Casein kinase 1delta (CK1delta) inhibitors

Abstract

The invention relates to pharmaceutical compositions having casein kinase 1 delta (CK1δ) inhibitors and to the use of the inhibitors in the treatment of neurodegenerative disorders such as Alzheimer's disease.

Claims

1. A method of treating Alzheimer's disease in a subject, comprising: administering a therapeutically effective amount of a pharmaceutical composition comprising a casein kinase delta (CK1d) inhibitor selected from any one of: 2-amino-3-[(thiophen-2-yl)carbonyl]indolizine-1-carboxamide (Compound 847), 2-amino-3-[(4-fluorophenyl)carbonyl]indolizine-1-carboxamide (Compound 987), 2-amino-3-benzoylindolizine-1-carboxanide (Compound 990), or 2-amino-1-[(4-fluorophenyl)carbonyl]-1H-indole-3-carboxamide (Compound 999), or a pharmaceutically acceptable salt or solvate thereof, to the subject; and treating Alzheimer's disease in the subject.

2. The method according to claim 1, wherein the casein kinase delta (CK1d) inhibitor is selected from any one of 2-amino-3-[(4-fluorophenyl)carbonyl]indolizine-1-carboxamide (Compound 987), 2-amino-1-[(4-fluorophenyl)carbonyl]-1H-indole-3-carboxamide (Compound 999), or a pharmaceutically acceptable salt or solvate thereof.

3. The method according to claim 1, wherein the casein kinase delta (CK1d) inhibitor is 2-amino-3-[(4-fluorophenyl)carbonyl]indolizine-1-carboxamide (Compound 987), or a pharmaceutically acceptable salt or solvate thereof.

4. The method of claim 1, wherein the pharmaceutical composition is a tablet, capsule, powder or liquid.

5. The method of claim 1, wherein the pharmaceutical composition is for oral administration.

6. The method of claim 1, wherein the pharmaceutical composition is for intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal administration.

7. The method of claim 1, wherein the pharmaceutical composition is a liquid.

8. The method of claim 1, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, carrier, buffer, or stabilizer.

9. The method of claim 1, wherein the pharmaceutical composition is a parenterally-acceptable aqueous solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effect of Compound 324 on the cell viability of SH-SY5Y-TMHT cells.

(2) FIG. 2 shows the effect of Compound 987 on the cell viability of SH-SY5Y-TMHT cells.

(3) FIG. 3 shows the effect of PF670462 on the cell viability of SH-SY5Y-TMHT cells.

(4) FIG. 4 including panels A and B shows reduction of phosphorylation of Serine 396. Panels A and B are sometimes referred to herein in the collective as FIG. 4.

(5) FIG. 5 shows the Western Blot measurement of pT231 (panel A) and total Tau (panel B) levels in SH-SY5Y-TMHT cells treated with selective CK1d inhibitors. Panels A and B are sometimes referred to herein in the collective as FIG. 5.

(6) In the present context, the term “pharmaceutically acceptable salt” is intended to indicate salts which are not harmful to the patient. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts and pharmaceutically acceptable alkaline addition salts. Acid addition salts include salts of inorganic acids as well as organic acids.

(7) Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.

(8) Representative examples of alkaline salts include, for example, sodium, potassium, lithium, calcium, magnesium or ammonium or organic bases such as, for example, methylamine, ethylamine, propylamine, trimethylamine, diethylamine, triethylamine, N,N-dimethylethanolamine, tris(hydroxymethyl)aminomethane, ethanolamine, pyridine, piperidine, piperazine, picoline, dicyclohexylamine, morpholine, benzylamine, procaine, lysine, arginine, histidine, N-methylglucamine.

(9) According to the invention, the compounds of formula (IB) can be in racemic forms, as well as in the form of pure enantiomers or non racemic (scalemic) mixture of enantiomers, including when the compounds of formula (IB) have more than one stereogenic centre. In case the compounds of formula (IB) have unsaturated carbon carbon double bonds, both the cis (Z) and trans (E) isomers and their mixtures belong to the invention.

(10) References herein to “halogen” means a fluorine, chlorine, bromine or iodine atom.

(11) References herein to “C.sub.1-6 alkyl” means any linear, branched hydrocarbon groups having 1 to 6 carbon atoms, or cyclic hydrocarbon groups having 3 to 6 carbon atoms. Representative examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, n-pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. References to “haloC.sub.1-6alkyl” mean a C.sub.1-6 alkyl group substituted by one or more halogen atoms as herein defined.

(12) References herein to “C.sub.1-6 alkylene” means a saturated divalent hydrocarbon chain having the specified number of member atoms. For example, C.sub.1-6 alkylene refers to a bond or an alkylene group having from 1 to 6 member atoms. Alkylene groups may be straight or branched. Representative branched alkylene groups have one or two branches. Alkylene includes methylene, ethylene, propylene (n-propylene and isopropylene) and butylene (n-butylene, isobutylene, and t-butylene).

(13) References herein to “C.sub.2-6 alkenyl” means any linear, branched hydrocarbon groups of 2 to 6 carbon atoms, or cyclic hydrocarbon group having 3 to 6 carbon atoms having at least one double bond. Representative examples of such alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl.

(14) References herein to “C.sub.2-6 alkynyl” means any linear, or branched hydrocarbon groups of 2 to 6 carbon atoms, having at least one triple bond. Representative examples of such alkynyl groups include ethynyl, propargyl and butynyl.

(15) References herein to ‘C.sub.1-6 alkoxy’ means an —O—C.sub.1-6 alkyl group wherein C.sub.1-6 alkyl is as defined herein. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy and the like.

(16) References herein to ‘C.sub.3-8 cycloalkyl’ means a saturated monocyclic hydrocarbon ring of 3 to 8 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl and the like.

(17) References herein to ‘aryl’ means a C.sub.6-12 monocyclic or bicyclic hydrocarbon ring wherein at least one ring is aromatic. Examples of such groups include phenyl, indyl or naphthyl and the like.

(18) References herein to “heteroatom” means a nitrogen, sulphur, or oxygen atom.

(19) References herein to “heterocyclyl” means a saturated or unsaturated non-aromatic ring containing from 1 to 4 heteroatoms as member atoms in the ring. Heterocyclyl groups containing more than one heteroatom may contain different heteroatoms. Heterocyclyl groups may be optionally substituted with one or more substituents as defined herein. Heterocyclyl groups are monocyclic ring systems or fused bicyclic or polycyclic ring systems or bicyclic structures known as heterocyclic “spiro” ring systems. In certain embodiments, heterocyclyl is saturated. In other embodiments, heterocyclyl is unsaturated and non-aromatic. Non-limiting examples of monocyclic heterocyclyl ring systems include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothienyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, morpholinyl, thiamorpholinyl, 1,3-dioxolanyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-oxathiolanyl, 1,3-oxathianyl, 1,3-dithianyl, and azetidinyl.

(20) References herein to “heteroaryl” means an aromatic ring containing from 1 to 4 heteroatoms as member atoms in the ring. Heteroaryl groups containing more than one heteroatom may contain different heteroatoms. Heteroaryl groups may be optionally substituted with one or more substituents as defined herein. Heteroaryl groups are monocyclic ring systems or are fused bicyclic or polycyclic ring systems. Monocyclic heteroaryl rings have 5 or 6 member atoms. Bicyclic heteroaryl rings have from 7 to 11 member atoms. Bicyclic heteroaryl rings include those rings wherein phenyl and a monocyclic heterocyclyl ring are attached forming a fused bicyclic ring system, and those rings wherein a monocyclic heteroaryl ring and a monocyclic cycloalkyl, cycloalkenyl, heterocyclyl, or heteroaryl ring are attached forming a fused bicyclic ring system. Non-limiting examples of heteroaryl includes pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furanyl, furazanyl, thienyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, tetrazinyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, pteridinyl, cinnolinyl, benzimidazolyl, benopyranyl, benzoxazolyl, benzofuranyl, isobenzofuranyl, benzothiazolyl, benzothienyl, furopyridinyl, and napthyridinyl.

(21) References herein to “heterocyclic ring system” mean either a heterocyclyl ring system or a heteroaryl ring system as herein before defined.

(22) Representative compounds of formula (IB) include the compounds as set forth below:

(23) TABLE-US-00001 Com- pound Num- ber Structure  2  3  26  27  28  30  31 0  32  33  35  47  48  51  54  57  58  59 0  60  63  64  78  84 113 123 127 128 129 0 145 155 156 157 171 172 173 204 206 207 0 210 225 227 233 235 236 241 242 244 249 0 269 285 288 303 307 308 309 310 311 312 0 314 315 316 320 324 325 333 336 351 357 0 358 359 360 373 374 375 384 385 386 387 0 388 389 390 391 396 399 400 401 402 404 0 405 407 408 409 410 411 414 424 425 427 00 428 01 437 02 448 03 456 04 457 05 458 06 482 07 484 08 485 09 489 0 490 491 495 496 497 498 505 507 516 519 0 524 526 553 559 560 568 570 575 585 590 0 594 596 597 601 602 609 615 616 618 626 0 627 638 649 653 669 692 693 694 703 705 0 709 712 716 719 725 734 738 740 746 749 0 753 754 756 758 759 767 770 777 778 784 0 785 790 792 796 800 801 804 805 808 819 0 821 827 828 831 833 838 844 847 857 858 0 869 872 875 877 891 910 912 926 933 952 00 955 01 962 02 963 03 969 04 987 05 990 06 999 07

(24) According to a further aspect of the invention, there is provided a compound of formula (IB) for use as a casein kinase 1 delta (CK1δ) inhibitor in the treatment of a neurodegenerative disorder, such as tauopathies.

(25) Compounds of formula 2-3, 26-28, 30-33, 35, 47-48, 51, 57-60, 63-64, 78, 84, 113, 123, 127-129, 145, 155-157, 171-173, 204, 206-207, 210, 225, 227, 233, 235-236, 241-242, 244, 249, 269, 285, 288, 303, 307-312, 314-316, 320, 324-325, 333, 336, 351, 357-360, 374-375, 384-391, 396, 399-402, 404-405, 407-411, 414, 424-425, 427-428, 437, 448, 456-457, 482, 484-485, 489-491, 495, 497-498, 505, 507, 516, 519, 524, 526, 553, 559-560, 568, 570, 575, 609, 615-616, 618, 626-627, 638, 653, 669, 692-694, 705, 709, 712, 716, 719, 725, 734, 738, 740, 746, 749, 753-754, 756, 758-759, 767, 770, 777, 784-785, 790, 792, 796, 800-801, 804-805, 808, 819, 821, 827-828, 831, 833, 838, 844, 847, 857-858, 869, 872, 875, 933, 952, 955, 969, 987, 990 or 999 are either commercially available or may be prepared in accordance with known synthetic procedures.

(26) According to a further aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (IB) for use in the treatment of a neurodegenerative disorder, such as tauopathies.

(27) The pharmaceutical compositions of the invention may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

(28) Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant.

(29) Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

(30) Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

(31) For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

(32) The compounds of formula (IB) are believed to be casein kinase 1 delta (CK1δ) inhibitors. Certain compounds of formula (IB) have inhibitory activity of greater than 5%, in particular greater than 10%, more particularly greater than 25%, yet more particularly greater than 50%, especially greater than 75%, such as greater than 90%. Such compounds may be useful in the treatment in neurodegenerative disorders such as tauopathies. Tauopathies are conditions which are characterised by neurofibrillary tangles or aggregates of the tau protein. Tauopathies are a recognised class of conditions known to those skilled in the art and include Alzheimer's disease, frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, corticobasal degeneration, multisystem atrophy (MSA), neurobasal degeneration with iron accumulation, type 1 (Hallervorden-Spatz), argyrophilic grain dementia, Down's syndrome, diffuse neurofibrillary tangles with calcification, dementia pugilistica, Gerstmann-Straussler-Scheinker disease, myotonic dystrophy, Niemann-Pick disease type C, progressive subcortical gliosis, prion protein cerebral amyloid angiopathy, tangle only dementia, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex, non-Guamanian motor neuron disease with neurofibrillary tangles/dementia, and Parkinson's disease. The intracellular tau deposits are usually neuronal or glial and are filamentous and generally in a hyperphosphorylated state as compared to the level of phosphorylation in tau from control human brain. In the case of AD, this hyperphosphorylated tau is often referred to a paired helical filament tau (PHF) tau because it is derived from the PHF. In one embodiment, the tauopathy comprises Alzheimer's disease.

(33) According to a further aspect of the invention, there is provided a method of treating a neurodegenerative disorder, such as tauopathies, which comprises administering a therapeutically effective amount of a compound of formula (IB).

(34) Biological Data

(35) 1. CK1δ Inhibition Assay

(36) The compounds of the invention may be tested for inhibition of casein kinase 1 delta (CK1δ) in accordance with the assay protocols described in US 2010/0152157, EP 1,636,375 or Hanger et al (2007) J. Biol. Chem. 282, 23645-23654. In particular, the assay was conducted in accordance with the following protocol:

(37) Reaction Buffer:

(38) Base Reaction buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 1% DMSO

(39) It should be noted that required cofactors are added individually to each kinase reaction.

(40) Reaction Procedure:

(41) 1. Prepare indicated substrate in freshly prepared Base Reaction Buffer as described above

(42) 2. Deliver any required cofactors to the substrate solution

(43) 3. Deliver indicated kinase into the substrate solution and gently mix

(44) 4. Deliver compounds in DMSO into the kinase reaction mixture

(45) 5. Deliver .sup.33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction

(46) 6. Incubate kinase reaction for 120 min. at room temperature

(47) 7. Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)

(48) 8. Wash filters extensively in 0.75% Phosphoric acid

(49) Kinase Information:

(50) CK1d—Genbank Accession #NP_620693

(51) Recombinant human full-length construct. GST-tagged, expressed in insect cells.

(52) Final concentration in assay=4 nM

(53) Substrate: CK1tide

(54) Substrate sequence: [KRRRAL[pS]VASLPGL]

(55) Final substrate concentration in assay=20 μM

(56) It should be noted that no additional cofactors are added to the reaction mixture.

(57) Compounds 30, 288, 314, 324-325, 336, 374, 391, 405, 615-616, 626, 705, 740, 753-754, 756, 759, 770, 784, 808, 819, 833, 844, 847, 869, 872, 875, 933, 952, 955, 969, 987, 990 and 999 were tested in the CK1δ inhibition assay and exhibited inhibition of greater than 5%.

(58) In particular, compounds 324-325, 405, 754, 847, 952, 987, 990 and 999 exhibited inhibition of greater than 50%.

(59) Yet more particularly, compounds 324, 952, 987, 990 and 999 exhibited inhibition of greater than 90%.

(60) 2. Measurement of Compound Effect on CK1d-Mediated Tau Phosphorylation

(61) The in vivo phosphorylation of Tau protein is complex with a number of putative protein kinases involved. It is widely accepted that the kinases GSK3b and CDK5 are significant players in the generation of PHF Tau, the pathogenic form found in neurofibrillary tangles in Alzheimer's disease. More recently, there has been growing evidence supporting the role of other kinases, particularly CK1δ in Tau hyperphosphorylation in vivo. Hanger et al. 2007 (J. Biol. Chem. 282, 23645-23654) identified 37 phosphorylation sites in human PHF Tau and were able to recapitulate these in vitro using recombinant tau and various purified kinase preparations. These studies identified that certain sites were uniquely phosphorylated by CK1δ and that certain other sites required CK1δ and another kinase with CK1δ providing upstream phosphorylation to render the targeted site available for the second kinase. Thus, to assess whether candidate compounds selective inhibit CK1δ activity either directly or through blocking its priming for other kinases a number of different screens have been developed. The general concept of these screens is provided in WO2005/001114.

(62) To measure the effect of putative CK1δ inhibitors on the levels of CK1δ-mediated phosphorylation selected-reaction monitoring assays were performed that provide quantitative relative measurement of phosphate group occupancy at specific sites in transgenic human and endogenous murine forms of Tau.

(63) The PhosphoTau SRM V2 assay measures total tau and relative phosphorylation levels at five of the most commonly studied sites on Tau and was obtained from Proteome Sciences plc (Cobham, England). None of the sites in the V2 assay is uniquely phosphorylated by CK1δ and there is a possibility that compound-induced inhibition of phosphorylation measured by this method may be achieved through promiscuous inhibition of other kinases such as GSK3b and/or CDK5. To address this limitation, Proteome Sciences has developed a V3 assay that measures total tau and two sites that are exclusively phosphorylated by CK1δ in addition to four others that have been shown to be phosphorylated in vitro by at least one other Tau kinase in addition to CK1δ. Table 1 lists the various sites covered and the candidate Tau kinases reported in Hanger et al. (2007).

(64) TABLE-US-00002 TABLE 1 Tau phosphorylation sites covered by Tau Phosphorylation SRM V2 and V3 assays Site number Candidate Kinases Assay V2 Ser181 GSK3b Ser199 CK2, GSK3b, PKA Thr231 GSK3b, PKA Ser262 CK1δ, GSK3b, PKA Ser396 CK1δ, CK2, GSK3b Assay V3 Ser46 CK1δ, GSK3b Thr50 CK1δ, GSK3b Ser113* CK1δ Ser396 CK1δ, CK2, GSK3b Ser404 CK1δ, CK2, GSK3b Ser433* CK1δ Numbering based on human 2N4R tau. *CK1d unique site
SH-SY5Y-TMHT Cell Line

(65) The SH-SY5Y-TMHT cell line (JSW Life Sciences, Graz, Austria) represents an in vitro model of tauopathy. The cell line is created by stably transfecting the human neuroblastoma derived SH-SY5Y cell line with a vector containing the full length human 2N4R Tau isoform which carries two common disease associated mutations (V337M/R406W). In recent studies (Flunkert et al. 2011 submitted, Loeffler et al. 2011 submitted) both the SH-SY5Y-TMHT cell line and a transgenic mouse line carrying the same human transgene were shown to express high levels of human Tau which becomes hyperphosphorylated at multiple epitopes previously demonstrated to be phosphorylated in various human tauopathies including Alzheimer's disease. Furthermore, in SH-SY5Y-TMHT cells exposed to different kinase inhibitors, including JNK-Inhibitor SP600125, and CK1 inhibitor IC261 levels of Tau phosphorylation at key pathogenic sites were reduced in patterns consistent with the known site-specificity of the targeted kinase. Thus, the SH-SY5Y-TMHT cell line is ideally suited to the screening of novel Tau kinase inhibitors.

(66) Compound Screening in SH-SY5Y-TMHT Cells

(67) SH-SY5Y-TMHT cells are kept in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine, 100 μg/ml Gentamycin, 300 μg/ml Geneticin G-418) for 2 days until 80-90% confluency. Cells are then differentiated in culture medium supplemented with 10 μM retinoic acid (RA) for 7 days changing medium every 2 to 3 days. Differentiated cells are seeded onto 6-well plates and 96-well plates at a cell density of 1.25×10.sup.6 and 8×10.sup.5 cells per well, respectively. On day 8 post-differentiation, the test compounds, reference compounds and vehicle control were added to the culture medium. After 6 h of compound exposure one plate of cells is subjected to a MTT assay to evaluate the effect of test and reference items on cell viability. Remaining wells are washed once with cold PBS and harvested in 300 μl RIPA-Buffer [50 mM Tris pH 7.4, 1% Nonident P40, 0.25% Na-deoxy-cholate, 150 mM NaCl, 1 mM EDTA, 1 μM NaF, 1 μM Na-ortho-vanadate, 80 mM Glycerophosphate, supplemented with freshly added protease (Calbiochem) and phosphatase (Sigma) inhibitor cocktail]. The cell suspension is transferred into a 1.5 ml tube and additionally lysed by sonication on ice. An aliquot of 20 μl is taken for the determination of the protein concentration (BCA assay). Subsequently, the lysates are snap frozen and stored at −80° C. until shipment.

(68) Two independent experiments in three (four) technical replicates are performed as depicted in Table 2.

(69) TABLE-US-00003 TABLE 2 Experiment Cells Treatment Concentration Evaluation ExpA SH- Vehicle — MTT BSA SY5Y- Compound 10-5-1-0.5-0.1- TauP TMHT 324 0.05 μM V2 Compound 10-5-1-0.5-0.1- TauP 987 0.05 μM V3 PF670462 1-0.5-0.1 μM ExpB SH- Vehicle — MTT BSA SY5Y Compound 10-5-1-0.5-0.1- TauP TMHT 324 0.05 μM V2 Compound 10-5-1-0.5-0.1- TauP 987 0.05 μM V3 PF670462 1-0.5-0.1 μM
Cell Viability Testing

(70) To determine compound activity, it is necessary to control for potential cell toxicity of all molecules. Viability of cultures is determined by the MTT assay. This assay allows the measurement of the mitochondrial dehydrogenase activity which reduces yellow MTT to dark blue formazan crystals. Since this reaction is catalyzed in living cells only this assay is used for the determination of cell viability. MTT solution is added to each well in a final concentration of 0.5 mg/ml. After 2 hours, the MTT containing medium is aspired. Cells are lysed in 3% SDS and the formazan crystals are dissolved in isopropanol/HCl. Optical density is measured with a plate-reader at wavelength 570 nm. Cell survival rate is expressed as optical density (OD). Values are calculated as percent of control values.

(71) Quantitative Determination of Total Protein Content

(72) Prior to assessment of specific Tau phosphorylation status the concentration of total protein in each cell lysate is determined using a standard BCA assay (Pierce Biotechnology, Rockford, USA). Briefly, 20 μl of cell lysate was used in the assay according to the manufacturer's instructions.

(73) Quantitative Determination of Total Tau & Phosphorylated Tau

(74) Mass Spectrometric Assays

(75) Total cell lysates from TMHT cell lines treated with Compound 324, Compound 987, PF670462 and relevant vehicle control respectively are first subjected to 1-dimensional SDS-PAGE to purify the protein fraction. Stacking gels are loaded with approximately 100 μg total protein based on BCA assay results. Gels are run until the total protein content forms a single discrete band in the stacking gel. Each protein band is then cut from the gel and digested with either trypsin or Asp-N and analysed using the PhosphoTau SRM assay V2 or V3 respectively. Each assay method quantifies the phosphorylation in pre-clinical material using a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Scientific, Hemel Hempstead, UK). Prior to SRM analysis phosphopeptides and pre-clinical samples were resolved by RP-chromatography (XBridge column, Waters, Manchester, UK) over a 9 minute gradient 0-30% ACN (buffer A; 0.1% FA, buffer B; ACN, 0.1% FA). Light and heavy versions for each peptide and phosphopeptide were monitored by several SRM transitions, using optimised S Lens values and collision energy settings. The area under the SRM LC peak was used to quantitate the amount of analyte present in each cell lysate as a single point reference to the signal of the heavy peptide spike. An 11 point calibration curve of light phosphopeptides with each point in the curve spiked with 100 fmol heavy phosphopeptides was also produced to determine assay characteristics (LOD, LOQ, precision and accuracy). For each specified tau population, the endogenous level of each tau phosphopeptide was quantified against its calibration curve (0.25-1000 fmol on column). Prior to LC-SRM analysis each tau population was spiked with 100 fmol of the heavy phosphopeptide standards. All data was processed using Pinpoint software (Thermo Scientific) and results reported as αg phospho-peptide/μg total protein.

(76) Western Blotting

(77) Lysates of treated cells were prepared in Laemmli buffer and 10 μg loaded into each lane of a 10% Nu-PAGE gel (Invitrogen, UK). Samples were run until the coomassie blue dye fromt was within 1 cm of the bottom of the gel. The separated proteins were transferred onto nitrocellulose and blots developed using antibodies specific for total tau (Polyclonal Rabbit Anti-Human Tau, Dako, UK (cat # A0024)) and phopho-Threonine 231 (Tau (Phospho-Thr231) Antibody, Signalway Antibody, USA (cat #11110)) respectively. In each case the bound antibody was detected using ECL Rabbit IgG, HRP-Linked (from donkey) (GE Healthcare, UK (cat # NA934))

(78) Results

(79) Effect of Test and Reference Compounds on the Cell Viability of SH-SY5Y-TMHT Cells

(80) Cell viability was determined in differentiated SH-SY5Y-TMHT cells by the MTT assay. Test and reference compounds were applied in a concentration range from 0.05 μM to 10 μM and from 0.1 μM to 1 μM, respectively. Upon 6 h of treatment, cell viability was evaluated. FIG. 1 shows the effect of Compound 324 on the cell viability of SH-SY5Y-TMHT cells wherein the graph represents effect of Compound 324 on cell viability of SH-SY5Y-TMHT cells in % of the vehicle control (VC, white bar). Statistical significance is indicated by *<0.05, **<0.01, ***<0.001 as determined by One-Way ANOVA. Data are shown from two independent experiments as group mean+/−SEM (n=8). It can be seen from FIG. 1 that Compound 324 exhibited a protective effect on the cell viability of SH-SY5Y-TMHT cells in a dose dependent manner although the effect was only statistically significant at a concentration of 10 μM. FIG. 2 shows the effect of Compound 987 on the cell viability of SH-SY5Y-TMHT cells wherein the graph represents effect of Compound 987 on cell viability of SH-SY5Y-TMHT cells in % of the vehicle control (VC, white bar). Statistical significance is indicated by *<0.05, **<0.01, ***<0.001 as determined by One-Way ANOVA. Data are shown from two independent experiments as group mean+/−SEM (n=8). It can be seen from FIG. 2 that Compound 987 decreased the cell viability in the lower and higher concentration range. At a concentration of 1 and 0.5 μM no cytotoxic effect was observed. FIG. 3 shows the effect of PF670462 on the cell viability of SH-SY5Y-TMHT cells wherein the graph represents effect of PF670462 on cell viability of SH-SY5Y-TMHT cells in % of the vehicle control (VC, white bar). Statistical significance is indicated by *<0.05, **<0.01, ***<0.001 as determined by One-Way ANOVA. Data are shown from two independent experiments as group mean+/−SEM (n=8). It can be seen from FIG. 3 that the reference compound PF 670462 only displayed a significant protective effect on the cell viability of SH-SY5Y-TMHT cells at a concentration of 0.5 μM.

(81) Protein Determination of SH-SY5Y-TMHT Cells Following Treatment

(82) Protein concentration of cell lysates of the treated SH-SY5Y-TMHT cells was determined using a standard BCA assay. Protein amount was determined from all samples in duplicates. The protein concentration of the samples was in the expected range according to the amount of cells seeded per 12-well plate ranging between 150-350 μg/ml.

(83) Determination of Compound Treatment Effect on Specific Phosphorylation Sites

(84) Mass Spectrometric Assay

(85) Testing of SH-SY5Y-TMHT cell lysates was performed using the PhosphoTau SRM assay V2 and V3. When the relative level of phosphorylation at each site is compared with the ratio in vehicle treated controls there was a distinct reduction in the level of phosphopeptide in cells treated with Compound 324 (data shown for 10 μM) and Compound 987 (data shown for 10 μM). An example showing reduction of phosphorylation on Serine 396 is shown in FIG. 4. This Figure shows mass spectrometric determination of CK1d-selective compounds on phosphorylation of Serine 396 in SH-SY5Y-TMHT cells. Panel A shows cells treated with Vehicle Control (VC) or Compound 324 (T.I.1_10 μM) and Panel B shows cells treated with Vehicle Control (VC) or Compound 987 (T.I.2_10 μM).

(86) In cells exposed to the vehicle control approximately 83% of Tau is phosphorylated at 5396. Treatment with 10 μM Compound 324 reduced this to 38% whilst 10 μM Compound 987 reduced pS396 levels to 24%. These results confirm the inhibition of pS396 by CK1d selective reagents.

(87) Western Blot Assay

(88) Levels of total Tau and Tau phosphorylated at Threonine 231 in SH-SY5Y-TMHT cell lysates treated with vehicle control, Compound 394 (10 μM), Compound 987 (10 μM) and PF670462 (5 μM) were quantified by Western Blotting. FIG. 5 shows the Western Blot measurement of pT231 (panel A) and total Tau (panel B) levels in SH-SY5Y-TMHT cells treated with selective CK1d inhibitors. As shown in FIG. 5, all three compounds reduced the detectable level of pT231 in Tau protein whereas this epitope was strongly present in vehicle-treated cells. There was no significant difference in the detectable levels of total Tau between the preparations other than for the PF670462-treated lysate which appeared to contain marginally less total Tau than the others. These results confirm the inhibition of pT231 by CK1d selective reagents.