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From dynamic combinatorial 'hit' to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy.

Ofori LO, Hoskins J, Nakamori M, Thornton CA, Miller BL - Nucleic Acids Res. (2012)

Bottom Line: We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro.Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA.Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14642, USA.

ABSTRACT
The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.

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Related in: MedlinePlus

Compound 11 improves MBNL1-dependent splicing in vivo. Asterisks (*) indicate statistical significance (P < 0.005); the dashed line is provided as a reference to splicing levels for HSALR mice in the absence of compound (n = 5 for compound-treated and control). The level of an MBNL1-independent splicing event (Capzb) is unaffected by 11. Error bars indicate SEM.
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gks298-F7: Compound 11 improves MBNL1-dependent splicing in vivo. Asterisks (*) indicate statistical significance (P < 0.005); the dashed line is provided as a reference to splicing levels for HSALR mice in the absence of compound (n = 5 for compound-treated and control). The level of an MBNL1-independent splicing event (Capzb) is unaffected by 11. Error bars indicate SEM.

Mentions: To examine effects on MBNL1-regulated splicing events we administered compounds 4, 9 or 11 to HSALR transgenic mice by daily intraperitoneal injection of 40 mg/kg for 5 days. In the initial experiment, analysis of splicing in hind limb (quadriceps) muscle showed that compound 4 produced a modest but statistically significant (P = 0.0379) improvement of Clcn1 splicing relative to saline-injected controls, whereas compound 9 did not (Figure 6). Statistically significant improvements in both Clcn1 and Atp2a1 (P = 0.0419 and P = 0.0223, respectively; Supplementary Figure S14) were observed following treatment with compound 11. To confirm these results, we carried out a further study in which five age-matched mice were treated with compound 11 via interperitoneal injection at 40 mg/kg once daily for 5 days, and compared with five age-matched controls. Once again, 11 provided a statistically significant improvement in Atp2a1 and Clcn1 splicing. Ttn, another transcript dependent on MBNL1-mediated splicing, also showed improvement (Figure 7). Splicing of Capzb, a developmentally regulated exon whose splicing does not depend on MBNL1, was not affected.Figure 6.


From dynamic combinatorial 'hit' to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy.

Ofori LO, Hoskins J, Nakamori M, Thornton CA, Miller BL - Nucleic Acids Res. (2012)

Compound 11 improves MBNL1-dependent splicing in vivo. Asterisks (*) indicate statistical significance (P < 0.005); the dashed line is provided as a reference to splicing levels for HSALR mice in the absence of compound (n = 5 for compound-treated and control). The level of an MBNL1-independent splicing event (Capzb) is unaffected by 11. Error bars indicate SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401475&req=5

gks298-F7: Compound 11 improves MBNL1-dependent splicing in vivo. Asterisks (*) indicate statistical significance (P < 0.005); the dashed line is provided as a reference to splicing levels for HSALR mice in the absence of compound (n = 5 for compound-treated and control). The level of an MBNL1-independent splicing event (Capzb) is unaffected by 11. Error bars indicate SEM.
Mentions: To examine effects on MBNL1-regulated splicing events we administered compounds 4, 9 or 11 to HSALR transgenic mice by daily intraperitoneal injection of 40 mg/kg for 5 days. In the initial experiment, analysis of splicing in hind limb (quadriceps) muscle showed that compound 4 produced a modest but statistically significant (P = 0.0379) improvement of Clcn1 splicing relative to saline-injected controls, whereas compound 9 did not (Figure 6). Statistically significant improvements in both Clcn1 and Atp2a1 (P = 0.0419 and P = 0.0223, respectively; Supplementary Figure S14) were observed following treatment with compound 11. To confirm these results, we carried out a further study in which five age-matched mice were treated with compound 11 via interperitoneal injection at 40 mg/kg once daily for 5 days, and compared with five age-matched controls. Once again, 11 provided a statistically significant improvement in Atp2a1 and Clcn1 splicing. Ttn, another transcript dependent on MBNL1-mediated splicing, also showed improvement (Figure 7). Splicing of Capzb, a developmentally regulated exon whose splicing does not depend on MBNL1, was not affected.Figure 6.

Bottom Line: We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro.Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA.Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14642, USA.

ABSTRACT
The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.

Show MeSH
Related in: MedlinePlus