WO2006075808A1

WO2006075808A1 - Pharmaceutical composition comprising a p25/cdk5 inhibitor for preventing or treating a neurodegenerative disease

Info

Publication number: WO2006075808A1
Application number: PCT/KR2005/000098
Authority: WO
Inventors: Sul-Hee Chung; Ilho Ha; Mi-Young Son; Hye-Won Lee
Original assignee: Inje University
Priority date: 2005-01-12
Filing date: 2005-01-12
Publication date: 2006-07-20
Also published as: KR100931928B1; KR20070094947A

Abstract

A pharmaceutical composition for preventing or treating a neurodegenerative disease comprises a compound inhibiting a P25/CDK (cycline-dependent kinase 5) complex as an active ingredient. Also disclosed is a method for screening a compound capable of preventing or treating a neurodegenerative disease, which comprises the steps of a) reacting a candidate compound with a P25/CDK5 complex and beta-site APP (amyloid precursor protein)-cleaving enzyme 1 (BACEl) or treating a mammalian cell expressing P25 with the candidate compound, and measuring BACE l phosphorylation; and b) comparing the measured BACEl phosphorylation with that of a control group employing no candidate compound, and identifying a compound reducing BACEl phosphorylation compared to the control group. The compound inhibiting the P25/CDK5 complex may be useful for preventing or treating a neurodegenerative disease including Alzheimer's disease because it inhibits BACEl phosphorylation and reduces the secretion of β-amyloid and the hyperphosphorylation of Tau.

Description

PHARMACEUTICAL COMPOSITION COMPRISING A P25/CDK5

INHIBITOR FOR PREVENTING OR TREATING

A NEURODEGENERATIVE DISEASE

Field of the Invention

The present invention relates to a pharmaceutical composition comprising a compound inhibiting a P25/CDK5 complex, and a method for screening the compound.

Background of the Invention

Alzheimer's Disease (AD), a typical form of a senile neurodegenerative disease, may lead to serious cognitive impairments, memory loss and behavior problems. AD is a chronic illness, which gradually and progressively exacerbates over an extended period of time. AD patients as well as their family members inevitably experience emotional distresses and financial burdens. But currently available treatments have provided only temporary alleviations of the symptoms. Therefore, a therapeutic development that cures or inhibits the progression of the disorder is imperative.

The present therapeutic developments related to AD have mainly focused on the abnormal functions of neurotransmitters. In particular, acetylcholinesterase inhibitors such as Aricept, Exelon, and Reminyl designed to target on the cholinergic neurons have been introduced in the market. Memantine, an antagonist against the receptor of glutamate, is another recent FDA-approved drug that controls the abnormal secretion of a neurotransmitter, glutamate. However, such drugs are ineffective to stop the progression of AD. Meanwhile, many studies have been carried out with targeting on β-amyloid (Aβ), a peptide that builds up an amyloid plaque in the brains of AD patients. Such studies aim for developing drugs capable of inhibiting the activity of β- or γ- secretase which mediates Aβ production, or degrading the produced Aβ. However, in this case, an interference with innate proteins in the human body may adversely affect normal physiological functions and cause undesirable side effects. Hence, it is preferable to develop a potential drug targeting toward a protein, which is synthesized only in the pathological state and closely related to the progression of a disease. The inhibition of such protein would effectively hinder a progression of the disease.

Recently, it has been reported that P25 accumulates in the brain of AD patients (Patrick, GN. et al, Nature 402: 615-622 (1999)). P25 is a c-terminal fragment of a CDK5 (cycline-dependent kinase 5)-activator, P35, and is also capable of activating CDK5. CDK5 is responsible for controlling neurogenesis and cyto-architecture during neurodevelopment, and involves in actin dynamics, microtubule stability, axon guidance, membrane transport and dopamine signaling. Extensively dispersed throughout the neuronal tissue, CDK5 also participates in the phophorylation of a protein containing S(or T)PX(X)K(or R or H) consensus motif, and has various substrates including Munclδ, APP, P35, PAKl, Tau, β-catenin, Nudel, DARPP32, Amphyphysinl and Synapsinl (Dhavan, R. and Tsai, L.H., Nature Review Molecular Cell Biology 2: 749-759 (2001)).

Lee et al. has discovered that the cleavage of P35 by calpain results in two protein fragments, PlO and P25, and, unlikely to P35, P25 expresses neurotoxicity (Lee M.S. et al, Nature 405: 360-364 (2000)). In contrast to P35, the absence of a membrane-binding myristoylation signal in P25 allows unrestricted, longer circulation of P25 inside a cytoplasm, resulting in a pathologic CDK5 upregulation. Hyperphosphorylation of Tau, one of cytoskeleton proteins, by CDK5 mediates weaker interactions between Tau and the cytoskeleton proteins and a formation of neurofibrillary tangles (NFT) which is a major pathological feature of AD. Aβ, associated with a senile plaque in AD, can trigger a cleavage of P35 into P25 (Lee M.S. et al, supra). Recent study has also demonstrated that P25 promotes enhanced Aβ secretion level (Lie, F. et al, FEBS Letters 547: 193-196 (2003)). Thus, it is suspected that P25 exists as a molecular link between the amyloid plaque and the neurofibrillary tangles. In addition, a P25/CDK5 complex has been confirmed to participate in neurodegeneration events including cytoskeletal collapse and neuronal death (Patrick, GN. et al, supra).

Meanwhile, increases in the amount and activity of BACEl (beta-site APP (amyloid precursor protein)-cleaving enzyme 1) protein, which is a protease involved in Aβ production, in the brain of AD patients have been reported (Yang, L-B. et al, Nature Medicine 9:3-4 (2003); Fulumoto, H. et al., Arch neurol. 59: 1381-1389 (2002)); however, the exact mechanism thereof has not been established.

The present inventors have discovered that an increase in P25 activates CDK5 resulting in BACEl phosphorylation as well as increases in β-secretase activity and Aβ secretion, and have accomplished the present invention by screening P25/CDK5 inhibitors and confirming that the inhibitors successfully inhibited phosphorylation of BACEl and Aβ secretion.

Summary of the Invention

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating a neurodegenerative disease, which comprises a compound inhibiting the P25/CDK5 complex. It is another object of the present invention to provide a method for screening the P25/CDK5 inhibitor.

It is a further object of the present invention to provide a method for preventing or treating a neurodegenerative disease in a mammal.

In accordance with one aspect of the present invention, there is provided a composition for preventing or treating a neurodegenerative disease, which comprises a P25/CDK5 inhibitor of formula (I) or (II) as an active ingredient.

(D wherein,

R¹ is hydrogen, linear or branched C]-C₄ alkoxy, or C]-C₄ alkylformamide substituted with a C₃~Cg cycloalkyl group;

R² is hydrogen, or linear or branched C]-C₆ alkyl; and R³ is hydrogen, linear or branched C]-C₆ alkyl, or saturated or unsaturated 5- or 6-membered heterocyclic compound comprising 1 to 3 heteroatoms; or

R² and R³ form, together with the carbon atoms where they are attached, a saturated or unsaturated 5- or 6-membered heterocyclic compound comprising 1 to 3 heteroatoms.

wherein, R¹, R², R³, R⁶, R⁷ and R⁸ are each independently hydrogen, halogen, linear or branched C]-C₆ alkyl, linear or branched C]-C₆ alkyl substituted with halogen, linear or branched C]-C₆ alkoxy, or linear or branched C]-C₆ alkylester; and

R⁴ and R⁵ are each independently hydrogen, fluoro, chloro, or linear or branched C , -C₄ alkyl .

In accordance with another aspect of the present invention, there is provided a method for screening a P25/CDK5 inhibiting compound which comprises the steps of a) reacting a candidate compound with a P25/CDK5 complex and BACEl or treating a mammalian cell expressing P25 with the candidate compound, and measuring BACEl phosphorylation, and b) comparing the measured BACEl phophorylation with that of a control group employing no candidate compound, and identifying a compound reducing BACEl phophorylation compared to the control group. In accordance with another aspect of the present invention, there is provided a method for preventing or treating a neurodegenerative disease by inhibiting BACEl phosphorylation in a mammal, which comprises administering the compound of formula (I) or (II) to the mammal.

Brief Description of the Drawings

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

Fig. IA: Autoradiography result of SDS-PAGE showing BACEl phosphorylation by a P25/CDK5 complex;

Fig. IB: Immunoblotting result illustrating specific reaction of phospho-specific BACEl antibody with BACEl phosphorylated at Thr²⁵²; Fig. 1C: Immunoblotting result showing that the phosphorylation of

BACEl at Thr²⁵² residue is mediated by CDK5;

Fig. 2A: Graph demonstrating an increased β-secretase activity due to in vitro BACEl phosphorylation by a P25/CDK5 complex;

Fig. 2B: Graph showing the effects of transient expressions of BACEl and phosphorylation-deficient mutant BACE1T252A on β-secretase activity in SK-N-BE2C cells;

Fig. 3 A: Graph depicting an increased β-secretase activity due to a transient expression of P25 in SK-N-BE2C cells;

Fig. 3B: Western blotting result showing protein expression profiles of PC-.2_tet._off cells, P25-3 and P25-5, which show stable expressions of P25;

Fig. 3C: Graph showing the amount of Aβ secretion in PC 12_tet._off cells, P25-3 and P25-5;

Fig. 4A: Immunohistochemical staining results showing the degrees of phosphorylation of BACEl in hippocampus, cortex and cerebellum of a wild type mouse and a P25-expressing transgenic mouse;

Fig. 4B: Western blotting result showing the expressions of P25, phosphorylated BACEl and actin in hippocampus, cortex and cerebellum of a wild-type mouse and a P25-expressing transgenic mouse;

Fig. 4C: Graph showing the level of phosphorylated BACEl quantified from the western blotting result of Fig. 4B with a densitometer;

Fig. 4D: Graph showing β-secretase activity in hippocampus, cortex and cerebellum of a wild-type mouse and a P25-expressing transgenic mouse;

Fig. 4E: Graph showing Aβ amount in hippocampus, cortex and cerebellum of a wild-type mouse and a P25-expressing transgenic mouse;

Fig. 5: Schematic illustration of novel pathway where BACEl phosphorylation by a P25/CDK5 complex increases BACE activity and Aβ secretion;

Fig. 6A: Immunoblotting result using P-Thr-Pro antibody to identify the phosphorylation of histone Hl by a P25/CDK5 complex;

Fig. 6B: ELISA result using histone Hl and P-Thr-Pro antibody to verify P25/CDK5 inhibiting activities of DSS30, DSS36 and their analogues; Fig. 7A: Western blotting result using BACEl and phospho-BACEl antibody to demonstrate P25/CDK5 inhibiting activities of DSS30, DSS36 and their analogues;

Fig. 7B: Western blotting result using Tau and phospho-Tau antibody to demonstrate P25/CDK5 inhibiting activities of DSS30, DSS36 and their analogues;

Fig. 8A: Western blotting result indicating inhibition of BACEl phophorylation by DSS30 and DSS36 compounds in P25-3 cells, and graph obtained by quantifying the result with a densitometer; and

Fig. 8B: Graphs showing the effects of DSS30, DSS36 and their analogues on inhibiting Aβ secretion in P25-3 cells.

Detailed Description of the Invention

As used herein, the term "degenerative disease" refers to a disease related to the accumulation of neurofibrillary tangles, P25 or β-amyloid in the brain, and examples thereof include, but not limited to, Alzheimer's Disease,

Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Niemann-Pick disease, dementia caused by cerebral ischemia and dementia caused by cerebral hemorrhage.

Preferable compounds of formula I, as an active ingredient of the inventive composition for preventing and treating a neurodegenerative disease, are those wherein R¹ is hydrogen, methoxy or cyclopropylmethylformamide; R² is hydrogen; and R³ is piperidine; or R² and R³ form a pyrrole or 1,4-dioxane ring, together with carbon atoms where they are attached. Among these, the most preferred compounds are DSS30, DSS301, DSS302, DSS303 and DSS304 as listed in Table 1. Preferable compounds of formula II, as an active ingredient of the inventive composition, are those wherein R¹, R², and R³ are each independently hydrogen, halogen, linear or branched C₁-C₄ alkoxy, or linear or branched C₁-C₄ alkylester; R⁴, and R⁵ are each independently hydrogen, or linear or branched C₁-C₄ alkyl; R⁶, R⁷ and R⁸ are each independently hydrogen, linear or branched C]-C₄ alkyl optionally substituted with halogen, or linear or branched Ci-C₄ alkylester. Among these, the most preferred compounds are DSS36, DSS361, DSS362, and DSS363 as listed in Table 1.

The present invention provides a method for screening a compound inhibiting a P25/CDK5 complex which comprises the steps of a) reacting a candidate compound with the P25/CDK5 complex and BACEl or treating a mammalian cell expressing P25 with the candidate compound, and measuring BACEl phosphorylation; and b) comparing the measured BACEl phosphorylation with that of a control group employing no candidate compound, and identifying a compound reducing BACEl phosphorylation compared to the control group.

In the above screening method, it is preferable to detect the degree of BACEl phosphorylation caused by a P25/CDK5 complex by employing an antibody that specifically binds to a phosphorylated BACEl or an radioisotope- labeled ATP. The antibody that specifically binds to a phosphorylated BACEl can be prepared through an immunization of an animal with an oligopeptide antigen which includes at the most 10 amino acids at each N- and C-terminal directions centering on Thr²⁵² in amino acid sequence of BACEl (SEQ ID NO: 6). In particular, an antibody prepared by employing peptide SLWY Thr²⁵²(PO₄)PIRR (SEQ ID NO: 2) as an antigen is preferred.

In an in vitro P25/CDK5 inhibitor screening, the P25/CDK5 inhibitors of the present invention listed in Table 1 such as DSS30, DSS36 or their structural analogues exhibit at least 50 percent of inhibitory activity against the phophorylation of histone Hl, BACEl and Tau by a P25/CDK5 complex (see

Figs. 6B, 7A and 7B).

It seems that the P25/CDK5 inhibitors of the present invention act in a non-competitive manner with ATP rather than bind to ATP binding sites of CDK5 as the currently developed inhibitors do.

Meanwhile, in a cell-based assay using P25-overexpressing cells, the P25/CDK5 inhibitors of the present invention, i.e., DSS303 and DSS36, inhibited Aβ secretion by more than 50 percent, as well as BACEl phosphorylation {see Figs. 8A and 8B).

In the present invention, the compounds that exhibit at least 10 percent, preferably more than 20 percent, inhibition of substrate phosphorylation by the

P25/CDK5 complex, as compared to that of the control group without the inhibitors, in the above in vitro and in vivo experiments were selected as the P25/CDK5 inhibitors.

From the above-mentioned test results of the present invention, a novel pathogenic pathway of AD is affirmed, wherein the accumulation of P25 in the brain of AD patients activates CDK5, the resulting P25/CDK5 complex phosphorylates and activates BACEl, the most important enzyme in Aβ production, thereby promoting increases in both β-secretase activity and Aβ secretion (see Fig. 5).

Not only can the inhibitors of the present invention prevent the progression of AD by inhibiting the phosphorylation by the P25/CDK5 complex and activity of BACEl, resulting in a decrease in Aβ secretion, but they can also successfully prohibit hyperphosphorylation of Tau, another substrate related to the pathological state of AD (Cruz, J. C. et al, Neuron 40: 471- 83(2003)). Consequently, the inventive inhibitors seem to be promising for treating AD patients based on their effective blockages of both amyloid plaque and neurofibrillary tangle (NFT) formations, the major pathological features of AD. In addition, these compounds show a potential for treating other neurodegenerative diseases associated with Tau hyperphosphorylation, as well as AD.

The inhibitors of the present invention also appears to be safe for patients afflicted with the neurodegenerative disease because they do not target on CDK5 that normally exists in a human body, but on the substrate binding site of P25 produced only during the pathological state. In this regard, in accordance with another aspect of the present invention, there is provided a method for inhibiting BACEl phosphorylation in a mammal comprising administering a compound of formula (I) or (II) to the mammal. Based on the method demonstrated in the present invention, the effect of treating or preventing a neurodegenerative disease in a mammal can be achieved through the inhibition of BACE 1 phosphorylation.

Meanwhile, the pharmaceutical composition of the present invention can be delivered through various routes including oral, transdermal, subcutaneous, intravenous and intramuscular administrations. The composition of the present invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a patient by employing any of the procedures well known in the art. The formulation may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like. Examples of carriers, excipients, and diluents suitable for the formulation are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The compositions may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. An effective daily dose of the inventive P25/CDK5 inhibitor may range from 0.01 to 1,000 mg/kg body weight, preferably from 0.05 to 200 mg/kg body weight, and can be administered in a single dose or in divided doses. However, it should be understood that the amount of the inventive P25/CDK5 inhibitor actually administered ought to be determined in light of various relevant factors including the condition to be treated, the selected route of administration, the age, sex and weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.

The following Examples are intended to further illustrate the present invention without limiting its scope.

Example 1 : Verification of the Mechanism of BACEl Phosphorylation by the P25/CDK5 Complex

It has been reported that the accumulations of BACEl and P25 in the brain of AD patients increase the activities of β-secretase and the P25/CDK5 complex (Yang, L-B. et al, Nature Medicine 9:3-4 (2003); Patrick, G. N. et al, Nature 402: 615-622 (1999)). In the present example, it was investigated as follows whether an abnormally increased activity of the P25/CDK5 complex in AD patients leads to BACEl phosphorylation, resulting in elevations of both β- secretase activity and Aβ secretion. Purified BACEl and the P25/CDK5 complex used in the following experiment were purchased from Invitrogen Corp. and Upstate Group, Inc. respectively.

(1) BACEl phosphorylation by the P25/CDK5 complex

(1-1) Isotope-based kinase assay

To 1.5 μg of purified BACEl and 40 ng of purified P25/CDK5 complex were added kinase buffer (20 mM MOPS (pH 7.0), 10 mM MgCl₂, 1 mM DTT, 20 μM sodium orthovanadate), 100 μM ATP and 5 μCi [γ-³²P]ATP, and the resulting mixture was adjusted to 30 μl with ddH₂O. The mixture was reacted for 1 hour at 30°C, and the reaction was completed by adding 7.5 μl of 5χ sample buffer (60 mM Tris-HCl (pH 6.8), 25 % glycerol, 2 % SDS, 14.4 mM 2- mercaptoethanol, and 0.1 % bromophenol blue). The reaction mixture was boiled for 5 min and subjected to 12 % SDS-PAGE gel electrophoresis. The gel was immersed in 10 % glycerol for 30 min, dried for 2 hours at 80⁰C, exposed to a film and then analyzed with FLA3000 (Fuji Photo Film Inc., Japan). The same procedure was repeated with the control group in the absence of B ACEl .

The result depicted in Fig. IA exhibited BACEl phosphorylation by the P25/CDK5 complex, and P25 autophosphorylation by CDK5 (*, Fig. IA).

(1-2) Phosphospecifϊc antibody-based kinase assay

A comparison among the amino sequences of BACEl of a human, a rat and a mouse reveals that a CDK5 phosphorylation consensus motif is present in Thr²⁵² (Thr-Pro-Ile-Arg; SEQ ID NO: 1). The following experiment was undertaken to investigate whether BACEl is phosphoryled at Thr²⁵² residue.

(Step 1) Production of BACEl phosphospecific antibody

An antibody against a phosphorylated peptide SLWYThr²⁵²(PO₄)PIRR (SEQ ID NO: 2) was manufactured by Peptron Inc. (Korea) as follows. 2 mg of the said phosphorylated peptide was conjugated with KLH (key-hole limpet hemocyanin). 400 μg each of the conjugated peptide was injected three times to a 10 to 12-week-old New Zealand white rabbit at intervals of three weeks. After a week from the day of the third injection, a blood sample was collected from the rabbit, and a serum was isolated therefrom. In order to isolate the phosphospecific antibody, an affinity resin was prepared by linking a phospho- peptide and a normal peptide (SLWYTPIRR; SEQ ID NO: 3) to a Sulfolink gel (Pierce Biotechnology, Inc.) at a concentration of 2 mg/ml, respectively, and blocking the remaining binding sites of the gel with L-cystein. The serum and 1 mM PMSF were initially added to the normal peptide affinity resin and allowed to bind at room temperature (RT) for 20 min, and the unbound serum was collected. The unbound serum was added to the phosphorylated peptide- conjugated affinity resin with an addition of 1 mM PMSF. After the binding for additional 20 min at RT, the resin was washed with 10 mM Tris-Cl (pH 7.5) and 500 mM NaCl buffers, and the unbound serum was eluted with 100 mM glycine (pH 2.5). The IgG present in the eluent was confirmed by ELISA to selectively bind only to the phosphorylated peptide but not to the normal peptide, and concentrated by Centricon to yield the phospho-specifϊc BACEl antibody.

(Step 2) Transient transfection of HEK293 cells and immunoblotting

8x 10⁵ cells/well of HEK293 cells (ATCC, CRL- 1573) were loaded on a

6-well plate containing DMEM (Gibco-BRL, Invitrogen Corp.) medium with 10 % FBS (Gibco-BRL, Invitrogen Corp.) and were cultured overnight at 37⁰C. Using Lipofectamine^® 2000 (Invitrogen Corp.), the cells were transfected according to the user's manual, as follows. BACEl cDNA was obtained from human brain cDNA library (Clontech Lab., Inc.) by the PCR amplification, sequenced, and cloned into pcDNAmychis vector (Invitrogen Corp.). A phosphorylation-deficient mutant of BACEl designated BACE1T252A was constructed by the site-directed mutagenesis method using BACEl cDNA as a template. BACEl cDNA or BACE1T252A cDNA was added to each well together with control DNA (pcDNAmychis, Invitrogen Corp.), P25 cDNA or CDK5 cDNA to a total amount of 6 μg DNA. The cells were cultured at 37°C with the supply of 5 % CO₂ for 24 hr and were washed once with Ix PBS (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, and 100 mM NaCl, pH 7.4). 200 μl of a lysis buffer [RIPA buffer (1 % NP-40, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 0.1 % SDS, and 0.5 % deoxycholic acid) containing 1 mM PMSF and protease inhibitor cocktail (Calbiochem, CN Biosicences, Inc.)] was added to each well to obtain cell lysates. The proteins in the cell lysates were quantified by BCA protein assay (Pierce Biotechnology, Inc.), and the cell lysates were subjected to 10 % SDS-PAGE gel electrophoresis with loading 50 μg proteins of the cell lysates in each lane. The resulting isolated bands were transferred to a PVDF membrane and blocked overnight with PBST (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, pH 7.4, and 0.1 % Tween^® 20) containing 10 % skim milk. The membrane was washed three times with PBST for 15 min each. Thereafter, a solution containing the above phosphospecifϊc-BACEl antibody diluted with 10 % BSA/PBST to a ratio of 1 : 1,000 was added to the membrane and reacted for 1 hr at RT. The membrane was further washed three times with I x PBST for 10 min each. A secondary anti-rabbit-HPR antibody (Amersham Biosciences) diluted with 10 % BSA/PBST to a ratio of 1 : 10,000 was added to the membrane and reacted for 1 hr at RT. Additional three-time washes were carried out with Ix PBST for 10 min each and, finally, the degree of BACEl phosphorylation was detected by ECL system (Amersham Biosciences).

The result indicated that the antibody generally reacted with phosphorylated BACEl, which was produced when BACEl was expressed with both CDK5 and P25. In contrast, the phosphorylation deficient mutant (BACE1T252A), wherein Thr²⁵² of BACEl is replaced by Ala, did not show any reactivity against the phosphorylated antibody even when expressed with both CDK5 and P25. Consequently, it can be seen that the phosphospecific antibody produced above specifically reacts with BACEl phosphorylated at Thr²⁵² {see Fig. IB).

(Step 3) In vitro BACEl phosphorylation and immunoblotting

To 320 ng of purified BACEl and 50 ng of purified P25/CDK5 complex were added a kinase buffer (20 mM MOPS (pH 7.0), 10 mM MgCl₂, 1 mM DTT, 20 μM sodium orthovanadate) and 200 μM ATP, and the mixture was adjusted to 30 μl with ddH₂O. The mixture was reacted at 3O⁰C for 0, 10, 20, 60, 120, and 180 min, respectively, and the reaction was completed by the addition of 7.5 μl of 5x sample buffer (60 mM of Tris-HCl (pH 6.8), 25 % glycerol, 2 % SDS, 14.4 raM 2-mercaptoethanol, and 0.1 % bromophenol blue). In the case of treating a CDK5 inhibitor 50 μM of roscovitine (Calbiochem, CN Biosciences, Inc.) was added to the reaction mixture in the same conditions as above and the mixture was reacted for 120 min. The reaction mixture was boiled for 5 min and subjected to 12 % SDS-PAGE gel electrophoresis. After transferring the isolated bands to PVDF membranes, the membranes were blocked overnight in PBST containing 10 % skim-milk and washed three times with PBST for 15min each. The phosphospecific-BACEl antibody prepared above and BACEl antibody (manufactured by Peptron Inc. using peptide RLPKKVFEAAVKSIK (SEQ ID NO: 4) corresponding to aa 296 to 310 of BACEl as an antigen) were diluted with RPMI 1640 medium (Gibco-BRL) containing 5 % FBS (Gibco-BRL, Invitrogen Corp.) to ratios of 1 :1,000 and 1 :2,500, respectively. The diluted solutions were then added to the membranes and reacted for 2 hr at RT. The membranes were washed three times with PBST for 15 min each. Goat anti-rabbit HRP conjugated antibody (Amersham Biosciences) was diluted with RPMI 1640 medium containing 5 % FBS to a ratio of 1 : 10,000 and was transferred to the membranes followed by 1 hr reaction at RT. The membranes were washed three times with PBST for 15 min each. Then, the degree of BACEl phosphorylation as well as the amount of BACEl was measured by employing ECL system (Amersham Biosciences).

As can be seen from Fig. 1C, BACEl phosphorylation detected by the phospho-specific antibody gradually increased over time prior to the decline to some extent observed after 2 hr. The inhibition of BACEl phosphorylation by a treatment of the CDK5 inhibitor, roscovitine, showed that CDK5 was responsible for the phosphorylation of BACEl at Thr²⁵².

(2) Increased activity of β-secretase due to the BACEl phosphorylation by P25/CDK5 complex

According to the reported crystal structure of BACEl (Hong et al.

Science 290: 150-3(2000)), Thr²⁵² has been found to be located near the active site of the enzyme. Therefore, it is expected Thr²⁵² can have an influence on the enzyme activity. The following study was undertaken to investigate whether phosphorylation of BACEl at Thr²⁵² residue by CDK5 affects the activity of β-secretase.

(2-1) Effect of in vitro BACEl phosphorylation on β-secretase activity

In order to phosphorylate BACEl, 2.2 μg of BACEl was added to 50 ng of purified P25/CDK5 complex, kinase buffer and 50 μM of ATP. The mixture was adjusted to 30 μl with adding ddH₂O and reacted for 2 hr at 30°C. In order to measure β-secretase activity, 100 μl of a mixture consisting of 10 μl of the obtained reaction mixture, 88 μl of BACEl FRET Assay buffer (50 mM sodium acetate, pH 4.5), and 2 μl of BACEl substrates (2 mM, R&D Systems Inc.) was added to each well of a 96-well black microwell plate (Corning Inc.), and only FRET assay buffer was added to the blank well. The plate was placed on a fluorometer (Molecular Devices Corp., SPECTRA MAX GerminiXS) and the β-secretase activity was measured under the conditions of excitation at 320 nm and emission at 405 nm. Acquired data was analyzed by SoftMax PRO 4.3 LS software. The same procedure was repeated with the control group without the P25/CDK5 complex. Further, the same procedure was repeated with the addition of 50 μM roscovitine in the phosphorylation reaction.

As can be seen from the result in Fig. 2 A, phosphorylated BACEl exhibited a significant increase in β-secretase activity by 21 % (N=3, p<0.01) as compared to the control group where no BACEl phosphorylation occurred. From the identical experiment repeated five times, β-secretase activity of phosphorylated BACEl was significantly increased by 20 ± 7 % (p<0.05) relative to the control group. In addition, Fig. 2A shows that roscovitine, a CDK5 inhibitor, effectively suppressed the elevation of β-secretase activity resulted from BACEl phosphorylation by 52 % (N=3, p<0.05). When the same experiment was repeated five times, roscovitine significantly suppressed the elevation of β-secretase activity by 62 ± 11 % (p<0.01). These results demonstrate that in vitro BACEl phosphorylation by the P25/CDK5 complex leads to a significant increase of β-secretase activity.

(2-2) Effect of the transient expression of BACEl phosphorylation deficient mutant on β-Secretase activity in SK-N-BE2C cell

SK-N-BE2C cells (ATCC, CRL-2268) were cultured in DMEM/F-12 medium containing 10 % FBS with a supply of 5 % CO₂ at 37⁰C. Using the same method described in step 2 of section (1-2), the control DNA (pcDNAmychis), BACEl cDNA or BACE1T252A was transiently transfected to SK-N-BE2C cell and a cell lysate was obtained therefrom. In each experiment, 50 μg protein of the cell lysate quantified by BCA protein assay (Pierce Biotechnology, Inc.) was mixed with BACEl FRET assay buffer (50 mM sodium acetate, pH 4.5) to a total volume of 198 μl. 2 μl of BACEl substrate (2 mM, R&D Systems Inc.) was added to the mixture, and β-secretase activity of the resulting mixture was measured by a fluorometer, as mentioned above in section (2-1).

As depicted in Fig. 2B, the transient transfection with BACEl cDNA caused 53 % increase of β-secretase activity (N=3, p<0.01), whereas the transient transfection with BACE1T252A cDNA showed β-secretase activity similar to the control group. These results indicate that phosphorylation of BACEl at Thr²⁵² by CDK5 plays an important role in the β-secretase activity.

(3) Involvement of P25 protein in increases of BACEl phosphorylation, β- secretase activity and Aβ secretion.

It has been reported that an increase of P25 has been found from the sporadic AD patients (Tseng, H.C. et al, FEBS Lett. 523: 58-62(2002)). Also, transient expression of P25 in SH-SY5Y, a neuronal cell line that stably expresses APP695sw, has promoted Aβ level (Lie, F. et al, FEBS Lett. 547: 193-196 (2003)). In the present example, it was examined whether the increase in the Aβ amount by P25 was caused by the phosphorylation of

BACEl and increased β-secretase activity. (3-1) Effect of transient expression of P25 on the β-secretase activity in SK-N- BE2C cells

SK-N-BE2C cells (ATCC, CRL-2268) were cultured in DMEM/F-12 medium containing 10 % FBS with a supply of 5 % CO₂ at 37⁰C. P25 cDNA was obtained from human brain cDNA library (Clontech Lab., Inc.) by the PCR amplification, sequenced, and then cloned into pcDNAmychis vector (Invitrogen Corp.). Using the same method described in Step 2 of section (1- 2), control DNA (pcDNAmychis) or P25 cDNA was transiently transfected to SK-N-BE2C cells and a cell lysate was obtained therefrom. In each experiment, 50 μg protein of the cell lysate quantified by BCA protein assay (Pierce Biotechnology, Inc.) was mixed with BACEl FRET assay buffer (50 mM sodium acetate, pH 4.5) to a total volume of 198 μl. 2 μl of BACEl substrate (2 mM, R&D Systems Inc.) was added to the mixture, and β-secretase activity of the resulting mixture was measured by a fluorometer, as mentioned above in section (2-1).

As depicted in Fig. 3A, the transient transfection of P25 cDNA caused 86 % increase of β-secretase activity (N=3, p<0.01). This result indicates that upregulation of P25 can lead to an increased β-secretase activity, and also suggest that the earlier reported event of increase in Aβ secretion by p25 is due to the enhanced β-secretase activity.

(3-2) The effect of stable expression of P25 in PC 12 cell on BACEl phosphorylation and Aβ secretion

In order to examine the effect of stable expression of P25 on BACEl phosphorylation and Aβ secretion, a cell which stably expressed P25 was established from PC 12 cell (ATCC CRL- 1721 ). PC 12 cell is a neuroendocrine cell and possesses the basic neuronal function of releasing neurotransmitters and basic neuronal protein network. It is one of the cell lines widely used in understanding various biological phenomena occurred in the brain. Cytotoxicity of P25 has been reported (Patrick, GN. et al, Nature 402: 615-622 (1999)) and, in the actual experiments, death of P25-expressing PC 12 cell lines was observed during the subculture process. In the present example, the effects of P25 on BACEl phosphorylation and Aβ activity were examined by employing a PC12_tet-off cell line (Clontech Lab., Inc.), which stably expresses P25 with a Tet-off system wherein the expression of P25 is controlled by doxycycline for the prevention of the cell death caused by the toxicity of P25.

(Step 1) Construction of PC12_tet._off cells stably expressing P25

PC12_te,_-Off cells (Clontech Lab., Inc.) were cultured in RPMI 1640 medium (Gibco-BRL, Invitrogen Corp.) containing 10 % FBS, 5 % HS (Gibco- BRL, Invitrogen Corp.) and 100 μg/ml of G418 with a supply of 5 % CO₂ at 37⁰C. In order to construct a cell line expressing P25 under Tet-off system, P25 cDNA synthesized in section (3-1) was subcloned into pTRE2pur vector (Clontech Lab., Inc.) to obtain pTRE2puro-P25 cDNA. Ix 10⁶ cells/well of PC12_tet-Off cells were incubated in 6-well plate for a day and a mixture of 4 μg of pTRE2puro-P25 cDNA and 12 μl of Lipofectamine 2000 (Invitrogen Corp.) was added to each well. 4 hr later, the medium was replaced by RPMI 1640 medium containing 10 % FBS, 5 % HS and 100 μg/ml G418. After 48 hr from the transfection, the medium was again replaced by RPMI 1640 medium containing 100 μg/ml of G418 (Clontech Lab., Inc.), 1 μg/ml of doxycycline (Clontech Lab., Inc.) and 3 μg/ml of puromycin (Clontech Lab., Inc.), and the medium replacement was continued once every 4 days. Dead cells were separated from living cells in 5 to 7 days. After several washings with RPMI 1640 medium, only living cells were cultured on a 150 mm dish and an isolated single cell colony formed therefrom was transferred serially onto 96-, 24-, 12-, and 6-well plates in order. For the protein expression, the clone was further cultured in RPMI 1640 medium lacking doxycycline for 3 days. P25 expression was observed by western blotting {see Step 2 described below) to select stable cells.

Consequently, two types of PC12_tet._off cell lines (P25-3 and P25-5, Fig. 3B) were obtained, which clearly over-expressed P25 when cultured in a medium lacking doxycycline.

(Step 2) Western analysis of BACEl phosphorylation in P25 stable cells

After culturing P25 stable PC12_tet._off cell lines (P25-3 and P25-5) in RPMI 1640 without doxycycline for 3 days, the cells were subjected to lysis according to the same method described in Step 2 of Section (1-2). The cell lysate was incubated on ice for 30 min and centrifuged at 4°C and 14,000 rpm for 10 min to obtain a supernatant. Then, the proteins in the supernatant were quantified by BCA protein assay (Pierce Biotechnology, Inc.). 8 μl of 5χ sample buffer was added to 100 μg of proteins from each cell line, and the mixture was adjusted to 40 μl with ddH₂O. After boiling for 5 min and running 12 % SDS-PAGE, the isolated bands were transferred to a PVDF membrane. The membrane was blocked overnight with PBST containing 10 % skim milk and washed three times with PBST for 15 min each. As the primary antibodies, the following antibodies were diluted with RPMI 1640 medium containing 5 % FBS (Gibco-BRL, Invitrogen Corp.) to the following dilution ratios: P25 antibody (c-19, Santa Cruz Biotechnology, Inc.) to 1 :700; CDK5 antibody (J-3, Santa Cruz Biotechnology, Inc.) to 1 : 1,000; phospho-APP antibody (Cell signaling Technology, Inc.) to 1 : 1,000; APP antibody (Cell signaling Technology, Inc.) to 1 : 1,000; phospho-B ACE 1 antibody to 1 :1,000; and BACEl antibody (M-83, Santa Cruz Biotechnology, Inc.) to 1 :700. The antibody dilutions were added to the membrane and reacted for 2 hr at RT. The membrane was washed three times with PBST for 15 min each. As the secondary antibodies, a goat anti-mouse HRP conjugated antibody (Amersham Biosicences) in case when the primary antibody was CDK5 antibody and a goat anti-rabbit HRP conjugated antibody (Amersham Biosciences) in case when the primary antibody was other than CDK5 antibody were respectively diluted to a ratio of 1 : 10,000 with RPMI 1640 medium containing 5 % FBS. The antibody dilutions were transferred to the membrane and reacted for 1 hr at RT. The membrane was washed three times with PBST for 15 min each, and the proteins on the membrane were detected with ECL plus system (Amersham Biosciences).

As demonstrated in Fig. 3B, the expression of P25 led to increases in phosphorylation of both APP (amyloid precursor protein) and BACEl . Thus, it can be seen from the result that CDK5 activation in the P25 stable cells results in the phosphorylation of BACEl as well as APP (Lie, F. et al., FEBS

Letters 547: 193-196 (2003)).

(Step 3) Measurement of Aβ amount in P25 stable cells

P25 stable PC12_tet-off cell lines (P25-3 and P25-5) were cultured in

RPMI 1640 medium without doxycycline for 3 days, and the cells were added to a 0.05 % Polyethylenimine-coated 6- well plate at a concentration of 1 x 10⁶ cells/well. 24 Hours after, the medium was replaced by 1 ml of conditioned medium (RPMI 1640 medium containing 5 % HS, 1 % FBS, and 0.5 % P/S), and the cells were cultured for 24 hr. The culture medium was collected, and Aβ amount thereof was measured by ELISA using an Aβ assay kit (IBL Co., Ltd.) according to the user's manual, as follows. 100 μl of the culture medium from P25 stable cell line or control cell line or a standard human Aβ was added to the wells of a 96-well plate pre-coated with antibody (anti-human Aβ(35-40) rabbit IgG; IBL Co., Ltd.) and was kept covered at 4°C for 24 hr. The wells were then washed seven times with a washing buffer (0.05 % Tween^®20 in phosphate buffer), and 100 μl each of anti-human Aβ( 11-28) mouse IgG-HRP conjugated antibody (IBL Co., Ltd.) was added to the wells. The reaction mixture was kept covered at 4°C for 24 hr, and the wells were washed nine times with the washing buffer. 100 μl of TMB (Tetra methyl Benzidine, Sigma-Aldrich, Inc) was added to each well, and the mixture was incubated in the dark for 15 min at RT. 100 μl of stop solution (1 N H₂SO₄) was added to each well, and the absorbance of each well was measured within 30 min with a microplate reader at 450 nm. The same procedures as above were repeated for the medium blank, test sample blank and reagent blank. Amount of Aβ was calculated by substituting into the graph drawn on the basis of the standard. The remaining cells were subjected to lysis by adding 120 μl of lysis buffer (RIPA buffer containing 1 mM PMSF and protease inhibitor cocktail (Calbiochem CN Biosciences, Inc.)) into each well, and the lysate was incubated on the ice for 30 min. The lysate was centrifuged at 4°C and 14,000 rpm for 10 min to obtain a supernatant and the proteins therein was quantified by BCA protein assay method (Pierce Biotechnology, Inc.). The measured values were normalized to Aβ(pg)/total protein(mg).

As can be seen from Fig. 3C, increases in Aβ amount were observed in P25 expressing PC 12_tet-off cells, P25-3 and P25-5.

(3-3) Examination of BACEl phosphorylation, β-secretase activity and Aβ amount in a human P25 expressing transgenic mouse

In order to find out whether the BACEl phosphorylation and an increased β-secretase activity observed in the cell lines also occur in animals, the following experiment was carried out using a human P25 expressing transgenic mouse (TgN(Eno2CDK5Rl)Udm, Jackson lab, USA).

(Step 1) Verification of the BACEl phosphorylation in a transgenic mouse expressing P25 by immunohistochemistry

Each of a 20-week old P25 TG (transgenic mouse) mouse and an age- matched wild mouse was anesthetized with isoflurane (Rhodia Inc.) for 20 sec, and their blood streams were washed out by perfusion of 5 ml of PBS, followed by perfusion of 10 ml PBS containing 4 % paraformaldehyde (Sigma-Aldrich, Inc.). Their brains were extracted and fixed overnight at 4°C in 100 ml of PBS containing 4 % paraformaldehyde. On the next day, the fixed brain samples were treated with 10 %, 15 % and 20 % sucrose for 1 hr each in order, and then with 20 % sucrose overnight at 4°C. The brains were embedded into OCT compound (Tissue-Tek, Sakura Finetek USA, Inc.), frozen by immersing in isopanthane (Yakuri Pure Chemicals Co. Ltd.) chilled with liquid nitrogen, and stored at -2O⁰C. Slices with the thickness of 16 μm were prepared using Cryostat (Microm, HM 505 N) at -20⁰C and placed on Superfrost^*/Plus slide (Fisher Scientific International). The slide was air-dried for 30 min at RT and stored at -2O⁰C. After drying for 30 min at RT, the slide was washed three times with PBS for 5 min each and blocked by incubating it with 100 μl of 3 % normal goat serum (Jackson ImmunoResearch Lab., Inc.) for 1 hr at RT. Phospho-BACEl antibody diluted with 100 μl of 1 % normal goat serum to a ratio of 1 :500 was added to the slide and incubated overnight at 4⁰C. After three washes with PBS for 15 min each, Cy 3 -conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Lab., Inc.) diluted with 100 μl of 1 % normal goat serum to a ratio of 1 :500 was added to the slide and incubated for 1 hr at RT, followed by three additional washes with PBS for 15 min each. The product was then mounted with an aqueous mounting solution (ResGen, Invitrogen

Corp.) and observed with a fluorescence microscope (Carl Zeiss, Axiophoto 2).

As can be seen from Fig. 4A, BACEl phosphorylation was significantly increased in the hippocampus of a P25 TG mouse and slightly increased both in the cortex and cerebellum thereof, as compared to a wild-type mouse. Three additional experiments with the P25 TG mice and wild-type mice in the same age led to the substantially same results. This result demonstrates that an increase in BACEl phosphorylation due to P25 expression also occurs in the P25 TG mouse as in the P25 stable cells.

(Step 2) Analysis of BACEl phosphorylation, β-secretase activity and Aβ amount in a P25 expressing transgenic mouse

A 26 week-old P25 TG mouse was subjected to cervical dislocation, and its brain was extracted and divided into the hippocampus, the cortex, and the cerebellum. The divided tissues were placed in an Eppendorf tube and quickly frozen in liquid nitrogen for 30 sec. 200 μl of RIPA buffer (1 % NP-

40, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 0.1 % SDS, and 0.5 % deoxycholic acid) containing 1 mM of PMSF (Sigma-Aldrich, Inc.) and protease inhibitor cocktail (Calbiochem, CN Biosciences, Inc.) was added to 100 mg of the brain tissue. The tissues were subjected to complete lysis by a homogenizer. After incubating on ice for 30 min at 4°C, the cell lysate was centrifuged at 14,000 rpm for 60 min to obtain a supernatant. The proteins in the supernatant were quantified by BCA protein assay (Pierce Biotechnology, Inc.). In order to analyze BACEl phosphorylation, 200 μg of the proteins from each tissue was subjected to the western blotting as described in Step 2 of section (3-2) by employing P25 antibody (1 :700, Santa Cruz Biotechnology, Inc.), phospho- BACEl antibody (1 : 1,000) and actin (1 :10,000, Sigma-Aldrich, Inc.).

After measuring western blotting bands of phospho-BACEl and actin with a densitometer, the results were normalized to measured phospho-BACEl value/actin value. In order to measure the β-secretase activity, 100 μg of proteins from each tissue was subjected to the BACE FRET assay as described in sections (2-1) and (2-2). For measuring Aβ amount, 4-fold volume of PBS containing 2 % SDS was added to the pellet obtained in the above centrifugation, and the tissues were subjected to lysis by a homogenizer. The lysate was centrifuged at 2O⁰C, 14,000 rpm for 60 min to obtain a supernatant, and the proteins in the supernatant were quantified by the BCA protein assay (Pierce Biotechnology, Inc.). Aβ assay was performed as described in Step 3 of section (3-2) by employing 50 or 100 μg of the proteins from each tissue, thereby determining the Aβ amount. As a control, the above procedure was repeated with a wild-type mouse in the same age. The ratios of the measured values of the P25 TG mouse relative to that of the control were plotted on the graph.

As depicted in Fig. 4B, the hippocampus and the cortex of the 26 week- old P25 TG mouse showed higher P25 expression, whereas the cerebellum exhibited only a slight expression. This result confirms the previously reported data (Ahlijanian M. K. et al, PNAS 97: 2910-2915(2000)). As compared to the wild-type mouse, BACEl phosphorylation was particularly elevated in the hippocampus and the cortex of the P25 TG mouse, but no variation in the cerebellum was observed. When the values obtained from the experiments repeated with 15 to 26-week old P25 TG mouse were compared with those of the wild-type mouse in the same age BACEl phosphorylation in the hippocampus of the P25 TG mouse significantly increased by 54 ± 10 % (N=4, P<0.05) than the wild-type mouse, which is consistent with the result of immunohistochemistry.

As also can be seen in Fig. 4C, β-secretase activity in the hippocampus of P25 TG mouse significantly increased by 56 ± 13 % (N=5, p<0.05) relative to the control, whereas little difference was observed in the cortex or cerebellum. Hence, it is considered that there is a direct and close relation between the phosphorylation of BACEl and β-secretase activity in the brain of the P25 TG mouse.

In addition, as illustrated in Fig. 4E, Aβ amount in the P25 TG mouse was significantly elevated in the hippocampus by approximately 36 ± 9.5 % (N=4, p<0.05) relative to the control. However, in the cortex and cerebellum, there seemed to be an increase in the Aβ amount without any statistical significance. These results are in agreement with the analytic results of

BACEl and β-secretase activity. Therefore, it can be concluded that P25 expression results in the increases in BACEl phosphorylation, β-secretase activity and Aβ amount in the P25 TG mouse, as in the P25 stable cell lines.

In the present Example 1, a novel pathway, wherein BACEl phosphorylation by P25/CDK5 increases β-secretase activity, thereby resulting in the increase of Aβ amount, has been confirmed through in vitro as well as in vivo experiments {see Fig. 5).

Example 2; Examination of Inhibitory Activity of P25/CDK5 Inhibitors by In vitro Phosphorylation Experiment and Cell-Based Assay

Inhibitory activities of compounds DSS30 and DSS36, and their analogues listed in Table 1 below against the P25/CDK5 complex were examined through in vitro phosphorylation experiment and cell-based assay, as follows.

DSS30 and its analogues (DSS301, DSS302, DSS303 and DSS304) were purchased from Maybridge Chemical Company Ltd.; DSS36 and DSS362, from Bionet Research Ltd.; and DSS361 and DSS363, from ChemBridge Corp..

The compounds were respectively stored in DMSO (Sigma-Aldrich, Inc.) at a concentration of 10 mM. Prior to experiment, the stock solution was diluted to make the final concentrations of the test compound and DMSO to 50 μM and 0.5 %, respectively.

Table 1

■

As an in vitro assay for studying P25/CDK5 inhibitory activity, three methods were carried out: (1) ELISA with a general P25/CDK5 substrate, histone Hl, and a phospho-specifϊc antibody; (2) Western blotting with AD related substrate of P25/CDK5, BACEl, and BACEl phospho-specific antibody; and (3) Western blotting with AD related substrate of P25/CDK5, Tau, and Tau phospho-specific antibody.

(1) Analysis of P25/CDK5 inhibitory activity using histone Hl and phospho- specific antibody

(1-1) Phosphorylation experiment with histone Hl and phospho-specific antibody The P25/CDK5 complex phosphorylates Thr(or Ser)-Pro-(X)_n-Lys(or Arg or His)(wherein, X is any amino acid and n is 1 or 2; SEQ ID NO: 5) site of a substrate. Thus, making use of phospho-threonine-proline antibody (P-Thr- Pro antibody; Cell signaling Technology, Inc.), which specifically recognizes the phosphorylated substrate, would allow determination of the activity of P25/CDK5 inhibitors. The specificity of P-Thr-Pro antibody for phosphorylated histone Hl was examined as follows.

20 ng of P25/CDK5 complex was added to 1 μg of histone Hl, kinase buffer, and 200 μM of ATP, and the mixture was adjusted to 30 μl with ddH₂O. The reaction mixture was incubated at 30°C for 2 hr, and the reaction was completed by adding 7.5 μl of 5x sample buffer. Thereafter, the reaction mixture was boiled for 5 min and subjected to 12 % SDS-PAGE. After transferring the isolated bands to a PVDF membrane, the membrane was blocked overnight by PBST containing 10 % skim-milk and washed three times with PBST for 15 min each. P-Thr-Pro antibody was diluted with RPMI 1640 medium containing 5 % FBS (Gibco-BRL, Invitrogen Corp.) to a ratio of 1 :2,500. The diluted solution was then added to the membrane and incubated at RT for additional 2 hr. The membrane was washed three times with PBST for 15min each. Goat anti-mouse HRP conjugated antibody (Amersham Biosciences) was diluted with RPMI 1640 medium with 5 % FBS to a ratio of 1 : 10,000 and the diluted solution was added to the membrane. After incubating at RT for 1 hr, the membrane was washed three times with PBST for 15 min each. Then, the degree of phosphorylation of histone Hl by the P25/CDK5 complex was detected utilizing ECL plus system (Amersham Biosciences). The same experiment was conducted in the absence of the P25/CDK5 complex to find out whether purified histone Hl was phosphorylated. Furthermore, the same experiment was performed in the presence of a CDK5 inhibitor, roscovitine (Calbiochem, CN Biosciences, Inc.), at a concentration of 50 μM in the phosphorylation step to confirm whether the phosphorylation of histone Hl was inhibited.

As can be seen from Fig. 6A, P-Thr-Pro antibody detected only phosphorylated form of histone Hl and, when roscovitine was treated, the phosphorylation of histone Hl was inhibited. Thus, P-Thr-Pro antibody seems to be appropriate in detecting the phosphorylation of histone Hl by the P25/CDK5 complex.

(1-2) Assay for the inhibition of P25/CDK5 by DSS30, DSS36 and their analogues using histone Hl and phospho-specific antibody

1 μg of histone Hl (1 mg/ml), 10 μl of NHS solution (Sigma-Aldrich, Inc.) and 50 μl of EDC solution (Sigma-Aldrich, Inc.) were mixed, and the mixture was adjusted to 100 μl by adding PBS (pH 7.4) thereto and put into each well of a 96-well plate. The plate was incubated overnight to coat the wells with histone Hl . After blocking by 100 μl of PBS containing 1 % BSA at 4°C for 4 hr, each well was washed two times with 200 μl of PBS (pH 7.4). 10 μl of a solution containing 500 μM inhibiting compound dissolved in 5 % DMSO (final cone, of the compound: 50 μM) was added to each well. Then, 20 ng of purified P25/CDK5, kinase buffer, 200 μM ATP were added to each well, and the reaction mixture was adjusted to 100 μl with ddH₂O. The mixture was incubated at 30°C for 2 hr, and the wells were washed five times with 200 μl of PBST and, then, with PBS (pH 7.4). P-Thr-Pro antibody (Cell signaling Technology, Inc.) was diluted with RPMI 1640 medium (Gibco-BRL, Invitrogen Corp.) containing 5 % FBS (Gibco-BRL, Invitrogen Corp.) to a ratio of 1 :1,000. 100 μl of the diluted solution was then added to each well and the reaction mixture was incubated overnight at 4°C. Each well was washed four times with 200 μl of PBST. Goat anti-mouse HRP conjugated secondary antibody (Zymed Lab., Inc.) was diluted with RPMI 1640 medium containing 5 % FBS to a ratio of 1 :4,000, and 200 μl of the resulting dilution was added to each well. After reacting at 37⁰C for 3 hr, each well was washed four times with 200 μl of PBST followed by the addition of 200 μl of ABTS substrate solution (Roche Diagnostics Corp.) and incubation at 37°C for 15 min. Then, absorbance of each well was measured at 405 nm with a plate reader. As a control, the same experiment was repeated in the absence of the test compound (Figs. 6B and 6C, No Inhibitor). Further, the value obtained from an identical experiment in the absence of ATP was determined as a background. In order to verify the inhibitory activity of the compound, a relative phosphorylation activity upon treatment with the compound was calculated by the following equation:

Phosphorylation activity (%) = {(Absorbance upon treatment with the compound - B ackground)/( Absorbance of the Control - Background)} x 100

As shown in Fig. 6B, DSS30 and DSS36 inhibited the activity of P25/CDK5 complex up to 50 %, at a concentration of 50 μM. In addition, four analogues of DSS30 exhibited similar or superior inhibitory activity as compared to DSS30. Among three analogues of DSS36, DSS363 displayed more effective inhibition than DSS36. Hence, it can be concluded that the structures of DSS30 and DSS36 are important to exhibit an inhibitory activity against the P25/CDK5 complex.

(2) Examination of the activity of a P25/CDK5 inhibitor using BACEl and phospho-specific BACEl antibody

In order to examine whether DSS30, DSS36 and their analogues inhibit the phosphorylation of BACEl by the P25/CDK5 complex, an experiment was conducted as follows using BACEl, which was discovered in the present invention as an AD related substrate.

320 ng of purified BACEl (Panvera, Invitrogen Corp.), 50 ng of purified P25/CDK5 complex, kinase buffer (20 mM MOPS (pH 7.0), 10 mM MgCl₂, 1 mM DTT, and 20 μM sodium orthovanadate) and 200 μM ATP were mixed together, and the mixture was adjusted to 30 μl with ddH₂O and then incubated at 30°C for 90 min. Then, the reaction was completed by adding 7.5 μl of 5χ sample buffer (60 mM Tris-HCl (pH 6.8), 25 % glycerol, 2 % SDS, 14.4 mM 2-mercaptoethanol, and 0.1 % bromophenol blue). A CDK5 inhibitor, roscovitine (Calbiochem, CN Biosciences, Inc.), or a test compound was added to the reaction mixture at a concentration of 50 μM, and the resulting mixture was incubated under the same condition as above. The reaction mixture was boiled for 5 min and subjected to 12 % SDS-PAGE. After transferring the isolated bands to a PVDF membrane, the membrane was blocked overnight by PBST containing 10 % skim milk and washed three times with PBST for 15 min each. The phospho-specific BACEl antibody and BACEl antibody (manufactured by Peptron Inc. using RLPKKVFEAAVKSIK (SEQ ID NO: 4) corresponding to a.a. 296-310 of BACEl as an antigen) were diluted with RPMI 1640 medium (Gibco-BRL, Invitrogen Corp.) containing 5 % FBS (Gibco-BRL, Invitrogen Corp.) to ratios of 1 : 1,000 and 1 :2,500, respectively. The diluted solutions were then added to the membrane and were incubated at RT for 1 hr. The membrane was washed three times with PBST for 15 min each. Goat anti-rabbit HRP conjugated antibody (Amersham Biosciences) was diluted with RPMI 1640 medium containing 5 % FBS to a ratio of 1 : 10,000 and added to the membrane. After incubating at RT for 1 hr, the membrane was washed three times with PBST for 15 min each. The degree of phosphorylation as well as the amount of BACEl was measured using ECL system (Amersham Biosciences).

As can be seen from Fig. 7 A, roscovitine inhibited BACEl phosphorylation. Further, all four analogues of DSS30 inhibited BACEl phosphorylation, wherein DSS301 and DSS303 showed the highest activity. Moreover, DSS36 also inhibited BACEl phosphorylation. Thus, it can be concluded that the structures of DSS30 and DSS36 are important in exhibiting an inhibitory activity against the P25/CDK5 complex, thereby prohibiting the phosphorylation of AD related substrate, BACEl .

(3) Examination of the activity of a P25/CDK5 inhibitor using Tau and phospho-specific Tau antibody

In order to examine whether DSS30, DSS36 and their analogues inhibit the phosphorylation of Tau by the P25/CDK5 complex, an experiment was conducted as follows using Tau, a known AD related P25/CDK5 substrate.

2.75 μg of purified Tau (Calbiochem, CN Biosciences, Inc.), 50 ng of purified P25/CDK5 complex, kinase buffer (20 mM MOPS (pH 7.0), 10 mM MgCl₂, 1 mM DTT, and 20 μM sodium orthovanadate) and 200 μM ATP were mixed together, and the mixture was adjusted to 30 μl with ddH₂O and then incubated at 30⁰C for 90 min. Then, the reaction was completed by adding 7.5 μl of 5 x sample buffer (60 mM Tris-HCl (pH 6.8), 25 % glycerol, 2 % SDS, 14.4 mM 2-Mercaptoethanol, and 0.1% Bromophenol Blue). A CDK5 inhibitor, roscovitine (Calbiochem, CN Biosciences, Inc.), or a test compound was added to the reaction mixture at a concentration of 50 μM, and the resulting mixture was incubated under the same condition as above. The reaction mixture were boiled for 5 min and subjected to 12 % SDS-PAGE gel electrophoresis. After transferring the isolated bands to a PVDF membrane, the membrane was blocked overnight by PBST containing 10 % skim-milk and washed three times with PBST for 15 min each. Phospho-specific Tau antibody (AT8, Innogenetics Inc.) and Tau antibody (AHB0042, BioSource International, Inc.) were diluted with RPMI 1640 medium (Gibco-BRL, Invitrogen Corp.) containing 5 % FBS (Gibco-BRL, Invitrogen Corp.) to ratios of 1 : 1,500 and 1 :2,500, respectively. The diluted mixtures were added to the membrane and reacted for 1 hr at RT. The membrane was washed three times with PBST for 15 min each. Goat anti-mouse HRP conjugated antibody (Amersham Biosciences) was diluted with RPMI 1640 medium containing 5 % FBS to a ratio of 1 : 10,000 and was added to the membrane. After incubating at RT for 1 hr, the membrane was washed three times with PBST for 15 min each. Then, the degree of phosphorylation as well as the quantity of Tau was measured using ECL system (Amersham Biosciences). As can be seen from Fig. 7B, roscovitine inhibited Tau phosphorylation.

Further, all four analogues of DSS30 inhibited Tau phosphorylation, wherein DSS303 showed the highest activity. Moreover, DSS36 and DSS363 also strongly inhibited Tau phosphorylation. Thus, it can be concluded that the structures of DSS30 and DSS36 are important in exhibiting an inhibitory activity against the P25/CDK5 complex, thereby inhibiting the phosphorylation of AD related substrate, Tau. (4) Cell-based assay of DSS30, DSS36 and their analogues using P25 overexpressing cell lines

The following experiment was performed to examine whether the inhibitors confirmed by the above in vitro assay actually inhibit the phosphorylation of BACEl by the P25/CDK5 complex and Aβ secretion in cells.

Construction of PC12_tet-o_ff cell lines which stably expresses P25, western blotting for analyzing BACEl phosphorylation in P25 stable cells, and measurement of Aβ amount are described in Steps 1 to 3 of Section (3-2), Example 1. P25-3 cells were cultured in RPMI 1640 without doxycycline for 3 days, and added to the wells of a 0.05 % polyethylenimine (Sigma-Aldrich, Inc)-coated 6 well plate at a concentration of 1 x 10⁶ cells/well. After 24 hr incubation, the medium was replaced by 1 ml conditioned medium (RPMI 1640 medium containing 5 % HS, 1 % FBS, and 0.5 % P/S). Roscovitine or an inhibitor compound was added thereto at a final concentration of 50 μM and the cells were cultured for 8 hr. Culture solutions were collected and Aβ amount thereof was measured by the method described in step 3 of section (3-2), Example 1. A cell lysate was prepared with the remaining cells and western blotting was conducted as described in step 2 of section (3-2), Example 1, to determine the degree of inhibition of BACEl phosphorylation.

As can be seen from Fig. 8A, DSS36 and DSS30 inhibited BACEl phosphorylation by 25 % and 12 %, respectively. Moreover, DSS303 and DSS36 reduced Aβ secretion by 72 % and 81 %, respectively (see Fig. 8B). Roscovitine, a positive control in the experiment, induces cell death when it is treated to cells for 24 hr at a concentration of 50 μM. Accordingly, in order to minimize the cell toxicity, the cells were treated with roscovitine only for 8 hr in the present example. In contrast to the 24 hr treatment of roscovitine showing 50 % inhibition of Aβ secretion, the 8 hr treatment of roscovitine resulted in the inhibition of BACEl phosphorylation but not in the reduction of Aβ secretion.

Example 2 has demonstrated the significance of the structures of DSS30 and DSS36 in exhibiting the inhibitory activity against the P25/CDK5 complex, through the in vitro phosphorylation experiments and the cell-based assay.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

What is claimed is:

1. A pharmaceutical composition comprising a compound of formula (I) or (II) as an active ingredient for preventing or treating a neurodegenerative disease:

wherein,

R¹ is hydrogen, linear or branched Ci-C₄ alkoxy, or C₁-C₄ alkylformamide substituted with a C₃-C₈ cycloalkyl group;

R² is hydrogen, or linear or branched C₁-C₆ alkyl; and

R³ is hydrogen, linear or branched C)-C₆ alkyl, or saturated or unsaturated 5- or 6-membered heterocyclic compound comprising 1 to 3 heteroatoms; or

wherein,

R¹, R², R³, R⁶, R⁷ and R⁸ are each independently hydrogen, halogen, linear or branched Cj-C₆ alkyl, linear or branched C₁-C₆ alkyl substituted with halogen, linear or branched C₁-C₆ alkoxy, or linear or branched C]-C₆ alkylester; and

R⁴ and R⁵ are each independently hydrogen, fluoro, chloro, or linear or branched Ci-C₄ alkyl.

2. The pharmaceutical composition of claim 1, wherein the compound is of formula (I) wherein R¹ is hydrogen, methoxy or cyclopropylmethylformamide; R² is hydrogen; and R³ is piperidine; or R² and R form a pyrrole or 1 ,4-dioxane ring, together with carbon atoms where they are attached.

3. The pharmaceutical composition of claim 2, wherein the compound of formula (I) are selected from the group consisting of the compounds having the following structural formulas:

DSS30 DSS301 DSS302

DSS303 DSS304

4. The pharmaceutical composition of claim 1, wherein the compound is of formula (II) wherein R¹, R², and R³ are each independently hydrogen, halogen, linear or branched Ci~C₄ alkoxy, or linear or branched Ci-C₄ alkylester; R⁴ and R⁵ are each independently hydrogen, or linear or branched C₁-C₄ alkyl; R , R and R are each independently hydrogen, linear or branched Ci~C₄ alkyl optionally substituted with halogen, or linear or branched C]-C₄ alkylester.

5. The pharmaceutical composition of claim 4, wherein the compound of formula (II) are selected from the group consisting of the compounds having the following structural formulas:

DSS36 DSS361

DSS362 DSS363

6. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Niemann-Pick disease, dementia caused by cerebral ischemia, and dementia caused by cerebral hemorrhage.

7. The pharmaceutical composition of claim 1, wherein the compound of formula (I) or (II) inhibits the phosphorylation of BACEl (beta-site APP- cleaving enzyme 1) and reduces the secretion of β-amyloid, thereby exhibiting a preventing or treating effect on a neurodegenerative disease.

8. A method for screening a compound capable of preventing or treating a neurodegenerative disease, which comprises the steps of a) reacting a candidate compound with a P25/CDK5 complex and BACEl or treating a mammalian cell expressing P25 with the candidate compound, and measuring BACEl phosphorylation; and b) comparing the measured BACEl phosphorylation with that of a control group employing no candidate compound, and identifying a compound reducing BACEl phosphorylation compared to the control group.

9. The method of claim 8, wherein the identified compound inhibits the BACEl phosphorylation due to a P25/CDK5 complex by more than 10 % as compared to the control group.

10. The method of claim 8, wherein the degree of BACEl phosphorylation due to a P25/CDK5 complex is detected by using an antibody specific for a phosphorylated BACE 1 or a radioisotope-labeled ATP.

11. The method of claim 10, wherein the antibody specific for the phosphorylated BACEl is synthesized by employing as an antigen an oligopeptide which include at the most 10 amino acids towards N- and C- terminals, respectively, centering on Thr²⁵² in the amino acid sequence of BACEl .

12. The method of claim 8, which further comprises the step of confirming whether the identified compound inhibits an increase in β-secretase activity or β-amyloid secretion.

13. A method for inhibiting BACEl phosphorylation in a mammal, comprising administering a compound of formula (I) or (II) to the mammal:

wherein,

R¹ is hydrogen, linear or branched Cj-C₄ alkoxy, or Ci-C₄ alkylformamide substituted with a C₃-Cg cycloalkyl group;

R² is hydrogen, or linear or branched C]-C₆ alkyl; and

R³ is hydrogen, linear or branched Ci-C₆ alkyl, or saturated or unsaturated 5- or 6-membered heterocyclic compound comprising 1 to 3 heteroatoms; or R² and R³ form, together with the carbon atoms where they are attached, a saturated or unsaturated 5- or 6-membered heterocyclic compound comprising 1 to 3 heteroatoms.

wherein,

R¹, R², R³, R⁶, R⁷ and R⁸ are each independently hydrogen, halogen, linear or branched Ci-C₆ alkyl, linear or branched C]-C_O alkyl substituted with halogen, linear or branched C]-C₆ alkoxy, or linear or branched C]-C₆ alkylester; and

R⁴ and R⁵ are each independently hydrogen, fluoro, chloro, or linear or branched C₁-C₄ alkyl.