piperidines and Muscular-Atrophy--Spinal

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. SMA results from insufficient survival motor neuron (SMN) protein due to alternative splicing. Antisense oligonucleotides, gene therapy and splicing modifiers recently received FDA approval. Although severe SMA transgenic mouse models have been beneficial for testing therapeutic efficacy, models mimicking milder cases that manifest post-infancy have proven challenging to develop. We established a titratable model of mild and moderate SMA using the splicing compound NVS-SM2. Administration for 30 d prevented development of the SMA phenotype in severe SMA mice, which typically show rapid weakness and succumb by postnatal day 11. Furthermore, administration at day eight resulted in phenotypic recovery. Remarkably, acute dosing limited to the first 3 d of life significantly enhanced survival in two severe SMA mice models, easing the burden on neonates and demonstrating the compound as suitable for evaluation of follow-on therapies without potential drug-drug interactions. This pharmacologically tunable SMA model represents a useful tool to investigate cellular and molecular pathogenesis at different stages of disease.

Topics: Animals; Animals, Newborn; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Kaplan-Meier Estimate; Mice; Mice, Transgenic; Motor Neurons; Muscular Atrophy, Spinal; Phenotype; Piperidines; Pyrazoles; Pyridazines; RNA Splicing; Survival of Motor Neuron 2 Protein; Time-to-Treatment

Topics: Amyotrophic Lateral Sclerosis; Benzamides; Edaravone; Humans; Muscular Atrophy, Spinal; Neuroprotective Agents; Oligonucleotides; Oligoribonucleotides, Antisense; Piperidines; Pyridines; Thiazoles; Thionucleotides

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron gene (SMN1) on chromosome 5q13. A second copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is truncated and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. In this study, we screened for the compounds that enhance SMN2 exon 7 inclusion by using SMN2 minigene-luciferase reporter system. We found that securinine can increase luciferase activity, indicating that securinine promoted SMN2 exon 7 inclusion. In addition, securinine increased full-length SMN2 mRNA and SMN protein expression in SMA patient-derived lymphoid cell lines. To investigate the mechanism of securinine effect on SMN2 splicing, we compared the protein levels of relevant splicing factors between securinine-treated and untreated cells. We found that securinine downregulated hnRNP A1 and Sam68 and upregulated Tra2-β1 expression. However, securinine, unlike HDAC inhibitors, did not enhance tra2-β1 gene transcription, indicating a post-transcriptional mechanism for Tra2-β1 upregulation. Furthermore, we treated SMA-like mice with securinine by i.p. injection and found that securinine treatment increased SMN2 exon 7 inclusion and SMN protein expression in the brain and spinal cord. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease.

Topics: Animals; Azepines; Cell Line; Central Nervous System Stimulants; Exons; Heterocyclic Compounds, Bridged-Ring; Heterogeneous-Nuclear Ribonucleoproteins; Lactones; Lymphoid Tissue; Mice; Muscular Atrophy, Spinal; Piperidines; Protein Splicing; RNA, Messenger; Serine-Arginine Splicing Factors; Survival of Motor Neuron 2 Protein

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure-property relationships including microsomal stability, cell permeability, and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models and a starting point for clinical development.

Topics: Alternative Splicing; Caco-2 Cells; Cell Membrane Permeability; Drug Design; Exons; Fibroblasts; Genes, Reporter; HEK293 Cells; Humans; Luciferases, Firefly; Male; Microsomes, Liver; Muscular Atrophy, Spinal; Piperidines; Promoter Regions, Genetic; Structure-Activity Relationship; Survival of Motor Neuron 1 Protein; Survival of Motor Neuron 2 Protein; Thiadiazoles; Thiazoles; Transcription, Genetic

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by death of motor neurons in the spinal cord that is caused by deletion and/or mutation of the survival motor neuron gene ( SMN1). Adjacent to SMN1 are a variable number of copies of the SMN2 gene. The two genes essentially differ by a single nucleotide, which causes the majority of the RNA transcripts from SMN2 to lack exon 7. Although both SMN1 and SMN2 encode the same Smn protein amino acid sequence, the loss of SMN1 and incorrect splicing of SMN2 have the consequence that Smn protein levels are insufficient for the survival of motor neurons. The therapeutic goal of our medicinal chemistry effort was to identify small-molecule activators of the SMN2 promoter that, by up-regulating gene transcription, would produce greater quantities of full-length Smn protein. Our initial medicinal chemistry effort explored a series of C5 substituted benzyl ether based 2,4-diaminoquinazoline derivatives that were found to be potent activators of the SMN2 promoter; however, inhibition of DHFR was shown to be an off-target activity that was linked to ATP depletion. We used a structure-guided approach to overcome DHFR inhibition while retaining SMN2 promoter activation. A lead compound 11a was identified as having high potency (EC50 = 4 nM) and 2.3-fold induction of the SMN2 promoter. Compound 11a possessed desirable pharmaceutical properties, including excellent brain exposure and long brain half-life following oral dosing to mice. The piperidine compound 11a up-regulated expression of the mouse SMN gene in NSC-34 cells, a mouse motor neuron hybrid cell line. In type 1 SMA patient fibroblasts, compound 11a induced Smn in a dose-dependent manner when analyzed by immunoblotting and increased the number of intranuclear particles called gems. The compound restored gems numbers in type I SMA patient fibroblasts to levels near unaffected genetic carriers of SMA.

Topics: Aminoquinolines; Animals; Biological Availability; Blood-Brain Barrier; Cell Line; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Fibroblasts; Folic Acid Antagonists; Heterozygote; Humans; Mice; Models, Molecular; Molecular Conformation; Muscular Atrophy, Spinal; Nerve Tissue Proteins; Permeability; Piperidines; Promoter Regions, Genetic; Quinazolines; RNA-Binding Proteins; SMN Complex Proteins; Spinal Muscular Atrophies of Childhood; Stereoisomerism; Structure-Activity Relationship; Survival of Motor Neuron 1 Protein; Survival of Motor Neuron 2 Protein; Tetrahydrofolate Dehydrogenase