TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to tryptamine, dopamine and quinolone derivatives and their use in delay aging and diseases of the human brain. The composition and methods use such a compound and may also be used in combination with a potentiator to delay aging.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

The present application includes a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 27, 2017, is named TECH2009WO_SeqList and is 8 kilobytes in size.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is described in connection with neurological conditions. The World Alzheimer's Report estimated that in 2015, 47.5 million people had AD-related dementia ("dementia") worldwide, and these numbers were expected to rise to 75.6 million by 2030 and to 131.5 million by 2050. Over 9.9 million new cases of AD-related dementia are diagnosed every year worldwide, which translates to 1 new case every 3.2 seconds. In high-income countries, such as the United States and Germany, seven in 10 persons aged 70 years and older will pass away with some form of dementia. Dementia has a huge economic impact on society, on the persons with dementia, and on their families and caretakers. The estimated total healthcare cost of dementia worldwide in 2015 was estimated at $818 billion, and the World Alzheimer' s Report foresees dementia as a trillion-dollar disease by 2018. While scientists are trying to determine causes of Alzheimer's Disease ("AD"), they have pinpointed several cellular changes that increase a person's risk for developing it, including synaptic loss and dysfunction, Αβ production and accumulation, inflammatory responses, phosphorylated tau formation and accumulation, cell cycle deregulation, and hormonal imbalance. Aging is the number one risk factor for AD. Mounting evidence indicates that Αβ plays a key role in AD pathophysiology. Intra-neuronal Αβ and Αβ deposits early in the disease process, and intracellular hyperphosphorylated tau and neurofibrillary tangles later in the disease process, have been found in postmortem brains from AD patients. In addition, Αβ has been found to induce synaptic dysfunction and mitochondrial oxidative damage, resulting in abnormal activation of redox-mediated events, as well as an abnormal elevation of cytoplasmic Ca2+, ultimately causing neuronal damage. AD pathogenesis has been linked to DNA damage. In mammals, including humans, an accumulation of oxidative DNA damage in particularly mutant DNA, including brain tissue, has been found in aging persons. One example is Patent Application Publication No. WO2008/074068, entitled, "Substituted quinoline derivatives as antiamyloidogeneic agents," discloses heterocyclic compounds, processes for their preparation and their use as pharmaceutical or veterinary agents, in particular for the treatment, amelioration and/or prophylaxis of conditions caused by or associated with unbalanced metal levels and/or oxidative stress, such as neurological conditions and cellular proliferative disorders, for example Alzheimer's disease, Parkinson's disease, Huntington's disease or brain cancer or tumors.

Another example is Patent Application Publication No. WO2002/058686, entitled, "Method of treatment of neurodegenerative disorders using pentaaza-macrocyclic ligand complexes," discloses pharmaceutical compositions and methods using such compositions for the treatment of neurodegenerative disorders. Such compositions contain a catalyst for the dismutation of superoxide, including superoxide dismutase enzyme (SOD) and low molecular weight organic ligand derived metal complexes that function as mimics of the enzyme (SOD mimetics or SODms).

SUMMARY OF THE INVENTION

The inventors designed a number of molecules with the aid of molecular docking software to reduce/prevent interactions between Αβ and Drp 1 ; Αβ and p-tau; Αβ and VDAC 1 . Specifically, the inventors implemented the use of four distinct scaffolding structures with molecular docking simulation to fit the active sites of each enzyme and extrapolate R-groups with high binding affinity against abnormal protein interactions. Thus, the present invention includes methods for identifying molecules that can delay aging process and prevent and/or stop neurodegeneration. The inventors discovered molecules that cause delayed aging and protect cells from oxidative insults. The inventors also identified specific molecule that can be modified to increase their effectiveness, and methods for using the same to treat neurodegenerative disease. It was found that the molecules possess certain features, e.g., (1) delayed aging, (2) protect human cells from oxidative insults, (3) reduce the toxicity of mutant proteins, (4) inhibit abnormal protein interactions in the brain cells, and/or (5) enhance cell survival.

In one embodiment, the present invention includes a molecule that inhibits the interaction of Αβ and Drp 1 proteins. In one aspect, the molecule inhibits the interaction of Αβ and Drpl proteins in nerve cells. In another aspect, the molecule inhibits mitochondrial, intracellular, and extracellular damage caused by the interaction of Αβ and Drpl in or about nerve cells. In another aspect, the molecule is water-soluble. In another aspect, the molecule is adapted for oral, intravenous, intramuscular, intraperitoneal, subcutaneous, parenteral, or pulmonary administration. In another aspect, the Αβ and Drpl proteins are human. In another aspect, the molecule delays age-dependent disease process in Alzheimer's, Huntington's, Parkinson's and ALS. In another aspect, the molecule at least one of: delays aging in neurons, protects neurons from oxidative insults, inhibits abnormal protein-protein interactions and protect neurons from mutant protein(s)-induced toxicities, and enhances cell survival. In another aspect, the molecule is selected from at least one of: phosphonium cation based structures; quinoline based alpha aminophosphonates; napthaline based alpha aminophosphonates, or hexahydropyramidine carboxylates structures. In another aspect, the composition is selected from at least one of:

lphosphonate):

MitoQ : (Phosphonium, [ 10-(4,5 -dimethoxy-2-methyl-3 ,6-dioxo- 1 ,4-cyclohexadien- 1 -yl)decyl]triphenyl-,

Dynasore: (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide) shown here in the non-limiting form of a hydrate:

Mdiv-1 : (3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(lH) -quinazolinone, 3-(2,4-Dichloro- 5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone):

In another aspect, the shaded regions are modified to at lease one of: increase the inhibition of Αβ and Drpl binding, increase crossing the blood-brain4oarrier, increase solubility, or increase the metabolic half-life of the molecule.

Another embodiment of the present invention includes a method for identifying a molecule that inhibits the interaction of Αβ and Drpl proteins comprising: obtaining a database of molecular coordinates for the regions of the Αβ and Drp 1 proteins that interact; obtaining a database of molecules comprising molecular coordinates; identifying the molecules from the database of molecules that fit between the regions in which the Αβ and Drp 1 proteins that interact; and testing in vitro the ability of the one or more molecules that fit between the regions in which the Αβ and Drpl proteins to prevent binding of Αβ and Drpl proteins. In one aspect, the method further comprises testing the inhibition of the one or more molecules to inhibit the interaction of Αβ and Drp l proteins in nerve cells. In another aspect, the method further comprises testing the molecule to inhibit mitochondrial, intracellular, and extracellular damage caused by the interaction of Αβ and Drp l in or about nerve cells. In another aspect, the molecule is water soluble. In another aspect, the method further comprises adapting the molecule for oral, intravenous, intramuscular, intraperitoneal, subcutaneous, parenteral, or pulmonary administration. In another aspect, the method further comprises testing the one or more molecules to delay age-dependent disease process in Alzheimer's, Huntington's, Parkinson's and ALS. In another aspect, the one or more molecules at least one of: delays aging in neurons, protects neurons from oxidative insults, inhibits abnormal protein-protein interactions and protect neurons from mutant protein(s)-induced toxicities, and enhances cell survival. In another aspect, the method further comprises selecting from at least one of: phosphonium cation based structures; quinoline based alpha aminophosphonates; napthaline based alpha aminophosphonates, or hexahydropyramidine carboxylates structures. In another aspect, the molecule tested is, or is based on, at least one of:

methylphosphonate):

MitoQ : (Phosphonium, [ 10-(4,5 -dimethoxy-2-methyl-3 ,6-dioxo- 1 ,4-cyclohexadien- 1 -yl)decyl]triphenyl-, methanesulfonate), shown here in the non-limiting form of a mesylate.

Dynasore: (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide) shown here in the non-limitin form of a hydrate:

Mdivi-1 : (3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(lH) -quinazolinone, 3-(2,4- Dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone):

In another aspect, the shaded regions are modified to at lease one of: increase the inhibition of Αβ and Drpl binding, increase crossing the blood-brain-barrier, increase solubility, or increase the metabolic half-life of the molecule.

Yet another embodiment of the present invention includes a method of treating a neuropathy caused by the interaction of Αβ and Drpl proteins comprising providing a molecule that inhibits the interaction of the Αβ and Drpl proteins. In one aspect, the molecule inhibits the interaction of Αβ and Drpl proteins in nerve cells. In another aspect, the molecule inhibits mitochondrial, intracellular, and extracellular damage caused by the interaction of Αβ and Drpl in or about nerve cells. In another aspect, the molecule is water soluble. In another aspect, the molecule is adapted for oral, intravenous, intramuscular, intraperitoneal, subcutaneous, parenteral, or pulmonary administration. In another aspect, the Αβ and Drp 1 proteins are human. In another aspect, the neuropathy is an age-dependent disease selected from Alzheimer's, Huntington's, Parkinson's disease or ALS. In another aspect, the molecule at least one of: delays aging in neurons; protects neurons from oxidative insults; inhibits abnormal protein-protein interactions and protect neurons from mutant protein(s)-induced toxicities; and enhances cell survival. In another aspect, the molecule is selected from at least one of: phosphonium cation based structures; quinoline based alpha aminophosphonates; napthaline based alpha aminophosphonates, or hexahydropyramidine carboxylates structures. In another aspect, the molecule is, or is based on, at least one of:

DDQ (diethyl(3,4-dihydroxyphenethylamine)(quinolin-4-yl)methylph osphonate):

Dynasore: (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide) shown here in the non-limiting form of a hydrate:

Mdivi-1 : (3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(lH) -quinazolinone, 3-(2,4- Dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone):

In another aspect, the shaded regions are modified to at lease one of: increase the inhibition of Αβ and Drpl binding, increase crossing the blood-brain-barrier, increase solubility, or increase the metabolic half-life of the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which: FIGS. 1A to 1C show the interaction of designed drug molecules against Αβ and Drpl Complex. FIG. 1A. Interaction of Drpl (PDB ID: 4hlu) and Αβ1-40 (PDBID: lba4) proteins. FIG IB. Inhibition of Drpl and Αβ40 peptide Interaction by DDQ. FIG 1C. Interaction of Drpl and designed drug molecules. The inventors designed 82 molecules based on existing mitochondrial division inhibiting drug molecules in AD and subjected to molecular docking studies. The inventors prepared Αβ and Drpl complex and introduced these molecular structures into this complex. Few of these molecules showed better docking score compared to existed drug molecules. Amongst DDQ is only involved in the prevention of Αβ-Drpl interaction by interacting at the active site such as ser8 and Leu34 of Αβ and ASN16 Glul6 of Drpl . And also DDQ showed better binding score (-10.8462) than existed drug molecules. DDQ is readily stopping the Drpl before forming the complex. DDQ exhibited binding interaction at Arg225 (C=0→0-P) and phenyl part of DDQ is also showing one arene cationic interaction at Arg225. Hence, among all designed molecules the inventors selected DDQ for detailed analysis but the methods and techniques apply equally to the four compounds without undue experimentation.

FIG. 2 shows a schematic of the synthetic procedure used to develop DDQ. FIG. 3 is a flowchart with the strategy of DDQ treatments in cell cultures. As shown in FIG. 3, the inventors studied five different groups of cells: (1) untreated SHSY5Y cells; (2) SHSY5Y cells treated (incubated) with DDQ (250 nM) for 24 hours; (3) SHSY5Y cells incubated with the Αβ1-42 peptide (20 μΜ final concentration) for 6 hours; (4) SHSY5Y cells incubated with Αβ1-42 for 6 hours, followed by DDQ treatment for 24 hours, and (5) SHSY5Y cells treated with DDQ for 24 hours, followed by Αβ1-42 incubation for 6 hours.

FIGS. 4A to 4C show the results of immunoblotting analysis mitochondrial dynamics, biogenesis and synaptic proteins. FIG. 4A. Representative Immunoblotting images (mitochondrial fission, fusion proteins and synaptic proteins) of DDQ, Αβ, Αβ+DDQ and DDQ+Αβ treated and untreated SHSY-5Y cells. FIG 4B. Representative Immunoblotting images (mitochondrial biogenesis proteins) of DDQ, Αβ, Αβ+DDQ and DDQ+Αβ treated and untreated SHSY-5Y cells. FIG. 4C. Quantitative densitometry analysis of mitochondrial dynamics and synaptic proteins. Quantitative densitometry analysis of mitochondrial biogenesis. Fission proteins levels were increased in cells treated with Αβ; and reduced in cells treated with DDQ, Αβ+DDQ and DDQ+Αβ treated cells. Whereas mitochondrial fusion proteins Mfnl and Mfn2 and synaptic proteins, synaptophysin and PSD95 were decreased in cells treated with Αβ, and enhanced in cells treated with DDQ, Αβ+DDQ and DDQ+Αβ treated cells.

FIGS. 5A and 5B show a co-immunoprecipitation analysis of Drpl and Αβ in SHSY5Y cells. FIG. 5A shows immunoprecipitation with the 6E10 antibody and immunoblotting with the 6E10 antibody, indicating that the specificity of 6E10 in the Co-IP analysis. In cells treated with DDQ+Αβ and Αβ+DDQ, 4 kDa Αβ levels were reduced relative cells treated Αβ alone. FIG. 5B shows Co-IP with Αβ antibody 6E10 and western blotting with Drpl antibody, indicating that Drpl interacts with 4 kDa Αβ. Reduced interaction between Αβ and Drpl was found in cells pretreated with DDQ and then Αβ added. Reduced interaction was strong in DDQ+Αβ cells compared to cells treated with Αβ alone.

FIG. 6 shows the co-immunoprecipitation analysis of mutant APPSwe/Ind cells treated with DDQ. The inventors' transfected mutant APPSwe cDNA construct into mouse neuroblastoma (N2a) cells. After 24 hrs of transfection, cells were treated with DDQ (250nM) for 24 hrs. Harvested mutant APPSwe/Ind cells treated and untreated with DDQ and prepared protein lysates and performed immunoprecipitation with Αβ (6E10) antibody and conducted immunoblotting analysis with 6E10 and Drpl antibodies. Lanes 1 and 2 represents IP with 6E10 and western blot with 6E10 and lanes 3 and 4 represents IP with 6E10 and western blot with Drpl antibody respectively. As shown in Figure, reduced levels of full-length APP and 4 kDa Αβ were found in lane 2 mutant APPSwe/Ind cells treated with DDQ compared to lane 1 of mutant APPSwe/Ind cells untreated with DDQ. Reduced levels of Drpl were found in lane 4 of mutant APPSwe cells treated with DDQ compared to lane 3 of mutant APPSwe/Ind cells untreated with DDQ. FIGS. 7A and 7B shows an immunofluorescence analysis. Immunofluorescence analysis of human neuroblastoma (SHSY5Y) cells treated with Αβ, DDQ, Αβ+DDQ and DDQ+Αβ relative to untreated cells. FIG. 7A shows representative immunofluorescence images of mitochondrial dynamic proteins and synaptic proteins. FIG. 7B shows quantitative immunofluorescence analysis of mitochondrial dynamics and synaptic proteins.

FIG. 8. Double labeling Immunofluorescence analysis of Drp l and Αβ. Double-labeling immunofluorescence analysis of Αβ (6E10 antibody) and Drpl in SHSY5Y cells. The localization of Drp l (green) and Αβ (red) and the colocalization of Drp l and Αβ (yellow, merged) at 60 ^χ the original magnification. Top panel, represents Αβ treated cells, middle panel shows Αβ+DDQ treated cells and the bottom panel shows DDQ+Αβ treated cells. As shown in FIGS. 7A and 7B, increased levels of Drp l and intra-neuronal Αβ (full-length APP) and colocalization of Drp l and Αβ in top panel, where as in the middle panel Αβ+DDQ cells, reduced Drp l and Αβ and also reduced colocalization and in the bottom panel Drpl and Αβ levels markedly reduced compared to top panel and also colocalization of Drp l and Αβ. These findings strongly suggest that DDQ 1) reduces Drp l and Αβ levels and also 2) inhibit the interaction of Drp l and Αβ in SHSY5Y cells. These findings agree with the Co-IP findings.

FIGS. 9A and 9B show sandwich ELISA analysis of Αβ40 and 42 in mutant APPSwe/Ind cells treated and untreated with DDQ. The inventors performed sandwich ELISA using protein lysates mutant APP cells treated and untreated with DDQ. FIG. 9A shows Αβ42 and FIG. 9B shows Αβ40. Significantly reduced levels of Αβ42 in mutant APPSwe/Ind cells treated with DDQ compared to mutant APPSwe/Ind cells untreated with DDQ. On the contrary, Αβ40 levels were significantly increased in mutant APPSwe/Ind cells treated with DDQ relative to mutant APPSwe/Ind cells untreated with DDQ.

FIG. 10 shows electron microscopy of SH-SY5Y cells. The inventors quantified mitochondrial architectures within the cell in all 5 groups to identify mitochondrial number and morphology. Average number of mitochondria per cell is shown in graphs. Error bars indicate the standard deviation. Mitochondrial number is significantly decreased in DDQ-treated SH-SY5Y cells, relative to untreated cells. On the contrary, mitochondrial number is significantly increased Αβ-treated SH-SY5Y cells. DDQ- pre and post-treated cells in the presence of Αβ showed reduced mitochondrial number compared to cells treated with Αβ alone. Mitochondrial length was measured for all groups of cells. Mitochondrial length was significantly reduced Αβ-treated SH-SY5Y cells. DDQ-pre and post-treated cells in the presence of Αβ showed increased mitochondrial length compared to cells treated with Αβ alone.

FIG. 1 1 shows mitochondrial function. Mitochondrial functional parameters in control human neuroblastoma (SHSY5Y) cells, in amyloid β (Αβ) incubated SHSY5Y cells, in SHSY5Y cells treated with DDQ and in SHSY5Y cells incubated with Αβ and then treated with DDQ and in SHSY5Y cells treated with DDQ and then incubated with Αβ (n=4). The inventors' analyzed mitochondrial functional data in two ways: (1) the control SHSY5Y cells were compared with the SHSY5Y cells treated with Αβ, DDQ, Αβ+DDQ and DDQ+Αβ and (2) Αβ incubated SHSY5Y cells were compared with Αβ+DDQ SHSY5Y cells and DDQ+Αβ treated cells. The inventors performed statistical analysis using ANOVA following the Dunnett correction, for: (a) H202 production, (b) lipid peroxidation, (c) cytochrome oxidase activity, (d) ATP levels, and (e) GTPase-Drpl activity.

FIG. 12 shows cell viability analysis. Cell viability of human neuroblastoma (SHSY5Y) cells, while treated with DDQ, Αβ, Αβ+DDQ and DDQ+Αβ relative to untreated cells. Cell viability of pre-treated DDQ and post-treated DDQ in Αβ incubated SHSY5Y cells relative to Αβ treated cells.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

An arising characteristic of neurodegenerative disease is the improper regulation of proteins responsible for mitochondrial fusion, fission, localization, membrane transport, and more in neurons. Recent research by the inventors has revealed the critical role of various enzymes specifically as mitochondrial regulators, including amyloid-β (Αβ), dynamin-related protein 1 (Drpl), phosphorylated tau (p-tau), and voltage- dependent anion channel protein 1 (VDAC1). The inventors have shown that interactions between combinations of these enzymes contribute to mitochondrial dysfunction and bioenergetic compromise that may cause neurodegeneration.

The present invention includes methods for identifying molecules that can delay aging process and prevent and/or stop neurodegeneration and novel compositions that delay aging and protect cells from oxidative insults, and method of using the same. It was found that the molecules identified were able to: (1) delay aging, (2) protect human cells from oxidative insults, (3) reduce the toxicity of mutant proteins, (4) inhibit abnormal protein interactions in the brain cells and (5) enhance cell survival. To date, no drugs have been developed to prevent and/or reduce abnormal protein interactions in Alzheimer's, Huntington's, and Parkinson's. The newly developed molecules inhibit these abnormal interactions and enhance cell survival and delay aging process.

Neurons are the building blocks of the nervous system, which includes the brain and spinal cord. Neurons normally don't reproduce or replace themselves, so when they become damaged or die they cannot be replaced by the body. Examples of neurodegenerative diseases are Alzheimer's, Huntington's disease, Parkinson's and amyotrophic lateral sclerosis (ALS). Various factors such as pathogenic mutations, genetic modifiers, abnormal interactions between disease causing proteins such amyloid beta or Αβ (in Alzheimer's), mutant huntingtin (in Huntington's), mutant SOD1 (in ALS) and parkin, DJ1 (in Parkinson's) and other diet and metabolism. In addition, aging is a major risk factor for neurodegenerative diseases.

Especially, in brain cells strong evidence indicates the amyloid beta precursor protein (APP), is the source of Αβ, the central player in the pathophysiology of the disease. This released Αβ induces synaptic dysfunction, mitochondrial oxidative damage, resulting in abnormal activation of redox-mediated events as well as elevation of cytoplasmic Ca ²⁺ , ultimately causing neuronal damage in AD. Recent evidence from our lab suggests mutant AD proteins Αβ and phosphorylated tau interacts mitochondrial fission protein, dynamin-related protein 1 (Drpl), induces mitochondrial fragmentation, mitochondrial dysfunction and synaptic damage in AD neurons. These abnormal interactions are age, mutant protein and disease progression dependent, indicating that age aging and abnormal protein interactions play a key role in AD development and pathogenesis. More recently, the present inventors found that mutant AD proteins Αβ and phosphorylated interacts with mitochondrial outer-membrane protein, voltage -dependent anion channel protein 1 (VDAC1), blocks mitochondrial permeability transition pores and causes gating of mitochondrial pore activities, ultimately leading to impairments in oxidative phosphorylation and low ATP production in AD neurons ⁹ . These abnormal protein-protein interactions lead to mitochondrial dysfunction, bioenergetics compromise and consequent synaptic dysfunction and loss and neuronal dysfunction in AD.

In AD, Αβ-induced synaptic dysfunction is a complicated process involving multiple pathways, components, and biological events, such as oxidative stress, kinase activation, and protein interactions. These complexities lead to the formation and accumulation of Αβ and phosphorylated tau, mitochondrial dysfunction, and ultimately neuronal death. Despite the numbers of clinical trials conducted to identify drug targets (i.e., molecules) that may reduce mutant protein toxicity in AD, such molecules remain unidentified. Recent research established that synaptic damage and mitochondrial dysfunction are early events in AD pathogenesis. Mitochondrial dysfunction is mainly due to the abnormal interaction of Drpl with Αβ, and with phosphorylated tau. Development of drugs capable of targeting underlying mechanisms of disease pathogenesis is needed in order to slow AD progression. The major focus of the proposed research effort is to develop a drug that can target the toxicity in involved in these interactions in order to prevent mitochondrial dysfunction and synaptic damage in AD progression.

Αβ, mitochondrial dysfunction, and AD: Extracellular canonical localization of Αβ has been identified in different subcellular compartments, including the endoplasmic reticulum; the Golgi apparatus (or the trans-Golgi network); early, late, and recycling endosomes; and the lysosome, where the Αβ are generated. In studies of postmortem brains from both AD patients and mouse models of AD, Αβ has also been found in mitochondria, and research from different independent research groups has clearly established that Αβ progressively accumulates in the mitochondria. Αβ has been found to induce mitochondrial dysfunction via different mechanisms. Αβ is taken up by mitochondria via the translocase of the outer membrane (TOM) complex and is imported into the inner membrane; Αβ alters the enzyme activity of the respiratory chain complexes I, and IV; Αβ affects mitochondrial dynamics by an impaired balance of fission and fusion; Αβ impairs mitochondrial permeability transition pore gating via the interaction of Αβ with VDAC1 ; Αβ induces decreased mitochondrial respiration; Αβ affects new mitochondrial biogenesis; and Αβ increases reactive oxygen species (ROS) generation.

Recent studies have shown that Drp l, which maintains and remodels mammalian mitochondria, interacts with Αβ and phosphorylates tau, leading to excessive mitochondrial fragmentation, impaired axonal transport of mitochondria, and ultimately, neuronal damage and cognitive decline.

Drp l structure and function: Based on published studies and NCBI databases, the human Drp l has been found to have several splice variants: variant 1 consists of 736 amino acids; in variant 2, exon 15 is spliced out; in variant 3, exons 15 and 16 are spliced out and have a total of 699 amino acids; variant 4 has 725 amino acids; variant 5, 710 amino acids; and variant 6, 749 amino acids. Drp l contains a highly conserved GTPase and is involved in various cellular functions. Similar to the human Drp l, the mouse Drp l has been found in multiple variants: variant 1 consists of 712 amino acids; in variant 2, exon 3 is spliced out; and in variant 3, exons 15 and 16 are spliced out. Recent research findings suggest that Drp l is involved in mitochondrial division, mitochondrial distribution, peroxisomal fragmentation, phosphorylation, SUMOlyation, and ubiquitination.

Drp l expression and mitochondrial dysfunction: Studies of Drp l in mammalian cells suggest that normal expression of Drp l is critical for normal mitochondrial dynamics and normal mitochondrial distribution and dendritic morphology in neurons. In AD and other neurodegenerative diseases, Drp 1 levels are altered via the interaction of Drp l with Αβ, leading to abnormal mitochondrial dynamics (increased fission and reduced fusion), in some cases with perinuclear clusters of mitochondria and disruption of inter-mitochondrial connectivity.

Drp l associated with Αβ: The present inventors have shown that mutant AD proteins, Αβ, and phosphorylated tau interact with Drp l, which induces mitochondrial fragmentation, mitochondrial dysfunction, and synaptic damage in neurons. These abnormal interactions are believed to be age- and disease-progression dependent, indicating that the interaction between aging and disease progression may play key roles in AD pathogenesis and development. Increasing evidence suggests that in AD, the accumulation of Αβ in synapses and synaptic mitochondria causes synaptic mitochondrial failure and synaptic degeneration.

Currently, there are no selective drug targets capable of preventing the abnormal interactions between Αβ and Drp l, and of delaying the age-dependent AD process (not only the AD process, but also other the processes of other neurological diseases, such as Huntington's, Parkinson's, and ALS). The present inventors have conducted studies, the goal of which is to develop drug molecules or molecular inhibitors capable of inhibiting or reducing abnormal interactions between Αβ and Drp 1 and of protecting neurons from multiple injuries caused by Αβ and Drp l interactions and Αβ- and Drp l -induced mitochondrial and synaptic toxicities. In these studies, the inventors identified four molecules that have demonstrated their capability to: ( 1) delay aging in neurons, (2) protect neurons from oxidative insults, (3) inhibit abnormal protein-protein interactions and protect neurons from mutant protein(s)-induced toxicities, and (4) enhance cell survival.

Very few molecules have been developed to prevent AD, but those that are the most promising are insoluble in water, rendering them problematic treatments due to water insolubility. As such, the present inventors developed water-soluble molecules that can reduce amyloid beta (Αβ) and Drp 1 levels, and that can reduce/prevent abnormal interactions between Αβ and Drp l in Alzheimer's disease (AD) affected neurons. The present invention targets: (1) increased production and accumulation of Αβ and increased expression of Drp l, and (2) abnormal interaction between Αβ and Drp l induce synaptic dysfunction and mitochondrial damage, resulting dysfunction of neurons affected by AD. The therapeutic compositions and methods disclosed herein involve treating AD-affected neurons with such molecules in order to reduce Αβ and Drp 1 levels, and to inhibit the interaction of Αβ and Drp 1.

To achieve this objective, the inventors conducted molecular docking studies and designed 82 scaffold structures. These structures were screened for existing molecules, using molecular docking software (MOE). The inventors developed the following criteria for selecting these 82 structures for further research as inhibitors of AD progression: 1) showed optimum binding energy values and 2) were capable of identifying and dissociating the binding sites of the abnormal interactions between Αβ and Drp 1. To begin, the inventors selected the best molecules of the 82, which exhibited the best optimum binding energy values and binding capabilities for these proposed in vitro and in vivo studies.

The following scaffold structures were designed to: ( 1) delay aging and (2) inhibit abnormal protein interactions. These designed structures screened for in silico studies using molecular docking software. Further, the inventors selected molecules that showed (1) optimum binding energy values and (2) identify and dissociate the binding sites of abnormal protein interactions - Αβ and Drpl, and phosphorylated tau and Drpl; Αβ and VDAC1 and phosphorylated tau and VDAC1 in AD neurons. A: Phosphonium cation based structures; B: Quinoline based alpha aminophosphonates; C: Napthaline based alpha aminophosphonates and D: Hexahydropyramidine carboxylates structures.

R- Aikyl/ «ryi R- Aikyl/ aryi R- Aikyl at l IV- Alkvi arvl

R*-Alkvl

A B C D

Molecular Docking results. The inventors prepared a protein complex of Αβ and Drpl by retrieving the crystal structures of Αβ (PDB ID: lba4) and dynamin-l-like protein (PDB ID: 4hlu) from a protein data bank. In Αβ and Drpl complex, Ser8, Leu34, Glyl20, Gly25 of Αβ are interacting with ASN98, ILU22, ASN12, Glu22 of Drpl respectively; the inventors predicted these sites as active interacting sites of Αβ and Drpl complex. Designed molecular structures were introduced into Αβ and Drpl complex in order to identify the interactions of ligands in the complex and identify their inhibitory properties against Αβ-Drp 1 interaction. Few molecules showed good binding score against this complex. Particularly, DDQ only interacted at specific interacting sites in the Αβ-Drpl complex and exhibited the best docking capability and received the best docking score than all other molecules. Correspondingly, DDQ is obstructing these Αβ and Drpl bindings by direct interactions at active sites such as ser8 and Leu34 of Αβ and ASN16 Glul6 of Drpl . DDQ is readily bound with Drpl independently before forming a complex. Therefore, the inventors selected DDQ as a selective target to treat against AD neuronal cells. The inventors synthesized DDQ by following protocol. Table 1 is a summary of molecular docking of the Drpl compounds.

Table 1. Molecular docking of the Drpl compounds.

DDQ (diethyl(3,4-dihydroxyphenethylamine)(quinolin-4-yl)methylph osphonate):

MitoQ (Phosphonium, [10-(4,5- dimethoxy-2-methyl-3,6-dioxo-l,4-cyclohexadien- l-yl)decyl]triphenyl-, methanesulfonate), in the non- limiting form of a mesylate.

Dynasore (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide) in the non- limiting form of a hydrate:

Mdivi-1 (3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(lH) -quinazolinone, 3-(2,4-Dichloro- 5-methox henyl)-2-sulfanyl-4(3H)-quinazolinone):

Each of the compounds can be formulated with one or more pharmaceutically acceptable salts, and if applicable, hydrates, solvates, tautomeric forms, stereoisomers, and prodrugs of the compounds described herein.

As used herein, the terms "pharmaceutically acceptable" or "physiologically acceptable" refer to compounds, salts, compositions, dosage forms and other materials which are useful in preparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

As used herein, the term "pharmaceutically acceptable salt" of a given compound refers to salts that retain the biological effectiveness and properties of the given compound, and which are not biologically or otherwise undesirable.

As used herein, the terms "pharmaceutically acceptable salts" or "physiologically acceptable salts" refer to salts with inorganic acids and salts with an organic acid. In addition, if the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare nontoxic pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethane sulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like. Likewise, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines (i.e., NH ₂ (alkyl)), dialkyl amines (i.e., HN(alkyl) ₂ ), trialkyl amines (i.e., N(alkyl) ₃ ), substituted alkyl amines (i.e., NH ₂ (substituted alkyl)), di(substituted alkyl) amines (i.e., HN(substituted alkyl) ₂ ), tri(substituted alkyl) amines (i.e., N(substituted alkyl) ₃ ), alkenyl amines (i.e., NH ₂ (alkenyl)), dialkenyl amines (i.e., HN(alkenyl) ₂ ), dialkenyl amines (i.e., N(alkenyl) ₃ ), substituted alkenyl amines (i.e., NH ₂ (substituted alkenyl)), di(substituted alkenyl) amines (i.e., HN(substituted alkenyl) ₂ ), tri(substituted alkenyl) amines (i.e., N(substituted alkenyl) ₃ , mono-, di- or tri-cycloalkyl amines (i.e., NH ₂ (cycloalkyl), HN(cycloalkyl) ₂ , N(cycloalkyl) ₃ ), mono-, di- or tri-arylamines (i.e., NH ₂ (aryl), HN(aryl) ₂ , N(aryl) ₃ ), or mixed amines, etc. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. The term "hydrate" refers to the complex formed by the combining of a compound and water.

As used herein, the term "solvate" refers to an association or complex of one or more solvent molecules and a compound of the invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethylsulfoxide, ethylacetate, acetic acid, and ethanolamine. As used herein, the terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" or "excipient" refer to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The compounds described herein, diethyl(3,4-dihydroxyphenethylamine)(quinolin-4- yl)methylphosphonate (DDQ) ; phosphonium, [ 10-(4,5 -dimethoxy-2-methyl-3 ,6-dioxo- 1 ,4-cyclohexadien- l-yl)decyl]triphenyl-, methanesulfonate) (MitoQ); (3-Hydroxy-naphthalene-2-carboxylic acid (3,4- dihydroxy-benzylidene)-hydrazide (Dynasore); and/or (3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2- thioxo-4(lH)-quinazolinone, 3-(2,4-Dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolin one) (Mdiv- 1), may be modified as will be known by the skilled artisan to target those regions of the molecules shown as clouds in FIG. 1A, for example, any of these regions may be modified or substituted with one or more of the following: alkyl, alkenyl, alkynyl, alkylene, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, aryl, heterocyclyl, heteroaryl, and/or heteroalkyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to alkyl, alkenyl, alkynyl, alkoxy, alkylthio, acyl, amido, amino, amidino, aryl, aralkyl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkylalkyl, guanadino, halo, halogen, haloalkyl, haloalkoxy, hydroxyalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, hydrazine, hydrazone, imino, imido, hydroxy, oxo, oxime, nitro, sulfonyl, sulfinyl, alkylsulfonyl, alkylsulfinyl, thiocyanate, sulfinic acid, sulfonic acid, sulfonamido, thiol, thioxo, N-oxide, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl

Synthesis of diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonat e. Synthesis of diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonat e (DDQ). To synthesize DDQ, the inventors followed two steps pudovik reaction. In the first step 4-(2-(quinolin-4- ylmethyleneamino)ethyl)benzene-l,2-diol was prepared by stirring 4-(2-aminoethyl)benzene- l,2-diol with quinoline-4-carbaldehyde at reflection temp 80 °C of THF. The inventors isolated the intermediates and 4-(2-(quinolin-4-ylmethyleneamino)ethyl)benzene-l,2-diol is selected for the second step reaction. In the second step, Dethylphosphite (DEP) was added to the solution of (Z)-4-(2-(quinolin-4- ylmethyleneamino)ethyl)benzene- l,2-diolin in drop-wise fashion, at room temperature in the presence of the S1O ₂ OSO ₃ H catalyst. The reaction mixture was stirred continuously for 4 hours at 65 °C. The reaction between DEP and Z)-4-(2-(quinolin-4-ylmethyleneamino)ethyl)benzene- l,2-diol was monitored by thin-layer chromatography, using silica gel as the adsorbent and a mixture of ethyl acetate and hexane (1 :2) as the eluent. After the reaction was complete, the mixture was quenched with water, and four samples of ethyl acetate (each at 10 mL) were then extracted. The samples were concentrated, using reduced pressure. The resulting mixture was purified by column chromatography, using a 100-200 mesh silica gel as the adsorbent and a mixture of ethyl acetate and hexane (1 :4) as the eluent, to derive the pure DDQ. Spectral data for diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonat e (DDQ). Yield: 82%; IR (KBr): 3509 and 3472 (20H), 3309 (N-H), 1270 (P = O), 1019 (P-C Ar ) cm ^"1 ; ^l H NMR (400 MHz, DMSO-d6): δ 1.18 (CH ₃ , 6H), 2.61-2.92 ((Ar-CH ₂ and CH ₂ -NH, 4H), 3.13 (NH, 1H), 4.12 (CH ₂ -CH _3, 4H), 4.82 (P-CH, 1H), 6.68-8.55 (9 ArH), 9.38 and 9.45 (Ar-OH, 2); ³¹ P NMR (161.9 MHz, DMSO-d6): δ 8.935;[ref.46] LC MS (%): m/z 431.7 (19%) [ΜΗ ^+' ], 430.9 (89%) [M ^+- ], 236.0 (100%) [ref. 47-48].

Based on the molecular docking results, synthesized DDQ is forwarded to screen its reducing effect of Αβ and Drpl levels and to determine its inhibiting capacity against Αβ and Drpl complex formation. So that, the inventors treated DDQ in human neuroblastoma (SHSY5Y) cells as shown in FIG. 3. These treated cells were used to quantify the effect of DDQ on gene expression and protein levels of synaptic, AD- related, and mitochondrial-related genes. The inventors determined the Αβ and Drpl interaction inhibitory property of DDQ and also quantified the number of mitochondria in the treated cells.

mRNA levels of mitochondrial dynamics, mitochondrial biogenesis and synaptic genes. Using the reagent TriZol (Invitrogen), the inventors isolated total RNA from all DDQ-treated and untreated cells. mRNA levels of mitochondrial dynamic (Drpl, Fisl, Mfnl and Mfh2), mitochondrial biogenesis genes (PGCla, Nrfl, Nrf2 and TFAM) and synaptic genes (PSD95, synaptophysin, synapsin 1, synapsin 2, synaptobrevin 1, synaptobrevin 2, synaptopodin, and GAP43) were measured by using Sybr-Green chemistry-based quantitative real time RT-PCR.

As shown in Table 2, in Αβ treated cells Drpl and in Fisl (mitochondrial fission genes) expression levels were significantly increased by 2.2 fold (P=0.004), and 1.7 fold (P=0.003) respectively, compared to untreated cells. In contrast, mRNA expression levels of mitochondrial fusion genes were significantly decreased (Mfnl by 2.3 fold (P=0.005) and Mfn2 by 2.6 fold (P=0.001)) in Αβ treated cells relative to untreated cells. This indicating the presence of abnormal mitochondrial dynamics in cells treated with Αβ. Drpl (2.2 fold decrease, P=0.01 in DDQ treated cells) and Fisl (4.4 fold decrease, P=0.001) mRNA levels were significantly decreased and fusion genes, Mfnl (1.7 fold P=0.02) and Mfh2 (2.3 fold P=0.002) mRNA levels are increased in DDQ treated cells relative to untreated cells. mRNA changes were significantly reduced for fission genes Drpl and Fisl, and increased for fusion genes Mfnl and Mfh2 in cells incubated with Αβ and then treated with DDQ relative to untreated cells. Similarly, cells pretreated with DDQ and incubated with Αβ relative to untreated cells, mRNA levels were unchanged for fission genes Drpl and Fisl, and fusion genes Mfnl and Mfn2.

Table 2. mRNA fold changes of mitochondrial structural, mitochondrial biogenesis and synaptic genes in Human Neuroblastoma (SHSY5Y) cells treated with DDQ, Αβ, Αβ+DDQ and DDQ+Αβ relative to the untreated SHSY5Y cells and cells treated with Αβ+DDQ and DDQ+Αβ relative to the Αβ-treated SHSY5Y cells.

Mitochondrial biogenesis genes (PGCla, Nrfl, Nrf2 and TFAM) expressions were significantly decreased in Αβ effected neuronal cells and healthy increase in DDQ treated cells relative to untreated cells. Biogenesis genes mRNA expression of Αβ effected neuronal cells was decreased as PGCla by 4.4 fold (P=0.001), Nrfl by 4.1 fold (P=0.004), Nrf2 by 2.8 fold (P=0.01) and TFAM by 4.6 fold (P=0.002)) relative to untreated cells. Biogenesis genes mRNA levels in DDQ treated cells relative were significantly increased as PGCla (1.5 fold decrease, P=0.02), Nrfl by 1.9 fold (P=0.01), Nrf2 by 2.7 fold (P=0.01) and TFAM by 1.7 fold (P=0.02) relative to untreated cells (Table 2). These observations indicate that DDQ increases mitochondrial biogenesis activity. Mitochondrial biogenesis genes PGCla (2.5 fold P=0.01), Nrfl (2.6 fold P=0.003), Nrf2 (2.1 fold P=0.03) and TFAM (2.3 fold P=0.01) levels were significantly increased in cells incubated with Αβ followed by treated with DDQ relative to Αβ treated cells. DDQ pretreated followed by Αβ incubated cells exhibited increased of mRNA levels for biogenesis genes (PGCla (3.9 fold P= 0.002), Nrfl (4.9 fold P= 0.0003), Nrf2 (2.6 P= 0.01) and TFAM (3.2 P= 0.002)) relative to Αβ treated cells. These results suggest that DDQ pretreatment prevented Αβ induced biogenesis toxicity.

In cells treated with Αβ compared with untreated cells, mRNA expression levels were decreased for synaptophysin by 3.7 fold (P=0.0002), PSD95 by 2.5 fold (P=0.004), synapsinl by 1.9 (P=0.02), synapsin2 by 1.4 (P=0.04), synaptobrevinl by 2.4 (P=0.003), synaptobrevin2 by 2.3 (P=0.01), synaptopodin by 2.3 (P=0.001) and GAP43 by 1.9 (P=0.001) indicating that Αβ reduces synaptic activity. mRNA levels were significantly increased for synaptophysin by 1.4 fold (P=0.04), PSD95 by 1.4 (P=0.03), Synapsin2 by 1.7 (P=0.01), synaptobrevin2 by 1.3, and GAP43 by 1.2 fold in DDQ treated cells relative to untreated cells. These observations indicate that DDQ boosts synaptic activity in healthy cells. In cells incubated with Αβ and then treated with DDQ relative to untreated cells, mRNA levels were increased. These observations indicate that DDQ rescued synaptic activity from Αβ induced toxicity. In cells pretreated with DDQ and incubated with Αβ relative to Αβ cells, mRNA levels were increased for synaptic mitochondrial biogenesis and mitochondrial fusion genes, indicating that DDQ enhances synaptic and mitochondrial fusion activities.

In cells incubated with Αβ and then treated with DDQ and pretreated with DDQ and incubated with Αβ relative to Αβ treated cells, the fold change of mitochondrial fission genes (Drpl and Fisl) were down- regulated and mitochondrial fusion genes (Mf l and Mfh2) were significantly upregulated. Similarly, synaptic gene expression levels were upregulated in cells incubated with Αβ and then treated with DDQ and pretreated with DDQ and incubated with Αβ relative to Αβ treated cells. This indicating that DDQ is protecting synaptic genes from Αβ.

Immunoblotting analysis. To determine the effects of Αβ on mitochondrial proteins and the useful effects of DDQ at the protein level, the inventors quantified mitochondrial proteins in five independent treatments of cells with Αβ, DDQ, Αβ+DDQ and DDQ+Αβ.

Comparison with untreated cells. In SHSY5Y cells treated with Αβ compared with untreated SHSY5Y cells, significantly increased proteins levels were found for Drpl (P=0.01) and Fisl (P=0.01) (Figure 4A, C). In contrast, decreased levels of mitochondrial fusion proteins, Mfhl (P=0.002) and Mfh2 (P=0.004) were found in cells incubated with Αβ compared with untreated cells. Synaptophysin (P=0.001) and PSD95 (P=0.01) levels were significantly reduced in Αβ incubated cells relative to untreated cells (Figure 4A, C).

Mitochondrial fission proteins, Drpl (P=0.03) and Fisl were significantly reduced and fusion protein Mfn2 P=0.004 was significantly increased in DDQ treated cells relative to untreated cells (Figure 4A, C). Mitochondrial biogenesis protein levels were significantly increased in DDQ treated cells relative to untreated cells (Figure 4B, D).

Mitochondrial biogenesis proteins PGCla (P=0.01), Nrfl (P=0.001), Nrf2 (P=0.01) and TFAM (P=0.01) levels were decreased in Αβ incubated cells relative to untreated cells. Interestingly, significant increase of mitochondrial biogenesis protein levels was observed in DDQ treated cells relative to untreated cells (Figure 4B and D).

Mitochondrial fission proteins Drpl (P=0.01) and Fisl (P=0.02) were reduced and fusion protein Mfnl (P=0.04) was significantly increased in Αβ+DDQ treated cells relative to untreated cells (Figure 4A, C). Synaptic proteins, synaptophysin (P=0.01) and PSD95 (P=0.04) levels significantly increased in Αβ+DDQ treated cells relative to untreated cells. Decreased levels of Drpl (P=0.004) were found in DDQ+Αβ treated cells relative to untreated cells (Figure 4A, C). Overall, these findings suggest that DDQ reduces fission activity and enhances fusion activity in the presence of Αβ.

Comparison with Αβ treated cells. As shown in Figure 4 (A, B), significantly reduced levels of fission protein, Drpl were found in cells treated with Αβ+DDQ (Drpl, P=0.01) and DDQ+Αβ (Drpl, P=0.001; Fisl, P=0.04) relative to Αβ treated cells. In contrast, fusion proteins were increased in Αβ+DDQ (Mfnl, P=0.01; Mfn2, P=0.01) and DDQ+Αβ (Mfnl, P=0.02; Mfn2, P=0.01) treated cells relative to Αβ treated cells.

In Αβ+DDQ cells exhibited increased mitochondrial biogenesis protein levels (Nrfl (P=0.04), Nrf2 (P=0.01) and TFAM (P=0.01) relative to Αβ treated cells. Similarly, DDQ pre-treated (DDQ+Αβ) cells showed significantly increased levels of mitochondrial biogenesis proteins (Nrfl (P=0.01), Nrf2 (P=0.01) and TFAM (P=0.004) relative to Αβ treated cells (Figure 4B, D).

Synaptic proteins were increased in Αβ+DDQ (synaptophysin, P=0.01; PSD95, P=0.03) and DDQ+Αβ (synaptophysin, P=0.04; PSD95, P=0.001) treated cells relative to Αβ treated cells (Figure 4A, C), indicating that DDQ enhances synaptic activity in the presence of Αβ in cells.

DDQ reduces Αβ and Drpl levels. To determine whether DDQ reduces Αβ and Drpl levels, the inventors conducted immunoblotting analysis, in cells treated with DDQ, Αβ+DDQ and DDQ+Αβ. As shown in Figure 5A, the inventors found reduced levels of 4 kDa Αβ in cells treated Αβ+DDQ and DDQ+Αβ relative to cells treated with Αβ alone. The inventors also found reduced levels of Drpl in cells treated DDQ, Αβ+DDQ and DDQ+Αβ relative to untreated and Αβ treated cells (Figure 4A).

Co-immunoprecipitation and immunoblotting analysis using DDQ treated SHSY5Y cells+Αβ. To determine whether DDQ reduces the interaction of Αβ with Drpl in Αβ incubated cells, the inventors performed co-immunoprecipitation analysis using the Drp 1 antibody, and immunoblotting analysis using Αβ recognizing 6E10 antibody and protein lysates of DDQ pre-treated, DDQ post-treated and Αβ incubated cells. As shown in Figure 5B, the inventors found reduced interaction between Αβ and Drpl in DDQ pre-treated, DDQ post-treated relative to Αβ incubated cells. Reduced interaction was strong in DDQ pre-treated than DDQ post-treated cells, indicating that prevention is better than treatment.

Co-immunoprecipitation and immunoblotting analysis of mutant APP _Swe /ind cells treated with DDQ. To determine whether DDQ reduces the interaction of Αβ with Drp 1 in mutant APP _Swe /ind cells, the inventors used mutant APP _Swe /ind cDNA construct transfected into mouse neuroblastoma (N2a) cells and further cells were treated with DDQ. The inventors performed immunoprecipitation with Αβ (6E10) antibody and immunoblotting analysis with 6E10 and the inventors also performed co-immunoprecipitation analysis with Αβ (6E10) antibody and immunoblotting analysis with Drp l antibody. As shown in Figure 6, the inventors found reduced levels of full-length APP and 4 kDa Αβ in lane 2 of mutant APP _Swe /ind cells treated with DDQ compared to lane 1 of mutant APP _Swe /ind cells untreated with DDQ. The inventors also found reduced levels of Drp 1 in lane 4 of mutant APP _{Swe Ind} cells treated with DDQ compared to lane 3 of mutant APP _{Swe Ind} cells untreated with DDQ. These findings further confirm that DDQ reduces full-length APP and 4 kDa Αβ and reduces interaction between Drp l and Αβ.

Immunofluorescence analysis of Drp l, synaptophysin and PSD95. To determine the effect of Αβ and DDQ on Drp l, synaptophysin and PSD95 levels and localizations, immunofluorescence analysis was performed in cells treated as shown in FIG. 7.

As shown in Figure 7A and B, the inventors found significantly increased Drp l (P=0.003) levels in Αβ treated cells relative to untreated cells, indicating that Αβ enhances fission activity in cells. In contrast, decreased Drp l levels were found in DDQ treated cells relative to untreated cells, but this was not significant. The synaptic proteins synaptophysin (P=0.002) and PSD95 (P=0.001) were significantly reduced in Αβ treated cells relative to untreated cells (Figure 7A and B).

Significantly reduced levels of fission protein Drp lwere found in cells treated with Αβ+DDQ (P=0.03) and DDQ+ Αβ (P=0.004) relative to Αβ treated cells (Figure 7A and B). In contrast, synaptic proteins were increased in Αβ+DDQ (synaptophysin, P=0.04; PSD95, P=0.03) and DDQ+Αβ (synaptophysin, P=0.004; PSD95, P=0.01) treated cells relative to Αβ treated cells (Figure 7A and B), indicating that DDQ enhances synaptic activity in the presence of Αβ in cells. Overall, the immunofluorescence findings agreed with the immunoblotting results.

Double -labeling immunofluorescence analysis of Drpl and Αβ. To determine whether Drp l localizes and interacts with Αβ, the inventors conducted double -labeling analysis of Drp l and Αβ in DDQ pre-treated, post-treated and untreated, Αβ incubated cells. As shown in Figure 8, the immunoreactivity of Drp l was colocalized with Αβ immunoreactivity (monomelic), indicating that Drpl interacts with Αβ.

Further, the inventors found reduced co-localization of Drp 1 with Αβ in DDQ pre-treated and post-treated Αβ incubated cells relative to Αβ incubated cells alone. Drpl and Αβ colocalization is markedly reduced in DDQ pre-treated cells than DDQ post-treated cells. These observations matched the immunoprecipitation findings of Drpl and 6E10. Overall, these observations suggest that DDQ reduces Drpl and Αβ interactions.

DDQ reduces soluble Αβ42 in mutant APP _Swe /ind cells treated with DDQ. To determine whether DDQ reduces Αβ levels, the inventors performed sandwich ELISA using protein lysates of mutant APP _Swe /ind cells treated with DDQ. As shown in Figure 9, the inventors found significantly decreased levels of Αβ42 in DDQ treated mutant APP _Swe /ind cells (P=0.01) relative to DDQ untreated mutant APP _Swe /ind cells. On the contrary, Αβ40 levels were significantly increased in DDQ treated mutant APP _Swe /ind cells (P=0.03) relative to DDQ untreated mutant APP _Swe /ind cells. These observations indicate that DDQ reduces Αβ42 levels in mutant APP _{Swe Ind} cells.

Transmission electron microscopy. To determine the effects of DDQ on mitochondrial number and morphology, and any rescual effects of DDQ on mitochondria in the untreated and Αβ treated cells, the inventors used TEM on untreated, DDQ, Αβ, Αβ+DDQ and DDQ+Αβ, treated SHSY5Y cells.

Mitochondrial number and length comparison of DDQ, Αβ, Αβ+DDQ and DDQ+Αβ treated cells with untreated cells. Mitochondrial number: As shown in Figure 10, the inventors found significantly increased number of mitochondria in Αβ treated cells relative to untreated cells (P=0.03), suggesting that Αβ treatment enhances mitochondrial number, in other words Αβ treatment enhances mitochondrial fragmentation. On the other hand, DDQ treatment reduced the number of mitochondria relative to untreated cells (P =0.003), suggesting that DDQ treatment reduces mitochondrial fragmentation. Interestingly, Αβ+DDQ and DDQ+Αβ treated cells have exhibited approximately equal number of mitochondria relative to untreated cells (Figure 10).

Mitochondrial length. The inventors also measured mitochondrial length in order to understand whether DDQ treatment alters mitochondrial length. As shown in Figure 10, the inventors found mitochondrial length is significantly increased in cells treated with DDQ relative to untreated cells. On the contrary, mitochondrial length is significantly reduced in Αβ treated cells (P=0.005) relative to untreated cells. Mitochondrial length is not significantly changed in Αβ+DDQ and DDQ+Αβ treated cells relative to untreated cells, indicating that DDQ preventing/rescuing mitochondrial length in the presence of Αβ.

Mitochondrial number and length comparison of Αβ+DDQ and DDQ+Αβ treated cells with Αβ treated cells. The number of mitochondria were significantly reduced in DDQ-pre (P=0.001) and post-treated (P=0.02) cells relative to cells treated with Αβ alone (Figure 10). Mitochondrial length is significantly increased in DDQ-pre (P=0.02) and post-treated (P=0.02) cells relative to cells treated with Αβ alone. These findings indicate that DDQ reduces excessive mitochondrial fragmentation and increased mitochondrial length in AD neurons.

Mitochondrial functional assays. H ₂ O ₂ production: As shown in FIG. 11, significantly increased levels of hydrogen peroxide (H ₂ O ₂ ) were found in mitochondria from cells incubated with Αβ (P=0.001). In measurements taken of H ₂ O ₂ from mitochondria isolated from cells treated with DDQ, significantly decreased levels of H ₂ O ₂ (P=0.01) were found relative to untreated cells. These findings suggest that Αβ increases free radical production and DDQ reduces H ₂ O ₂ in the presence of Αβ. Significantly increased levels were found in cells incubated with Αβ+DDQ (P=0.01) and DDQ+Αβ (P=0.03) relative to untreated cells. When the data were compared between cells incubated with Αβ and Αβ+DDQ (P=0.04) and DDQ+Αβ (P=0.002) cells, H ₂ O ₂ levels were significantly reduced, indicating that DDQ reduces H ₂ O ₂ levels in the presence of Αβ (Figure 11).

Lipid peroxidation: Significantly increased levels of lipid peroxidation (4-hydroxy-nonenol) were found (P=0.002) in Αβ treated relative to untreated cells (FIG. 11). However, significantly decreased levels were found in the DDQ treated cells (P=0.04) relative to untreated cells. The inventors also found significantly reduced levels of lipid peroxidation in Αβ+DDQ (P=0.04) and DDQ+Αβ (P=0.04) relative to cells incubated with Αβ alone, indicating that DDQ reduces lipid peroxidation levels in the presence of Αβ (Figure 11).

ATP production: As shown in Figure 11, significantly decreased levels of ATP were found in cells that were incubated with Αβ (P=0.01) relative to untreated cells. Significantly increased levels of ATP were found in cells treated with DDQ compared with untreated cells. Significantly increased levels were found in cells incubated with Αβ+DDQ (P=0.03) relative to untreated cells (Figure 11). Significantly increased ATP levels were found in Αβ+DDQ (P=0.04) and DDQ+Αβ treated (P=0.01) cells relative to Αβ incubated cells, indicating DDQ increases ATP levels in the presence of Αβ.

Cytochrome oxidase activity: Significantly decreased levels of cytochrome oxidase activity were found in cells that were incubated with Αβ (P=0.04) (Figure 11). However, significantly increased levels of cytochrome oxidase activity were found in DDQ treated cells relative to untreated cells. Cytochrome oxidase activity levels were unchanged in Αβ+DDQ and DDQ+Αβ cells relative to untreated cells. Similar to ATP levels, cytochrome oxidase activity levels were increased in Αβ+DDQ and DDQ+Αβ treated (P=0.03) cells relative to Αβ incubated cells (FIG. 11), indicating DDQ increases cytochrome oxidase activity levels in the presence of Αβ. GTPase-Drp l activity: To determine whether Αβ affects GTPase-Drpl activity in cells that treated with Αβ incubation, the inventors measured GTPase-Drp l activity from Drp l IP elutes of all cell treatments. Interestingly, the inventors also found significantly increased Drp 1 enzymatic activity in Αβ treated cells (P=0.01) relative to untreated cells. The inventors found decreased levels were found in the DDQ treated cells (P=0.04) relative to untreated cells. Significantly increased Drpl enzymatic activity was observed in cells incubated with Αβ+DDQ (P=0.04) relative to untreated cells. Drp l enzymatic activity was reduced in cells treated like Αβ+DDQ (P=0.05) and DDQ+Αβ (P=0.02) relative to cells incubated with Αβ, indicating that DDQ reduces GTPase-Drpl activity in the presence of Αβ (Figure 1 1).

Cell viability. Significantly decreased levels of cell viability were found in Αβ treated cells (P=0.01) (Figure 12) relative to untreated cells. Cell viability was also significantly increased in cells treated with DDQ (P=0.01) compared with untreated cells. Cell viability levels were unchanged in cells treated with Αβ+DDQ and DDQ+Αβ relative to untreated cells. Significantly increased cell viability levels were found in cells treated with DDQ+Αβ (P=0.03) relative to Αβ incubated cells, suggesting that DDQ increases cell viability in the presence of Αβ.

The present inventors have developed a robust and reproducible method for identifying drug molecules that reduce Drp 1 and Αβ levels and also to inhibit abnormal interaction between Drp 1 and Αβ that reduce excessive mitochondrial fragmentation and maintain mitochondrial function and synaptic activity in AD neurons. The elevated levels of Αβ and increased expressions of mitochondrial fission protein Drp l, and abnormal interactions between Αβ and Drp l, have been found to induce synaptic dysfunction and mitochondrial damage, causing neuronal dysfunction in AD neurons. As the target to develop aqua- soluble drug molecule, that is capable of reducing Αβ and Drp l levels in AD neurons and also that can inhibit Αβ and Drp l interaction, the inventors have designed 82 molecular crystal structures based on existing drug molecules and screened by molecular docking studies. The inventors designed these molecules with multiple functions including anti-inflammatory, anti-antioxidant, pro-longevity, and anti- amyloid functions. For the first time, the inventors selected DDQ as a novel drug target because, it bound at Αβ and Drp l interacting sites to inhibit Αβ and Drp l complex formation and also showed better docking score than other designed molecules and existing molecules such as MitoQ, Mdivi l and SS31. Additionally, DDQ is formulated to dissolve in water. DDQ is obstructing Αβ and Drp 1 binding sites by direct interactions at active sites of Αβ (ser8 and Leu34) and Drp l (ASN16 and Glul6) (FIG. IB). DDQ is readily bound with Drp l (independently before forming a Drp l- Αβ complex), leaving less Drp l binding sites with Αβ - meaning DDQ interfere with Drp l and Αβ interactions (FIG. 1C). FIG. 2 shows a schematic of the synthetic procedure used to develop DDQ. The inventors examined the protective effects of DDQ in healthy human neuroblastoma cells, and also in neurons incubated with Αβ42. The inventors studied preventive (DDQ+Αβ) and intervention (Αβ+DDQ) effects of DDQ against Αβ in AD neurons. The inventors measured mR A and protein levels of mitochondrial dynamics, biogenesis and synaptic genes using real time RT-PCR, immunoblotting and immunofluorescence analysis. The inventors also assessed mitochondrial function by measuring H ₂ O ₂ , lipid peroxidation, cytochrome oxidase activity, GTPase-Drpl activity and mitochondrial ATP. Further, the inventors studied cell viability using the MTT assay. Mitochondrial number and morphology was studied using transmission electron microscopy.

Mitochondrial fission protein levels were increased and fusion, biogenesis and synaptic proteins levels were decreased in Αβ treated neurons relative to untreated neurons, indicating the toxicity of Αβ. On the contrary, DDQ enhanced fusion activity; reduced fission machinery; and increased mitochondrial biogenesis & synaptic activities. DDQ pre- and post-treated of Αβ incubated cells showed appropriate mitochondrial dynamics and synaptic activities similar to untreated neurons. Likewise, DDQ pre- and post-treated of Αβ incubated neurons showed reduced abnormal mitochondrial dysfunction, maintained cell viability and synaptic activity, relative to Αβ treated neurons. Further, the protective effects of DDQ were stronger in pretreated neurons than in post-treated neurons, as such, DDQ works better in prevention than treatment in AD-like neurons. Mitochondrial count is significantly decreased in DDQ-treated neurons relative to untreated neurons. These findings strongly suggest that DDQ is a promising molecule to treat AD neurons.

DDQ reduces the levels of Αβ and Drpl and interaction between Αβ and Drpl . Using co- immunoprecipitation, immunoblotting and double-labeling immunofluorescence analyses, the inventors studied Drpl and Αβ and their interactions. The inventors found that Αβ interacts with Drpl in Αβ incubated cells, and this interaction is gradually reduced by pre- and post-treatment of DDQ and the reduction of Drpl and Αβ is stronger in DDQ pre-treated cells than post-treated. Further, the inventors also found reduced levels of Αβ and Drp 1 in DDQ treated AD (Αβ+DDQ and DDQ+Αβ) neurons (Figure 5).

Using co-immunoprecipitation and immunoblotting analysis and mutant APP _Swe /ind cells, the inventors also studied Drpl and full-length APP and Αβ levels, and also Drpl interaction with Αβ. The inventors found reduced levels of Drpl, full-length APP and Αβ in DDQ-treated mutant APP _Swe /ind cells relative to DDQ untreated APP _Swe /ind cells, indicating that DDQ reduces Drpl, full-length APP and Αβ levels in mutant APP _{Swe Ind} cells (Figure 6).

The double-labeling immunofluorescence analysis (Figure 8.) strongly agreed with the co-IP data. The previous findings state that these interaction/co localization increases as AD progresses (10). The double- labeling immunofluorescence analysis of Αβ and Drp l in DDQ pre-treated and post-treated in the presence Αβ showed reduced Αβ and Drp l colocalization relative to neurons incubated with Αβ alone.

The reduced interaction between Αβ and Drp 1 may be due to the reduced synthesis/production of Αβ and Drp l in DDQ pre- and post-treated cells. This reduced interaction of Αβ with Drp l in DDQ pre-and post- treated cells may reduce mitochondrial fragmentation and keep mitochondria in normal count, normal length and normal function and further it may protect to neuronal cells from Αβ attack. These findings lead to the conclusion that DDQ reduced Αβ and Drp 1 levels and also prevented/recused the interaction of Αβ and Drp l ; and inhibit mitochondrial fragmentation in neurons affected by AD. The current findings of Figure 5 (SHSY5Y cells pre- and post-treated DDQ and Αβ) and Figure 6 (mutant APP _Swe /ind cells treated with DDQ) strongly suggest that DDQ reduces the synthesis/production of full-length APP, Αβ and Drp 1 and these reduced levels of full-length APP, Αβ and Drp 1 are one of the possible reasons for reduced interaction between Αβ and Drp l in AD neurons.

Further, as expected DDQ is obstructing Αβ and Drp l binding sites by direct interactions at active sites of Αβ (ser8 and Leu34) and Drp l (ASN16 and Glul6) (Figure 1). DDQ is readily bound with Drp l (independently before forming a Drp l- Αβ complex), leaving less Drp l binding sites with Αβ. However, additional research is needed to determine the precise effects of DDQ's role in reducing physical interaction between Αβ and Drp 1.

DDQ reduces Αβ42 in mutant APP _Swe /ind cells. To determine whether DDQ reduces Αβ42 levels, the inventors measured both Αβ42 and Αβ40 in mutant APP _Swe /ind cells treated and untreated with DDQ. Interestingly, Αβ42 levels were significantly reduced and Αβ40 levels were significantly increased in DDQ-treated mutant APP _Swe /ind cells (Figure 9) relative to DDQ-untreated APP _Swe /ind cells. These observations are interesting and may have therapeutic value for AD. It is possible that DDQ blocks/reduces the activity of epsilon cleavage site (that is responsible for Αβ42 production) at C-terminal region of Αβ in mutant APP _Swe /ind cells. Additional research is still needed in order to determine how DDQ reduces the levels of Αβ42 and increases Αβ40 levels in AD neurons.

DDQ maintains mitochondrial function and cell viability. To determine differences in mitochondrial function among DDQ-treated and untreated cells (as shown Figure 3), the inventors assessed mitochondrial function assays in all treated and untreated cells. The parameters included H ₂ O ₂ production, lipid peroxidation, ATP production, GTPase Drp l enzymatic activity and cell viability. In Αβ treated cells, mitochondrial function was found to be defective and cell viability was also reduced. These observations agree with others on Αβ induced defective mitochondrial function and cell viability. Interestingly, DDQ treated cells showed enhanced mitochondrial function (Figure 1 1) and increased cell viability (Figure 12), implying that DDQ treated cells exhibited increased mitochondrial ATP, cytochrome oxidase activity and cell viability, and reduced free radicals and oxidative stress. These observations strongly suggest that DDQ reduces cellular toxicity and boosts mitochondrial function and promotes cell longevity.

In summary, for the first time, the inventors designed and synthesized DDQ based on the best docking score and its binding interactions with Αβ and Drpl complex. In AD neurons treated with DDQ, the inventors found reduced levels of mitochondrial fission gene expressions and proteins and increased levels of mitochondrial fusion, biogenesis and synaptic gene expressions and proteins relative to neurons incubated with Αβ alone indicating that DDQ is protective against Αβ- and Drpl-induced toxicities. Hence, it is proved that DDQ have protective effects on neuronal cells and it protects against Αβ induced mitochondrial and synaptic toxicities in AD neurons. Further it is required to do more preclinical using AD mouse models and clinical studies using AD patients of DDQ to determine it's the preventive effects against Αβ induced neuronal toxicities in AD affected animal and human models.

Chemicals and Reagents: Chemicals for synthesis of DDQ were procured from Sigma- Aldrich and Merck were used as such, without further purification. All solvents used for spectroscopic and other physical studies were reagent grade and were further purified by literature methods. Infrared spectra (IR) were obtained on a Perkin-Elmer Model 281-B spectrophotometer. Samples were analyzed as potassium bromide disks. Absorptions were reported in wave numbers (cm ¹ ). ¾ and ³¹ P NMR spectra were recorded as solutions in DMSO-de on a Bruker AMX 400 MHz spectrometer operating at 400 MHz for ¾ and 161.9 MHz for ³¹ P NMR. The ¾ chemical shifts were expressed in parts per million (ppm) with reference to tetramethylsilane (TMS) and ³¹ P chemical shifts to 85% H ₃ PO ₄ . LCMS mass spectra were recorded on a Jeol SX 102 DA/600 Mass spectrometer. Αβ 1-42 peptide was purchased from Anaspec, Fremont, CA, USA. Dulbecco's Modified Eagle Medium/F- 12 (DMEM/F12), penicillin/streptomycin, Trypsin-EDTA and fetal bovine serum were purchased from GIBCO (Gaithersberg, MD). SHSY5Y cells. SHSY5Y cells were purchased from American Tissue Type Collection (ATCC), Virginia, USA.

Molecular docking. Molecular docking simulations were generated and prepared, using MOE software. The crystal structures of Αβ (PDB ID: lba4) and dynamin-l-like protein (PDB ID: 4hlu) were retrieved from the Protein Data Bank. PDB structure of Drpl was loaded into the MOE working environment, ignoring all heteroatoms and water molecules. When receptor was loaded into MOE molecular modeling software, the heteroatoms and water molecules were removed, and polar hydrogens were added to relieve any close contact between the X and Y-axis. Protonation of the 3D structure was carried out for all of the atoms, in the implicit solvated environment at 300 K that had a pH of 7 and a salt concentration of 0.1. Electrostatic potential was applied to a cut-off value of 1.5 A at a dielectric value of 1. A non-bonded cut- off value of 8 A was applied to the Leonard-Jones terms. After protonation, the completed structure was energy-minimized, using the MMFF94x force field at a gradient cut off value of 0.05. Molecular dynamic simulations were carried out at a constant temperature of 300 K for a heat time of 10 picoseconds. All simulations were carried out, over a total of 2000 picoseconds. The time step was considered 0.001, and the temperature relaxation time was set to 0.2 picoseconds. The position, velocity, and acceleration were determined and the data collected and saved every 0.5 picoseconds.

PDB structure of Αβ was constructed in the MOE working environment and subjected to energy minimization. MMFF94x force fields were included, and the related potential energy terms were enabled for all bonded interactions, Van der Waals interactions, and electrostatic interactions and restraints. The non-bonded cut-off value was enabled between 8- 10 A. A generalized born implicit salvation model was enabled, all parameters were fixed, the gradient was set to 0.05, and the partial charges of the force field were enabled in order to run calculations during the minimization process. Dynamic simulations were carried out, using the Nose-Poincare-Anderson equational algorithm. Consequently, the inventors formed Αβ and Drp l complex, which further used as receptor to find the binding interactions designed molecules.

The 3D structures of all designed structures were constructed in the MOE working environment and subjected to energy minimization. MMFF94x force fields were included, and the related potential energy terms were enabled for all bonded interactions, Van der Waals interactions, and electrostatic interactions and restraints. The non-bonded cut-off value was enabled between 8-10 A. A generalized Born implicit salvation model was enabled, all parameters were fixed, the gradient was set to 0.05, and the partial charges of the force field were enabled in order to run calculations during the minimization process. Dynamic simulations were carried out, using the Nose-Poincare-Anderson equational algorithm. The temperature for the proteins was set to 30 K and was increased to 300 K for run-time temperature. Heat time and cool time were set to 0 picoseconds. The site for the Prediction of Binding Site for Ligand Activity of the crystallographic structure of Drp l was defined. The MOE dock module was used to dock the compounds into specified binding sites along with the reference compound exemestane. Exemestane was found in contact with 3S7S, determined by alpha PMI (Principle Moments of Inertia) placement methodology, where Poses were generated by aligning principal moments of inertia and ligand conformations to a randomly generated subset of alpha spheres in the receptor site. Thirty docked conformations were generated for each ligand and ranked by an alpha HB scoring function, which is a linear combination of the geometric fit of the ligand to the binding site and hydrogen bonding effects. From all the receptor-ligand complexes, the conformation with the lowest docking score was chosen for additional analysis.

In vitro biological studies of DDQ. DDQ exhibited a good molecular docking score and better binding interactions compared to the other molecules those the inventors developed. Therefore, the inventors decided to quantify the biological effects of DDQ in AD pathogenesis. Therefore, the inventors treated AD neurons with DDQ and quantified the effect of DDQ on gene expression levels of synaptic, AD- related, and mitochondrial-related genes. For these treatments, the inventors performed the following protocols: The inventors treated SHSY5Y cells in five different groups as explained in Figure 3.

Figure 3 illustrates the experimental strategy of the cell culture work and including treatments. The cells were grown in a medium (1 : 1 DMEM and F12, 10% FBS, lx penicillin, and streptomycin) at 37°C in a humified incubator with a 5% CO ₂ environment. After seeding were allowed to grow for 24-48 hrs or until 80% confluence in six-well plates and used for experiments. The inventors used five different groups of cells - (1) untreated SHSY5Y cells; (2) SHSY5Y cells treated with DDQ (250 nM final concentration) for 24 hrs; (3) SHSY5Y cells incubated with Αβ peptide 1-42 (20uM final concentration) for 6 hrs; (4) SHSY5Y cells treated Αβ for 6 hrs +DDQ for 24 hrs and (5) SHSY5Y cells treated DDQ for 24 hrs and Αβ for 6 hrs. Half a million SHSY5Y cells were suspended per well into six-well plates. The inventors used Αβ peptide 1-42 and DDQ 250 nM in the experiments. After treatments, the inventors harvested cells, conducted experiments to measure the levels mRNA using Sybr-Green based real-time RT-PCR, proteins using immunoblotting and immunofluorescence analysis and cell viability using MTT assay. The inventors counted the number of mitochondria by electron microscopy.

Data were compared two ways - (1) untreated cells versus cells treated with Αβ, DDQ, DDQ+Αβ and Αβ+DDQ, and (2) Cells treated with Αβ versus DDQ+Αβ and Αβ+DDQ

Quantification of mitochondrial dynamics, biogenesis and synaptic genes expression using real-time RT- PCR. Using the reagent TriZol (Invitrogen), the inventors isolated total RNA from control and experimental groups (Figure 3). Using primer express Software (Applied Biosystems), the inventors designed the oligonucleotide primers for the housekeeping genes β-actin, mitochondrial structural genes; fission (Drpl and Fisl); fusion genes (MFN1, MFN2); mitochondrial biogenesis genes PGCla, Nrfl, Nrf2 and TFAM; and synaptic genes (PSD95, synaptophysin, synapsin 1, synapsin 2, synaptobrevin 1, synaptobrevin 2, synaptopodin, and GAP43). The primer sequences and amplicon sizes are listed in Table 3. Using SYBR-Green chemistry -based quantitative real-time RT-PCR, the inventors measured mRNA expression of the genes mentioned above as described by Manczak et al. [10].

Table 3. Summary of real-time RT-PCR oligonucleotide primers used in measuring mRNA expression in mitochondrial and synaptic genes in untreated-SHSY5Y, DDQ-SHSY5Y, Αβ-SHSYSY, Ap+DDQ-SHSY5Y and DDQ+A -SHSY5Y treated cell line. Gene DNA Sequence (5'-3') PCR SEQ ID

Product NO:

Size

Drpl Forward Primer TGGGCGCCGACATCA 54 1

Reverse Primer GCTCTGCGTTCCCACTACGA 2

Fisl Forward Primer TACGTCCGCGGGTTGCT 54 3

Reverse Primer CCAGTTCCTTGGCCTGGTT 4

MFN1 Forward Primer TCTCCAAGCCCAACATCTTCA 62 5

Reverse Primer ACTCCGGCTCCGAAGCA 6

MFN2 Forward Primer TGGTGAGGTGCTATCTCGGA 72 7

Reverse Primer AACAGAGCTCTTCCCACTGC 8

Synaptic genes

Synaptophysin Forward Primer CATTCAGGCTGCACCAAGTG 59 9

Reverse Primer TGGTAGTGCCCCCTTTAACG 10

PSD95 Forward Primer GGACATTCAGGCGCACAAG 58 11

Reverse Primer TCCCGTAGAGGTGGCTGTTG 12

Synapsin 1 Forward Primer TGAGGACATCAGTGTCGGGTAA 64 13

Reverse Primer GGCAATCTGCTCAAGCATAGC 14

Synapsin 2 Forward Primer TCCCACTCATTGAGCAGACATACT 63 15

Reverse Primer GGGAACGTAGGAAGCGTAAGC 16

Synaptobrevin 1 Forward Primer TGCTGCCAAGCTAAAAAGGAA 68 17

Reverse Primer CAGATAGCTCCCAGCATGATCA 18

Synaptobrevin 2 Forward Primer CGGAAGAGTCAGTCTCCATTGG 64 19

Reverse Primer CACCTGCAGATAATGTCGTGCTA 20

Neurogranin Forward Primer AGCCGGACGACGACATTCTA 79 21

Reverse Primer AAACTCGCCTGGATTTTGGC 22

GAP43 Forward Primer CTGAGGAGGAGAAAGACGCTGTA 57 23

Reverse Primer TCCTGTCGGGCACTTTCC 24

Synaptopodin Forward Primer TCCTGCGCCCTGAACCTA 70 25

Reverse Primer GACGGGCGACAGAGCATAGA 26

GAPDH Forward Primer TTCCCGTTCAGCTCTGGG 59 27

Reverse Primer CCCTGCATCCACTGGTGC 28

Mitochondrial Biogenesis genes

PGCl Forward primer GCAGTCGCAACATGCTCAAG 83 29

Reverse primer GGGAACCCTTGGGGTCATTT 30

Nrfl Forward primer AGAAACGGAAACGGCCTCAT 96 31

Reverse primer CATCCAACGTGGCTCTGAGT 32

Nrf2 Forward primer ATGGAGCAAGTTTGGCAGGA 96 33

Reverse primer GCTGGGAACAGCGGTAGTAT 34

TFAM Forward primer TCCACAGAACAGCTACCCAA 84 35

Reverse primer CCACAGGGCTGCAATTTTCC 36

Housekeeping Genes

Beta Actin Forward Primer AGACCTGTACGCCAACACAG 72 37

Reverse Primer TCTGCATCCTGTCGGCAAT 38

Immunoblotting analysis. To determine whether DDQ, or Αβ alters the protein levels of mitochondrial and synaptic genes that showed altered mRNA expressions in the real-time RT-PCR, the inventors performed immunoblotting analyses of protein lysates from cells of control and experimental treatments in independent cells treatments (n=3) as described in Manczak et al. [13]. Details of proteins, dilutions of antibodies used for immunoblotting analysis was given in Table 4.

Table 4. Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial structural and synaptic proteins in the SHSY5Y cell treated with DDQ, Αβ, Αβ+DDQ and DDQ+Αβ.

SHSY5Y cells, mutant APP _Swe/ in _d cells and co-immunoprecipitation analysis. SHSY5Y cells: To determine whether Αβ interact with Drpl, the inventors used protein lysates from Αβ incubated (DDQ pre-treated, post-treated and untreated) SHSY5Y cells.

Mutant APP _Swe /ind cells: The inventors purchased mutant APP _Swe cDNA clone (pCAX-APP swe/ind) from Addgene - https://www.addgene.org and verified expression of mutant APP APP _Swe /ind cDNA and further sub-cloned into a mammalian expression vector (Arubala P. Reddy - unpublished observations). The inventors transfected mutant APP _Swe /ind cDNA into mouse neuroblastoma (N2a) cells for 24 hrs and after transfection, cells were treated with DDQ (250nM) for 24 hrs. The inventors harvested mutant APP _Swe /ind cells treated and untreated with DDQ and prepared protein lysates and performed co-immunoprecipitation using Αβ (6E10) antibody and conducted immunoblotting analysis with 6E10 and Drpl antibodies.

The inventors performed co-immunoprecipitation (co-IP) assays using the Dynabeads Kit for Immunoprecipitation (Invitrogen). Briefly, 50 of Dynabeads containing protein G was incubated with 10 μg 6E10 or 10 μg of the Drpl antibodies (both mono- and polyclonal antibodies; Santa Cruz), with rotation, for 1 h at room temperature. The inventors used all the reagents and buffers provided in the kit. Details of antibodies used for co-IP and western blotting are given Table 5. The Dynabeads-Αβ complex was washed three times with a washing buffer and was then incubated overnight with 400 μg of protein at 4°C, with rotation. The incubated Dynabead antigen/antibody complexes were washed again 3 times with a washing buffer, and an immunoprecipitant was eluted from the Dynabeads, using a NuPAGE LDS sample buffer. The Αβ and Drp 1 IP elute was loaded onto a 4-20 gradient gel, followed by western blot analysis of Αβ and/or Drpl antibodies. The inventors also cross-checked the results by performing co-IP experiments, using both anti-Αβ and anti-Drpl antibodies [10].

Table 5. Summary of antibody dilutions and conditions used in the co-immunoprecipitation.

Immunofluorescence analysis. To study immune-reactivity of proteins of interest, cells were grown on coverslips using regular cell culture medium and treated as shown in strategy Figure 3 and performed immunofluorescence analysis using the antibodies in these treated and untreated cells as described by Manzcak et al [13]. Details of proteins, dilutions of antibodies used for immunofluorescence analysis was given in Table 6. Cells were washed with warm PBS, fixed in freshly prepared 4% paraformaldehyde in PBS for 10 min, and then washed with PBS and permeabilized with 0.1% Triton-XlOO in PBS. They were blocked with 1% blocking solution (Invitrogen) for 1 hr at room temperature. All neurons were incubated overnight with primary antibodies. The neurons were incubated with appropriate secondary antibodies. The cells were washed neurons 3 times with PBS, and slides were mounted. Photographs were taken with a multiphoton laser scanning microscope system (ZeissMeta LSM510). To quantify the immunoreactivity of proteins of interest, for each treatment 10-15 photographs were taken at x20 magnification.

Table 6. Summary of antibody dilutions and conditions used in the immunohistochemistry/ immunofluorescence analysis of Drpl, Synaptophysin and PSD95 in the SHSY5Y cell treated with DDQ, Αβ, Αβ+DDQ and DDQ+Αβ.

Double labeling immunofluorescence analysis of Drpl and Αβ. To determine the interaction between

Drpl and Αβ, the inventors conducted double-labeling immunofluorescence analysis, using an anti- Drplantibody (rabbit polyclonal, Santa Cruz Biotechnology) and 6E10 (Covance). As described earlier, the sections from AD patients and control subjects were deparaffinized and treated with sodium borohydrate to reduce autofluorescence.

For the first labeling, the Αβ incubated (DDQ pre-treated, post-treated and untreated) cells were incubated overnight with the anti-Drpl antibody (1 :200) at room temperature. On the day after this primary antibody incubation, the sections were washed with 0.5% Triton in PBS. They were then incubated with a secondary biotinylated anti-rabbit antibody at a 1 :400 dilution (Vector Laboratories, Burlingame, CA, USA) or a secondary biotinylated anti -mouse antibody (1:400) for 1 h at room temperature. They were incubated for 1 h with labeled streptavidin, an HRP solution (Molecular Probes). The cells were washed three times each with PBS for 10 min, at pH 7.4, and treated with Tyramide Alexa488 for 10 min at room temperature.

For the second labeling, the cells were blocked for 1 h with a blocking solution containing 0.5% Triton in PBS+10% donkey serum+1% BSA. Then they were incubated overnight with 6E10 ( 1 :200 dilution, Covance) at room temperature. Next, they were incubated with the donkey anti-mouse secondary antibody labeled with Alexa 594 for 1 h at room temperature. They were cover-slipped with Prolong Gold and photographed with a confocal microscope [10].

Transmission electron microscopy. To determine the effects of DDQ on the numbers of mitochondria and any rescue effects of DDQ on mitochondria in the mutant SHSY5Y neurons, the inventors used TEM on untreated and treated SHSY5Y cells (as shown in fig 3). [Batches 1-5] . All SHSY5Y cells batches 1-5 were fixed in 100 um sodium cacodylate (pH 7.2), 2.5% glutaraldehyde, 1.6% paraformaldehyde, 0.064% picric acid and 0.1% ruthenium red. They were gently washed and post-fixed for 1 h in 1% osmium tetroxide plus 08% potassium ferricyanide, in 100 mm sodium cacodylate, pH 7.2. After a thorough rinsing in water, the SHSY5Y cells were dehydrated, infiltrated overnight in 1 : 1 acetone :Epon 812 and infiltrated for 1 h with 100% Epon 812 resin. They were then embedded in the resin. After polymerization, 60 to 80 nm thin sections were cut on a Reichert ultramicrotome and stained for 5 min in lead citrate. They were rinsed and post-stained for 30 min in uranyl acetate and then were rinsed again and dried. Electron microscopy was performed at 60 kV on a Philips Morgagne TEM equipped with a CCD, and images were collected at magnifications of ^χ 1000-37 000. The numbers of mitochondria were counted in the SHSY5Y cells batches 1-5, and statistical significance was determined, using one-way ANOVA.

Mitochondrial function assays. Hydrogen peroxide production. Using an Amplex® Red H ₂ O ₂ Assay Kit (Molecular Probes, Eugene, OR), the inventors measured the production of H ₂ O ₂ in independent experiments (n=4) of SHSY5Y neurons treated 1) with and 2) without DDQ, DDQ treated and then incubated with Αβ, as described in Manczak et al. [13] .

Cytochrome oxidase activity. Cytochrome oxidase activity was measured in the mitochondria isolated from SHSY5Y cells of control and experimental treatments (n=4), as described in Manczak et al. ³³ . Enzyme activity was assayed spectrophotometrically using a Sigma Kit (Sigma-Aldrich) following manufacturer's instructions.

ATP levels. ATP levels were measured in mitochondria isolated from SHSY5Y neurons of control and experimental treatments (n=4) using the ATP determination kit (Molecular Probes) as described in Manczak et al. [13] . Lipid peroxidation assay. Lipid peroxidates are unstable indicators of oxidative stress in neurons. 4- hydroxy-2-nonenol (HNE) is the final product of lipid peroxidation that was measured in the cell lysates from SHSY5Y cells of control and experimental treatments (n=4), using an HNE-His ELISA Kit (Cell BioLabs, Inc., San Diego, CA) as described in Manczak et al. [13].

Cell viability test (MTT assay). Mitochondrial respiration, an indicator of cell viability, was assessed in the SHSY5Y cells from control and experimental treatments (n=4), using the mitochondrial-dependent reduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan as described in Manczak et al. [13].

Statistical analyses. Statistical analyses were conducted in 2 ways: 1. untreated cells versus cells treated with Αβ, DDQ, DDQ+Αβ and Αβ+DDQ and 2. Cells treated with Αβ versus DDQ+Αβ and Αβ+DDQ for mRNA and protein levels, cell viability and mitochondrial functional parameters H ₂ O ₂ , cytochrome oxidase activity, lipid peroxidation, ATP production and cell viability using appropriate statistical analysis.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of or "consisting of. As used herein, the phrase "consisting essentially of requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation, "about", "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, 35 U.S.C. 112, paragraph (f), or equivalent thereto, as it exists on the date of filing hereof unless the words "means for" or "step for" are explicitly used in the particular claim.

For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

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