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Mutations of the mitochondrial genome Treatment

Mutations of the mitochondrial genome Treatment. Dimitra Smaragda Kyriakouli. The “Magic Circle”. DiMauro,S and Schon,E, NEJM 348 , 2659. Disorders due to mtDNA mutations. DiMauro,S and Schon,E, NEJM 348 , 2660. Clinical Features of mtDNA-associated diseases.

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Mutations of the mitochondrial genome Treatment

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  1. Mutations of the mitochondrial genomeTreatment Dimitra Smaragda Kyriakouli

  2. The “Magic Circle” DiMauro,S and Schon,E, NEJM 348, 2659

  3. Disorders due to mtDNA mutations DiMauro,S and Schon,E, NEJM 348, 2660

  4. Clinical Features of mtDNA-associated diseases Chinnery, P and Schon,E, JNNP 74, 1193

  5. Treatment Approaches • Vitamin cocktails [1] and exercise regimes [2] ineffective • Partially and temporary rescue biochemical and clinical defects • Overall aim = Permanently reduce mutated mtDNA proportion to SUBTHRESHOLD LEVELS … so THERAPY STILL REMAINS ELUSIVE [1] B. Marriage, M.T. Clandinin and D.M. Glerum, J. Am. Diet. Assoc. 103 (2003): 1029-1038. [2] T. Taivassalo, K. Fu, T. Johns, D. Arnold, G. Karpati and E.A. Shoubridge, Hum Mol Genetics. 8 (1999): 1047-1052

  6. Treatment Approaches • Vitamin cocktails [1] and exercise regimes [2] ineffective • Partially and temporary rescue biochemical and clinical defects • Overall aim = Permanently reduce mutated mtDNA proportion to SUBTHRESHOLD LEVELS … so THERAPY STILL REMAINS ELUSIVE • Manipulation of the heteroplasmy levels via an antigenomic approach [3] • Antigenomic molecule selectively inhibit propagation of mutated mtDNA species • Replication of the WT species only • Overtime, the cellular defect rescued and in theory at least disease progression reversed [1] B. Marriage, M.T. Clandinin and D.M. Glerum, J. Am. Diet. Assoc. 103 (2003): 1029-1038. [2] T. Taivassalo, K. Fu, T. Johns, D. Arnold, G. Karpati and E.A. Shoubridge, Hum Mol Genetics. 8 (1999): 1047-1052 [3] P.M. Smith, G.F. Ross, R.W. Taylor, D.M. Turnbull and R.N. Lightowlers, BBA-Bioenergetics. 1659 (2004): 232-239

  7. Properties of the ANTIGENOMIC AGENT • Hydrophilic (happily reside within cells), • Lipophilic (import) BUT not too lipophilic (bound within membranes) • Bind specifically and strongly to intended target (singly mismatch significantly affect binding ability) • Inhibit mtDNA polymerase γ

  8. Properties of the ANTIGENOMIC AGENT • Hydrophilic (happily reside within cells), • Lipophilic (import) BUT not too lipophilic (bound within membranes) • Bind specifically and strongly to intended target (singly mismatch significantly affect binding ability) • Inhibit mtDNA polymerase γ Cell Membrane Cross Oligomer (CMCO)

  9. Cell Membrane Cross Oligomer (CMCO)

  10. Cell Membrane Cross Oligomer (CMCO) Nielsen PNA monomer Ugichem CMCO monomer

  11. = Nielsen PNA monomer = N = Ugichem CMCO monomer = U The promising CMCO patterns • 1st PATTERN • 1:1 N:U backbone monomers • 2nd PATTERN • 1:1 N:U backbone monomers • Cluster of N monomers in the middle

  12. O- O- OH O- O- NO2 NO2 NO2 NO2 NO2 = Nielsen PNA monomer = N = Ugichem CMCO monomer = U NO2 NO2 NO2 NO2 NO2 = 2,4 DiNitroPhenol = DN pH=7.4 The promising CMCO patterns • 1st PATTERN • 1:1 N:U backbone monomers • 2nd PATTERN • 1:1 N:U backbone monomers • Cluster of N monomers in the middle deprotonated protonated

  13. O- O- OH O- O- NO2 NO2 NO2 NO2 NO2 = Nielsen PNA monomer = N = Ugichem CMCO monomer = U NO2 NO2 NO2 NO2 NO2 = 2,4 DiNitroPhenol = DN pH=7.4 The promising CMCO patterns “CROSSING” “TRAP” • 1st PATTERN • 1:1 N:U backbone monomers • 2nd PATTERN • 1:1 N:U backbone monomers • Cluster of N monomers in the middle deprotonated protonated

  14. O- NO2 NO2 OUTER MT MEMBRANE MATRIX INNER MT MEMBRANE INTERMEMBRANE SPACE pH=7.4 PLASMA MEMBRANE + pH=7.4 Δψm=30-60 mV - NUCLEUS

  15. O- O- NO2 NO2 NO2 NO2 OUTER MT MEMBRANE MATRIX INNER MT MEMBRANE INTERMEMBRANE SPACE pH=7.4 DIFFUSION DIFFUSION PLASMA MEMBRANE + pH=7.4 Δψm=30-60 mV - NUCLEUS

  16. O- O- O- O- O- NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 O- NO2 NO2 OUTER MT MEMBRANE MATRIX INNER MT MEMBRANE INTERMEMBRANE SPACE pH=7.4 DIFFUSION DIFFUSION PLASMA MEMBRANE + pH=7.4 Δψm=30-60 mV - CYTOSOLIC ACCUMULATION NUCLEUS

  17. O- O- NO2 NO2 NO2 NO2 mtDNA CYTOPLASM DIFFUSION pH=7.4 pH=7.4 OUTER MT MEMBRANE INNER MT MEMBRANE RNA MT MATRIX INTERMEMBRANE SPACE PLASMA MEMBRANE

  18. O- NO2 NO2 mtDNA CYTOPLASM pH=7.4 H+ pH=7.4 OUTER MT MEMBRANE ETC pH=7.4 H+ pH=7.8 INNER MT MEMBRANE RNA MT MATRIX INTERMEMBRANE SPACE PLASMA MEMBRANE

  19. OH O- NO2 NO2 NO2 NO2 mtDNA CYTOPLASM H+ OUTER MT MEMBRANE ETC pH=7.4 H+ pH=7.8 INNER MT MEMBRANE RNA MT MATRIX INTERMEMBRANE SPACE PLASMA MEMBRANE

  20. O- NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c H+ OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  21. O- NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  22. OH NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  23. OH NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  24. OH NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  25. O- NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV H+ - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  26. O- NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV H+ - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  27. O- NO2 NO2 INTERMEMBRANE SPACE H+ H+ pH=7.4 H+ H+ H+ e- Cyt c OUTER LEAFLET e- H+ e- e- CoQ H+ H+ H+ e- H+ + IINNER MT MEMBRANE CxI CxIII Δψ CxIV ΔpH INNER LEAFLET CxII CxV - O2 H+ H+ H2O ADP H+ MT MATRIX ATP pH=7.8

  28. O- NO2 NO2 mtDNA CYTOPLASM H+ OUTER MT MEMBRANE ETC pH=7.4 H+ pH=7.8 INNER MT MEMBRANE RNA MT MATRIX - Δψm=150-180 mV + INTERMEMBRANE SPACE PLASMA MEMBRANE

  29. O- NO2 NO2 INTERMEMBRANE SPACE mtDNA RNA INNER MT MEMBRANE MT MATRIX

  30. O- O- NO2 NO2 NO2 NO2 INTERMEMBRANE SPACE WT RNA WT mtDNA MUT mtDNA MUT RNA INNER MT MEMBRANE MT MATRIX

  31. O- O- NO2 NO2 NO2 NO2 INTERMEMBRANE SPACE Ribosomes POLγ WT RNA WT mtDNA MUT mtDNA MUT RNA INNER MT MEMBRANE MT MATRIX

  32. O- O- NO2 NO2 NO2 NO2 INTERMEMBRANE SPACE WT RNA WT mtDNA MUT mtDNA mtDNA MUT RNA INNER MT MEMBRANE MT MATRIX

  33. O- O- NO2 NO2 NO2 NO2 INTERMEMBRANE SPACE WT RNA WT mtDNA MUT mtDNA mtDNA MUT RNA INNER MT MEMBRANE MT MATRIX

  34. O- O- NO2 NO2 NO2 NO2 INTERMEMBRANE SPACE WT RNA WT mtDNA MUT mtDNA mtDNA MUT RNA INNER MT MEMBRANE MT MATRIX

  35. TIME CLINICAL DEFECTS (DISEASE) NO CLINICAL DEFECTS REDUCTION OF MUTANT mtDNA

  36. EXPERIMENTAL PROCEDURES Certain key points in this hypothesis 1. Binding Ability and Specificity 2. Inhibition of mtDNA Replication 3. Cellular Uptake and Subcellular Localisation 4. Cytotoxicity 5. Effect ? • Antisense • Antigenomic

  37. 1. SPECIFICITY AND BINDING ABILITY OF CMCOS • Kinetic Studies: • Thermal Melt Analysis (Tm) • Real-time Biomolecular Interaction Analysis (Gold Standard of Surface Plasmon Resonance) (KD) • Electrophoretic Mobility Shift Assay (EMSA) (KD)

  38. %Hyperchromicity 100 50 0 Tm 1. SPECIFICITY AND BINDING ABILITY OF CMCOSTHERMAL MELT • Record hyperchromic effect as a function of T (Melting curve) • Tm = 50% of DNA/CMCO complex changes to free DNA, CMCO molecules : info on binding ability and specificity

  39. O- NO2 NO2 Tm1 Tm1 THERMAL MELT CMCO 1ST SET 1st set CMCOTm= Tm1=63C,equivalent KD=10-6M

  40. 1. SPECIFICITY AND BINDING ABILITY OF CMCOSREAL-TIME BIOMOLECULAR INTERACTION ANALYSIS • Surface Plasmon Resonance = Optical phenomenon, can determine the binding affinity of two molecules and specificity (e.g. fully complementary ssDNA/CMCO, singly mismatched ssDNA/CMCO) • One molecule (LIGAND i.e. bio-ssDNA) immobilised onto sensor surface, the other (ANALYTE i.e. CMCO) injected through FC • Accumulation of ANALYTE/LIGAND complex on surface cause increase in REFRACTIVE INDEX • Measured in real time • Result plotted as RU against time (SENSORGRAM) • Determine KD (in M) values (i.e. CMCO Concentration for 50% of ssDNA to be bound)

  41. ASSOCIATION (ka) DISSOCIATION (kd) O- NO2 NO2 SPR ANALYSIS CMCO 1ST SET 1:1 Langmuir – KD determination KD approx. 10-8M (Chi2=35) with fully complementary ssDNA sequence – Empirically 10-6 KD cannot be determined with the singly mismatched ssDNA sequence

  42. O- NO2 NO2 1. SPECIFICITY AND BINDING ABILITY OF CMCOSEMSA

  43. O- NO2 NO2 1. SPECIFICITY AND BINDING ABILITY OF CMCOSEMSA 0 0 22 55 97 U3 U4 U5 U6 U7 B0 94 U2 100 6 100 100 78 45 3 D1 D2 D3 D4 D5 D6 D7 B1 B2 B3 B4 B5 B6 B7 55% BOUND – 10pmoles/reaction CMCO i.e. KD=0.7μΜ=0.7x10-6M i.e approx. 10-6M

  44. 561 40nt 171nt 712 2. IN VITRO INHIBITION OF mtDNA REPLICATION • (Altered version of the minimal mtDNA replisome by Falkerberg et al. [5]) [5] J.A. Korhonen, X. Hoi Pham, M. Pellegrini and M. Falkenberg, EMBO Journal. 23 (2004): 2423-2439

  45. 561 40nt 171nt 712 B 0 100 100 76 86 21 U1 U2 U3 U4 U5 U6 100 0 0 24 14 79 D1 D2 D3 D4 D5 D6 2. IN VITRO INHIBITION OF mtDNA REPLICATION p. 167 LB5 24% inhibition when 10pmoles/reaction CMCO i.e. Ki=1.67mM

  46. 3. CELLULAR CMCO UPTAKE AND ITS SUBCELLULAR LOCALISATION • Three cell lines - HeLa cells, - 143B parental cells, and - CHO K1 cells • 24hrs exposure to biotinylated cmcos • Visualise - bio-cmco: fluorescein avidin (GREEN) - mitochondria: Mitotracker (RED) - nuclei: Dapi (BLUE) • Leica • Metamorph software to analyse the captured images

  47. CELLULAR CMCO UPTAKE AND ITS SUBCELLULAR LOCALISATION – 1ST SET CMCO O- NO2 NO2 HeLa – 10μΜ CMCO Dapi MitoTracker

  48. CELLULAR CMCO UPTAKE AND ITS SUBCELLULAR LOCALISATION – 1ST SET CMCO O- NO2 NO2 HeLa – 10μΜ CMCO Dapi MitoTracker Fluorescein

  49. CELLULAR CMCO UPTAKE AND ITS SUBCELLULAR LOCALISATION – 1ST SET CMCO O- NO2 NO2 HeLa – 10μΜ CMCO Dapi MitoTracker Fluorescein

  50. CELLULAR CMCO UPTAKE AND ITS SUBCELLULAR LOCALISATION – 1ST SET CMCO O- NO2 NO2 HeLa – 10μΜ CMCO

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