Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

Moorwood, Catherine; Lozynska, Olga; Suri, Neha; Napper, Andrew D.; Diamond, Scott L.; Khurana, Tejvir S.

doi:10.1371/journal.pone.0026169

Cited by 65 publications

(70 citation statements)

References 59 publications

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“…Mice treated at the higher dose showed little to no improvements in the dystrophic pathology. A similar trend in dose response was observed earlier during a drug-screen for natural compounds that activate the utrophin A promoter (46). With the use of a luciferase reporter promoter assay in cultured myogenic C 2 C 12 cells, it was discovered that RSV could activate the utrophin A promoter, with the maximum fold change in luciferase activity observed at a relatively low dose of 6.25 M. These dose-response effects noted in earlier studies mirror much of what we observed in our investigation.…”

Section: MDXsupporting

confidence: 82%

“…Following the 6-wk treatment period, mice were euthanized by CO 2 asphyxiation and cervical dislocation and the tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAST) muscles were harvested and immediately frozen in liquid nitrogen or in melting isopentane cooled with liquid nitrogen for histological and immunofluorescence analyses. For the second RSV protocol, treated mdx mice were given a standard control diet, supplemented with RSV (ϳ 500 mg·kg Ϫ1 ·day Ϫ1 ) based on earlier work (29,46), and the duration was extended to 12 wk. This was considered a high dose and a long duration (HDLD).…”

Section: Methodsmentioning

confidence: 99%

“…Because of the promising results described above, we decided to perform an additional series of experiments where both the daily RSV dose and treatment duration were increased in an effort to determine if greater exposure to RSV yielded a more robust induction of therapeutic skeletal muscle plasticity. The dose was increased to ϳ500 mg·kg Ϫ1 ·day Ϫ1 , based in part, on recent literature (29,46), while the duration was extended to 12 wk (HDLD). Thus male mice of 6 -7 wk of age were once again divided into three groups, based on genetic background and diet: 1) WT-CTL, 2) MDX-CTL, and 3) MDX-RSV-HDLD.…”

Section: Rsv-mdsd Induces Sirt1 and Pgc-1␣ Signaling Andmentioning

confidence: 99%

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Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis

Ljubicic

Burt

Lunde

et al. 2014

American Journal of Physiology-Cell Physiology

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Ljubicic V, Burt M, Lunde JA, Jasmin BJ. Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1␣ axis. Am J Physiol Cell Physiol 307: C66 -C82, 2014. First published April 24, 2014; doi:10.1152/ajpcell.00357.2013.-Slower, more oxidative muscle fibers are more resistant to the dystrophic pathology in Duchenne muscular dystrophy (DMD) patients as well as in the preclinical mdx mouse model of DMD. Therefore, one therapeutic strategy for DMD focuses on promoting expression of the slow, oxidative myogenic program. In the current study, we explored the therapeutic potential of stimulating the slow, oxidative phenotype in mdx mice by feeding 6-wk-old animals with the natural phenol resveratrol (RSV; ϳ100 mg·kg Ϫ1 ·day Ϫ1 ) for 6 wk. Sirtuin 1 (SIRT1) activity and protein levels increased significantly, as well as peroxisome proliferatoractivated receptor-␥ coactivator-1␣ (PGC-1␣) activity, in the absence of alterations in AMPK signaling. These adaptations occurred concomitant with evidence of a fast, glycolytic, to slower, more oxidative fiber type conversion, including mitochondrial biogenesis and increased expression of slower myosin heavy chain isoforms. These positive findings raised the question of whether increased exposure to RSV would result in greater therapeutic benefits. We discovered that an elevated RSV dose of ϳ500 mg·kg Ϫ1 ·day Ϫ1 across a duration of 12 wk was clearly less effective at muscle remodeling in mdx mice. This treatment protocol failed to influence SIRT1 or AMPK signaling and did not result in a shift towards a slower, more oxidative phenotype. Taken together, this study demonstrates that RSV can stimulate SIRT1 and PGC-1␣ activation, which in turn may promote expression of the slow, oxidative myogenic program in mdx mouse muscle. The data also highlight the importance of selecting an appropriate dosage regimen of RSV to maximize its potential therapeutic effectiveness for future application in DMD patients. DMD; SIRT1; utrophin A; PGC-1␣; AMPK DUCHENNE MUSCULAR DYSTROPHY (DMD) is a life-limiting, progressive muscle wasting disease that causes the loss of muscle function and independence. Genetic disruption of the dystrophin gene prevents the synthesis of the full-length dystrophin protein in skeletal muscle, which is the primary cause of the pathology (53). In the absence of dystrophin, the recruitment of the dystrophin-associated protein complex to the sarcolemma is impaired thereby creating a pathophysiological cascade with numerous adverse downstream events, including compromised sarcolemmal integrity, increased intracellular calcium, defective mitochondrial morphology, myofibrillar degradation, and ultimately death of the fibers (12). Over time, repeated cycles of muscle degeneration/regeneration result in the exhaustion of the muscle progenitor pool and the failure of regenerative capacity. Multiple experimental approaches to treat DMD are currently under investigation, including exon skipping and stop codo...

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Section: MDXsupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Rsv-mdsd Induces Sirt1 and Pgc-1␣ Signaling Andmentioning

confidence: 99%

See 1 more Smart Citation

Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis

Ljubicic

Burt

Lunde

et al. 2014

American Journal of Physiology-Cell Physiology

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“…Our results are similar to those found in other studies that have been used in preclinical and clinical trials. For example, the nonsteroidal antiinflammatory drug nabumetone upregulates utrophin protein expression 1.2‐fold in C2C12 cells 26. SMTC1100, a drug tested in phase I clinical trials, upregulated utrophin twofold in human DMD cells and showed an ∼75% increase in expression in the diaphragm of mdx mice 27…”

Section: Discussionmentioning

confidence: 99%

Anisomycin Activates Utrophin Upregulation Through a p38 Signaling Pathway

Hadwen

Farooq

Witherspoon

et al. 2018

Clinical Translational Sci

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Duchenne muscular dystrophy is a recessive X‐linked disease characterized by progressive muscle wasting; cardiac or respiratory failure causes death in most patients by the third decade. The disease is caused by mutations in the dystrophin gene that lead to a loss of functional dystrophin protein. Although there are currently few treatments for Duchenne muscular dystrophy, previous reports have shown that upregulating the dystrophin paralog utrophin in Duchenne muscular dystrophy mouse models is a promising therapeutic strategy. We conducted in silico mining of the Connectivity Map database for utrophin‐inducing agents, identifying the p38‐activating antibiotic anisomycin. Treatments of C2C12, undifferentiated murine myoblasts, and mdx primary myoblasts with anisomycin conferred increases in utrophin protein levels through p38 pathway activation. Anisomycin also induced utrophin protein levels in the diaphragm of mdx mice. Our study shows that repositioning small molecules such as anisomycin may prove to have Duchenne muscular dystrophy clinical utility.

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“…29,30 However, recent studies show that regulation of utrophin translation and mRNA stability via UTR-mediated mechanisms is of great importance in the control of utrophin protein levels. Indeed, the repressive posttranscriptional regulation of utrophin may be part of the reason that utrophin upregulating therapies have not yet reached the clinic.…”

Section: Discussionmentioning

confidence: 99%

A Cell-Based High-Throughput Screening Assay for Posttranscriptional Utrophin Upregulation

et al. 2013

Self Cite

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Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease caused by mutations in the dystrophin gene. Utrophin is a homologue of dystrophin that can compensate for its absence when overexpressed in DMD animal models. Utrophin upregulation is therefore a promising therapeutic approach for DMD. Utrophin is regulated at both transcriptional and posttranscriptional levels. Transcriptional regulation has been studied extensively, and assays have been described for the identification of utrophin promoter-targeting molecules. However, despite the profound impact that posttranscriptional regulation has on utrophin expression, screening assays have not yet been described that could be used to discover pharmaceuticals targeting this key phase of regulation. We describe the development and validation of a muscle cell linebased assay in which a stably expressed luciferase coding sequence is flanked by the utrophin 5′-and 3′-untranslated regions (UTRs). The assay was validated using the posttranscriptional regulation of utrophin by miR-206. The assay has a Z′ of 0.7, indicating robust performance in high-throughput format. This assay can be used to study utrophin regulatory mechanisms or to screen chemical libraries for compounds that upregulate utrophin posttranscriptionally via its UTRs. Compounds identified via this assay, used alone or in a synergistic combination with utrophin promoter-targeting molecules, would be predicted to have therapeutic potential for DMD.

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Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

Cited by 65 publications

References 59 publications

Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis

Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis

Anisomycin Activates Utrophin Upregulation Through a p38 Signaling Pathway

A Cell-Based High-Throughput Screening Assay for Posttranscriptional Utrophin Upregulation

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