Is there a possibility of computational drug     design for neurological disorders?           Jack Tuszynski     Departmen...
Collaborators:T. Craddock, U. AlbertaS. Hameroff, U. ArizonaH. Freedman, Univ. Algavre•Priel, Israel Inst Advanced Studies...
The Human Brain:             a computer cluster of computers    1011 neurons in our brains    1015 synapses operating at...
Microtubule: biological functions•Part of the cytoskeleton:  Compressional strength•Required for mitosis•Intracellular tra...
Challenge: integration of  various levels in a hierarchyBuilding a bridge between  the molecular level (cytoskeleton)  t...
Dendritic spine has microtubules interacting with              membrane receptors.
Microtubules in dendrites :   Self-assemble to extend dendrites    (and axons)   Form synaptic connections   Linked to ...
Tubulin interacts with hundreds       of other proteins
MTs and Neurodegenerative            diseases   A common feature: a deteriorating cytoskeleton   Bioengineered cytoskele...
Alzheimer’s disease   Both the neuronal and cognitive consequences of    Both the neuronal and cognitive consequences of ...
Parkinson’s and Huntington’s                diseases   Mutations in genes for α-synuclein and parkin proteins lead    to ...
   Stroke and brain injury – The cytoskeleton is disrupted    following ischemia due to blood hemorrhage, occlusion, or  ...
Microtubules   Essential cytoskeletal components involved in structural    support, motility, cell division       , intra...
Tubulin’s Crystallographic StructureCommon Tubulin Structures1JFF   3.5Å e-crystallogr.      on               taxol1TUB   ...
Tubulin Structural MotifsGTP/GDP   M Loop   H3 Helix   T3 Loop   T7 Loop   Taxol
Longitudinal Bonds              Dimer-dimer binding                 Calculate free energy:                     3D-RISM    ...
Lateral Bondsweaker
Microtubule Lattices                  =>Nogales et al Nature (1998)PDB: 1TUB, 1JFF                              A Lattice ...
Carboxy-terminal tails                                              Although tubulin has been                            ...
Gamma tubulin dipole stabilizes            tubulin ring   Gamma    tubulin    ring    Slide courtesy of N. Dyer, U. Warwick
Microtubule Reconstruction         In Silico
Charge distribution on a microtubule-end              +end                               red/blue= negative/positive      ...
Drug Binding Sites on MTs
From Sequence to Tubulin                     Structure: HomologyMRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGA...
All alpha, beta and gamma        Tubulin IsotypesComputationally Reconstructed
Modes of oscillation depend on the tubulin isotype
C-terminal tails dynamics   C-terminal tails exist in multiple states    that    dynamically oscillate on the order of ~G...
The electrical circuit model of  ion conduction along MTsBulkOuter sheet              α   β   α   βInner sheetLumen
Modes of Computation/Signaling in                  MTs C-termini states (4 per dimer) Electron hopping (4 per dimer) Co...
Capacity of Human Memory? Von Neumann (1950) – 3x1020 bitsTotal life experience -we agree Anatomists (1970’s) – 1013-101...
Phosphorylation sites on tubulin:a molecular code for memory andconsciousness? Travis J. A. Craddock, Jack A. Tuszynski, S...
Memory – good or bad? (PTSD, OCD) Science News Memories Selectively, Safely Erased In Mice ScienceDaily (Oct. 23, 2008) Se...
Ca2+/Calmodulin Kinase II     (CaMKII) and Tubulin   Calmodulin regulated enzyme systems associated    with microtubules ...
Calmodulin kinase complex CaMKII as memory read/write device
CaMKII Activation Calcium ions (Ca2+) bind calmodulin (CaM) Ca2+/CaM complex binds with CaMKII to activate it CaMKII fu...
If all microtubule phosphorylation sites in the brain utilized, thisalone could lead up to 1019 bits per brain (short term...
Not just memory encoding but also      information processing:     AND gate       XOR gate
Brain’s memory code
Anesthetic-Microtubule               Interactions?   Hypothesis: The microtubule (MT)    network in dendrites is related ...
General Anesthetics (GAs)   A drug that brings about:    •   Analgesia: blocking the conscious sensation of pain    •   H...
GAs Possess Dissimilar StructureInhaled                        Intravenous                                   Propofol     ...
Anesthetics and MT - post 1973   1979 – Livingston et. al.: low concentration of    halothane increases MT/axon while hig...
Autodock   Performs and ranks search of different ligand    conformations, using:    •   Monte Carlo Simulated Annealing ...
Blind Docking Simulations   Accurate predictions are those that place the correct    binding site and confirmation in the...
Blind Docking Results for Tubulin   Investigated 1JFF   Sites found:    •   Halothane 54    •   Sevoflurane 145    •   E...
Focused Docking Results   Indicate colchicine and vinblastine binding    regions are preferred binding regions for    hal...
Intensive Docking - Body   23 distinct binding spots    found on the minimized    tubulin structure:    • Taxol/vinblasti...
Intensive Docking – C Termini Tails   50 conformations of the    TUBB2 and TUBA6    C-terminal tails    investigated   v...
Results - Intensive Docking -Tails   Halothane-tail interaction may limit tail flexibility    interfering with polymeriza...
Computational Results:    Anesthetic Binding Sites on Tubulin   Predicted binding pockets found in taxol,    vinblastine,...
Caffeine     THC Marijuana        LSD               Adrenaline Nicotine   MDMA Ecstasy      Chocolate             Methamph...
Alzheimer’s zincdyshomeostasis hypothesis         Craddock TJA, Tuszynski JA, Chopra D,         Casey N, Goldstein LE, Ham...
Mathematical Model
Tauopathy in Alzheimer’s       disease
Dynamics of MAP-MT Network    (parallel computer architecture):           long-term memory?Memory loss: Alzheimer’s diseas...
AD Mouse model brains
GBM and γ-tubulin    γ-tubulin found to be overexpressed    βIII-tubulin also overexpressed• γ-tubulin shows "prominent ...
Results: γ-tubulin
Colchicine binding site: gamma     Autodock: -(5-6) kcal/mol
Drug Binding to Inhibit Aberrant  Protein’s Activity is the key                  Cavity                                   ...
How Do We Solve Our Puzzles?
SCREENINGFLOWCHART
Conclusions   The human brain has an amazing computational    potential (supercomputer cluster)   The neuron (and other ...
Jack Tuszynski
Jack Tuszynski
Jack Tuszynski
Jack Tuszynski
Jack Tuszynski
Jack Tuszynski
Jack Tuszynski
Jack Tuszynski
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Jack Tuszynski

  1. 1. Is there a possibility of computational drug design for neurological disorders? Jack Tuszynski Departments of Physics and Oncology University of Alberta Edmonton, Canada
  2. 2. Collaborators:T. Craddock, U. AlbertaS. Hameroff, U. ArizonaH. Freedman, Univ. Algavre•Priel, Israel Inst Advanced StudiesV. Rezania, Macewan U.N. Woolf, UCLAH. Cantiello, MGH/Buenos AiresR. Tanzi, HarvardD. Chopra, Chopra Institute
  3. 3. The Human Brain: a computer cluster of computers 1011 neurons in our brains 1015 synapses operating at about 10 impulses/second (CPUs have 108 transistors) Approximately 1016 synapse operations per second i.e. at least 10 PF ( Blue Gene performs at 1015 FLOPS=1 PETAFLOP) Total energy consumption of the brain is about 25 watts (Blue Gene requires 1.5 MW) Is there anything special inside each neuron? YES another computer that has both classical and quantum processors http://www.merkle.com/brainLimits.html
  4. 4. Microtubule: biological functions•Part of the cytoskeleton: Compressional strength•Required for mitosis•Intracellular transport: Motor proteins•Cell motility: Cilia and flagella• Nerve cells
  5. 5. Challenge: integration of various levels in a hierarchyBuilding a bridge between the molecular level (cytoskeleton) the membrane level (synaptic activity, AP)
  6. 6. Dendritic spine has microtubules interacting with membrane receptors.
  7. 7. Microtubules in dendrites : Self-assemble to extend dendrites (and axons) Form synaptic connections Linked to ion-channels and synaptic receptors Organized in parallel structures – interconnected by MAP-2 Appear in mixed polarity
  8. 8. Tubulin interacts with hundreds of other proteins
  9. 9. MTs and Neurodegenerative diseases A common feature: a deteriorating cytoskeleton Bioengineered cytoskeletal protein products can stabilize, or destabilize, the existing cytoskeletal matrix, and prevent neuronal degeneration resulting from multiple causes. Products include modified tubulins and actins derived from known animal and plant sequences, gene vectors for modified tubulins and actins, biohybrid tubulins and actins, coated microtubules and actin filaments, and biologically- inspired cytoskeletal-like nanomaterials (e.g., modified nanotubes).
  10. 10. Alzheimer’s disease Both the neuronal and cognitive consequences of Both the neuronal and cognitive consequences of cytoskeletal protein disruption Cortical neurons in AD brain accumulate hyperphosphorylated tau, a MAP, which triggers the formation of neurofibrillary tangles. Neurons in AD demonstrate impaired axonal transport and compromised MT matrixes, even in the absence of neurofibrillary tangles. Preventing neurofibrillary tangle accumulation protects against dementia or cognitive impairment. Stabilizing the microtubule cytoskeleton can prevent or diminish the neural degradation associated with AD
  11. 11. Parkinson’s and Huntington’s diseases Mutations in genes for α-synuclein and parkin proteins lead to familial Parkinson’s, and these proteins also contribute to sporadic cases Altered α-synuclein and parkin proteins result in impaired axonal transport of dopamine-containing vesicles. Dopamine is released and degraded into toxic by-products that kill dopamine-containing neurons in the nigrostriatal pathway. Stabilizing the cytoskeleton, responsible for axonal transport, is expected to prevent this type of cytotoxicity. Huntington’s chorea: an autosomal dominant disorder caused by mutations in huntingtin protein, characterized by polyglutamine repeat expansion. Polyglutamine repeats in huntingtin protein disrupt its binding to microtubules resulting in impaired axonal transport. Stabilized MTs could remedy this condition.
  12. 12.  Stroke and brain injury – The cytoskeleton is disrupted following ischemia due to blood hemorrhage, occlusion, or injury. Stabilizing the cytoskeleton is expected to counter these effects. Epilepsy – Microtubule-associated protein, MAP2, shows decreased phosphorylation in parts of brain where epileptic seizure activity is prevalent. This is indicative of impaired cytoskeletal dynamics. Stabilizing the cytoskeleton is expected to aid in the recovery of damaged brain Amyotropic lateral sclerosis – Axonal transport is compromised in this movement disorder as a result of cytoskeletal disruption. Stabilizing the cytoskeleton would ameliorate these conditions. Charcot-Marie-Tooth disease – An underlying cause of the impaired axonal transport may be stalled microtubules that assume a hyperstabilized state due to mutated dynamin2 protein.
  13. 13. Microtubules Essential cytoskeletal components involved in structural support, motility, cell division , intracellular transport MT composition: • One alpha and one beta tubulin unit come together to form a dimer • Long protofilaments formed from these dimers. • MT has cylindrical form from assembly of a number (13) of protofilaments Characterized by dynamic polymerization/depolymerization
  14. 14. Tubulin’s Crystallographic StructureCommon Tubulin Structures1JFF 3.5Å e-crystallogr. on taxol1TUB 3.7Å stabilized Zn2+ sheets1TVK 2.9Å e-crystallogr. on taxol / stabilized Zn2+1SA0 4.2Å sheets 3.5Å Tuszynski at. al., Roy Soc of London Phil e-crystallogr. on EpA Tr A, 356(1743), p.1897 stabilized Zn2+ sheets McKean et. al., J. Cell Sci. 114, 2723-2733 X-ray crystallogr. on (2001) colchicine & stathmin like domain complex
  15. 15. Tubulin Structural MotifsGTP/GDP M Loop H3 Helix T3 Loop T7 Loop Taxol
  16. 16. Longitudinal Bonds Dimer-dimer binding Calculate free energy: 3D-RISM implicit solvation Adaptive biasing for fo explicit solvation Conformational change stronger Catastrophe Use ABF to force bend
  17. 17. Lateral Bondsweaker
  18. 18. Microtubule Lattices =>Nogales et al Nature (1998)PDB: 1TUB, 1JFF A Lattice B Lattice
  19. 19. Carboxy-terminal tails  Although tubulin has been crystallized, the carboxy- terminal tails could not be assigned because of flexibility  Functions of the tails include: • Facilitate binding of MAP proteins to MT’s • Enhance motor protein processivity • Increase the rate of MT diassembly α• Physical properties: – 10 residues for alpha tail, 18 residues for beta β – High proportion of negatively charged residues = Glu or Asp – Multiple isotypes of tubulin show main differences in tails – Often post-translationally modified, including polyglutamylation
  20. 20. Gamma tubulin dipole stabilizes tubulin ring Gamma tubulin ring Slide courtesy of N. Dyer, U. Warwick
  21. 21. Microtubule Reconstruction In Silico
  22. 22. Charge distribution on a microtubule-end +end red/blue= negative/positive Charge density: ~ 20e– per monomer (250 – 32500 e–/µm) Distributed more on the outer surface than in the inner core with ratio ~ 2:1 Kim et el, BioPhys J, (2008)
  23. 23. Drug Binding Sites on MTs
  24. 24. From Sequence to Tubulin Structure: HomologyMRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGKHVPRAVFVDLEPTVIDEVRTGTY Primary: amino acid sequenceRQLFHPEQLITGKEDAANNYARGHYTIGKEIIDLVLDRIRKLADQCTGLQGFSVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQVSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYTNLNRLIGQIVSSITASLRFDGALNVDLTEFQTNLVPYPRGHFPLATYAPVISAEKAYHEQLSVAEITNACFEPANQMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTKRTIQFVDWCPTGFKVGINYEPPTVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEGMEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEYTertiary: 3D-folding Secondary: α-helix and β- sheet Quaternary: multimeric arrangement
  25. 25. All alpha, beta and gamma Tubulin IsotypesComputationally Reconstructed
  26. 26. Modes of oscillation depend on the tubulin isotype
  27. 27. C-terminal tails dynamics C-terminal tails exist in multiple states that dynamically oscillate on the order of ~GHz α β α β Lumen
  28. 28. The electrical circuit model of ion conduction along MTsBulkOuter sheet α β α βInner sheetLumen
  29. 29. Modes of Computation/Signaling in MTs C-termini states (4 per dimer) Electron hopping (4 per dimer) Conformational changes/GTP states(2 per dimer)Total: 32 states/ dimer100 kB/MT or 1 GB/neuron100 billion neurons: 1020 bits/brainat 10 nanosec transitions: 1028 flops=104 yottaflops!!!Recall: BlueGene 1014 flopsIs there a way to store information?
  30. 30. Capacity of Human Memory? Von Neumann (1950) – 3x1020 bitsTotal life experience -we agree Anatomists (1970’s) – 1013-1015 synapses allowing 1016 syn-ops/sec Landauer (1986*) – 109 bits • assumed we retain 2 bits/sec of visual, verbal, tactile, musical memory! • Human lifetime ~ 2.5 billion secondsThomas K. Landauer "How Much Do People Remember? Some Estimatesof the Quantity of Learned Information in Long-term Memory" Cognitive Science10, 477-493, 1986
  31. 31. Phosphorylation sites on tubulin:a molecular code for memory andconsciousness? Travis J. A. Craddock, Jack A. Tuszynski, Stuart Hameroff. Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation? PLoS Computational Biology, 2012; 8 (3): e1002421 DOI:
  32. 32. Memory – good or bad? (PTSD, OCD) Science News Memories Selectively, Safely Erased In Mice ScienceDaily (Oct. 23, 2008) Selected set of memories can be rapidly and specifically erased from the mouse brain in a controlled and inducible manner. Dr JZ Tsien“… downstream of NMDA receptor….αCaMKII involved in memory storage and recall.Tsien developed a … pharmacologic inhibitor to instantly turn αCaMKII off and on in amouse…to study exactly what happened if he threw off the natural balance during theretrieval stage.“
  33. 33. Ca2+/Calmodulin Kinase II (CaMKII) and Tubulin Calmodulin regulated enzyme systems associated with microtubules and may regulate calcium effects on cytoskeleton. CaMKII phophorylates tubulin in the c-terminal region resulting in a conformational change reducing its capacity to assemble. CaMKII acts as a “memory molecule” or “molecular memory switch” CaMKII is required in both short and long term memory and LTP, the latter involving mRNA-based CaMKII synthesis. CaMKII correlates with memory.
  34. 34. Calmodulin kinase complex CaMKII as memory read/write device
  35. 35. CaMKII Activation Calcium ions (Ca2+) bind calmodulin (CaM) Ca2+/CaM complex binds with CaMKII to activate it CaMKII functions include transfer of phosphate groups and chemical energy CaMKII is a serine/threonine kinase CaMKII is a dodecamer with: 12 kinase regions, 6 above center, 6 below
  36. 36. If all microtubule phosphorylation sites in the brain utilized, thisalone could lead up to 1019 bits per brain (short term memory?)
  37. 37. Not just memory encoding but also information processing: AND gate XOR gate
  38. 38. Brain’s memory code
  39. 39. Anesthetic-Microtubule Interactions? Hypothesis: The microtubule (MT) network in dendrites is related to memory, and interaction with anesthetics can influence consciousness and alter memory formation. Anesthetics natural probe for functional sites of consciousness Memory formation and learning rely on normal MT cytoskeleton functioning Postoperative Cognitive Dysfunction (POCD) Exacerbation of diseases (Alzheimer’s, FTD, Schizophrenia) http://www.brainleadersandlearners.com/wp- following anesthesia content/uploads/2008/09/blog-brain-business2.jpg content/uploads/2008/09/blog-brain-business2.jp
  40. 40. General Anesthetics (GAs) A drug that brings about: • Analgesia: blocking the conscious sensation of pain • Hypnosis: producing unconsciousness  main focus • Amnesia: preventing memory formation  main focus • Paralysis: preventing unwanted movement or muscle tone• Potency of anesthetics inversely proportional to lipid solubility (Meyer-Overton Relation).• Potency of inhaled anesthetics described by the Minimum Alveolar Concentration (MAC)
  41. 41. GAs Possess Dissimilar StructureInhaled Intravenous Propofol Ketamine Etomidate
  42. 42. Anesthetics and MT - post 1973 1979 – Livingston et. al.: low concentration of halothane increases MT/axon while high concentration lowers MT/axon in unmyelinated axons from rat sciatic nerves. 1981 – Vergara et. al: halothane modifies colchicine-tubulin binding in rat brain. 2004 – Futterer et. al.: tubulin altered out to 3 days by desflurane. 2007 – Kalenka et. al.: tubulin altered out to 28 days by sevoflurane in rat brain. 2007 – Pan et. al.: halothane binds specifically to tubulin in humans, tubulin is prominently changed by halothane and isoflurane in rat brain. 2008 - Pan et. al.: of ~500 detectable proteins, tubulin among the ~2% affected by halothane, and ~1% altered by isoflurane (1 of 3 affected by both)
  43. 43. Autodock Performs and ranks search of different ligand conformations, using: • Monte Carlo Simulated Annealing • Genetic Algorithm • Lamarckian Genetic Algorithm  most efficient, reliable, & successful
  44. 44. Blind Docking Simulations Accurate predictions are those that place the correct binding site and confirmation in the lowest energy rank. Blind Docking has ~71% accuracy Focused Docking has ~84% accuracy Intensive Docking studies are currently under investigation.
  45. 45. Blind Docking Results for Tubulin Investigated 1JFF Sites found: • Halothane 54 • Sevoflurane 145 • Enflurane 124 • Desflurane 125 Binding Energies: • -1.0 to -3.0 kcal/mol Blue - Halothane Yellow - Desflurane Red - Sevoflurane Orange - Enflurane
  46. 46. Focused Docking Results Indicate colchicine and vinblastine binding regions are preferred binding regions for halothane and halogenated ethers. This relates to MT depolymerization and macrotubule formation by halothane. Predict halogentated ethers to also depolymerize MT and form macrotubules. The taxol region is expected to be involved in MT stabilization.
  47. 47. Intensive Docking - Body 23 distinct binding spots found on the minimized tubulin structure: • Taxol/vinblastine binding region • non-hydrolyzable GTP binding site • Others not near definable sites Binding energies between -2.1 and -3.5 kcal/mol indicating weak binding.
  48. 48. Intensive Docking – C Termini Tails 50 conformations of the TUBB2 and TUBA6 C-terminal tails investigated various binding locations along tail binding energies between -1.7 and -2.7 kcal/mol
  49. 49. Results - Intensive Docking -Tails Halothane-tail interaction may limit tail flexibility interfering with polymerization mechanisms. Would also have an affect on the binding of microtubule-associated-proteins.
  50. 50. Computational Results: Anesthetic Binding Sites on Tubulin Predicted binding pockets found in taxol, vinblastine, colchicine, and GDP regions. Some predicted sites in contact with surface Tryptophans Some sites near surface Serines/Threonines in C- terminal region. Predicted sites not near the 390-394 positive patch Additionally, due to protofilament interactions, found binding regions in and around nanopores Some are transient and depend on conformation
  51. 51. Caffeine THC Marijuana LSD Adrenaline Nicotine MDMA Ecstasy Chocolate Methamphetamine Xanax Heroin Rohypnol (Date rape) Alcohol
  52. 52. Alzheimer’s zincdyshomeostasis hypothesis Craddock TJA, Tuszynski JA, Chopra D, Casey N, Goldstein LE, Hameroff SR, Tanzi RE (2012) The Zinc Dyshomeostasis Hypothesis of Alzheimers Disease. PLoS ONE 7(3): e33552. doi:10.1371/journal.pone.0033552
  53. 53. Mathematical Model
  54. 54. Tauopathy in Alzheimer’s disease
  55. 55. Dynamics of MAP-MT Network (parallel computer architecture): long-term memory?Memory loss: Alzheimer’s disease: MAP tau connection
  56. 56. AD Mouse model brains
  57. 57. GBM and γ-tubulin γ-tubulin found to be overexpressed βIII-tubulin also overexpressed• γ-tubulin shows "prominent diffuse cytoplasmic localization beyond the pericentriolar region”• βIII-tubulin associated with microtubules• Aberrant expression of βIII-tubulin and γ-tubulin may be linked to malignant changes in glioma cells 65 Katsetos et al., Neurochem Res (2007) 32:1387–1398
  58. 58. Results: γ-tubulin
  59. 59. Colchicine binding site: gamma  Autodock: -(5-6) kcal/mol
  60. 60. Drug Binding to Inhibit Aberrant Protein’s Activity is the key Cavity Cavity Drug / Ligand Protein Cavity
  61. 61. How Do We Solve Our Puzzles?
  62. 62. SCREENINGFLOWCHART
  63. 63. Conclusions The human brain has an amazing computational potential (supercomputer cluster) The neuron (and other cells) performs complex computation and acts as an evolvable computer which communicates with up to 100,000 nearby computers Microtubules are elementary computational elements ( 1GB chips) performing parallel processing and RAM storage functions Microtubules are involved in numerous neurological disorders and are targets for drug development

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