WO2017182581A1 - A method for evaluating a candidate compound (t) for treating myotonic dystrophy type i (dm1) - Google Patents

Abstract

The invention relates to a method for evaluating a candidate compound (T) for treating Myotonic Dystrophy Type I (DM1). The disclosed method comprises the use a Muscleblind-Like Splicing Regulator 1 (MBNL1) polypeptide and a CUG_n RNA. The MBNL1 polypeptide is contacted with the CUG_n RNA in the presence of the candidate compound (T) to be evaluated. The inhibition of binding of the MBNL1 polypeptide to the CUG_n RNA by the candidate compound (T) is determined.

Description

A method for evaluating a candidate compound (T) for treating Myotonic Dystrophy Type I (DM1)

Description

The present invention relates to a method for evaluating a candidate compound (T) for treating nucleotide repeat diseases such as Myotonic Dystrophy Type I (DM1 ).

Myotonic dystrophy type I (DM1 ) is a disabling neuromuscular disease affecting multiple organ systems, predominantly skeletal muscle, with no causal treatment available. This disease is caused by expanded CTG triplet repeats in the 3' UTR of the Myotonic Dystrophy Protein Kinase (DMPK) gene, whereby the disease severity is roughly correlated to the repeat expansion size. On the RNA level these expanded CUG repeats (CUG_n) form hairpin structures, which lead to ribonuclear inclusions. More specifically, the RNA with expanded CUG repeats sequesters splicing-factors, such as muscleblind-like splicing regulator 1 (MBNL1 ). Lack of available MBNL1 leads to mis-regulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1 .

Myotonic dystrophy type I (DM1 ) is one of the most common neuromuscular diseases with relatively high prevalence numbers of about 1 :8'000 in the adult population. This autosomally dominant inherited disease affects multiple organs, most prominently the skeletal muscle, with wasting, weakness and an inability to relax (myotonia). Currently, there is no effective treatment for this disabling disease. The pathomechamism of DM1 is linked to a CTG_n expansion in the 3' UTR of the Myotonic Dystrophy Protein Kinase (DMPK) gene leading to a toxic gain-of-function RNA. The mutant DMPK transcript is entrapped within nuclei of affected cells, where it forms aggregates (foci) with splicing factors such as muscleblind-like splicing regulator 1 (MBNL1 ).

Bound to mutant DMPK CUG_n RNA, MBNL1 is no longer available for correct splicing of its target pre-mRNAs. Thus, the splicing of a multitude of pre-mRNAs is mis-regulated, including the skeletal muscle chloride channel (CLCN 1 ), the insulin receptor (INSR), sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 1 (SERCA1 ) and cardiac troponin T type 2 (TNNT2) pre-mRNA. Interestingly, the mis-splicing of some pre-mRNAs can directly be linked to a certain disease symptom, e.g. in the case of the CLCN 1 pre-mRNA. It is developmental^ regulated by MBNL1 ; after birth MBNL1 promotes the exclusion of the alternative exon 7a from the CLCN 1 pre-mRNA. Hence, MBNL1 sequestration by CUG_n RNA causes inclusion of exon 7a, leading to a shift in the open reading frame and to premature termination of translation. As a result of this, the functional CLCN 1 protein is decreased and the resting chloride conductance is reduced, which leads to myotonic discharges characteristic of DM1.

To date, most therapeutic strategies towards DM1 focused either on the development of agents degrading the toxic RNA or blocking its pathogenic interaction with proteins. Antisense oligonucleotides targeting the DMPK-CUG_n transcripts have been shown to reverse the toxic RNA effect in vitro and in vivo. Viral overexpression of MBNL1 also resulted in a reversal of the toxic RNA effect in vivo.

Compared to the antisense oligonucleotide and gene-therapy approaches, an advantage of a suitable small molecule drug is its potential to penetrate into all tissues affected in DM1 patients, as well as its likely oral bioavailability. A variety of small molecules have been described that interfere with the MBNL1-CUG_n RNA complex and improve DM1 associated molecular defects in vitro and in some cases also in vivo. Several approaches were successful in identifying small molecules, such as screening of known nucleic acid-binders, rational design of small molecules based on the structure of CUG_n RNA, rational design of oligomers of CUG_n RNA binders by modular assembly, combinatorial chemistry, and high throughput screening. Increasingly, molecules were identified that act on other targets than the MBNL1-CUGn RNA complex. These molecules include gene transcription modulators, kinase modulators, or Ras farnesyltransferase inhibitors.

Based on this background, it is the objective of the present invention to provide simple and efficient means for determining the therapeutic potential for Myotonic Dystrophy Type I (DM1 ) of a target compound.

According to a first aspect the invention relates to a method for evaluating a candidate compound (T) for treating Myotonic Dystrophy Type I (DM1 ) comprising the steps of:

a) providing a Muscleblind-Like Splicing Regulator 1 (MBNL1 ) polypeptide and a

b) contacting the MBNL1 polypeptide with the CUG_n RNA in the presence of the candidate compound (T) in a reaction volume;

c) determining the inhibition of binding of the MBNL1 polypeptide to the CUG_n RNA by the candidate compound (T).

In the context of the present specification, CUG_n refers to a triplet of nucleotides that is repeated n times. The repeats follow each other directly without any other nucleotides between the triplets. For example, CUG₃ would refer to the sequence CUGCUGCUG (SEQ ID No 36) and CUG₇₈ would consist of 78 CUG triplets.

In the context of the present specification, CTG_n refers to a triplet of nucleotides that is repeated n times. The repeats follow each other directly without any other nucleotides between the triplets. For example, CTG₃ would refer to the sequence CTGCTGCTG (SEQ ID No 38) and CTG₇₈ would consist of 78 CTG triplets

In some embodiments, the MBNL1 polypeptide has an identity of >85%, >90%, >95%, or >99% compared to MBNL1 of SEQ ID NO 01 or MBNL1 of SEQ ID NO 37 or compared to an ortholog polypeptide of SEQ ID NO 1 or SEQ ID NO 37. Particularly, the MBNL1 polypeptide is able to bind said CUG_n RNA, wherein particularly said MBNL1 polypeptide is characterized by a binding affinity of at least 80%, 85%, 90%, 95% or 99% compared to the MBNL1 Polypeptide of SEQ ID NO 01 or SEQ ID NO 37.

SEQ ID NO 01 :

MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKY LHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPY LGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAH PADSTMI DTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQAKIKAAQYQ

SEQ ID NO 37 (MBNL1 , homo sapiens, Q9NR56)

MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKY LHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPY LGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAH PADSTMI DTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMTQS AVKSLKRPLEATFDLGI PQAVLPPLPKRPALEKTNGATAVFNTGI FQYQQALANMQLQQHTAFLPPVP MVHGATPATVSAATTSATSVPFAATATANQI PI ISAEHLTSHKYVTQM

In the context of the present specifications the terms sequence identity, identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA.

Unless otherwise stated, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs using default parameters (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses default settings such as: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 1 1 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear.

In the context of the present specification, the term ortholog refers to a gene and its corresponding polypeptide that evolved by vertical descent from a single ancestral gene. In other words these genes/polypeptides share a common ancestor and were divided when a species diverged into two separate species. The copies of a single gene in the two resulting species are then referred to as orthologs. To ascertain that two genes are orthologs a person skilled in the art can carry out a phylogenetic analysis of the gene lineage by comparing the aligned nucleotide or amino acid sequences of genes or polypeptides.

In some embodiments, n of CUG_n is 13 to 500, in particular n is 35 to 400, more particular n is 50 to 350, most particular n is 78.

In some embodiments, the CUG_n NA is attached to a first label, in particular a first affinity label, a first fluorescent label or an AlphaScreen acceptor or donor bead, more particular an affinity label such as biotin.

In some embodiments, the CUG_n RNA is synthesized using modified nucleotides comprising a first label.

In some embodiments, the CUG_n RNA is synthesized using modified CTPs comprising a first label

In some embodiments, the CUG_n RNA is synthesized using modified UTPs or TTPs comprising a first label

In some embodiments, the CUG_n RNA is synthesized using a mixture of modified GTPs, CTPs, TTPs and/or UTPs comprising a first label.

In some embodiments, the CUG_n RNA is synthesized using a mixture of modified nucleotides comprising a first label and unmodified nucleotides.

In some embodiments, the CUG_n RNA is synthesized using a mixture of modified nucleotides comprising a first label and unmodified nucleotides with a ratio between 1 :2 to 1 :10.

In some embodiments, the CUG_n RNA is synthesized using a mixture of modified nucleotides comprising a first label and unmodified nucleotides with a ratio between 1 :3 to 1 :8.

In some embodiments, the CUG_n RNA is synthesized using a mixture of modified nucleotides a first label and unmodified nucleotides with a ratio between 1 :4 to 1 :6.

In some embodiments, the first label is an affinity label, particularly biotin. In some embodiments, the first label is a fluorescent dye, particularly the fluorescent dye acts as an acceptor or donor for time-resolved fluorescence energy transfer

In some embodiments, the first label is usable as an AlphaScreen donor bead or an AlphaScreen acceptor bead.

In the context of the present specification, the term "AlphaScreen" refers to an Amplified Luminescence Proximity Homogeneous Assay. An AlphaScreen assay essentially comprises two components, a donor bead and an acceptor bead. The donor bead comprises a photosensitive compound such as phtalocyanine that is able to release singlet oxygen after excitation by a specified wavelength of light. The acceptor bead comprises compounds such as thioxene, anthracene or rubrene that emits light of a shorter wave length after reaction with the released singlet oxygen. The acceptor bead has to be within a radius of approximately 200 nm around the donor bead to be able to efficiently react with the singlet oxygen provided by the donor bead. In consequence, the acceptor bead only emits light if it is in close proximity to the donor bead.

In some embodiments, a fluorescently labeled ligand, particularly a streptavidin comprising ligand is used to label the CUG_n RNA via its first label.

In some embodiments, the CUG_n RNA is immobilized on a surface in contact with said reaction volume, particularly via said first label.

In some embodiments, said MBNL1 polypeptide is attached to a second label, in particular a second affinity label, a fluorescent label or an AlphaScreen acceptor bead or an AlphaScreen donor bead, more particular an affinity tag such as a his tag.

In some embodiments, the second label is attached to the MBNL1 polypeptide by an antibody specific for the MBNL1 polypeptide.

In some embodiments, said MBNL1 polypeptide is labeled, in particular fluorescently labeled using an antibody specific for its second label.

In some embodiments, determining said inhibition comprises determining the amount of said MBNL1 polypeptide bound to said CUG_n RNA.

In some embodiments, said amount of said MBNL1 polypeptide is determined by contacting said MBNL1 polypeptide bound to said CUG_n RNA with a first ligand specifically reactive to said MBNL1 polypeptide or said second label, and determining the amount of said first ligand bound to said MBNL1 polypeptide or said second label.

In some embodiments, said amount of said first ligand bound to said MBNL1 polypeptide or said second label is determined by contacting said first ligand with a second ligands specifically reactive to said first ligand, wherein said second ligand comprises a third label, and determining the amount of said third label.

In some embodiments, said third label is an optical label or an enzymatic activity.

In some embodiments, the enzymatic activity is determined by measuring the amount of a substrate of the enzyme, wherein particularly said substrate is TMB (3, 3', 5,5'- Tetramethylbenzidine).

In some embodiments, n of CUG_n is > 12.

In some embodiments, n of CUG_n is > 35.

In some embodiments, n of CUG_n is >50.

In some embodiments, n of CUG_n is between 50 and 500.

In some embodiments, n of CUG_n is between 50 and 100.

In some embodiments, n of CUG_n is 78. In other words, a CUG₇₈ RNA is used.

In some embodiments, the target compound is a small molecule, particularly characterized by a molecular weight below 3000 Da.

According to a second aspect the invention, a nucleic acid molecule consisting of or comprising a nucleic acid sequence characterized by SEQ ID NO 02 (CTG₇₈ DNA), in particular for use as an intermediate in a method for evaluating a candidate compound (T) for treating Myotonic Dystrophy Type I (DM1 ), is provided.

According to a third aspect the invention, a nucleic acid molecule consisting of or comprising a nucleic acid sequence characterized by SEQ ID NO 03 (CUG₇₈ RNA), in particular for use as in a method for evaluating a candidate compound (T) for treating Myotonic Dystrophy Type I (DM1 ), is provided

According to a fourth aspect the invention, a kit-of parts is provided, comprising:

a nucleic acid sequence characterized by SEQ ID NO 03 and

- a MBNL1 polypeptide with an identity of ≥85%, ≥90%, >95%, or >99% compared to:

• MBNL1 of SEQ ID NO: 01 or SEQ ID NO 37

• an ortholog polypeptide of SEQ ID NO 01 or SEQ ID NO 37. Examples:

Identification of small molecules of natural origin which disrupt the MBNL1 -CUG78 RNA complex in vitro

A collection of 70 isolated natural compounds and a library containing 2128 extracts from plants and fungi were screened with a novel in vitro CUG₇8-MBNL1 inhibition assay (Fig. 1A). For the screening assay the inventors used in vitro transcribed CUG₇₈ RNA (SEQ ID No 03) and purified recombinant MBNL1 -HIS (Y. Yuan et al., Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs. Nucleic Acids Research 35, 5474-5486 (2007)).

Compounds were co-incubated with recombinant MBNL1-HIS in 96-well plates containing immobilized CUG₇₈ RNA, and then MBNL1-CUG₇₈ complex inhibition was measured by antibody detection of RNA-bound MBNL1 -HIS.

From the compound library the isoquinoline alkaloid berberine was identified (Fig. 1 B) as a complex formation inhibitor with an IC₅₀ of 86.3 ± 5.8 μΜ (Fig. 1 C). Another alkaloid, isaindigotone, showed weak inhibitory activity at 100 μΜ concentration. From the extract library 21 extracts were identified that inhibited MBNL1 -CUG₇₈ complex formation by at least 40%, as compared to no-compound controls, at a concentration of 100 μg ml. In order to identify the active compounds in the extracts, an approach referred to as HPLC-based activity profiling was used. It combines separation of complex mixtures with spectroscopic data recorded on-line, and with biological information obtained in parallel from time-based microfractionation and subsequent bioassay (O. Potterat, M. Hamburger, Combined Use of Extract Libraries and HPLC-Based Activity Profiling for Lead Discovery: Potential, Challenges, and Practical Considerations. Planta Med SO, 1 171-1 181 (2014)).

In addition, off-line microprobe NMR analysis was used to fully establish the structure of active compounds. The ten extracts with the strongest inhibitory activity were fractionated using this approach, and the resulting 29 or 30 fractions per extract were retested in the CUG₇₈-MBNL1 inhibition assay. The alkaloid harmine (Fig. 1 B) was identified as an active constituent in a methanolic extract from roots of Peganum harmala (Nitrariaceae) (Fig. 2).

Besides, two closely related diterpenquinones, methylenetanshinquinone and 1 ,2- dihydrotanshinquinone, were detected in the active fractions of an ethyl acetate extract from roots of Salvia miltiorrhiza (Lamiaceae). A commercial sample of harmine had an IC₅₀ of 132.4 ± 9.3 μΜ (Fig. 1 C), whereas the two diterpenquinones, also commercially obtained, showed weak inhibitory activity at 100 μΜ concentration. The inhibitory activity of the remaining eight extracts could be assigned to tannins, since the activity was lost after filtration of the extracts through a polyamide cartridge (data not shown; R. A. Collins, T. B. Ng, W. P. Fong, C. C. Wan, H. W. Yeung, Removal of polyphenolic compounds from aqueous plant extracts using polyamide minicolumns. Biochemistry and Molecular Biology International 45, 791-796 (1998)). Based on the structure of the isoquinoline alkaloid berberine was the most active compound from the screening. Furthermore, the synthetic alkaloid coralyne (Fig. 1 B), a fully planar berberine derivative, was identified as a strong MBNL1 -CUG₇₈ complex inhibitor with an IC₅₀ of 17.8 ± 0.2 μΜ, (Fig. 1 C). As reference compounds, Hoechst 33258 and neomycin B, two known nucleic acid-binders, were tested and had IC5₀ values of 195.5 ± 3.0 μΜ and 5.3 ± 0.6 μΜ, respectively.

Pentamidine, a lead compound for DM1 (M. B. Warf, M. Nakamori, C. M. Matthys, C. A. Thornton, J. A. Berglund, Pentamidine reverses the splicing defects associated with myotonic dystrophy. Proceedings of the National Academy of Sciences of the United States of America 106, 18551-18556 (2009)), had no inhibitory effect on complex formation, even at a concentration as high as 500 μΜ.

Identified alkaloids improve mis-splicing in a human myoblast cell model of DM1

The alkaloids identified in the in vitro screening assay were tested for their ability to rescue mis-splicing in human DM1 myoblasts. The alternative splicing of the insulin receptor (INSR) and cardiac troponin T type 2 (TNNT2) pre-mRNA in two human fibroblast cell lines containing a doxycycline inducible MYOD construct was investigated (S. Chaouch et al., Immortalized Skin Fibroblasts Expressing Conditional MyoD as a Renewable and Reliable Source of Converted Human Muscle Cells to Assess Therapeutic Strategies for Muscular Dystrophies: Validation of an Exon-Skipping Approach to Restore Dystrophin in Duchenne Muscular Dystrophy Cells. Human Gene Therapy 20, 784-790 (2009)).

The wild type (WT) cell line contained a CUG₅ repeat in the 3' UTR of the DMPK gene, whereas the DM1 cell line contained a CUG₁₃₀o repeat. These cell lines were differentiated into myoblasts by addition of doxycycline, which induced MYOD expression. As indicators of differentiation, MYOD and desmin expression were confirmed with IHC staining (data not shown). Differentiated DM1 myoblasts were treated for one day with the identified alkaloids, followed by RNA isolation, reverse transcription, and quantitative PCR (qPCR). DMSO- treated WT and DM1 cells served as controls. For the quantification of alternative splicing, a qPCR-based method was developed that makes use of two primer pairs. One primer pair detected all alternative splicing variants of a pre-mRNA and the other primer pair detected only those variants which included an investigated alternative exon. Berberine rescued the mis-splicing of the TNNT2 pre-mRNA but had a negative effect on the INSR pre-mRNA splicing (Fig. 3A). TNNT2 pre-mRNA splicing was rescued by 62.1 ± 3.2% (20 μΜ), 75.1 ± 2.8% (40 μΜ) and 86.2 ± 0.8% (80 μΜ) through berberine treatment (Fig. 3A). In contrast to berberine, harmine improved the mis-splicing of the TNNT2 pre-mRNA by 53.3 ± 3.5% (20 μΜ), 76.8 ± 1 .6% (40 μΜ) and 66.1 ± 1 .2% (80 μΜ) (Fig. 3B).

Furthermore, it also improved mis-splicing of the INSR pre-mRNA by 55.4 ± 3.3% at a concentration of 80 μΜ (Fig. 3B). The synthetic berberine derivative coralyne and the two diterpenequinones, identified together with harmine during the extract screening, showed no effect on mis-splicing in the DM1 cell model (data not shown). The alternative splicing results obtained with the qPCR method were confirmed with classical RT-PCR and visualization of two alternatively spliced isoforms of the INSR and TNNT2 pre-mRNA on 3% agarose gels (Fig. 3C,D).

To investigate the selectivity of berberine and harmine, their effect on alternative splicing of two genes known to be alternatively spliced, but independently of MBNL1 , i.e. ATE1 and FHL1 (Fig 3E,F) was tested (A. Mykowska, K. Sobczak, M. Wojciechowska, P. Kozlowski, W. J. Krzyzosiak, CAG repeats mimic CUG repeats in the misregulation of alternative splicing. Nucleic Acids Research 39, 8938-8951 (201 1 )).

Exon 7 inclusion in the ATE1 pre-mRNA, which was close to 33% for the WT cell line and close to 40% in the DM1 cell line was analyzed. Exon 5 inclusion in the FHL1 pre-mRNA was close to 0.1 - 0.2% for both the WT and the DM1 cell line. Compared to untreated WT and DM1 control cells, treatment of DM1 cells with berberine and harmine (both at 20 μΜ and 80 μΜ) did not show any significant difference in alternative splicing of both genes.

Harmine reduces CUG_n repeat foci formation in a human myoblasts cell model for DM1

To examine whether the alkaloids reduced the sequestration of MBNL1 by CUG_n RNA, foci formation was investigated in the same human cell lines used in the cellular splicing assay. Immunofluorescence staining in both the WT and the DM1 cell line showed that MBNL1 was mainly localized to the nuclei. In the DM1 cells, punctuate staining of MBNL1 within the nucleus could be co-localized in foci with Cy3-CAG₁₀ probe staining. Upon addition of 80 μΜ harmine, a clear reduction in the intensity and quantity of the foci (Fig. 4A) was observed.

However, 40 μΜ concentration of harmine was not sufficient to reduce foci formation. Berberine did not reduce foci formation, neither at 40 μΜ, nor at 80 μΜ (Fig 4A). Quantification of foci in a total of 150 randomly chosen nuclei per condition showed a clear decrease in foci number for 80 μΜ harmine-treated DM1 myoblasts to 0.9 ± 0.2 foci per nucleus, compared to DMSO-treated DM1 myoblasts with 4.7 ± 0.3 foci per nucleus (Fig. 4 B). MBLN1 staining gave a rather diffuse, nuclear and cytoplasmic localization, indicative of the release of MBNL1 from the toxic CUG_n RNA and its redistribution. 80 μΜ berberine treatment in contrast seemed to increase the number of foci to 5.8 ± 0.9 foci per nucleus (Fig. 4B).

Cellular toxicity evaluation of berberine and harmine

Before testing the identified alkaloids in vivo, their cellular toxicity in a cell viability assay was evaluated. C2C12 mouse myoblasts were treated with the alkaloids at concentrations ranging from 1 to 900 μΜ and the concentrations were determined at which half of the cells remained viable after two days of compound incubation, i.e. toxicity IC₅₀ (Tox IC₅₀) values. Berberine yielded a Tox IC₅₀ value of 212.1 ± 18.3 μΜ, whereas harmine gave a value of 123.3 ± 4.6 μΜ. The IC₅₀ values from the CUG₇8-MBNL1 inhibition assay (Fig. 1A) and the Tox IC₅₀ values were relatively close to each other for both alkaloids. However, both of the compounds showed an effect on alternative splicing in the cell model at concentrations significantly lower than the IC₅₀ and Tox IC₅₀ values, e.g. at 20 μΜ (Fig 3). Mitomycin C was measured as a reference compound and yielded a Tox IC₅₀ of 20.4 ± 1 .6 μΜ.

Identified alkaloids ameliorate mis-splicing of the CLCN1 pre-mRNA in the HSA^LR DM1 mouse model

HSA^LR mice were treated, a DM1 model containing a CTG25₀ repeat expressed under an actin promoter, with the identified alkaloids (A. Mankodi et al., Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289, 1769-1772 (2000)). The compounds were administered in a short treatment protocol, consisting of 2 injections at an interval of 12h. 2-4 hours after the second injection, mice were sacrificed, and quadriceps muscle dissected for splicing and protein analysis.

Groups of a minimum of three mice were either treated with vehicle or with compounds at two or three dose levels. The compounds were tested for their ability to restore mis-splicing of the CLCN1 (A. Mankodi et al., Expanded CUG repeats trigger aberrant splicing of CIC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Molecular Cell 10, 35-44 (2002); N. Charlet-B et al., Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Molecular Cell 10, 45-53 (2002) and SERCA1 (S.-i. Hino et al., Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1 . Human Molecular Genetics 16, 2834-2843 (2007)) pre-mRNA.

MBNL1 promotes the exclusion of the alternatively spliced exon 7a of the CLCN1 pre-mRNA and promotes the inclusion of exon 22 of the SERCA1 pre-mRNA. WT mice at the age of 10 to 12 weeks showed a CLCN1 pre-mRNA exon 7a inclusion of 5.0 ± 0.5%, whereas in the HSA^LR line the inclusion level was elevated to 40.9 ± 2.5%. The level of SERCA1 pre-mRNA exon 22 inclusion in the WT mice was at 83.9 ± 2.9%. In the HSA mice exon 22 inclusion was decreased to 24.8 ± 2.7%. Splicing was analyzed by qPCR, analogously to the splicing analysis in human myoblasts. Compared to vehicle-treated control mice, treatment of HSA^LR mice with 20 mg/kg berberine i.p. showed a clear effect on CLCN1 pre-mRNA as well as on SERCA1 pre-mRNA splicing, but was lethal to two out of four treated mice. Treatment with 5 mg/kg and 10 mg/kg was better tolerated but did not result in any significant splicing improvement (data not shown).

Two close derivatives of berberine were tested, dihydroberberine (DHB) and palmatine, with higher reported LD₅₀ values, i.e. lower toxicity. DHB improved the mis-splicing of the CLCN1 pre-mRNA at the higher dose of 10 mg/kg by 32.5% (P=0.0008, Student's t-test), whereas 5 mg/kg showed no statistical significant effect (Fig. 5A).

The CLCN1 splicing improvement by 10 mg/kg DHB was confirmed by classical RT-PCR and analysis of splicing isoforms on a 3% agarose gel (Fig. 5D).

Palmatine treatment improved the CLCN1 pre-mRNA mis-splicing at a dose of 40 mg/kg by 34.8% (P=0.0009), and at a dose of 25 mg/kg by 25.3% (P=0.0017), whereas 10 mg/kg did not show a significant effect (Fig. 5B).

Harmine treatment at a dose of 40 mg/kg decreased CLCN1 pre-mRNA exon 7a inclusion by 31.2% (P=0.0003) and did not significantly improve mis-splicing at a dose of 20 mg/kg (Fig. 5C).

DHB, palmatine and harmine had no significant effect on SERCA1 pre-mRNA mis-splicing (Fig. 5). Only the initially tested 20 mg/kg berberine had shown an improvement of SERCA1 pre-mRNA mis-splicing.

Alkaloid treatment increases CLCN1 protein levels in the HSA^LR DM1 mouse model

It was examined by Western immunoblot analysis whether the high dose alkaloid treatments, which had evoked improved CLCN1 pre-mRNA mis-splicing, also increased protein levels of functional full-length CLCN1 channel in vivo. Using a rabbit polyclonal antibody against the N-terminus of full-length CLCN1 (N. Charlet-B ef a/., Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Molecular Cell 10, 45-53 (2002)), the CLCN1 channel was detected at the expected size of 130 kD in nuclear/membrane fractions of quadriceps muscle of WT and HSA^LR mice.

As a protein loading control, we used Lamin B. CLCN1 protein levels in quadriceps muscle of four vehicle-treated HSA^LR mice were decreased by 29.1 % (P=0.0002, Student's t-test, 3 x n=4) compared to four vehicle-treated WT mice (Fig. 6). Treatment of HSA^LR mice with 10 mg/kg DHB increased CLCN1 protein levels by 25.0% (P=0.0296, n=4), compared to levels of vehicle treated HSA mice (Fig 6). Both the palmatine and harmine high dose treatments of 40 mg/kg did not increase the CLCN1 protein levels in HSA^LR mice.

Compounds

Compounds and extracts screened in this study were part of natural product libraries established at the Division of Pharmaceutical Biology of the University of Basel. One library contained 70 pure natural compounds as 10 mM solutions in DMSO and a second library consisted of 2128 extracts from plants and fungi archived as 10 mg/ml solutions in DMSO (O. Potterat, M. Hamburger, Combined Use of Extract Libraries and HPLC-Based Activity Profiling for Lead Discovery: Potential, Challenges, and Practical Considerations. Planta Med 80, 1 171-1 181 (2014)). Harmine hydrochloride was purchased TCI Europe (#H0002). Berberine chloride (#B3251 ), palmatine chloride hydrate (#361615), coralyne sulfoacetate (#S424536) were ordered from Sigma Aldrich. Dihydroberberine (#80429) was purchased from PhytoLab GmbH. Methylenetanshinquinone (#QP-393) and 1 ,2-dihydrotanshinquinone (#QP-1 166) were obtained from Quality Phytochemicals LLC, East Brunswick, NJ, MBNL1 Preparation

MBNL1 cDNA (isoform with amino acids 1-382) was used. The pGEX-6P-MBNL1-N-His (amino acids 1-253; SEQ ID No 01 ) construct used in this study was cloned according to Yuan et al. (Y. Yuan et al., Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs. Nucleic Acids Research 35, 5474-5486 (2007)).

Using BL21 expression cells, protein expression was induced with 0.1 mM isopropyl-p-D- thiogalactosid (IPTG) at an OD₆₀₀ = 0.8-1 , for 3^1 h at 30 °C and 180 rpm. Bacteria were pelleted at 7Ό00 rpm for 10 min at 4°C, the supernatant was discarded and the pellet frozen. The pellet lysed in lysis buffer (50 mM Tris [pH 8.5], 150 mM NaCI, 5 mM dithiotreitol (DTT) and 10% glycerol) containing 1 mg/mL of lysozyme, 10 μg/ml DNAse, and protease inhibitors 0.5 g/ml aprotinin, 1 g ml pepstatin, 2 g/ml leupeptin and 1 mM phenylmethanesulfonylfluoride (PMSF). N-octyl-p-D-glucopyranoside was added to a final concentration of 7.3 mg/ml and the lysate was rocked for 15 min at RT. After three times of freezing in liquid nitrogen and thawing in hand-hot water, the cell extract was centrifuged at 4°C for 10 min at 13Ό00 rpm and the supernatant, containing GST-MBNL1-HIS, was collected. GST-MBNL1-HIS protein was bound to His-Select Nickel Affinity Gel beads (Sigma) overnight at 4 °C in equilibration buffer (50 mM Tris [pH 7.4], 150 mM NaCI, 1 mM DTT, 10% glycerol). Beads were washed 1 x with equilibration buffer and 1x with equilibration buffer containing 10 mM imidazole. Protein was eluted from beads with elution buffer (equilibration buffer + 500 mM imidazole). Eluted protein was then bound to Glutathione Sepharose 4B beads (GE Healtcare) by incubation in equilibration buffer for 4h at 4°C. Beads were washed 1 x with equilibration buffer. MBNL1-HIS was cleaved from beads in cleavage buffer (10 mM Tris [pH 7.4], 50 mM NaCI, 1 mM DTT) overnight at 4°C with PreScission Protease (GE Healthcare). The eluate was collected, protein concentration determined with the NanoDrop spectrophotometer and a BCA assay (Sigma). The purity of MBNL1 -HIS was evaluated by means of SDS-PAGE. MBNL1 -HIS aliquots were snap frozen in liquid nitrogen and stored at -70°C.

CUG₇« NA Preparation

A CAG₇₈ repeat was used from a blood sample of a patient, who gave informed consent. Genomic DNA was isolated from the patient sample with the Puregene Blood Core Kit B (Qiagen) according to the manufacturer's protocol. A genomic fragment located in the DMPK gene, containing the CAG₇₈ repeat, was PCR amplified with HOT Start DNA Polymerase (Solis BioDyne) by addition of Solution S. The following primers were used for PCR: DMPK forward - ⁵CAG CTC CAG TCC TGT GAT CC³ (SEQ ID NO 04) and DMPK reverse - ⁵CTG GCC GAA AGA AAG AAA TG^3' (SEQ ID NO 05). The amplicon was agarose gel purified by means of the QIAquick Gel Extraction Kit (Qiagen). The purified DNA fragment was cloned by TA-cloning with the pCR ll-TOPO plasmid vector in Dh5a bacterial cells. The plasmid was purified with the QIAprep Spin Miniprep Kit (Qiagen) and used as template for PCR amplification with the following primers: T7 CUG forward - ⁵TAA TAG GAC TCA CTA TAG GCA GCT CCA GTC CTG TGA TCC³ (SEQ ID NO 06) and T7 CUG reverse - ⁵TAA TAC GAC TCA CTA TAG GCT GGC CGA AAG AAA GAA ATG³ (SEQ ID NO 07). The amplified DNA was purified with the QIAquick PCR Purification Kit (Qiagen) and the concentration measured with the NanoDrop spectrophotometer. 200 ng of DNA was used as a template for in vitro RNA transcription by means of the MEGAscript T7 transcription kit (Ambion). CUG₇₈ RNA was biotin-labeled by addition of 1.875 mM biotin-14-CTP (37.5 nmol), 6.625 mM CTP (1 12.5 nmol), and 7.5 mM ATP, GTP and UTP to the transcription reaction. RNA quality and purity was tested by visualization of RNA on denaturing 8 M urea/TBE 5% polyacrylamide gels.

Biochemical Screening Assay: CUG₇«-MBNL1 inhibition assay

All wash steps were performed at RT with 150 μΙ wash buffer per well (25 mM Tris [pH 7.4], 80 mM NaCI, 1 mM MgCI₂, 0.5 mM DTT, 0.05% Tween-20, 1.5 mg/ml BSA, DEPC-treated water). The incubation steps were performed with 50 μΙ incubation buffer per well (wash buffer + 25 U/ml RNasin) at 30°C on a BIOSAN plate shaker at 300 rpm. Reacti-Bind NeutrAvidin coated 96-well plates (Pierce) were prewashed once. Wash buffer was removed and of incubation buffer containing 25ng of biotinylated CUG₇₈ RNA was added per well. After incubation for 1 h the plates were washed twice. Wash buffer was removed and 45 μΙ incubation buffer containing 300 ng of MBNL1 -HIS was added followed by the addition of 5 μΙ of 10x concentrated compound or 5 μΙ of DMSO/water for controls. After washing wells twice, mouse anti-HIS antibody (1 :2000, GE Healthcare, #27-4710-01 ) was incubated for 1 h. Another two wash steps preceded 1 h incubation with goat anti-mouse-HRP antibody (1 :8000, Jackson ImmunoResearch Laboratories, #1 15-035-174). After two final wash steps, 70 μΙ of TMB substrate (Thermo Scientific) was added to the wells at RT. The colorimetric reaction was performed at 30 °C, 700 rpm for 3 to 5 min and stopped with 70 μΙ 0.15 M H₂S0₄ per well. The optical density was read at 450 nm wavelength with a Molecular Devices plate reader. Inhibition curves were fitted with Prism software and the IC₅₀ values determined.

Time-resolved fluorescence energy transfer (TR-FRET) assay:

For HTRF binding assays 5ul biotinylated (CUG)78 (50 nM final), 4 μΙ MBNL1 (50 nM final), 4 μΙ anti-His-Tb, 4 μΙ streptavidin-d2, and 2 μΙ compounds were mixed in binding buffer composed of 25 mM Tris [pH 7.4], 80 mM NaCI, 1 mM MgCI₂, 0.5 mM DTT, 0.05% Tween- 20, 1 .5 mg/ml BSA, DEPC-treated water. After 0.5 to 4 hours incubation at room temperature, the fluorescence was measured using an TECAN Flourometer.

HPLC-based activity profiling of extracts

HPLC-based activity profiling was performed on a Waters 2695 Alliance Separation Module equipped with a Waters 996 Photodiode Array (PDA) detector and a C18 SunFire column (3.0 x 150 mm; 3.5 μιτι; Waters). Mobile phase consisted of 0.1 % formic acid in H₂0 (A) and MeCN (B), and a gradient of 5 to 95% B in 30 minutes was applied. The flow rate was 0.5 ml/min. 900 μg of extract were injected in three portions and time-based microfractions were collected into a deep well 96-well microtiter plate (30 fractions of 60 sec each). The microtiter plate was then dried in a Genvac EZ-2 evaporating system at 35°C overnight. For screening, the dried fractions were taken up in 16 μΙ of DMSO, and 1 or 2 μΙ were used in the assay.

Cell Splicing Assay

The two used human fibroblast cell lines (WT and DM1 ) with an inducible MyoD construct for differentiation into myoblasts were kindly provided by Denis Furling, Universite Pierre et Marie Curie-Paris, Paris, France (S. Chaouch et a/., Immortalized Skin Fibroblasts Expressing Conditional MyoD as a Renewable and Reliable Source of Converted Human Muscle Cells to Assess Therapeutic Strategies for Muscular Dystrophies: Validation of an Exon-Skipping Approach to Restore Dystrophin in Duchenne Muscular Dystrophy Cells. Human Gene Therapy 20, 784-790 (2009)). These cell lines were grown in monolayers in growth medium (DMEM with GlutaMAX, 10% FBS, 30 mM HEPES, 50 μg ml Gentamycin) at 37 °C under 5% C0₂. 50Ό00 cells per well were seeded in 0.1 % gelatin-coated 6-well plates and grown for 2.5 to 3 days. Cells were washed 1x with DMEM, then differentiation medium (= growth medium + 5 μg ml Doxycycline) was added.

After 24h of differentiation the cells were washed with DMEM. The myoblast culture was then treated for 24 h with the compounds of interest or DMSO in differentiation medium. The cells were washed 1 x with PBS and total RNA was extracted with TRI reagent (Sigma) according to the manufacturer's protocol. The resulting RNA pellet was taken up in 20 μΙ of RNase free water and the concentration of the isolated RNA was determined with a NanoDrop spectrophotometer. 500 ng of RNA was used for reverse transcription which was performed with the Superscript III First-Strand Synthesis System for RT-RCR (Invitrogen) according to the manufacturer's protocol with random hexamer primers. Of the resulting cDNA 1 μΙ was used as template for PGR, with gene specific primers, with a 7^~ _m of 61 °C, and 40 amplification cycles. Two PGR methods were used to quantify the splicing of marker genes:

(1 ) RT-PCR: In this protocol PGR was performed as described above with the following primer pairs: INSR forward - ⁵CCA AAG ACA GAC TCT CAG AT^3' (SEQ ID NO 08) and reverse - ⁵AAC ATC GCC AAG GGA CCT GC^3' (SEQ ID NO 09); TNNT2 forward - ⁵ATA GAA GAG GTG GTG GAA GAG TAG³ (SEQ ID NO 10) and reverse - ⁵GTC TCA GCC TCT GCT TCA GCA TCC^3' (SEQ ID NO 1 1 ). Amplification was performed with HOT Start DNA Polymerase (Solis BioDyne) and an Applied Biosystems PGR machine. The PCR products were run on 3% agarose gels stained with RedSafe (iNtRON). Gels were imaged with a Gel Doc XR+ (Bio-Rad) and the band densities were quantified by the ImageJ software.

(2) RT-qPCR: In this protocol PCR was performed as described above with two primer pairs for each gene, one primer pair only amplifying the splicing variant containing the alternative exon (exon) and the other primer pair amplifying two splicing variants (pan): INSR forward - ⁵GAC CTG GTG TCC ACC ATT CG³ (SEQ ID NO 12) and reverse (exon) - ⁵CAC CAG TGC CTG AAG AGG TT³ (SEQ ID NO 13) and reverse (pan) - ⁵ ACG AAA ACC ACG TTG TGC AG³ (SEQ ID NO 14) ; TNNT2 forward - ⁵CTG CTG TTC TGA GGG AGA GC^3' (SEQ ID NO 15) and reverse (exon) - ⁵TCG TCC TCT CTC CAG TCC TC³ (SEQ ID NO 16) and reverse (pan) - ⁵ CTG CTC CTC CTC CTC GTA CT³ (SEQ ID NO 17). As gene expression control β- actin was used with the following primers: forward - ⁵CCA ACC GCG AGA AGA TGA^3' (SEQ ID NO 18) and reverse - ⁵CCA GAG GCG TAG AGG GAT AG^3' (SEQ ID NO 19). Amplification was performed with HOT FIREPol EvaGreen qPCR Mix (Solis BioDyne) on an Applied Biosystems qPCR machine. The amplification efficiencies of the individual reactions were determined via standard curves and the splicing quantification was done with Excel software. MBNL1 -independent splicing

The effect of the tested alkaloids on MBNL1-independent splicing events was analyzed using the same RT-qPCR protocol (2), with primers for two alternatively spliced genes, ATE1 and FHL1 (38). ATE1 forward - ⁵GGG TTT CCA GGC TCA AGG TC^3' (SEQ ID NO 20) and reverse (exon) - ⁵TGA ACT GCG AAC TTG GTG GA^3' (SEQ ID NO 21 ) and reverse (pan) - ⁵TGT GTG ATG CAT TCT CTG GTA A^3' (SEQ ID NO 22). FHL1 forward - ⁵ATG CCG ATT GCT TTG TGT GT^3' (SEQ ID NO 23) and reverse (exon) - ⁵CTG GGT GGC TCA CTC TTG AC^3' (SEQ ID NO 24) and reverse (pan) - ⁵TCT TGC ATC CAG CAC ACT TCT^3' (SEQ ID NO 25). FISH and Immunofluorescence

Human fibroblasts were seeded on 0.3% gelatin-coated coverslips in 24-well cell culture dishes (15Ό00 cells/well) and grown for up to 24 h in growth medium at 37°C under 5% C0₂. After a wash step with DMEM, cells were differentiated in differentiation medium for 24 h. Another wash step with DMEM preceded the compound addition in differentiation medium and incubation for 24 h. Cells were washed in PBS and fixed for 10 min in 4% paraformaldehyde phosphate buffered solution. Cells were washed 3x in PBS for 5 min and permeabilized for 5min in PBS, 0.2% Triton X-100. For the probing with Cy3-CAG₁₀ coverslips were prewashed in 2xSSC, 30% formamide for 10 min at RT. Hybridization was performed for 2 h at 37 °C with 1 ng/μΙ Cy3-CAGi₀ in incubation buffer (30% formamide, 2X SSC, 2 μg/ml BSA, 66 μg/ml yeast tRNA, 2 mM vanadyl complex, 0.05% Triton X-100). Coverslips were then washed once in 2xSSC, 30% formamide buffer for 30min at 37°C, once with 1xSSC for 30 min at RT and twice with PBS, 0.05% Triton X-100 for 10min each.

Coverslips were then incubated for 15 min at RT in blocking buffer (3% BSA, PBS, 0.05% Triton X-100) followed by overnight incubation at 4 °C with 1 :5000 diluted MBNL1 antibody (A2764, kind gift from C. Thornton) in PBS, 0.05% Triton X-100 and washed twice in PBS 0.05% Triton X-100 for 10 min. Goat anti-rabbit DyLight 488 antibody (Jackson Imunoresearch Laboratories, #1 1 1 -485-144) was added at a dilution of 1 :750 in PBS for 1 h in PBS 0.05% Triton X-100. Coverslips were then washed twice in PBS 0.05% Triton X-100 for 10min. Cells were stained with 1 :20Ό00 DAPI (4,6 diamino-2-phenylindole dihydrochloride) for 5min in PBS 0.05% Triton X-100 and washed twice in PBS 0.05% Triton X-100 for 10min. Coverslips were then mounted on glass slides with FluorSave reagent (Calbiochem) and dried for at least one day before imaging.

Cell Viability Assay

C2C12 mouse myoblasts were plated in 96-well plates in growth medium, 4000 cells in 100 μΙ/well, and grown overnight at 37 °C under 5% C0₂. 65 μΙ of medium was removed and 35 μΙ of 2x concentrated compound in growth medium added and incubated for 48 h. Compound concentrations ranged from 1 to 900 μΜ. As a reference compound Mitomycin C (#M0440, Sigma) was used. After compound incubation 14 μΙ of CellTiter-Blue reagent (Promega), containing the dye Resazurin, was added per well and incubated at 37°C under 5% C0₂ for 1.5 h. Viable cells convert Resazurin to Resorufin, which is fluorescent. The fluorescence was then measured by means of an Infinite F500 plate reader (Tecan), with an excitation wavelength of 535 nm and an emission wavelength of 590 nm. Signal-concentration curves were fitted with Prism® software to determine Tox IC₅₀ values.

Treatment in Mice

HSA^LR transgenic mice in line 20b were kindly provided by Charles Thornton, University of Rochester, USA. FVB/N WT control mice were obtained from the animal facility of the Department of Biomedicine, University Hospital Basel, Switzerland. Age- and gender- matched groups of WT or HSA^LR mice were treated by intraperitoneal (i.p.) injections of compounds or vehicle. We used male mice at an age of 10 to 12 weeks. Considering the short in vivo half-life times of isoquinoline alkaloids such as berberine (Y. N. Zhao et a/., A new approach to investigate the pharmacokinetics of Traditional Chinese Medicine YL2000. American Journal of Chinese Medicine 32, 921 -929 (2004)) and beta-carboline alkaloids such as harmine (G. Zetler, G. Back, H. Iven, PHARMACOKINETICS IN RAT OF HALLUCINOGENIC ALKALOIDS HARMINE AND HARMALINE. Naunyn-Schmiedebergs Archives of Pharmacology 285, 273-292 (1974)), we performed a short in vivo treatment protocol with two i.p. injections at an interval of 12 h and sacrificed the mice 2-4 h after the second injection. Harmine hydrochloride was administered in saline, Dihydroberbererine and Palmatine hydrochloride in 5% DMSO/PBS. The quadriceps, tibialis anterior and gastrocnemius muscles of mice were dissected for analysis. For splicing and protein analysis quadriceps muscle was powdered after freezing in liquid nitrogen and aliquots stored at - 80°C. Animal studies were conducted in accordance with the Animal Research Authorities of the canton of Basel, Switzerland.

Mouse Splicing Assay

Powdered quadriceps muscle tissue was taken up in TRI reagent (Sigma) and was grinded with a rotor-stator Polytron for 30 sec at 4°C. Extracellular matrix material and debris was then removed by a centrifugation step at 12Ό00 rpm, 4°C for 10 min. The supernatant was transferred into a new tube and RNA was extracted according to the TRI reagent (Sigma) manufacturer's protocol. Reverse transcription, PCR, and splicing analysis was performed as described earlier for the cell splicing assay. In the RT-PCR protocol (1 ) the following primers were used: CLCN1 forward - ⁵GGA ATA CCT CAC ACT CAA GGC C³ (SEQ ID NO 26) and reverse - ⁵CAC GGA ACA CAA AGG CAC TGA ATG T^3' (SEQ ID NO 27); SERCA1 forward - ⁵GCT CAT GGT CCT CAA GAT CTC AC^3' (SEQ ID NO 28) and reverse - ⁵GGG TCA GTG CCT CAG CTT TG³ (SEQ ID NO 29)^'. In the RT-qPCR protocol (2) the following primer pairs were used: CLCN1 forward (exon) ⁵ GGG CGT GGG ATG CTA CTT TG^3' (SEQ ID NO 30) and forward (pan) - ⁵CTG ACA TCC TGA CAG TGG GC^3' (SEQ ID NO 31 ) and reverse - ⁵AGG ACA CGG AAC ACA AAG GC^3' (SEQ ID NO 32); SERCA1 forward - ⁵ GCC CTG GAC TTT ACC CAG TG^3' (SEQ ID NO 33) and reverse (exon) - ⁵ACG GTT CAA AGA CAT GGA GGA^3' (SEQ ID NO 34) and reverse (pan) - ⁵ CCT CCA GAT AGT TCC GAG CA³ (SEQ ID NO 35). Western Immunoblot Detection of Clcn-1 Protein from Mouse Muscle

Proteins were extracted from powdered mouse quadriceps muscle according to Dimauro et. al. (I. Dimauro, T. Pearson, D. Caporossi, M. J. Jackson, A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue. BMC research notes 5, 513-513 (2012)) to obtain the nuclear/membrane fraction. Instead of using NET buffer, RIPA+ buffer was used (50mM Tris HCL [pH 8.0], 150mM NaCI, 1 % NP-40, 0.5% sodium deoxycholate, 1 % Triton X100, 0.1 % SDS, 10% glycerol), containing protease and phosphatase inhibitor tablets. Protein concentrations were determined with a BCA assay (Sigma). 10 ug per sample were separated by Tris-glycine SDS-PAGE on a 8% gel and analyzed by Western immunoblot using Protran BA85 nitrocellulose membranes (GE Healthcare), rabbit polyclonal anti-CLCN1 antibody (1 :1000, kind gift from Thomas Cooper, Baylor College of Medicine, USA) ( 13) and HRP-tagged goat anti-rabbit secondary antibody (1 :10000, Jackson Immunoresearch, #1 1 - 035-003). To detect Lamin B, goat polyclonal anti-Lamin B antibody (1 :1 Ό00, Santa Cruz Biotechnology, #sc-6216) and HRP-tagged swine anti-goat antibody (1 :10Ό00, Life Technologies, #ACI3404) were used. All antibodies were incubated in TBS, 3% BSA, 0.1 % Tween-20, 0.08% SDS. Protein bands were detected with the ECL technique using LumiGLO (KPL) chemiluminescent substrate. Membranes were exposed to Super RX films (Fuji).

Description of the figures

Fig. 1A: shows a CUG₇₈-MBNL1 displacement screening assay: Biotinylated CUG₇₈

RNA is immobilized and incubated with MBNL1 and compounds. RNA-bound MBNL1 is detected via a primary anti-HIS and a secondary anti-mouse-HRP antibody. A substrate is added to give an optical signal, correlating to the amount of RNA-bound MBNL1. In between all five incubation steps, washing is performed; shows the molecular structure of the identified alkaloids berberine, harmine, and coralyne;

shows MBNLI-CUG7₈ RNA complex inhibition curves with berberine, harmine, and coralyne;

shows HPLC-based activity profiling of the methanol extract of Peganum harmala. Shown on the y-axis are the online HPLC-UV trace at 254 nm and the OD45₀ signal from the CUG₇₈-MBNL1 inhibition assay for each of the 29 fractions (min 1 to 30, time on x-axis). The activity of the fractions at minutes 8 and 9 could be assigned to the alkaloid harmine. Based on on-line MS and UV spectroscopic data, the other major peak eluting at 6.6 min was tentatively assigned to harmol. The activity in the early eluting fraction 2-3 min did not correspond to any peak and was not further investigated;

show representative RT-qPCR splicing data for WT and DM1 control cells as well as treated DM1 human myoblast cells: Berberine improves the mis- splicing of the TNNT2 pre-mRNA but has a detrimental effect on the INSR pre- mRNA splicing (Fig. 3A); Harmine improves mis-splicing of the TNNT2 pre- mRNA and also of the INSR pre-mRNA at the highest concentration (Fig. 3B); Alternative splicing of INSR and TNNT2 pre-mRNA analyzed by RT-PCR and visualized on 3% agarose gels. Shown are the two alternative splicing isoforms for both genes in untreated WT and DM1 control cells and DM1 cells treated with berberine (Fig. 3C) and harmine (Fig. 3D). The WT splicing isoform represents the predominant mature splicing variant, whereas the DM1 isoform represents the fetal splicing variant; Berberine and harmine do not affect alternative splicing of MBNL1 -independently regulated ATE1 and FHL1 pre-mRNAs in treated DM1 myoblasts, compared to untreated WT and DM1 control myoblasts (Fig. 3E and Fig. 3F);

shows FISH and immunofluorescence: Harmine reduces the amount and intensity of foci at 80μΜ concentration whereas berberine does not reduce foci. For each condition a foci staining, MBNL1 staining as well as a merge of both with a nuclear DAPI staining is shown. Vehicle treated control WT and DM1 cells as well as compound treated (80 μΜ) cells are shown;

shows Quantification of the number of CUG_n RNA foci in human DM1 cells either treated with DMSO or the indicated alkaloids at 80 μΜ;

shows representative in vivo RT-qPCR splicing data for vehicle treated WT and HSA^LR mice as well as compound treated HSA^LR mice (quadriceps muscle). Shown are the splicing of the CLCN1 channel and SERCA1 . All three alkaloids improve the splicing of the CLCN1 channel whereas the mis- splicing of SERCA1 is not improved: Dihydroberberine (DHB) treatment (Fig. 5A); Palmatine treatment (Fig. 5B); Harmine treatment (Fig. 5C); Alternative splicing of the CLCN1 pre-mRNA analyzed by RT-PCR and visualized on 3% agarose gels. Shown are the two alternative splicing isoforms in untreated WT FVB/N and DM1 HSA^LR control mice and HSA^LR mice treated with 10 mg/kg dihydroberberine (DHB) (Fig. 5D);

Fig. 6A shows Western immunoblot showing CLCN1 protein levels of vehicle treated

WT mice (a), vehicle treated HSA^LR mice (b), and compound treated HSA^LR mice (c-e). Lamin B was used as loading control;

Fig. 6B shows Quantification of CLCN1 band intensities normalized to Lamin B. The means of four replicates for each condition including the standard deviation are shown;

Claims

Claims:

1 . A method for evaluating a candidate compound (T) for treating Myotonic Dystrophy Type I (DM1 ) comprising the steps of:

a) providing a Muscleblind-Like Splicing Regulator 1 (MBNL1 ) polypeptide and a CUG_n RNA;

b) contacting said MBNL1 polypeptide with said CUG_n RNA in the presence of said candidate compound (T) in a reaction volume;

c) determining the inhibition of binding of said MBNL1 polypeptide to said CUG_n RNA by said candidate compound (T).

2. The method according to claim 1 , wherein the MBNL1 polypeptide amino acid sequence has an identity of >85%, >90%, >95%, or≥99% compared to

- to SEQ ID NO: 01 or SEQ ID NO 37, or

- an ortholog polypeptide of SEQ ID NO 01 or SEQ ID NO 37; characterized in that said MBNL1 polypeptide is able to bind said CUG_n RNA.

3. The method according to any one of the previous claims, characterized in that n is 13 to 500, in particular n is 35 to 400, more particular n is 50 to 350, most particular n is 78.

4. The method according to any one of the previous claims, characterized in that said CUG_n RNA is attached to a first label, in particular a first affinity label, a first fluorescent label or an AlphaScreen acceptor or donor bead, more particular an affinity label such as biotin.

5. The method according to any one of the previous claims, characterized in that said CUG_n RNA is synthesized using a mixture of modified nucleotides comprising a first label and unmodified nucleotides with a ratio of 1 :2 to 1 :10, in particular 1 :3 to 1 :6, more particular 1 :4 to 1 :8.

6. The method according to any one of the previous claims, characterized in that said MBNL1 polypeptide is attached to a second label, in particular a second affinity label, a fluorescent label or an AlphaScreen acceptor or donor bead, more particular an affinity tag such as a his tag.

7. The method according to any one of the previous claims, characterized in that determining said inhibition comprises determining the amount of said MBNL1 polypeptide bound to said CUG_n RNA.

8. The method according to claim 7, characterized in that said amount of said MBNL1 polypeptide is determined by contacting said MBNL1 polypeptide bound to said CUG_n RNA with a first ligand specifically reactive to said MBNL1 polypeptide or said second label, and determining the amount of said first ligand bound to said MBNL1 polypeptide or said second label.

9. The method according to claim 8, characterized in that said amount of said first ligand bound to said MBNL1 polypeptide or said second label is determined by contacting said first ligand with a second ligand specifically reactive to said first ligand, wherein said second ligand comprises a third label, and determining the amount of said third label.

10. The method according to any one of the previous claims, characterized in that said third label is an optical label or an enzymatic activity.

1 1. The method according to any one of the previous claims, characterized in that the target compound is a small molecule, particularly characterized by a molecular weight below 3000 Da.

12. A nucleoid acid molecule consisting of or comprising a nucleic acid sequence characterized by SEQ ID NO 02 (CTG₇₈ DNA).

13. A nucleoid acid molecule consisting of or comprising a nucleic acid sequence characterized by SEQ ID NO 03 (CUG₇₈ RNA).

14. A kit-of-parts comprising:

a nucleic acid sequence characterized by SEQ ID NO 03 and a MBNL1 polypeptide amino acid sequence with an identity of >85%,

≥90%,≥95%, or >99% compared to:

• MBNL1 of SEQ ID NO 01 or SEQ ID NO 37; or

• an ortholog polypeptide of SEQ ID NO 01 or SEQ ID NO 37.