WO2020147508A1

WO2020147508A1 - Polypeptide for inhibiting tumor metastasis and bone tumors, and use thereof

Info

Publication number: WO2020147508A1
Application number: PCT/CN2019/126726
Authority: WO
Inventors: 胡国宏; 连丞; 彭方理; 李晓逊
Original assignee: 中国科学院上海营养与健康研究所
Priority date: 2019-01-17
Filing date: 2019-12-19
Publication date: 2020-07-23
Also published as: CN111440227B; CN111440227A

Abstract

The present invention relates to a polypeptide for inhibiting tumor metastasis and bone tumors, and a use thereof. Specifically, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof has the structure of formula I or formula III. The polypeptide of the present invention or a pharmaceutically acceptable salt thereof can significantly inhibit (a) tumor metastasis; and/or (b) bone tumors; and/or (c) osteoclast differentiation and maturation.

Description

Polypeptide for inhibiting tumor metastasis and bone tumor and its application

Technical field

The invention relates to the field of biomedicine, in particular to a polypeptide for inhibiting tumor metastasis and bone tumors and its application.

Background technique

According to statistics from the British "Cancer and Skeleton" magazine, more than 250,000 people worldwide die of breast cancer each year. Tumor metastasis is the main clinical challenge leading to the death of cancer patients. Bone metastasis of breast cancer causes bone destruction, hypercalcemia, fractures, nerve compression syndrome, pain, etc., which bring greater pain to the patient.

At present, the main clinical drugs used to treat bone metastasis are bisphosphonates, neutralizing antibodies and small molecule inhibitors, all of which act to achieve therapeutic effects by inhibiting the differentiation and maturation of osteoclasts. Although bisphosphonates can reduce bone pain, the duration of treatment is not clear and there is no evidence to prove that it is helpful to extend the life of patients. Neutralizing antibody drugs include Denosumab, which inhibits RANKL. Neutralizing antibodies and small molecule inhibitors are difficult to popularize because of their high prices. Therefore, the development of low-cost, safe, and high-efficiency drugs is of great significance.

Giant cell tumors of bone account for about 6% of all primary bone tumors. The age of onset is 20-40 years old. The incidence of women is higher than that of men. At the same time, the incidence of Asian countries is higher than that of European and American countries. Giant cell tumor of bone originates from the mesenchymal tissue in the bone marrow and is mainly composed of three cell types: spindle-shaped stromal cells, monocytes and multinucleated giant cells. Multinucleated giant cells have many properties similar to osteoclasts and are considered to be the main effector cells responsible for osteolysis. Therefore, giant cell tumor of bone is a kind of osteolytic tumor, which has similar osteolytic phenomena and symptoms caused by osteoclasts to the osteolytic metastases of breast cancer and other tumors. Similar to bone metastases, the current treatments for giant cell tumor of bone are mainly bisphosphonates and denosumab.

Therefore, there is an urgent need in this field to develop a safe and effective polypeptide drug.

Summary of the invention

The purpose of the present invention is to provide a safe and effective polypeptide medicine.

In the first aspect of the present invention, there is provided an isolated polypeptide or a pharmaceutically acceptable salt thereof, characterized in that the polypeptide or a pharmaceutically acceptable salt thereof has the structure shown in Formula I:

X1-QLVAG-X2 Formula I

In the formula,

X1 is none or any peptide;

X2 is none or any peptide;

Wherein, the length of the polypeptide or a pharmaceutically acceptable salt thereof is ≤100aa, preferably ≤70aa, more preferably ≤50aa, more preferably, ≤40aa, more preferably, ≤30aa, more preferably, ≤20aa ; More preferably, it is 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa.

And the polypeptide or a pharmaceutically acceptable salt thereof has (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumor; and/or (c) inhibiting osteoclast differentiation and maturation activity.

In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, colon cancer, multiple myeloma, kidney cancer, pancreatic cancer, melanoma, lymphoma, thyroid cancer, or a combination thereof .

In another preferred embodiment, the tumor metastasis is selected from the group consisting of breast cancer bone metastasis, lung cancer bone metastasis, gastric cancer bone metastasis, liver cancer bone metastasis, colon cancer bone metastasis, multiple myeloma bone metastasis, kidney cancer bone metastasis, Pancreatic cancer bone metastasis, melanoma bone metastasis, lymphoma bone metastasis, thyroid cancer bone metastasis, or a combination thereof.

In another preferred embodiment, the bone tumor is selected from the following group: osteoclast bone tumor, giant cell tumor of bone, aneurysmal bone cyst, bone fibrous dysplasia, or a combination thereof.

In another preferred embodiment, the peptide fragment includes a tag protein.

In another preferred example, the length of X1 is 1-80aa, preferably, 1-30aa, more preferably, 1-20aa, more preferably, 1-10aa.

In another preferred example, the length of X2 is 1-65aa, preferably, 1-30aa, more preferably, 1-20aa, and more preferably, 1-10aa.

In another preferred embodiment, the X1 or X2 includes natural or unnatural amino acids.

In another preferred embodiment, the polypeptide has a cell penetrating element.

In another preferred embodiment, the length of the cell penetrating element is 4-20 amino acids, preferably 5-15 amino acids.

In another preferred example, a cyclic peptide is formed between X1 and X2.

In another preferred example, at least one pair of disulfide bonds are optionally formed between X1 and X2.

In another preferred embodiment, the polypeptide is an N-mer.

In another preferred embodiment, the N-mer has the following structure of formula II:

-(X1-QLVAG-X2-L1) _n- (II);

Wherein, X1 and X2 are defined as described above; L1 is no or connecting peptide; n is 1-10, preferably, 1-7, more preferably, 1-5; each "-" is independently a connecting peptide or Peptide bond.

In another preferred example, the length of L1 is 1-30aa, preferably, 1-20aa, and more preferably, 1-10aa.

In another preferred embodiment, the polypeptide or a pharmaceutically acceptable salt thereof has the structure of Formula III:

X1a-X2a-X3a-X4a-X5a-X6a-X7a-X8a-X9a-QLVAG-X1b-X2b-X3b-X4b-X5b-X6b (III);

among them,

X1a is none or D;

X2a is none or T or I;

X3a is none or H or K or T;

X4a is none or I or V;

X5a is none or I or L;

X6a is none or K or D or R;

X7a is none or A;

X8a is none or Q or K or H;

X9a is none or S or Y or C;

X1b is none or I;

X2b is none or K;

X3b is none or Y;

X4b is none or F or Y;

X5b is none or L or M;

X6b is none or T;

In another preferred embodiment, the sequence of the polypeptide is shown in SEQ ID NO.: 1-8.

In another preferred embodiment, the polypeptide of Formula I or Formula III has ≥50%, ≥60%, ≥70%, ≥80% compared with the polypeptide of SEQ ID NO.: 1-8, ≥90% identity (or homology).

In another preferred embodiment, the polypeptide represented by formula I or formula III retains at least ≥50%, ≥60%, ≥70%, ≥80 of the biological activity of the polypeptide shown in SEQ ID No.: 1-8 %, ≥90%, ≥100%, such as 80-500%, preferably 100-400%.

In another preferred embodiment, the biological activity refers to (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation activity.

In another preferred embodiment, the polypeptide is artificially synthesized.

In another preferred embodiment, the polypeptide is not the polypeptide shown in SEQ ID NO.: 1-8.

In another preferred embodiment, the polypeptide is selected from the following group:

(a) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1-8;

(b) The amino acid sequence shown in SEQ ID NO:1-8 is formed by the substitution, deletion or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and A polypeptide derived from (a) having the activity of (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation.

In another preferred embodiment, the polypeptide is a polypeptide shown in SEQ ID NO.: 1-8 through 1-3, preferably 1-2, more preferably 1 amino acid substitution or deletion; and/ or

It is formed by adding 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids.

In another preferred example, the length of the polypeptide is 5-150 amino acids, preferably, 5-100 aa, more preferably, 5-90, more preferably, 5-40, more preferably, 5-25, more preferably, 5-20, more preferably, 5-15, more preferably, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In another preferred embodiment, the derivative polypeptide retains ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥100%, such as 80-500%, preferably 100-400 % SEQ ID NO: 1-8 (a) inhibit tumor metastasis; and/or (b) inhibit bone tumor; and/or (c) inhibit osteoclast differentiation and maturation activity.

In another preferred embodiment, the identity of the derivative polypeptide with SEQ ID NO: 1-8 is ≥50%, preferably, ≥60%, more preferably, ≥70%, more preferably, ≥80% , More preferably, ≥90%.

The present invention also provides (a) inhibition of tumor metastasis; and/or (b) inhibition of bone tumors; and/or (c) inhibition of osteoclast differentiation and maturation activity, dimers and multimers of polypeptides represented by formula I or formula III In a polymeric form, the dimer and multimeric forms have (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation activity.

In the second aspect of the present invention, a fusion protein is provided, including:

(a) The polypeptide of the first aspect of the present invention or a pharmaceutically acceptable salt thereof;

(b) A peptide fragment fused with the polypeptide described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the peptide segment includes a carrier protein.

In another preferred embodiment, the carrier protein is selected from the following group: Fc fragment, human serum albumin (HSA), CTP, transferrin, or a combination thereof.

In another preferred embodiment, the peptide segment is modified.

In another preferred embodiment, the modification includes polyethylene glycol (PEG) modification.

In the third aspect of the present invention, an isolated nucleic acid is provided, which encodes the polypeptide of the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

In the fourth aspect of the present invention, a pharmaceutical composition is provided, including:

(a) A therapeutically effective amount of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof; and

(b) A pharmaceutically acceptable carrier or excipient.

In another preferred example, the polypeptide retains ≥70%, 75%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200 % SEQ ID NO: 1-8 (a) inhibit tumor metastasis; and/or (b) inhibit bone tumor; and/or (c) inhibit osteoclast differentiation and maturation activity.

In another preferred embodiment, the drug is administered by a mode selected from the group consisting of intravenous, intratumor, intracavity, subcutaneous or hepatic artery administration (such as injection, drip, etc.).

In another preferred embodiment, the pharmaceutical preparation is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried agents.

In another preferred embodiment, the pharmaceutical preparation is an injection.

In another preferred embodiment, the polypeptide is administered to the mammal at a dose of 0.01-100 mg/kg body weight (each time or daily).

In the fifth aspect of the present invention, there is provided a use of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof for preparing a composition or preparation, and the composition or preparation is used in (a) Inhibit tumor metastasis; and/or (b) inhibit bone tumor; and/or (c) inhibit osteoclast differentiation and maturation.

In another preferred embodiment, the composition includes a pharmaceutical composition.

In another preferred example, the bone tumor is selected from the following group: osteoclast bone tumor, giant cell tumor of bone, aneurysmal bone cyst, bone fibrous dysplasia, or a combination thereof.

In the sixth aspect of the present invention, there is provided a method for screening (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting the differentiation and maturation activity of osteoclasts, comprising the steps :

(a) Mix the CTSB protein with the test substance, and determine the binding condition of the test substance and the CTSB protein;

Wherein, if the test substance binds to the CTSB protein, it indicates that the test substance that binds to the CTSB protein is a candidate substance.

In another preferred example, the method further includes step (b): administering the candidate substance determined in step (a) to a non-human mammal, and determining its (a) tumor metastasis to the non-human mammal; and /Or (b) bone tumor; and/or (c) inhibition or treatment of osteoclast differentiation and maturation.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the test substance is selected from the group consisting of polypeptides, compounds, or combinations thereof.

In the seventh aspect of the present invention, there is provided a method of (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting the differentiation and maturation of osteoclasts, which includes the steps of: Administration of a therapeutically effective amount of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and/or the fusion protein according to the second aspect of the present invention, and/or the pharmaceutical combination according to the fourth aspect of the present invention Things.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes rodents (such as mice, rats, rabbits) and primates (such as monkeys).

In the eighth aspect of the present invention, a method for treating bone tumors is provided, which includes the steps of: administering to a subject in need a therapeutically effective amount of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and/or The fusion protein according to the second aspect of the present invention, and/or the pharmaceutical composition according to the fourth aspect of the present invention.

In another preferred embodiment, the subject is a human or non-human mammal.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

BRIEF DESCRIPTION

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Figure 1: A schematic diagram showing the structure of CST6 (also known as Cystatin E/M) and related peptides, and the binding of CST6 to its downstream target protease CTSB.

Figure 1A. Schematic diagram of the structure of the cysteine protease inhibitor Cystatin E/M (CST6) and its effective truncated GQ86, DQ51, GM-30, AY-11 and truncated negative control DR-9, where CST6, GQ86 and DQ51 are obtained by prokaryotic expression and purification; DR-9, GM-30 and AY-11 are obtained by artificial synthesis (Gill Biochemical Company).

Figure 1B. Alignment of partial sequences of CST6 in mammals. The yellow part is the conserved sequence, and the red box is the key QLVAG site for functioning. The serial number of human CST6 protein in Genbank is AAH31334.1, and other serial numbers have been indicated.

Figure 1C. CST6 protein structure diagram. The key site QLVAG that binds to the CST6 target protein CTSB is shown in red. W135 locus is shown in pink.

Figure 1D. The structure diagram of the binding of CTSB and CST6 family protein CSTA. The active cleft site of the CTSB protein (top left) is shown in purple. The QLVAG homology region of CSTA (bottom right) and CST6 is shown in red; the homology site of W135 of CST6 is shown in pink.

Figure 1E. Binding structure diagram of CTSB and its inhibitor CA-074. CA-974 is displayed in red.

Figure 2: Shows that CST6 functions by inhibiting downstream CTSB.

Figure 2A. Cathepsin L (CTSL) inhibitor Z-FY(t-Bu)-DMK (abbreviated as FY) inhibited the CTSL enzymatic activity of the osteoclast precursor cell Raw264.7. CTSL enzyme activity was detected by BioVision's kit (Catalog#K142-100).

Figure 2B. CTSL inhibitor Z-FY(t-Bu)-DMK (abbreviated as FY) cannot inhibit the differentiation of Raw264.7 into mature osteoclasts. Add Z-FY(t-Bu)-DMK or its solvent control DMSO to Raw264.7 cell culture medium, and then observe osteoclast differentiation by TRAP staining. The multinucleated giant TRAP+ positive cells are mature osteoclasts.

Figure 2C. CTSB inhibitor CA-074 Me inhibits the enzymatic activity of CTSB in osteoclast precursor cells. CTSB enzyme activity was detected by BioVision's kit (Catalog#K140-100).

Figure 2D. CA-074 Me, an inhibitor of Cathepsin B, can inhibit osteoclast differentiation. CA-074 Me or its solvent control DMSO was added to Raw264.7 cell culture medium, and then osteoclast differentiation was observed by TRAP staining. The multinucleated giant TRAP+ positive cells are mature osteoclasts.

Figure 3: Shows that CST6 recombinant protein and GQ86 polypeptide have the function of inhibiting CTSB enzyme activity.

Figure 3A. Western blotting experiments to identify recombinantly expressed CST6 wild-type and mutant (mutated with CTSB binding key sites) protein and GQ86 and DQ51 polypeptides. Both protein and peptide carry 6 tandem His tags. His antibody was used for western blotting.

Figure 3B. Coomassie brilliant blue staining experiment to identify recombinantly expressed CST6 wild-type and mutant (mutant that binds to the key site of CTSB) proteins and GQ86 and DQ51 polypeptides.

Figure 3C. The recombinantly expressed CST6 protein and polypeptide GQ86 can inhibit the enzymatic activity of CTSB.

Figure 4: Shows that the CST6 recombinant protein and GQ86 and DQ51 polypeptides have the function of inhibiting the differentiation and maturation of osteoclasts, but the CST6 mutant protein cannot inhibit the differentiation of osteoclasts.

Figure 4A. In vitro induction of osteoclasts from primary mouse bone marrow cells. CST6 mutant protein (CST6-mutant), CST6 recombinant protein, GQ86, and DQ51 were added to primary mouse bone marrow cells at concentrations of 8nM, 16nM, and 32nM, respectively, and TRAP staining was performed 7 days after the induction of differentiation. The black arrow points to a wine-red multinucleated cell with more than three nuclei as a mature osteoclast. Scale bar, 100μm.

Figure 4B. Corresponding mature osteoclast count.

Figure 5: Shows that synthetic short peptides GM-30 and AY-11 containing active sites can inhibit osteoclast differentiation in vitro, but short peptides DR-9 that do not contain active sites cannot inhibit osteoclast differentiation.

Figure 5A. In vitro induction of osteoclasts from primary mouse bone marrow cells. GQ86, DR-9, GM-30 and AY-11 were added to the primary mouse bone marrow cells at a concentration of 32 nM. After 7 days of differentiation, the TRAP staining results showed that the black arrow points to wine-red multinucleated cells with more than three nuclei. Each cell is a mature osteoclast. Scale bar, 100μm.

Figure 5B. Corresponding mature osteoclast count. *, p<0.05.

Figure 6: Shows that CST6 recombinant protein and GQ86 and DQ51 polypeptides inhibit the bone metastasis of breast cancer tumors in mice, but the CST6 mutant protein cannot inhibit bone metastasis. A mouse model of bone metastasis was constructed by injecting breast cancer cell line SCP2 into the left ventricle of the mouse. SCP2 cells are labeled with F-Luciferase, which can quantify bone metastasis through bioluminescence in vivo imaging. The protein and peptide administration concentration is 1 mg/kg/day, and the administration method is tail vein injection.

Figure 6A. Bioluminescence in vivo imaging (top) and X-ray (bottom) analysis of control and bone metastasis after CST6 or GQ86 administration. The white arrow points to the site of bone loss.

Figure 6B. Quantification of bone metastasis signals in control and CST6 and GQ86 administration mice within four weeks. *, p<0.05.

Figure 6C. Body weight changes of control and CST6 and GQ86 administration mice in the first and fourth weeks. ns, the statistical difference is not significant; *, p<0.05.

Figure 6D. Survival curve of control and CST6 and GQ86 mice within 38 days. *, p<0.05 compared with control.

Figure 6E. Bioluminescence in vivo imaging (top) and X-ray and micro-CT (bottom) analysis of control and CST6 mutant, GQ86 and DQ51 administration of bone metastases in mice at the fifth week. The white arrow points to the site of bone loss.

Figure 6F. Quantification of bone metastasis signals in mice administered with CST6-Mutant, GQ86 and DQ51 within four weeks. ns, the statistical difference is not significant; **, p<0.01.

Figure 6G. Body weight changes of mice administered with CST6-Mutant, GQ86 and DQ51 in the first and fourth weeks. ns, the statistical difference is not significant; *, p<0.05.

Figure 6H. Survival curves of mice administered with CST6-Mutant, GQ86 and DQ51 within 38 days. *, p<0.05 compared with control.

Figure 7. CST6&GQ86 acute toxicology experiment. Among them, n=2, the LD50 of CST6 was 126.61 mg/kg, and the LD50 of GQ86 was 142.23 mg/kg calculated by the modified Kou’s method.

Figure 8: Shows the therapeutic effects of CST6, GQ86, DQ51 and GM30 on giant cell tumor of bone. The conditioned medium and corresponding protein or peptide drugs from the primary cell samples of patients with giant cell tumor of bone were added to the primary mouse bone marrow for in vitro culture, and the ability of the giant cell tumor of bone samples to induce osteoclasts was analyzed.

Figure 8A. The experiment of the cells from the patient sample of giant cell tumor of bone No. 2 inducing osteoclasts from primary mouse bone marrow. While adding the conditioned medium of giant cell tumor cells of bone, 32nM CST6, GQ86 and GM30 were added respectively. After 7 days of induction of differentiation, the TRAP staining results showed that the wine-red multinucleated cells indicated by the black arrows are mature osteoclasts. CST6, GQ86 and GM30 all inhibit the osteoclast differentiation induced by giant cell tumor of bone. Scale bar, 100μm.

Figure 8B. Mature osteoclast count corresponding to Figure 8A.

Figure 8C. The experiment of inducing osteoclasts from primary mouse bone marrow by cells from patient sample of giant cell tumor of bone No. 4. The experimental method is the same as Figure 8A.

Figure 8D. Mature osteoclast count corresponding to Figure 8C.

detailed description

After extensive and in-depth research, the inventors prepared for the first time a class of CST6 protein-derived products that have (a) inhibit tumor metastasis (such as breast cancer bone metastasis); and/or (b) inhibit bone tumors (such as bone giant cells). Tumor); and/or (c) a small molecule polypeptide (such as peptide DQ51, GM30, etc.) with a molecular weight of less than 16kD (such as 6kD or 3kD) that inhibits the differentiation and maturation of osteoclasts. Specifically, the present invention integrates a variety of different technologies such as protein polypeptide production technology, and successfully developed an effective (a) inhibition of tumor metastasis (such as breast cancer bone metastasis); and/or (b) inhibition of bone tumors (such as bone giant cells) Tumor); and/or (c) a polypeptide that inhibits osteoclast differentiation and maturation, and the polypeptide of the present invention is safe and has little toxic and side effects on biological tissues. On this basis, the present inventor has completed the present invention.

CST6 protein

CST6, also known as Cystatin E/M, is a member of the cysteine protease inhibitor superfamily. In terms of structure and function, it has greater similarity with Cystatin type Ⅱ proteins, and is also a tightly bound semi- As a cystine protease inhibitor, human CST6 acts as a protease inhibitor, exists in various human body fluids and exosomes, and is encoded and expressed by the CST6 gene. Like most cystatin genes, the three exons of the human CST6 gene are separated by two introns. Exon 1 is 294 bp long and contains the 5'-untranslated region (5'-UTR) of the coding sequence and the initiation ATG codon. Exon 2 is 126bp long. Exon 3 is 188bp long, contains a TGA stop codon, 3'-UTR and a typical aataaa polyadenylation signal, followed by 20bp. The lengths of intron 1 and intron 2 are 541 and 365 bp, respectively. The human CST6 gene is transcribed into a messenger RNA (mRNA) containing 607 nucleotides (nt), with no other transcription products. The transcript consists of 53 nt 5'-UTR, 447 nt coding sequence and 107 nt 3'-UTR.

The accession number of the gene sequence of wild-type human CST6 protein is NM_001323, and the accession number of its protein sequence is NP_001314.

In addition, the accession number of the gene sequence of the wild-type mouse CST6 protein is NM_028623, which has 85% homology with the gene sequence of the wild-type human CST6 protein, and the accession number of the protein sequence is NP_082899, which is similar to wild-type human CST6. The homology of the protein sequence of the protein is 70%.

Active peptide

In the present invention, the terms "polypeptide of the present invention", "small peptide of the present invention", "short peptide CST6" or "peptide CST6" are used interchangeably, and all refer to having (a) inhibiting tumor metastasis; and/or (b) Inhibiting bone tumors; and/or (c) A protein or polypeptide of an amino acid sequence (formula I, formula III) that inhibits osteoclast differentiation and maturation activity. In addition, the term also includes variants conforming to Formula I and Formula III with CTSB inhibitory activity. These variants include (but are not limited to) the addition of one or several (usually within 5, preferably within 3, and more preferably within 2) amino acids at the N-terminal. For example, in the art, the substitution of amino acids with similar or similar properties usually does not change the function of the protein. Adding one or several amino acids to the N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention or pharmaceutically acceptable salts thereof in monomer and multimeric forms.

The present invention also includes active fragments, derivatives and analogs of the polypeptides of the present invention. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the activity of inhibiting CTSB protein. The polypeptide fragments, derivatives or analogs of the present invention can be (i) a polypeptide with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing the polypeptide of the present invention with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid The sequence is fused to the polypeptide sequence to form a polypeptide (the protein fused to the leader sequence, secretory sequence, or 6His tag sequence). According to the teachings herein, these fragments, derivatives and analogs are within the scope of those skilled in the art.

A preferred type of active derivative means that compared with the amino acid sequence of formula I and III, there are at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is composed of similar or similar properties. Amino acids are replaced to form polypeptides. These conservative variant polypeptides are best produced by amino acid substitutions according to Table 1a.

Table 1a

最初的残基Initial residue	代表性的取代Representative substitution	优选的取代Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Lys；ArgGln; His; Lys; Arg	GlnGln
Asp(D)Asp(D)	GluGlu	GluGlu
Cys(C)Cys(C)	SerSer	SerSer

Gln(Q)Gln(Q)	AsnAsn	AsnAsn
Glu(E)Glu(E)	AspAsp	AspAsp
Gly(G)Gly(G)	Pro；AlaPro; Ala	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe	LeuLeu
Leu(L)Leu(L)	Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr	LeuLeu
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	SerSer	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala	LeuLeu

The invention also provides analogs of the polypeptides of the invention. The difference between these analogs and the natural polypeptide of the present invention may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). For example, Cys can form disulfide bonds with unnatural Hcy. It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Some commonly used unnatural amino acids are listed in Table 1b below.

Table 1b

Modified (usually without changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

In a preferred example, the polypeptide of the present invention has at least one internal disulfide bond (introduced intrachain disulfide bond). Surprisingly, the existence of the internal disulfide bond not only does not affect its inhibitory activity, but also helps to extend the half-life and increase the inhibitory activity. Generally, it can be formed by conventional methods in the art, such as combining cysteine or homocysteine sulfhydryl groups under oxidizing conditions to form disulfide bonds.

A preferred polypeptide of the present invention includes SEQ ID NO.: 1-8.

The polypeptides of the present invention also include polypeptides modified from the polypeptides shown in SEQ ID NO.: 1-8.

The polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base. These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and in the form of esters, carbamates, or other conventional "prodrugs".

Coding sequence

The invention also relates to polynucleotides encoding the polypeptides of the invention. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA can be a coding strand or a non-coding strand. The sequence of the coding region encoding the mature polypeptide may be the same as the sequence of the coding region or a degenerate variant. The full-length nucleotide sequence of the polypeptide of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the polypeptide (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

The present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the polypeptide coding sequence of the present invention.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies or antibody fragments specific to the polypeptides of the present invention, especially monoclonal antibodies.

In the context of two nucleic acids or polypeptides, the term "substantially identical" refers to two or more sequences or subsequences that have at least about 80%, such as at least about 85 %, about 90%, about 95%, about 98%, or about 99% of the nucleotide or amino acid residues are identical to a specific reference sequence, as determined by using the following sequence comparison method and/or by visual inspection.

Preparation

The polypeptide of the present invention can be a recombinant polypeptide or a synthetic polypeptide. The polypeptide of the present invention can be chemically synthesized or recombinant. Correspondingly, the polypeptide of the present invention can be artificially synthesized by conventional methods, or can be produced by recombinant methods.

A preferred method is to use liquid phase synthesis technology or solid phase synthesis technology, such as Boc solid phase method, Fmoc solid phase method or a combination of the two methods. Solid-phase synthesis can quickly obtain samples, and an appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide. For example, the preferred solid phase carrier in the Fmoc system is the Wang resin connected with the C-terminal amino acid in the peptide. The Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; 25% hexahydropyridine is used /Dimethylformamide is treated at room temperature for 20 minutes to remove the Fmoc protecting group and extend from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After the synthesis is completed, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protective group is removed. The crude peptide can be separated by ether precipitation after the resin is filtered off. After lyophilizing the resulting product solution, the desired peptide is purified by gel filtration and reverse phase high pressure liquid chromatography. When using the Boc system for solid-phase synthesis, the preferred resin is PAM resin connected with the C-terminal amino acid in the peptide. The PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl phenylacetamide; synthesized in Boc In the system, in the cycle of deprotection, neutralization, and coupling, the protective group Boc is removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane. The peptide chain condensation is completed Afterwards, treated with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0°C for 1 hour to cut the peptide chain from the resin while removing the protective group. With 50-80% acetic acid (containing A small amount of mercaptoethanol) to extract the peptide, the solution is lyophilized and further separated and purified with molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide. Various couplings known in the field of peptide chemistry can be used Reagents and coupling methods to couple each amino acid residue, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate can be used (HBTU) for direct coupling. For the synthesized short peptide, its purity and structure can be confirmed by reversed-phase high performance liquid phase and mass spectrometry analysis.

In a preferred example, the polypeptide of the present invention is prepared by solid-phase synthesis according to its sequence, purified by high performance liquid chromatography to obtain high-purity target peptide freeze-dried powder, and stored at -20°C.

Another method is to use recombinant technology to produce the polypeptide of the present invention. Through conventional recombinant DNA technology, the polynucleotide of the present invention can be used to express or produce a recombinant polypeptide of the present invention. Generally speaking, there are the following steps:

(1) Use the polynucleotide (or variant) of the present invention encoding the polypeptide of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify proteins from culture medium or cells.

The recombinant polypeptide can be expressed in the cell or on the cell membrane or secreted out of the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Because the polypeptide of the present invention is relatively short, it is possible to consider connecting multiple polypeptides in series to obtain a multimeric expression product after recombinant expression, and then forming the required small peptides by methods such as restriction enzyme digestion.

Cell penetrating element

As used herein, the terms "cell penetrating element" and "cell penetrating peptide" are used interchangeably and both refer to the ability to effectively penetrate the inhibitory polypeptide into the cell without any damage to the cell without affecting the inhibitory polypeptide. Active small peptides.

Pharmaceutical composition and method of administration

On the other hand, the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient . The amount of the polypeptide of the present invention or its pharmaceutically acceptable salt is usually 10 micrograms to 100 mg/dose, preferably 100 to 1000 micrograms/dose.

For the purpose of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof. In addition, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be used singly or together with other therapeutic agents (for example, formulated in the same pharmaceutical composition).

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.

The pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol. In addition, these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.

Generally, the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.

Once the composition of the invention is formulated, it can be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The objects to be prevented or treated can be animals; especially humans.

When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the use situation. It is preferably an intravenous preparation or an intratumor injection.

These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic Isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a usual manner according to the dosage form.

For example, the preparation of eye drops can be carried out by dissolving the polypeptide of the present invention or a pharmaceutically acceptable salt thereof together with the basic substance in sterile water (a surfactant is dissolved in sterile water) to adjust the osmotic pressure And the pH to the physiological state, and appropriate pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants, and thickeners can be added optionally, and then completely dissolved.

The pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation. For example, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained-release polymers, ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the polypeptide of the present invention or its pharmaceutically acceptable salt as an active ingredient can be adjusted according to the weight, age, sex, and degree of symptoms of each patient to be treated. Reasonably determine.

The main advantages of the present invention include:

(a) The polypeptide of the present invention and its derivative polypeptides have small molecular weight, small toxic and side effects on biological tissues, and high safety.

(b) The polypeptide of the present invention can effectively inhibit tumor metastasis; and/or bone tumor; and/or osteoclast differentiation and maturation.

(c) It can be prepared by solid-phase synthesis, with high purity, large output and low cost.

(d) The polypeptide of the present invention has good stability.

(e) The polypeptide of the present invention has high specificity.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow the conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacturer The suggested conditions.

Unless otherwise specified, the reagents or materials used in the embodiments of the present invention are all commercially available products.

Methods of peptide screening:

Through the SWISS-MODLE homology modeling method of the free web interface of ExPASy server (http://swissmodel.expasy.org), the binding area obtained by the crystal structure analysis of the combination of CTSB or CTSL and CST6 is used as the activity pocket of the respective enzymes. And based on this area, through molecular docking software (such as Zdock, reference: PLoS One.2011; 6(9): e24657; surfex, reference: J.Med.Chem.2003:46(4):499-511 ) Screen out polypeptide fragments with high affinity for active pockets.

The polypeptide sequences obtained by screening are shown in the following table:

Example 1

The cloning of CST6 (Cystatin E/M), GQ86 and DQ51 genes and the construction of their prokaryotic expression system 1. Primer design: According to the cDNA sequence of human CST6 provided by NCBI, in accordance with the general primer design principles and the requirements of pET28a(+) cloning, Primer5.0 was used to design RT-PCR primers CST6-F and CST6-R using the coding region as a template to directly amplify the CST6 mature peptide coding sequence.

The 5'-end primer (CST6-F) of the human CST6 gene cDNA sequence (used to clone the human CST6 gene mature peptide coding sequence):

5'-CATGCCATGGCGCGTTCGAACCTCC-3' (SEQ ID NO.: 9); wherein the 5'-end has an Ncol restriction site;

The 3'-end primer (CST6-R) of the cDNA sequence of the human CST6 gene (used to clone the mature peptide coding sequence of the human CST6 gene):

5'-CCGCTCGAGCATCTGCACACAGTTGTGC-3' (SEQ ID NO.: 10); the primer contains a restriction site Xhol and a stop codon.

2. Extraction of total RNA: Digest the human breast cancer cell line MDA-MB-231 from the culture dish with trypsin, collect the cells in a 1.5ml EP tube, and then lyse with 1ml Trizol. Vibrate for 30s. Add 0.2mL chloroform, shake vigorously for 30s, and room temperature for 2min. Centrifuge at 13000-13300rpm at 4°C for 15min. Suck the upper colorless water phase and transfer it to another EP tube. Add an equal volume of isopropanol and place it at -20°C for 30 minutes. Centrifuge at 13000-13300 rpm for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% ethanol, and shake. Centrifuge at 13000-13300rpm at 4°C for 5min. Discard the supernatant, suck up the remaining liquid, and dry at room temperature for 5 min. The precipitate is dissolved in 40-100μL DEPC water. Take out 1μL and measure its OD260/OD280. Store the extracted RNA in a refrigerator at -80°C.

3. Reverse transcription synthesis of single-stranded cDNA: Takara (D2680A) reverse transcription PCR kit is used for the synthesis of single-stranded cDNA. The PCR reaction system and procedures are as follows:

The reverse transcription PCR program is as follows:

42℃ 10min

95℃ 2min

4. PCR amplification of CST6

Using the primers CST6-F and CST6-R to use the single-stranded cDNA synthesized by reverse transcription as a template, the Q5 enzyme amplifies the target gene fragment. The amplification system and procedures are as follows:

PCR program:

5. CST6 product recovery

Prepare 1% agarose gel macroporous gel, add the PCR product of CST6 into the gel tank for electrophoresis detection, voltage 120V; 15-30min later, tap the gel under the tapping machine to recover the target fragments, use ordinary agarose gel DNA recovery reagents Box (Tiangen Biochemical Technology Co., Ltd.) to recover the product; use the restriction endonuclease produced by NEB company for restriction enzyme digestion, water bath 37 ℃ overnight or digestion for two hours, according to the characteristics of the enzyme, refer to the NEB website restriction instruction plan; restriction enzyme digestion After completion, use PCR product recovery kit (Tiangen Biochemical Technology Company) to purify and recover. Both CST6 product and pET28a(+) plasmid are double digested

The digestion system is as follows:

6. Connect the PCR product of CST6 and pET28a(+)

Use T4 ligase (NEB) to ligate the purified vector DNA and insert DNA according to the ratio of the number of molecules of 1:10 at 16°C overnight.

The connection system is as follows

7. Transform competent bacteria and pick out single clones

1) 50μl of DH5α competent bacteria, thawed on ice for 10min.

2) Add 20 μl of the ligation product to 50 μl of DH5α competent bacteria, and incubate on ice for 30 minutes.

3) Open the water bath to 42°C in advance, put the incubated competent bacteria mixture in the water bath for 90 seconds, and then immediately place it on ice for 2 minutes.

4) Add 400 μl of antibiotic-free LB liquid medium to the placed competent bacteria, and shake the bacteria in a constant temperature shaker at 37°C for 45 minutes.

5) After centrifugation at 1000g for 5 minutes, the supernatant is sucked off, leaving 50-100ul of the bacterial solution. After pipetting it evenly, spread it evenly on the Kanar-resistant LB solid plate with a coating rod.

6) The preparation of LB is shown in the table below

7) After the colonies on the plate grow to a size of about 0.5 mm in diameter visible to the naked eye, pick up the monoclonal colonies with a small pipette tip and pipette them into the EP tube pre-added with 400 μl of kana-resistant LB liquid medium.

8) Shake the bacteria in a constant temperature shaker at 37°C and 250 rpm for 2 hours.

8. Bacterial liquid identification, preservation and plasmid extraction

1) Bacteria detection uses the laboratory's self-made pTaq enzyme, the PCR program is the same as that of gene fragment amplification, and the PCR system is as follows:

2) 1% agarose gel electrophoresis of the PCR products, after UV DNA imaging, take 100μl of the corresponding bacterial solution of the positive clone and send it for sequencing.

3) Add the bacteria solution with correct sequencing to a centrifuge tube of 5ml Kanak-resistant LB medium, and shake the bacteria overnight in a constant temperature shaker at 37°C.

4) The next day, take 500 μl of bacterial solution and store it in 50% glycerol, and store it in a refrigerator at -80°C.

5) Use a small amount of plasmid extraction kit to extract the plasmid from the remaining bacterial liquid, and measure the concentration with nanodrop.

9. GQ86 and DQ51 use the same method as CST6 to construct GQ86/pET28a(+) and DQ51/pET28a(+) plasmids (Figure 1A). The only difference is the use of different primers, where the primer GQ86-F:

5'-CATGCCATGGGAGAACTCCGGGACCTGTCG-3' (SEQ ID NO.: 11); wherein the 5'-end has an Ncol restriction site;

The primer GQ86-R:

5'-GCCTCGAGCTGCTGCGCCCCTGCTG-3' (SEQ ID NO.: 12); the primer contains a restriction site Xhol and a stop codon.

The primer DQ51-F:

5’-CATGCCATGGACACGCACATCATCAAGGCG-3’; (SEQ ID NO.: 13) where the 5’-end has an Ncol restriction site;

The primer DQ51-R:

5'-GCCTCGAGCTGCTGCGCCCCTGCTG-3' (SEQ ID NO.: 14); the primer contains a restriction site Xhol and a stop codon.

Example 2

Prokaryotic expression and purification of CST6/pET28a(+), GQ86/pET28a(+) and DQ51/pET28a(+) recombinant plasmids

1. Approximately 200 ng of the validated CST6/pET28a(+), GQ86/pET28a(+) and DQ51/pET28a(+) recombinant plasmids were added to 50μl of competent E. coli BL21(DE3) cells, and incubated on ice for 30min. The water bath is opened to 42°C in advance, and the incubated competent bacterial mixture is placed in the water bath to heat shock for 90 seconds, and then immediately placed on ice for 2 minutes. Add 400 μl of antibiotic-free LB liquid medium to the placed competent bacteria, and shake the bacteria in a constant temperature shaker at 37°C for 45 minutes. After centrifuging the bacterial solution at 1000 g for 5 minutes, the supernatant was sucked off, leaving 50-100ul of the bacterial solution, which was blown evenly, and then spread evenly on the Kanar-resistant LB solid plate with a coating rod. When the colonies on the plate grow to a size of about 0.5 mm in diameter visible to the naked eye, pick up the monoclonal colonies with a small pipette tip, and pipette into the EP tube pre-added with 400 μl of kana-resistant LB liquid medium. Shake the bacteria in a constant temperature shaker at 37°C and 250 rpm for 2 hours.

2. Transfer 400μl of bacterial solution to 500ml LB liquid medium (containing 50mg/ml kanamycin) and shake culture at 37°C. When OD600=0.5, add IPTG with a final concentration of 1mM and induce expression at 18°C for 10hr, 10000 The cells were harvested by centrifugation at ×g for 10 min. Then follow the Novagen pET system instructions (TB055) and His fusion tag protein purification kit instructions (TB054) inclusion body method to purify the protein. Dialysis once for 3 hours, 2 to 3 times; dialysis buffer is TGE buffer (50mM Tris-HCl; 0.5mM EDTA; 50Mm NaCl; 5-50% glycerol; pH 8.0). The protein was detected by Western blotting with His antibody to detect whether it contained the target protein (Figure 3A), the whole protein was detected by Coomassie brilliant blue staining (Figure 3B), and the concentration of purified protein was detected by BCA. Finally, use endotoxin high-efficiency removal and purification resin (Yusheng Biotechnology, 20518ES10) to remove endotoxin, and store at -80℃ for later use.

Example 3

1. Homology analysis of active site

The MEGA software was used to align the sequence of CST6 in some mammals. The key functional site of CST6, QLVAG, is very conserved in mammals (Figure 1B).

2. Protein function binding prediction

Use the ProteinWorkshop tool of the PDB public Web page (https://www.rcsb.org/) to analyze the protein structure and protein-protein, protein-small molecule interaction structure, and it can be found that the key site QLVAG in the CST6 protein forms a prominent ring structure (Figure 1C), its homologous site on the same family protein CSTA can be docked with the active cleft site of CTSB (Figure 1D), and the CTSB inhibitor CA-074 can form a similar docking with the same active cleft site of CSTB ( Figure 1E).

Example 4

1. Enzyme activity detection

CTSB enzyme activity was detected by BioVision's kit (Catalog#K140-100). CTSL enzyme activity was detected by BioVision's kit (Catalog#K142-100) (Figure 2A, 2C). Shows that inhibition of CTSB enzyme activity, but not CTSL enzyme activity, can inhibit osteoclast differentiation and maturation (Figure 2B, 2D). Combined with the experiment in Figure 3C, it is confirmed that CTS6 inhibits the function of downstream CTSB through its key active site.

Example 5

1. Osteoclast differentiation and maturation experiment

1) After 4 weeks to 6 weeks, BALB/c mice were sacrificed and the femurs and tibias were taken and rinsed with PBS three times.

2) Cut off both ends of the hind limb bones, and suck the α-MEM culture solution with an insulin syringe to flush out the bone marrow cells in the hind limb bones.

3) Filter the bone marrow cell suspension with a 40μm filter, centrifuge at 1500g at 4°C for 5 minutes, remove the supernatant, add an appropriate amount of culture medium to neutralize, add the red blood cell lysate to split the red blood cells for 10 minutes.

4) Centrifuge at 1500g for 5 minutes at 4°C, transfer the cells into a culture dish, and routinely culture them overnight with α-MEM medium (Invitrogen, A1049001) (containing 20% FBS).

5) Spread sterile glass slides on a 24-well plate, count the suspended bone marrow cells cultured overnight, and pave the 24-well plate with 1 million cells per well. Add α-MEM culture medium (containing 20% FBS, RANKL 50-100ng/ml, M-CSF 25ng/ml), and conditioned medium of primary cultured cells of patients with giant cell tumor of bone (final concentration is 10-20%) .

6) The cells are changed once every three days, and the experiment ends on the sixth day. The medium used is the same as before.

7) For TRAP staining, refer to the relevant kit instructions (Sigma, 387A-1KT). After drying, the slides are sealed with neutral resin.

8) Observe and count the cells that are wine-red and have more than three nuclei on the slide (Figure 4A, Figure 5A, Figure 8A and Figure 8C) and count (Figure 4B, Figure 5B, Figure 8B and Figure 8D). Regardless of adding self-purified protein (Figure 4 (4A-4B)) or artificially synthesized (Figure 5 (5A-5B)) peptides containing QLVAG sequence have the ability to inhibit the differentiation and maturation of osteoclasts, and for two clinical cases Osteolytic giant cell tumor samples of bone also have the ability to inhibit the differentiation and maturation of osteoclasts (Figure 8 (8A-8D)).

Example 6

1. Mouse left ventricular injection

1) Anesthesia: 1% sodium pentobarbital is injected intraperitoneally for anesthesia, and the required anesthetic dose is calculated at the amount of 30-40 mg/kg for each nude mouse. Sterilize the front chest wall with 75% alcohol. Touch the most obvious part of the apex of the heart, about 3mm to the left of the sternum and the second intercostal space, aim at the center and enter the needle at 45 degrees to the body. If you can see bright red blood spurting out, it means The needle has entered the left ventricle. After slowly pushing the cell suspension in, the needle is quickly withdrawn.

2) The amount of injected cells is: inject at a concentration of 5.0×10 ⁵ cells/ml and at a volume of 0.1 ml/head. D-Luciferin was injected at the bottom of 0.1ml/mouse at a dose of 5mg/ml (nude mice intravenous injection is the same), and Berthold Imaging System imaging was performed weekly (Figure 6A, Figure 6E) and photographed. After the injection, continue to raise, eat freely, observe the life state and general conditions of the nude mice; closely observe the vital signs and state of the nude mice within 24 hours after the injection.

2. Administration

CST6, GQ86 and DQ51 were administered by tail vein injection at a concentration of 1 mg/kg, and the administration cycle was once a day. The results of the experiment were presented in two times. The first time was administered with TGE buffer-Control, CST6 and GQ86 (Figure 6A-6D); the second time was administered with TGE buffer-Control, CST6-Mutant, GQ86 and DQ51 (Figure 6E- 6H). Berthold Imaging System imaging every week (Figure 6A, Figure 6E), take pictures, use the built-in software Indigo2.0 to quantify the fluorescence signal (Figure 6B, Figure 6F), weigh its weight (Figure 6C, Figure 6G), and finally count 40 Survival curve within days (Figure 6D, Figure 6H). It can be found that after administration of the polypeptide containing the QLVAG sequence, the signal of breast cancer bone metastasis is significantly weakened, and the body weight and survival curve of the corresponding experimental animals have a certain effect, which can inhibit the bone metastasis of breast cancer.

Example 7

Acute Toxicology Experiment

Take 10 BALB/c mice and divide them into 5 groups with 2 as a group, half of the male and female. CST6 and GQ86 were administered via tail vein injection at concentrations of 25mg/kg, 50mg/kg, 90mg/kg, 120mg/kg and 200mg/kg. Observe the symptoms of experimental animals within 24 hours after one administration and record the number of deaths. Finally, using the modified Kou’s method, the median lethal dose (LD50) of CST6 was 126.61 mg/kg, and the median lethal dose (LD50) of GQ86 was 142.23 mg/kg (Figure 7).

The results show that the polypeptide of the present invention has low toxicity and good safety.

All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

An isolated polypeptide or a pharmaceutically acceptable salt thereof, characterized in that the polypeptide or a pharmaceutically acceptable salt thereof has the structure shown in formula I:

X1-QLVAG-X2 Formula I

In the formula,

X1 is none or any peptide;

X2 is none or any peptide;

Wherein, the length of the polypeptide or a pharmaceutically acceptable salt thereof is ≤100aa, preferably ≤70aa, more preferably ≤50aa, more preferably, ≤40aa, more preferably, ≤30aa, more preferably, ≤20aa ；

And the polypeptide or a pharmaceutically acceptable salt thereof has (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumor; and/or (c) inhibiting osteoclast differentiation and maturation activity.
The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the tumor is selected from the group consisting of breast cancer, lung cancer, gastric cancer, liver cancer, colon cancer, multiple myeloma, kidney cancer, pancreas Cancer, melanoma, lymphoma, thyroid cancer, or a combination thereof.
The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the tumor metastasis is selected from the group consisting of breast cancer bone metastasis, lung cancer bone metastasis, gastric cancer bone metastasis, liver cancer bone metastasis, colon cancer bone metastasis Metastasis, multiple myeloma bone metastasis, kidney cancer bone metastasis, pancreatic cancer bone metastasis, melanoma bone metastasis, lymphoma bone metastasis, thyroid cancer bone metastasis, or a combination thereof.
The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the polypeptide or a pharmaceutically acceptable salt thereof has the structure of Formula III:

X1a-X2a-X3a-X4a-X5a-X6a-X7a-X8a-X9a-QLVAG-X1b-X2b-X3b-X4b-X5b-X6b

(III);

among them,

X1a is none or D;

X2a is none or T or I;

X3a is none or H or K or T;

X4a is none or I or V;

X5a is none or I or L;

X6a is none or K or D or R;

X7a is none or A;

X8a is none or Q or K or H;

X9a is none or S or Y or C;

X1b is none or I;

X2b is none or K;

X3b is none or Y;

X4b is none or F or Y;

X5b is none or L or M;

X6b is none or T;

Wherein, the length of the polypeptide or a pharmaceutically acceptable salt thereof is ≤100aa, preferably ≤70aa, more preferably ≤50aa, more preferably, ≤40aa, more preferably, ≤30aa, more preferably, ≤20aa ；

And the polypeptide or a pharmaceutically acceptable salt thereof has (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumor; and/or (c) inhibiting osteoclast differentiation and maturation activity.
A fusion protein, characterized in that it comprises:

(a) The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof;

(b) A peptide fragment fused with the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.
An isolated nucleic acid, characterized in that the nucleic acid encodes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition, characterized in that it comprises:

(a) A therapeutically effective amount of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; and

(b) A pharmaceutically acceptable carrier or excipient.
A use of the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that it is used to prepare a composition or preparation, and the composition or preparation is used for (a) inhibiting tumor metastasis; and/or (b) Inhibiting bone tumors; and/or (c) Inhibiting osteoclast differentiation and maturation.
A method for screening (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting the differentiation and maturation activity of osteoclasts, characterized by comprising the steps of:

(a) Mix the CTSB protein with the test substance, and determine the binding condition of the test substance and the CTSB protein;

Wherein, if the test substance binds to the CTSB protein, it indicates that the test substance that binds to the CTSB protein is a candidate substance.
A method of (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation, characterized in that it comprises the steps of: administering a therapeutically effective amount to a subject in need The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, and/or the fusion protein of claim 5, and/or the pharmaceutical composition of claim 7.