INHIBITORS AND THEIR USES

Abstract

The invention relates to inhibitors of CDK12 (cyclin-dependent kinase 12), and there use in the treatment or prevention of a disorder in a subject caused by the generation of repeat expansion transcripts.

Claims

1. An inhibitor for use in the treatment or prevention of a disorder in a subject caused by the generation of repeat expansion transcripts, wherein the inhibitor is an inhibitor of CDK12 (cyclin-dependent kinase 12).

2. The inhibitor for use according to claim 1, wherein the repeat expansion transcript results in the transcript being retained in the nucleus.

3. The inhibitor for use according to claim 1 or claim 2, wherein the disorder comprises any disorder selected from the group comprising Myotonic Dystrophy type 1, Myotonic Dystrophy type 2, Fragile X associated tremor/ataxia syndrome, amylotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9ORF72), Huntington's Disease like 2, Huntington's Disease, Spinocerebellar Ataxia Types 1, 2, 3, 6, 7, 8, 10, 31, 17, Dentatorubral-pallidoluysian atrophy and Spinal and Bulbar Muscular Atrophy.

4. The inhibitor for use according to any preceding claim, wherein the repeat expansion transcript comprises RNA from a CTG DNA repeat; a CCTG DNA repeat; a CGG DNA repeat; a GGGGCC DNA repeat; a ATTCT DNA repeat; a TGGAA DNA repeat or a CAG DNA repeat.

5. The inhibitor for use according to any preceding claim, wherein the inhibitor is specific for CDK12.

6. The inhibitor for use according to any preceding claim, wherein the inhibitor is not an inhibitor of CDK9 activity or availability.

7. The inhibitor for use according to any of claims 1 to 4, wherein the inhibitor comprises an inhibitor of CDK12 expression.

8. The inhibitor for use according to claim 7, wherein the inhibitor comprises an oligonucleotide capable of inhibiting CDK12 expression.

9. The inhibitor for use according to claim 8, wherein the oligonucleotide comprises a sequence substantially complementary to CDK12 mRNA transcript.

10. The inhibitor for use according to any of claims 1 to 6, wherein the inhibitor comprises a molecule capable of binding to CDK12 and/or capable of blocking binding of CDK12 to its target molecule.

11. The inhibitor for use according to any of claim 1 to 6 or 10, wherein the inhibitor comprises a molecule capable of preventing CDK12 binding to cyclin K.

12. The inhibitor for use according to any of claim 1 to 6, 10 or 11, wherein the inhibitor comprises a molecule capable of preventing CDK12 phosphorylating Ser2 on the c-terminal domain of RNA polymerase II.

13. The inhibitor for use according to any of claims 10 to 12, wherein the binding of the inhibitor to CDK12 is at, or adjacent to, the CDK12 active site, such that the active site is blocked.

14. The inhibitor for use according to any of claims 10 to 13, wherein the binding of the inhibitor to CDK12 is at amino acid position, 727-1020.

15. The inhibitor for use according to any of claims 10 to 14, wherein the binding of the inhibitor to CDK12 is at a C terminal domain extension that extends around the N and C terminal lobes and contacts bound ATP.

16. The inhibitor for use according to any of claims 10 to 15, wherein the binding of the inhibitor to CDK12 is at any one or more of the ATP contact residues selected from Thr737, Lys756, Glu814, Met816 and Asp819.

17. The inhibitor for use according to any of claims 1 to 6, or 10 to 16, wherein the inhibitor comprises a small molecule, oligonucleotide, peptide or protein capable of binding to CDK12.

18. The inhibitor for use according to any of claims 1 to 6, or 10 to 17, wherein the inhibitor comprises a pyrazolo[1,5b]pyridazine core structure, and is capable of inhibiting CDK12 activity.

19. The inhibitor for use according to any of claims 1 to 6, or 10 to 18, wherein the inhibitor comprises a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof: ##STR00019## wherein: R.sup.1 is H, OH, O, C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynl, C.sub.1-6haloalkyl, halogen, CN, OC.sub.1-6alkyl; R.sup.2 is H, OH, O, C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynl, C.sub.1-6haloalkyl, halogen, CN, OC.sub.1-6alkyl or a five or 6 membered cycloaryl, cycloalkyl or heterocycl having one, two or three heteroatoms selected from O, S and N, for example benzene, morpholinyl, piperidine, piperazine; R.sup.3 is C.sub.3-6cycloalkyl, for example cyclopropyl, ##STR00020## wherein: R.sup.4 is H, OH, O, C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynl, C.sub.1-6haloalkyl, halogen, CN, OC.sub.1-6alkyl; R.sup.5 is H; OH; O; C.sub.1-6alkyl; C.sub.2-6alkenyl; C.sub.2-6alkynl; C.sub.1-6haloalkyl; halogen; CN; OC.sub.1-6alkyl; -; C.sub.1-6alkyl-N-(X)(Y); a five or six membered cycloaryl, cycloalkyl or heterocycl having one, two or three heteroatoms selected from O, S and N and said cycloaryl, cycloalkyl or heterocycle being optionally substituted with a C.sub.1-3alkyl, for example N-methylpiperazinyl; or OC.sub.1-6alkyl-N(X)(Y); wherein X is H or C.sub.1-6alkyl, and Y is H or C.sub.1-6alkyl; and wherein the alkyl groups are optionally substituted by one or more OH groups; and R.sup.6 is H, OH, O, C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynl, C.sub.1-6haloalkyl, halogen, CN, OC.sub.1-6alkyl.

20. The inhibitor for use according to claim 19, wherein: R.sup.1 is H; R.sup.2 is H; R.sup.3 is C.sub.3-6cycloalkyl, for example cyclopropyl, ##STR00021## wherein: R.sup.4 is H, CN, OC.sub.1-6alkyl, C.sub.1-6haloalkyl; R.sup.5 is H; C.sub.1-6alkyl-N-(X)(Y); a five or six membered cycloaryl, cycloalkyl or heterocycl having one, two or three heteroatoms selected from O, S and N and said cycloaryl, cycloalkyl or heterocycle being optionally substituted with a C.sub.1-3alkyl, for example N-methylpiperazinyl, OC.sub.1-6alkyl-N(X)(Y); wherein X is H or C.sub.1-6alkyl, and Y is H or C.sub.1-6alkyl; and wherein the alkyl groups are optionally substituted by one or more OH groups; and R.sup.6 is H or OC.sub.1-6alkyl.

21. The inhibitor for use according to claim 19, wherein: R.sup.1 is H; R.sup.2 is H; R.sup.3 is cyclopropyl, ##STR00022## wherein: R.sup.4 is H, CN, OCH.sub.3, CF.sub.3; R.sup.5 is H, CH.sub.2N(CH.sub.3).sub.2, N-methylpiperazinyl, OCH.sub.2CH(OH)CH.sub.2N(CH.sub.3).sub.2; and R.sup.6 is H or OCH.sub.3.

22. The inhibitor for use according to any claim 19, wherein: R.sup.1 is H, R.sup.2 is H, R.sup.3 is cyclopropyl, ##STR00023## wherein: R4 is H; R5 is CH.sub.2NEt.sub.2, or OCH.sub.2CH(OH)CH.sub.2N(CH.sub.3).sub.2; and R6 is H; or R4 is CN, OCH.sub.3; R5 is H; and R6 is H; or R4 is CF.sub.3; R5 is N-methylpiperazinyl; and R6 is H; or R4 is OCH.sub.3; R5 is H; and R6 is OCH.sub.3.

23. The inhibitor for use according to claim 19, wherein the inhibitor of Formula (I) is of the following formula: ##STR00024## ##STR00025##

24. The inhibitor for use according to claims 1 to 6, or 10 to 16, wherein the inhibitor comprises a compound of Formula (X) or a pharmaceutically acceptable salt or solvate thereof: ##STR00026##

25. The inhibitor for use according to claims 1 to 6, or 10 to 16, wherein the inhibitor comprises a compound of Formula (XI) or a pharmaceutically acceptable salt or solvate thereof: ##STR00027##

26. The inhibitor for use according to claims 1 to 6, or 10 to 16, wherein the inhibitor comprises a compound of Formula (XII) or a pharmaceutically acceptable salt or solvate thereof: ##STR00028##

27. The inhibitor for use according to claims 1 to 6, or 10 to 16, wherein the inhibitor comprises a compound of Formula (XIII) or a pharmaceutically acceptable salt or solvate thereof: ##STR00029##

28. The inhibitor for use according to claims 1 to 6, or 10 to 16, wherein the inhibitor comprises a compound of Formula (XIV) or a pharmaceutically acceptable salt or solvate thereof: ##STR00030##

29. The inhibitor for use according to any preceding claim, wherein the inhibitor is administered, or arranged to be administered, in combination with at least one other therapeutic agent.

30. The inhibitor for use according to claim 29, wherein the one other therapeutic agent comprises an oligonucleotide, such as siRNA or miRNA or equivalents thereof; or wherein the at least one other therapeutic agent may comprise a small molecule, drug, pro-drug, peptide, protein, antibody, nucleotide or vaccine.

31. The inhibitor for use according to claim 29, wherein the at least one other therapeutic agent comprises a sodium channel blocker; a CNS stimulant drug; dehydroepiandrosterone (DHEA); creatine supplementation; mecasermin rinfabate (IPLEX, combination of recombinant insulin-like growth factor 1 and its binding protein, BP-3); pentamidine; a bisamidinium inhibitor; lomofugin; or dilomofungin; or combinations thereof.

32. The inhibitor for use according to any preceding claim, wherein the use is in combination with an oligonucleotide that targets the DMPK gene.

33. The inhibitor for use according to any preceding claim, wherein the inhibitor is administered, or arranged to be administered, intermittently.

34. Use of a CDK12 inhibitor in the preparation of a medicament for the treatment or prevention of a disorder caused by the generation of repeat expansion transcripts in a subject.

35. A method of treatment or prevention of a disorder caused by the generation of repeat expansion transcripts in a subject, comprising administering an inhibitor of CDK12 to the subject.

36. The method according to claim 35, wherein the method further comprises a subsequent administration of at least one other therapeutic agent.

37. A method of screening for a therapeutic agent for a disorder caused by the generation of repeat expansion transcripts comprising: providing a molecule for screening; detecting if the molecule inhibits the activity of CDK12, wherein detection of inhibition of CDK12 selects the molecule as a potential candidate therapeutic agent.

38. A composition comprising a CDK12 inhibitor and a pharmaceutically acceptable carrier.

39. The composition according to claim 38 further comprising at least one other therapeutic agent.

40. The inhibitor, method, use, or composition substantially as described herein, optionally with reference to the accompanying figures.

Description

[0102] There now follows by way of example only a detailed description of the present invention with reference to the following drawings.

[0103] FIG. 1: Screening the PKIS compound collection identified 6 inhibitors that reduce nuclear foci. (A-F) Six inhibitors cause reduced nuclear foci across an 11 point dilution. Graphs show percentage of nuclear foci relative to DMSO treated cells. All six compounds share a pyrazolo[1,5b]pyridazine core structure.

[0104] FIG. 2: Analysis of the PKIS inhibition profiles to identify common kinase targets. (A) Loading plot of 2-compound partial least squares model. Kinase activities correlating with the nuclear foci assay are labelled, with cyclin-dependent kinases highlighted with *. (B-G) Plots show the percentage inhibition of the kinase target at 0.1 M concentration of compounds, grouped according to activity in the nuclear foci assay, including the six active compounds. Compounds with less than 25% inhibitory active activity on the kinase are not included on the graphs.

[0105] FIG. 3: Focussed screening of CDK family inhibitors. 12 point dilution graphs plotting percentage of nuclear foci relative to DMSO treated cells for (A) SNS-032 (B) AT7519 (C) PD0332991 (D) R-roscovitine (E) dinaciclib (F) CDK9 inhibitor II.

[0106] FIG. 4: Chemoproteomics target deconvolution. IC50 values were generated by affinity capturing of kinases from K562 or A204 cell extract using beads derivatized with SNS-032, in the presence of different concentrations of free competing compound or vehicle (DMSO). pIC50 values are plotted against the pIC50 in the foci inhibition assay for each compound. A good correlation of kinase binding affinity with the inhibitory activity on foci is observed for CDK5, CDK7, CDK9, and CDK12. r: Pearson correlation coefficient; p: p-value (calculated probability).

[0107] FIG. 5: CDK9 and CDK12 protein expression in DM. Western blot of vastus lateralis muscle biopsy samples in non-DM and DM1 patients for (A) CDK9 and (B) CDK12. Both blots are normalised to -tubulin. (C) Histogram to quantify levels of CDK9 protein normalised to -tubulin. (D) Histogram to quantify levels of CDK12 protein normalised to -tubulin. (E) Immunohistochemistry of CDK12 in non-DM and DM1 fibroblast cells. (F) Quantification of the number of CDK12 nuclear granules. (G) Immunohistochemistry and in situ hybridisation shows co-localisation of repeat expansion foci with CDK12 nuclear granules (CDK12 in green/bright spots, repeat expansion RNA (Arrow)) (H) CDK12 protein knockdown by shRNA results in reduced CDK12 protein granules (green) and a subsequent reduction in CUG repeat expansion RNA foci (red/Arrow).

[0108] FIG. 6: Inhibitor treatment as a therapeutic for DM. (A) Ethidium bromide stained gel showing RT-PCR products from nuclear (N) and cytoplasmic (C) RNA fractions following amplification and BpmI digest of a fragment of DMPK. GAPDH is used as a loading control. (B) Histograms showing the relative proportions of nuclear mutant DMPK transcripts compared to wild type DMPK transcripts. The relative proportions of the mutant and wild type DMPK transcripts in DM1 fibroblast cells were assessed following treatment with dinaciclib (1 m) for 24 hours. (C-F) Histograms show percentage of cells in the population with 0, <2, <5 and 5+ foci per nucleus (C) Untreated DM1 cells. (D) DM1 cells treated with SNS-032 for 2 hours. (E) DM1 cells treated with SNS-032 for 2 hours with 48 hours recovery in growth media (F) DM1 cells treated with SNS-032 for 2 hours with 72 hours recovery in growth media.

[0109] FIG. 7: Inhibitor treatment in a DM1 mouse model (A) Combined myotonia grade scores in quadriceps, gastrocnemius and paraspinal muscles from HSALR mice following vehicle and dinaciclib compound treatment (n=6 per treatment group) (B) Myotonia grades by muscle type in dinaciclib and vehicle treated HSALR mice. (C) Ethidium bromide stained gel to assess ATP2A1 splice isoforms in quadriceps and gastrocnemius muscle samples from vehicle and dinaciclib treated mice (D) Histogram showing the relative proportion of exon 22 exclusion and inclusion in vehicle and dinaciclib treated HSALR mice (E-F) Laminin stain demonstrates a reduction in centralised nuclei in muscle fibres of dinaciclib treated mice compared to vehicle control animals.

[0110] FIG. 8: Screening the PKIS collection identifies the CMGC kinase family. Plots to analyse the PKIS inhibition profiles of kinases identified from the partial least squares model as possible cellular targets comparing active versus inactive compounds. Compounds with less than 25% inhibition are not included on the graphs.

[0111] FIG. 9: Kinobead profiling of 11 compounds. Kinobeads profiling of a set of 11 compounds which represent a range of activities in the nuclear foci assay. Target profiles were generated by adding each compound to K562 cell extract at a concentration of 2 M followed by incubation with kinobeads and quantification of bead-bound proteins. A: Values indicate target binding compared to a DMSO control where a value of 1 represents 100% binding and therefore no target inhibition and a value of 0 indicates 0% binding and 100% inhibition of the target kinase. B: Shows the structure of each compound tested.

[0112] FIG. 10: Comparison of protein binding profiles for immobilised inhibitors SNS-032 and AT7519.

[0113] FIG. 11: Examples of dose-response competition binding curves for different compound/target combinations. An affinity matrix was generated by immobilisation of SNS-032 to sepharose beads and affinity capturing was performed from K562- or A204 cell extract in the presence of vehicle (DMSO) or different concentrations of inhibitor as indicated. IC50 concentrations and inflection points of the dose-response competition curves are indicated by dotted lines.

[0114] FIG. 12: CDK12 protein knockdown by siRNA and shRNA. (A) CDK12 immunohistochemistry following knockdown with scrambled and CDK12 shRNA (B) Quantification of CDK12 protein granules within the nucleus in CDK12 shRNA cells shows a 56% reduction in CDK12 granule number compared to scrambled shRNA (C) In situ hybridization analysis following knockdown with scrambled and CDK12 shRNA (D) Quantification of CUG repeat RNA foci within the nucleus in CDK12 shRNA cells shows a 69% reduction compared to scrambled shRNA (E) Histogram show percentage of cells in the population with 0, 1-2, 3-4 and 5+ foci per nucleus, in scrambled and CDK12 shRNA treated cells. (F) CDK12 immunohistochemistry following knockdown with scrambled and CDK12 siRNA (G) Quantification of CDK12 granules within the nucleus in CDK12 siRNA cells shows a 47% reduction in CDK12 granule number compared to scrambled siRNA (H) siRNA validation by western blot analysis for CDK12 and -tubulin (I) Histogram of intensity scan of western blot analysis showed a 34% reduction in CDK12 protein compared to scrambled controls. Data normalized to -tubulin. (J) In situ hybridization analysis following knockdown with scrambled and CDK12 siRNA (K) Quantification of CUG repeat RNA foci within the nucleus in CDK12 siRNA cells shows a 46% reduction compared to scrambled siRNA (L) Histograms show percentage of cells in the population with 0, 1-2, 3-4 and 5+ foci per nucleus, in scrambled and CDK12 siRNA treated cells.

[0115] FIG. 13: IC50 values of previously reported CDK inhibitors.

[0116] FIG. 14: pIC50 values generated by affinity capturing with the SNS-032 affinity matrix in K562 cell extract for the different CDK inhibitors added to the cell extracts.

INTRODUCTION

[0117] Myotonic dystrophy is caused by a CTG repeat expansion within the 3 untranslated region of the DMPK gene, leading to the formation of distinct nuclear foci. The involvement of kinases has been linked to the pathophysiology of the condition but to date a definitive kinase target for drug development has not been identified. It has been observed herein that CDK12 is elevated in DM cell lines and in DM patient muscle biopsies. Repeat expansion transcripts accumulate at the periphery of nuclear speckles and CDK12 co-localises with these nuclear speckles. It has been found that inhibition of CDK12 leads to the dispersal of DM-associated nuclear foci and degradation of repeat expansion transcripts.

[0118] Results and Discussion

[0119] Screening the PKIS Collection

[0120] Using a previously reported assay the PKIS collection was screened for compounds that reduce nuclear foci and the compounds were then analysed for their known selectivity profiles to identify the common kinase targets (Ketley, A. et al. (2014). Hum Mol Genet, 23: 1551-1562). DM1 fibroblasts were treated with compounds in an 11 point dilution series from 2011M-19 nM for 24 hours. Following treatment, fluorescent in situ hybridisation was performed with a cy3 labelled CAG10 probe, to visualize nuclear foci and cells were analysed on a Molecular Devices plate reader with customised MetaExpress software (Ketley, A. et al. (2014). Hum Mol Genet, 23: 1551-1562). Compounds that reduced nuclear foci in a concentration dependent manner, compared to DMSO treated cells were identified and prioritized for further study. Six compounds that share a pyrazolo[1,5b]pyridazine core were found to reduce the number of nuclear foci following 24 hour treatment of DM cells (FIG. 1).

[0121] Target Deconvolution

[0122] The known selectivity profile of the six active compounds was examined to identify common kinase targets. The pIC50 values generated from the foci assays were compared to the compound inhibition profiles against 224 kinase targets (Drewry, D. H. et al. (2014). Current topics in medicinal chemistry, 14: 340-342). A partial least squares (PLS) model was used to cluster the data which suggested that the common target was likely to be a member of the CMGC (cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSKs) and CDK-like kinases) family (FIG. 2A). Indeed, these compounds were originally designed to target CDK family members (Stevens, K. L. et al. (2008). Bioorganic & medicinal chemistry letters, 18: 5758-5762) and the inhibitors active in the nuclear foci assay all showed significant activity across many members of the CDK family. The kinase inhibition profiles for the six hit compounds were compared to their activity in the nuclear foci assay, which suggested that CDK1, CDK2, CDK3, CDK5 and CDK6 are unlikely to be responsible for foci formation (FIG. 2B-G). Of the targets covered by the PKIS collection, CDK4 appeared more likely to play a role (FIG. 2E) but other CDK family members required consideration as they are absent from the PKIS annotations.

[0123] Focussed Screening of Known CDK Family Inhibitor Molecules

[0124] Next, additional small molecule CDK inhibitors with well-annotated selectivity profiles were tested (FIG. 13) in the nuclear foci assay. This subset of inhibitors displayed differential activities in the nuclear foci assay and a diverse range of potencies across the CDK family members (FIG. 3). To confirm involvement of the CDK family the kinobeads methodology was employed, which is based on sepharose beads derivatized with a combination of promiscuous kinase inhibitors (Bantscheff, M. et al. (2007). Nat Biotechnol, 25: 1035-1044; Werner, T. et al. (2012). Analytical chemistry, 84: 7188-7194; and Kruse, U. et al. (2011). Leukemia, 25: 89-100) to profile 10 compounds, which represent a range of activities in the nuclear foci assay. Target profiles were generated by adding each compound to K562 erythroleukemia cell extract at a concentration of 2 M, followed by incubation with two variations of kinobeads and quantification of bead-bound proteins. The first type of bead was developed for the profiling of tyrosine and serine/threonine kinases of the eukaryotic protein kinase family (Bantscheff, M. et al. (2007). Nat Biotechnol, 25: 1035-1044), whereas the second type of bead captures additional kinases from the P13K/lipid kinase family (Bergamini, G. et al. (2012). Nature chemical biology, 8: 576-582). The profiling results clearly indicated that the most active compounds in this subset exhibited shared activities only across the CDK family (FIG. 9A), consistent with the PKIS data.

[0125] To refine the possible target kinases, the IC50 values were compared for the nuclear foci active inhibitors and the nuclear foci inactive inhibitors. Small molecules SNS-032 (also known as BMS 387032) and AT-7519 demonstrate high activity in the nuclear foci assay and display the highest potency against CDK2 and CDK9 (FIGS. 3a and 3b and FIG. 13). However CDK2 cannot be responsible for nuclear foci reduction due to significant overlap in the IC50 values of active and inactive compounds against this target. Likewise CDK1, CDK4, CDK5, CDK6 and CDK7 can all be discounted for similar reasons (FIG. 13).

[0126] The analysis of this subset of known CDK inhibitor compounds confirms the results of the PKIS screen but raises the possibility that the target responsible for nuclear foci reduction is a less well described CDK family member. To investigate this possibility four additional CDK inhibitors, with a range of potencies, were tested to determine the potential involvement of CDK9 as a possible target (FIG. 3E-F and FIG. 13). Interestingly, dinaciclib and SNS-032 have the same IC50 value against CDK9, however, dinaciclib displays a significantly increased activity in the nuclear foci assay. Furthermore, CDK9 Inhibitor II (FIG. 3F), which is a specific but less potent CDK9 inhibitor, with an IC50 value of 350 nM, reduced nuclear foci at the highest concentration whereas two other compounds; PD0332991 and R-roscovitine, with comparable CDK9 IC50 values (400 nM and 500 nM, respectively) are inactive in the nuclear foci assay at the concentrations tested (FIGS. 3C and 3D). These data suggest an additional CDK family member as the target for nuclear foci reduction.

[0127] Chemoproteomics Target Resolution

[0128] To determine the specific CDKs responsible for the reduction of nuclear foci it was sought to expand the target coverage within the CDK family by the immobilization of two of the active compounds containing a suitable secondary amine; SNS-032 and AT7519 (FIG. 10). Beads derivatized with either compound showed good coverage of the CDK family, including family members which are not present in commercial kinase panels. For an in depth chemoproteomics study profiling inhibitor selectivity across the CDK family, dose-response competition-binding profiles were generated for all active and one of the inactive compounds (PD0332991) in K562 cells or in A204 rhabdomyosarcoma cells. To confirm that the K562, A204 and DM1 cell lines express the same kinases a whole proteome analysis was conducted. All CDK/PCTK proteins identified by profiling were also identified in the cells used for the phenotypic foci assay as follows:

[0129] CDK family member proteins identified by whole proteome analysis of DM fibroblasts (protein accession numbers for CDK family proteins).

[0130] CDK1: P06493

[0131] CDK2: CAA43807.1

[0132] CDK4: CAG47043

[0133] CDK5: CAG33322

[0134] CDK6: Q00534

[0135] CDK7: P50613

[0136] CDK9: AAF72183

[0137] CDK10: AAH25301

[0138] CDK12: Q9NYV4.2

[0139] CDK13: Q14004.2

[0140] PCTK1: Q00536

[0141] PCTK2: Q00537.2

[0142] The resulting dataset comprises IC50 curves for 12 CDK family kinases (FIG. 11, FIG. 14). The best correlation across the compound set of kinase IC50 values with the inhibitory activity on foci was observed for CDK12, followed by CDK9 (FIG. 4). The IC50 values for CDK9 and CDK12 are closely in line with their observed activity in the foci assay, whereas the IC50 values for CDK2 and CDK7 appear too low to implicate them as targets. Consistent with the previous analysis, presented in FIG. 3E, dinaciclib was the most active inhibitor against the CDK family, in particular for CDK12, suggesting it may be the most likely kinase target.

[0143] CDK12 in DM Pathogenesis

[0144] Thus far the experiments point to CDK12 as the most likely kinase to have an association with repeat expansion foci. To examine the potential involvement in DM pathogenesis, the endogenous levels of CDK12 were assessed, in addition to CDK9, in vastus lateralis muscle biopsy samples from four DM1 and four healthy volunteers using Western blots. No significant difference in CDK9 protein level compared to that in controls could be detected (FIGS. 5A and 5C). However, there was a clear increase in levels of CDK12 in DM biopsies versus those from healthy volunteers, with 48% more CDK12 protein detected in DM samples (p=0.0025) (FIGS. 5B and 5D).

[0145] To understand the relationship between nuclear foci and CDK12 in DM pathophysiology, the cellular location of the protein was established by immunohistochemistry in DM and non-DM fibroblasts. Consistent with previously published data in non-DM cells CDK12 was localised in the nucleus in granular structures in both DM and non-DM cells (Ko, T. K. et al. (2001). Journal of cell science, 114: 2591-2603) (FIG. 5E). Quantification of these structures in 400 cells showed that the number of granules was elevated in DM cells, with an average number of 18.74 (3.88), compared to 12.07 (2.27) in non-DM cells (p<0.0001) (FIG. 5f). The size of CDK12 granules was not significantly different in non-DM and DM cells. This increase in number is consistent with the increased overall levels of CDK12 protein detected by Western blot. Next immunohistochemistry of CDK12 was conducted followed by in situ hybridisation to detect the repeat expansion RNA foci. Co-localisation of the repeat expansion foci with CDK12 protein was found. Quantification using customised MetaXpress software revealed that 100% of repeat expansion foci co-localised with CDK12 protein (FIG. 5g). To understand the impact of increased CDK12 protein in DM and the relationship between CDK12 protein granules and repeat expansion foci shRNA and siRNA were used to reduce the number of CDK12 granules in DM cells. The effect on repeat expansion foci was then assessed. Following lentiviral infection expressing three shRNAs against CDK12 a 56% reduction in the number of CDK12 granules and a 69% reduction in repeat expansion foci compared to control cells was observed (FIG. 5H and FIG. 12A-E). This was verified by siRNA knockdown of CDK12 and quantification by Western blot analysis, which confirmed a 34% reduction in protein levels. This resulted in a 46% reduction in CDK12 protein granules and a 47% reduction in repeat expansion RNA foci indicating that specific inhibition of CDK12 and dissolution of CDK12 protein from nuclear granules leads to a dispersal of repeat expansion foci (FIG. 12F-L).

[0146] Inhibitor Treatment as a Therapeutic for DM

[0147] As an association between CDK12 and nuclear foci has been established, and nuclear foci comprise repeat expansion transcripts, it was sought to establish the effect of inhibitor treatment on the level of repeat expansion transcripts. For this an RT-PCR assay was employed that utilises a Bpml polymorphism to distinguish between wild-type and mutant DMPK transcripts (FIG. 6A) (Hamshere, M. G. et al. (1997). Pro Natl Acad Sci USA, 94: 7394-7399). Following treatment with dinaciclib, analysis of nuclear and cytoplasmic cell extracts showed that the repeat expansion transcripts were still retained within the nuclear fraction. However, quantification using Genescan analysis showed a 59% decrease in the relative proportion of repeat expansion transcripts compared to wild type transcripts in the nucleus (FIG. 6B). These data indicate that exposure to dinaciclib, leads to preferential loss of the repeat containing transcript, which in turn suggests that targeting CDK12 provides a viable option for DM treatment development.

[0148] Loss of the repeat transcript may result from incomplete transcription of the expanded transcript following inhibitor treatment or it may be due to CDK12 removal from the repeat expansion transcript with subsequent dissociation of the nuclear foci and degradation of the mutant transcript. If the latter is correct it would suggest that nuclear foci protect repeat expansion transcripts from degradation and once released, they may be vulnerable to cellular or targeted degradation. Thus, a possible two-hit therapy regime is proposed in which short treatment with a CDK12 inhibitor is used to disperse foci and expose repeat expansion transcripts to degradation via endogenous processes or by antisense oligonucleotides (Mulders, S. A. et al. (2009). Proc Natl Acad Sci USA, 106: 13915-13920).

[0149] As CDK12 is a transcription-regulating kinase associated with nuclear foci in DM cells, it was sought to establish the effect of inhibiting this target on the kinetics of nuclear foci formation and dispersal. To do this DM1 fibroblast cells were exposed to the two most potent foci reducing compounds, dinaciclib and SNS-032, for different lengths of time from 2 hours to 48 hours. Both compounds produced a significant reduction in foci but this was most rapid in the case of SNS-032, which was effective following just 2 hours of treatment. Continuous exposure to transcription-regulating inhibitors, would not be a viable therapy option for DM, thus the effect of short term treatment on nuclear foci was examined. DM1 fibroblasts were exposed to SNS-032 for 2 hours, after which time the cells were washed thoroughly and allowed to recover in complete growth media. Quantification of nuclear foci showed that 68% of untreated DM1 cells have more than 5 foci and only 5% have no detectable foci (FIG. 6C). When cells are treated with SNS-032 for 2 hours, with no recovery time, this distribution shifts to 28% of cells with more than 5 foci and 10% cells with no foci (FIG. 6D). However, following 48 and 72 hours of recovery following exposure to SNS-032, the proportion of cells without nuclear foci increases further to 14% and 36%, respectively (FIGS. 6E and 6F). Taken together this data suggests that a short (2 hr) treatment with inhibitor, followed by a prolonged (72 hr) recovery leads to a significant reduction in numbers of nuclear foci and a preferential reduction in mutant transcripts, and therefore that pulsatile treatment could be an efficacious approach to DM therapy.

[0150] To establish the in vivo effect of this inhibitor HSA.sup.LR mice were treated by intraperitoneal injection for a 28 day treatment period comprising 12 injections in total. Following inhibitor treatment the mice were analysed by EMG analysis to assess the functional effect on myotonia and demonstrated a significant improvement in the myotonia grade score across the four muscle types tested; quadriceps, gastrocnemius, tibialis anterior and the lumbar paraspinals (p=0.0021, n=6) (FIG. 7A-B). Molecular analysis demonstrated an improvement in the inclusion of exon 22 for the splice isoforms of ATP2A1 (FIG. 7C-D) and a reduction in the presence of centralised nuclei within muscle fibres following inhibitor treatment (FIG. 7E-F).

[0151] Materials and Methods

[0152] Cell Culture

[0153] Fibroblast cells were grown in Dulbecco's Modified Eagles Medium (DMEM) with penicillin and streptomycin, and 10% fetal calf serum (Sigma).

[0154] In Situ Hybridization Protocol

[0155] Cells were exposed to compounds for 24 hrs after which in situ hybridization was performed to identify foci using a Cy3 labelled (CAG).sub.10 probe. Plates were analysed on a Molecular Devices Micro High Content Imaging system, with nine fields imaged per well to give approximately 100 cells per well, per compound treatment. The nuclear area was identified by Hoechst stain and the number, size and intensity of foci was determined by scoring adjacent pixels that were 80 grayscales or more above background.

[0156] Preparation of Cell Extracts

[0157] K562 and A204 cells were obtained from ATCC and cultured in RPMI medium containing 10% FCS. Cells were expanded to 1,5106 cells/ml. A204 cells were cultured in McCoy's 5A medium containing 15% FCS. Cells were expanded to 100% confluency. Cells were harvested and subjected to 3 washes with ice-cold PBS. Aliquots were snap frozen in liquid nitrogen and stored at 80 C. Cell extracts were prepared as described (Bantscheff, M. et al. (2011). Nat Biotechnol, 29: 255-265).

[0158] Chemoproteomics

[0159] Affinity profiling was performed as described previously (Bantscheff, M. et al. (2007). Nat Biotechnol, 25: 1035-1044 and Bantscheff, M. et al. (2011). Nat Biotechnol, 29: 255-265). Sepharose beads were derivatized with SNS-032 at a concentration of 1 mM to generate a bead matrix, or Kinobeads were used as a matrix for profiling. Beads (35 l in case of Kinobeads or 5 l in case of SNS-032) were washed and equilibrated in lysis buffer at 4 C. for 1 h with 1 ml (5 mg) K562 cell extract, which was pre-incubated with compound or buffer. Beads were transferred to disposable columns (MoBiTec), washed extensively with lysis buffer and eluted with SDS sample buffer. Proteins were alkylated, separated on 4-12% NuPAGE (Invitrogen), stained with colloidal Coomassie, and quantified by isobaric mass tagging and LC-MS/MS.

[0160] Peptide and Protein Identification and Quantification

[0161] Sample preparation and labeling with TMT isobaric mass tags was performed essentially as described (Bantscheff, M. et al. (2011). Nat Biotechnol, 29: 255-265). For mass spectrometric analyses samples were dried in vacuo and resuspended in 0.1% formic acid in water and aliquots of the sample were injected into a nano-LC system coupled to a mass spectrometer: Eksigent 1D+ coupled to LTQ-OrbitrapXL mass spectrometer, Waters nanoAcquity coupled to Orbitrap Elite mass spectrometer, or Ultimate 3000 RSLC nano coupled to Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were separated on custom 50 cm75 M (internal diameter) reversed-phase columns (Reprosil) at 40 C. Gradient elution was performed from 3% acetonitrile to 40% acetonitrile in 0.1% formic acid over 120-270 min. LTQ-Orbitrap XL was operated with Xcalibur 2.0, Orbitrap Elite and Q Exactive instruments were operated with Xcalibur 2.2 software. Intact peptides were detected in the LTQ-OrbitrapXL/Orbitrap Elite at 30.000 resolution (measured at m/z=400), in the Q Exactive at 70.000 resolution (m/z=200). Internal calibration was performed with LTQ-OrbitrapXL using the ion signal from (Si(CH3)20)6H+ at m/z 445.120025. Data-dependent tandem mass spectra were generated for up to ten peptide precursors (LTQ-OrbitrapXL/Orbitrap Elite six precursor, Q Exactive ten) using a combined CID/HCD (LTQ-Orbitrap XL) approach or using HCD only (Orbitrap Elite/Q Exactive) at a resolution of 15.000/17.500. For CID up to 5,000 ions (LTQ-Orbitrap XL) were accumulated in the ion trap (maximum ion accumulation time=150 msec), for HCD up to 50.000 ions (LTQ-OrbitrapXL, maximum ion accumulation time=350 msec), up to 30.000 ions (Orbitrap Elite, maximum ion accumulation time=150 msec) and 1e6 ions (Q Exactive, maximum ion accumulation time=60 msec) were accumulated in the HCD cell. Mascot 2.3 and 2.4 (Matrix Science) was used for protein identification using 10 p.p.m. mass tolerance for peptide precursors and 0.6 Da (CID) or 20 mDa (HCD) tolerance for fragment ions. Carbamidomethylation of cysteine residues and TMT modification of lysine residues were set as fixed modifications and methionine oxidation, N-terminal acetylation of proteins and TMT modification of peptide N-termini were set as variable modifications. The search database consisted of a customized version of the International Protein Index database combined with a decoy version of this database created using a script supplied by Matrix Science. Criteria for protein quantification were: a minimum of 2 sequence assignments matching to unique peptides was required (FDR for quantified proteins <<0.1%), Mascot ion score >10, signal to background ratio of the precursor ion >4, signal to interference >0.5 (Savitski, M. M. et al. (2010). Journal of the American Society for Mass Spectrometry, 21: 1668-1679). Reporter ion intensities were multiplied with the ion accumulation time yielding an area value proportional to the number of reporter ions present in the mass analyser. Peptide fold changes were corrected for isotope purity as described and adjusted for interference caused by co-eluting nearly isobaric peaks as estimated by the signal-to-interference measure (Savitski. M. M. et al. (2013). Journal of proteome research, 12: 3586-3598). Protein quantification was achieved using a sum-based bootstrap algorithm (Savitski, M. M. (2011). Analytical chemistry, 83: 8959-8967).

[0162] Assay for Repeat Expansion Transcripts

[0163] Reverse transcription was performed using 1 g total RNA from compound-treated and untreated cells. PCR was carried out using 1/20 of the synthesized cDNA with primers N11, 5-CACTGTCGGACATTCGGGAAGGTGC and 133, 5GCTTGCACGTGTGGCTCAAGCAGCTG. For Genescan analysis primer N11 was labelled with FAM. Amplification was performed with a Tm of 58 C. The PCR product was subsequently heated to 95 C. for 2 minutes followed by cooling to 4 C. For Bpml restriction digestion analysis of DMPK PCR products, 8 l of PCR mixture was digested overnight with restriction enzyme Bpml (NEB) in a total reaction volume of 20 l at 37 C. The final products were analysed by electrophoresis at 90V with 3% agarose gels and the density of bands quantified using ImageJ software or by fragment analysis on an AB1377 sequencer followed by Genescan quantification.

[0164] Western Blots and Detection

[0165] Western blotting was performed using a commercial NuPage system (Invitrogen, UK) according to the manufacturer's instructions. The primary antibodies used in this study were human CDK9 (Abcam, 1:1000 dilution), human CDK12 (Abcam, 1:400 dilution), human -tubulin and human Lamin B (both obtained from Santa Cruz and used at dilutions of 1:500). Anti-mouse IgG-horseradish peroxidise (HRP) was used as the secondary antibody. ImageJ software was used for the quantification of bands on western blots.

[0166] Colocalisation Studies

[0167] Cells were grown on coverslips for 24 hours before being fixed and permeabilised with 50:50 ice cold acetone:methanol. Cells were blocked in 5% BSA with 5% sheep serum. Anti-CDK12 antibody (Abcam) was used at 1:1000 dilution at 4 C. overnight followed by staining with Alexafluor-488 anti-mouse secondary antibody (1:500). Cells were incubated in 4% PFA for 5 minutes, followed by 15 minutes in pre-hybridisation solution (40% formamide, 10% 20xSSC, 50% DEPC water) and incubated with a cy3 labelled CAGio probe overnight at 37 C. Coverslips were mounted on slides using Vectorshield Mounting Media with DAPI. Images were acquired using a Zeiss 710 confocal microscope and analysed using LSM image browser.

[0168] siRNA Synthesis

[0169] The siRNA oligonucleotides were synthesized on an ABI 394 DNA/RNA synthesizer. Columns (SynBase CPG 1000, RNA: 0.2 mol), standard 2-OTBDMS RNA-phosphoramidites and reagents for the synthesizer were purchased from Link Technologies Ltd., MeNH.sub.2 solution (33 wt. % in ethanol) was obtained from Fluka, NEt3.3HF, N-methylpyrrolidinone (NMP) were purchased from Aldrich, illustra Nap-10 columns were obtained from GE Healthcare Europe GmbH. Dichloromethane and acetonitrile were freshly distilled from CaH.sub.2 before use on the synthesizer.

[0170] The siRNA oligonucleotides were synthesized using a standard 0.2 M scale protocol, but with a 10 min coupling time for each nucleotide addition step. The polymer-bound oligoribonucleotide was transferred from the synthesis column to a 1.5 mL microfuge tube and suspended in MeNH.sub.2 solution (1 mL). The mixture was heated to 65 C. for 10 min, cooled to room temperature (water/icebath) and centrifuged for 1 min (10 000 g). The supernatant was separated from the CPG beads, the beads were washed with RNase free water (2>0.25 mL), all supernatants were combined and dried (2 h under nitrogen stream, then freeze dried). The oligoribonucleotide was resuspended in anhydrous NEt.sub.3.3HF/NEt.sub.3/NMP solution (250 l of a solution of 1.5 mL NMP, 750 l NEt3 and 1.0 mL NEt.sub.3.3HF), heated to 65 C. for 1.5 h, cooled to room temperature and quenched with 3M NaOAc solution (25 L). n-BuOH (1 mL) was added to the mixture, which was then thoroughly mixed, cooled to 70 C. for 1-2 h (dry ice) to encourage further precipitation and centrifuged for 30 min (4 C., 13 000 g). The supernatant was removed, the pellet washed with 70% EtOH (2500 L) and then dried in vacuo (30 min). The dry precipitate was dissolved in RNase free water (1 mL) and desalted using a Nap-10 column following the standard protocol. The resulting solution was freeze dried overnight leaving the oligoribonucleotide as a white foam/powder.

[0171] CDK12 siRNA Knockdown

[0172] Scrambled: 5 ACGUGACACGUUCGGAGAAUU and CDK12: 5 CGAAAUAAUGAUGUUGGCACCAGUU siRNA sequences. Cells were electroporated on day 1 and day 4 with 800 nM of scrambled or CDK12 siRNA using the Amaxa Nucleofector system. Cells were collected on day 7 for immunohistochemistry, in situ hybridisation and western blot analysis.

[0173] CDK12 shRNA Knockdown

[0174] Cells were plated at 40% confluency the day before infection in 96 well format. Lentiviral titre (SantaCruz sc-44343-V) was added at an MOI of 10 in 5 g/ml polybrene diluted in DMEM media. Cells were spin inoculated by centrifugation at 2500 rpm for 30 minutes. Following 24 hours incubation the virus was removed and replaced with fresh DMEM media. The infection was repeated on day 4 and cells were collected on day 7 for immunohistochemistry and in situ hybridisation analysis.

TABLE-US-00003 CDK12sequence 1020304050 MPNSERHGGKKDGSGGASGTLQPSSGGGSSNSRERHRLVSKHKRHKSKHS 60708090100 KDMGLVTPEAASLGTVIKPLVEYDDISSDSDTFSDDMAFKLDRRENDERR 110120130140150 GSDRSDRLHKHRHHQHRRSRDLLKAKQTEKEKSQEVSSKSGSMKDRISGS 160170180190200 SKRSNEETDDYGKAQVAKSSSKESRSSKLHKEKTRKERELKSGHKDRSKS 210220230240250 HRKRETPKSYKTVDSPKRRSRSPHRKWSDSSKQDDSPSGASYGQDYDLSP 260270280290300 SRSHTSSNYDSYKKSPGSTSRRQSVSPPYKEPSAYQSSTRSPSPYSRRQR 310320330340350 SVSPYSRRRSSSYERSGSYSGRSPSPYGRRRSSSPFLSKRSLSRSPLPSR 360370380390400 KSMKSRSRSPAYSRHSSSHSKKKRSSSRSRHSSISPVRLPLNSSLGAELS 410420430440450 RKKKERAAAAAAAKMDGKESKGSPVFLPRKENSSVEAKDSGLESKKLPRS 460470480490500 VKLEKSAPDTELVNVTHLNTEVKNSSDTGKVKLDENSEKHLVKDLKAQGT 510520530540550 RDSKPIALKEEIVTPKETETSEKETPPPLPTIASPPPPLPTTTPPPQTPP 560570580590600 LPPLPPIPALPQQPPLPPSQPAFSQVPASSTSTLPPSTHSKTSAVSSQAN 610620630640650 SQPPVQVSVKTQVSVTAAIPHLKTSTLPPLPLPPLLPGDDDMDSPKETLP 660670680690700 SKPVKKEKEQRTRHLLTDLPLPPELPGGDLSPPDSPEPKAITPPQQPYKK 710720730740750 RPKICCPRYGERRQTESDWGKRCVDKFDIIGIIGEGTYGQVYKAKDKDTG 760770780790800 ELVALKKVRLDNEKEGFPITAIREIKILRQLIHRSVVNMKEIVTDKQDAL 810820830840850 DFKKDKGAFYLVFEYMDHDLMGLLESGLVHFSEDHIKSFMKQLMEGLEYC 860870880890900 HKKNFLHRDIKCSNILLNNSGQIKLADFGLARLYNSEESRPYTNKVITLW 910920930940950 YRPPELLLGEERYTPAIDVWSCGCILGELFTKKPIFQANLELAQLELISR 9609709809901000 LCGSPCPAVWPDVIKLPYFNTMKPKKQYRRRLREEFSFIPSAALDLLDHM 10101020103010401050 LTLDPSKRCTAEQTLQSDFLKDVELSKMAPPDLPHWQDCHELWSKKRRRQ 10601070108010901100 RQSGVVVEEPPPSKTSRKETTSGTSTEPVKNSSPAPPQPAPGKVESGAGD 11101120113011401150 AIGLADITQQLNQSELAVLLNLLQSQTDLSIPQMAQLLNIHSNPEMQQQL 11601170118011901200 EALNQSISALTEATSQQQDSETMAPEESLKEAPSAPVILPSAEQTTLEAS 12101220123012401250 STPADMQNILAVLLSQLMKTQEPAGSLEENNSDKNSGPQGPRRTPTMPQE 12601270128012901300 EAAACPPHILPPEKRPPEPPGPPPPPPPPPLVEGDLSSAPQELNPAVTAA 13101320133013401350 LLQLLSQPEAEPPGHLPHEHQALRPMEYSTRPRPNRTYGNTDGPETGFSA 13601370138013901400 IDTDERNSGPALTESLVQTLVKNRTFSGSLSHLGESSSYQGTGSVQFPGD 14101420143014401450 QDLRFARVPLALHPVVGQPFLKAEGSSNSVVHAETKLQNYGELGPGTTGA 1460147014801490 SSSGAGLHWGGPTQSSAYGKLYRGPTRVPPRGGRGRGVPY

[0175] The kinase domain of CDK12 is from amino acid position, 727-1020 (underlined). CDK12 has an additional C terminal domain extension that extends around the N and C terminal lobes and contacts bound ATP (underlined and italics). This is unique to CDK12 and is not present in CDK9.

[0176] The contact residues with ATP are Thr737, Lys756, Glu814, Met816 and Asp819 (highlighted by bold type)

TABLE-US-00004 CDK12transcriptsequence (SEQIDNO:1) 1 gtgtgactgggtctgtgtgagggagagagtgtgtgtggtgtggaggtgaaacggaggcaa 61 gaaagggggctacctcaggagcgagggacaaagggggcgtgaggcacctaggccgcggca 121 ccccggcgacaggaagccgtcctgaaccgggctaccgggtaggggaagggcccgcgtagt 181 cctcgcagggccccagagctggagtcggctccacagccccgggccgtcggcttctcactt 241 cctggacctccccggcgcccgggcctgaggactggctcggcggagggagaagaggaaaca 301 gacttgagcagctccccgttgtctcgcaactccactgccgaggaactctcatttcttccc 361 tcgctccttcaccccccacctcatgtagaagggtgctgaggcgtcgggagggaggaggag 421 cctgggctaccgtccctgccctccccacccccttcccggggcgctttggtgggcgtggag 481 ttggggttgggggggtgggtgggggttgctttttggagtgctggggaacttttttccctt 541 cttcaggtcaggggaaagggaatgcccaattcagagagacatgggggcaagaaggacggg 601 agtggaggagcttctggaactttgcagccgtcatcgggaggcggcagctctaacagcaga 661 gagcgtcaccgcttggtatcgaagcacaagcggcataagtccaaacactccaaagacatg 721 gggttggtgacccccgaagcagcatccctgggcacagttatcaaacctttggtggagtat 781 gatgatatcagctctgattccgacaccttctccgatgacatggccttcaaactagaccga 841 agggagaacgacgaacgtcgtggatcagatcggagcgaccgcctgcacaaacatcgtcac 901 caccagcacaggcgttcccgggacttactaaaagctaaacagaccgaaaaagaaaaaagc 961 caagaagtctccagcaagtcgggatcgatgaaggaccggatatcgggaagttcaaagcgt 1021 tcgaatgaggagactgatgactatgggaaggcgcaggtagccaaaagcagcagcaaggaa 1081 tccaggtcatccaagctccacaaggagaagaccaggaaagaacgggagctgaagtctggg 1141 cacaaagaccggagtaaaagtcatcgaaaaagggaaacacccaaaagttacaaaacagtg 1201 gacagcccaaaacggagatccaggagcccccacaggaagtggtctgacagctccaaacaa 1261 gatgatagcccctcgggagcttcttatggccaagattatgaccttagtccctcacgatct 1321 catacctcgagcaattatgactcctacaagaaaagtcctggaagtacctcgagaaggcag 1381 tcggtcagtcccccttacaaggagccttcggcctaccagtccagcacccggtcaccgagc 1441 ccctacagtaggcgacagagatctgtcagtccctatagcaggagacggtcgtccagctac 1501 gaaagaagtggctcttacagcgggcgatcgcccagtccctatggtcgaaggcggtccagc 1561 agccctttcctgagcaagcggtctctgagtcggagtccactccccagtaggaaatccatg 1621 aagtccagaagtagaagtcctgcatattcaagacattcatcttctcatagtaaaaagaag 1681 agatccagttcacgcagtcgtcattccagtatctcacctgtcaggcttccacttaattcc 1741 agtctgggagctgaactcagtaggaaaaagaaggaaagagcagctgctgctgctgcagca 1801 aagatggatggaaaggagtccaagggttcacctgtatttttgcctagaaaagagaacagt 1861 tcagtagaggctaaggattcaggtttggagtctaaaaagttacccagaagtgtaaaattg 1921 gaaaaatctgccccagatactgaactggtgaatgtaacacatctaaacacagaggtaaaa 1981 aattcttcagatacagggaaagtaaagttggatgagaactccgagaagcatcttgttaaa 2041 gatttgaaagcacagggaacaagagactctaaacccatagcactgaaagaggagattgtt 2101 actccaaaggagacagaaacatcagaaaaggagacccctccacctcttcccacaattgct 2161 tctcccccaccccctctaccaactactacccctccacctcagacaccccctttgccacct 2221 ttgcctccaataccagctcttccacagcaaccacctctgcctccttctcagccagcattt 2281 agtcaggttcctgcttccagtacttcaactttgcccccttctactcactcaaagacatct 2341 gctgtgtcctctcaggcaaattctcagccccctgtacaggtttctgtgaagactcaagta 2401 tctgtaacagctgctattccacacctgaaaacttcaacgttgcctcctttgcccctccca 2461 cccttattacctggagatgatgacatggatagtccaaaagaaactcttccttcaaaacct 2521 gtgaagaaagagaaggaacagaggacacgtcacttactcacagaccttcctctccctcca 2581 gagctccctggtggagatctgtctcccccagactctccagaaccaaaggcaatcacacca 2641 cctcagcaaccatataaaaagagaccaaaaatttgttgtcctcgttatggagaaagaaga 2701 caaacagaaagcgactgggggaaacgctgtgtggacaagtttgacattattgggattatt 2761 ggagaaggaacctatggccaagtatataaagccaaggacaaagacacaggagaactagtg 2821 gctctgaagaaggtgagactagacaatgagaaagagggcttcccaatcacagccattcgt 2881 gaaatcaaaatccttcgtcagttaatccaccgaagtgttgttaacatgaaggaaattgtc 2941 acagataaacaagatgcactggatttcaagaaggacaaaggtgccttttaccttgtattt 3001 gagtatatggaccatgacttaatgggactgctagaatctggtttggtgcacttttctgag 3061 gaccatatcaagtcgttcatgaaacagctaatggaaggattggaatactgtcacaaaaag 3121 aatttcctgcatcgggatattaagtgttctaacattttgctgaataacagtgggcaaatc 3181 aaactagcagattttggacttgctcggctctataactctgaagagagtcgcccttacaca 3241 aacaaagtcattactttgtggtaccgacctccagaactactgctaggagaggaacgttac 3301 acaccagccatagatgtttggagctgtggatgtattcttggggaactattcacaaagaag 3361 cctatttttcaagccaatctggaactggctcagctagaactgatcagccgactttgtggt 3421 agcccttgtccagctgtgtggcctgatgttatcaaactgccctacttcaacaccatgaaa 3481 ccgaagaagcaatatcgaaggcgtctacgagaagaattctctttcattccttctgcagca 3541 cttgatttattggaccacatgctgacactagatcctagtaagcggtgcacagctgaacag 3601 accctacagagcgacttccttaaagatgtcgaactcagcaaaatggctcctccagacctc 3661 ccccactggcaggattgccatgagttgtggagtaagaaacggcgacgtcagcgacaaagt 3721 ggtgttgtagtcgaagagccacctccatccaaaacttctcgaaaagaaactacctcaggg 3781 acaagtactgagcctgtgaagaacagcagcccagcaccacctcagcctgctcctggcaag 3841 gtggagtctggggctggggatgcaataggccttgctgacatcacacaacagctgaatcaa 3901 agtgaattggcagtgttattaaacctgctgcagagccaaaccgacctgagcatccctcaa 3961 atggcacagctgcttaacatccactccaacccagagatgcagcagcagctggaagccctg 4021 aaccaatccatcagtgccctgacggaagctacttcccagcagcaggactcagagaccatg 4081 gccccagaggagtctttgaaggaagcaccctctgccccagtgatcctgccttcagcagaa 4141 cagacgacccttgaagcttcaagcacaccagctgacatgcagaatatattggcagttctc 4201 ttgagtcagctgatgaaaacccaagagccagcaggcagtctggaggaaaacaacagtgac 4261 aagaacagtgggccacaggggccccgaagaactcccacaatgccacaggaggaggcagca 4321 gcatgtcctcctcacattcttccaccagagaagaggccccctgagccccccggacctcca 4381 ccgccgccacctccaccccctctggttgaaggcgatctttccagcgccccccaggagttg 4441 aacccagccgtgacagccgccttgctgcaacttttatcccagcctgaagcagagcctcct 4501 ggccacctgccacatgagcaccaggccttgagaccaatggagtactccacccgaccccgt 4561 ccaaacaggacttatggaaacactgatgggcctgaaacagggttcagtgccattgacact 4621 gatgaacgaaactctggtccagccttgacagaatccttggtccagaccctggtgaagaac 4681 aggaccttctcaggctctctgagccaccttggggagtccagcagttaccagggcacaggg 4741 tcagtgcagtttccaggggaccaggacctccgttttgccagggtccccttagcgttacac 4801 ccggtggtcgggcaaccattcctgaaggctgagggaagcagcaattctgtggtacatgca 4861 gagaccaaattgcaaaactatggggagctggggccaggaaccactggggccagcagctca 4921 ggagcaggccttcactgggggggcccaactcagtcttctgcttatggaaaactctatcgg 4981 gggcctacaagagtcccaccaagagggggaagagggagaggagttccttactaacccaga 5041 gacttcagtgtcctgaaagattcctttcctatccatccttccatccagttctctgaatct 5101 ttaatgaaatcatttgccagagcgaggtaatcatctgcatttggctactgcaaagctgtc 5161 cgttgtattccttgctcacttgctactagcaggcgacttacgaaataatgatgttggcac 5221 cagttccccctggatgggctatagccagaacatttacttcaactctaccttagtagatac 5281 aagtagagaatatggagaggatcattacattgaaaagtaaatgttttattagttcattgc 5341 ctgcacttactgatcggaagagagaaagaacagtttcagtattgagatggctcaggagag 5401 gctctttgatttttaaagttttggggtgggggattgtgtgtggtttctttcttttgaatt 5461 ttaatttaggtgttttgggtttttttcctttaaagagaatagtgttcacaaaatttgagc 5521 tgctctttggcttttgctataagggaaacagagtggcctggctgatttgaataaatgttt 5581 ctttcctctccaccatctcacattttgcttttaagtgaacactttttccccattgagcat 5641 cttgaacatactttttttccaaataaattactcatccttaaagtttactccactttgaca 5701 aaagatacgcccttctccctgcacataaagcaggttgtagaacgtggcattcttgggcaa 5761 gtaggtagactttacccagtctctttccttttttgctgatgtgtgctctctctctctctt 5821 tctctctctctctctctctctctctctctctctctctctctgtctcgcttgctcgctctc 5881 gctgtttctctctctttgaggcatttgtttggaaaaaatcgttgagatgcccaagaacct 5941 gggataattctttactttttttgaaataaaggaaaggaaattcagactcttacattgttc 6001 tctgtaactcttcaattctaaaatgttttgttttttaaaccatgttctgatggggaagtt 6061 gatttgtaagtgtggacagcttggacattgctgctgagctgtggttagagatgatgcctc 6121 cattcctagagggctaataacagcatttagcatattgtttacacatatatttttatgtca 6181 aaaaaaaaacaaaaacctttcaaacagagcattgtgatattgtcaaagagaaaaacaaat 6241 cctgaagatacatggaaatgtaacctagtttagggtgggtatttttctgaagatacatca 6301 atacctgaccttttttaaaaaaataattttaaaacagcatactgtgaggaagaacagtat 6361 tgacatacccacatcccagcatgtgtaccctgccagttcttttagggatttttcctccaa 6421 agagatttggatttggttttggtaaaaggggttaaattgtgcttccaggcaagaactttg 6481 ccttatcataaacaggaaatgaaaaagggaagggctgtcaggatgggataatttgggagg 6541 cttctcattctggcttctatttctatgtgagtaccagcatatagagtgttttaaaaacag 6601 atacatgtcatataatttatctgcacagacttagaccttcaggaaacataggttaagccc 6661 ccttttacaaagaaaaagtaaacatacttcagcatcttggagggtagttttcaaaactca 6721 agtttcatgtttcaatgccaagttcttattttaaaaaataaaatctacttataagagaaa 6781 ggtgcattacttaaaaaaaaaaaactttaaagaaatgaaagaagaaccctcttcagatac 6841 ttacttgaagactgttttcccctgttaatgagatatagctagatatcggtgtgtgtattt 6901 ctttattattctctggtttttgatctggccttgcctccagggccaaacactgatttagaa 6961 agagagccttctagctattttggcattgatggctttttataccagtgtgtccagttagat 7021 ttactaggcttactgacatgctattggtaaatcgcattaaagttcatctgaaccttctgt 7081 ctgttgacttcttagtcctcagacatgggcctttgtgttttagaatatttgaatttgagt 7141 tattgggccccactccctgttttttattaaagaacgtgagcctgggatactttcagaagt 7201 atctgttcaatgaaaaaaagttggtttcccatcaaatatgaataaaattctctatatatt 7261 tcattgtattttggttatcagcagtcatcaataatgtttttccctcccctctcccacctc 7321 ttatttttaattatgccaaatatcctaaataatatacttaagcctccattccctcatccc 7381 tactagggaagggggtgagtgtatgtgtgagtgtatgtgtatgtatgatcccatctcacc 7441 cccacccccattttgggagtcttttaaaatgaaaacaaagtttggtagttttgactattt 7501 ctaaaagcagaggagaaaaaaaaacttatttaaatatcctggaatctgtatggaggaaga 7561 aaaggtatttgttaatttttcagttacgttatctataaacatgatggaagtaaaggtttg 7621 gcagaatttcaccttgactatttgaaaattacagacccaattaattccattcaaaagtgg 7681 ttttcgttttgttttaattattgtacaatgagagatattgtctattaaatacattatttt 7741 gaacagatgagaaatctgattctgttcatgagtgggaggcaaaactggtttgaccgtgat 7801 catttttgtggttttgaaaacaaatatacttgacccagtttccttagttttttcttcaac 7861 tgtccataggaacgataagtatttgaaagcaacatcaaatctatacgtttaaagcagggc 7921 agttagcacaaatttgcaagtagaacttctattagcttatgccatagacatcacccaacc 7981 acttgtatgtgtgtgtgtatatataatatgcatatatagttaccgtgctaaaatggttac 8041 cagcaggttttgagagagaatgctgcatcagaaaagtgtcagttgccacctcattctccc 8101 tgatttaggttcctgacactgattcctttctctctcgtttttgacccccattgggtgtat 8161 cttgtctatgtacagatattttgtaatatattaaatttttttctttcagtttataaaaat 8221 ggaaagtggagattggaaaattaaatatttcctgttactataccacttttgctccattgc 8281 att