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Possibility of Fullerene as Therapeutic Agent in

Alzheimer's

PRESENTED BY:Vidushi SharmaMtech 1st year, Department of Chemistry,

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CO

NT

EN

TS i. Introduction

Fullerene Alzheimer’s Disease

ii. Fullerene: Possible Therapeutic Agentiii. Anti- Amyloid Beta activityiv. Radical Scavengingv. Acetylcholine esterase inhibitionvi. Discussionvii. Futureviii. Conclusion

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INTRODUCTIONFullerene (Buckministerfullerene)

•Carbon allotrope discovered in 1985 by Harry Kroto and his

team.

•60 equivalent carbon atoms arranged in spherical pattern

composed of 12pentagons and 20 hexagons

•Diameter of about 1nm to 10nm.

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Applications in Medical sciences:

•Drug Delivery

•Diagnostic

•Probe

•Enzyme inhibition

•Site specific DNA photocleavage

•ROS quenching

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ALZHEIMER’S DISEASE:

A progressive neurodegrading disease characterized by memory loss and loss of cognitive functions .

Two hallmark pathologies: Extracellular plaque deposits of the β-amyloid peptide (Aβ) Neurofibrillary tangles of the microtubule binding protein tau.

Other mechanisms which signify AD: Oxidative stress Metal Homeostasis Dysfunction of acetylcholine esterase Mitochondrial dysfunction

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Why Fullerene derivatives are being suggested as potential

therapeutic agents in AD?

Hydrophobic core

Membrane penetrating tendency

ROS quenching

Inhibitory action

DNA photocleavage

Low toxicity

Potential to get variously functionalized

FULLERENE: AD THERAPEUTIC AGENT

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ANTI AMYLOID BETA ACTIVITY

Amino acid sequence of amyloid beta. (Highlighted motif is a hydrophobic core)Sisodia et al, The FASEB Journal, Vol. 9 March 1995

Ameloid Beta deposits in brain have been accounted as one of the early symptoms of AD.

Ameloid beta are 39 to 43 amino acid sequence long proteins with a trans-membrane and an extracellular domain.

Ameloid beta is formed from Ameloid Precursor Protein (APP) due to incorrect cleavage by secretase enzyme at cell surface.

Mutations in APP sequence- secretase cleave at a different position resulting in fibrillogenic ameloid beta.

Aggregation by nucleation and growth process

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Ameloid beta plagues are prominantly found in hippocampus and neocortex regions of brain.

Plaques:-

Blocking vital cell to cell signaling, transport

Cause dys-functioning of mitochondria (disrupting membrane)

ROS generation

Lipid peroxidation

Inhibition of key enzymes in respiratory cycle

Form high affinity complex with copper ions to generate hydrogen peroxide

Neuronal apoptosis

Figure: Shows brain severely suffering from AD. Dark regions shows the aggregated plagues in brain and shrunken shape represents damaged neurons.(www.alz.org/braintour/healthy_vs_alzheimers.asp)

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Fullerene + Functional group = Soluble derivative

Jeong et al. in 2003 had shown that DMF inhibits amyloid fibrils greatly via Thioflavin T (ThT) fluorescence assay

No details on possible interaction behind this inhibition.

Xiaoying et. al in 2014 have provided a study on possible interaction of fullerene derivative 1,2-dimethoxymethano fullerene (DMF) with insoluble amyloid fibrils.

Three binding domains : Central Hydrophobic core (17CVFFA21), Turn site (27NKGAI31) C-terminal β sheet site (31IIGLMVGGVVI41).

These regions have stronger hydrophobicity, aromatic residues and concave surfaces which facilitates the binding of DMF molecule to these regions.

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Figure: (a) The structure of DMF. (b) The initial conformation of the Aβ42 protofibril with a DMF molecule placed at three different positions. In the three initial states, the minimum distance between DMF and Aβ42 is 2 nm. For DMF molecules, carbon atoms are in gray, oxgen atoms are in red, and CH3 (united-atom) is in green. For Aβ42, different colors represent different residues types: blue, positively charged; red, negatively charged; green, polar; gray, hydrophobic. Xiaoying et. al in 2014

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Figure: Concave surface in turn region provides a potential binding site for DMF molecule. Binding in turn region disrupts the salt bridges in fibrils and causing their disintegration. Xiaoying et. al in 2014

Podolski et. Al, 2007

Bobylev et al. 2010, fullerenol (FL), sodium fullerenolate (NaFL) and NaPCF respectively.

Bobylev et al. 2011, Nitroderivatives of fullerene C60. (a methyl ester of LN[(2nitroglyceryl) fullerenyl] proline; (c) methyl ester of L N[(2,3dinitroglyceryl) fullerenyl] proline; (e) 2nitroxyethyl ester of LN([2(nitroxy) ethyl] fullerenyl) proline.

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Figures: SEM images of Aggregated Amyloid beta fribrils. Podolski et. al, 2007

Figures: SEM images of Aggregated Amyloid beta fribrils incubated with fullerenols. Image shows disaggregation of plaques on action of fullerene derivates. Podolski et. al, 2007

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A B C D E

A- Aggregated FibrillsB- fullerenol (FL)C- NaFLD- NaPCFE- Control

Graph: Thioflavin T (ThT) fluorescence assay on Aggregated amyloid beta fibrills after treatment with fullerene derivatives. Bobylev et al. 2010

Tan-Yi Lu et al, 2011 : PEG-fullerene derivative Lee et al, 2011 a pentoxifylline derivative (PTX-fullerene)

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RADICAL SCAVENGINGOxidative stress :earliest symptom of the disease

Excessive generation of Reactive Oxygen Species (ROS) in brain due to simultaneous dys-functionaling of a number of mechanisms in AD brain.

Zhi-You et al., 2007 : OS results in major damage to proteins and lipids by increasing lipid peroxidation and causing unwanted oxidation of proteins as well as nucleic acids

Julie K. Anderson, 2004, Zhao et al.,2013 : Effects of oxidative stress on neurons in AD brain

Ranjana et al. 2012; Padurariu et al., 2013: sources of ROS:Amyloid β accumulation, Mitochondrial dysfunction Metal homeostasis

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Zhao et al., 2013: Studies performed in Tg 19959 transgenic mice model and Tg2576 mouse model

Amyloid β accumulationActivation of N-methyl D-aspartate receptorCalcium concentrations in neuronal cellsMitochondira dysfunction (ATP generation and Krebs cycle, Adam-vizi et.al, 2010)ROS generationROS Increases β and γ secretase JNK pathway activated Amyloid β aggregation

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Mitochondrial dysfunction:Amyloid β accumulation localized to mitochondrial regions Damage to mitochondrial metabolism Disrupting its lipid polarityIncreasing its membrane permeability Release of cytochrome cDisturbance in electron transport chain Release of reactive oxygen species (ROS).

Amyloid β accumulation causes nitration and inactivation of MnSOD which is a primary antioxidant enzyme present ion brain.

Figure: Dys-functioning in Mitochondria , results in generation of ROS species.

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Figure: Type I and Type II photosensitization reactions by fullerene to generate free radicals (Markovic et.al, 2008)

Figure: Surface coated soluble fullerene,scavenging capability depends on the porosity of surfactant coating (Markovic et.al, 2008)

Addition of extra functional side groups decreases the π electron delocalization by breaking π bonds and hence decreses the role of fullerene as a photosensitizer

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Fullerene scavenges ROS species by three mechanisms:

Physical Quenching is a weak mechanism where singlet oxygen is deactivated by converting its electronic excitation energy to vibrational energy of oxygen and fullerene. In derivatized fullerene molecule exhibits this type of ROS quenching mechanism which is relatively weak with rate constant of or depending upon the solvent. This quenching increases with addition of functional groups in the fullerene cage. Its been noted that fullerols are best quenchers and their ROS quenching ability increases with each addition of –OH group.

Chemical quenching is mostly seen for Hydroxyl radicals as they react with Carbon atoms attached to π double bond in cage. Thus as double bond decreases on addition of functional groups, tendency of fullerene derivative to quench Hydroxyl radicals decreases.

Catalytic quenching occurs for superoxide anion through catalytic dismutation. Due to attachment of some carboxyl groups on ring, generation of some electron deficient regions occur on cage which then take part in half reactions as catalyst to deactivate superoxide anion (Bensasson et al., 2000).

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Figure: Structure of the C3 tris malonic acid C60 derivative, showing the paired carboxylic acid groups attached to the three cyclopropane carbons on the C60 molecule.

Figure: C3 decreases mitochondrial superoxide anion production by cortical astrocytes. Cultured astrocytes were loaded with dihydroethidium (DHE), then treated with vehicle (H2O) or C3 for 1 h. DHE is oxidized to ethidium, which is detected as increasing nuclear Fuorescence in the astrocyte monolayer. C3 decreases basal superoxide production by astrocytes, which derives primarily from the mitochondrial electron transport chain . (Dugan et.al, 2001)

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Figure: Fullerene derivatives. Metallo-fullerenols showed maximum ROS scavenging activity. Yin et. al, 2009 , Biomaterials .

Bar Graph: Hydrogen peroxide scavenging action of above fullerene derivatives in A549 mice cells. Yin et al., 2009 in their study showed that metallo fullerols with a metal atom present endohedrally in cage exhibits greater antioxidant tendency by increasing the electron affinity and polarizibility of the fullerene cage. He also reported that these endohedral fullerols showed less tendency to aggregate as compared to fullerols alone.

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ACETYLCHOLINE ESTERASE INHIBITION

Acetylcholine esterase (ACh)

Loss of cholinergic neurotransmission by acetylcholine in cortex and hippocampus

Amyloid β aggregates (Munoz et al., 1999)

Figure: Neurotransmission in Synapsis. Image taken from http://neuroanatomyblog.tumblr.com/post/35372201455/lostinretrosynthesis-acetylcholine-ach-cycle

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Figure: Histochemical staining of AChE bound to amyloid Fibrils. Munoz et al., 1999

Graph: Efect of increasing concentrations of AChE (25, 100 and 250 nM) on the formation of fibrils nby using 70 nmol amyloid beta. Munoz et al., FEBS Letters 450 (1999) 205^209

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Gonclaves et. Al, 2015: five fullerene (C60) derivatives designed to act as new Human acetylcholinesterase (HssAChE) inhibitors by blocking its fasciculin II (FASII) binding site.

Residues: Tyr72, Asp74, Trp286, Gln291, Tyr341 and Pro344

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Positively charged fullerene derivatives have affinity HssAChE entrane

Pyridine ring interacts with active site. Derivative 3 and 5 showed best inhibition (Gonclaves et. Al, 2015)

Cavity of enzyme is hydrophobic, ideal for fullerenes to enter

Most effective inhibitors have tertiary amines or quaternary ammonium salts- interacts with catalytic triad: Ser200, Glu327, His440 and Trp84 at base of gorge. (Pastorin et. Al, 2006)

Figures: Left to right are Molecular structures of bis-N,N dimethylfulleropyrrolidinium salts, trans forms while last one is cis form. It is shown that trans form with farther functional groups have better orientation towards Trp84 and Trp 279 for both primary and secondary active site. Pastorin et al, Org. Biomol. Chem., 2006, 4, 2556–2562

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DISCUSSIONChemical and physical properties can be modified as per our requirements by surface functionalization.

Easy object to circulate in blood vessels around nervous system

Lipophilicity of its carbon cage can make it possible for fullerene to cross Blood Brain Barrier (BBB) (Ekkabut et. al, 2008, Nature)

Cause target specific DNA cleavage can be utilized to not merely pause the progression of AD but also stop it by causing mutations in AD causing genes.

Might have a toxic effect due to its tendency to generate oxygen radical on being photosensitized, however, unless a light source is provided, fullerene has no such effect.

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Figure: Permeation of fullerene through a lipid membrane. (Ekkabut et. al, 2008, Nature)

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Complications observed over choice of functionalized group for fullerene to act as therapeutic agent in AD:

Groups ROS scavenging

AChE inhibition

Toxicity Deposition Clearance from body

Hydroxyl Good Mildly High Low

Carbonyl Good No activity No Toxicity Moderate Low

Cationic grp Good activity Highly toxiz High Low

Metallo-C60 Good Less toxic Moderate Better

Surfactant coated

Coating dependent

doubtfull No toxicity

Yamago et al, 1995, Tsuchiya et al, 1996, Tang et al, 2007, Tzoupis et al, 2011, Saathoff et al, 2011: compile some basic data about the biodistribution , toxicity and clearance of fullerene derivatives on basis of their respective studies.

Fullerene molecule – no toxic effect, its choice of functional groups might impart a significant toxicity to the fullerene derivative.

Anionic groups - very less toxic Cationic groups - greater toxicity (Tang et.al, 2007)

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Mode of Fullerene intake:

•Orally - not adsorbed in body neither metabolized and was found to have been excreted via faeces and urine almost entirely•On intravenous injection - widely adsorbed in liver, kidney, bones and tissues and showed extremely slow clearance rate. (Yamago et al, 1995)

Bar graph: Fecal excretion of radioactive C14 labelled Fullerene derivative after oral (blue) and intravenous (red) dosing of compound in rats. (Yamago et al, 1995, Chemistry & Biology 1995, Vol 2 No 6 )

Thus, keeping the depository tendency of fullerene in mind, it is likely that even if toxicity of molecule is reduced, molecule might still have some degree of harmful effects in long run which are not yet brought to lime light.

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Work can be done in designing a single derivative with multi-functionalities in adequate amount to counter all mechanisms responsible for progression of AD.

Since all these discussed therapies are to cease progression of AD once it has started, efforts can be made to design a therapy using fullerene to cure AD and stop it from arising.

There are some genetic causes for amyloid-β aggregation and tau hyperphosphorylation which have been clearly understood to this date (Sisodie et al, 1995, Ballatore et al, 2007) and can be subjected to a gene therapy mediated by fullerene derivatives since it is well known fullerene also has property to perform site specific DNA cleavage.

FUTURE WORK:

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With many complications such as choice of functional group, toxicity, bio-distribution, clearance, membrane permeability, absence of a reliable method to generate exactly defined functional derivatives of fullerene and little knowledge of its possible long term side effects; Fullerene has a long way yet to go to be an AD therapeutic, possibility of which cannot be ruled out.

CONCLUSION

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