Utrophin Modulators Research Articles

Molecular and Biochemical Therapeutic Strategies for Duchenne Muscular Dystrophy.

Significant progress has been achieved in understanding Duchenne muscular dystrophy (DMD) mechanisms and developing treatments to slow disease progression. This review article thoroughly assesses primary and secondary DMD therapies, focusing on innovative modalities. The primary therapy addresses the genetic abnormality causing DMD, specifically the absence or reduced expression of dystrophin. Gene replacement therapies, such as exon skipping, readthrough, and gene editing technologies, show promise in restoring dystrophin expression. Adeno-associated viruses (AAVs), a recent advancement in viral vector-based gene therapies, have shown encouraging results in preclinical and clinical studies. Secondary therapies aim to maintain muscle function and improve quality of life by mitigating DMD symptoms and complications. Glucocorticoid drugs like prednisone and deflazacort have proven effective in slowing disease progression and delaying loss of ambulation. Supportive treatments targeting calcium dysregulation, histone deacetylase, and redox imbalance are also crucial for preserving overall health and function. Additionally, the review includes a detailed table of ongoing and approved clinical trials for DMD, exploring various therapeutic approaches such as gene therapies, exon skipping drugs, utrophin modulators, anti-inflammatory agents, and novel compounds. This highlights the dynamic research field and ongoing efforts to develop effective DMD treatments.

3D-QSAR Modeling on 2-Pyrimidine Carbohydrazides as Utrophin Modulators for the Treatment of Duchenne Muscular Dystrophy by Combining CoMFA, CoMSIA, and Molecular Docking Studies.

The 3D-QSAR models were developed using CoMFA and CoMSIA techniques to investigate essential molecular fields, optimization strategies, and structure-activity relationships for utrophin-modulating compounds. The data set (71 molecules) was divided into two training and test sets using the hierarchical clustering approach. The training set was aligned based on the most active compound. The built and optimized models based on the PLS approach provided acceptable results. The results were q 2 = 0.528 and r 2 = 0.776 for CoMFA and q 2 = 0.600 and r 2 = 0.811 for CoMSIA models. According to the statistical results, it was found that both the CoMFA models with and without regional focusing and also the CoMSIA model have good estimation ability. Molecular docking was also performed with high-activity compounds (as ligands) and target receptors (protein), and its results, together with the results of 3D-QSAR, give new insights for the design of compounds with higher biological activity. Finally, based on the overall results, the design of new compounds with higher utrophin modulation activity was carried out.

Discovery of Quinazoline and Quinoline-Based Small Molecules as Utrophin Upregulators via AhR Antagonism for the Treatment of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease caused by the absence of a dystrophin protein. Elevating utrophin, a dystrophin paralogue, offers an alternative therapeutic strategy for treating DMD, irrespective of the mutation type. Herein, we report the design and synthesis of novel quinazoline and quinoline-based small molecules as potent utrophin modulators screened via high throughput In-Cell ELISA in C2C12 cells. Remarkably, lead molecule SG-02, identified from a library of 70 molecules, upregulates utrophin 2.7-fold at 800 nM in a dose-dependent manner, marking the highest upregulation within the nanomolar range. SG-02's efficacy was further validated through DMD patient-derived cells, demonstrating a significant 2.3-fold utrophin expression. Mechanistically, SG-02 functions as an AhR antagonist, with excellent binding affinity (Kd = 41.68 nM). SG-02 also enhances myogenesis, as indicated by an increased MyHC expression. ADME evaluation supports SG-02's oral bioavailability. Overall, SG-02 holds promise for addressing the global DMD population.

Structure-activity relationships of 2-pyrimidinecarbohydrazides as utrophin modulators for the potential treatment of Duchenne muscular dystrophy

A therapeutic approach that holds the potential to treat all Duchenne muscular dystrophy (DMD) patient populations is utrophin modulation. Ezutromid, a first generation utrophin modulator which was later found to act via antagonism of the arylhydrocarbon receptor, progressed to Phase 2 clinical trials. Although interim data showed target engagement and functional improvements, ezutromid ultimately failed to meet its clinical endpoints. We recently described the identification of a new class of hydrazide utrophin modulators which has a different mechanism of action to ezutromid. In this study we report our early optimisation studies on this hydrazide series. The new analogues had significantly improved potency in cell-based assays, increased sp3 character and reduced lipophilicity, which also improved their physicochemical properties. A representative new analogue combining these attributes increased utrophin protein in dystrophic mouse cells showing it can be used as a chemical tool to reveal new insights regarding utrophin upregulation as a strategy for DMD therapeutic intervention.

Utrophin modulator drugs as potential therapies for Duchenne and Becker muscular dystrophies.

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin‐deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co‐localise in human foetal muscle, in the dystrophin‐competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.

Discovery and mechanism of action studies of 4,6-diphenylpyrimidine-2-carbohydrazides as utrophin modulators for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a fatal disease with no cure, caused by lack of the cytoskeletal protein dystrophin. Upregulation of utrophin, a dystrophin paralogue, offers a potential therapy independent of mutation type. The failure of first-in-class utrophin modulator ezutromid/SMT C1100 in Phase II clinical trials necessitates development of compounds with better efficacy, physicochemical and ADME properties and/or complementary mechanisms. We have discovered and performed a preliminary optimisation of a novel class of utrophin modulators using an improved phenotypic screen, where reporter expression is derived from the full genomic context of the utrophin promoter. We further demonstrate through target deconvolution studies, including expression analysis and chemical proteomics, that this compound series operates via a novel mechanism of action, distinct from that of ezutromid.

Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.

Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 μM, solubility: 477 μM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.

2-Arylbenzo[d]oxazole Phosphinate Esters as Second-Generation Modulators of Utrophin for the Treatment of Duchenne Muscular Dystrophy.

Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27, led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid.

Duchenne muscular dystrophy (DMD) is a fatal muscle‐wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first‐in‐class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid

AbstractDuchenne muscular dystrophy (DMD) is a fatal muscle‐wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first‐in‐class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Synthesis of SMT022357 enantiomers and in vivo evaluation in a Duchenne muscular dystrophy mouse model

Following on from ezutromid, the first-in-class benzoxazole utrophin modulator that progressed to Phase 2 clinical trials for the treatment of Duchenne muscular dystrophy, a new chemotype was designed to optimise its physicochemical and ADME profile. Herein we report the synthesis of SMT022357, a second generation utrophin modulator preclinical candidate, and an asymmetric synthesis of its constituent enantiomers. The pharmacological properties of both enantiomers were evaluated in vitro and in vivo. No significant difference in the activity or efficacy was observed between the two enantiomers; activity was found to be comparable to the racemic mixture.

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase.

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ± 0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.

Isolation, Structural Identification, Synthesis, and Pharmacological Profiling of 1,2-trans-Dihydro-1,2-diol Metabolites of the Utrophin Modulator Ezutromid.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2/mdx Mice

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by mutations in the dystrophin gene. DMD boys are wheelchair-bound around 12 years and generally survive into their twenties. There is currently no effective treatment except palliative care, although personalized treatments such as exon skipping, stop codon read-through, and viral-based gene therapies are making progress. Patients present with skeletal muscle pathology, but most also show cardiomyopathy by the age of 10. A systemic therapeutic approach is needed that treats the heart and skeletal muscle defects in all patients. The dystrophin-related protein utrophin has been shown to compensate for the lack of dystrophin in the mildly affected BL10/mdx mouse. The purpose of this investigation was to demonstrate that AAV9-mediated micro-utrophin transgene delivery can not only functionally replace dystrophin in the heart, but also attenuate the skeletal muscle phenotype in severely affected D2/mdx mice. The data presented here show that utrophin can indeed alleviate the pathology in skeletal and cardiac muscle in D2/mdx mice. These results endorse the view that utrophin modulation has the potential to increase the quality life of all DMD patients whatever their mutation.

Neurology Care, Diagnostics, and Emerging Therapies of the Patient With Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is the most common form of childhood muscular dystrophy. A mutation in the DMD gene disrupts dystrophin (protein) production, causing damage to muscle integrity, weakness, loss of ambulation, and cardiopulmonary compromise by the second decade of life. Life expectancy has improved from mid-teenage years to mid-20s with the use of glucocorticoids and beyond the third decade with ventilator support and multidisciplinary care. However, Duchenne muscular dystrophy is associated with comorbidities and is a fatal disease. Glucocorticoids prolong ambulation, but their side effects are significant. Emerging investigational therapies have surfaced over the past decade and have rapidly been tested in clinical trials. Gene-specific strategies include nonsense readthrough, exon skipping, gene editing, utrophin modulation, and gene replacement. Other mechanisms include muscle regeneration, antioxidants, and antifibrosis and anti-inflammatory pathways. With potential therapies emerging, early diagnosis is needed to initiate treatment early enough to minimize morbidity and mortality. Newborn screening can be used to significantly improve early diagnosis, especially for gene-specific therapeutics.

Current and Emerging Therapies for Duchenne Muscular Dystrophy.

The purpose of this review is to summarize the current and emerging therapies for Duchenne muscular dystrophy (DMD). Coinciding with new standardized care guidelines, there are a growing number of therapeutic options to treat males with DMD. Treatment of the underlying pathobiology, such as micro-dystrophin gene replacement, exon skipping, stop codon read-through agents, and utrophin modulators showed variable success in animal and human studies. Symptomatic therapies to target muscle ischemia, enhance muscle regeneration, prevent muscle fibrosis, inhibit myostatin, and reduce inflammation are also under investigation. DMD is a complex, heterogeneous degenerative disease. The pharmacological and technological achievements made in recent years, plus timely supportive interventions will likely lead to an improved quality of life for many individuals with DMD.

P.393 - Computational alignment of duplex immunohistochemically-stained muscle sections in support of therapies for Duchenne muscular dystrophy

Continuous expression of utrophin protein by a utrophin modulator could be a disease modifying treatment for Duchenne muscular dystrophy (DMD) patients regardless of their dystrophin mutation. At least 3 biomarkers (a fibre identification biomarker, a regeneration biomarker, and utrophin biomarker) are needed to separate utrophin expression resulting from therapeutic intervention with a utrophin modulator from the pathophysiologic utrophin expression associated with muscle fibre regeneration. Technical challenges of multiplex assay development and analytical validation for 3 or more biomarkers limit the utility of such assays in clinical trials. Having successfully completed the analytical validation of utrophin-laminin α2 (merosin) and developmental myosin heavy chain (MHCd)-laminin α2 duplex immunohistochemical-Computational Tissue Analysis (IHC-cTA™) based assays, we have developed a fibre-based image analysis software to co-register consecutive and non-consecutive serial biopsy sections. The co-registration assay was developed utilising DMD, Becker muscular dystrophy (BMD), and control muscle biopsy sections obtained from the Wellstone Muscular Dystrophy Cooperative Research Center stained with each duplex assay highlighted above. Co-registration quality was numerically evaluated, and visually assessed by a veterinary pathologist. We demonstrate that the cTA™ approach reproducibly detects biomarker signal features (e.g., mean signal intensities), tissue morphometrics (e.g., fibre minimum diameter), and fibre classification (e.g., utrophin+/MHCd-) in whole-slide images of muscle cryosections at an individual muscle fibre resolution. This facilitates understanding of architectural and functional relationships between multiple biomarkers throughout biopsy specimens, and may provide valuable assessment of therapeutic efficacy in on-going and future clinical trials.

P.308 - Discovery of small molecule utrophin modulators for the therapy of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD, although various promising approaches (e.g. exon skipping, read through of stop codons, gene therapy) are being developed. By transcriptionally reprogramming the temporal and spatial expression of the dystrophin-related protein utrophin, we aim to develop a pharmacological therapy applicable to all DMD patients by targeting the primary defect. In partnership with Summit Therapeutics, the utrophin modulator ezutromid (formerly SMT C1100), which demonstrates reduced dystrophic symptoms in the mdx mouse, has progressed to human clinical trials. As a potential First-In-Class molecule, ezutromid is currently being evaluated in an open label phase 2 trial in DMD patients. A series of second generation utrophin modulators which are structurally similar to ezutromid, but with improved physicochemical and metabolism profiles have been evaluated in the mdx mouse, and results were published recently. Novel utrophin modulator chemotypes have also been discovered using an alternative in vitro dystrophin null myoblast screening assay where the reporter gene has been directly knocked into a utrophin exon. New structural classes which increase utrophin expression in both murine and human DMD myoblasts have been identified and are now being optimised. Initial evidence suggests that some of these small molecules modulate utrophin transcription through an alternative regulatory mechanism to ezutromid. These new compounds exhibit favourable properties and are well tolerated in the mdx mouse. SAR studies are underway, with the objective to improve compound effectiveness and exposure.

P.403 - PhaseOut DMD: a Phase 2, proof of concept, clinical study of utrophin modulation with ezutromid

This study investigates the hypothesis that Duchenne muscular dystrophy (DMD) can be treated by stimulating utrophin expression in muscles to replace missing or defective dystrophin. Ezutromid (SMT C1100) is an orally-available, small-molecule, utrophin modulator that enhances utrophin production in vitro and in the mdx mouse. In previous Phase I patient studies, ezutromid was shown to be safe and well tolerated. PhaseOut DMD is a Phase 2, open-label, study assessing the activity and safety of ezutromid, administered orally to ambulatory boys with DMD, aged 5–10 years. This is a multi-centre study in the UK and the US. It includes a 28-day Baseline and Screening Phase, a 48-week open-label Treatment Phase and a 30-day Safety Follow-up Phase. In Cohort 1, approximately 30 patients will receive 2.5 g ezutromid bid as a microfluidised aqueous oral suspension (F3). In Cohort 2 (US sites only) approximately 10 patients will receive 1 g ezutromid bid as a powder for oral suspension (F6). The study investigates the relationships between ezutromid levels and utrophin modulation, muscle markers of regeneration, and long-term impact on functional measures. MRI measurements of muscle adiposity and inflammation at 12 week intervals are a primary endpoint, along with plasma concentrations of ezutromid and its metabolites. Changes between baseline and 24 or 48 weeks in utrophin protein expression and in biomarkers of muscle regeneration are secondary endpoints, as are safety parameters. Functional tests, including the 6-minute walking distance (6MWD) test and the North Star Ambulatory Assessment (NSAA) global score, are exploratory endpoints. PhaseOut DMD is expected to complete enrolment in Q2 2017 and to report interim findings in Q1 2018. An extension of the study is planned. Results of this study are expected to facilitate the design of a pivotal study for the treatment of DMD. Details of the protocol and baseline data will be presented.

P.235 - Circulating miRs biomarkers for therapeutic monitoring in utrophin based DMD therapy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular pathology that results in progressive muscle weakness, paralysis and premature death. The disorder is caused by mutations which result in the absence of dystrophin, a protein localized underneath the sarcolemma. In collaboration with summit therapeutics plc, the Davies Lab has developed a promising therapeutic approach applicable to all DMD patients based on the modulation of utrophin, a structural and functional paralogue of dystrophin, to compensate its absence and ameliorate the dystrophic phenotype. One small molecule, ezumtromid is currently in clinical trial. Muscle biopsies are highly invasive so there is an urgent need to develop new approaches to measure efficacy of utrophin modulation clinical trials. Our project intends to decipher the miRome of serum from different murine models including wild-type, the dystrophic mdx, the transgenic mdx-Fiona mice expressing high levels of utrophin and the double knockout utrophin/dystrophin (dko) in order to provide a comprehensive description of myomiRs in these different utrophin contexts. Diverse miRNAs including miR-1, miR-133 and miR-206 have been previously described as highly elevated biomarkers in serum of mdx mice as well in DMD patients. Interestingly, their levels decreased in exon skipping therapy and were correlated to dystrophin restoration. These observations demonstrate the important role of circulating miRNAs as reliable biomarkers to monitor disease progression and response to treatment in DMD. A molecular profile of these miRNAs in mdx model and its modification by utrophin expression will allow us to compare utrophin modulation with other DMD therapies. Overall, our results should facilitate the clinical translation of utrophin modulation DMD therapy.