ABSTRACT
Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.
Subject(s)
Muscular Atrophy, Spinal/drug therapy , Piperazines/pharmacology , RNA Precursors/metabolism , RNA Splicing/drug effects , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Biflavonoids/pharmacology , Cell-Free System , Computational Biology , Epoxy Compounds/pharmacology , Exons/genetics , Fibroblasts , HEK293 Cells , HeLa Cells , Humans , Ligands , Macrolides/pharmacology , Muscular Atrophy, Spinal/genetics , Piperazines/chemical synthesis , Protein Binding , Protein Structure, Quaternary , Proteomics/methods , RNA Precursors/genetics , RNA, Messenger/genetics , Spliceosomes/drug effects , Spliceosomes/metabolism , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 2 Protein/geneticsABSTRACT
Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.
Subject(s)
Alternative Splicing/drug effects , Muscular Atrophy, Spinal/drug therapy , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , RNA, Messenger/genetics , Survival of Motor Neuron 2 Protein/genetics , Animals , Exons/drug effects , Humans , Mice , Muscular Atrophy, Spinal/genetics , Pyrimidinones/pharmacokinetics , Pyrimidinones/therapeutic useABSTRACT
Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.
Subject(s)
Alternative Splicing/drug effects , Coumarins/administration & dosage , Isocoumarins/administration & dosage , Longevity/drug effects , Muscular Atrophy, Spinal/drug therapy , Pyrimidinones/administration & dosage , Small Molecule Libraries/administration & dosage , Survival of Motor Neuron 2 Protein/genetics , Administration, Oral , Animals , Cells, Cultured , Coumarins/chemistry , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Isocoumarins/chemistry , Mice , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Pyrimidinones/chemistry , RNA, Messenger/genetics , Sequence Deletion , Small Molecule Libraries/chemistry , Survival of Motor Neuron 2 Protein/metabolismABSTRACT
We characterized the lability and bioaccessibility of Zn, Pb, and Cd in size-fractionated mine waste at the Tar Creek Superfund Site (Oklahoma) to assess the potential for metal transport, exposure, and subsequent bioavailability. Bulk mine waste samples contained elevated Zn (9100 +/- 2500 ppm), Pb (650 +/- 360 ppm), and Cd (42 +/- 10 ppm), while particles with the greatest potential for windborne transport and inhalation (< 10 microm) contained substantially higher concentrations, up to 220 000 ppm Zn, 16 000 ppm Pb, and 530 ppm Cd in particles < 1 microm. Although the mined ore at Tar Creek primarily consisted of refractory metal sulfides with low bioavailability, sequential extractions and physiologically based extractions indicate that physical and chemical weathering have shifted metals into relatively labile and bioaccessible mineral phases. In < 37 microm mine waste particles, 50-65% of Zn, Pb, and Cd were present in the "exchangeable" and "carbonate" sequential extraction fractions, and 60-80% of Zn, Pb, and Cd were mobilized in synthetic gastric fluid, while ZnS and PbS exhibited minimal solubility in these solutions. Our results demonstrate the importance of site-specific characterization of size-fractionated contemporary mine waste when assessing the lability and bioavailability of metals at mine-waste impacted sites.
