WO2006090555A1

WO2006090555A1 - Akt ACTIVATING AGENT

Info

Publication number: WO2006090555A1
Application number: PCT/JP2006/301326
Authority: WO
Inventors: Atsumi Nitta; Toshitaka Nabeshima
Original assignee: National University Corporation Nagoya University
Priority date: 2005-02-25
Filing date: 2006-01-27
Publication date: 2006-08-31
Also published as: JPWO2006090555A1

Abstract

Novel use of Leu-Ile. There is provided an Akt activating agent comprising as an active ingredient any of the following compounds: (a) peptides composed of Leu and Ile, (b) products of modification of peptides composed of Leu and Ile, and (c) pharmaceutically acceptable salts of compounds (a) or (b).

Description

Akt activator

Technical field

[0001] The present invention relates to an Akt activator. The Akt activator of the present invention can be used for the treatment or prevention of diseases involving Akt through the activity of Akt.

Background art

[0002] In recent years, the number of drug abusers has increased in the younger generation who will lead the future of Japan, and drug addiction is becoming a major social problem. Therefore, elucidation of the mechanism of drug dependence formation and development of treatment methods are urgent issues. Previous studies have reported that glial cell line-derived neurotrophic factor (GDNF) suppresses drug dependence (Green- Sadan et al., 2 003; Messer, 2000 (Non-Patent Documents 1 and 2). )). However, since GDNF is a high molecular protein and cannot be passed through the blood-brain barrier and is degraded by proteases in the blood, it is difficult to obtain a therapeutic effect by administration from the terminal. Therefore, we examined the therapeutic effects on drug dependence using the low molecular weight hydrophobic dipeptide Leu-Ile (Nitta et al., 2004 (Non-patent Document 3)) that induces the production of GDNF.

When Leu-lie was administered to mice given the most commonly abused stimulant methamphetamine in Japan, Leu-Ile suppressed the increase in place preference and momentum caused by methamphetamine, and was already formed. It has also been shown to have an inhibitory effect on increased place preference and momentum (Nitta et al, 2003 (Non-Patent Document 4)). These studies suggested that Leu-Ile could be a drug-dependent treatment. However, it has not yet been elucidated what protein Leu-Ile binds to in vivo and what kind of signal pathway it produces and induces GDNF.

GDNF has a protective effect on fetal mesoderm-derived dopaminergic neurons, motor neurons, hippocampal neurons, cerebral cortex neurons, etc. that are not only drug dependent (Houeno u et al "1996; Lin et al" 1993 Trupp et al "1997; Wang et al" 1997 (Non-Patent Documents 5 to 8)), and application as a therapeutic agent for Parkinson's disease and spinal cord injury is also expected. According to a survey by the Ministry of Health, Labor and Welfare, the number of Parkinson's disease patients in Japan alone is 141,000. In 2014, the number of patients with spinal cord injury was 100,000 (the 2001 survey on the actual condition of children with physical disabilities and persons with disabilities). Have a degenerative disease.

The Ministry of Education, Culture, Sports, Science and Technology started the “Regenerative Medicine Realization Project” in FY2003 to aim at the practical application of new treatment methods using regenerative medicine for these diseases and lifestyle-related diseases such as arteriosclerosis. did. If regenerative medicine is realized, it can be an innovative medical technology that fundamentally changes the conventional medicine. In order to realize regenerative medicine using “stem cells”, it is necessary to elucidate the mechanisms related to cell growth and regulation. Neurotrophic factors such as GDNF, which are also closely related to early neurogenesis, are thought to be important regulators of neural stem cell proliferation and differentiation (Lee et al., 2005; Riaz et al. 2004; Roussa and Krieglstein., 2004; Tzeng et al., 2004; Yoo et al, 2003 (Non-Patent Documents 9 to 13)).

Non-Patent Document 1: Green- Sadan T, Kinor Ν, Roth-Deri I, Geffen- Aricha R, Schindler CJ, Yadid G: Transplantation of glial cell line-derived neurotrophic factor-expressing c ells into the striatum and nucleus accumbens attenuates acquisition of cocaine self— a dministration in rats. Eur J Neurosci, 18: 2093—2098 (2003)

Non-Patent Document 2: Messer CJ, Eisch AJ, Carlezon WA Jr, Whisler K, Shen L, Wolf DH, Westphal H, Collins F, Russell DS, Nestler EJ: Role for GDNF in biochemical and b ehavioral adaptations to drugs of abuse. Neuron, 2b: 247-257 (2000)

Non-Patent Document 3: Nitta A, Nishioka H, Fukumitsu H, Furukawa Y, Sugiura H, Shen L, F urukawa S: Hydrophobic dipeptide Leu—lie protects against neuronal death by indu cing brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis. J Neurosci Res, 78: 250—258 (2004)

Non-Patent Document 4: Nitta A, Yamada Y, Nakajima A, Noda Y, Yamada K, Nabeshima T: C andidate genes or compounds for therapeutic tools against methamphetamine and / or morphine-induced dependence. Folia Pharmacologica Japonica, 122: 81-83 (2003) Non-Patent Document 5: Houenou LJ, Oppenheim RW, Li L, Lo AC, Prevette D: Regulation of spinal motoneuron survival by GDNF during development and following injury. Tissue Res, 286: 219-223 (1996)

Non-Patent Document 6: Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F: GDNF: a glial eel 1 line-derived neurotropnic factor for midbrain dopaminergic neurons. Science, 260: 1130-1132 (1993)

Non-Special Reference 7: Trupp M, Belluardo N, Funakoshi H, Ibanez CF: Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret p roto— oncogene, and GDNF receptor-alpha indicates multiple mechanisms of tropnic actions in the adult rat CNS.J Neurosci, 17: 3554-3567 (1997)

Non-Patent Document 8: Wang Y, Lin SZ, Chiou AL, Williams LR, Hoffer BJ: Glial cell line- der ived neurotrophic factor protects against ischemia— induced injury in the cerebral cor tex. J Neurosci, 17: 4341-4348 (1997 )

Non-Patent Document 9: Lee CS, Tee LY, Dusenbery S, Takata T, Golden JP, Pierchala BA, Gottlieb DI, Johnson EM Jr, Choi DW, Snider JB: Neurotrophin and GDNF family liga nds promote survival and alter excitotoxic vulnerability of neurons derived from muri ne embryonic stem cells. Exp Neurol, 191: 65-76 (2005)

Non-Patent Document 10: Riaz SS, Theofilopoulos S, Jauniaux E, Stern GM, Bradford HF: The differentiation potential of human foetal neuronal progenitor cells in vitro. Brain Res Dev Brain Res, 153: 39—51 (2004)

Special Article 11: Roussa E, Krieglstein K: GDNF promotes neuronal differentiation an d dopaminergic development of mouse mesencephalic neurospheres. Neurosci Lett, 361: 52-55 (2004)

Non-Special Terms 12: Tzeng SF, Tsai MJ, Hung S, Cheng H: Neuronal morphological change of size-sieved stem cells induced by neurotrophic stimuli. Neurosci Lett, 367: 2 3-28 (2004)

Non-Patent Document 13: Yoo YM, Kim YJ, Lee U, Paik DJ, Yoo HT, Park CW, Kim YB, Lee SG, Kim WK, Yoo CJ: Neurotrophic factor in the treatment of Parkinson disease. N eurosurg Focus, 15: 1 (2003)

Disclosure of the invention Problems to be solved by the invention

[0004] GDNF has a neuroprotective action and is expected to be applied to the treatment of neurodegenerative diseases. However, since GDNF cannot cross the blood-brain barrier when administered peripherally and is degraded by proteases in the blood, it cannot be expected to act on the brain. Therefore, it is desirable to develop a drug that can effectively increase the amount of GDNF produced in the brain or nerve tissue by oral administration. In search of such compounds, the present inventors have found Leu-Ile (previously reported). However, it is completely unknown how Leu-Ile induces GDNF production!

Therefore, the present invention aims to provide a new application of Leu-Ile by clarifying its action point and action mechanism.

Means for solving the problem

[0005] The present inventors have conducted research for the purpose of elucidating the GDNF production regulation mechanism of Leu-Ile, and have obtained the following findings.

(1) Leu-Ile is cell membrane permeable.

(2) When FITC-labeled Leu-Ile was reacted with mouse brain homogenization solution, a 70 KD protein was bound.

(3) As a result of mass spectrometry of Leu_Ile binding protein, Hsc70 was identified.

(4) was examined for binding activity to the crystal oscillator biosensor with Hsc70 and Hsc70 highly homologous Hsp70 and Leu- lie, Hsc70 is ^{Kd = 1.83 X 10- 8 M,} Hsp70 is K d = 1.24 X bound to Leu-Ile and specifically in strength of 10- ⁸ M.

(5) The molecular potential was used to predict the binding site of Hsc70 and Leu-Ile, and the mutual potential energy was calculated. As a result, -119.044 kCal for the ATPase domain and -76.711 kCal for the substrate-binding domain, C-terminal domain It was suggested that Leu-lie binds at a strength of -64.337 kCal, and that H sc70 is likely to bind to the ATPase domain.

(6) In cultured hippocampal neurons treated with Leu-Ile, NF-κΒ was translocated into the nucleus and activated.

(7) In cultured hippocampal neurons treated with Leu-Ile, NF-κB inhibitors sulfasalazine and Leu-Ile were allowed to act simultaneously, in which nerve cell death was suppressed and GDNF production was induced. In the cell population, the effect of Leu-Ile was suppressed.

(8) In the cultured hippocampal neurons treated with Leu-Ile, the degree of phosphorylation of CREB increased compared to the control group.

(9) When CREB antisense oligonucleotide was allowed to act on cultured hippocampal neurons, Leu-Ile's GDNF production-inducing effect was suppressed.

(10) In cultured hippocampal neurons treated with Leu-Ile, there was no change in CaMKII, PKC-?

(11) In cells treated with the PI3K inhibitors LY294002 and Leu-Ile simultaneously, phosphorylation of Akt and CREB induced by Leu-Ile was not suppressed. On the other hand, in cells treated with geldanamycin, an inhibitor of Hsp90, and Leu-Ile simultaneously, phosphorylation of Akt and CREB induced by Leu-Ile was suppressed.

[0006] Based on the above findings, Leu-Ile binds to Hsc70 and activates NF-κB and CREB via the Hsp90 / Akt pathway (the pathway in which Akt is activated by binding to Hsp90). It was thought to induce the production of GDNF. It was found that Leu-Ile induces GDN F production through the Hsp90 / Akt signaling pathway, and Hsc70 and Hsp90 / Akt could be identified as new target genes responsible for the regulation of GDNF expression.

[0007] As described above, it has been clarified that Leu-Ile induces GDNF production through a signal pathway via Hsp90 / Akt. That is, it has been found that Leu-Ile has an action of activating Akt, and as a result of this action, production of GDNF is induced.

The present inventors have noted that Leu-Ile has Akt activity. The ability to activate Akt means that it can regulate various signaling pathways involving Akt. Therefore, Leu-Ile is effective for diseases that can have therapeutic and prophylactic effects by regulating the gnnal pathway involved in Akt! /.

The present invention has been completed based on the above knowledge and consideration, and provides the following configuration.

The present invention is an Akt activator comprising any one of compounds (a) to (c) as an active ingredient.

(a) a peptide comprising Leu and lie; (b) a modified form of a peptide comprising Leu and lie;

(c) A pharmaceutically acceptable salt of ( _a ) or (b).

In one embodiment of the present invention, the peptide as an active ingredient is Leu-lie.

The Akt activator of the present invention is preferably used for the treatment or prevention of Parkinson's disease, spinal cord injury, drug dependence, Alheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia, rheumatism, or schizophrenia. The

Another embodiment of the present invention provides a food containing the Akt activator.

In still another aspect, the present invention includes a step of administering the Akt activator to a living body, including Parkinson's disease, spinal cord injury, drug dependence, Alzheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia, rheumatism Or a method for preventing or treating schizophrenia.

In the present specification, the following abbreviations are used as necessary.

AP2: activator protein 2, CEB: cytoplasmic extraction buffer and REB: cAMP respo nse element binding protein, CsA: cyclosporin A, DMEM: Dulbecco's modified Eagle e's medium ^ DTT: dithiothreitol, EDTA: ethylenediamine tetracetic acid, egr- 1: ear ly growth response- 1, egr- 2: earlygrowth response- 2, F12HAM: Ham's nutrient mixture F12, FBS: fetal bovine serum, FGF: fibloblast growth factor FITC: Fluoresce in isothiocyanate isomer 1, FK506: tacrolimus FKBP: FK506 binding protein, GCF: GC factor GDNF: glial cell line-derived neurotrophic factor GFR-a? : GDNF fa mily receptor- a, IL-1: interleukin— lbeta, MALDI: Matrix-assisted laser desorpti on / ionization ^ MNF: myocyte nuclear factor MRE: metal response element MS: Mass spectrometry MTT: methyl thiazol tetrazolium, NCBI: National Center for Biot echnology Information ^ NEB: nuclear extraction buffer NF— κ B: nuclear factor— kap pa B, NRSE: neural-restrictive silencer elements ODN: oligodeoxynucleotide, PBS: phosphate buffer s aline PDB: protein data bank, PDVF: polyvinylidene fluoride PI 3K: phosphatidylinositol-3 kinase PMSF: phenylmethansulfonylfluoride, RCSB: Res earch Collaboratory for Structural Bioinformatics, SDS: sodium dodecyl sulfate Six2: sine oculis— related homeobox 2 homology SPl: trans-acting transcription factor 1, TBS: Tris buffer saline TFA: trifluoroacetic acid, TNF-: tumore necrosis factor— alpha TOF: time of flight-time of flight, Tyr: tyrosine USF: Up-stream stimulatory factor YY1: yin yang— 1.

Brief Description of Drawings

[Fig. L] Results of Leu-Ile cell membrane permeability experiment. A: Add only FITC-labeled Leu-Ile or FITC to the supernatant of cultured neurons, and examine the dose dependence of these cell membrane permeabilities by measuring the fluorescence intensity of FITC in the cells 30 minutes later. did. B: FITC-labeled Leu-Ile (10 g / ml) was added to cultured neurons, and the time dependence of cell membrane permeation after addition was examined. C: FITC-labeled Leu-Ile (10 μg / ml) and various concentrations of Leu-Ile were added together to cultured neurons, and the intracellular FITC fluorescence intensity was measured after 30 minutes. -Ile suppressed the increase in fluorescence intensity caused by FI TC-labeled Leu-Ile supplement. D:-, FITC (10 g / ml) and various concentrations of Leu-Ile were added to cultured neurons together, and the fluorescence intensity increased even after measuring intracellular FITC fluorescence intensity after 30 minutes The power was restrained.

[Fig. 2] Results of Leu-Ile binding protein isolation experiment. (A): Lane 1: Leu-lie fluorescently labeled on the N-terminal side and mouse brain lysate were allowed to act at 4 ° C or 37 ° C. Lane 2: Leu-Ile fluorescently labeled on the C-terminal side and mouse brain lysate were allowed to act at 4 ° C or 37 ° C. These electrophoretic images were read with a fluorescence scanner. (B): Mouse brain lysate was reacted with Leu-Ile-conjugated Affigel 10 (lane 1) or! /, Nana! /, Affigel 10 (lane 2), and Leu-Ile binding protein was Purified. The Leu-Ile binding protein was subjected to silver staining after electrophoresis and mass analysis was performed. Lane l; Leu-Ile binding protein. Lane 2; control. The arrow indicates the protein subjected to mass analysis. (C): The sample used for mass analysis was subjected to Western blotting of Hsc70.

[Fig. 3] Results of measuring the binding affinity between Leu-Ile and Hsc70 or Hsp70 with a quartz crystal. (A): Leu-Ile, Pro-Leu or lie-Pro was adsorbed on a quartz crystal unit and fixed in PBS. Thereafter, Hsc70, Hsp70, or heat-denatured Hsc70 or Hsp70 at 25 ° C. was added to PBS. The frequency at that time was counted. The dissociation constant was calculated with the software AQUA

[Fig. 4] Shows the binding site between Leu-lie and Hsc. Ascase domain of Hsc70 (Flaherty et al., 1990) And Leu-Ile combine.

[Fig. 5] Number of non-activated NF-κB cells and the amount of inactivated NF-κB in the nucleus. (A): Leu-lie (10 μg / ml), TNF-α (100 ng / ml) and GDNF (50 ng / ml) were allowed to act on the cells for 30 minutes. (A-a): Cells in which typical NF-κB is not activated. (A-b): Typical NF-κB-active cells. (A-c): percentage of cells in which NF-κΒ is not activated. For each culture dish, 8 fields were counted (75 x 55 m each) and examined using 4 culture dishes. * P <0.05 vs. control (Sheffe test). (B): Leu-Ile (10 μg / ml), TNF-a (100 ng / ml) and GDNF (50 ng / ml) were allowed to act on the cells for 30 minutes, and Westamp lot was performed. Typical experimental results are shown. The strength was also examined (n = 4), and the average standard deviation was shown. * P <0.05 vs. control (Sheffe test).

[Fig. 6] Effect of NF-κΒ on cell survival and GDNF expression in cultured neurons. (A) Leu-Ile (10 g / ml) was allowed to act on cultured neurons for 3 days in the presence or absence of sulfasalazine (25 M). The cell survival effect was examined by the MTT method. Leu-Ile increased the number of viable cells, but sulfasalazine suppressed its effect of Leu-Ile. Values are expressed as (n = 6) standard deviation. * P <0.05 versus control (Sheffe test). (B): Leu-Ile (10 μg / ml) was allowed to act for 1 day in the presence or absence of sulfasalazine. The expression level of GDNF was examined in the stamp lot. Typical experimental results are shown. The experimental results are shown in average standard deviation. Leu-Ile induced GDNF expression, and sulfasalazine suppressed its action. Values are expressed as (n = 4) mean standard deviation. * P <0.05 vs. control (Sheffe test).

[Fig. 7] CREB phosphorylation and GDNF expression level. (A): Leu-Ile (10 μg / ml) was allowed to act on cultured neurons for 0, 10, 20 or 30 minutes, and phosphorylated CREB was measured by Western blot. Phosphorylated CREB increased from 20 minutes after the action of Leu-Ile and was maintained until 30 minutes. A typical Western plot result is shown. (B): Antisense or sense nucleotide of CREB was allowed to act on cultured neurons (ODN) for 24 hours. The amount of CREB protein was measured by Western plot. (C): Leu-Ile (10 g / ml) was allowed to act for 24 hours in the presence or absence of antisense or sense oligonucleotide of CREB. The expression level of GDNF was examined by Western blot. The ability of Leu-Ile to increase the expression level of GDNF Antisense oligonucleotide suppressed its action. Values are expressed as (n = 4) mean standard deviation. * P <0.05 vs. control (0 hour) (Sheffe test).

FIG. 8 shows the effect of Leu-Ile on the expression of GDNF protein and GDNF mRNA. A: The expression level of GDNF protein after 24 hours when Leu-Ile, Pro-Leu or Ile-Pro (10 μg / ml) was added to cultured neurons. Only Leu-Ile increases GDNF protein expression. B: Expression level change of GDNF mRNA after addition of Leu-Ile. The expression level of GDNF mRNA increased 12 or 18 hours after adding Leu-Ile.

[Fig. 9] Effect of Leu-Ile on CREB and Akt signaling. (A): Leu-Ile (10 μg / ml) was allowed to act on cultured neurons for 0, 10, 20 or 30 minutes. The degree of phosphorylation of Akt, ERK, CaMKII, and PKC-γ was measured by Western blot. (B), (C): Leu-Ile and PI3 k inhibitor or Hsp90 inhibitor were allowed to act for 30 minutes. (B): The degree of Akt phosphate was measured by Western plot. (C): The degree of CREB phosphorylation was measured by Western blot. Shown are the results of a typical Western plot experiment (top). The band intensity was measured and digitized (lower). Values are expressed as (n = 4) mean standard deviation. * P 0.05 vs. control (0 hour) (¾neffe test).

[Figure 10] Expected Leu-lie action pathway.

[Figure 11] Results of mascot search. The amino acid fragments obtained as a result of mass analysis were analyzed with mascot software. Ten peptides matched the sequence of Hsc70.

[FIG. 12] Partial nucleotide sequence of human GDNF promoter II, exon 1 and first intron. The 1st to 174th bases are exon 1. The transcription factor binding site is underlined. These amino acid sequences are listed in GenBank AF053749. Abbreviations: activator protein 2, P- 2), early growth response- 1, egr- 1), early growth response- 2 (egr- 2), C factor (GCF), myocyte nuclear factor (MNF), metal response element -A (MRE-A), metal response element— B (MRE— B), trans-acting transcription factor 1 (SP1), Up-stream stimulatory factor (USF), yin yang-1 (YY1).

BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, according to the conventional notation, peptides are represented such that the left end is the amino end and the right end is the carboxy terminus. In the case of amino acid residue strength forms, the indication that the form is L may be omitted. In addition, unless otherwise stated, in this specification, dipeptides are expressed. Therefore, “peptide” is used as a term including peptides of various lengths.

[0010] A first aspect of the present invention relates to an Akt activator. As used herein, “Akt activator” refers to a substance having an action of activating Akt. Akt is a serine Z threonine kinase, also called Protein Kinase B (PKB), which is activated in the PI3 kinase pathway. Akt has been reported to be involved in various phenomena such as cell cycle and insulin metabolism.

Akt activity up-regulates the signaling pathway via Akt. For example, NF-κB and CREB are activated by the activity of Akt. Therefore, Akt activator can be regarded as activator of NF-κB and CREB. On the other hand, the activity of NF-κΒ and CREB stimulates the production of BDNF. Therefore, the Akt activator can be excluded as a BDNF production inducer.

[0011] The Akt activator of the present invention can exert an effect on the treatment and prevention of diseases associated with Akt through the regulation of Akt activity. Therefore, the Akt activator of the present invention can be applied to “diseases in which a preventive or therapeutic effect can be obtained by regulating Akt”. “Disease in which preventive or therapeutic effects can be obtained by modulating Akt” can be restated as “disease characterized by abnormal Atk activity”. That is, the diseases (target diseases) that are the subject of the present invention include diseases caused as a result of Akt activity deviating from the normal range. For example, Parkinson's disease, spinal cord injury, Alzheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia, rheumatism, or schizophrenia fall under such diseases.

[0012] The study by the present inventors has revealed that the active ingredient of the present invention described in detail below induces the production of GDNF via Akt. From this fact, the Akt activator of the present invention can induce the production of GDNA via Akt. Therefore, a disease in which a prophylactic effect or a therapeutic effect is obtained by the production induction of GDNF is one of the preferable target diseases of the present invention. For example, Parkinson's disease, spinal cord injury, Alzheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia, rheumatism, or schizophrenia fall under such diseases.

There are some reports on the relationship between AKt or GDNF and diseases (Tas SW, Remans PH, Reeaquist KA, lak PP. Signal transduction pathways and transcription factors as th erapeutic targets in inflammatory disease: towards innovative antirheumatic therapy. Curr Pharm Des. 2005; 11 (4): 581—611., Rickle A, Bogdanovic N, Volkman I, Winblad B, Ravid R, Cowburn RF. Akt activity in Alzheimer's Neuroreport. 2004 Apr 29; 15 (6): 955— 9.) Force S. Disease and other neurodege nerative disorders.

In this specification, the term “disease” is used interchangeably with a word indicating an abnormal state such as a disease, illness, or disease state.

In one embodiment, the Akt activator of the present invention includes a peptide (dipeptide) comprising Leu and lie as an active ingredient. Leu (Leucine) is also called 2-aminoisocabronic acid, and its L form is represented by the following chemical formula.

[Chemical 1]

COOH

H ₂ NCH

CH ₂

I

CHaCH

_{On the} other hand, lie (isoleucine) is also known as 2-amino-3-methyl-n-valeric acid, and its form is represented by the following chemical formula.

[Chemical 2]

C00H

Thigh CH

CH ₃ CH

CH ₂

I

CH ₃

In the peptide of the present invention, these two amino acid residues are preferably linked in the order of leucine and isoleucine from the _N- terminal side to the c-terminal side.

In the present invention, it is preferred that each amino acid residue is in the L form, but the amino acid residue Some or all of the groups may be in D form.

[0014] In general, a compound obtained by modifying a part of a certain compound may have properties and characteristics similar to those of the compound before modification. That is, the modification may not affect the specific properties of the compound. In consideration of this, even a modified product obtained by modifying the above peptide can be used as an active ingredient in the present invention as long as the Akt activity is maintained.

Therefore, in another embodiment of the Akt activator of the present invention, a modified form of the peptide (hereinafter referred to as “peptide modified form”) is used as an active ingredient. In the present invention, the “modified peptide” refers to a part of the basic structure (dipeptide) composed of Leu and lie (dipeptides) may be substituted with other atomic groups or the like. A compound having a structure different from the basic structure at least in part by adding a modification such as addition of a molecule of this type is known to those skilled in the art or consists of Leu and lie using means for β. Modifications such as substitutions based on dipeptides can be designed. Moreover, it is considered easy for those skilled in the art to prepare a target modified product using well-known or β means based on the profitable design and investigate its properties and actions.

[0015] Representative examples of the modified peptide in the present invention include peptide derivatives in which a part of the side chain (atom or atomic group) is substituted with another atom or atomic group in each amino acid residue. . Such peptide derivatives can be prepared by any manufacturing process designed to yield the peptide derivative as a final product. Therefore, when the target peptide derivative is a peptide in which a part (for example, an atomic group that is a part of a side chain) is apparently substituted by a specific atomic group, Peptide derivatives may be produced by a substitution reaction using the apparently basic peptide as a starting material and using the specific atomic group, or, for example, a suitable substitution reaction using a peptide having another structure as a starting material. Etc. (may be a plurality of steps in some cases). Therefore, for example, in the case of a derivative of a Leu-Ile dipeptide, the Leu-Ile dipeptide may not be used as a starting material. Other atoms or atomic groups herein include hydroxyl groups, halogens (fluorine, chlorine, fluorine, iodine, etc.), alkyl groups (methyl group, ethyl group, _n -propyl group, isopropyl group, etc.), hydroxyalkyl groups (Hydroxymethyl group, hydroxyethyl group, etc.), alkoxy group (methoxy group, ethoxy group, etc.), isyl group (formyl group, acetyl group, malol group, benzoyl group, etc.), etc. .

[0016] The modified peptide of the present invention includes those in which the functional group in the constituent amino acid residue is protected by an appropriate protecting group. As the protecting group used for such purposes, an acyl group, an alkyl group, a monosaccharide, an oligosaccharide, a polysaccharide and the like can be used. Such a protecting group is linked by an amide bond, an ester bond, a urethane bond, a urea bond, or the like according to the peptide site to which the protecting group is bound, the kind of the protecting group to be used, or the like.

[0017] It is also possible to form the modified peptide of the present invention by attaching (linking) amino acids. However, it is considered preferable that the number of amino acids added is not so large in terms of solubility and bioavailability. Specifically, the number of amino acids to be added is, for example, 1 to 9, preferably 1 to 5, more preferably 1 to 3, and most preferably 1 or 2. It should be noted that amino acids may be applied on both sides of the basic peptide (or modified peptide).

[0018] As a further example of the modified peptide of the present invention, one modified with a sugar chain attachment can be mentioned. Various peptides classified as alkylamines, alkylamides, sulfiers, sulfonamides, amides, amides, amino alcohols, esters, amino aldehydes, etc. by replacing the N-terminal or C-terminal with other atoms Derivatives are also included in the modified peptide of the present invention.

[0019] A peptide derivative constituted by combining the various modification methods described above may be used as the modified peptide of the present invention.

[0020] In still another embodiment of the Akt activator of the present invention, the peptide or a salt of the modified peptide is used as an active ingredient. The salt of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and is a salt (inorganic acid salt) with hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, boric acid, formic acid, acetic acid, lactic acid, fumaric acid. Examples thereof include salts (organic acid salts) with maleic acid, tartaric acid, citrate, and the like. These salts can be prepared by conventional means. [0021] Peptides, modified peptides, or these! /, Any of their salts (hereinafter collectively referred to as "peptides") typically contained in the Akt activator of the present invention are typically The use of a compound that is linear but has a cyclic structure is not excluded. That is, a peptide or the like that forms a cyclic structure partially or entirely by linking side chains of amino acid residues or the like can be used as an active ingredient of the Akt activator of the present invention.

[0022] The peptide and the like in the present invention can be produced by a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method). However, when the peptide of the present invention exists in nature, it can also be prepared by operations such as extraction and purification. Examples of sources for obtaining the peptide of the present invention include animal cells (including humans), plant cells, body fluids (blood, urine, etc.) and the like.

The peptide of the present invention may be prepared using a genetic engineering technique. That is, the peptide of the present invention can also be prepared by introducing a nucleic acid encoding the peptide of the present invention into an appropriate host cell and recovering the peptide expressed in the transformant. The recovered peptide is purified as necessary. The recovered peptide or the like can be subjected to an appropriate substitution reaction to be converted into a desired modified peptide.

Among peptides and the like in the present invention, for example, Leu-Ile is commercially available and can be obtained from a domestic chemical company (KOKUSAN CHEMICAL Co., Ltd. Tokyo, Japan).

[0023] Of course, the Akt activator of the present invention can be used as a drug for a specific disease, for example, as a food (food additive) for reducing the risk of suffering from a specific disease. It can also be used as a research reagent for studying the onset mechanism or progress mechanism of a specific disease. That is, the Akt activity modulator of the present invention can be provided in the form of a drug, food (food additive), research reagent, and the like.

[0024] Formulation when provided as a drug can be performed according to a conventional method. When formulating, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emollients, suspensions, soothing agents, stabilizers, preservatives, preservatives) Agent, physiological saline, etc.). As the excipient, lactose, starch, sorbitol, D-manntol, sucrose and the like can be used. As the disintegrant, starch, carboxymethyl cellulose, calcium carbonate, or the like can be used. Buffers such as phosphate, kenate, acetate, etc. Can be used. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methinorescenellose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, polypropylene alcohol, diethylin sulfite, ascorbic acid and the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl paraben, and the like can be used. Examples of antiseptics that can be used include salt benzalkonium, nonoxybenzoic acid, and chlorobutanol.

[0025] The dosage form for formulation is not particularly limited, and can be prepared, for example, as tablets, powders, fine granules, granules, force capsules, syrups, injections, external preparations, suppositories, and the like.

The drug of the present invention thus formulated is administered orally or parenterally depending on its form.

(Intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal injection, etc.) can be applied to the patient. The content of the active ingredient (peptide or the like) in the drug of the present invention generally varies depending on the dosage form. For example, it is about 0.001 wt% to about 90 wt% so as to achieve a desired dose.

[0026] In another aspect of the present invention, target diseases using the above drugs (Parkinson's disease, spinal cord injury, Alzheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia, rheumatism, schizophrenia, etc.) Prevention methods or treatment methods (hereinafter, these two methods are collectively referred to as “treatment methods”). The therapeutic method of the present invention includes the above peptides, peptide modifications, or these! / Includes the step of administering a drug containing any salt as an active ingredient to the living body. The administration route is not particularly limited, and examples thereof include oral, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, and transmucosal.

[0027] The dose of the drug varies depending on symptoms, patient age, sex, weight, and the like, but those skilled in the art can appropriately set an appropriate dose. For example, when using a drug containing Leu-Ile as an active ingredient, the amount of active ingredient per day for adults (body weight of about 60 kg) is about 0.1 to about 1000 mg, preferably about lmg to about lOOmg. The dose can be set to be The administration schedule is, for example, once to several times a day, once every two days. Times, or once every three days. In setting the administration schedule, it is possible to consider the patient's medical condition and the duration of drug effect.

[0028] In a further aspect of the present invention, a food (or food-added carotenoid) containing the Akt activator of the present invention is provided. Examples of food here include milk and soft drinks. In the case of food-added calories, it can be provided in the form of powder, granule powder, tablet, etc. When using an Akt activator as such food or food additive, the amount added should be expected to have a therapeutic or prophylactic effect. The amount to be added can be determined in consideration of the medical condition, health status, age, sex, weight, etc. of the subject.

Leu-Ile has high solubility and is tasteless and odorless. In addition, Leu-Ile is a substance that is constantly present in the living body, so it can be said that its safety is high. In this way, Leu-Ile has a special characteristic that is preferred as a substance added to food.

[0029] In one embodiment of the present invention, the Akt activator of the present invention is provided as a research reagent. In this case, the label may be modified in advance. It can also be provided in the form of a kit combining various reagents necessary for the use of the Akt activator or a reaction container.

Example 1

[0030] 1. Identification of Leu-Ile binding protein in mouse brain

1-1 Purpose

Cyclosporin A (CsA) and tacrolimus (FK506), which are used to prevent rejection after organ transplantation, are immunophilin ligands that bind to endogenous immunophilin and exhibit immunosuppressive action. CsA and tacrolimus have been reported to suppress neurodegeneration in a variety of pathological models with only immunosuppressive action, and research has been conducted even for their usefulness as neuroprotective drugs (Ogawa et al., 1993; Sharkey and Butcher., 1994; Shiga et al., 1992; Tanaka and Ogawa., 2004) o However, for patients with neurodegenerative diseases, these drugs may also have neuroprotective effects, but at the same time immune function As a result, a major problem remains. This has led to studies on non-immunosuppressive immunophilin ligands that have only neuroprotective effects (Gold., 2000; Gold and Nutt., 2002; Herdeg an et al., 2000; Snyder et al "1998). Leu-Ile is a peptide discovered in search of non-immunosuppressive immunophilin ligands. Leu-Ile is similar to the structure of the immunophilin binding site of tacrolimus and has been shown to induce the production of GDNF in the same way as takuguchi rim. However, it has been found that it does not bind to FK506 binding proteinl2 (FKBP12), which is a binding protein of tacrolimus, suggesting that Leu-Ile may induce GDNF production by a different action from tacrolimus. (Nitta et al., 2002; 2004) ₀

Therefore, in the following, in order to investigate how Leu-Ile induces GDNF production, we identified the binding protein of Leu-Ile in the brain using mouse brain homogenization solution.

[0031] 1-2 Experimental Materials and Methods

1-2-1 animals

In the experiment, 7-week-old ICR male mice (Japan SLC, Shizuoka) were used. This study was conducted based on the Nagoya University School of Medicine Animal Experiment Fund † and Principles of Laboratory Animal Care (National Institutes of Health Publication 85-23, 1985).

[0032] 1-2-2 Demonstration experiment of Leu-Ile permeating cell membrane

Add FITC-labeled Leu-Il to the culture supernatant of cultured neurons to a concentration of 0-1.5 μg / ml, remove the supernatant after 10-20 minutes, wash with physiological buffer, and wash the cells. The material was peeled and collected with a rubber policeman. After centrifugation, the intensity of fluorescence (excitation wavelength: 485 nm, fluorescence wavelength: 518) in the supernatant was measured with a plate reader for fluorescence measurement. Calibration curves for FITC-labeled Leu-Ile and FITC were prepared for each experiment.

[0033] 1-2-3 Binding experiments between fluorescein isothiocyanate (FITC) -labeled Leu-lie and mouse brain protein

Under anesthesia with pentobarbital, 7-week-old ICR male mice were also removed with ^ | homogenization buffer ((0.25M NaCl, 5mM ethylenediamine tetracetic acid (EDTA), 1% TritonX—100, 0.25% dehydroacetic acid sodium salt Add 20 mM Tris-HCl (pH7.4)) containing monohydrate, ImM phenylmethansulfonylfluoride (PMSF), 1 ^ g / ml leupeptin, 1 ^ g / ml aprotinin, 5 ^ g / ml pepstatin A) for 30 seconds. Homogenization was performed with a homogenizer (heat systems, NY, USA). This solution was centrifuged at 10,000g for 4 hours at 4 ° C, and the supernatant was recovered. Was used as a brain homogenizing solution in the experiment. Leu—lie (Thermo Electron Corporation, Ulm, Germany) (10 mg / ml) with N- and C-terminal ends of FITC and monthly ¾ homomonise solution (total protein 6 mg / ml) at 4 ° C or 37 Sample buffer (125 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 4% sodium dodecyl sulfate (SDS), 10% sucrose, 0.004% bromophenol blue) after reacting at ° C for 3 hours Boiled at 95 ° C for 5 minutes and electrophoresed 30 / zg protein using 10% polyacrylamide gel. FluorImager595 (Molecular Dynamics, CA, USA) was used to detect the fluorescent band.

[0034] 1-2-4 Purification of Leu-lie binding protein by Affige 卜 10

Agarose beads £ ¾6 卜 10 (810 '1 ^ 0, CA, USA) were packed into a 30 ml syringe (Terumo, Tokyo) (diameter 22 mm, height 26.5 mm) to make a column. The column was washed with 100 ml of 0.1 M sodium bicarbonate solution (pH 8.5) to wash the beads, and Leu-Ile (Kokusan Kagaku, Tokyo) dissolved in 0.1 M sodium bicarbonate solution (pH 8.5) (30 mg / ml) and the beads were reacted at 4 ° C for 3 hours. After washing the beads with 0.1 M phosphate buffer (pH 7.4) and removing Leu-Ile that did not bind to the beads, 20 ml of brain homogenization solution (6 mg / ml) similar to 2-2-2 was added. In addition to the force ram, the reaction was carried out at 4 ° C with beads conjugated with Leu-Ile. After washing the beads with 100 ml of 0.1 M phosphate buffer (pH 7.4), the protein bound to Leu-Ile was eluted with 0.17 M glycine-HCl solution (pH 3). The same operation was performed using beads not bound with Leu-Ile as a control. This was electrophoresed using a 5-20% gradient gel (BIORAD) and silver stained using the silver staining “Daiichi” kit (Daiichi Kagaku, Tokyo).

[0035] 1-2-5 trypsin treatment and mass spectrometry of Leu-Ile binding protein

The gel stained with 1-2-4 was washed three times with distilled water for 10 minutes, and a gel piece containing the target protein was cut out using a scalpel. The gel pieces were reacted with 100 μl of decolorizing solution (18.5 mg / ml Na CI, 18.5 mg / ml CuS04, 18.5 mg / ml Na2S203) for 5 minutes and washed with distilled water until the gel pieces became transparent. After dehydration with acetonitrile, the gel pieces were dried in a vacuum centrifuge for 5 minutes. 30 1 10 mM dithiothreitol solution was infiltrated into the gel piece and reacted at room temperature for 30 minutes, then the dithiothreitol solution was removed, 100 μm iodoacetamide was added 30 μ \, light-shielded with aluminum foil, and alkylated at room temperature for 30 minutes . Acetonit to gel pieces again Add rile, dehydrate and dry, infiltrate with 10 μl trypsin (0.01 μg / ul) (Promega, WI, USA), add 100 mM 100 mM ammonium bicarbonate solution, 37 ° It was made to react with C. (1) 100 mM bicarbonate solution containing 5% acetonitrile and 0.1% trifluoroacetic acid (TFA), (2) 100 mM bicarbonate solution containing 50% acetonitrile, 0.1% TFA, ( 3) 100 mM ammonium bicarbonate solution containing 80% acetonitrile and 0.1% TFA was sequentially added to elute the peptide fragments from the gel pieces. The eluate was dried and dissolved in an aqueous solution containing 5% acetonitrile, 0.1% formic acid and 0.1% TFA, and the sequence of the peptide fragment was analyzed using a mass spectrometer Q-Tof2 (Micromass, Manchester, UK). Analytical results were searched using a mascot search (Matnx Science, MA, U¾A) and a database search of enter for Biotechnology Information (N CBInr: http://www.ncbi.nlm.nih.gov/Entrez/) ( Perki ns et al., 1999). The score calculation is based on the Mowse algorithm, and when the score is 44 or higher, it is statistically reliable with a risk factor of 5% or less (Pappin et al, 1993). 1-2-6 Western blotting

An equal volume of sample buffer was boiled at 95 ° C for 5 minutes in the sample used for mass spectrometry, and electrophoresis was performed using 5-20% gradient gel (BIO RAD).

Transfer protein from polyacrylamide gel to polyvinylidene fluoride (PDVF) membrane (Millipore, MA, USA) by semi-dry transfer method, and then gently agitate the membrane in blocking buffer (KPL, MD, USA) 2 Blocked for hours at room temperature.

Next, the primary antibody (rat anti-Hsc70 antibody 3; i rabbit anti-Hsp70 antibody (Stressgen, B, and then Canada) was used to react once at 4 ° C. After washing with Tris buffer saline (TBS) (10 mM Tris-HC1, 137 mM NaCl, 0.1% Tween-20) three times for 15 minutes, secondary antibody (peroxidase labeled goat anti-rat IgG (H + L ) antibody ¾7t ^ peroxidase labeled goat anti-rabbit IgG (H + L) antibody (Kirkegaard & Perry Laboratries, MD, USA)) for 2 hours at room temperature, then washed 3 times with TBS, ECLTM (Amersham Pharmacia Biotec, UK) was allowed to react for 1 minute and developed into a hyperfilm (Amersham Bioscience, NA, UK) using an FPM100 (Fuji Photo Film, Tokyo) processor. ATTO Densito Graph (Atoichi, Tokyo) was used. [0037] 1-2-7 Examination of binding force between heat shock cognate protein 70 (Hsc70) and heat shock protein 70 (Hsp70) and Leu- lie by quartz crystal biosensor

The quartz crystal microbalance method is changed by molecular bond 'dissociation, polymerization' decomposition. By detecting the increase or decrease in the absolute weight of the quartz crystal by the change in frequency, the molecular reaction can be measured at the nanogram level without labeling with a fluorescent substance. (Motomiya et al., 2003; Okahata and Furusawa., 2004; Sauerbrey., 1959). The lens sensor chip (Y-Siam, Tokyo) was washed twice with Piranha solution (7.5% H202, 25% H2S04) for 5 minutes, and then 5 mM 3,3-dithiodipropionic acid solution was placed on the 100 μ1 sensor chip at room temperature. Let stand for 30 minutes. After washing with distilled water, 100 μ1 of an equal volume mixture of l- (3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (100 mg / ml) and N-hydroxysuccinimide (100 mg / ml) is placed on the sensor chip. The mixture was allowed to stand at room temperature for 30 minutes and washed with distilled water.

Dipeptide is prepared by placing 100 μ \ phosphate buffer containing Leu-Ile, Pro-Leu (domestic chemistry) or lie-Pro (domestic chemistry) (10 g / ml) on the sensor chip for about 1 hour at room temperature. Was immobilized on a sensor chip. Pro-Leu and lie-Pro are the same hydrophobic dipeptides as Leu-Ile, but do not show GDNF production-inducing action (Nitta et al, 2004), so they were used as negative controls.

After setting the sensor chip with the dipeptide immobilized on AFFINIX Q (Y-Siam, Tokyo), stabilizing the frequency in phosphate buffer, Hsp70 (Stressgen) (1.79, 3.57, 7.14, 14.3, 28.6 nM) or Hsc70 (Stressgen) (1.79, 3.57, 7.14, 14.3, 28.6 nM) was added successively. The frequency change was monitored by AFFINIX Q User Analysis (Y-Siam) to visualize the degree of binding of Hsp70 or Hsc70 to the dipeptide. For the frequency force, a concentration-binding saturation curve was obtained, and the dissociation constant (Kd value) was calculated.

[0038] 1-2-8 Prediction of binding sites of Hsc70 and Leu-Ile using molecular simulation

Hsc70 is divided into 44 KD ATPase domain, 18 KD substrate binding domain, and 10 KD C-terminal domain from the N-terminal side. The ability to predict which site of Leu-Ile binds to / by Hsc70 by using Nyeon Soft-Fu ~~ (MuE; Chemical and omputing GroupQuebec, and ana da). Research and ollaboratory for Structural Bioinformatics (S and SB) Protein data bank (PDB) (http://pdbbeta.rcsb.org/pdb/Welcome·do) force 3D structure data of each domain (3HSC (Flaherty et al. 1990), 7HSC (Morshauser et al, 1999) and 1UOD (Chou et al, 2003)), and using MO E (molecular force field: MMFF94x, cut-off: 9.5 nm) with Hsc70 and Leu-Ile. The binding site was analyzed.

[0039] 1-3 results

1-3-1 Leu-lie membrane permeability

It was proved that FITC-labeled Leu-Ile permeates the cell membrane. As shown in Fig. 1A, the fluorescence intensity of intracellular FITC increased with increasing concentration of FITC-labeled Leu-Ile. We also examined the relationship with the passage of time and found that FITC-labeled Leu-Ile was taken up into cells very quickly after 120 minutes of addition to cultured cells, and further uptake was observed. No longer (Figure 1B). FITC itself can penetrate cell membranes FITC-labeled Leu-I le is easier to penetrate. FITC-labeled Leu-Ile membrane permeability To determine whether it is caused by Leu-Ile, not FITC, add excess Leu-Ile to the culture supernatant at the same time as FITC-labeled Leu-Ile or FITC. I'm sorry. When FITC-labeled Leu-Ile was used, the fluorescence intensity of FITC incorporated into cells decreased in a dose-dependent manner with Leu-Ile, and cell membrane permeability was suppressed (Fig. 1C). When the Leu-Ile was added at the same time, FITC intracellular uptake was not suppressed. From these results, it was shown that Leu-Ile has cell membrane permeability.

[0040] 1-3-2 Examination of FITC-labeled Leu-Ile binding protein in the brain

A mixture of Leu-Ile labeled with FITC at the N or C terminus and mouse brain homogenized solution was electrophoresed, and an image was obtained using a fluorescence scanner (Fig. 2A). Compared to Leu-Ile labeled with FITC on the C-terminal side, the force of binding of more proteins to Leu-Ile labeled on the N-terminal side Both N and Leu-Ile labeled with FITC on the C-terminal side A protein of 70 KD size was bound. In addition, each bond was almost unaffected by the temperature difference between 4 ° C and 37 ° C.

[0041] 1-3-3 Identification of Leu-lie binding protein

The eluate purified with agarose beads Affigd-10 was electrophoresed and silver stained. As a result, a band with a size of 70 KD was recognized as in the experiment conducted in 1-3-1. (Fig. 2B). It is considered that this is a protein that specifically binds to Leu-Ile, digested with trypsin, and then 7 minutes later, Matrix-assisted laser aesorption / ionization- time of flight / time of flight— Mas s spectrometry (MALDI- Protein mass spectrometry was performed using TOF / TOF-MS). When searching the NCBInr database using mascot search, the peptide chain strength was consistent with the sequence of 10 peptide shock cognate protein 70 (Hsc70) and scored 96, indicating that Hsc70 is a Leu-Ile binding protein. The possibility was extremely high! (Fig. 11). As a result of Western blotting of the eluate purified with Affige® 10 using an antibody of Hsc70, a band thought to be Hsc70 was detected (FIG. 2C). On the other hand, no force band was detected by Western blotting for Hsp70 having 81% homology with Hsc70 (data not shown). Hsc70 is a protein that is expressed constantly. Hsp70 is known to be induced by exposure to stress such as heat ischemia (Dworniczak and Mirault., 1987). Since the brain used in the test was unstressed, it is thought that the binding between Hsp70 and Leu-Ile could not be observed.

[0042] 1-3 -4 Examination of binding force of Hsc70 and Hsp70 with Leu-lie using quartz crystal biosensor

The binding activity of Hsc70 and Hsp70 and Leu-Ile was measured using a quartz crystal biosensor. When Hsc70 or Hsp70 was reacted with a crystal oscillator with Leu-Ile, Pro-Leu, or lie-Pro, the frequency of only those with Leu-Ile binding decreased (Fig. 3A). 9 The addition of heat-denatured Hsc70 and Hsp70 incubated at 5 ° C for 5 minutes did not reduce the frequency, so the heat-denatured Hsc70 and Hsp70 do not bind to Leu-Ile! (Figure 3A). Result of obtaining the dissociation constant of Hsc70 or Hsp70 and Leu-Ile, Hsc7 0 is ^{Kd = 1.83 X 10- 8 M,} Hsp70 is found to bind Leu- lie in the intensity of Kd = 1.24 X 10- ⁸ M (Figure 3B). Quartz crystal biosensor experiments have shown that not only Hsc70 but also Hsp70 binds to Leu-Ile.

[0043] 1-3-5 Prediction of binding sites of Hsc70 and Leu-Ile using molecular simulation

Hsc70 is divided into 44 KD ATPase domain, 18 KD substrate binding domain and 10 KD C terminal domain from the N-terminal side. et al., 2003; Flaherty et al., 1990; Morshauser et al., 1999). The domain of Leu—lie bound to and analyzed using molecular simulation software (MOE), and the mutual potential energy was calculated. As a result, the ATPase domain was -119.044 kCal, and the substrate binding domain was -It was shown that Leu-lie binds to 76.711 kCal and C-terminal domain with a strength of -64.337 kCal (Fig. 4).

1 -4 Discussion

In an experiment in which FITC-conjugated Leu-Ile was reacted with brain homogenization solution, it was shown to specifically bind to 70 KD protein eu-Ile. At this time, even if the reaction temperature was changed from 4 ° C to 37 ° C, there was no difference in the degree of binding, so these bonds are not considered to be enzymatic reactions that are sensitive to temperature. . Mass spectrometry of a 70KD protein that specifically binds to Leu-Ile identified Hsc70. In an experiment using a quartz crystal biosensor, not only Hsc70 but also Hsp70 specifically binds to Leu-Ile. The possibility was suggested.

Tacrolimus is thought to suppress the dephosphorylation effect of calci-eurin by binding to FKBP12 and to express immunosuppressive activity by suppressing the production of interleukin2 (Gr iffith et al., 1995; Schreiber et al., 1991). Leu-Ile is a force obtained as an analog of the partial structure of tacrolimus. FKBP12 does not bind and does not have calci-eurin inhibitory activity. Therefore, Leu-Ile does not suppress immune activity. (Nitta et al., 2004) ₀

It has also been suggested that an important factor for the neuroprotective action of tacrolimus is FKBP52 (Hsp56) (Gold et al., 1999). In this study, the force shown to bind Hsc70 and Hsp70 to Leu-lie FKBP52 and Hsc70 and Hsp70 are reported to bind to steroid receptors together with Hsp90 to form a complex (Bagchi et al, 1991; Mc Laughlin et al., 2002), tacrolimus and Leu-Ile may induce neuroprotective effects by exerting some effect on these complexes.

Hsc70 and Hsp70 belong to the heat shock protein 70 family and have a similar function to prevent protein misfolding and aggregation because they have 81% amino acid homology (Dworniczak and Mirault., 1987; Young et al., 2003) By using the energy of ATP there is a report that is delivered to the Hsp90 (Bagchi et al, 19 91 ;.. McLaughlin et al, 2002) 0 In the analysis using the molecular simulation strongly to the ATPase domain of 0 Leu-Ile is Hsc7 The possibility of binding indicates that Leu-Ile is involved in the protein transfer between Hsc70 and Hsp90 using the energy of ATP! Since Hsp90 functions to regulate intracellular signals related to cell cycle, cell proliferation and apoptosis (Zhang and Burrows et al., 2004), Leu-lie is responsible for inducing GDNF production and nerve activity through signal pathways mediated by Hsp90. It is considered that a protective action is being developed.

In immune cells, Hsp70 has the function of suppressing the activity of IκB kinase and suppresses the transcription of tumor necrosis factor- (TNF-) by suppressing the activity of NF-κB. (Beere et al., 2004; Feinstein et al., 1996; Yoo et a 1., 2000) o From this, Leu-lie suppresses the action of Hsc70 and Hsp70, and the transcription factor of GDNF However, it was considered that GDNF production was induced by activating NF-κB. Therefore, in the following, we focused on the signal pathway regulated by Hsc70 and the transcription factor of GDNF, and examined the relationship between Hsc70 and the signal inducing GDNF production.

Example 2

2. Leu-Ile induces GDNF production in cultured rat hippocampal neurons

2- 1 Purpose

GDNF was obtained from rat glial cells (B49) as a trophic factor for fetal substantia nigra donominin neurons and was expected to be applied as a therapeutic drug for Parkinson's disease caused by substantia nigra dopamine neurons (Lin et al, 1993). . Later, it became clear that GNDF had a strong survival effect on spinal motor nerves and sympathetic nerves (Henderson et al, 1994). It became apparent that it plays an important role in the control of migration to the enteric nervous system and organ formation (Moore et al., 1996; Pichel et al, 1996; Sanchez et al., 1996.).

GDNF, a homodimeric secreted protein, binds to the GDNF family receptor-a (GFR al) on the cell membrane, forms a complex with the receptor RET, and transmits a signal into the cell. The ligand GDNF was found in 1993 as a neurotrophic factor (Lin et al, 1993). However, its receptor, RET, was discovered as an oncogene in 1985 (Takahashi et al., 1985), and since long ago, detailed studies on diseases caused by RET mutations and intracellular signaling pathways of RET Has been done. Diseases caused by RET mutations include multiple endocrine adenomas and familial medullary thyroid carcinoma. Pathological changes include neoplastic lesions in the thyroid, parathyroid and adrenal medulla, or neural crest cell-derived Abnormalities in ectoderm (Airaksinen et al., 1999; Mulligan et al., 1994; Romeo et al "1994; Santoro et al., 1990) o In this way, the RET gene is a proto-oncogene. This may support that GDNF / RET signals are strongly involved in cell survival, proliferation and differentiation.

RET is a transmembrane tyrosine kinase receptor, and three isoforms (RET9, RET43, RET51) with different intracellular domain lengths signal in the cell (Lorenzo et al "1995; Myers et al. al "1995; Takahashi et al., 1989). When the ligands GDNF ゝ neurtu rin, artemin, and persephin bind to RET, RET forms a dimer and activates intracellular signals by autophosphorylating tyrosine residues (T yr) (Grimm et al. al., 2001; Schl essinger, 2000). When RET is stimulated by GDNF, Tyr905, Tyrl015, Tyrl062 and Tyrl096 of RET are autophosphorylated (Coulpier et al., 2002; Ichihara et al., 2004). Of these four tyrosine residues, Tyrl062 is phosphatidylinosito.活性 It is considered that the neuroprotective effect of GDNF / RET is expressed by activating the 3 kinase (PI3K) / Akt pathway (Besset et al., 2000; Coulpier et al., 2002; Encinas et al Hayashi et al., 2000; Kobayashi and Matsuoka., 2000; Takahashi., 2001).

Elucidation of the intracellular signal pathway activated by GDNF / RET has been progressed by previous studies, but the transcriptional regulatory mechanism that promotes the production of GDNF is not yet well understood. . Substances that induce GDNF production include TNF-a, inflammatory site force-in such as interleukin-1? (IL-1?), Nutritional factors such as fibloblast growth factor (FGF), and excitatory amino acids. (Marco et al., 2002; Taylor et al., 2003; Verity et al, 1999) o It has also been found that GDNF is also rapidly induced by injury such as spinal cord injury (Satake et al., 2000). However, the expression regulation pattern of GDNF varies depending on the glial cell or nerve cell, for example, IL-1? Is a glioblastoma U- The force that stimulates the production of GDNF when acting on 87MG It has been reported that the action of IL-1? On the neuroblastoma SK-N-AS suppresses the production of GDNF (Verity et al, 1999 ). Such a complex expression pattern of GDNF is thought to be regulated by its promoter region (Tanaka et al., 2001).

GDNF promoter region includes transcription regulators such as NF-κB, CREB, trans-acting transcription factor 1 (SP1), sine oculis-related homeobox 2 homolog (Six2), and silencers that suppress transcription neura 卜 restrictive There is a silencer elements (NRSE) binding site (Brodbeck et al., 2004; Tanaka et al., 2000; Woodbury et al., 1998), and these factors influence transcription in complex ways. (Figure 12). For example, constant GDNF expression may involve SP1, and induction of GDNF expression in the neural tube or somite at the developmental stage may involve Six2 force disorder or GDNF induction by site force-in may involve NF-κΒ. Known (Baecker et al "1999; Brodbeck et al., 2004; Grimm et al" 1998; Maruyama et al., 2004; Tanaka et al., 2001). The GDNF production-inducing effect by Leu-lie is thought to be expressed by the activation of any of these transcriptional regulators. In the above 1., it was shown that Hsp70 and Hsc70 are Leu-Ile binding proteins, but in the following, Leu-Ile induces G DNF production via any signal after Hsp70 and Hsc70 bind. NF-κ Β (Υοο et al, 2000), whose activity is suppressed by inducing the expression of Hsp70, which has a deep relationship with damage and site force-in, and neuroprotection However, the focus was on deep CREB.

[0046] 2-2 Experimental materials and methods

2- 2-1 animals

In the experiment, Wistar ST rat pregnancy day 17 (Japan SLC) was used. This study was conducted based on the Nagoya University School of Medicine Animal Experiment Fund † and Principles of Laboratory Animal Care (Nationa 1 Institutes of Health Publication 85-23, 1985).

[0047] 2-2-2 Rat Hippocampal Neuron Culture Method

Under anesthesia with pentobarbital, fetuses from the 17th day of pregnancy were removed from Wistar ST rats, and the hippocampus was removed in ice-cooled L15 medium (Sigma Aldrich Japan, Tokyo). After trypsin (Invitrogen, NY, USA) was allowed to act at 37 ° C for 15 minutes, fetal calf serum (fe tal bovine serum: FBS) to stop the reaction, phosphate buffer saline (PBS) (1.3 mmol / NaCl, 81 mmol / 1 Na2HP04, 26.8 mmol / 1 KCl, 14.7 mmol / 1 KH2P04) (pH 7.4 ) Was added to the container containing the hippocampus and shaken gently to remove the PBS. Add 10 ml of Dulbecco s modified Eagles medium / Ham s nutrient mixture F12 (DMEM / F12HAM) (Sigma Aldrich Japan) to this, and dissociate the cells by repeating pipetting 10 times to obtain 96 wells. The culture was started at a cell density of 250,000 cells / cm ² using a plate or a 6 cm petri dish (NALGE NUNC Internatial, Tokyo). DMEM / F12HAM with N2 supplement (Invitrogen) after 1 day incubation at 37 ° C, 5% CO

2

Cells replaced with serum-free medium and further cultured for 1 day were used for immunostaining, western blotting, or methyl thiazol tetrazolium (MTT) experiments.

[0048] 2-2-3 NF-κ Β immunohistochemical staining using cultured hippocampal neurons

In hippocampal neurons cultured in 2-2-2, Leu-lie (10 μg / ml), TNF-α (100 ng / ml) (benefit from Dainippon Pharmaceutical (Osaka)) or GDNF (50 ng / ml) ) (Given from Amgen, CA, USA) for 2 hours. Cells were fixed with -20 ° C methanol for 20 minutes and washed 3 times with 4 ° C PBS for 15 minutes. Cells were incubated with PBS solution containing 2% bovine serum albumin (BSA) and 10% FBS at 37 ° C for 1 hour and diluted 1000 times with PBS rabbit anti p65 / NF-κB antibody (Lockland , PA, USA) at 4 ° C. As a negative control, PBS alone was added to the cells and left at 4 ° C. The cells were washed 3 times with PBS for 15 minutes, then reacted with Alexa Fluor 546 goat anti-rabbit IgG (H + L) (Molecular Probes, OR, USA) diluted 1000-fold with PBS for 3 hours, and PBS for 15 minutes. Washed 3 times. Stained cells were sealed with a cover glass and observed with a fluorescence microscope axioscop 2 plus (Zeiss, NY, USA) (Zhu et al., 2004).

[0049] 2-2 -4 Isolation of the nucleus of cultured hippocampal neurons

Leu-Ile (10 g / ml) was allowed to act on hippocampal neurons cultured in a 6 cm petri dish for 30 minutes, and after removing the culture supernatant, the cultured hippocampal neurons were washed with PBS and cytoplasmic extraction buffer (CEB) (10 The reaction was carried out on ice for 5 minutes with mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT). Protease inhibitors (1 μg / ml leupeptin, 1 μg / ml pepstatin, 20 μg / ml phosphoramidon, 0.2 mg / ml EDTA, 2 μg / ml aprotinin , 0.5 mM PMSF) containing 0.4% Nonidet P-40 / CEB 300 μl for 5 minutes, cells were gently peeled off with a rubber policeman and collected, and centrifuged at 1,000 g for 5 minutes. The precipitate obtained by centrifugation was washed with CEB containing a protease inhibitor, nuclear extraction buffer (NEB) (20 mM Tris—HC1 (pH 7.9), 0.4 M NaCl, 1.5 mM MgC12, 1.5 mM EDTA, 1 mM DTT, 25% glycerol) was stirred well and left on ice for 10 minutes. This solution was centrifuged at 16,000 g for 5 minutes and the supernatant was recovered as a nuclear extract (Yoo et al, 2000).

[0050] 2-2-5 CREB antisense oligonucleotide supplementation suppresses CR EB expression in cultured hippocampal neurons

CREB antisense oligonucleotide corresponding to the start codon region of CREB (5'-GCT CCA GAG TCC ATG GTC AT-3 ': SEQ ID NO: 1) or CREB sense oligonucleotide (5'-AT GAC CAT GGA CTC TGG AGC- 3 ': 1 μg of SEQ ID NO: 2) was cultivated in a 6-cm dish and transferred to a sea urchin strain, and introduced with Lipofectamine Plus transfection reagent (Invitro gen) (Gonzalez et al., 1989 Sato- Bigbee and DeVries., 199 6). Leu-Ile (10 μg / ml) added to cells transfected with CREB antisense oligonucleotide and cultured for 24 hours (Afshari et al., 2001; Johnson et al., 2000), Western blotting of GDNF Used as a sample.

[0051] 2-2-6 Sample preparation and Western blotting from cultured hippocampal neurons

Akt, CaMKII, ERK and PKC-? Western blotting using cultured hippocampal neurons treated with Leu-lie (10 μg / ml) for 10, 20 or 30 minutes, and Leu-Ile (10 μg / ml) for Western blotting of GDNF The cells were used for 1 day. Also, PI3K inhibitor LY294002 (30 μΜ) (Sigma Aldrich Japan) or geldanamycin (10 μΜ) (Sigma Aldrich Japan), which inhibits Hsp90 complex formation, and Leu-lie were allowed to act simultaneously for 30 minutes. Cells were used for Western blotting of Akt and CREB. Remove the culture supernatant of cultured hippocampal neurons treated with Leu-Ile, wash the cells with PBS, precipitation buffer (20 mM Tris—HC1 (pH 7.6), 150 mM NaCl, 1 mM sodiu m orthovanadate, 2 mM EDTA, 50 mM NaF, 1% Nonidet P—40, 1 mM PMSF, 20 μg / ml aprotinin, 20 μg / ml leupeptin,, 20 μg / ml pepstatin) And recovered. Disrupt cells for 30 seconds with a homogenizer, and then 4 ° C 10,000 The mixture was centrifuged at g for 1 hour, and the supernatant was used as a sample for Western blotting.

The amount of protein in the sample was measured with DC Protein Assey Kit II (BIO-RAD, CA, USA). An equal volume of sample buffer to the diluted sample was boiled at 95 ° C for 5 minutes and electrophoresed on a 10% polyacrylamide gel.

After transferring the protein from the polyacrylamide gel to the PDVF membrane by the semi-dry transfer method, the membrane was blocked in a blocking buffer (KPL, MD, USA) for 2 hours at room temperature with gentle agitation.

Next, the primary antibody (mouse mon oclonai anti-GDNF antibody (R & D systems, MN, USA), rabbit anti-phospho-Ca, diluted 1000 times with blocking buffer in PDVF membrane)

MKII antibody (Promega, WI, USA), rabbit ant to NF-κB antibody (Lockland), mouse monoclonal anti-CREB antibody (Santa Cruz Biotechnology, CA, USA), rabbit ant

1-phospho-CREB antibody (Cell Signaling Technology, MA, USA), rabbit anti-pho spho- Akt (Ser2448) (Cell Signaling Technology), anti-Akt antibody (Cell Signaling Technology), anti-phospho-PKC-? Antibody (Cell signaling Technology), mouse monoclonal anti-phospho-ERK antibody (Cell signaling Technology), rat anti-Hsc70 antibody or anti-Hsp90 antibody (Stressgen)) were reacted at 4 ° C. After washing 3 times with TBS (10 mM Tris-HCl, 137 mM NaCl, 0.1% Tween-20) for 15 minutes, secondary antibody (peroxidase labeled goat anti-mouse IgG (H + L) antioody 7 peroxidase labeled goat anti-rabbit IgG (H + L) antibody (Kirkegaard & Perry Laboratries, MD, USA)) for 2 hours at a temperature. Thereafter, the plate was washed 3 times with TBS and reacted with ECL ™ (Amersham Pharmacia Biotec, UK) for 1 minute. Development was performed on a hyperfilm (Amersham Bioscience, NA, UK) using a FPM100 (Fuji Photo Film, Tokyo) processor. ATTO Densito Graph (Atoichi, Tokyo) was used for intensity analysis of the band developed on Hyperfilm.

2- 2--7 MTT Atsey

MTT Atssey is a method for measuring the amount of color developed by soluble crystals of purple formazan product formed by reduction of yellow MTT by mitochondria in living cells. Since the mitochondria's MTT reducing power is proportional to the number of viable cells, the cell viability can be measured by measuring the absorbance of the product, and it is widely used for counting the number of viable cells in culture experiments. (Liptay et al., 1999; Uludag and Sefton., 1990).

In addition to Leu-Ile (10 μg / ml) and NF-κB inhibitor sulfasala zine dOO ju M) (Sigma Aldrich Japan) dissolved in DMEM / F12HAM in addition to cultured hippocampal neuronal cells cultured in 96-well plates Cultured for 3 days. 10 μl of cocoon solution (Sigma Aldrich Japan) (10 mg / ml) was added and incubated at 37 ° C. for 1 hour, and then the supernatant was removed. A purple formazan product crystal formed by reduction of MTT was solubilized by adding 200 ul of 50% dimethylformamide, 20% SDS at pH 4.7, and a microplate reader with a wavelength of 570 nm (Model 450, BIO 'RAD ) To measure the absorbance. Since the amount of color development was proportional to the number of viable cells, the viability was determined with the absorbance of control cells as 100%.

[0053] 2-2-8 Quantification of GDNF mRNA

For quantification of GDNF mRNA, a real-time RT-PCR method using iCycler System (Bio-Rad) was used. RNeasy (registered trademark) Mini Kit (Quiagen) was used to extract mRNA from cultured neurons. Reverse transcription from 1 μg of mRNA to cDNA was performed using oligo primers and Superscriptll RT (Life Technologies). Of the 20 μ1 reaction product, 1 μ1 was used for PCR. Platinum (registered trademark) Quantitative PCR SuperMix-UDG (Invitrogen) was used for the PCR reaction. As a control, quantification of ribosomal mRNA (TaqMan Ribosaomal RNA control Reagents; Applied Biosystem) was also corrected for variations between samples. For detection of GDNF, 5′-AGCTGCCAGCCCAGAGAATT-3 ′ (bp 288-307) (SEQ ID NO: 3) and 5 and GCACCCCCGATTTTTGC-3 ′ (bp 354-370) (SEQ ID NO: 4) were used as forward and reverse primers, respectively. The probe for detection was dye probe being 5 and CAGAGGGAAAGGTCGCAGAGGCC-3 ′ (bp 309-331) (SEQ ID NO: 5) was used.

[0054] 2-2-9 Statistical analysis

All results are shown as mean values and standard errors. For statistical analysis, one-way analysis of variance was performed. If a significant difference was observed, Scheffe's multiple comparison test was performed. When the risk rate is 5% or less, there is a significant difference.

[0055] 2-3 results

2-3-1 Translocation of NF—κ 移行 into the nucleus by Leu—lie Leu-Ile, TNF-a, or GDNF was allowed to act on cultured hippocampal primary neurons, immunostained with anti-NF-κΒ antibody, and the number of cells measured with a fluorescence microscope was 4500 in a unit area of 0.66 mm ² Cells, of which cells are not stained, 7.9 ± 1.31% in the control group, 3.1 ± 0.83% in the group treated with Leu-Ile, 2.1 ± 0.54% in the group treated with TNF-α It was 2.4 ± 0.59% in the group treated with GDNF. In cells treated with Leu-Ile, TNF-a, and GDNF, the number of cells that were not stained was significantly reduced compared to the control group (Fig. 5A). In immunostaining, NF-κB nuclear translocation was observed in many cells even in controls. As previously reported, TNF-a and GDNF in the positive controls showed nuclear translocation of NF-κΒ. And Leu-lie has also been shown to promote NF-kappa intranuclear translocation (Hayashi et al., 2000; Yoo et al., 2000; Zhu et al., 2004) ₀ When only the nucleus was isolated and the amount of NF-κB in the nucleus was examined by Western blotting, the amount of NF-κΒ present in the nucleus in cells treated with Leu-Ile was controlled. Compared to 196 ± 16% (Fig. 5 Β).

[0056] 2-3-2 Effects of NF-κΒ inhibitors on Leu-Ile cell death suppression and GDNF production induction

Cultured neurons die over time after the start of culture, and on day 3, the number of cells decreases to 58.6 ± 2.7% at the start of culture. As a result of the assay, the number of cells in the group to which Leu-Ile (10 μg / ml) was added increased to 119 ± 1.4% compared to the control. There was no difference in the number of viable cells between the cells treated with sulfasalazine (100 μΜ), an inhibitor of NF-κΒ, simultaneously with Leu-Ile, and the control group. The effect of Leu-Ile was significantly suppressed. (Figure 6A). In addition, as a result of Western blotting of GDNF, the GDNF expression level increased to 188 ± 13% in the cells treated with Leu-lie (10 μg / ml) compared to the control, sulfasalazine (100 M). In cells treated with Leu-lie (10 g / ml), the expression level of GDNF did not increase. In cells treated with sulfasalazine alone, the expression level of GDNF was not different from the control (Fig. 6B). Since sulfasalazine also suppressed Leu-Ile's neuroprotective action and GDNF production-inducing action, it was suggested that Leu-Ile's neuroprotective action and GDNF production-inducing action may be mediated by NF-κB.

[0057] 2-3-3 CREB phosphate action and GDNF production-inducing action by Leu-Ile Leu-Ile was allowed to act on cultured hippocampal neurons, and the degree of CRE B phosphate was examined using the Western blotting method. In contrast, CREB phosphorylation was induced in 164 ± 14% (Fig. 7A).

By applying CREB antisense oligonucleotide to cultured hippocampal neurons, the expression level of CREB is reduced to 63 ± 13% (Fig. 7B). Leu-Ile was allowed to act on these cells, and then Western blotting of GDNF was performed. Compared with the control, the expression level of GDNF was reduced to 72 ± 10% in cells treated with CREB antisense oligonucleotide alone. In addition, in cells treated only with Leu-Ile (10 g / ml), the GDNF expression level increased to 188 ± 13% compared to the control cells. Cells treated with Leu-Ile together with CREB antisense oligonucleotide. However, it decreased to 69 ± 9.2% compared to cells treated with Leu-Ile alone. Cells treated with CREB sense oligonucleotide did not differ in the degree of phosphorylation of CREB compared to control (FIG. 7C). In cells where CREB antisense oligonucleotides acted and CREB expression was reduced, the GDNF production-inducing action was suppressed, suggesting that CREB may be involved in Leu-Ile's GDNF production-inducing action. It was.

When Leu-Ile, Pro-Leu or Ile-Pro (10 μg / ml) was added to cultured neurons, the increase in GDNF protein expression after 24 hours was examined. Increased GDNF protein expression (Fig. 8A). Furthermore, it was found that the expression of GDNF mRNA was increased 12 or 18 hours after adding Leu-Ile by using real-time RT-PCR (FIG. 8B). These experimental results are consistent with our previous report (Nitta et al, 2004).

2-3-4 Leu-Ile-mediated CREB phosphorylation and GDNF production induction through Hsp90 / Akt Cultured hippocampus treated with Leu-Ile to elucidate molecules that activate CREB transcription factors As a result of investigating the degree of phosphorylation of Akt, CaMKIU PKC-? And ERK using nerve cells, the degree of phosphorylation of Akt increased to 154 ± 8.6%. This experiment was conducted for CaMKIU PKC-? And ERK. Under the conditions, the degree of phosphate was not increased (Fig. 9A). In the cells treated with LY294002 (30 μΜ), an inhibitor of PI3 K, and Leu-Ile at the same time for 30 minutes, the Akt phosphate induced by Leu-Ile was not suppressed. Complex formation of Hsp90 In cells treated with geldanamycin (10 μΜ) and Leu-Ile simultaneously, Leu-Ile Inhibited Akt phosphorylation by 52 ± 7.5% (FIG. 9B). In addition, in cells treated with LY294002 and Leu-Ile simultaneously for 30 minutes, the phosphorylation of CREB induced by Leu-Ile was not suppressed. In cells treated with geldanamycin and Leu-Ile simultaneously for 30 minutes, 50 ± 8.2 % (Fig. 9C).

2 -4 Discussion

In this study, the effect of Leu-Ile on the survival of cultured hippocampal primary neurons was measured by MTT assay, and the effect of Leu-Ile on the expression level of GDNF was examined using Western blotting. Similar to the previous report (Nitta et al, 2004) where the number of viable cells was examined by direct counting and the expression level of GDNF was examined using an enzyme immunoassay, Leu-Ile has an effect of inhibiting cell death and inducing GDNF production. It was confirmed that it had.

Activity of transcription factors NF-κB and CREB having binding sequences in the promoter region of GDNF was observed in cells treated with Leu-Ile. In addition, Leu-Ile was not observed to induce GDNF production in cells treated with NF-κΒ inhibitor sulfas alazine or CREB antisense oligonucleotide. And was thought to induce GDNF production via CRE 介.

Previous studies have reported that TNF-a and FK960 induce GDNF via NF-κΒ or CREB (Koyama et al., 2004; Maruyama et al., 2004). Activation of NF-κΒ via PI3K / Akt and activation of CREB via ER K are also observed on the signal pathway that is activated by NF (Feng et al., 1999; Hayashi et al., 2000). Since cultured hippocampal neurons express both GDNF, GFR- and RET, they act on the induced GDNF force RET and activate NF-κΒ and CREB via PI3K / Akt and ERK. In addition, there is a possibility that positive feedback control that induces the production of GDNF works (Lenhard et al "1998; 2002) ₀

Therefore, it has been reported that CREB is activated (Shaywitz and Greenberg., 1999; Wang et al., 1999). It was investigated whether ERK, Akt, CaMK and PKC are activated by Leu-Ile. As a result, only Akt phosphate was observed within 30 minutes when Leu-Ile was absorbed and CREB activity was observed. In addition, in order to investigate whether Akt phosphate induced by Leu-Ile is mediated by PI3K, Leu-Ile and PI3K inhibitor LY294002 were simultaneously administered to cells. When activated, the Akt phosphate chain induced by Leu-Ile was not suppressed. GDNF I RET signals in human neuroblastoma TGW cells and early neuroectodermal cancer cells SK-N-MC promote CREB phosphorylation via ERK and Akt phosphorylation via PI3K. Has been reported to promote (Hayashi et al, 2000). In this study, ERK was not phosphorylated even when Leu-Ile was allowed to act, and since Akt was phosphorylated without PI3K, the CREB phosphorylation observed when Leu-Ile was allowed to act for a short time. The acid was thought to be activated through a pathway different from the GDN F / RET signal.

In addition to PI3K, Hsp90 has been reported as a factor that regulates phosphorylation of Akt, and the signal pathway of Hsp90 I Akt regulates the phosphorylation of CREB (Doong et al. al., 2003; Du and Montminy., 1998). As a result of investigating the degree of Akt and CREB phosphorylation using geldanamy cin, which inhibits Hsp90 complex formation, Lekt-Ile-induced phosphorylation of Akt and CREB phosphorylation was suppressed. It was thought that CREB was activated through the Hsp90 / Akt signaling pathway to induce GDNF production.

Hsp90 and Hsc70 form a complex and are known to regulate protein delivery and activity (Bagchi et al., 1991; McLaughlin et al., 2002). And the possibility of changing the binding ability of Hsp90. In this study, it was not possible to show how the interaction between Hsp90 and Hsc70 changes when Leu-Ile is applied. However, there are reports that Hsp90 and Hsc70 form a complex, and exchange various proteins and regulate the activity. Therefore, Leu-Ile changes the properties of these complexes. Hsp90 is thought to activate Akt (Fig. 10).

3. Summary

The following knowledge was obtained from the above results.

(1) Leu-Ile is cell membrane permeable.

(4) was examined for binding activity of Hsc70 and Hsc70 and highly homologous Hsp70 and Leu-Ile using quartz crystal biosensor, Hsc70 is ^{Kd = 1.83 X 10- 8 M,} Hsp70 is K bound to Leu-Ile and specifically in strength of d = 1.24 X 10- ⁸ M.

(7) In cultured hippocampal neurons treated with Leu-Ile, a group of cells in which neuronal cell death was suppressed and GDNF production was induced NF-κB inhibitors sulfasalazine and Leu-Ile were allowed to act simultaneously Then, the effect of Leu-Ile was suppressed.

[0061] From the above findings, it was considered that Leu-Ile binds to Hsc70 and activates NF-κΒ and CREB through the Hsp90 / Akt pathway to induce GDNF production. It was found that Leu-Ile induces GDNF production through the Hsp90 / Akt signaling pathway, and Hsc70 and Hsp90 / Akt could be identified as new target genes responsible for the regulation of GDNF expression.

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Industrial applicability

The Akt activator of the present invention can be used as a drug or food for a disease for which a preventive or therapeutic effect can be obtained by regulating Akt, or as a reagent for studying the onset mechanism or progress mechanism of the disease. The active ingredient of the present invention has the advantage that it is easy to prepare because the basic structure is a very simple dipeptide. The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

The contents of papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

Claims

The scope of the claims

[1] An Akt activator comprising any one of the compounds (a) to (c) as an active ingredient:

(a) a peptide comprising Leu and lie;

(b) a modified form of a peptide comprising Leu and lie;

(c) A pharmaceutically acceptable salt of (a) or (b).

[2] The Akt activator according to claim 1, wherein the peptide is Leu-lie.

[3] The Akt activator according to claim 1 or 2, wherein Akt is activated through a pathway in which Akt is activated by the binding of Hsp90.

[4] Parkinson's disease, spinal cord injury, Alzheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia

The Akt activator according to any one of claims 1 to 3, which is used for the treatment or prevention of rheumatism or schizophrenia.

[5] A food containing the Akt activator according to any one of claims 1 to 3.

[6] A method for preventing or treating Parkinson's disease, spinal cord injury, Alzheimer's disease, hydrocephalus, brain injury, cerebral infarction, dementia, rheumatism, or schizophrenia, comprising the following steps: (A) Claims 1 to 4. A step of administering the Akt activator according to any one of 3 to a living body.