Discovery of a CNS penetrant small molecule SMN2 splicing modulator with improved tolerability for spinal muscular atrophy

Figures

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  Figure 1. TEC-1 is structurally similar to SMN-C3 or risdiplam and modulates SMN2 splicing. (a) Pharmacophore model and alignment of TEC-1 (green), risdiplam (pink), and SMN-C3 (blue) with MOE2018.01. Pharmacophore annotation: Aromatic and other pi-system rings (orange), H-bond donor heavy atom (magenta), H-bond acceptor heavy atom (pale blue). (b) Structure of TEC-1. (c) Structure of SMN-C3. (d) Structure of risdiplam. (e) qPCR analysis of FL-SMN2 transcripts in SMA type II fibroblasts (GM03813) exposed to each compound for 24 h. The amounts of mRNA were normalized to those of GAPDH. (f) qPCR analysis of Δ7 transcripts of GM03813 cells exposed to each compound for 24 h. The amounts of mRNA were normalized to those of GAPDH. Data are from two biologically independent samples in e, f. a-d were derived by by Axcelead Drug Discovery Partners, Inc.

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  Figure 2. TEC-1 does not substantially affect FOXM1 splicing. (a) Schematic representation of exon construction and exon inclusion of FOXM1 mRNA splicing. (b) RT-PCR analysis of FOXM1 variants of GM03813 fibroblasts exposed to each compound for 24 h. RT-PCR products of FOXM1 variants were separated by agarose gel electrophoresis and visualized with ethidium bromide. Standard FOXM1 fragments corresponding to three isoforms (Oligo STD) are shown. Molecular weight markers are shown on the right (bp). The full-length gel is presented in Supplementary Fig. 1e. (c–e) qPCR analysis of each FOXM1 variant of GM03813 cells exposed to each compound for 24 h. The amounts of each variant were normalized to those of GAPDH. Data are from two biologically independent samples in (c–e).

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  Figure 3. TEC-1 does not strongly impact GALC cryptic splicing. (a) qPCR analysis of GALC transcripts in GM03813 cells treated with each compound for 24 h. The amounts of GALC mRNA were normalized to those of GAPDH. (b) Schematic representation of exon construction and cryptic exon inclusion of GALC mRNA splicing. (c) RT-PCR analysis of GALC transcripts in GM03813 cells treated for 6 h with both compounds and CHX. RT-PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. Standard GALC fragments (Oligo STD) with or without the cryptic exon are shown. Molecular weight markers are shown on the right (bp). The full-length gel is presented in Supplementary Fig. 2c. (d) qPCR analysis of the GALC cryptic exon in GM03813 cells treated for 6 h with both compounds and CHX. Aberrant GALC mRNA amounts with cryptic exons were quantified and normalized to those of GAPDH. (e) GALC enzymatic assay from human oligodendroglioma (Hs683) cells treated with each compound for 3 days. The amounts of standardized product (fluorogenic HMU-β-D galactopyranoside) were normalized to those of total protein. (f) Ratio of psychosine and sphingosine in Hs683 cells treated with…

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  Figure 4. TEC-1 does not strongly affect HTT cryptic splicing and protein abundance unlike SMN-C3. (a) qPCR analysis of HTT transcripts in GM03813 cells treated with each compound for 24 h. The amounts of HTT mRNA were normalized to those of GAPDH. (b) Schematic representation of exon construction and cryptic exon inclusion or exclusion of HTT mRNA splicing. (c) RT-PCR analysis of HTT transcripts in GM03813 cells treated for 6 h with compounds and CHX. RT-PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. Standard HTT fragments with or without the cryptic exon (Oligo STD) are shown. Molecular weight markers are shown on the right (bp). Full-length gels are presented in Supplementary Fig. 5b. (d) qPCR analysis of cryptic HTT transcripts of GM03813 cells treated for 6 h with compounds and CHX. The amounts of HTT with the cryptic exon were quantified and normalized to those of GAPDH. Values less than the quantitation limit were presumed to be 0. (e) Full-length (FL)-HTT protein levels of GM03813 fibroblasts treated with each compound for 3 days. Following western blot analyses, the signals of FL-HTT protein were analyzed and were normalized to…

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  Figure 5. TEC-1 displays disease-modifying effects in motor neurons derived from SMA patient iPSCs (GM24468). (a) qPCR analysis of the SMN transcripts of motor neurons treated with TEC-1 for 24 h. The amounts of mRNA, including (FL-SMN2), and excluding (Δ7) exon 7, were normalized to those of GAPDH. (b) SMN protein levels in motor neurons treated with TEC-1 for 3 days. SMN protein in cell lysates was quantified with homogeneous time resolved fluorescence (HTRF) and normalized to total protein. (c) The level of FL-SMN protein. The SMN density in western blots was normalized to GAPDH density. Full-length blots are presented in Supplementary Fig. 10. (e) The levels of ChAT protein. The ChAT density in western blots was normalized to GAPDH density. Full-length blots are presented in Supplementary Fig. 10. Data in (a–d) represent means ± SEM of three independent assessments per concentration. *p < 0.05, **p < 0.01, ***p < 0.001 as assessed by one-way ANOVA followed by Dunnett’s test using DMSO-treated cells as a control (100%).

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  Figure 6. TEC-1 rescues the phenotype in a murine model of SMA. (a) Appearance of a vehicle-treated SMA mouse model, a 20 mg/kg TEC-1-treated SMA mouse model, and a vehicle-treated heterozygous mouse (HET) at P13. The murine model of SMA was treated from P2 to P23 once daily with vehicle or TEC-1 by intraperitoneal administration (i.p.) at 2, 6, or 20 mg/kg, and thereafter once daily by oral administration (p.o.) or i.p. with 2, 6, or 20 mg/kg. (b) Body weight from P2 through P65. (c–f) Righting reflex of SMA mice. The mean time for mice to right themselves after being put onto their backs, assessed at P6, P11, and P16, total duration. #p < 0.05, ##p < 0.01 and ###p < 0.001 (vs. HET/Hetero, vehicle) as assessed by Student’s t-test (unpaired, two-tailed). *p < 0.05, **p < 0.01 and ***p < 0.001 as assessed by one-way ANOVA followed by Dunnett’s test using vehicle-treated SMA mice as a control. Individuals who died had a value of 60 s (maximum). The number of mice (N) is shown in the graph. (g) Kaplan–Meier survival curves from birth to P65. Numbers at the right indicate survivors at P65 mice per group. The log-rank (Mantel-Cox) test and Bonferroni test were used for comparisons. *p < 0.05,…