Scavenger receptor-targeted polypeptide ligand, non-covalent dual-targeting molecule and pharmaceutical use thereof

Abstract

The present application relates to the field of biomedicine, and in particular to polypeptide ligands targeting scavenger receptors, non-covalent dual-targeting molecules and pharmaceutical uses. In particular, the present application relates to a linear polypeptide comprising a plurality of glutamic acid alanine units (‑EA‑), and also relates to molecules formed by non-covalent interactions between the above-mentioned linear polypeptide and a stapled peptide, and pharmaceutical uses of the linear polypeptide or the molecules. The polypeptide ligands of the present invention and the stable non-covalent dual-targeting molecules formed with the stapled peptide can be used to treat diseases such as psoriasis that are related to IL‑17A overexpression. At the same time, the polypeptide ligands or non-covalent dual-targeting molecules of the present invention can target and bind to scavenger receptors, have the potential ability of cell or tissue targeting and selectivity, and can be used for targeted therapy.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a polypeptide ligand targeting a scavenger receptor, a non-covalent dual-targeting molecule, and pharmaceutical uses. Background Art

[0002] At present, the development of inhibitors against disease targets is still the mainstream drug development strategy. However, the occupancy-driven paradigm adopted by target inhibitors faces defects that cannot be ignored. For example, it can only inhibit targets with catalytic activity, but it is helpless against targets that play non-catalytic or skeletal functions in the occurrence and development of diseases. This has led to the emergence of many undruggable targets. In addition, when the target is overexpressed or mutated in response to the action of the inhibitor, or the ligand of the target is overexpressed, drug resistance is generated, which greatly reduces the efficacy of the drug.

[0003] Targeted protein degradation strategies eliminate the pathogenic activity of target proteins by directly degrading them, making up for the shortcomings of inhibitor strategies in drug development. At present, the mainstream targeted protein degradation strategies are mainly through the proteolysis-targeted chimera (PROTAC) technology that hijacks the ubiquitin-proteasome degradation system, the lysosome-targeted chimera (LYTAC) technology that hijacks the endosome-enzyme degradation system, and the autophagy-lysosome-targeted chimera (AUTAC) technology. They achieve the degradation of target proteins by designing covalent chimeras that can act on target proteins and degradation-inducing molecules (ubiquitin ligases / membrane surface lysosome targeting receptors / autophagy-inducing proteins) at the same time to form a ternary complex of target protein-chimera molecules-degradation-inducing molecules. However, the current protein targeted degradation strategies also face challenges. On the one hand, the design of covalent chimeric molecules, especially PROTAC molecules, needs to consider multiple factors such as the spatial distance, orientation, and flexibility of the linker between the two ligands, which determines whether the target protein can be successfully ubiquitinated. Therefore, the matching of the coupling site, length, and flexibility of the linker requires a lot of work to optimize and screen. On the other hand, some receptors need to bind to multivalent ligands to effectively function, such as the M6PR and ASGPR receptors in the LYTAC technology. The synthesis process of multivalent / polysaccharide ligands such as M6Pn and Tri-GalNAc that bind to them is complicated, which to some extent limits the development of the corresponding target protein degradation technology in practical applications.

[0004] In addition, the lysosomal targeting receptors currently being developed for targeted protein degradation (TPD) strategies are relatively limited, mainly M6PR, ASGPR, CXCR7 and Integrinαvβ3. Some of them are widely expressed on different types of cells, and some can be highly expressed on the surface of hepatocytes or tumor cells. The development of protein degradation strategies that are selective for cells or tissues is the next stage of TPD development. Therefore, the development of more types of lysosomal targeting receptors is very important to meet the degradation needs of different diseases and targets. Recently, there have also been reports of using scavenger receptors SRs as targeting receptors to develop dendritic DNA chimeras for cancer treatment.

[0005] Scavenger receptor A (SR-A) is a pattern recognition receptor widely distributed on the surface of immune cells, such as macrophages, monocytes, dendritic cells, microglia, etc. It can recognize and induce disease-associated molecules (DAMPs) and damage-associated molecules (PAMPs) in the body to be internalized by immune cells such as macrophages and cleared through the lysosomal pathway, thereby stabilizing the body's internal environment homeostasis. Scavenger receptors prefer to recognize ligands with multiple negative charges, such as lipopolysaccharide (LPS), oxidized or acetylated low-density lipoprotein (Ac / Ox-LDL), lipid phosphate (LTA) and β-glucan. And because it is specifically expressed on specific cells, scavenger receptors are expected to achieve cell- or tissue-specific clearance of disease-related proteins. In addition, although scavenger receptors have recently been shown to be useful for targeted cancer therapy, they still have great untapped potential in the treatment of immune-related diseases, such as immune diseases caused by macrophage aggregation. Studies have shown that the enrichment of macrophages in skin lesions, especially at the interface between the dermis and epidermis, is an important feature of psoriasis and plays an important role in psoriasis-like skin inflammation. SUMMARY OF THE INVENTION

[0006] The present invention develops a scavenger receptor-dependent non-covalent dual-targeted protein degradation strategy, including designing a polypeptide ligand SRAL that can target the scavenger receptor, carrying it with a polypeptide 17Abp that has affinity for IL-17A, to form a stable non-covalent dual-targeted molecule SAncb (SRAL&17Abp non-covalent bispecific molecule) to achieve targeted degradation of IL-17A.

[0007] The present invention can avoid the complicated optimization work faced in the construction of covalent chimeric molecules. The ligand molecules can be designed with structural modules that can interact with each other to drive the two ligands to form a stable dual-targeted non-covalent binding system, which can play a role in specific removal of target proteins by forming a target receptor with the target protein and lysosome.

[0008] The polypeptide ligand SRAL of the present invention and the stable non-covalent dual-targeting molecule SAncb formed by the polypeptide 17Abp can be used to treat diseases related to IL-17A overexpression, such as psoriasis. At the same time, the polypeptide ligand or non-covalent dual-targeting molecule of the present invention can target and bind to scavenger receptors, have the potential ability of cell or tissue targeting and selectivity, and can be used for the treatment of targeted immune-related diseases.

[0009] Therefore, the present application provides the following invention:

[0010] In the first aspect, the present application provides a linear polypeptide comprising n glutamic acid alanine units (-EA-), where n is 3 to 50; optionally, the polypeptide further comprises a modification group located at the C-terminus or N-terminus, wherein the modification group comprises a polycyclic group or an alkanoyl group; optionally, there is a spacer between the modification group and the n glutamic acid alanine units (-EA-), and the spacer is an aminoalkanoic acid.

[0011] In a second aspect, the present application provides a molecule formed by the above-mentioned linear polypeptide and a stapled peptide through non-covalent interaction, wherein the stapled peptide comprises a main chain amino acid sequence selected from the following:

[0012] (a) the amino acid sequence shown as IHVTIPADLWDWINK (SEQ ID NO: 1);

[0013] (b) an amino acid sequence having at least 80%, 86.7% or 93% sequence identity to SEQ ID NO: 1; or

[0014] (c) an amino acid sequence having 1, 2 or 3 amino acid residues added or substituted compared to SEQ ID NO: 1;

[0015] wherein the amino acid residues at positions i and i+3, or the amino acid residues at positions i and i+4, or the amino acid residues at positions i and i+7 of the main chain of the stapled peptide are coupled to form a ring via a linker, and the linker comprises at least one amino acid;

[0016] The i is an integer greater than or equal to 1, and preferably an integer less than or equal to 8.

[0017] In a third aspect, the present application provides a method for preparing the molecule, comprising contacting the linear polypeptide and the stapled peptide for a certain period of time.

[0018] In a fourth aspect, the present application provides a pharmaceutical composition or formulation comprising the linear polypeptide or the molecule.

[0019] In a fifth aspect, the present application also provides the use of the linear polypeptide or the molecule in the preparation of a drug or a preparation. DETAILED DESCRIPTION OF THE INVENTION

[0021] Definition of terms

[0022] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps involved in this article are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0023] In the present invention, "molecule" refers to a whole composed of constituent atoms bonded together in a certain bonding order and spatial arrangement, and the atoms can be bonded to each other through covalent or non-covalent interactions (such as hydrogen bonds, ionic bonds or π conjugation effect (π-π stacking)).

[0024] In the present invention, "receptor" refers to a protein, which is mostly located on the plasma membrane of the target organ or in the nucleus / cytosol. The corresponding information molecule that binds to it is called "ligand". Different ligands can bind to their corresponding receptors, activate the information transmission system in the cell, and lead to changes in cell function.

[0025] In the present invention, SRAL is a linear polypeptide comprising a plurality of (eg, 3 to 50) glutamic acid and alanine units (-EA-), which can specifically bind to scavenger receptors.

[0026] In the present invention, "stapled peptide" refers to a polypeptide modified by a stapling strategy, and the "stapling strategy" refers to cross-linking the side chains of the polypeptide anchor residues with the side chains or the side chains with the terminal groups into a ring, and "staples" the polypeptide backbone with a side chain ring similar to a staple to stabilize the secondary structure conformation of the polypeptide. The stapling strategy can make the polypeptide form a stable helical conformation in advance, thereby reducing the "entropy penalty" in the target binding process.

[0027] In the present invention, 17Abp is a polypeptide that can bind to IL-17A. The polypeptide can specifically bind to IL-17A and inhibit the interaction between IL-17A and its receptor IL-17RA. An exemplary amino acid sequence of 17Abp can be shown as SEQ ID NO: 1. In the present invention, the term "17Abp" is intended to include variants of 17Abp, and the "variant" refers to a polypeptide whose amino acid sequence is different from the amino acid sequence of 17Abp by one or more (e.g., 1, 2, 3 or more) amino acids (e.g., conservative amino acid substitutions) or has at least 60%, 70%, 80%, 86.7%, 93%, 96% or higher identity, and it has the same function as 17Abp, and the "function" can be one or more of the following functions: i) binding to IL-17A, ii) inhibiting the interaction between IL-17A and its receptor IL-17RA.

[0028] In the present invention, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Usually, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0029] In the present invention, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, another amino acid residue from the same side chain family can be selected to replace the corresponding amino acid residue. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0030] In the present specification, unless otherwise specified, the numbers indicating the positions of amino acid residues in an amino acid sequence are assigned sequentially toward the C-terminal direction, with the N-terminal amino acid residue being 1.

[0031] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, fillers, diluents, binders, wetting agents, disintegrants, lubricants, surfactants, preservatives, colorants, flavoring agents, fragrances, effervescent agents, emulsifiers, flocculants, deflocculants, antibacterial agents, and solubilizers.

[0032] In the present invention, "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0033] The amount of the drug administered to the subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, weight and tolerance to the drug, and also depends on factors such as the type of preparation and the mode of administration of the drug, and the administration cycle or time interval. Those skilled in the art can determine the appropriate dosage based on these and other factors.

[0034] The inventors used the multi-negatively charged ligand preference of SR-A to design a new type of multi-negatively charged peptide SRAL targeting SR-A, which is composed of multiple glutamic acid alanine units (-EA-). SRAL can form a non-covalent dual-targeting molecule SAncb (SRL&17Abp non-covalent bispecific molecule) with the polypeptide 17Abp that can specifically bind to IL-17A through non-covalent interaction. The evaluation results of the ability of SAncb to target IL-17A degradation at the cellular level showed that compared with Chimera, SAncb can efficiently promote IL-17A to be internalized by cells and degraded via the lysosomal pathway. This process is dependent on the scavenger receptor SR-A. Therefore, the present application provides the following inventions:

[0035] Linear peptides

[0036] The present application provides a linear polypeptide, which comprises n glutamic acid alanine units (-EA-), where n is 3 to 50; optionally, the polypeptide further comprises a modification group located at the C-terminus or N-terminus, wherein the modification group comprises a polycyclic group or an alkanoyl group; optionally, there is a spacer between the modification group and the n glutamic acid alanine units (-EA-), and the spacer is an aminoalkanoic acid.

[0037] 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0038] In some embodiments, the relative molecular mass of the linear polypeptide is 700-12000, for example, 700-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000, 9000-10000, 10000-11000 or 11000-12000.

[0039] In certain embodiments, a modification group is attached to the C-terminus or N-terminus of the linear polypeptide.

[0040] In certain embodiments, the modifying group comprises a polycyclic group.

[0041] In certain embodiments, the modifying group comprises a 5-6-membered heterocycle and a 5-6-membered heterocycle, for example, -carbonyl-(C1-C6 alkyl)-(5-6-membered heterocycle and a 5-6-membered heterocycle). In certain embodiments, the modifying group comprises a 5-6-membered nitrogen-containing heterocycle and a 5-6-membered sulfur-containing heterocycle, for example, -carbonyl-(C1-C6 alkyl)-(5-6-membered nitrogen-containing heterocycle and a 5-6-membered sulfur-containing heterocycle). The 5-6-membered heterocycle and a 5-6-membered heterocycle, a 5-6-membered nitrogen-containing heterocycle and a 5-6-membered sulfur-containing heterocycle are optionally substituted.

[0042] In certain embodiments, the modifying group is biotin, an exemplary chemical structure of which is as follows:

[0043]

[0044] In certain embodiments, the modifying group comprises a dibenzo 5-6 membered cycloalkyl group, for example -carbonyl-(C 1-6 In certain embodiments, the modifying group is fluorenylmethoxycarbonyl (Fmoc), and its exemplary chemical structure is as follows:

[0045]

[0046] In certain embodiments, the modifying group comprises a dibenzo 5-6 membered heterocyclic group, for example, a dibenzo 5-6 membered oxygen-containing heterocyclic group. The dibenzo 5-6 membered heterocyclic group and the dibenzo 5-6 membered oxygen-containing heterocyclic group are optionally substituted.

[0047] In certain embodiments, the modifying group is selected from fluorescein isothiocyanate (FITC) and rhodamine B (RB), and their exemplary chemical structures are as follows:

[0048]

[0049]

[0050] In certain embodiments, the modifying group may be a fluorescent group, including but not limited to FITC, AMC, FAM, Rhodamine B, TAMRA, Cy3, Cy5, Cy7.

[0051] In certain embodiments, the modifying group is an alkanoyl group, such as an alkanoyl group containing 1-18 carbon atoms, including but not limited to acetyl.

[0052] In the present invention, "optionally substituted" means that the group is optionally substituted by one or more groups selected from the following: deuterium atoms, halogen, hydroxyl, cyano, amino, nitro, carboxyl, carbonyl, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, C 3-8 Cycloalkyl or -N(C1-C6 alkyl)2. In certain embodiments, there is a spacer between the modified group of the linear polypeptide and n glutamic acid alanine units (-EA-) to avoid mutual influence between the modified group and the -EA-unit. The spacer can be an aminoalkanoic acid, such as an aminoalkanoic acid containing 6-18 carbon atoms, such as 6-aminohexanoic acid.

[0053] The linear polypeptides include but are not limited to the linear polypeptides represented by the following formulas:

[0054]

[0055]

[0056] In the above formulae, n is preferably 10 to 15 (eg, 10, 11, 12, 13, 14 or 15).

[0057] Non-covalent dual targeting molecules

[0058] The present application provides a molecule formed by non-covalent interaction between the linear polypeptide described above and a stapled peptide, wherein the stapled peptide comprises a main chain amino acid sequence selected from the following:

[0059] (a) the amino acid sequence shown as IHVTIPADLWDWINK (SEQ ID NO: 1);

[0060] (b) an amino acid sequence having at least 80%, 86.7% or 93% sequence identity to SEQ ID NO: 1; or

[0061] (c) an amino acid sequence having 1, 2 or 3 amino acid residues added or substituted compared to SEQ ID NO: 1;

[0062] The amino acid residues at positions i and i+3, or the amino acid residues at positions i and i+4, or the amino acid residues at positions i and i+7 of the main chain of the stapled peptide are coupled into a ring through a linker, and the linker comprises at least one amino acid; i is an integer greater than or equal to 1, preferably an integer less than or equal to 8.

[0063] In some embodiments, i is 7 or 10.

[0064] In the present invention, the amino acid contained in the linker can be an L-amino acid or a D-amino acid, and can be a natural amino acid or a non-natural amino acid. In some preferred embodiments, the amino acid contained in the linker is a natural α-amino acid. In some preferred embodiments, the amino acid contained in the linker is an L-amino acid.

[0065] In addition, various types of amino acids can be used to form linkers, such as polar amino acids or non-polar amino acids, basic amino acids or acidic amino acids, hydrophobic amino acids or hydrophilic amino acids, amino acids with side chains or amino acids without side chains. The amino acids included in the linker include but are not limited to:

[0066] Non-polar amino acids (hydrophobic amino acids): glycine (Gly) alanine (Ala) valine (Val) leucine (Leu) isoleucine (Ile) phenylalanine (Phe) tryptophan (Trp) methionine (Met) proline (Pro);

[0067] Polar amino acids (hydrophilic amino acids):

[0068] 1) Polar uncharged / polar neutral amino acids: threonine (Thr), serine (Ser), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), tyrosine (Tyr);

[0069] 2) Basic amino acids (positively charged amino acids) lysine (Lys), arginine (Arg), histidine (His);

[0070] 3) Acidic amino acids (negatively charged amino acids): aspartic acid (Asp), glutamic acid (Glu).

[0071] Furthermore, the side chains carried by different amino acid linkers have different sizes and properties, some of which can play an important regulatory role in polypeptides and can also be used as handles for secondary modification for further functional derivatization. In some preferred embodiments, the amino acids contained in the linker are selected from Glu, Ala, Lys, and Ser.

[0072] In other embodiments, amino acids without side chains can be used to form the linker. In some embodiments, the amino acid contained in the linker is Gly.

[0073] The inventors found that mutating some sites on the 17Abp sequence is beneficial to eliminating the negative impact of the stapled peptide side chain loop itself on the polypeptide helical conformation, and can improve the target affinity and resistance to enzymatic hydrolysis.

[0074] In some embodiments, the 17Abp backbone amino acid sequence has 1, 2 or 3 substitutions (eg, conservative substitutions or non-conservative substitutions) of amino acid residues compared to SEQ ID NO: 1. In some preferred embodiments, the substitution is at position 7, position 10, position 14 or any combination thereof.

[0075] In some preferred embodiments, the substitution is selected from: the amino acid residue at position 7 is substituted with Glu, the amino acid residue at position 10 is substituted with Ala, the amino acid residue at position 14 is substituted with Lys, or any combination thereof.

[0076] In some more preferred embodiments, the substitution is selected from: the amino acid residue at position 7 is replaced by Ala to Glu, the amino acid residue at position 10 is replaced by Trp to Ala, and the amino acid residue at position 14 is replaced by Asn to Lys.

[0077] Particularly preferred substitutions include, but are not limited to:

[0078] (1) The amino acid residue at position 7 is replaced by Ala to Glu;

[0079] (2) the amino acid residue at position 10 was replaced by Ala from Trp;

[0080] (3) The amino acid residue at position 14 was replaced from Asn to Lys;

[0081] (4) the amino acid residue at position 7 is replaced by Ala to Glu, and the amino acid residue at position 10 is replaced by Trp to Ala;

[0082] (5) the amino acid residue at position 7 is replaced by Ala to Glu, and the amino acid residue at position 14 is replaced by Asn to Lys;

[0083] (6) the amino acid residue at position 10 is replaced by Trp to Ala, and the amino acid residue at position 14 is replaced by Asn to Lys;

[0084] (7) the amino acid residue at position 7 is replaced by Ala to Glu, the amino acid residue at position 10 is replaced by Trp to Ala, and the amino acid residue at position 14 is replaced by Asn to Lys;

[0085] (8) The amino acid residue at position 7 is substituted from Ala to Glu, the amino acid residue at position 14 is substituted from Asn to Lys, and the amino acid residue at position 1, 2, 3, 4, 5, 6, 10, 11 or 12 is substituted with Ala.

[0086] In some particularly preferred embodiments, the stapled peptide comprises a backbone amino acid sequence selected from the group consisting of:

[0087] IHVTIPEDLWDWIKK (SEQ ID NO: 2)

[0088] IHVTIPEDLADWIKK (SEQ ID NO: 3)

[0089] IHVTIPEDLKDWINK (SEQ ID NO: 4)

[0090] IHVTIPADLEDWIKK (SEQ ID NO:5).

[0091] Furthermore, the N-terminus and / or C-terminus of the main chain of the stapled peptide of the present invention may also be modified. In some embodiments, the N-terminus of the stapled peptide main chain is acetylated to reduce the hydrolysis of the peptide by exonucleases and increase the half-life of the peptide.

[0092] The coupling reaction for forming the stapled peptide of the present invention can be an amidation reaction. In some embodiments, the linker contained in the stapled peptide couples the amino acid residues at positions i and i+3, or the amino acid residues at positions i and i+4, or the amino acid residues at positions i and i+7 of the stapled peptide backbone into a ring through a peptide bond.

[0093] In some embodiments, the side chain of the amino acid residue at the i-th position of the stapled peptide main chain may contain a free carboxyl group, the side chain of the amino acid residue at the i+3-th position, the i+4-th position or the i+7-th position of the stapled peptide main chain may contain a free amino group, and the amino acid contained in the linker forms peptide bonds with the free carboxyl group and the free amino group, respectively.

[0094] In other embodiments, the side chain of the amino acid residue at the i-th position of the stapled peptide main chain may contain a free amino group, the side chain of the amino acid residue at the i+3-th, i+4-th or i+7-th position of the stapled peptide main chain may contain a free carboxyl group, and the amino acid contained in the linker forms peptide bonds with the free amino group and the free carboxyl group, respectively.

[0095] The stapled peptides include but are not limited to the stapled peptides represented by the following formulas:

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] The above-mentioned stapled peptide can be prepared by a method comprising the following steps:

[0102] (1) providing the main chain of the linker and the stapled peptide;

[0103] (2) Using the linker, the amino acid residues at positions i and i+3, or the amino acid residues at positions i and i+4, or the amino acid residues at positions i and i+7 of the main chain of the stapled peptide are coupled to form a ring.

[0104] Staple peptides can be synthesized by solid phase synthesis using a peptide synthesizer. A suitable organic solvent, such as a DMF solution, can be used; a suitable deprotection reagent (such as a DMF solution containing 20% ​​piperidine) can be used; an excess of amino acids (such as 4 times the equivalent) and a suitable condensation reagent (such as HATU / HOBt) and an appropriate amount of base (such as DIEA) can be used to carry out condensation reactions for an appropriate period of time for multiple syntheses. If necessary, after the condensation of large sterically hindered amino acids such as proline, isoleucine, threonine and valine, the condensation cycle of an amino acid can be repeated multiple times. The α-amino group can be protected by a 9-fluorenylmethoxycarbonyl protecting group ( α N-Fmoc) amino acids, such as Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-Glu(OAll)-OH, Fmoc-Lys(Dde)-OH.

[0105] The following route can be used to synthesize amino acid linkers:

[0106]

[0107] Exemplary synthetic methods include:

[0108] 1) The α-amino group is protected by a 9-fluorenylmethoxycarbonyl protecting group ( α Deprotection of amino acids containing N-Fmoc;

[0109] 2) reacting the deprotected amino acid with an excess of allyl chloroformate to obtain an amino acid linker in which the α-amino group is protected by an allyloxycarbonyl group.

[0110] The deprotection reaction can be carried out at room temperature in the presence of piperidine. Suitable solvents include, but are not limited to, dichloromethane.

[0111] The reaction of amino acid with allyl chloroformate can be carried out in the presence of a base (such as sodium bicarbonate). Suitable solvents include, but are not limited to, a tetrahydrofuran / water mixed solvent.

[0112] After obtaining the stapled peptide backbone and amino acid linker, coupling to form a ring can be achieved by amidation reaction. Exemplary methods include:

[0113] 1) removing the protecting group of the amino group on the amino acid side chain at an anchor point on the main chain, for example, using hydrazine hydrate to remove the Dde protecting group of the amino group to obtain a naked amino group;

[0114] 2) Connecting the amino acid linker whose α-amino group is protected by an allyloxycarbonyl group to the exposed amino group in the previous step through an amidation reaction;

[0115] 3) removing the allyloxycarbonyl protecting group on the amino acid linker to obtain a naked amino group, and removing the carboxyl protecting group on the amino acid side chain of another anchor point on the main chain to obtain a naked carboxyl group;

[0116] For example: treatment with tetrakistriphenylphosphine palladium to remove the allyloxycarbonyl protecting group on the amino acid linker and simultaneously remove the allyl protecting group on the amino acid side chain at another anchoring point on the backbone;

[0117] 4) The amino and carboxyl groups exposed in the previous step are condensed through an amidation reaction to form a stapled peptide.

[0118] Optionally, the method further comprises: removing other protecting groups on the main chain, and / or purifying the stapled peptide.

[0119] In the non-covalent molecules of the present invention, the linear polypeptide SRL targeting SR-A and the stapled peptide 17Abp targeting IL-17A are stably combined together through non-covalent interactions (such as ionic bonds and / or π-π stacking). The type of non-covalent interaction is related to the charge and chemical structure (especially the terminal modification structure of the polypeptide) of SRL and 17Abp respectively. In certain embodiments, the N-terminus of the linear polypeptide and the N-terminus of the stapled peptide carry the same modification group, such as acetyl, FITC or Fmoc. When the N-termini of the linear polypeptide and the stapled peptide are both modified with Fmoc, the Fmoc molecule can increase the stability and compactness of the non-covalent interaction through π-π stacking, thereby forming more effective SAncb molecules and improving the endocytosis efficiency of IL-17A. In certain embodiments, the N-terminus of the linear polypeptide and the N-terminus of the stapled peptide may carry different modification groups.

[0120] The affinity of the stapled peptide 17Abp to the linear polypeptide SRL can be tested, for example, by surface plasmon resonance (SPR) technology. In certain embodiments, the affinity is not less than 4.25 μM.

[0121] In addition, 17Abp and SRL can also produce a fluorescence resonance energy transfer (FRET) effect, proving that the two polypeptides are close to each other in the solution and the distance between them is very close (eg, less than 10 nm).

[0122] On the other hand, the present application also provides a method for preparing the non-covalent dual-targeting molecule, comprising contacting the linear polypeptide and the stapled peptide for a certain time (e.g., 8 to 12 hours). An exemplary method includes incubating the linear polypeptide and the stapled peptide in a liquid phase (e.g., a cell culture medium or a buffer solution) for 8 to 12 hours. In some embodiments, the co-incubation is carried out at room temperature. In some embodiments, the linear polypeptide and the stapled peptide are each co-incubated at a concentration of 400 to 800 μM (e.g., 400 μM, 500 μM, 600 μM, 700 μM, or 800 μM).

[0123] Pharmaceutical compositions and pharmaceutical uses

[0124] In one aspect, the present application provides a pharmaceutical composition comprising a linear polypeptide or a non-covalent dual targeting molecule of the present invention. Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. Pharmaceutical carriers that can be used in the present invention include, but are not limited to, fillers, diluents, binders, wetting agents, disintegrants, lubricants, surfactants, preservatives, colorants, flavoring agents, fragrances, effervescent agents, emulsifiers, flocculants, deflocculating agents, antibacterial agents, and solubilizers.

[0125] The pharmaceutical composition of the present invention can be prepared into various suitable dosage forms, including but not limited to: oral dosage forms, injection dosage forms (such as dosage forms suitable for subcutaneous injection, intramuscular injection or intravenous injection), inhalation dosage forms, mucosal administration dosage forms or local administration dosage forms. In certain embodiments, the pharmaceutical composition is prepared into oral dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, micropills or microtablets.

[0126] In one aspect, the present application provides use of the linear polypeptide or non-covalent dual-targeting molecule of the present invention in the preparation of a medicament for preventing or treating a disease associated with overexpression of IL-17A and / or associated with a scavenger receptor.

[0127] Scavenger receptor A (SR-A) is selectively expressed on the surface of immune cells, such as macrophages, monocytes, dendritic cells, microglia, etc. The linear polypeptide or non-covalent dual-targeting molecule of the present invention can target and bind to scavenger receptors, and thus has the potential ability of cell or tissue targeting and selectivity, and can be used for targeted therapy.

[0128] In certain embodiments, the disease involves cells that specifically express scavenger receptors, including but not limited to macrophages, monocytes, dendritic cells, microglia, and the like.

[0129] In certain embodiments, the disease can be an inflammatory or immune-related disease, such as autoinflammatory diseases (AIDs) and autoimmune diseases (ADs), such as ankylosing spondylitis (AS), rheumatoid arthritis (RA), psoriasis (Ps), psoriatic arthritis (PsA), multiple sclerosis (MS), Crohn's disease (CD).

[0130] In one aspect, the present application provides a preparation containing a linear polypeptide or a non-covalent dual targeting molecule of the present invention. In certain embodiments, the preparation is used to promote the uptake, degradation and / or clearance of IL-17A by cells that specifically express scavenger receptors, including but not limited to macrophages, monocytes, dendritic cells, microglia, etc.

[0131] In one aspect, the present application provides the use of the linear polypeptide or non-covalent dual-targeting molecule of the present invention in the preparation of a preparation, wherein the preparation is used to promote the uptake, degradation and / or clearance of IL-17A by cells, wherein the cells specifically express scavenger receptors, and the cells include but are not limited to macrophages, monocytes, dendritic cells, microglia, etc.

[0132] The preparation of the present invention can be administered in vivo or in vitro; for example, the preparation is administered to a subject to promote the uptake, degradation and / or clearance of IL-17A by cells in the subject, or the preparation is administered to in vitro cells (e.g., cell lines) to promote the uptake, degradation and / or clearance of IL-17A by cells in vitro. In certain embodiments, the preparation is used in a method comprising the following steps: contacting the preparation with cells for a certain period of time (e.g., 0.5 to 48 hours, such as 0.5 to 1 hour, 1 to 6 hours, 6 to 12 hours, 12 to 18 hours, 18 to 24 hours, 24 to 36 hours, or 36 to 48 hours).

[0133] In one aspect, the present application provides a method for treating and / or preventing a disease in a subject, comprising administering a therapeutically and / or preventively effective amount of a linear polypeptide, a non-covalent dual-targeting molecule or a pharmaceutical composition of the present invention to a subject in need thereof. The subject may be a mammal, such as bovine, equine, porcine, canine, feline, rodent, primate; wherein a particularly preferred subject is a human. The disease may be the disease described above.

[0134] In one aspect, the present application provides the use of a linear polypeptide of the present invention and a stapled peptide as defined above in combination for the preparation of a medicament for preventing or treating a disease associated with overexpression of IL-17A and / or associated with scavenger receptors. In certain embodiments, the treatment is targeted therapy.

[0135] In certain embodiments, the disease involves cells that specifically express scavenger receptors, including but not limited to macrophages, monocytes, dendritic cells, microglia, etc. The disease may be the diseases listed above.

[0136] In one aspect, the present application provides a method for treating and / or preventing a disease in a subject, the method comprising administering to the subject a linear polypeptide of the present invention and a stapled peptide as defined above, preferably, the linear polypeptide and the stapled peptide are present in the same formulation and in contact with each other. In certain embodiments, the treatment is targeted therapy.

[0137] Beneficial Effects

[0138] The present application provides a scavenger receptor-dependent SAncb non-covalent bimolecular targeted protein degradation system, which successfully achieves targeted degradation of IL-17A in vitro and in vivo. SAncb exerted significant anti-inflammatory activity in a psoriasis mouse model by clearing IL-17A, improved the psoriasis symptoms of mice, and showed a certain potential for disease treatment. It has also been successfully demonstrated that SR-A can be developed as a new lysosomal targeted degradation receptor, expanding the application of targeted degradation strategies in more disease types. It is worth mentioning that, unlike the traditional strategy of developing inhibitor drugs for IL-17A, this application demonstrates the feasibility of using macrophages to clear IL-17A to improve psoriasis symptoms, and provides a new drug design idea for the treatment of IL-17A-related diseases such as psoriasis.

[0139] Hereinafter, the embodiments of the present application are specifically disclosed in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there may be cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide SRL3 (FITC).

[0141] Figure 2 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (FITC).

[0142] Figure 3 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide chimera (FITC).

[0143] Figure 4 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRAL (biotin).

[0144] Figure 5 The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRAL (RB).

[0145] Figure 6 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Ac).

[0146] Figure 7This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Glu).

[0147] Figure 8 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Fmoc).

[0148] Fig. 9 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (Ac2).

[0149] Fig.10 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRAL (Fmoc).

[0150] Fig.11 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRAL (Ac2).

[0151] Fig.12 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRL1 (FITC).

[0152] Fig.13 This is the result of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide SRL2 (FITC).

[0153] Fig.14 The results are for evaluating the cellular entry ability of SRL peptide ligands. The concentration of the peptide incubated with cells was 10 μM.

[0154] Fig.15 The results of the co-localization experiment of peptide ligand and lysosome, the concentration of peptide incubated with cells was 10μM. The red signal is the lysosome tracer; the green signal is the peptide; the blue signal is the cell nucleus, and the yellow signal is the co-localization signal of lysosome and peptide.

[0155] Fig.16 The figure shows the uptake of exogenous IL-17A by macrophages. The concentration of the peptide incubated with the cells was 50 μM and the concentration of IL-17A was 0.5 μg / ml.

[0156] Fig.17 a shows the effect of different incubation concentrations of SAncb on the IL-17A internalization efficiency, the IL-17A concentration was 0.5 μg / ml; Fig.17b shows the effect of different incubation times of SAncb on the IL-17A internalization efficiency, the concentration of SAncb incubated with cells was 50 μM, and the concentration of IL-17A was 0.5 μg / ml. (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0157] Fig.18 a shows the co-localization result of IL-17A and lysosome, the red signal is IL-17A, the green signal is lysosome, the white signal is the cell nucleus, and the yellow signal is the co-localization signal of IL-17A and lysosome. Fig.18 b shows the effect of lysosomal inhibitor LPT on IL-17A degradation. The concentration of SAncb incubated with cells was 50 μM, and the concentration of IL-17A was 0.5 μg / ml (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0158] Fig.19 The co-localization results of IL-17A, SAncb and lysosomes are shown. IL-17A-647 is an interleukin-17A protein labeled with a red fluorescent molecule, showing a red signal; both polypeptides in the SAncb system are labeled with FITC molecules at the end, showing green; the lysosome tracer lysotracker 567 is defined as a blue signal in this experiment.

[0159] Fig. 20 a shows that the internalization of IL-17A by macrophages is an energy-dependent process; Fig. 20 b shows that the uptake of IL-17A by macrophages is mainly through clathrin-mediated endocytosis and macropinocytosis. The concentration of SAncb incubated with cells was 50 μM, and the concentration of IL-17A was 0.5 μg / ml. (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0160] Fig.21 a shows the effect of SR-A inhibitor on cellular uptake of IL-17A; Fig.21 b shows the uptake of IL-17A by cells with different SR-A expression levels. The concentration of SAncb incubated with cells was 50 μM, and the concentration of IL-17A was 0.5 μg / ml (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0161] Fig. 22 The effect of SAncb preincubation time on IL-17A internalization efficiency is shown.

[0162] Fig.23 a shows the affinity results between 17Abp and SARL evaluated by MST; Fig.23 b shows the FRET phenomenon between 17Abp and SARL, the black line is 17Abp (FITC), the red line is SAncb (FITC / RB), and the blue line is SRAL (RB).

[0163] Fig.24 The co-localization and FRET phenomenon of the peptide SRAL and the peptide 17Abp in the cell are shown. The red signal is SRAL labeled with rhodamine B, the green signal is 17Abp labeled with FITC, and the purple signal is the FRET signal of the two.

[0164] Fig.25 The effect of SAncb with different N-terminal modifications on IL-17A endocytosis is shown. The concentration of the peptide incubated with cells was 50 μM, and the concentration of IL-17A was 0.5 μg / ml (n=3, **P<0.01, ***P<0.001, ****P<0.0001).

[0165] Fig.26 Flowchart for animal experiments and control group settings.

[0166] Fig. 27 a shows the severity scores of psoriasis lesions in mice, Fig. 27 b shows the weight changes of mice. Fig. 27 c shows the spleen index of mice (n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0167] Fig.28 Shown are the skin lesions of mice and the results of skin H&E staining analysis.

[0168] Fig.29 The changes of IL-17A protein levels in mouse serum are shown (n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0169] Fig.30 The changes of IL-17A protein levels in mouse skin lesions are shown (n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0170] Fig.31 The changes in the gene levels of IL-17A and its downstream inflammatory factors in mouse skin are shown (n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0171] Fig.32 The HE staining results of the heart, liver, spleen, lung and kidney of the experimental mice are shown (Scale bar: 100 μm).

[0172] Fig.33 The process of SRAL and 17Abp forming a SAncb bimolecular system through non-covalent interaction to achieve SR-A receptor-mediated targeted degradation of IL-17A is demonstrated exemplarily. DETAILED DESCRIPTION

[0173] I. Materials and Methods

[0174] Chemical reagents and consumables: All purchased reagents were used directly without purification. The sources of reagents include Sigma-Aldrich, TCI, Adamas, J&K, Acros, Hi-Tech, Gill Biochemical, Anergy, J&K, etc. All GR grade solvents, such as ethyl acetate, dichloromethane, acetone, etc., were purchased from Tongguang Company. HPLC grade solvents were purchased from J&K Company, Sigma, Oceanpak, Acros, etc. Ether, tetrahydrofuran, toluene, dichloromethane and DMF (N, N-dimethylformamide) were purchased and purified by PURE (Innovative Technology, Inc.) solvent purification system for drying and purification. The crude peptide solution was filtered with a filter membrane before analysis and purification. The filter membrane model for analysis was Bulk 13mm syringe filter with 0.2μm GHP membrane, the filter model for preparation is BulkGHP 13mm syringe filter with0.45μm GHP membrane.

[0175] The brands of Nα-Fmoc protected amino acid compounds used for solid phase synthesis of peptides include GLBiochem, Bomeijie, CSI Biotech, and bio), BiDe Pharmaceuticals, NovaBiochem and Anaiji and other companies purchased: Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, FmocCys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OAll)-OH, Fmoc-Gly-OH, FmocHis(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, FmocVal-OH.

[0176] The resin Fmoc-Rink Amide-MBHAResin used for solid phase synthesis of peptides was purchased from Gill Biochemical Company with a loading rate of 0.355 mmol / g.

[0177] The sample bottles used for peptide sample analysis were purchased from Anpu Laboratory Technology Co., Ltd. (Shanghai); the analytical chromatographic columns were Agilent Eclipse XDB-C18 column (5 μm, 4.6×150 mm) and Exsil Pure 300C18 column (5 μm, 20×150 mm); the preparative chromatographic column was Exsil Pure 300C18 column (10 μm, 20×250 mm).

[0178] Biological reagents and consumables: Recombinant mouse interleukin 17A was purchased from Novoprotein; macrophage RAW264.7 was purchased from the National Biomedical Experimental Cell Resource Bank; culture medium and PBS buffer were purchased from Beijing Zhongke Maichen Company; fetal bovine serum was purchased from PAN Biotech, Germany; cell culture consumables were purchased from Thermo and Corning; permeabilization solution (00-8333-56) was purchased from eBioscience; 4% tissue cell fixative was purchased from Feimo Bio; PE anti-IL-17A flow cytometry antibody was purchased from Biolegend; IL-17A red fluorescent labeling kit His-tag Labeling KitRED-tri-NTA 2nd Generation was purchased from Nanotemper; Lysotracer 567 was purchased from BioTec; Trizol (15596026) was purchased from Thermo Fisher; qPCR reagents were purchased from Shanghai Yisheng Company, and PCR 8-tube set was purchased from Fisher; Leupeptin, Poly I and Poly C was purchased from Sigma-Aldrich; chlorpromazine, EIPA, and methyl-β-cyclodextrin were purchased from Yuanye Biotechnology; imiquimod cream was purchased from Sichuan Mingxinlidi Company; methotrexate was purchased from Sigma; and the ELISA kit for mouse IL-17A was purchased from Dongge Boye Company.

[0179] Instruments: The analytical HPLC of peptides was acquired by Waters Alliance e2695 HPLC equipped with Waters2489 UV / Visible (UV / Vis) Detector or Agilent Technologies 1260Infinity LC equipped with 1260VWD Detector; the preparation of peptides was performed by Hanbang Technology NP7005CHPLC equipped with Hanbang Technology NU3010C UV detector or Waters 1525Binary HPLC equipped with Waters 2489UV / VisDetector. The synthesis of peptides was performed by solid phase peptide automatic synthesizer CS Bio peptide synthesizer (CX136XT) using a synthesis mode based on Fmoc protecting group. The circular dichroism signal of the peptide was collected from the instrument Jasco J-810 circular dichroism spectropolarimeter using a quartz cuvette with a light path of 1 mm; the flow experiment used a Cytoflex six-laser flow cytometer; the confocal microscope used a Zeiss super-resolution laser confocal (LSM880+airyscan); the qPCR experiment used an ABI fluorescence quantitative PCR instrument; the fluorescence resonance energy transfer experiment and ELISA experiment were measured using the microplate reader BioTek SynergyNeo2 multi-function microplate detection system. The protein extraction from mouse skin tissue was performed using the Shanghai Jingxin tissue grinder.

[0180] Data were processed using GraphPad software.

[0181] II. General Steps

[0182] 2.1 Fmoc-SPPS post-treatment - preparation of N-terminal acetyl-protected peptides

[0183] After the solid phase synthesis is completed, the resin loaded with the peptide is transferred to the peptide synthesis tube with DCM, and 5 mL of DMF / acetic anhydride / DIEA (8 / 1 / 1, v / v / v) mixed solution is added. After shaking on a shaker for 5 minutes, the reaction liquid is discharged, and the remaining resin in the synthesis tube is washed 3 times in the order of DCM-DMF-DCM, and then dried on a vacuum water pump for 15 minutes before proceeding to the next step.

[0184] 2.2 Fmoc-SPPS post-treatment - preparation of peptide amide derivatives

[0185] After solid phase synthesis is completed, transfer the peptide-loaded resin to the peptide synthesis tube with DCM, drain the solvent on the vacuum water pump, add 8mL TFA / TIS / H2O (95 / 2.5 / 2.5, v / v / v) mixed solution to remove the peptide from the resin and remove the protective group on the side chain of the peptide, shake on a shaker for 2 hours, transfer the liquid to a glass bottle, blow dry the solution with a nitrogen stream, and wash the remaining solid with ice ether three times. Then use a water / acetonitrile mixed solution containing 5% acetic acid to fully dissolve the washed solid, filter with a filter membrane to obtain a clear solution, and then analyze and purify it.

[0186] 2.3 Fmoc-SPPS post-treatment - preparation of N-terminal FITC or rhodamine B labeled peptides

[0187] After solid phase synthesis is completed, transfer the peptide-loaded resin to a peptide synthesis tube with DCM, add 5 ml of DCM to fully swell the resin, then add 10 eq of DIEA and 1.2 eq of FITC or Rhodamine B, wrap the peptide synthesis tube with tin foil to protect it from light, shake it on a shaker overnight and drain the reaction liquid, wash the remaining resin three times in the order of DCM-DMF-DCM, and then dry it on a vacuum water pump for 15 minutes before proceeding to the next step.

[0188] 2.4 Internalization of IL-17A by macrophages

[0189] Prepare 800μM aqueous solutions of 17Abp and SRAL respectively, then take an equal volume of the two polypeptide master solutions and add them to the same EP tube, blow and mix with a gun tip, and prepare a 400μM mixed liquid SAncb, which is placed in a 4°C refrigerator for overnight incubation. At the same time, macrophages RAW264.7 were plated into 48-well plates at a number of 1 million cells per well, with 200μl DMEM culture medium per well, and cultured overnight until the cells adhered to the wall. After 12h of cell culture and polypeptide pre-incubation, IL-17A protein was added to the SAncb system, and the two were diluted with culture medium to concentrations of 50μM and 0.5μg / ml, respectively. Subsequently, it was used to replace the culture medium in the 48-well plate, and SAncb and IL-17A proteins were allowed to continue to incubate with cells for 6h. At the end of incubation, each well of cells was washed three times with PBS, and then gently blown off with a gun and transferred to an EP tube. After fixation, membrane permeabilization, and staining with anti-IL-17A fluorescent antibody, the signal intensity of intracellular IL-17A was analyzed by flow cytometry.

[0190] 2.5 Colocalization of lysosomes

[0191] 200,000 RAW264.7 cells were plated into confocal dishes and cultured overnight until they adhered to the wall. Then, the IL-17A protein was labeled with IL-17A-647 with red fluorescent molecules according to the instructions of His-tag labeling kit RED-tri-NTA. After the SAncb sample was prepared as described in 2.4, it was diluted with culture medium to a concentration of 50 μM and 0.5 μg / ml, respectively, together with the IL-17A-647 molecule, and incubated with the cells for 6 hours. Before the end of the incubation, the nuclear dye Hoechst 33342 and lysotracker were added to the dish 20 minutes in advance. At the end of the incubation, each dish of cells was washed three times with phenol red-free culture medium, and the co-localization was observed using a confocal microscope.

[0192] 2.6 Lysosomal degradation assay

[0193] Macrophage RAW264.7 cells were plated in 48-well plates at a number of 1 million cells per well, with 200 μl DMEM medium per well, and cultured overnight until the cells adhered. After 6 hours of co-culture with 50 μM and 0.5 μg / ml SAncb and IL-17A as described in 2.4, the culture medium was replaced with fresh culture medium without protein and peptide and continued to be cultured for 2 hours or 4 hours to allow the intracellular IL-17A to be fully degraded. Among them, the 2h and 4h experimental groups were each subdivided into two mutual control groups, one group added 0.1 mg / ml LPT, and the other group did not, in order to study whether the lysosomal inhibitor LPT could inhibit the degradation of IL-17A. The 0h group was a positive control that was not further cultured. After the second incubation, the cells in each well were washed three times with PBS, and then gently blown off with a gun and transferred to an EP tube. After fixation, membrane permeabilization, and staining with anti-IL-17A fluorescent antibody, the signal intensity of intracellular IL-17A was analyzed by flow cytometry.

[0194] 2.7 Verification of endocytosis mechanism

[0195] Macrophage RAW264.7 was plated in a 48-well plate at a number of 1 million cells per well and cultured overnight until the cells adhered to the wall. After the endocytosis inhibitors 100mM NaN3, 20μM EIPA, 0.5mM Me-β-cyclodextrin and 3μg / ml chlorpromazine were pre-incubated with the cells for half an hour, the SAncb and IL-17A samples prepared as described in 2.4 were added and continued to be incubated with the cells for 6 hours. At the end of the incubation, the cells in each well were washed three times with PBS, then gently blown off with a gun and transferred to an EP tube. After fixation, membrane permeabilization, and Anti-IL-17A fluorescent antibody staining, the signal intensity of intracellular IL-17A was analyzed by flow cytometry.

[0196] 2.8 Verification of scavenger receptor-mediated TPD

[0197] Macrophage RAW264.7 was plated in a 48-well plate at a number of 1 million cells per well and cultured overnight until the cells adhered. Prepare SAncb and IL-17A samples as described in 2.4, add appropriate amounts of Poly I and Poly C, dilute with culture medium to a concentration gradient of 0, 125, 250, 500, 1000 μg / ml, and at the same time ensure that the corresponding SAncb and IL-17A at each concentration are 50 μM and 0.5 μg / ml, respectively. Incubate with cells for 6 hours. At the end of incubation, wash each well of cells three times with PBS, then gently blow it down with a gun and transfer it to an EP tube. After fixation, membrane permeabilization, and Anti-IL-17A fluorescent antibody staining, the signal intensity of intracellular IL-17A was analyzed by flow cytometry.

[0198] 2.9 Affinity analysis of peptide 17Abp and SRAL - surface plasmon resonance (SPR) experiment

[0199] The affinity analysis of peptide 17Abp and SRAL was detected by Biacore 8K surface plasmon resonance detector. A biotin molecule was coupled to the N-terminus of peptide SARL, and the peptide was coupled to the chip through its interaction with the streptavidin molecule on the chip. The sites on the chip that were not coupled with peptides were then blocked with free biotin solution. Then, different concentrations of 17Abp solutions were flowed through the chip, and the response signals were measured. The affinity data KD value was calculated by Biacore 8KEvaluation Software.

[0200] 2.10 Fluorescence resonance energy transfer

[0201] N-terminal rhodamine B-modified SRAL and FITC-modified 17Abp were mixed in equal volumes to a final concentration of 320 μM and 160 μM, respectively. SAncb (RB / FITC) was prepared by pre-incubation overnight, and the presence of FRET was detected using a BioTek SynergyNeo2 microplate reader. First, the two separate polypeptides (320 μM SRAL and 160 μM 17Abp) were excited with an excitation wavelength of FITC (488 nm), and their emission wavelength spectra were plotted. Then, the sample SRAncb (RB / FITC) was excited with the same excitation wavelength, and an emission wavelength spectrum was plotted. The signal increase and decrease of the three emission wavelength spectra were compared.

[0202] 2.11 Animal experiments

[0203] This experiment has been reviewed and approved by the Experimental Animal Welfare Ethics Committee of the Biomedical Ethics Committee of Peking University (approval number: LA2022226). Female Balb / c mice aged 8-10 weeks were selected, and a 2cm×3cm hairless area was shaved on their backs. The psoriasis model was established by applying 5% imiquimod cream (62.5mg / d) to the shaved backs of the mice for 7 consecutive days. During this period, the experimental group mice were injected with SAncb every day, recorded as the SAncb group (15mg / kg / d, this concentration was calculated based on the dose of 17Abp, and the concentration of SRAL was consistent with that of 17Abp, and the two polypeptides were pre-incubated for 12h before administration). The negative control group was applied with vaseline, recorded as the Vehicle group (62.5mg / d). The positive control group was only applied with imiquimod cream, recorded as the Saline group. Both groups of mice were injected with the same volume of normal saline as the SAncb group. The control group injected with only 17Abp (15 mg / kg / d) was recorded as the 17Abp group to explore the difference in the therapeutic effect of IL-17A clearance compared with IL-17A inhibition. In addition, the positive treatment group control group was injected with commercial methotrexate (1 mg / kg / 2d), recorded as the MTX group. There were 5 mice in each group. For 7 consecutive days, the mice were weighed at 8:00 am every day, the back skin condition was recorded by taking photos, and the symptoms were evaluated. The scores were scored according to the degree of erythema, dandruff, and skin thickness, each between 0-4 points. Skin thickness was measured with a vernier caliper. The three index scores were added as the PASI (Psoriasis Area and Severity Index) score. Subsequently, imiquimod cream or vaseline was applied to its exposed back. After 6 hours, the drugs were administered sequentially by intraperitoneal injection starting at 2 pm. Whole blood of mice was obtained by orbital bleeding on the 3rd and 8th days respectively. The mice were killed on the 8th day and their back skin was cut for later use. The spleen of the mice was removed and weighed, and the spleen index was calculated.

[0204] 2.12 Evaluation of cytokine levels in mouse serum

[0205] After obtaining whole blood from mice, the blood was allowed to stand at room temperature for two hours, and then centrifuged at 6000 rpm for 15 minutes. The upper clear yellow serum was aspirated and its IL-17A content was evaluated using an ELISA kit.

[0206] 2.13 Extraction and evaluation of cytokines in mouse skin

[0207] 100 mg of skin was collected from each mouse, chopped into pieces in an EP tube, and 1 ml of normal saline was added. The skin was placed in a tissue grinder and each sample tissue was fully ground under the same conditions. The skin was then centrifuged at 1000 g for 15 min, and the supernatant was taken and the IL-17A content was evaluated using an ELISA kit.

[0208] 2.14 Tissue section preparation and H&E staining

[0209] (1) Sampling and fixation: Take the skin tissue and organs (heart, liver, spleen, lung, kidney) of mice and soak them in 4% tissue fixative for 12 hours to denature and coagulate the proteins in the tissues, avoid the autolysis and decomposition of dead cells, and maintain the morphology and structure of cells and tissues. (2) Tissue dehydration: Soak the tissues in 30%, 50%, 70%, 80%, 95%, and 100% ethanol in gradient concentrations for 3 minutes each to gradually remove the water in the tissues. Then, after standing in 100% ethanol for 1 hour, soak the tissues in a mixture of ethanol and xylene (1 / 1, v / v) for another 45 minutes, and then soak them in xylene to replace the residual alcohol in the tissues and make the tissues transparent. Soak in xylene twice, 45 minutes each time. (3) Paraffin embedding: Place the dehydrated and transparent tissue in a wax dissolving box to keep it warm temporarily, then place the tissue in a metal embedding box of appropriate size, inject paraffin liquid to seal it completely, and make sure that the surface of the tissue to be cut stands at the bottom of the embedding box. Then quickly place the tissue soaked in paraffin liquid on a cold table to cool and solidify into a block. (4) Tissue sectioning: Fix the paraffin block with embedded tissue on a microtome, cut it into thin slices with a thickness of 5μm, then unfold it in deionized water at 43°C, take the slice with a hydrophobic slide, and bake it in a 60°C oven until the water evaporates. (5) Dewaxing, hydration and H&E staining: Use an automatic dewaxing, hydrating and staining instrument for dewaxing, hydration and H&E staining, then place it in a sealing machine and seal it with resin.

[0210] 2.15 Evaluation of mRNA levels of target genes using qPCR technology

[0211] (1) mRNA extraction: Weigh 100 mg of mouse skin tissue and place it in a 5 ml EP tube. Add 1 ml Trizol reagent and soak for 5 minutes. Grind evenly with a grinding rod and let stand for 10 minutes. Pipette 500 μL of supernatant into a 1.5 ml EP tube, add 100 μL of chloroform, mix by inverting twice, and centrifuge at 12000 g at 4 °C for 15 minutes. Pipette the colorless aqueous phase (about 300 μL) on the upper layer and transfer it to a 1.5 ml EP tube. Add 300 μL of isopropanol to precipitate mRNA, mix by inverting once, centrifuge again at 12000 g at 4 °C for 10 minutes, discard the supernatant, and white mRNA solid can be observed attached to the bottom. Continue to add 100 μL of 75% ethanol to each tube, centrifuge at 7500 g at 4 °C for 5 minutes, discard the supernatant, and wait for the ethanol to evaporate naturally until the white solid becomes colorless and transparent. Add 20 μL of RNAse-free water and heat in a water bath for 10 minutes to fully dissolve the mRNA. Use Nanodrop to quantify the mRNA concentration. (2) cDNA synthesis: Add 3 μL of 5×g DNA digester Mix and 2 μg of extracted mRNA to a sterile PCR tube and make up to 15 μL with dd water. Add 5 μL ⅢSuperMixplus, mix well and incubate at 42℃ for 2 minutes on a PCR instrument to complete the synthesis of cDNA. The cDNA can be stored in a -20℃ refrigerator or proceed to the next step. (3) qPCR quantitative experiment: Take 20μL cDNA, add 180μL deionized water, and vortex to mix. Add 10μL to each well of the eight-row PCR tube. qPCR SYBR Green Master Mix (High Rox Plus), 0.4 μL primers and 8 μL cDNA, finally made up to 20 μL with deionized water, and quantitative experiments were performed on ABI fluorescent quantitative PCR instrument.

[0212] Primer sequences:

[0213] IL-6-Forward Primer:5'-TAGTCCTTCCTACCCCAATTTCC-3'

[0214] IL-6-Reverse Primer:5'-TTGGTCCTTAGCCACTCCTTC-3'

[0215] CCL20-Forward Primer:5'-GCCTCTCGTACATACAGACGC-3'

[0216] CCL20-Reverse Primer:5'-CCAGTTCTGCTTTGGATCAGC-3'

[0217] IL-22-Forward Primer:5'-GGTGACGACCAGAACATCCA-3'

[0218] IL-22-Reverse Primer:5'-CAGCAGGTCCAGTTCCCCAAT-3'

[0219] IL-22-Reverse Primer:5'-CAGCAGGTCCAGTTCCCCAAT-3'

[0220] IL-17A-Reverse Primer:5'-TTTAACTCCCTTGGCGCAAAA-3'

[0221] IL-17A-Reverse Primer:5'-CTTTCCCTCCGCATTGACAC-3'

[0222] Example 1 Preparation and characterization of polypeptides

[0223] 1. Peptide SRL3 (FITC) or SRAL (FITC) (n = 15)

[0224]

[0225] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 25-40%, using Exsil Pure300C18 column). The retention time of the product was 9.50-11.30 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain the peptide SRL3 (FITC) (12.6 mg, total yield 7.2%), and the product was a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRL3 (FITC) are shown in Figure 1 Left: UV trace (linear concentration gradient of mobile phase B was 25-40% in 30 minutes, using Agilent C18 column), t R = 10.68 min); right: mass spectrometry data, calculated C 147 H 205 N 33 O 66 Relative molecular mass of S: 3520.34Da (isotope average), [M+2H] 2+m / z=1761.17,[M+3H] 3+ m / z=1174.45. Detection results: 1761.75, 1174.77.

[0226] 2. Peptide 17Abp (FITC)

[0227]

[0228] After removing the protecting group, the crude polypeptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-55%, using Exsil Pure300C18 column). The retention time of the product was 25.00-26.00 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain polypeptide 17Abp (FITC) (12.6 mg, total yield 6.5%), and the product was a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide 17Abp (FITC) are shown in Figure 2 Left: UV trace (linear concentration gradient of mobile phase B was 30-55% in 30 minutes, using Agilent C18 column), t R = 25.43 minutes); right: mass spectrometry data, calculated C 116 H 165 N 25 O 28 Relative molecular mass of S: 2388.20Da (isotope average), [M+2H] 2+ m / z=1195.10,[M+3H] 3+ m / z=797.07. Detection result: 1195.31, 797.07.

[0229] 3. Peptide chimera (FITC) (for control)

[0230]

[0231] After removing the protecting group, the crude polypeptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-60%, using Exsil Pure300C18 column). The retention time of the product was 16.50-18.30 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain polypeptide chimera (FITC) (5.3 mg, total yield 1.9%), and the product was a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide chimera (FITC) are shown in Figure 3Left: UV trace (linear concentration gradient of mobile phase B was 30-60% in 30 minutes, using Agilent C18 column), t R = 17.95 min); right: mass spectrometry data, calculated C 242 H 356 N 56 O 89 Relative molecular mass of S: 5502.48Da (isotope average), [M+3H] 2+ m / z=1835.16,[M+4H] 3+ m / z=

[0232] 1376.62Test results: 1836.15, 1377.40.

[0233] 4. Peptide SRAL (biotin)

[0234]

[0235] After removing the protecting group, the crude polypeptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 25-40%, using Exsil Pure300C18 chromatographic column). The retention time of the product was 15.00-17.30 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain the polypeptide SRAL (biotin) (13 mg, total yield 8.0%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRAL (biotin) are shown in Figure 4 Left: UV trace (linear concentration gradient of mobile phase B was 25-40% in 30 minutes, using Agilent C18 column), t R = 16.03 min); right: mass spectrometry data, calculated C 130 H 197 N 33 O 62 Relative molecular mass of S: 3244.30Da (isotope average), [M+2H] 2+ m / z=1623.15,[M+3H] 3+ m / z=1077.43 Detection result: 1623.72, 1077.22

[0236] 5. Peptide SRAL (RB)

[0237]

[0238] After removing the protecting group, the crude polypeptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-60%, using Exsil Pure300C18 column). The retention time of the product was 11.00-12.00 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain the polypeptide SRAL (RB) (mg, total yield 6.2%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the polypeptide SRAL (RB) are shown in Figure 5 Left: UVtrace (linear concentration gradient of mobile phase B from 30% to 60% in 30 minutes, using an Agilent C18 column), t R = 11.33 min); right: mass spectrometry data, calculated C 154 H 224 N 34 O 63 Relative molecular mass: 3557.54Da (isotope average), [M+2H] 2+ m / z=1779.77,[M+3H] 3+ m / z=1186.85, 890.39. Detection results: 1779.98, 1186.98, 890.63.

[0239] 6. Peptide 17Abp (Ac)

[0240]

[0241] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-55%, using Exsil Pure300C18 column). The retention time of the product was 15.00-16.25 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain peptide 17Abp (Ac) (12 mg, total yield 12.4%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide 17Abp (Ac) are shown in Figure 6 Left: UV trace (linear concentration gradient of mobile phase B was 30-55% in 30 minutes, using Agilent C18 column), t R = 15.85 min); right: mass spectrometry data, calculated C 91 H 145 N 23 O 23 Relative molecular mass: 1929.30Da (isotope average), [M+2H]2+ m / z = 965.65, [M+3H] 2+ m / z=644.10; detection results: 965.38, 643.86.

[0242] 7. Peptide 17Abp (Glu)

[0243]

[0244] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-55%, using Exsil Pure300C18 column). The retention time of the product was 16.25-17.75 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain peptide 17Abp (Glu) (16 mg, total yield 16.0%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide 17Abp (Glu) are shown in Figure 7 Left: UV trace (linear concentration gradient of mobile phase B was 30-55% in 30 minutes, using Agilent C18 column), t R = 17.43 minutes); right: mass spectrometry data, calculated C 90 H 140 N 22 O 25 Relative molecular mass: 1930.24Da (isotope average), [M+2H] 2+ m / z=966.12; detection result: 965.45.

[0245] 8. Peptide 17Abp (Fmoc)

[0246]

[0247] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 40-55%, using Exsil Pure300C18 column). The retention time of the product was 25.00-27.25 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain peptide 17Abp (Fmoc) (15 mg, total yield 6.8%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide 17Abp (Fmoc) are shown in Figure 8Left: UV trace (linear concentration gradient of mobile phase B was 40-55% in 30 minutes, using Agilent C18 column), t R = 25.45 minutes); right: mass spectrometry data, calculated C 110 H 164 N 24 O 25 Relative molecular mass: 2221.23Da (isotope average), [M+2H] 2+ m / z=1111.62,[M+3H] 3+ m / z=741.41; detection results 1112.52, 741.98.

[0248] 9. Peptide 17Abp (Ac2)

[0249]

[0250] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-60%, using Exsil Pure300C18 column). The retention time of the product was 15.00-17.00 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain peptide 17Abp (Ac2) (11 mg, total yield 5.4%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptide 17Abp (Ac2) are shown in Fig. 9 Left: UV trace (linear concentration gradient of mobile phase B was 30-60% in 30 minutes, using Agilent C18 column), t R = 16.20 min); right: mass spectrometry data, calculated C 97 H 156 N 24 O 24 Relative molecular mass: 2041.17Da (isotope average), [M+2H] 2+ m / z=1021.59,[M+3H] 3+ m / z=681.39; detection results 1021.87, 681.72.

[0251] 10. Peptide SRAL (Fmoc)

[0252]

[0253] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-60%, using Exsil Pure300C18 column). The retention time of the product was 16.00-18.00 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain the peptide SRAL (Fmoc) (30 mg, total yield 8.9%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRAL (Fmoc) are shown in Fig.10 Left: UV trace (linear concentration gradient of mobile phase B was 30-60% in 30 minutes, using Agilent C18 column), t R = 17.20 min); right: mass spectrometry data, calculated C 141 H 204 N 32 O 63 Relative molecular mass: 3353.37Da (isotope average), [M+2H] 2+ m / z=1677.69,[M+3H] 3+ m / z=1118.79; detection results 1678.60, 1118.79.

[0254] 11. Peptide SRAL (Ac2)

[0255]

[0256] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 20-40%, using Exsil Pure300C18 column). The retention time of the product was 16.00-18.00 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain the peptide SRAL (Ac2) (21 mg, total yield 6.6%), and the product was a fluffy solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRAL (Ac2) are shown in Fig.11 Left: UV trace (linear concentration gradient of mobile phase B was 20-40% in 30 minutes, using Agilent C18 column), t R = 18.63 min); right: mass spectrometry data, calculated C 128 H 196 N 32 O 60 Relative molecular mass: 3173.32Da (isotope average), [M+2H] 2+m / z=1587.66; detection result 1588.86.

[0257] 12. Peptide SRL1 (FITC) (for control)

[0258]

[0259] After removing the protecting group, the crude peptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-60%, using Exsil Pure300C18 column). The retention time of the product was 20.00-22.00 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain the peptide SRL1 (FITC) (22.5 mg, total yield 20.6%), and the product was a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the peptide SRL1 (FITC) are shown in Fig.12 Left: UV trace (linear concentration gradient of mobile phase B was 30-60% in 30 minutes, using Agilent C18 column), t R = 20.91 min); right: mass spectrometry data, calculated C 110 H 124 N 20 O 25 Relative molecular mass of S: 2156.88Da (isotope average), [M+2H] 2+ m / z=1079.44, detection result: 1079.54.

[0260] 13. Peptide SRL2 (FITC) (for control)

[0261]

[0262] After removing the protecting group, the crude polypeptide was dissolved in 16 ml of acetonitrile / water (50 / 50, v / v) solution and purified by reverse phase high performance liquid chromatography (RP-HPLC) (the linear concentration gradient of mobile phase B was 30-60%, using Exsil Pure300C18 column). The retention time of the product was 26.90-28.30 minutes. The product fraction was collected and lyophilized under reduced pressure to obtain polypeptide SRL2 (FITC) (23.5 mg, total yield 17.0%), and the product was a fluffy yellow solid. The results of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of polypeptide SRL2 (FITC) are shown in Fig.13 Left: UV trace (linear concentration gradient of mobile phase B was 30-60% in 30 minutes, using Agilent C18 column), t R= 27.03 min); right: mass spectrometry data, calculated C 139 H 176 N 26 O 33 Relative molecular mass of S: 2769.26Da (isotope average), [M+2H] 2+ m / z=1385.63,[M+3H] 3+ m / z = 924.09 Detection results: 1385.92, 924.37. Signal 1191.43 is the raw material before coupling with FITC.

[0263] Example 2 Evaluation of the ability of linear peptides to enter macrophages and co-localize with lysosomes

[0264] Flow cytometry was used to evaluate the ability of the following three peptides to enter macrophages.

[0265]

[0266] The results are as follows Fig.14 As shown, compared with the other two peptides reported in the literature, the peptide SRL3 targeting SR-A receptor of the present invention showed an absolute advantage in cell entry, while the peptide SRL2 targeting SR-B showed the lowest cell entry efficiency.

[0267] In order to determine whether the three polypeptides escaped from the endosomes and diffused in the cytoplasm or successfully entered the lysosomes after being taken up by macrophages, confocal microscopy was used to observe whether the polypeptide ligands could co-localize with the lysosomes after entering the cells. First, the N-termini of the three polypeptides were labeled with FITC (6-aminohexanoic acid as a spacer), and then they were added to the culture medium and incubated with the cells for a period of time. After washing away the fluorescently labeled polypeptides, the confocal microscope was used to observe ( Fig.15 ). The green polypeptide signal is in the form of dots, indicating that it is not diffused in the cytoplasm, but exists in the organelles. In addition, the green signals of the three polypeptides are highly superimposed with the red lysosome signal, presenting a yellow co-localization signal, indicating that the three polypeptides can be transported to the lysosome after entering the cell. Combining the results of the cell entry experiment and the co-localization experiment, it can be seen that the polypeptide SRL3 (SRAL) targeting the SR-A receptor of the present invention has a more superior performance.

[0268] Example 3 Verification of SAncb-mediated IL-17A degradation via SR-A

[0269] 1. Verify that SAncb promotes IL-17A internalization by macrophages

[0270] In order to determine whether the two polypeptide ligands can promote the internalization of IL-17A by macrophages, the inventors designed five experimental groups: chimera, a chimeric polypeptide covalently linked to 17Abp and SARL, a control group mixture of equal volumes of SARL and 17Abp, a control group SARL, 17Abp and a control group without added polypeptides. The polypeptides were all in the form of N-terminal FITC modification. Equal concentrations of commercial IL-17A were added to the five groups, and after incubation with macrophages for 6 hours, the amount of IL-17A entering the cells was evaluated by flow cytometry. The flow cytometric results showed that neither 17Abp nor SARL could promote the large-scale internalization of IL-17A when acting alone, which shows the importance of simultaneously targeting the SR-A receptor and the target protein. However, unexpectedly, the mixture can effectively promote the uptake of IL-17A by macrophages. The intracellular signal of IL-17A in the chimera group was close to that in the control group, indicating that it can hardly help transport IL-17A into cells ( Fig.16 This suggests that the two polypeptide ligands in the mixture may form a dual-targeting system (named SAncb (SRL&17Abp non-covalent bispecific molecule)) through non-covalent interactions, which can capture IL-17A and bind to the SR-A receptor at the same time, thereby transporting IL-17A into cells.

[0271] To further confirm that SAncb can promote the internalization of IL-17A by macrophages, the inventors studied the effects of the incubation concentration and incubation time of SAncb in the culture medium. The results showed that the uptake of IL-17A by macrophages was dependent on the concentration of SAncb, that is, the higher the concentration of SAncb, the stronger the IL-17A signal transported into macrophages. And when the concentration of SAncb reached 50 μM, the uptake efficiency reached a plateau ( Fig.17 a). As the SAncb incubation time increased, the intracellular signal of IL-17A reached a peak at 6 h. After that, as time went by, the uptake efficiency gradually decreased to the same level as the blank group signal ( Fig.17 b). The inventors speculate that during the rising phase of the intracellular IL-17A signal, the concentration of IL-17A in the culture medium is high, so the uptake amount is greater than the intracellular degradation amount. Afterwards, as the incubation time increases, the IL-17A concentration in the culture medium decreases, and at this time the uptake amount is less than the intracellular degradation amount, resulting in a decrease in the intracellular IL-17A signal. This result also shows to a certain extent that macrophages degrade IL-17A after taking it into the cell, providing evidence for the effectiveness of the targeted protein degradation strategy.

[0272] 2. Verify the degradation of IL-17A by macrophages

[0273] In order to determine whether IL-17A can undergo lysosomal degradation after being taken up by macrophages, the inventors first studied the co-localization of IL-17A transported into cells and lysosomes. Confocal microscopy results ( Fig.18 a) shows that the experimental group with added SAncb molecules can observe the co-localization of IL-17A-647 and lysosomes, while the control group did not observe red IL-17A signals in the cells, indicating that in the absence of SAncb, exogenous IL-17A cannot be directly taken up by macrophages ( Fig.19 The lysosomal inhibitor LPT was used to investigate whether the lysosomal hydrolase activity would affect the degradation of IL-17A.

[0274] In another experiment, the inventors first cultured macrophages with a culture medium containing SAncb and IL-17A for 6 hours to allow them to fully absorb the IL-17A in the culture medium. The culture medium was then replaced with a fresh culture medium containing or without LPT, and the cells were continued to be cultured for 0 hours, 2 hours and 4 hours (where cells at 0 hours served as a positive control), and finally the remaining amount of IL-17A in the cells was evaluated. The results showed that after the culture medium was replaced and culture continued, the content of IL-17A in the cells decreased over time. The degradation of IL-17A in cells treated with LPT was significantly reduced compared to cells not treated with LPT at the same time, indicating that low lysosomal enzyme activity would delay the degradation of IL-17A ( Fig.18 b) In summary, SAncb molecules promote the endocytosis of IL-17A by macrophages, which then transport it to lysosomes for degradation, thereby achieving the clearance of IL-17A.

[0275] 3. Verify the endocytosis mechanism of SAncb-induced IL-17A uptake

[0276] Next, in order to verify what kind of endocytic mode IL-17A undergoes during endocytosis and degradation, the inventors first selected low temperature conditions (4°C) and ATP inhibitors (NaN3) to explore whether this process is an energy-consuming active uptake process. Fig. 20 As shown in a, both low temperature and ATP inhibitors can significantly reduce the amount of IL-17A endocytosis by macrophages (****P<0.0001), and the inhibitory effect of NaN3 is more significant (**P<0.01). This shows that under the action of SAncb, the endocytosis of IL-17A by macrophages is an energy-dependent process. Next, in the study of the specific pattern of endocytic behavior, the inventors selected three endocytosis inhibitors to carry out experiments, including the lipid valve pathway inhibitor methyl-β-cyclodextrin (Me-β-CD), the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA), and the clathrin pathway inhibitor chlorpromazine (CPM). Results ( Fig. 20 b) shows that the three inhibitors can reduce the cellular uptake of IL-17A to varying degrees, among which EIPA and CPZ have more significant inhibitory effects (****P

[0277] <0.0001), indicating that clathrin-mediated endocytosis and macropinocytosis are the main processes for macrophages to take up IL-17A. The above results show that the process of SAncb molecules inducing macrophages to take up IL-17A is an energy-dependent endocytic pathway dominated by clathrin-mediated and macropinocytosis. This is similar to the SR-A-mediated endocytic behavior.

[0278] 4. Verification of scavenger receptor SR-A mediated cellular uptake of IL-17A

[0279] Although the above results have proved that the endocytosis mode of IL-17A under the action of SAncb is similar to the SR-A mediated pathway, more direct evidence is needed to prove that the SR-A receptor is involved in this SAncb-induced protein degradation. Therefore, the inventors selected Poly I, a specific inhibitor of SR-A, to explore whether the inhibition of SR-A would reduce the endocytosis of IL-17A by cells, with Poly C as the negative control group. The flow cytometry results showed that the endocytosis efficiency of IL-17A decreased with the increase of Poly I concentration, but was not affected by Poly C (21a). In addition, the results of the study on the endocytosis efficiency of IL-17A in four cell lines with different SR-A expression levels showed that the endocytosis amount of IL-17A was positively correlated with the SR-A expression level on the cell surface (Table 21b). This suggests that SAncb molecules may be able to use this difference to achieve cell or tissue-specific targeted protein degradation. Based on the above two experimental results, it can be considered that SAncb promotes the endocytosis and degradation of IL-17A through a lysosomal targeted degradation process mediated by the SR-A receptor.

[0280] Example 4 Verification of the molecular mechanism of SAncb degradation of IL-17A

[0281] Cell experiments showed that the longer the pre-incubation time of SAncb molecules, the higher the efficiency of IL-17A internalization ( Fig. 22 ), from which it can be inferred that it takes a certain amount of time to form a stable non-covalent interaction between two polypeptides.

[0282] In order to verify the interaction between the two polypeptides, the inventors first used surface plasmon resonance (SPR) technology to test the affinity between 17Abp and SARL. The results showed that 17Abp had an affinity of up to 4.25 μM for SARL polypeptide ( Fig.23 a). In addition, 17Abp and SARL can also produce FRET effect ( Fig.23 b), proving that the two polypeptides are close to each other in the solution, and the distance is very close (less than 10nm). This also proves from another perspective that the two polypeptides have the possibility of forming a non-covalent binding body. At the same time, confocal microscopy also observed that the two molecules can co-localize in the lysosome after entering the cell, and FRET phenomenon was also observed in the cell ( Fig.24 ). This result indicates that the two polypeptides are endocytosed into the cell together at a very close distance and are finally located in the lysosome. Based on the above experimental conclusions, the inventors believe that 17Abp and SARL can form a tight dual-targeting SAncb system through non-covalent interactions, which can mediate the uptake and degradation of IL-17A by macrophages.

[0283] Example 5 Evaluation of the ability of polypeptide ligands modified with different groups to promote IL-17A internalization

[0284] Using 6-aminohexanoic acid as a spacer, this example designed three peptide ligand pairs with N-terminal acetyl Ac modification, FITC modification, and Fmoc modification, namely SAncb(Ac2), SAncb(FITC), and SAncb(Fmoc), to evaluate their ability to promote IL-17A endocytosis. Fig.25 As shown, the three SAncb molecules can significantly promote the endocytosis of IL-17A by macrophages compared with the control group, among which the Fmoc-modified polypeptide has the best effect. The inventors speculate that the Fmoc molecules at the N-termini of the two polypeptides can increase the stability and compactness of this non-covalent interaction through π-π stacking, thereby forming more effective SAncb molecules and improving the endocytosis efficiency of IL-17A.

[0285] Example 6 SAncb treats psoriasis in mice by clearing IL-17A

[0286] 1 Design and process of animal experiments

[0287] After verifying the feasibility of SAncb to clear IL-17A at the cellular level, this example further explored the IL-17A clearing ability of the co-assembly targeted degradation strategy in vivo in a mouse psoriasis model and whether mouse psoriasis can be treated by clearing IL-17A. Here, the N-terminal Fmoc-modified SAncb non-covalent molecular system that performed best in cell experiments was selected for research. A psoriasis model was established by applying imiquimod cream to the back of mice after shaving for 7 consecutive days ( Fig.26). During this period, the experimental group mice were injected with SAncb (referred to as SAncb group) every day, and the petrolatum negative control group (referred to as Vehicle group) and IMQ positive control group (referred to as Saline group) mice were injected with an equal volume of normal saline. The control group injected with only 17Abp (referred to as 17Abp group) was used to explore the difference in the therapeutic effect of IL-17A clearance compared with IL-17A inhibition. In addition, the positive treatment group was set to be injected with commercial methotrexate (referred to as MTX group).

[0288] 2. Evaluation of the therapeutic effect of SAncb on psoriasis in mice

[0289] During the 7-day modeling and drug administration, we scored the degree of skin lesions on the back of mice ( Fig. 27 a), including the degree of skin thickening, erythema and severity of dandruff. And their cumulative score PASI (Psoriasis Area and Severity Index) was calculated. The results showed that the SAncb group and the MTX group had equivalent efficacy in improving the three types of psoriasis lesions, while 17Abp only played a certain role in alleviating skin thickening. The body weight of mice treated with IMQ cream decreased to varying degrees, which is one of the manifestations of the disease. In the experiment, it was observed that SAncb played a better effect in alleviating the weight loss of mice than the MTX treatment group ( Fig. 27 b). Spleen swelling is also one of the inflammatory manifestations caused by psoriasis. After the mice were killed on the eighth day, the spleen index of each group of mice was evaluated (spleen weight / mouse body weight, mg / g). The results showed that compared with the Saline group, SAncb treatment could significantly improve the degree of spleen swelling in mice (****P<0.0001), and even better than the improvement effect of MTX (*P<0.05). However, 17Abp alone did not alleviate the spleen swelling of mice ( Fig. 27 c). In general, SAncb showed better effects than MTX in improving weight loss and spleen swelling in mice. This shows to some extent that SAncb has lower toxicity than MTX, reflecting the advantages of peptide molecules in biocompatibility.

[0290] According to the pictures of mouse back skin and their corresponding H&E staining results ( Fig.28), it can be visually observed that the back skin of the mice in the Saline group had obvious dandruff, erythema and thickened skin. The results of tissue staining showed that the stratum corneum was significantly thickened and incompletely keratinized, the spinous skin was significantly thickened, the epidermal ridges were elongated, the blood vessels at the top of the dermal papillae were dilated, and Munro bodies were present. Whether from a macroscopic or microscopic level, the mice in the Saline group showed obvious psoriasis symptoms, and the situation of the mice in the 17Abp group was similar. In contrast, the above symptoms in the SAncb and MTX treatment groups were significantly alleviated.

[0291] To evaluate whether SAncb can clear IL-17A in psoriasis mice, we measured the levels of IL-17A protein in serum and skin ( Fig.29 ). The results showed that the IL-17A protein concentration in the serum of SAncb group mice was significantly lower than that of Saline group mice on the fourth and seventh days (*P<0.05), while 17Abp alone could not reduce the concentration of IL-17A. This indicates that the SAncb strategy can also successfully eliminate the target protein IL-17A in vivo.

[0292] The evaluation of skin IL-17A content showed that compared with the Saline group, the MTX group and SAncb group could significantly reduce the IL-17A content (***P<0.001, **P<0.01), while 17Abp alone had no significant change ( Fig.30 ). In summary, SAncb successfully reduced the levels of IL-17A in mouse serum and skin, demonstrating the feasibility of SAncb's protein degradation strategy in vivo.

[0293] Subsequently, this study evaluated the anti-inflammatory activity of the SAncb system in vivo by measuring the changes in the gene expression levels of IL-17A and its downstream related inflammatory factors (IL-6, CCL20, IL-22) in mouse skin. The results showed that compared with the Saline group, both SAncb and MTX could significantly reduce IL-

[0294] The mRNA levels of 17A (****P<0.0001), IL-6 (***P<0.001), CCL-20 (****P<0.0001) and IL-22 (****P<0.0001) were significantly decreased when 17Abp acted alone. However, the mRNA levels of CCL20 (***P<0.001) and IL-22 (*P<0.05) were only decreased when 17Abp acted alone, and the effect was significantly weaker than that of SAncb ( Fig.31 ). Therefore, there is reason to believe that SAncb can achieve the effect of downregulating the expression level of IL-17A and its downstream cytokine genes by clearing IL-17A protein. And its anti-inflammatory effect is significantly better than the effect of 17Abp inhibitor alone.

[0295] After verifying the feasibility of SAncb in treating psoriasis by removing IL-17A in vivo, this example continued to evaluate its in vivo toxicity. H&E staining of various important organs (heart, liver, spleen, lung and kidney) of mice showed ( Fig.32 ), compared with the control group injected with normal saline only, the organs of mice in the SAncb group, MTX group and 17Abp group did not show abnormal changes. This result shows that SAncb has good biocompatibility and does not cause systemic toxicity to mice.

[0296] In summary, the present invention designs a two-component non-covalent peptide targeted protein degradation strategy dependent on the scavenger receptor SR-A, and uses it to remove IL-17A. First, utilizing the multi-negatively charged ligand preference of SR-A, the present invention designs the SRAL polypeptide ligand targeting SR-A. Then, SRAL and 17Abp are pre-incubated to interact with each other to form a SAncb non-covalent bimolecular system. The results of evaluating the ability of SAncb to target IL-17A degradation at the cellular level show that compared with Chimera, SAncb can efficiently promote IL-17A to be internalized by cells and degraded via the lysosomal pathway. At the same time, the present invention also verifies that this process is dependent on the scavenger receptor SR-A.

[0297] In the subsequent verification of the formation mechanism of the SAncb non-covalent bimolecular system, SPR and FRET results proved that there was an interaction between 17Abp and SRAL.

[0298] Furthermore, the present invention verifies in a psoriasis mouse model that SAncb molecules can reduce the content of IL-17A protein in mouse serum and skin lesions, and achieve targeted degradation of IL-17A in vivo. At the same time, SAncb molecules show improvement in the symptoms of psoriasis in mice (dandruff, erythema, epidermal thickening and histopathological manifestations), and show a superior ability to slow down the weight loss of mice and reduce the degree of spleen swelling in mice compared to the treatment control group MTX, indicating the advantages of the SAncb polypeptide system in biocompatibility. SAncb can also downregulate the mRNA expression level of IL-17A and its downstream inflammatory factors, and its anti-inflammatory activity is significantly higher than that of the IL-17A inhibitory polypeptide 17Abp acting alone. In addition, SAncb molecules have good biocompatibility and will not cause toxic damage to the organs of mice (heart, liver, spleen, lungs, kidneys, etc.).

[0299] Fig.33 The process of SRAL and 17Abp forming a SAncb bimolecular system through non-covalent interaction to achieve SR-A receptor-mediated targeted degradation of IL-17A is demonstrated exemplarily.

[0300] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A molecule formed by a linear polypeptide and a stapled peptide through non-covalent interaction, wherein the linear polypeptide comprises n glutamic acid and alanine units; the polypeptide further comprises a modification group located at the C-terminus or N-terminus, wherein the modification group comprises a polycyclic group or an alkanoyl group; there is a spacer between the modification group and the n glutamic acid and alanine units, wherein the spacer is an aminoalkanoic acid; The linear polypeptide is selected from the linear polypeptides represented by the following formulas: SRAL (FITC) SRAL (RB) in, RB stands for rhodamine SRAL (Fmoc) SRAL (Ac2) AC is acetyl; In the above formulas, n is 15; The stapled peptide is selected from any of the stapled peptides represented by the following formulae: Staple peptide 17Abp (FITC) Stapled Peptide 17Abp (Fmoc) Staple peptide 17Abp (Ac2) ; The N-terminus of the linear polypeptide and the N-terminus of the stapled peptide carry the same modification group.

2. A method of making the molecule of claim 1, comprising contacting the linear polypeptide and the stapled peptide for a certain period of time.

3. A pharmaceutical composition comprising the molecule of claim 1; Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

4. Use of the molecule according to claim 1 in the preparation of a medicament for preventing or treating diseases associated with overexpression of IL-17A and / or scavenger receptors; The disease is psoriasis.

5. The use according to claim 4, wherein the treatment is targeted therapy.

6. A preparation comprising the molecule of claim 1; the preparation is used to promote the uptake, degradation and / or clearance of IL-17A by cells, wherein the cells specifically express scavenger receptors; and the cells are macrophages.

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