JP5754008B2 - CaMKII inhibitory peptide and CaMKII inhibitor containing the same - Google Patents

Description

The present invention relates to a peptide that inhibits the activity of Ca ²⁺ / calmodulin-dependent protein kinase (CaMKII) and a CaMKII inhibitor containing the same.

Ca ²⁺ / calmodulin-dependent protein kinase ^{(Ca 2+ / calmodulin-dependent protein} kinase; CaMK) is activated by Ca ²⁺ / calmodulin that Ca ²⁺ is bound to calmodulin is Ca ²⁺ binding protein A generic term for serine / threonine kinases. There are three families of CaMK (CaMKI, CaMKII, and CaMKIV), each of which plays a different role in vivo. Among them, CaMKII is abundant in the brain and is known to play an important role in the control of higher nerve functions such as synthesis and secretion of neurotransmitters and induction of long-term potentiation of neurotransmission. On the other hand, it has been shown that abnormal activation of CaMKII is closely related to the onset of neurological diseases such as Parkinson's disease, cognitive impairment, and convulsions. Furthermore, it is known to be involved in the development of various human diseases such as induction of heart failure, osteoporosis, and promotion of carcinogenesis through inhibition of apoptosis induction, not limited to neurological diseases.

  As a CaMKII inhibitor, a peptide AIP derived from the sequence of an autophosphorylation site present in its regulatory domain: Autocamtide 2 Related Inhibitory Peptide, SEQ ID NO: 15: Lys-Lys-Ala-Leu-Arg-Arg- Gln-Glu-Ala-Val-Asp-Ala-Leu (KKALRQRQAVDAL) is known (Non-patent Document 1). For example, Patent Documents 1 to 3 propose peptides having an amino acid sequence similar to the amino acid sequence of AIP as CaMKII inhibitors.

On the other hand, structural analysis of CaMKII has been performed in recent years, and its extremely unique higher-order structure has been revealed. In CaMKII, 12 molecules are assembled in a disk shape centering on a region called an association domain, and each kinase domain (catalytic domain) of CaMKII is arranged in a ring shape around this disk-shaped core structure. doing. Although the Ca ²⁺ absence exist stably in this state, the intracellular Ca ²⁺ concentration increases due to a variety of stimuli, packing undone when Ca ²⁺ is calmodulin bound is supplied, 12 weight It is believed that each kinase in the body is activated sequentially in a coordinated manner.

Japanese Patent No. 2963986 Special table 2001-509808 gazette JP 2005-504829 A WO2006 / 001542 Publication

Biochem Biophys Res Commun. 1995 Jul 26; 212 (3): 806-12

  However, in the case of the peptides of AIP and other patent documents 1 to 3, it is difficult to examine in detail the importance of each position in the amino acid sequence and the inhibition mechanism, and the purpose is to control in vivo kinetics, improve membrane permeability, stability, etc. There is a problem that it is difficult to add various functions and improve the functions. If this point can be overcome, it is expected that development of excellent therapeutic agents for the various important diseases will be greatly promoted.

  On the other hand, the present inventors have established a new method for identifying a peptide that inhibits strong binding based on multivalent interaction (cluster effect), a multivalent peptide library method (see above patent). Reference 4). The peptide library method has the great feature that the relative importance of the amino acid at each position of the identified peptide sequence can be quantified.

  Therefore, the present inventors have assumed that the screening of peptide sequences that act on clustered CaMKII may be facilitated by applying the multivalent peptide library method to CaMKII. And an attempt to obtain a series of excellent CaMKII inhibitory peptides.

  The present invention has been made in view of the circumstances as described above, and an object of the present invention is to provide a CaMKII inhibitory peptide having a novel amino acid sequence different from AIP and a CaMKII inhibitor containing the same.

 In order to solve the above problems, the CaMKII inhibitory peptide of the present invention is characterized by being a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 6.

  In the CaMKII-inhibiting peptide of the present invention, the peptide is preferably a multivalent peptide having a plurality of amino acid sequences of any one of SEQ ID NOs: 1 to 6.

  In the CaMKII inhibitory peptide of the present invention, the following molecular nucleus in which three lysines are bound,

And a peptide containing any one of the amino acid sequences of SEQ ID NOS: 1 to 6 is bonded to each -NH at the end of the molecular nucleus portion directly or via a spacer. Is preferred.

  The CaMKII inhibitor of the present invention is characterized by containing the above-mentioned CaMKII inhibitory peptide.

  The CaMKII-inhibiting peptide of the present invention is a peptide having a novel amino acid sequence, and various functions can be added according to the purpose. Moreover, the CaMKII inhibitor containing this CaMKII inhibitory peptide can be used for the treatment of diseases caused by abnormal activation of CaMKII.

It is the figure which illustrated the structure of the multivalent type peptide library. It is a figure which shows the result of the inhibitory effect of each synthetic peptide of M1-M6. It is a figure which shows the inhibitory effect of the synthetic peptide of M1-M6 with respect to PKA. It is a figure which shows the inhibitory effect of the synthetic peptide of M1-M6 with respect to PKC. It is a figure which shows the inhibitory effect of tetravalent peptide T1-T6 with respect to the kinase activity of CaMKII (alpha) -KD. It is a figure which shows the inhibitory effect of tetravalent peptide T1-T6 with respect to the kinase activity of CaMKII (alpha) -KD.

The CaMKII inhibitory peptide of the present invention can inhibit CaMKII activity by binding to CaMKII with a strong binding affinity. Specifically, the CaMKII inhibitory peptide of the present invention has the following amino acid sequence:
Sequence number 1: Arg-Arg-Arg-Leu-Leu-Leu-Leu-Leu-Ala-Arg-His-His
(RRRRLLLLLARHH)
Sequence number 2: Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-His-His
(RRILLLLLARHH)
Sequence number 3: Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-Leu-His
(RRILLLLLARLH)
Sequence number 4: Arg-Ile-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Leu-Leu-His
(RIILLLLALLLH)
Sequence number 5: Arg-Ile-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-Leu-His
(RIILLLLLARLH)
Sequence number 6: Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Leu-Leu-His
(RRILLLLALLALLH)
It is a monovalent or polyvalent peptide containing one or more of any of the above. The amino acid sequences of SEQ ID NOs: 1 to 6 are those identified by screening shown in Examples described later.

As long as the CaMKII-inhibiting peptide does not impair the binding affinity with CaMKII, the N-terminal and / or C-terminal of the amino acid sequences of SEQ ID NOs: 1 to 6 are considered 1 Modification molecules such as the above amino acids and modification groups can be appropriately added. For example, a peptide containing a sequence of three lysines (KKK) can be added to the C-terminus of the amino acid sequences of SEQ ID NOs: 1 to 6 as amino acids for ensuring the water solubility of the peptide. The length of the CaMKII inhibitory peptide can be exemplified by the number of amino acids in the range of about 10-20. In addition, a modifying molecule for adjusting the molecular length can be added to the C-terminus of the amino acid sequences of SEQ ID NOs: 1 to 6 in accordance with the molecular structure of CaMKII. As a modified molecule for adjusting the molecular length, for example, one having a chain length of about 4 to 10 carbon atoms is preferable. Specifically, amino hexanoic acid [NH _2- (CH ₂ ) ₅ -COOH] or the like is used. It can be illustrated.

  When preparing a multivalent peptide in which the CaMKII-inhibiting peptide includes a plurality of amino acid sequences of SEQ ID NOs: 1 to 6, for example, the amino acid sequence of any of SEQ ID NOs: 1 to 6 is attached to the end of the molecular nucleus where a plurality of lysines are bound. A plurality of peptides can be bound. Specifically, examples of the molecular nucleus include the following structures formed by combining three lysines.

A tetravalent CaMKII-inhibiting peptide can be produced by binding a peptide containing any one of SEQ ID NOs: 1 to 6 to the terminal -NH of this molecular nucleus. In this case as well, modified molecules such as amino acids can be added to the N-terminal and C-terminal of the amino acid sequences of SEQ ID NOs: 1 to 6. For example, aminohexanoic acid [NH _2- (CH ₂ ) ₅ -COOH] or the like is introduced as a spacer between the molecular nucleus and the C-terminal of the amino acid sequences of SEQ ID NOs: 1 to 6, or three consecutive lysines. It is considered that a sequence (KKK) to be introduced is introduced.

  Since the CaMKII-inhibiting peptide includes any one of the amino acid sequences of SEQ ID NOs: 1 to 6, the CaMKII-inhibiting peptide exhibits excellent binding affinity for CaMKII and can inhibit the activity of CaMKII. In particular, in the case of a multivalent peptide comprising a plurality of amino acid sequences of any one of SEQ ID NOs: 1 to 6, it exhibits excellent binding affinity for CaMKII. Further, the amino acid sequences of SEQ ID NOs: 1 to 6 included in the CaMKII inhibitory peptide are novel sequences different from the conventionally known AIP and the like, and control of in vivo kinetics, membrane permeability, improvement of stability, etc. It can be used for studies for adding various functions according to the purpose.

  The CaMKII inhibitor of the present invention contains the above CaMKII inhibitory peptide as an active ingredient. The CaMKII inhibitor of the present invention is prepared and stored by mixing with any pharmaceutically acceptable carrier, excipient or stabilizer in addition to the CaMKII inhibitory peptide. Acceptable carriers, excipients, or stabilizers can be appropriately selected depending on the dosage form and administration route, provided that they are non-toxic to patients at the dosages and concentrations used. For example, buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride Phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; serum albumin , Gelatin, or protein such as immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, including lucose, mannose, or dextrin, and other carbohydrates such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn − Protein complexes) or TWEEN (trade name), PLURONICS (trade name), and nonionic surfactants such as polyethylene glycol (PEG).

  Moreover, the CaMKII inhibitor of the present invention may have sustained release properties. As a sustained-release agent, for example, a semipermeable matrix (for example, in the form of a film or a microcapsule) of a solid hydrophobic polymer. Examples of sustained release matrices are polyester hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate. , Non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D) -3-hydroxy Butyric acid or the like can be used as appropriate.

  The thus-prepared CaMKII inhibitor can be systemically administered, for example, orally, locally, or intravenously, depending on the type and symptoms of the disease. The dosage can be determined according to the weight, symptoms, etc. of the patient.

  The CaMKII inhibitor of the present invention can be used for the treatment / prevention of neurological diseases such as Parkinson's disease, cognitive impairment, and convulsions caused by abnormal activation of CaMKII, for example. In addition, it can be used for the treatment of various human diseases such as heart failure, osteoporosis, and carcinogenesis through inhibition of apoptosis, and suppression of onset.

(1) Preparation of CaMKII CaMKIIα, which is one of CaMKII isozymes, was used to target its kinase domain. CaMKIIα kinase domain (KD) with a his-tag (CaMKIIα-KD) was prepared and immobilized on Ni-beads. For the large-scale preparation of CaMKII, a baculovirus expression system was used, and it was confirmed that CaMKIIα-KD immobilized on the beads had a purity of 90% or more.
(2) Design of multivalent peptide library suitable for CaMKIIα FIG. 1 shows the structure of a multivalent peptide library. As shown in [Chemical Formula 1], this multivalent peptide library has a molecular nucleus having three lysines bonded at the center, and has a structure branched into four. Then, amino hexanoic acid [NH ₂ — (CH ₂ ) ₅ —COOH] represented by “U” in FIG. 1 is bonded to the end of the molecular nucleus as a spacer. The spacer length is designed to be optimized for CaMKII. And from the edge part of this spacer, it is designed in the tetravalent peptide library which four peptides containing the library part shown by XXX in FIG. 1 couple | bonded. Met-Ala (MA) is bound to the end of each library part. This MA was introduced at the time of screening, and it is not always necessary to add it to the CaMKII inhibitory peptide of the present invention. The library portion is simply indicated by XXXX for convenience, and the number of amino acids and the like will be described in detail below.
Regarding the design of the library part, the present inventors: 1) In many substrates that have been known to be phosphorylated by CaMKIIα, the amino acid at position -5 from the position of Ser / Thr to be phosphorylated Is often Leu. 2) Substituting another amino acid for Leu corresponding to this position in the AIP sequence significantly reduces the inhibitory effect. Inferred to be important for high affinity binding.
Therefore, the sequence of the library part of the multivalent peptide library (indicated as “XXXX” in FIG. 1) is XXXXLXXXAXXX (SEQ ID NO: 13: Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Ala -Xaa-Xaa-Xaa) (X part (Xaa) indicates a degenerate position consisting of a mixture of 19 amino acids other than Cys). In the sequence, phosphorylation is prevented by fixing the 0 position corresponding to the phosphorylation position of the substrate to Ala (A), and the library part and CaMKIIα-KD are fixed by fixing the -5 position to Leu (L). Designed to increase binding affinity. In addition, Ala-Lys-Lys-Lys (AKKK) was introduced between the library portion and the spacer. These three Lys are introduced to ensure the water solubility of the multivalent peptide library.

(3) Multivalent peptide library screening The screening method is in accordance with the method described in the patent document (WO2006 / 001542). Using the above-mentioned multivalent peptide library (240 μg) optimized for CaMK IIα, it was incubated with CaMKIIα-KD (200 μg) immobilized on Ni-beads at 4 ° C. for 18 hours (composition of reaction solution; 50 mM HEPES -NaOH (pH = 7.5), 1 mM EGTA, 20 mM β-glycerophosphate, 0.02% NP-40, 1 mM DTT, 10 μM ATP, final volume 300 μl). The beads were washed with PBS and 0.5% NP-40 PBS and eluted with 30% acetic acid to prepare a fraction that strongly binds to CaMKIIα-KD. This fraction was subjected to amino acid sequencing, and the molar ratio of 19 kinds of detected amino acids was calculated for each degenerate position. Furthermore, the original multivalent peptide library (SEQ ID NO: 13: Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Xaa-Ala-Xaa-Xaa-Xaa as the library part) For the peptide library, amino acid sequencing was performed in the same manner, and the detection molar ratio of amino acids at each degenerate position was calculated. For each amino acid at each degenerate position, the numerical value obtained from the screening was divided by the numerical value obtained from the original multivalent peptide library to quantify how many times the selectivity was improved. Furthermore, when the numerical values of all 19 amino acids at a certain degenerate position were added, they were corrected to be 19. At this time, if there is no difference in selectivity between amino acids, the value is 1. When this value exceeded 1.3, it was judged that strong selectivity was observed.

  Two screenings using a multivalent peptide library optimized for CaMKIIα were performed, and the amino acid selected at each X position was determined. Table 1 shows the results.

  As a result, it was shown that Leu was selected very strongly at positions -1 to -4, Arg was selected at position -8, and His was selected at position +3. In position +1 and +2, two amino acids, Arg and Leu, were selected in common. On the other hand, at positions -7, -6, and +2, there were some differences in the amino acids selected between them.

  From the above results, the following six amino acid sequences (SEQ ID NOs: 1 to 6) were identified as CaMKIIα binding motifs as sequences covering these information.

Sequence number 1: Arg-Arg-Arg-Leu-Leu-Leu-Leu-Leu-Ala-Arg-His-His
(RRRRLLLLLARHH)
Sequence number 2: Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-His-His
(RRILLLLLARHH)
Sequence number 3: Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-Leu-His
(RRILLLLLARLH)
Sequence number 4: Arg-Ile-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Leu-Leu-His
(RIILLLLALLLH)
Sequence number 5: Arg-Ile-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-Leu-His
(RIILLLLLARLH)
Sequence number 6: Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Leu-Leu-His
(RRILLLLALLALLH)
(4) Inhibitory activity measurement Based on the above amino acid sequences (SEQ ID NOs: 1 to 6) obtained as a result of screening, the following peptides (SEQ ID NOs: 7 to 12) containing the above amino acid sequences (SEQ ID NOs: 1 to 6) were obtained. Synthesized and examined the effect as a CaMKIIα inhibitory peptide.

SEQ ID NO: 7
Met-Ala-Arg-Arg-Arg-Leu-Leu-Leu-Leu-Leu-Ala-Arg-His-His-Ala-Lys-Lys-Lys
(MARRRLLLLARHHAKKK)
SEQ ID NO: 8
Met-Ala-Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-His-His-Ala-Lys-Lys-Lys
(MARRILLLLARAHHAKK)
SEQ ID NO: 9
Met-Ala-Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-Leu-His-Ala-Lys-Lys-Lys
(MARRILLLLLARLHAKKK)
SEQ ID NO: 10
Met-Ala-Arg-Ile-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Leu-Leu-His-Ala-Lys-Lys-Lys
(MARIILLLLALLALLHAKKK)
SEQ ID NO: 11
Met-Ala-Arg-Ile-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Arg-Leu-His-Ala-Lys-Lys-Lys
(MARIILLLLLARLHAKKK)
SEQ ID NO: 12
Met-Ala-Arg-Arg-Ile-Leu-Leu-Leu-Leu-Leu-Ala-Leu-Leu-His-Ala-Lys-Lys-Lys
(MARILLLLALLALLHAKKK)
These synthetic peptides (SEQ ID NOs: 7 to 12) have Met-Ala (MA) added to the N-terminus of the CaMKIIα binding motif (SEQ ID NOs: 1 to 6) obtained as a result of the above screening, and SEQ ID NOs: 14: Ala-Lys-Lys-Lys (AKKK) is added. The N-terminal MA is added corresponding to the structure of the multivalent peptide library, but can be omitted. In addition, C-terminal AKKK (SEQ ID NO: 14: Ala-Lys-Lys-Lys) is introduced to ensure the water solubility of the synthetic peptide. Furthermore, in order to correspond to the molecular structure of CaMKIIα, the molecular chain length was adjusted by adding amino hexanoic acid [NH _2- (CH ₂ ) ₅ -COOH] to the C-terminus of the synthetic peptides shown in SEQ ID NOs: 7-12 It was used. Hereinafter, the synthetic peptide of SEQ ID NO: 7 added with amino hexanoic acid [NH _2- (CH ₂ ) ₅ -COOH] is referred to as M1 (monomer 1), and similarly, the synthetic peptide of SEQ ID NOs: 8-12 Those added with amino hexanoic acid [NH ₂ — (CH ₂ ) ₅ —COOH] are described in order as M2 (monomer 1) to M6 (monomer 6).

  The inhibitory effect on the kinase activity of CaMKIIα-KD was examined using the synthetic peptides M1 to M6 and the synthetic substrate AIP. In addition, the effect of protein kinase A (PKA) and protein kinase C (PKC), which are general kinases present in cells, was also examined.

  The results of the inhibitory effect of the synthetic peptides M1 to M6 on the kinase activity of CaMKIIα-KD are shown in FIG. As can be seen from FIG. 2, although the inhibitory activity of M4 was attenuated at a high concentration, the other synthetic peptides were all shown to exhibit strong inhibitory activity, and the effect was similar to that of AIP.

  On the other hand, the inhibitory effect of the synthetic peptide of M1 to M6 on PKA is shown in FIG. 3, and the inhibitory effect of the synthetic peptide of M1 to M6 on PKC is shown in FIG. As shown in FIGS. 3 and 4, none of the peptides showed an inhibitory effect on PKA and PKC.

As described above, the CaMKIIα inhibitory peptides, M1 to M6, which have a novel sequence completely different from AIP and specifically strongly inhibit CaMKIIα can be identified by the multivalent screening method. By obtaining M1 to M6, it becomes possible to study various functional additions and functional improvements in accordance with the purpose, such as control of in vivo kinetics, membrane permeability and stability, and abnormal activation of CaMKII The development of excellent therapeutic agents for various important diseases involved is greatly promoted.
(5) Inhibitory Effect of Multivalent CaMKII Inhibitory Peptide CaMKIIα shown in SEQ ID NOs: 1 to 6 in four library parts (indicated as “XXXX” in FIG. 1) of the multivalent peptide library shown in FIG. A tetravalent peptide into which any of the binding motifs was introduced was prepared. These tetravalent peptides are described as T1 to T6 in order from the one containing SEQ ID NO: 1. In addition, this tetravalent peptide (T1-T6) has the molecular nucleus shown below,

Has a structure similar to that of a tetravalent peptide in which a peptide containing any one of the amino acid sequences of SEQ ID NOS: 7 to 12 is bound to each -NH.

  And the inhibitory effect with respect to the kinase activity of CaMKII (alpha) -KD was examined using the tetravalent peptides T1-T6 and the synthetic substrate AIP.

  The results of the inhibitory effect of the tetravalent peptides T1 to T6 on the kinase activity of CaMKIIα-KD are shown in FIGS. In FIG. 5, the horizontal axis is converted into the number of moles of CaMKIIα binding motif (SEQ ID NOs: 1 to 6) contained in the tetravalent peptide (T1 to T6). In FIG. 6, the horizontal axis represents the number of moles of tetravalent peptides (T1 to T6).

  As can be seen from FIGS. 5 and 6, it was confirmed that the tetravalent peptide introduced with any of the CaMKIIα binding motifs shown in SEQ ID NOs: 1 to 6 has an inhibitory effect on CaMKIIα activity. In particular, when the concentration of the CaMKIIα binding motif contained in the tetravalent peptide (T1 to T6) and the concentration of the tetravalent peptide (T1 to T6) are low (for example, 1 μM to 10 μM), inhibition equal to or higher than that of AIP The effect was confirmed.

Claims (4)

  A CaMKII inhibitory peptide, which is a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 6.   The CaMKII inhibitory peptide according to claim 1, wherein the peptide is a multivalent peptide having a plurality of amino acid sequences of any one of SEQ ID NOs: 1 to 6. The following molecular core with three lysine bonds:
And a peptide containing any one of the amino acid sequences of SEQ ID NOS: 1 to 6 is bonded to each -NH at the end of the molecular nucleus portion directly or via a spacer. The CaMKII-inhibiting peptide according to claim 2, wherein   A CaMKII inhibitor comprising the CaMKII inhibitory peptide according to any one of claims 1 to 3.