Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Abstract

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3 UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG) n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG) n -MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG) n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ␤-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases. Myotonic dystrophy type I (DM1)4 is the most common muscular dystrophy in the adult population, with a relatively high prevalence of about 1:8000 (1). This autosomal dominantly inherited disease affects multiple organs, most prominently the skeletal muscle, with wasting, weakness, and an inability to relax (myotonia) (1). Currently, there is no effective treatment for this disabling disease. The pathomechanism of DM1 is linked to a CTG n expansion in the 3Ј UTR of the dystrophia myotonica protein kinase (DMPK) gene (2, 3), leading to a toxic gain-of-function RNA (4, 5). The mutant DMPK transcript is entrapped within nuclei of affected cells, where it forms aggregates (foci) with splicing factors such as muscleblind-like 1 (MBNL1) (6, 7). Bound to mutant DMPK (CUG) n RNA, MBNL1 is no longer available for correct splicing of its target pre-mRNAs (8, 9). Thus, the splicing of a multitude of pre-mRNAs is misregulated, including the skeletal muscle chloride channel (CLCN1), the insulin receptor (INSR), sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATPase 1 (SERCA1), and cardiac troponin T type 2 (TNNT2) pre-mRNA (10 -16). Interestingly, the missplicing of some pre-mRNAs can be linked directly to a certain disease symptom, e.g. myotonia in the case of the CLCN1 pre-mRNA. MBNL1 sequestration by (CUG) n RNA causes inclusion of alternative exon 7a, leading to a shift in the open reading frame and to premature termination of translation (12, 1...

Autoregulation of the human splice factor kinase CLK1 through exon skipping and intron retention

“…We also treated DU145 cells with the drug harmine, a multifunctional alkaloid derived from the plant Peganum harmala that has a range of effects including antimicrobial, antitumour, and hallucinogenic (Patel et al, 2010;Moloudizargari et al, 2013). Harmine is also a potent inhibitor of the splice factor DYRK1A (Göckler at al., 2009), and it causes changes in alternative splicing in vivo (Herrendorf et al, 2016). DU145 cells were treated with 1-5M harmine for six hours.…”

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

“…HSA LR mice also recapitulate DM1 molecular characteristics such as foci accumulation, MBNL1 sequestration, and splicing abnormalities (5,7,8). Therapeutic strategies have mainly focused on targeting DM1-associated mis-splicing and mRNA toxicity (9)(10)(11), although a more complete understanding of pathogenic pathways would clearly be of interest for the development of alternative or additional therapeutic options. Recently, deregulation of cellular processes and signaling pathways important for maintaining proper muscle homeostasis has been reported in DM1.…”

Small Molecules Which Improve Pathogenesis of Myotonic Dystrophy Type 1

“…Aromatic alkaloids can interact with RNA, and indeed berberine and harmine (Table 1 ) have been reported to bind RNA structures ( 78 , 79 ). In DM1 myoblasts, harmine reduced foci, nevertheless it did not improve the histology in gastrocnemius muscle of the HSA LR mice, as the percentage of fibers with internalized nuclei was not altered by the treatment of the mice with the compound ( 77 ). However, although harmine increased the levels of MBNL1 in DM1 myoblasts and enhanced MBNL1-dependent alternative splicing of cTNT E5, INSR E11, and Clcn1 E7a (Table S1 in Supplementary Material), a similar effect was found in wild-type myoblasts, suggesting that inhibition of the CUG–MBNL1 complex is not the primary MoA of this alkaloid.…”

“…However, only a few studies have reported utilization of molecules of natural origin ( 40 , 76 ). Recently, a set of plant-derived alkaloids was identified as small molecules with an anti-DM1 effect ( 77 ). Using a novel CUG 78 –MBNL1 complex inhibition assay, a collection of isolated natural compounds and extracts from plants and fungal strains was screened.…”