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O01 Identification of new chemical compounds with upregulate utrophin for the therapy of Duchenne muscular dystrophy
Neuromuscular Disorders 22S1 (2012) S3–S5
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Abstracts, UK Neuromuscular Translational Research Conference 2012 Oral presentations
Thursday 22
nd
March 2012
O01 Identification of new chemical compounds with upregulate utrophin for the therapy of Duchenne muscular dystrophy R.J. Fairclough1 , S.E. Squire1 , A.C. Potter1 , D.S. Powell1 , S.G. Davies2 , C.J.R. Bataille2 , G.M. Wynne2 , A.J. Russell2,3 , K.E. Davies1 . 1 MRC Functional Genomics Unit, University of Oxford, Department of Physiology Anatomy and Genetics, South Parks Road, OX1 3PT, UK; 2 Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK; 3 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK DMD is a devastating X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no effective treatment. By pharmacologically upregulating the dystrophin-related protein utrophin, we aim to develop a therapy applicable to all DMD patients by targeting the primary defect and restoring sarcolemmal stability in DMD. In partnership with Summit plc, our previous drug screen lead to the development of SMT C1100 – the first drug to enter clinical trials based on utrophin upregulation for the therapy of DMD. This provided crucial proofof-principle for the strategy we have developed. We now aim to build on our previous results and perform a new drug screen based on improved in vitro and in vivo screening tools. A new screening assay based on immortalised myoblasts from the utrophin luciferase (LUmdx) knock-in mouse model enables us to screen the utrophin promoter in its genomic context. This should better mimic the in vivo situation and also enable identification of compounds which upregulate utrophin through regulatory pathways outside of the 10 kb promoter A fragment that formed the basis of our previous screen. Compounds (7000) have been specifically selected from a 25000 member library to enhance the hit rate whilst screening substantially fewer compounds. Selection criteria included similarity, yet structural distinction, to known utrophin upregulators, along with chemical scaffolds containing motifs of known transcriptional activators. Screening has identified new structural classes with utrophin upregulating capabilities together with compounds demonstrating enhanced activity compared to our current positive controls.
0960-8966/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
O02 Dysregulated mitophagy and mitochondrial transport in severe dominant optic atrophy due to OPA1 mutations C. Liao‡ 1 , N. Ashley‡ 1 , K. Morten1 , K. Phadwal1 , A. Williams2 , I. Fearnley2 , L. Rosser3 , J. Lowndes4 , C. Fratter5 , D. Ferguson6 , L. Vay8 , G. Quaghebeur8 , L. Macleod1 , A. Gabriel1 , S. Downes1 , K. Simon1 , M. Votruba7,8 , J. Poulton10 . 1 NIHR Translational Immunology Lab Biomedical Research Centre, Nuffield Department of Medicine, Oxford, UK; 2 Departments of Paediatrics and Ophthalmology, Northampton General Hospital, Northampton, UK; 3 Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK; 4 Department of Clinical Genetics, Churchill Hospital, Oxford, UK; 5 Molecular Genetics Laboratories, Churchill Hospital, Oxford, UK; 6 Nuffield Department of Pathology, Oxford, UK; 7 Cardiff Eye Unit, University Hospital, Wales, UK; 8 Department of Neuroradiology, West Wing, John Radcliffe Hospital, Oxford, UK; 9 School of Optometry and Vision Sciences, Cardiff University, Cardiff, UK; 10 Nuffield Dept Obstetrics and Gynaecology, The Women’s Centre, Oxford OX3 9DU, UK OPA1 mutations are the commonest cause of dominantly inherited optic atrophy (DOA), the Opa1 protein being essential for normal mitochondrial fusion. In mouse DOA, autophagy (recycling of spent cellular components) is dysregulated in retinal ganglion cells (RGCs), but mitophagy (mitochondrial recycling) has not been directly implicated. Profound down-regulation of OPA1 using RNAi caused mitochondrial fragmentation, loss of mitochondrial DNA, impaired mitochondrial function and perinuclear clustering (co-localising with Golgi and microtubule organising centre) in control fibroblasts. We identified a boy with a very severe DOA phenotype, presenting aged 2, developing an axonal sensory ataxic neuropathy and cognitive regression aged 10. Cranial MRI showed very small optic nerves. We identified the well established c.2708_2711delTTAG mutation in the OPA1 gene. He also had a c.661G>A OPA1 variant in trans, resulting in a Glu221Lys substitution. Abnormal fibroblast mitochondrial morphology was associated with the c.2708_2711delTTAG mutation. We validated and used novel ImageStream technology to quantitate mitophagy in primary cells, showing increased co-localisation of mitochondria and autophagomes, consistent with increased mitophagy in the patient’s fibroblasts. This was confirmed by western blotting. Ours is the first example of abnormal mitophagy in a human genetic disease. Furthermore, genetic background appears to influence penetrance. We suggest that OPA1 mutations cause constitutive mitochondrial fragmentation and dysregulated mitophagy. When OPA1 is profoundly reduced, increased mitophagy causes mitochondrial DNA depletion. RGCs may be particularly susceptible to mitochondrial DNA depletion and to impaired plus ended microtubule mediated transport, both of which may impair the supply of energy to sites of high energy usage. ‡ Equal first authors.
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Abstracts, UK Neuromuscular Translational Research Conference 2012 Oral presentations
Thursday 22
nd
March 2012
O01 Identification of new chemical compounds with upregulate utrophin for the therapy of Duchenne muscular dystrophy R.J. Fairclough1 , S.E. Squire1 , A.C. Potter1 , D.S. Powell1 , S.G. Davies2 , C.J.R. Bataille2 , G.M. Wynne2 , A.J. Russell2,3 , K.E. Davies1 . 1 MRC Functional Genomics Unit, University of Oxford, Department of Physiology Anatomy and Genetics, South Parks Road, OX1 3PT, UK; 2 Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK; 3 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK DMD is a devastating X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no effective treatment. By pharmacologically upregulating the dystrophin-related protein utrophin, we aim to develop a therapy applicable to all DMD patients by targeting the primary defect and restoring sarcolemmal stability in DMD. In partnership with Summit plc, our previous drug screen lead to the development of SMT C1100 – the first drug to enter clinical trials based on utrophin upregulation for the therapy of DMD. This provided crucial proofof-principle for the strategy we have developed. We now aim to build on our previous results and perform a new drug screen based on improved in vitro and in vivo screening tools. A new screening assay based on immortalised myoblasts from the utrophin luciferase (LUmdx) knock-in mouse model enables us to screen the utrophin promoter in its genomic context. This should better mimic the in vivo situation and also enable identification of compounds which upregulate utrophin through regulatory pathways outside of the 10 kb promoter A fragment that formed the basis of our previous screen. Compounds (7000) have been specifically selected from a 25000 member library to enhance the hit rate whilst screening substantially fewer compounds. Selection criteria included similarity, yet structural distinction, to known utrophin upregulators, along with chemical scaffolds containing motifs of known transcriptional activators. Screening has identified new structural classes with utrophin upregulating capabilities together with compounds demonstrating enhanced activity compared to our current positive controls.
0960-8966/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
O02 Dysregulated mitophagy and mitochondrial transport in severe dominant optic atrophy due to OPA1 mutations C. Liao‡ 1 , N. Ashley‡ 1 , K. Morten1 , K. Phadwal1 , A. Williams2 , I. Fearnley2 , L. Rosser3 , J. Lowndes4 , C. Fratter5 , D. Ferguson6 , L. Vay8 , G. Quaghebeur8 , L. Macleod1 , A. Gabriel1 , S. Downes1 , K. Simon1 , M. Votruba7,8 , J. Poulton10 . 1 NIHR Translational Immunology Lab Biomedical Research Centre, Nuffield Department of Medicine, Oxford, UK; 2 Departments of Paediatrics and Ophthalmology, Northampton General Hospital, Northampton, UK; 3 Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK; 4 Department of Clinical Genetics, Churchill Hospital, Oxford, UK; 5 Molecular Genetics Laboratories, Churchill Hospital, Oxford, UK; 6 Nuffield Department of Pathology, Oxford, UK; 7 Cardiff Eye Unit, University Hospital, Wales, UK; 8 Department of Neuroradiology, West Wing, John Radcliffe Hospital, Oxford, UK; 9 School of Optometry and Vision Sciences, Cardiff University, Cardiff, UK; 10 Nuffield Dept Obstetrics and Gynaecology, The Women’s Centre, Oxford OX3 9DU, UK OPA1 mutations are the commonest cause of dominantly inherited optic atrophy (DOA), the Opa1 protein being essential for normal mitochondrial fusion. In mouse DOA, autophagy (recycling of spent cellular components) is dysregulated in retinal ganglion cells (RGCs), but mitophagy (mitochondrial recycling) has not been directly implicated. Profound down-regulation of OPA1 using RNAi caused mitochondrial fragmentation, loss of mitochondrial DNA, impaired mitochondrial function and perinuclear clustering (co-localising with Golgi and microtubule organising centre) in control fibroblasts. We identified a boy with a very severe DOA phenotype, presenting aged 2, developing an axonal sensory ataxic neuropathy and cognitive regression aged 10. Cranial MRI showed very small optic nerves. We identified the well established c.2708_2711delTTAG mutation in the OPA1 gene. He also had a c.661G>A OPA1 variant in trans, resulting in a Glu221Lys substitution. Abnormal fibroblast mitochondrial morphology was associated with the c.2708_2711delTTAG mutation. We validated and used novel ImageStream technology to quantitate mitophagy in primary cells, showing increased co-localisation of mitochondria and autophagomes, consistent with increased mitophagy in the patient’s fibroblasts. This was confirmed by western blotting. Ours is the first example of abnormal mitophagy in a human genetic disease. Furthermore, genetic background appears to influence penetrance. We suggest that OPA1 mutations cause constitutive mitochondrial fragmentation and dysregulated mitophagy. When OPA1 is profoundly reduced, increased mitophagy causes mitochondrial DNA depletion. RGCs may be particularly susceptible to mitochondrial DNA depletion and to impaired plus ended microtubule mediated transport, both of which may impair the supply of energy to sites of high energy usage. ‡ Equal first authors.