Figure 1. In vitro activity of SMT C1100. (A) SMT C1100 dose response in murine H2k-mdx utrnA-luc cells expressing the human utrophin promoter linked to a luciferase reporter gene. Cells were treated with compound for 48 h in standard growth medium containing 0.3% DMSO. The chart shows relative luminescence (RLU) in relation to five different doses of SMT C1100. A Four Parameter Logistic Model was used to generate an EC50. Points represent a mean 6S.E. of three experiments and are typical of the results for all batches of SMTC1100. The structure for SMTC1100 is shown; (B) SMT C1100 significantly increased mRNA copy number of the utrophin transcript in SkMC cells. In this assay Gene Expression Assay 4326315 was used for b- actin detection and Gene Expression Assay Hu01125984_m1 was used for utrophin transcript detection (both Applied Biosystems). Cells were exposed to SMT C1100 in standard media with 1% DMSO (vehicle) for SMT C1100 Reduces mdx Dystrophic Symptoms 72 hours with six biological replicates. *p = 0.01 relative to vehicle only; (C) Utrophin protein levels in human DMD cell line treated with SMT C1100 (1 mM) or vehicle (0.1% DMSO). Blots were stained with anti- utrophin (MANCHO3; 1:100) and ECL HRP-conjugated anti-mouse antibody (GE Healthcare). Bands were quantified using Image J and arbitrary units represent utrophin levels corrected for equal loading by a-actinin immunostaining. Results represent a mean 6 S.E based on n = 3. {p = 0.00683; 1p,0.001; #p,0.005 relative to vehicle-treated cells. doi:10.1371/journal.pone.0019189.g001 1 = mild - Focal expansion of the inter-bundle connec- tive tissue; mild focal expansion of the intra-bundle space may be present. 2 = moderate - Multiple foci of expansion of the connective tissue component in the inter-bundle area; focal intra-bundle increases in connective tissue between individual muscle fibres. 3 = severe - Multiple large foci of connective tissue in the inter-bundle region extending into the intra-bundle connective tissue with expansion of the inter-bundle and intra-bundle space. In addition two 42-bit color images were captured with a Zeiss AxioCam HR digital camera on a Zeiss Axioskop 2 microscope utilizing AxioVision 4.4 software (Zeiss) of each muscle on the H&E stained slides. Once the images were captured they were white balanced in Adobe Photoshop (Adobe). The proportion of centrally nucleated fibers was determined by analyzing the images and counting the number of centrally located nuclei; a total of two hundred cells per muscle were evaluated. Students’ two-tailed t- test was used to compare the groups with significance set at p,0.05. Statistical Analyses Significance was calculated using the Student’s t test with a two- tailed distribution assuming unequal sample variance. Multiple statistical comparisons between groups, was performed by one-way ANOVA, with Bonferroni’s t test post-hoc correction for allowing a better evaluation of intra- and inter-group variability and avoiding false positive. Results In vitro upregulation of utrophin SMT C1100 was identified from an iterative analoging approach from initial hits identified using a human muscle specific utrophin A promoter cell-based assay. Myoblasts (mdx) were cloned from H-2K-tsA586mdx with an IFN/tsSV40 T-Ag transgene in order to control proliferation and fusion [13]. The screening line named H2K-mdx utrnA-luc contains a stably integrated reporter consisting of 8.4 kb of the human utrophin promoter linked to a luciferase reporter gene. The region of the utrophin promoter contained all the motifs known to control utrophin expression [19,20]. This high throughput screening assay identified a number of luciferase-inducing compounds that also have the ability to increase the transcription of the endogenous mouse UTRN, thus identifying compounds with both human and mouse activity eventually leading to the final optimized compound, SMT C1100 whose chemical structure is shown in Fig. 1A. SMT C1100 shows a maximal increase of four to five-fold compared to vehicle with an EC50 of 0.4 mM (Fig. 1A). In vitro dosing of human myoblasts with SMT C1100 leads to a 25% increase in utrophin mRNA (Fig. 1B) when compared to vehicle- only dosing after three days of treatment. Treatment of human PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19189