WO2006075808A1 - Composition pharmaceutique comprenant un inhibiteur de p25/cdk5 permettant de prevenir ou de traiter une maladie neurodegenerative - Google Patents

Composition pharmaceutique comprenant un inhibiteur de p25/cdk5 permettant de prevenir ou de traiter une maladie neurodegenerative Download PDF

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WO2006075808A1
WO2006075808A1 PCT/KR2005/000098 KR2005000098W WO2006075808A1 WO 2006075808 A1 WO2006075808 A1 WO 2006075808A1 KR 2005000098 W KR2005000098 W KR 2005000098W WO 2006075808 A1 WO2006075808 A1 WO 2006075808A1
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bacel
branched
linear
phosphorylation
compound
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PCT/KR2005/000098
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English (en)
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Sul-Hee Chung
Ilho Ha
Mi-Young Son
Hye-Won Lee
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Inje University
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Priority to KR1020077017681A priority Critical patent/KR100931928B1/ko
Priority to PCT/KR2005/000098 priority patent/WO2006075808A1/fr
Publication of WO2006075808A1 publication Critical patent/WO2006075808A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound inhibiting a P25/CDK5 complex, and a method for screening the compound.
  • AD Alzheimer's Disease
  • 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.
  • 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.
  • AD Alzheimer's disease
  • 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.
  • a ⁇ ⁇ -amyloid
  • Such studies aim for developing drugs capable of inhibiting the activity of ⁇ - or ⁇ - secretase which mediates A ⁇ production, or degrading the produced A ⁇ .
  • an interference with innate proteins in the human body may adversely affect normal physiological functions and cause undesirable side effects.
  • 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.
  • CDK5 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 Muncl ⁇ , APP, P35, PAKl, Tau, ⁇ -catenin, Nudel, DARPP32, Amphyphysinl and Synapsinl (Dhavan, R. and Tsai, L.H., Nature Review Molecular Cell Biology 2: 749-759 (2001)).
  • a ⁇ associated with a senile plaque in AD, can trigger a cleavage of P35 into P25 (Lee M.S. et al, supra).
  • P25 promotes enhanced A ⁇ secretion level (Lie, F. et al, FEBS Letters 547: 193-196 (2003)).
  • P25 exists as a molecular link between the amyloid plaque and the neurofibrillary tangles.
  • a P25/CDK5 complex has been confirmed to participate in neurodegeneration events including cytoskeletal collapse and neuronal death (Patrick, GN. et al, supra).
  • BACEl beta-site APP (amyloid precursor protein)-cleaving enzyme 1) protein, which is a protease involved in A ⁇ 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 ⁇ -secretase activity and A ⁇ secretion, and have accomplished the present invention by screening P25/CDK5 inhibitors and confirming that the inhibitors successfully inhibited phosphorylation of BACEl and A ⁇ secretion.
  • composition for preventing or treating a neurodegenerative disease which comprises a P25/CDK5 inhibitor of formula (I) or (II) as an active ingredient.
  • R 1 is hydrogen, linear or branched C]-C 4 alkoxy, or C]-C 4 alkylformamide substituted with a C 3 ⁇ Cg cycloalkyl group;
  • R 2 is hydrogen, or linear or branched C]-C 6 alkyl
  • R 3 is hydrogen, linear or branched C]-C 6 alkyl, or saturated or unsaturated 5- or 6-membered heterocyclic compound comprising 1 to 3 heteroatoms; or
  • R 2 and R 3 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.
  • R 1 , R 2 , R 3 , R 6 , R 7 and R 8 are each independently hydrogen, halogen, linear or branched C]-C 6 alkyl, linear or branched C]-C 6 alkyl substituted with halogen, linear or branched C]-C 6 alkoxy, or linear or branched C]-C 6 alkylester; and
  • R 4 and R 5 are each independently hydrogen, fluoro, chloro, or linear or branched C , -C 4 alkyl .
  • 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.
  • 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.
  • 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 Thr 252 ;
  • Fig. 1C Immunoblotting result showing that the phosphorylation of
  • Fig. 2A Graph demonstrating an increased ⁇ -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 ⁇ -secretase activity in SK-N-BE2C cells;
  • Fig. 3 A Graph depicting an increased ⁇ -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 te t. off cells, P25-3 and P25-5, which show stable expressions of P25;
  • Fig. 3C Graph showing the amount of A ⁇ 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 ⁇ -secretase activity in hippocampus, cortex and cerebellum of a wild-type mouse and a P25-expressing transgenic mouse;
  • Fig. 4E Graph showing A ⁇ 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 A ⁇ 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;
  • Fig. 8B Graphs showing the effects of DSS30, DSS36 and their analogues on inhibiting A ⁇ secretion in P25-3 cells.
  • the term "degenerative disease” refers to a disease related to the accumulation of neurofibrillary tangles, P25 or ⁇ -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 R 1 is hydrogen, methoxy or cyclopropylmethylformamide; R 2 is hydrogen; and R 3 is piperidine; or R 2 and R 3 form a pyrrole or 1,4-dioxane ring, together with carbon atoms where they are attached.
  • 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 R 1 , R 2 , and R 3 are each independently hydrogen, halogen, linear or branched C 1 -C 4 alkoxy, or linear or branched C 1 -C 4 alkylester; R 4 , and R 5 are each independently hydrogen, or linear or branched C 1 -C 4 alkyl; R 6 , R 7 and R 8 are each independently hydrogen, linear or branched C]-C 4 alkyl optionally substituted with halogen, or linear or branched Ci-C 4 alkylester.
  • 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.
  • 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 Thr 252 in amino acid sequence of BACEl (SEQ ID NO: 6).
  • an antibody prepared by employing peptide SLWY Thr 252 (PO 4 )PIRR (SEQ ID NO: 2) as an antigen is preferred.
  • 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
  • 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.
  • the P25/CDK5 inhibitors of the present invention i.e., DSS303 and DSS36, inhibited A ⁇ secretion by more than 50 percent, as well as BACEl phosphorylation ⁇ see Figs. 8A and 8B).
  • 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.
  • 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 A ⁇ 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.
  • NFT neurofibrillary tangle
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kinase buffer (20 mM MOPS (pH 7.0), 10 mM MgCl 2 , 1 mM DTT, 20 ⁇ M sodium orthovanadate), 100 ⁇ M ATP and 5 ⁇ Ci [ ⁇ - 32 P]ATP, and the resulting mixture was adjusted to 30 ⁇ l with ddH 2 O.
  • the mixture was reacted for 1 hour at 30°C, and the reaction was completed by adding 7.5 ⁇ l of 5 ⁇ sample buffer (60 mM Tris-HCl (pH 6.8), 25 % glycerol, 2 % SDS, 14.4 mM 2- mercaptoethanol, and 0.1 % bromophenol blue).
  • 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 80 0 C, 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 .
  • An antibody against a phosphorylated peptide SLWYThr 252 (PO 4 )PIRR was manufactured by Peptron Inc. (Korea) as follows. 2 mg of the said phosphorylated peptide was conjugated with KLH (key-hole limpet hemocyanin). 400 ⁇ g 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.
  • KLH key-hole limpet hemocyanin
  • 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.
  • 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-specif ⁇ c BACEl antibody.
  • Step 2 Transient transfection of HEK293 cells and immunoblotting
  • DMEM Gibco-BRL, Invitrogen Corp.
  • FBS Gibco-BRL, Invitrogen Corp.
  • 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 ⁇ g DNA.
  • the cells were cultured at 37°C with the supply of 5 % CO 2 for 24 hr and were washed once with Ix PBS (80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , and 100 mM NaCl, pH 7.4).
  • 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 ⁇ g 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 Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.4, and 0.1 % Tween ® 20) containing 10 % skim milk.
  • PBST 80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.4, and 0.1 % Tween ® 20
  • the membrane was washed three times with PBST for 15 min each. Thereafter, a solution containing the above phosphospecif ⁇ c-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).
  • kinase buffer (20 mM MOPS (pH 7.0), 10 mM MgCl 2 , 1 mM DTT, 20 ⁇ M sodium orthovanadate) and 200 ⁇ M ATP, and the mixture was adjusted to 30 ⁇ l with ddH 2 O.
  • the mixture was reacted at 3O 0 C for 0, 10, 20, 60, 120, and 180 min, respectively, and the reaction was completed by the addition of 7.5 ⁇ l 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).
  • 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.
  • 50 ⁇ M 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.
  • 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.
  • 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 Thr 252 .
  • Thr 252 has been found to be located near the active site of the enzyme. Therefore, it is expected Thr 252 can have an influence on the enzyme activity.
  • the following study was undertaken to investigate whether phosphorylation of BACEl at Thr 252 residue by CDK5 affects the activity of ⁇ -secretase.
  • BACEl In order to phosphorylate BACEl, 2.2 ⁇ g of BACEl was added to 50 ng of purified P25/CDK5 complex, kinase buffer and 50 ⁇ M of ATP. The mixture was adjusted to 30 ⁇ l with adding ddH 2 O and reacted for 2 hr at 30°C.
  • SK-N-BE2C cells (ATCC, CRL-2268) were cultured in DMEM/F-12 medium containing 10 % FBS with a supply of 5 % CO 2 at 37 0 C.
  • the control DNA pcDNAmychis
  • BACEl cDNA or BACE1T252A was transiently transfected to SK-N-BE2C cell and a cell lysate was obtained therefrom.
  • 50 ⁇ g 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 ⁇ l.
  • 2 ⁇ l of BACEl substrate (2 mM, R&D Systems Inc.) was added to the mixture, and ⁇ -secretase activity of the resulting mixture was measured by a fluorometer, as mentioned above in section (2-1).
  • SK-N-BE2C cells (ATCC, CRL-2268) were cultured in DMEM/F-12 medium containing 10 % FBS with a supply of 5 % CO 2 at 37 0 C.
  • 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.
  • 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.
  • 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 ⁇ g/ml of G418 with a supply of 5 % CO 2 at 37 0 C.
  • RPMI 1640 medium Gibco-BRL, Invitrogen Corp.
  • HS Gibco- BRL, Invitrogen Corp.
  • P25 cDNA synthesized in section (3-1) was subcloned into pTRE2pur vector (Clontech Lab., Inc.) to obtain pTRE2puro-P25 cDNA.
  • Ix 10 6 cells/well of PC12 tet-Off cells were incubated in 6-well plate for a day and a mixture of 4 ⁇ g of pTRE2puro-P25 cDNA and 12 ⁇ l 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 ⁇ g/ml G418.
  • the medium was again replaced by RPMI 1640 medium containing 100 ⁇ g/ml of G418 (Clontech Lab., Inc.), 1 ⁇ g/ml of doxycycline (Clontech Lab., Inc.) and 3 ⁇ g/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.
  • Step 2 Western analysis of BACEl phosphorylation in P25 stable cells
  • 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.
  • 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.
  • 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).
  • 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 10 6 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 A ⁇ amount thereof was measured by ELISA using an A ⁇ assay kit (IBL Co., Ltd.) according to the user's manual, as follows.
  • 100 ⁇ l of the culture medium from P25 stable cell line or control cell line or a standard human A ⁇ was added to the wells of a 96-well plate pre-coated with antibody (anti-human A ⁇ (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 ⁇ l each of anti-human A ⁇ ( 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.
  • TMB Tetra methyl Benzidine
  • stop solution (1 N H 2 SO 4
  • Amount of A ⁇ was calculated by substituting into the graph drawn on the basis of the standard.
  • the remaining cells were subjected to lysis by adding 120 ⁇ l 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 A ⁇ (pg)/total protein(mg).
  • lysis buffer containing 1 mM PMSF and protease inhibitor cocktail (Calbiochem CN Biosciences, Inc.)
  • Step 1 Verification of the BACEl phosphorylation in a transgenic mouse expressing P25 by immunohistochemistry
  • 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 -2O 0 C.
  • Slices with the thickness of 16 ⁇ m were prepared using Cryostat (Microm, HM 505 N) at -20 0 C and placed on Superfrost * /Plus slide (Fisher Scientific International). The slide was air-dried for 30 min at RT and stored at -2O 0 C.
  • Step 2 Analysis of BACEl phosphorylation, ⁇ -secretase activity and A ⁇ 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 ⁇ l of RIPA buffer (1 % NP-
  • the lysate was centrifuged at 2O 0 C, 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.).
  • a ⁇ assay was performed as described in Step 3 of section (3-2) by employing 50 or 100 ⁇ g of the proteins from each tissue, thereby determining the A ⁇ amount.
  • 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.
  • Example 1 a novel pathway, wherein BACEl phosphorylation by P25/CDK5 increases ⁇ -secretase activity, thereby resulting in the increase of A ⁇ amount, has been confirmed through in vitro as well as in vivo experiments ⁇ see Fig. 5).
  • DSS30 and its analogues 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 ⁇ M and 0.5 %, respectively.
  • DMSO Sigma-Aldrich, Inc.
  • 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.
  • P-Thr-Pro antibody detected only phosphorylated form of histone Hl and, when roscovitine was treated, the phosphorylation of histone Hl was inhibited.
  • P-Thr-Pro antibody seems to be appropriate in detecting the phosphorylation of histone Hl by the P25/CDK5 complex.
  • 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 ⁇ l 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 ⁇ l 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 ⁇ l of the resulting dilution was added to each well.
  • each well was washed four times with 200 ⁇ l of PBST followed by the addition of 200 ⁇ l 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
  • DSS30 and DSS36 inhibited the activity of P25/CDK5 complex up to 50 %, at a concentration of 50 ⁇ M.
  • four analogues of DSS30 exhibited similar or superior inhibitory activity as compared to DSS30.
  • DSS363 displayed more effective inhibition than DSS36.
  • 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 MgCl 2 , 1 mM DTT, and 20 ⁇ M sodium orthovanadate) and 200 ⁇ M ATP were mixed together, and the mixture was adjusted to 30 ⁇ l with ddH 2 O and then incubated at 30°C for 90 min.
  • kinase buffer (20 mM MOPS (pH 7.0)
  • 10 mM MgCl 2 10 mM MgCl 2
  • 1 mM DTT 1 mM DTT
  • 20 ⁇ M sodium orthovanadate 20 ⁇ M sodium orthovanadate
  • reaction was completed by adding 7.5 ⁇ l of 5 ⁇ sample buffer (60 mM Tris-HCl (pH 6.8), 25 % glycerol, 2 % SDS, 14.4 mM 2-mercaptoethanol, and 0.1 % bromophenol blue).
  • 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.
  • a test compound was added to the reaction mixture at a concentration of 50 ⁇ M, 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.
  • 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.
  • roscovitine inhibited BACEl phosphorylation.
  • all four analogues of DSS30 inhibited BACEl phosphorylation, wherein DSS301 and DSS303 showed the highest activity.
  • DSS36 also inhibited BACEl phosphorylation.
  • reaction was completed by adding 7.5 ⁇ l 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 ⁇ M, 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.
  • 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.
  • DSS30 inhibited Tau phosphorylation, wherein DSS303 showed the highest activity.
  • DSS36 and DSS363 also strongly inhibited Tau phosphorylation.
  • 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.
  • PC12 te t-o ff cell lines which stably expresses P25, western blotting for analyzing BACEl phosphorylation in P25 stable cells, and measurement of A ⁇ 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 10 6 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 ⁇ M and the cells were cultured for 8 hr. Culture solutions were collected and A ⁇ 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.
  • DSS36 and DSS30 inhibited BACEl phosphorylation by 25 % and 12 %, respectively.
  • DSS303 and DSS36 reduced A ⁇ 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 ⁇ M. Accordingly, in order to minimize the cell toxicity, the cells were treated with roscovitine only for 8 hr in the present example.
  • 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.

Abstract

L'invention concerne une composition pharmaceutique permettant de prévenir ou de traiter une maladie neurodégénérative, qui comprend un composé inhibant un complexe P25/CDK5(kinase 5-cycline-dépendante) comme ingrédient actif. L'invention concerne une méthode permettant de cribler un composé capable de prévenir ou de traiter une maladie neurodégénerative, qui consiste: a) à faire réagir un composé candidat avec un complexe P25/CDK5 et l'enzyme de clivage 1 (BACE1) d'APP (protéine précurseur amyloïde) en site bêta ou à traiter une cellule de mammifère exprimant P25 à l'aide du composé candidat, et à mesurer phosphorylation BACE1; et b) à comparer la phosphorylation mesurée de BACE1 avec celle d'un groupe témoin n'utilisant pas de composé candidat, et à identifier un composé réduisant la phosphorylation de BACE1 par rapport au groupe témoin. On peut utiliser le composé inhibant le complexe P25/CDK5 pour prévenir ou traiter une maladie neurodégénérative, notamment, la maladie d'Alzheimer du fait qu'il inhibe la phosphorylation de BACE1 et limite la sécrétion de β-amyloïde et l'hyperphosphorylation de Tau.
PCT/KR2005/000098 2005-01-12 2005-01-12 Composition pharmaceutique comprenant un inhibiteur de p25/cdk5 permettant de prevenir ou de traiter une maladie neurodegenerative WO2006075808A1 (fr)

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PCT/KR2005/000098 WO2006075808A1 (fr) 2005-01-12 2005-01-12 Composition pharmaceutique comprenant un inhibiteur de p25/cdk5 permettant de prevenir ou de traiter une maladie neurodegenerative

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US8728472B2 (en) 2009-06-03 2014-05-20 The Board Of Regents Of The University Of Texas System Antibodies that bind selectively to P25 and uses therefor
WO2016022465A1 (fr) * 2014-08-04 2016-02-11 Drexel University Nouveaux composés et procédés pour traiter ou atténuer un trouble ou une maladie à médiation par il-1r au moyen de ces composés
EP4061356A4 (fr) * 2019-11-19 2024-03-20 Nibn The Nat Institute For Biotechnology In The Negev Ltd Nouveaux dérivés de benzothiophène et leur utilisation pour stimuler le renouvellement mitochondrial

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KR101713421B1 (ko) * 2015-07-03 2017-03-09 울산대학교 산학협력단 뇌신경질환의 진단 또는 치료용 조성물

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Publication number Priority date Publication date Assignee Title
US8728472B2 (en) 2009-06-03 2014-05-20 The Board Of Regents Of The University Of Texas System Antibodies that bind selectively to P25 and uses therefor
WO2016022465A1 (fr) * 2014-08-04 2016-02-11 Drexel University Nouveaux composés et procédés pour traiter ou atténuer un trouble ou une maladie à médiation par il-1r au moyen de ces composés
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US11345696B2 (en) 2014-08-04 2022-05-31 Drexel University Compounds and methods of treating or ameliorating an IL-1R-mediated disease or disorder using same
EP4061356A4 (fr) * 2019-11-19 2024-03-20 Nibn The Nat Institute For Biotechnology In The Negev Ltd Nouveaux dérivés de benzothiophène et leur utilisation pour stimuler le renouvellement mitochondrial

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