of the product by gel filtration chromatography, SDS-PAGE analysis of the target peak, and concentration of the liquid in the collection tube corresponding to the target protein.

7. The long-acting IL-1 1 antagonist compound according to claim 5, wherein The structure of the fatty acid is shown as follows: 。 8. A method of preparing the long-acting IL-11 antagonist compound of claim 4, comprising the steps of, The method comprises the following steps: The IL-11 mutein cysteine mutant protein is prepared by the following method, comprising the steps of: ultrafiltration and concentration to a final volume of 1-2 ml, addition of TCEP, mixing at room temperature for 10-30 min, addition of a fatty acid side chain, reaction at room temperature for about 8-15 h, purification of the product by gel filtration chromatography, SDS-PAGE analysis of the target peak, and concentration of the liquid in the collection tube corresponding to the target protein.

9. Use of the long-acting IL-11 antagonist compound of claim 4 in the preparation of a drug for treating organ fibrosis.

10. Use according to claim 9, characterized in that, The organ fibrosis comprises kidney fibrosis, lung fibrosis, heart fibrosis and liver fibrosis.

Images