Patent Application
20230220005
The present invention relates to S100A8-inhibiting peptides and a disease therapeutic agent containing the same.
Currently, a top cause of death in our country is cancer-related death, and many of them are metastasis to distant organs. In the process of cancer development, it has been found that there is an inflammation-like microenvironment present at the surrounding tumors together with abnormal activation associated with mutation at gene level in cancer cells themselves.
As described above, it is suggested that the microenvironment caused by inflammation plays an important role, and the present inventor has confirmed the presence of pre-metastatic lung microenvironment (Non-Patent Literature 1). In addition, the present inventor has identified S100A8 (S100 calcium binding protein A8) as a pre-metastatic lung microenvironment forming factor in lung metastasis, and has reported that S100A8 enhances vascular permeability and promotes lung metastasis of cancer (Non-Patent Literature 2). Furthermore, the present inventor has reported that S100A8 acts as a toll-like receptor 4 (TLR4) endogenous ligand, that the TLR4 inhibitor Eritoran remarkably suppresses the development of a subcutaneous tumor and inhibits both the recruitment of myeloid cells and tumor angiogenesis, that the Carboxyl-terminal side of S100A8 is involved in binding between a TLR4/MD-2 complex and S100A8, and the like (Non-Patent Literature 3).
From the findings found so far, it is expected that if S100A8 can be inhibited, it acts on both cancer microenvironment and pre-metastatic microenvironment and can suppress development of cancer, but no peptide that directly inhibits S100A8 has been found so far.
The present invention has been made in view of the above circumstances, and an object thereof is to provide novel peptides capable of inhibiting S100A8 and a disease therapeutic agent containing the peptide.
In order to solve the above problem, the S100A8-inhibiting peptide of the present invention includes:
(A) a peptide of 5 to 10 residues in length containing the fifth alanine (Ala) from the N-terminus in an amino acid sequence of SEQ ID NO: 1, or
(B) a peptide consisting of an amino acid sequence of SEQ ID NO: 2.
The disease therapeutic agent of the present invention is characterized by containing the S100A8-inhibiting peptide.
Since the S100A8-inhibiting peptide of the present invention binds to a TLR4/MD-2 complex with high affinity, it can inhibit the activity of S100A8. Therefore, the composition containing the S100A8-inhibiting peptide of the present invention acts on cancer microenvironment and pre-metastatic microenvironment, and can suppress development of cancer, and thus is useful as a therapeutic agent against metastatic cancer or the like. In addition, the disease therapeutic agent containing the S100A8-inhibiting peptide of the present invention can treat other diseases (psychiatric diseases, lifestyle-related diseases, rheumatoid arthritis, 2019 novel coronavirus infection (COVID-19), Crohn's disease, ulcerative colitis, cystic fibrosis, allergic dermatitis) in which S100A8 is involved, and the like.
An embodiment the S100A8-inhibiting peptide (S100 calcium binding protein A8-inhibiting peptide) of the present invention will be described.
The S100A8-inhibiting peptide of the present invention includes:
(A) a peptide of 5 to 10 residues in length containing the fifth alanine (Ala) from the N-terminus in an amino acid sequence of SEQ ID NO: 1, or
(B) a peptide consisting of an amino acid sequence of SEQ ID NO: 2.
Specifically, the peptide (A) is preferably a 10-residue peptide consisting of an amino acid sequence of SEQ ID NO: 1. Examples of other peptides include peptides of the following SEQ ID NOs: 3 to 12 and the like.
In addition, the S100A8-inhibiting peptide of the present invention is preferably a divalent peptide containing two identical peptides among the peptides (A) and (B). Furthermore, the S100A8-inhibiting peptide of the present invention is preferably a tetravalent peptide containing four identical peptides among the peptides (A) and (B).
Forms and synthesis methods of the bivalent peptide or the tetravalent peptide are not particularly limited. Specifically, for example, regarding synthesis of a peptide, condensation and removal of a protecting group can be performed according to a conventionally known method. For example, a peptide solid phase synthesis method, an Fmoc peptide synthesis method or the like can be adopted, and the synthesis can also be performed by a commercially available peptide synthesizer.
Preferably, the peptide synthesis method on a sheet already proposed by the present inventor (Patent Literature 1) can be considered. A synthetic core structure of a divalent peptide or a synthetic core structure of a tetravalent peptide can be formed from an amino group on a sheet of cellulose, resin or the like. Specifically, for example, Fmoc-Lys and Fmoc-His represented by Chemical Formula 1 shown below can be used to form a divalent or tetravalent synthetic core structure. In consideration of interactions with other amino acids and the like, Fmoc-Lys is preferable. A branch point can be provided by introducing Fmoc-Lys, and amino acid synthesis from the branch point can be designed to be divalent.
In addition, in the case of forming a tetravalent synthetic core structure, it is considered that peptide Fmoc-Lys is continuously synthesized twice. The synthetic core structure is not specifically limited as long as it is a structure capable of extending a divalent or tetravalent peptide.
Furthermore, a spacer can be appropriately introduced into the divalent peptide or the tetravalent peptide. The spacer is not specifically limited, and for example, Ahx (amino hexanoic acid [NH2—(CH2)5—COOH]) can be exemplified. For cutting out the synthetic peptide, for example, trifluoroacetic acid or the like can be appropriately used.
In addition, when the S100A8-inhibiting peptide of the present invention is in the form of a tetravalent peptide, the following molecular core structure (Chemical Formula 2) formed by binding three lysines (Lys) can be included.
The S100A8-inhibiting peptide of the present invention can be exemplified by a tetravalent peptide in which the peptide (A) or (B) is bonded to each of the four amino groups located at the end of the above molecular core structure directly or via a spacer.
As a more preferred embodiment, for example, in Chemical Formula 3 shown below, a tetravalent peptide in which any one of the peptides (A) or (B) is incorporated into each of four XXXX parts located at the end of the molecular core structure composed of three lysines (Lys) is exemplified.
In Chemical Formula 3, the position at which the peptide (A) or (B) is incorporated is described as WOW for convenience.
In addition, Chemical Formula 3 exemplifies a form in which a spacer is bonded to each of the four amino groups located at the end of the molecular core structure, but the peptide (A) or (B) can also be directly bonded to each of the four amino groups without interposing a spacer. When the spacer is bound, specific molecules and lengths are not limited as long as a binding property to the TLR4/MD-2 complex is not impaired, and they can be appropriately designed. As the spacer, for example, a spacer having an amino acid at the terminal and a chain length of about 4 to 10 carbon atoms is preferable, and in particular, amino hexanoic acid [NH2—(CH2)5—COOH] (aminocaproic acid) represented in Chemical Formula 3 can be preferably exemplified. Also, examples of the amino acid contained in the spacer include alanine (A).
This form of the S100A8-inhibiting peptide (tetravalent peptide) can also be prepared by a known method such as using a peptide synthesizing device or the like. For example, the incorporated peptide (A) or (B) can be synthesized by sequentially adding amino acids to the tetravalent core structure, and can be conveniently bulk synthesized by the same method as that for monovalent peptide synthesis.
In addition, the S100A8-inhibiting peptide may have a modified molecule at each terminal of the peptide incorporated into the XXXX part of Chemical Formula 3. Since a positive charge is generated when NH2 is exposed at the terminal of the peptide, from the viewpoint of charge regulation, it is also considered that a molecule having no charge or a hydrophobic molecule is bound to each terminal as a modified molecule. In addition, when a therapeutic agent containing the S100A8-inhibiting peptide of the present invention is orally administered, NH2 at the terminal can also be protected by an acetyl group for the purpose of stabilization for suppressing degradation by protease in the gastrointestinal tract. As described above, the modified molecule at the terminal of the peptide (A) or (B) can be appropriately selected according to a desired effect, and may be modified by phosphorylation, methylation, adenylation, sugar chain addition, or the like.
Furthermore, the S100A8-inhibiting peptide can also include, for example, a basic membrane penetrating sequence including amino acids such as histidine (His), lysine (Lys), arginine (Arg), and the like.
The S100A8-inhibiting peptide of the present invention binds to the TLR4/MD-2 complex with high affinity. That is, the S100A8-inhibiting peptide of the present invention has excellent competitive activity for binding between the TLR4/MD-2 complex and S100A8. Therefore, the composition containing the S100A8-inhibiting peptide of the present invention acts on cancer microenvironment and pre-metastatic microenvironment, and can suppress development of cancer, and thus is useful as a therapeutic agent for metastatic cancer.
In addition, it has been reported so far that S100A8 is involved not only in cancer diseases but also in overexpression of S100A8 in synovial fluid of patients with rheumatoid arthritis (Odink et al., Nature 330, 80, 1987), macrophage-derived S100A8 controls activation of adipocytes (Sekimoto, et al., PNAS, 112, E2058, 2015), activation of innate immunity via TLR4 induces depression-like behavior (Nie, et al., Neuron, 99, 464, 2018), increased expression of S100A8 is observed in bronchoalveolar lavage fluid of patients infected with COVID-19 (Zhou et al., Cell Host and Microbe, 2020), and the like. Therefore, the disease therapeutic agent containing the S100A8-inhibiting peptide of the present invention can treat diseases such as metastatic cancer, psychiatric diseases, lifestyle diseases, rheumatoid arthritis, COVID-19, Crohn's disease, ulcerative colitis, cystic fibrosis, and allergic dermatitis.
An administration form of the disease therapeutic agent containing the S100A8-inhibiting peptide of the present invention is not particularly limited, and may be oral administration or parenteral administration. Examples of the parenteral administration include injection administration such as intramuscular injection, intravenous injection, subcutaneous injection, percutaneous administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, rectal), and the like.
The disease therapeutic agent of the present invention may use the S100A8-inhibiting peptide as it is as an active ingredient, or may be formulated by adding a pharmaceutically acceptable carrier, excipient, additive, or the like. Examples of the dosage form include liquid preparations (for example, injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, granules, granules, capsules, syrups, troches, inhalants, ointments, eye drops, nasal drops, ear drops, cataplasms, and the like.
The disease therapeutic agent of the present invention can be formulated by a conventional method using, for example, an excipient, a binder, a disintegrant, a lubricant, a dissolving agent, a solubilizing agent, a coloring agent, a flavoring agent, a stabilizing agent, an emulsifier, an absorption promoting agent, a surfactant, a pH adjusting agent, an antiseptic agent, an antioxidant and the like as appropriate.
Examples of the component to be used for formulation include purified water, saline, phosphate buffer, dextrose, pharmaceutically acceptable organic solvents such as glycerol and ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohol, stearyl alcohol, stearic acid, human serum albumin, and the like.
The disease therapeutic agent of the present invention can be administered to mammals including humans (for example, mouse, rat, guinea pig, rabbit, dog, horse, monkey, pig, and the like), and in particular, the dosage when administered to humans varies depending on the symptoms, age, sex, weight and sensitivity difference of the patient, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but for example, 100 μg to 1000 mg can be administered once or in several divided doses.
The S100A8-inhibiting peptide and the disease therapeutic agent involving S100A8 of the present invention are not limited to the above embodiments, and can be appropriately designed as long as the binding property to the TLR4/MD-2 complex and the S100A8 inhibitory effect are not impaired.
Hereinafter, the present invention will be described with reference to Examples, but the S100A8-inhibiting peptide and the disease therapeutic agent of the present invention are not limited to the following Examples at all.
So far, it has been reported that mouse S100A8 binds to a TLR4/MD-2 complex on the carboxyl terminal side (Non-Patent Literature 3), and based on this report, full length human S100A8 was divided into three parts, and it was revealed that the carboxyl terminal side binds to the TLR4/MD-2 complex as with mouse S100A8 (
As a result, Peptide #3 (SEQ ID NO: 13: Phe-Gln-Glu-Phe-Leu-Ile-Leu-Val-Ile-Lys (FQEFLILVIK)) was found to have high affinity for TLR4/MD-2 (
Pre-purified S100A8 protein was immobilized on an ELISA plate. Non-specific binding was blocked with blocking buffer in the same manner as in Example 1. Peptides #1 to #6 were added simultaneously with TLR4/MD-2, and the mixture was reacted. Each well was washed with PBS-Tween 20, then incubated with an anti-TLR4 antibody (Abcam) and an anti-rabbit IgG-HRP labeled antibody (GE Healthcare) subsequently, and finally a reaction with TMB as an HRP substrate was terminated with an equivalent of 1 N sulfuric acid. Bound TLR4/MD-2 was quantified by measuring the absorbance at OD 450 nm. Peptide #3 and Peptide #4 were found to have competitive inhibitory activity on the binding between S100A8 and the TLR4/MD-2 complex (
The TLR4/MD-2 complex and S100A8 were mixed in a tube. At this time, 10 times the molar amount of each of Peptides #1 to #6 was added, and immunoprecipitation was performed with an anti-TLR4 antibody. The immunoprecipitate was washed several times with a washing buffer, and the final pellet was electrophoresed by SDS-PAGE. Then, S100A8 and TLR4 were detected by Western blotting using an anti-S100A8 antibody and an anti-TLR4 antibody. As a result, it became clear that S100A8 was not detected only in the presence of Peptide #3, that is, Peptide #3 had competitive inhibitory activity on the binding of S100A8 to the TLR4/MD-2 complex (
Peptide3A1, Peptide3A2, Peptide3A3, Peptide3A4, Peptide3A5, Peptide3A6, Peptide3A7, Peptide3A8, Peptide3A9, and Peptide3A10 in which each amino acid of Peptide #3 (SEQ ID NO: 13: Phe-Gln-Glu-Phe-Leu-Ile-Leu-Val-Ile-Lys) was substituted with alanine were synthesized. In these peptides, the description of “A1 to A10” indicates a position from the N-terminal side in an amino acid sequence of SEQ ID NO: 13, that is, a position substituted with alanine.
In addition, acetylation was introduced at the amino terminus of these peptides, and amidation modification was introduced at the carboxyl terminus. These 10 peptides were immobilized on an ELISA plate (Thermo Fisher Scientific Inc.; Maxisorp) to verify binding with TLR4/MD-2.
The results are shown in
Peptide3A5 (SEQ ID NO: 1: Phe-Gln-Glu-Phe-Ala-Ile-Leu-Val-Ile-Lys) had higher affinity for TLR4/MD-2 than Peptide #3, the original sequence derived from S100A8. On the other hand, binding of Peptide3A6, Peptide3A7, Peptide3A8, and Peptide3A10 to TLR4/MD-2 was reduced.
Human colorectal cancer SW480 cells express TLR4, and induce IL-8 by S100A8 stimulation. In order to verify inhibitory activity of peptides isolated so far, after pretreating divalent Peptide #3 or divalent Peptide3A5, the cells were stimulated with S100A8 recombinant protein, and the mRNA expression of IL-8 after 5 hours was analyzed. The divalent Peptide #3 and the divalent Peptide3A5 were prepared according to the method of Patent Literature 1 described above.
As a result, it was confirmed that the divalent peptides of Peptide #3 and Peptide3A5 remarkably inhibited the expression of mRNA of IL-8 induced by S100A8 (
In order to verify whether the competitive inhibitory activity changes in the case of a multivalent type, first, S100A8 was immobilized on an ELISA plate, then monovalent Peptide3A5 and divalent Peptide3A5 were added at various concentrations simultaneously with the TLR4/MD-2 complex, and the amount of TLR4/MD-2 bound to S100A8 was measured to verify competitive inhibitory activity of each peptide. As a result, it was found that the divalent Peptide3A5 showed the competitive inhibitory activity at a lower concentration as compared with the monovalent type (
As shown in
First, two weeks after transplantation of SW480 cells subcutaneously in a mouse, the size of the subcutaneous tumor was measured, and confirmed to be equivalent tumor size. The peptide was administered to the abdominal cavity at the indicated concentrations twice a week, and the size of the tumor was measured over time. After two weeks, the size of the subcutaneous tumor was measured, and the subcutaneous tumor was collected. As shown in
In order to identify an essential region in the Peptide3A5 sequence, a series of partial sequence peptides based on the amino acid sequence of Peptide3A5, which were obtained by deleting the amino acid from the amino terminal side or the carboxyl terminal side, were spot-synthesized as divalent peptides on a cellulose membrane sheet using the method of Patent Literature 1. Several kinds of affinity peptides with high affinity to TLR4/MD-2 were obtained by reacting the sheet with TLR4/MD-2 (
The results are shown in
In
As shown in
Among these peptides, as a candidate having both a high affinity and a competitive inhibitory activity,
were identified. ILVIK (SEQ ID NO: 2) was identified as a candidate because in the verification using a monovalent form binding was reduced when the carboxyl-terminal lysine (K) was changed to alanine, and there was a high possibility that synthesis of long chain was difficult.
A competition assay was performed using divalent ILVIK (SEQ ID NO. 2), divalent Peptide3A5 (SEQ ID NO. 1), and tetravalent ILVIK. The tetravalent ILVIK was also synthesized using the method of Patent Literature 1. As shown in
The divalent ILVIK was identified as an S100A8-inhibiting peptide candidate by chain length screening, and influence of the divalent ILVIK on mouse subcutaneous tumors was examined in the same manner as in Example 5 (
Since it was found that proliferation of subcutaneous tumors was suppressed by the divalent ILVIK which was obtained as an S100A8-inhibiting peptide candidate by chain length screening, an effect of tetravalent ILVIK was further examined in the same manner as in Example 5 (
First, two weeks after transplantation of SW480 cells subcutaneously in a mouse, the size of the subcutaneous tumor was measured, and confirmed to be equivalent tumor size. The tetravalent ILVIK was administered to the abdominal cavity at the indicated concentrations (1 mg/kg, 5 mg/kg, 10 mg/kg) twice a week, and the size of the tumor was measured over time. After two weeks, the size of the subcutaneous tumor was measured, and the subcutaneous tumor was collected. The tetravalent ILVIK suppressed the development of mouse subcutaneous tumors in a concentration-dependent manner (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-096367 | Jun 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/020095 | 5/26/2021 | WO |
