JP7138909B2 - VEGF binding inhibitory peptide - Google Patents

Description

The present invention relates to VEGF binding inhibitory peptides.

Vascular Endothelial Growth Factor (VEGF) is a protein that regulates angiogenesis. Angiogenesis plays an important role in the formation of the circulatory system and the construction of many tissues during the embryonic period of vertebrates. Participate in formation, etc. It is also involved in cancer proliferation and metastasis, pathogenesis and promotion of rheumatoid arthritis, diabetic retinopathy, and the like, and angiogenesis is regarded as important in these physiological and pathological conditions. When VEGF binds to the extracellular domain of the VEGF receptor, the receptor dimerizes, tyrosine kinases in the intracellular domain are activated, and signals are transmitted downstream.

VEGF-A, one of the VEGF family members, is the most potent angiogenesis promoter, and it binds or interacts with the transmembrane tyrosine kinase receptor VEGFR (VEGFR-1, VEGFR-2) to exert a scientific effect. Therefore, by inhibiting the binding or interaction between VEGF and VEGFR, angiogenesis is suppressed, and cancer growth and metastasis are suppressed, rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration, etc. expected to connect.

By the way, as an antibody-like peptide stabilized in vivo, a peptide having a helix-loop-helix (HLH) structure (HLH peptide) is disclosed in Patent Document 1 and the like. A peptide having a helix-loop-helix structure has an N-terminal amino acid sequence (N-terminal helix: A block), a C-terminal amino acid sequence (C-terminal helix: C block), A block and C block. has a linker (loop: B block) that connects The A block and C block each form an α-helical coiled-coil structure due to the presence of loops. Although this peptide has a low-molecular-weight structure, it has a stable secondary structure in solution, and it is easy to introduce functional groups with different chemical properties into the portion of the molecule exposed to the solvent side.

Patent document 2 discloses a VEGF binding inhibitor that inhibits binding or interaction between vascular endothelial growth factor (VEGF) and transmembrane tyrosine kinase receptor (VEFGR) using HLH peptide. This inhibitor is a fusion peptide in which thioredoxin is bound to the N-terminus of a peptide of HLH structure, and exhibits excellent ability to inhibit VEGF binding.

However, since this fusion peptide is fused with thioredoxin, it is expected to have a large molecular weight and poor absorbability into the body. Furthermore, according to subsequent research by the inventors, this fusion peptide does not bind to the VEGFR-binding site of VEGF, but the inhibition of the binding or interaction between VEGF and VEGFR by this fusion peptide is due to the steric hindrance of the thioredoxin moiety. presumed to be due to In addition, there are also problems such as higher production costs compared to low-molecular-weight products in terms of production, and stable and low-molecular peptidic VEGF-binding inhibitors that bind to the VEGFR-binding site of VEGF are desired. rice field.

JP-A-10-245397 JP 2014-047156 A

The problem to be solved by the present invention is to obtain a peptide that exhibits strong binding to VEGF and binds to the site of VEGF that binds to VEGFR, and to provide a peptidic VEGF binding inhibitor that has a low-molecular-weight structure. be.

The peptide according to the present invention is the peptide represented by SEQ ID NO:1.

The peptide of the present invention inhibits the binding or interaction between VEGF and VEGFR by binding to the VEGF-side site involved in the binding of VEGF and VEGFR, thereby providing a highly stable peptidic VEGF binding inhibitor. be.

FIG. 1 is a diagram showing amino acids frequently appearing at randomized sites in HLH peptides obtained by secondary screening. FIG. 2 shows CD spectra of peptides VS42 and VS42-1. FIG. 3 shows the degree of dissociation of peptide VS42-1 to VEGF. FIG. 4 shows inhibition of binding of peptides VS42 and VS42-1 to VEGF. FIG. 5 shows the cell growth inhibitory action of peptide VS42-1.

Logically, the peptide according to the present invention is a helix in which two α-helices each consisting of 14 amino acid residues on the N-terminal side and the C-terminal side are linked via a peptide consisting of 6 or 7 glycine residues. - have a loop-helix structure (HLH structure); In the peptide having this HLH structure, the two α-helices form a stable HLH structure due to the hydrophobic interaction of the leucine side chains existing inside, and furthermore, cysteines are introduced at the N-terminus and C-terminus. Intramolecular disulfide bridges further improve the stability of the conformation (Suzuki, N., Fujii, I., Optimization of the loop length for folding of a helix-loop-helix peptide, Tetrahedron Lett. 40, 6013 ( 1999), Fujii I, Takaoka Y., Suzuki K., Tanaka, T., A Conformationally Purified α-Helical Peptide Library, Tetrahedron Lett. 42, 3323 (2001)). In the present invention, a peptide having such an HLH structure is preferred, but the peptide does not necessarily have to have such an HLH structure.

A peptide according to the present invention has the amino acid sequence shown in SEQ ID NO:5. X in this amino acid sequence, X18 (amino acid at the 18th residue from the N-terminal amino acid) is a basic amino acid, and X19 (amino acid at the 19th residue from the N-terminal amino acid) is a hydrophobic amino acid. , the 20th amino acid from the N-terminal amino acid (X20), the 21st amino acid from the N-terminal amino acid (X21), and the 23rd amino acid from the N-terminal amino acid (X23) are acidic amino acids. be. Here, the basic amino acid is an amino acid having a positively charged side chain, and is preferably H, R or K. A hydrophobic amino acid is an amino acid with an uncharged side chain, preferably V, L or I among these. An acidic amino acid is an amino acid with a negative charge on its side chain, preferably E or D.

Further, the peptide according to the present invention is a peptide having the amino acid sequence shown in SEQ ID NO: 5, in which X18 is F or Q in addition to a basic amino acid, and X20 is an acidic amino acid other than E, G or L , X21 can be S in addition to the acidic amino acids E and D, and X23 can be N, H, S, W as well as the acidic amino acids D and E. Also in these peptides, X18 is H or R, X19 is V or L, X20 is E or L, X21 is E or D, X23 is D, S or E, Further preferably, X18 is H, X19 is V or L, X20 is E or L, and X21 and X23 are E or D, respectively. Among the amino acids appearing in the peptides obtained in the secondary screening (see Table 2), the amino acids shown in these are the amino acids appearing with high frequency. More specifically, the peptides shown in SEQ ID NOS: 6 to 20 are preferred, and a particularly preferred peptide has the amino acid sequence of SEQ ID NO: 6 (VS42-1: CAAELAALEAELAALEGHVEEADFPWGKLNNLIEKLWQLKQAC).

In the present invention, the amino acids that constitute the peptide may be natural amino acids, derivatives of natural amino acids, or non-natural amino acids. Preferably, the amino acids that make up the peptide are natural amino acids. Derivatives of natural amino acids include, but are not limited to, for example, hydroxyl group-introduced amino acids such as hydroxyproline and hydroxylysine, and amino group-introduced amino acids such as diaminopropionic acid. Examples of non-natural amino acids include α,α-disubstituted amino acids (α-methylalanine, etc.), N-alkyl-α-amino acids, D-amino acids, β-amino acids, α -Hydroxy acids, etc.; amino acids whose side chains differ from natural types (norleucine, homohistidine, etc.), homoamino acids with extra methylenes in side chains (homophenylalanine, homohistidine, etc.) and carboxylic acid functional groups in side chains Examples include, but are not limited to, amino acids substituted with sulfonic acid groups (cysteic acid, etc.).

In addition, the peptide according to the present invention is preferably a cyclic peptide formed by disulfide bonding of the N-terminal cysteine and the C-terminal cysteine C of the amino acid sequences shown in the respective SEQ ID NOs. Cyclization stabilizes the three-dimensional structure of the peptide.

Acyclic peptides among the above peptides can be synthesized according to various known peptide synthesis methods. Examples thereof include liquid phase synthesis methods such as Fmoc solid phase synthesis method and fragment condensation method. A solid-phase synthesis method is preferably used because the operation is simple. A cyclic peptide can also be obtained by obtaining a non-cyclic peptide and then cyclizing it. For example, it can be cyclized by forming a disulfide bond between two cysteines in the amino acid sequence by a known technique. It can also be cyclized by forming a thioester or thioether bond in the molecule by a known technique. Further, cyclization may be performed by intramolecular condensation of a carboxy group and an amino group by a known technique such as a high-dilution method.

Acyclic peptides can be produced not only by chemical synthesis methods such as the above-mentioned solid-phase synthesis method and liquid-phase synthesis method, but also by using genetic engineering techniques. For example, a nucleic acid having a nucleotide sequence encoding the amino acid sequence is incorporated into an appropriate expression vector, and the resulting expression vector is used to transform an appropriate host cell (eg, mammalian cell, insect cell, E. coli). Then, after culturing the host cell under conditions suitable for expression of the peptide, the peptide may be isolated from the culture.

Peptides that inhibit the binding or interaction with VEGF-VEGFR show an inhibitory effect on the growth of vein endothelial cells in animals including mammals, particularly in humans. Therefore, the peptide according to the present invention acts as an angiogenesis inhibitor, and various anticancer agents, chronic rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration, etc. It is expected to be used as a therapeutic drug for diseases. In particular, peptides having a dissociation constant (K _D ) for VEGF of 350 nM or less, preferably 100 nM or less, more preferably 50 nM or less, as measured by the plasmon resonance method, are used as these therapeutic agents.

The pharmaceutical composition according to the present invention contains the above peptide as an active ingredient. The pharmaceutical composition of the present invention may contain, in addition to an effective amount of the peptide, a pharmacologically acceptable formulation aid. Auxiliaries can be, for example, excipients, binders, disintegrants, lubricants, coating agents, flavoring agents, solubilizers. The composition is provided in a form (dosage form) that can be applied orally or parenterally to animals including humans. The dosage form may be, for example, tablets, granules, powders, liquids, injections, and suppositories.

The dosage of the peptide according to the present invention is appropriately determined by those skilled in the art according to sex, body weight, age, race, symptoms, and the like. The lower limit of the dosage is, for example, 0.001 μg/kg body weight, 0.01 μg/kg body weight, 0.1 μg/kg body weight, 0.001 mg/kg body weight, 0.01 mg/kg body weight, 0.05 mg/kg body weight, and may be 0.1 mg/kg body weight. Also, the upper limit may be, for example, 1000 mg/kg body weight, 100 mg/kg body weight, 10 mg/kg body weight, 5 mg/kg body weight, or 1 mg/kg body weight.

Hereinafter, the present invention will be described in more detail based on the following examples, but it goes without saying that the present invention is not limited to these.

[Primary screening]
Two primary libraries L1 and L2 composed of peptides having the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 were generated by phage surface display. The phage surface display method is a known method (Japanese Patent Application Laid-Open No. 2014-245397), and here, a method of displaying peptides on the minor coat protein of M13 phage was used. In the primary library L1, 6 amino acid residues that are not involved in the formation of the three-dimensional structure in the C-terminal side helix structure and 9 amino acid residues in the loop portion are randomized. In the randomization, BNS codons were used so that hydrophobic amino acids Y, W, F, and L were selected in the loop portion, and P and G were selected to facilitate loop formation. Also, the NDK codon was used in the helix structure so that proline, which destabilizes the helix structure, is not selected. In the primary library L2, nine amino acid residues in the loop portion are randomized, and in the helix portion, all the amino acid residues randomized in the primary library L1 are fixed to alanine. is.

Primary screening was performed from the two resulting primary libraries. _VEGF165 was used for the _screening among four isoforms ( _VEGF121 , VEGF165, _VEGF189 , _VEGF206 ) of VEGF-A. Screening is (1) contact with VEGF, (2) removal of phages that did not bind to VEGF, (3) collection of phages detached from VEGF by VEGFR2, (4) amplification by infection with E. coli, (5) VEGF and A series of operations of contacting was used as one round, and phages displaying peptides with high binding properties to VEGF were concentrated (biopanning). As a result, by performing four rounds of manipulation, as shown in Table 1, a peptide ( Peptide VS41) was obtained from the primary library L2, and a peptide (peptide VS42) having the amino acid sequence shown in SEQ ID NO: 4 was obtained at a high frequency. In addition, since these peptides are peptides released from VEGF due to competition with VEGFR2, it can be said that they inhibit the VEGF-VEGFR2 interaction.

Peptide VS42, which appeared at a higher frequency, was synthesized by the Fmoc solid-phase synthesis method, and the dissociation constant (K _D ) for VEGF was measured, and the dissociation constant K _D was 350 nM. From this, it is concluded that the peptide VS42 inhibits the VEGF-VEGFR2 interaction by binding to the VEGFR-interacting site of VEGF.

[Secondary screening]
Since the results obtained from the primary screening showed a common amino acid sequence (FPWG) in the loop portion between peptide VS42 and peptide VS41, randomization was performed on the other portions of the loop portion while retaining this portion. Tried. Based on the amino acid sequence of peptide VS42, a secondary library consisting of peptides having the amino acid sequence shown in SEQ ID NO: 5 was constructed using the yeast surface display method. A yeast surface display method is also known (Boder, ET, Wittrup, KD Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol, 16, 553-557 (1997)). Obtained.

For the resulting secondary library, biotin-labeled VEGF and magnetic beads coated with streptavidin or anti-biotin antibody that binds to biotin are used, and peptide-presenting yeast bound to VEGF is collected on a column fixed to a magnetic stand. By doing so, biopanning similar to the primary screening was performed. By performing three rounds of manipulation, peptides showing high binding to VEGF as shown in Table 2 were obtained. Here, as shown in FIG. 1, the 18th residue (X18) is a basic amino acid (H, R, K) with a positive charge on the side chain, and the 19th residue (X19) is a non-polar side chain. Hydrophobic amino acids with chains (V, L, I), and many acidic amino acids (E, D) with negative charges on the side chains were confirmed at the 20th, 21st, and 23rd residues (X20, X21, X23). . Peptide VS42 obtained in the primary screening does not have charged amino acids in the loop portion, but by replacing these peptides with charged amino acids like the above amino acids, the binding to VEGF is increased. concluded.

[Function evaluation]
Next, the peptide (VS42-1) having the amino acid sequence shown in SEQ ID NO: 6 that appeared with high frequency was synthesized by the Fmoc solid-phase synthesis method, the circular dichroism (CD) spectrum was measured, and the dissociation constant for VEGF (K _D ) was measured. Furthermore, inhibitory activity against VEGF-VEGF receptor (VEGF2) interaction was determined. For comparison, the CD spectra and dissociation constants of the peptide (YT1-S) shown in SEQ ID NO: 21 having a stable HLH structure that does not show binding to VEGF, and the peptide VS42 obtained in the primary screening was measured.

(Maintenance of three-dimensional structure)
CD spectra were measured by calculating the average residue ellipticity (θ) using circular dichroism. The results are shown in FIG. 2. Peptide VS42-1 maintained a stable helical structure although the helical structure was slightly distorted.

(Affinity for VEGF)
The dissociation constant was calculated from a sensorgram plotting the equilibrium value of Resonance Unit (RU) against the concentration of peptide VS42-1 by the surface plasmon resonance (SPR) method using a sensor chip on which VEGF was immobilized. As a result, the dissociation constant was approximately 26 nM as shown in FIG.

(Inhibitory activity against VEGF-VEGFR2 interaction)
The inhibitory activity _IC50 of each peptide was measured by the SPR method using a sensor chip on which VEGF receptors were immobilized. A mixed solution of each peptide and VEGF was used as a sample, and the inhibitory activity IC ₅₀ of each peptide against the VEGF-VEGFR2 interaction was determined from the plot of the equilibrium value of Resonance Unit (RU) against the concentration of the sample. Measurement was performed by adding each peptide in the range of 800 nM to 0.78125 nM to 10 nM VEGF, and the RU value for each added concentration was calculated from a sensorgram plotted. As a result, as shown in FIG. 4, the _IC50 of peptide VS42 was 200 nM and that of peptide VS42-1 was 35 nM.

(cell growth inhibition)
Peptide VS42-1 was subjected to a cell proliferation inhibition test using Human Umbilical Vein Endothelial Cells (HUVEC). HUVEC expresses VEGFR2 on the cell surface and proliferates by binding with VEGF. HUVEC seeded at 5.0×10 ³ cells/well was added with 10 nM VEGF and a sample solution adjusted to a range of 161.6 μM to 4.93×10 ⁻³ μM, and cultured at 37° C. for 48 hours. After culturing, color was developed with a WST-1 reagent, and absorbance at 450 nm was measured. Curve fitting is performed on a graph plotting absorbance against peptide concentration, with the absorbance when cultured with only VEGF as a control of 100% and the absorbance when cultured without adding peptide or VEGF as a control of 0%. The inhibitory activity _IC50 was determined by As a result, as shown in FIG. 5, the inhibitory activity _IC50 of peptide VS42-1 was 181 nM. In addition, the inhibitory activity _IC50 of peptide VS42 was 1200 nM.

From these facts, peptide VS42-1 strongly inhibits VEGF-VEGFR2 interaction and can be used as a VEGF binding inhibitor. In addition, each peptide from peptide VS42-2 to peptide VS42-15 obtained in the secondary screening shared the amino acid sequence of peptide VS42-1 with AXFPW on the C-terminal side of the two helix portions and the loop portion. It can be said that VEGF-VEGFR2 interaction inhibitory activity is observed in the same manner as VS42-1, which has an amino acid sequence and was found to have activity to inhibit VEGF-VEGFR2 interaction. In addition, as described above, in the loop part, X18 has a positively charged basic amino acid (H, R, K) in the side chain, and X20, X21, X23 has a negatively charged amino acid in the side chain (E, D ) increases binding to VEGF. Therefore, peptides according to the present invention, such as peptide SV41, in which AXFPW in the helix and loop portions are conserved, can be used as VEGF binding inhibitors.

INDUSTRIAL APPLICABILITY According to the present invention, a novel antibody-like inhibitor of VEGF binding is provided, and is expected to be used as an anticancer agent based on its angiogenesis inhibitory action.

Claims (9)

A peptide having the amino acid sequence of SEQ ID NO:5.
However, in the same amino acid sequence,
The 18th amino acid (X18) from the N-terminal amino acid is a basic amino acid,
The 19th amino acid (X19) from the N-terminal amino acid is a hydrophobic amino acid,
The 20th amino acid (X20) from the N-terminal amino acid is an acidic amino acid,
The 21st amino acid (X21) from the N-terminal amino acid is an acidic amino acid,
The 23rd amino acid (X23) from the N-terminal amino acid is an acidic amino acid. In the amino acid sequence of SEQ ID NO: 5,
the 18th amino acid (X18) from the N-terminal amino acid is H, R or K;
The 19th amino acid (X19) from the N-terminal amino acid is V, L or I,
the 20th amino acid (X20) from the N-terminal amino acid is E or D;
the 21st amino acid (X21) from the N-terminal amino acid is E or D;
the 23rd amino acid (X23) from the N-terminal amino acid is E or D;
The peptide of claim 1, which is A peptide having the amino acid sequence of SEQ ID NO:5.
However, in the same amino acid sequence,
the 18th amino acid (X18) from the N-terminal amino acid is H, R, F, K or Q;
The 19th amino acid (X19) from the N-terminal amino acid is V, L or I,
the 20th amino acid (X20) from the N-terminal amino acid is E, G or L;
the 21st amino acid (X21) from the N-terminal amino acid is E, D or S;
The 23rd amino acid (X23) from the N-terminal amino acid is D, N, E, H, S or W. In the amino acid sequence of SEQ ID NO: 5,
The 18th amino acid (X18) from the N-terminal amino acid is H or R,
The 19th amino acid (X19) from the N-terminal amino acid is V or L,
the 20th amino acid (X20) from the N-terminal amino acid is E or L;
the 21st amino acid (X21) from the N-terminal amino acid is E or D;
The 23rd amino acid (X23) from the N-terminal amino acid is D, S or E
4. The peptide of claim 3, which is In the amino acid sequence of SEQ ID NO: 5,
The 18th amino acid (X18) from the N-terminal amino acid is H,
The 19th amino acid (X19) from the N-terminal amino acid is V or L,
the 20th amino acid (X20) from the N-terminal amino acid is E or L;
the 21st amino acid (X21) from the N-terminal amino acid is E or D;
The 23rd amino acid (X23) from the N-terminal amino acid is E or D
4. The peptide of claim 3, which is A peptide having an amino acid sequence of any of SEQ ID NOS:6-20. 7. The peptide according to any one of claims 1 to 6, wherein the N-terminal cysteine and the C-terminal cysteine in the amino acid sequence are disulfide bonded. A pharmaceutical composition comprising an effective amount of the peptide of any one of claims 1-7. 9. The pharmaceutical composition according to claim 8, which is an anti-cancer composition, an anti-rheumatoid arthritis composition, an anti-diabetic retinopathy composition, or an anti-age-related macular degeneration composition.

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