The role of muscleblind-like proteins in myotonic dystrophy

Myotonic dystrophy (DM) is a progressive multisystemic genetic disorder which is inherited as an autosomal dominant trait. There are two subtypes of the disorder, DM type 1 and DM type 2. DM type1 is caused by an expansion of a CTG repeat located in the 3' untranslated region of the DMPK gene on chromosome 19q13.3, whereas DM type 2 is caused by a CCTG expansion in intron 1 of ZNF9 gene located on chromosome 3q. The mutant RNAs containing the expanded CTG/CCTG repeats alters the activity of various alternative splicing factors like Muscleblind-like (MBNL) proteins, which are sequestered in the ribonuclear foci in nucleus by the expanded mutant transcripts resulting in a number of splicing defects observed in DM patients. In first part of my thesis, I have assessed the nuclear and cytosolic distribution of MBNL proteins in both normal and DM cells. In both DM1 and DM2 cells the amount of nuclear MBNL1 was found to be at least 50% greater than seen in normal cells. In addition to this, I studied the distribution of MBNL1 protein in nuclear and cytosolic fractions of DM cells before and after treatment with compounds chromomycin A3, gemcitabine, IMOX, RO 31-8220 and hypericin which were highlighted in the primary screen. Treatment with the compounds produced a significant reduction in the proportion of nuclear MBNL1 compared to DMSO treated cells in DM fibroblast and myoblasts. In second part of this thesis I have examined the effect of MBNL1/2 down regulation on both RNA and MBNL1 foci in DM cells. MBNL1 and MBNL2 double knockdown resulted in a 40% increase of nuclear RNA foci than observed in scrambled siRNA treated cells, though a significant reduction was observed in case of MBNL (protein) foci. Also, MBNL 1 and MBNL2 down regulation did not result in the release of mutant transcript from the nucleus to the cytoplasm in KB-Telo MyoD (DM) cells as seen in BpmI restriction polymorphism assay. However, it had a degradative effect on the mutant DMPK transcript

The role of muscleblind-like proteins in myotonic dystrophy

Myotonic dystrophy (DM) is a progressive multisystemic genetic disorder which is inherited as an autosomal dominant trait. There are two subtypes of the disorder, DM type 1 and DM type 2. DM type1 is caused by an expansion of a CTG repeat located in the 3' untranslated region of the DMPK gene on chromosome 19q13.3, whereas DM type 2 is caused by a CCTG expansion in intron 1 of ZNF9 gene located on chromosome 3q. The mutant RNAs containing the expanded CTG/CCTG repeats alters the activity of various alternative splicing factors like Muscleblind-like (MBNL) proteins, which are sequestered in the ribonuclear foci in nucleus by the expanded mutant transcripts resulting in a number of splicing defects observed in DM patients. In first part of my thesis, I have assessed the nuclear and cytosolic distribution of MBNL proteins in both normal and DM cells. In both DM1 and DM2 cells the amount of nuclear MBNL1 was found to be at least 50% greater than seen in normal cells. In addition to this, I studied the distribution of MBNL1 protein in nuclear and cytosolic fractions of DM cells before and after treatment with compounds chromomycin A3, gemcitabine, IMOX, RO 31-8220 and hypericin which were highlighted in the primary screen. Treatment with the compounds produced a significant reduction in the proportion of nuclear MBNL1 compared to DMSO treated cells in DM fibroblast and myoblasts. In second part of this thesis I have examined the effect of MBNL1/2 down regulation on both RNA and MBNL1 foci in DM cells. MBNL1 and MBNL2 double knockdown resulted in a 40% increase of nuclear RNA foci than observed in scrambled siRNA treated cells, though a significant reduction was observed in case of MBNL (protein) foci. Also, MBNL 1 and MBNL2 down regulation did not result in the release of mutant transcript from the nucleus to the cytoplasm in KB-Telo MyoD (DM) cells as seen in BpmI restriction polymorphism assay. However, it had a degradative effect on the mutant DMPK transcript

High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM

Targeting the 5' untranslated region of SMN2 as a therapeutic strategy for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN2) gene. All patients have at least one copy of a paralog, SMN2, but a C-to-T transition in this gene results in exon 7 skipping in a majority of transcripts. Approved treatment for SMA involves promoting exon 7 inclusion in the SMN2 transcript or increasing the amount of full-length SMN by gene replacement with a viral vector. Increasing the pool of SMN2 transcripts and increasing their translational efficiency can be used to enhance splice correction. We sought to determine whether the 5' untranslated region (5' UTR) of SMN2 contains a repressive feature that can be targeted to increase SMN levels. We found that antisense oligonucleotides (ASOs) complementary to the 5' end of SMN2 increase SMN mRNA and protein levels and that this effect is due to inhibition of SMN2 mRNA decay. Moreover, use of the 5' UTR ASO in combination with a splice-switching oligonucleotide (SSO) increases SMN levels above those attained with the SSO alone. Our results add to the current understanding of SMN regulation and point toward a new therapeutic target for SMA

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12