WO2012088100A2 - Triclabendazole and fenbendazole for cell protection - Google Patents

Abstract

Triclabendazole (TCBZ) and fenbendazole (FBDZ) have been discovered to extend the lifespan of yeast cells (a model for apoptosis), increase protection for cells from oxidative stress, and protect against cell death caused by Parkinson's disease-related protein alpha- synuclein. Both triclabendazole and fenbendazole can be used to extend the lifespan of eukaryotic cells, protect cells from oxidative stress, and treat various neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, and Huntington's diseases) that involve toxic protein aggregates. Derivatives or metabolites of either TCBZ or FBDZ would have similar effects. Known metabolites of TCBZ include a sulfoxide (TCBZ-SO) and a sulfone (TCBZ- S02). Similar metabolites of FBDZ include a sulfoxide (FBDZ-SO) and a sulfone (FBDZ- S02).

Description

TRICLABENDAZOLE AND FENBENDAZOLE FOR CELL PROTECTION

Stephan N. Witt, Yong J. Lee, and Shaoxiao Wang

File No. Witt 10S 10W

[0001] The benefit of the filing dates of provisional U.S. application Serial Number 61/425,536 filed 21 December 2010 and of provisional U.S. application Serial Number 61/528,870 filed 30 August 2011 is claimed under 35 U.S.C. § 119(e) in the United States, and is claimed under applicable treaties and conventions in all countries.

[0002] This invention was made with government support under grant number NS057656 from the National Institutes of Neurological Disorders and Stroke of the National Institutes of Health. The Government has certain rights in this invention.

TECHNICAL FIELD

[0003] This invention pertains to the use of triclabendazole and fenbendazole to extend the lifespan of eukaryotic cells, protect cells from oxidative stress, and treat various neurodegenerative diseases based on abnormal protein aggregation (e.g., Alzheimer's, Parkinson's, and Huntington's diseases.)

BACKGROUND ART

Benzimidazoles

[0004] Benzimidazoles are heterocyclic aromatic organic compounds consisting of a fusion of benzene and imidazole. Examples of benzimidazoles include triclabendazole, albenazole, flubendazole, and fenbendazole, whose structures are shown in Fig. 1. The benzimidazoles, including triclabendazole, are well known for their anthelmintic activity. Triclabendazole (TCBZ), 5-Chloro-6(2,3-dichlorophenoxy)-2-methylthio-lH-benzimidazole, is used as an antihelmintic to treat liver fluke (Fasciola hepatica) infections in cattle and humans, usually given orally [3,9,13]. It is approved for veterinary purposes in the U.S.A. TCBZ is thought to inhibit β-tubulin from F. hepatica [14]. A mutation in tubulin was shown to confer resistance against TCBZ [19], which suggests that tubulin may be the target of this drug. Fenbendazole (FBDZ), methyl N-(6-phenylsulfanyl-lH-bensoimidazol-2- yl)carbamate, is a benzimidazole compound used as a veterinary antihelmintic in a number of animals, including poultry, swine and cattle. It is used to control nematodes such as Ascaridia, Heterakis and Capillaria in poultry and in swine and is often administered in the feed or in the drinking water. Abendazole, methyl[6-(propylthio)-lH-benzoimidazol- 2yl)carbamate, is a broad spectrum antihelminitic and is used as a treatment for a variety of worm infestations.

Cell Death and Degeneration

[0005] Degeneration and/or death of cells in the nervous system are major factors in many diseases and medical conditions. Such diseases and conditions include traumatic brain and spinal cord injuries, stroke, neural perfusion secondary to Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD) and other "poly Q" disease neurodegenerative diseases based on proteinopathies (as described below). There is a need for drugs to help prevent or decrease such cell death and degeneration.

[0006] Certain compounds are known to be useful as neuroprotective agents, for example, insulin-like growth factor 1 (IGF-1) (U.S. Patent No. 5,804,550), and the N- terminal tripeptide Gly-Pro-Glu (GPE) (U.S. Patent No. 6,187,906). A derivative of GPE; cyclic Pro-Gly ("cPG"), a diketopiperazine, has been shown to be neuroprotective and neuroregenerative. cPG was found to prevent toxic neural degeneration and cell death and to promote neurite outgrowth in neurons (International Publication No: WO 03/039487). Diketopiperazine analogues of thyrotropin-releasing hormone (TRH) are also known to be neuroprotective (International Publication No. WO 99/40931). Other known neuroprotective agents include, for example, insulin-like growth factor-II (IGF-II), transforming growth factor-.beta.l, activin, growth hormone, nerve growth factor, growth hormone binding protein, and/or IGF-binding proteins. Proteinopathies

[0007] Many neurodegenerative diseases, defined here as "proteinopathies," result from the accumulation of toxic aggregated proteins in neurons with age. Examples of such proteinopathies include amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, polyglutamine expansion diseases (poly Q diseases) including Huntington's disease (HD) and tauopathies, which include AD, frontotemporal dementia associated with tau-immunoreactive inclusions (FTD-tau), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) [23,24]. Many common diseases and the proteins are listed in Table 1. Poly Q diseases are diseases where a polyglutamine segment of varying length is covalently attached to a protein. Examples include Huntington's disease (HD), and 8 other polyQ diseases, (where Q stands for glutamine).

Table 1; Diseases with Examples of Associated Proteins

[0008] A common feature of the above proteinopathies is that various proteins (Αβ and tau, alpha-synuclein, polyglutamine expansion proteins, TDP-43 and others) exhibit complex aggregation behavior, often linked with oxidative stresses. These proteins can form soluble, β-sheet-rich oligomeric structures that are thought to be toxic; and with time and increasing protein concentration, these structures convert into insoluble amyloid fibers, which may also be toxic. Very often, for example in PD and HD, the amyloid fibers form inclusions in the cytoplasm of neurons [23]. In some cases, individuals have mutations in one of these proteins, and mutations often accelerate the rate of aggregation and the onset of the disease, e.g., PD. Idiopathic or sporadic PD typically occurs after 65 years of age and is thought to be causally linked to the aggregation of alpha-synuclein (a-syn). However, for individuals who express the mutant A53T a-syn, the age of onset is much less, around ~30 years of age. In vitro experiments with purified A53T have shown that A53T aggregates much faster than wild-type a-syn [25]; thus, the rapid age of onset in individuals who express this mutant is likely due to the enhanced rate of aggregation of the mutant.

[0009] There are reports that link oxidative stress to the aggregation of a-syn in PD and the polyglutamine-expanded huntingtin protein in HD [26,27]. Neurons expressing either of these proteins, whether soluble and functional or aggregated and non-functional, are susceptible to oxidative stress and the proteins often aggregate more when oxidized [32]. Even a disease like diabetes (type 1 or 2) may be considered a proteinopathy [28], and insulin siganaling is known to be important to aging [29] .

Parkinson 's Disease

[0010] Parkinson's disease is a movement disorder of increasing occurrence in aging populations. Parkinson's disease is a common disabling disease of old age affecting about one percent of the population over the age of 60 in the United States. Symptoms include pronounced tremor of the extremities, bradykinesia, rigidity and postural change. A perceived pathophysiological cause of Parkinson's disease is progressive destruction of dopamine producing cells in the basal ganglia which comprise the pars compartum of the substantia nigra located in the brain stem. Loss of dopamineric neurons results in an excess of acetylcholine relative to dopamine. In some patients with a history of Parkinson's disease, the symptoms may be episodic, with periods of time that are relatively symptom free, and other periods where symptoms worsen. The main treatment for Parkinson's disease is with L- dopa (levo-dihydroxy-phenylalanine), a precursor to dopamine. L-dopa has a disadvantage in causing adverse side effects, including nausea, vomiting, postural hypotension, confusion or, when the treatment is continued extended periods of time, dyskinesia.

[0011] Currently the classes of drugs available to treat Parkinson's are the following: carbidopa/levodopa therapy; dopamine agonists; anticholinergics; and MAO-B inhibitors. L- dopa is the most potent medication for Parkinson's, and is combined with carbidopa to prevent nausea and vomiting. It is a dopamine precursor which can cross the blood-brain barrier to be converted to dopamine in the brain. Dopamine agonists stimulate the human brain similar to dopamine, but are generally not as effective as L-dopa in treating Parkinson's disease. Examples of dopamine agonists include pramipexole, ropinirole, rotigotine, bromocriptine, and apomorphine. Anticholinergics act by decreasing the amount of acetylcholine in the brain, and help maintain a more normal dopamine to acetylcholine ratio. Anticholinergics are helpful for certain symptoms, including tremor and dystonia. Examples of anticholinergics include trihexyphenidyl, benztropine, benzhexol, orphenedrine, and procyclidine. MAO-B inhibitors work by blocking an enzyme in the brain that breaks down L-dopa. Examples of MAO-B inhibitors include selegiline and rasagiline. Other drugs have been used to treat symptoms of Parkinson's including amantadine and rivastigmine. Many of the above drugs also show adverse side effects. (See also, U.S. Patent Nos. 6,620,415 and 7,776,876).

[0012] U.S. Patent Publication No. 2009/0105317 describes that albendazole, a benzimidazole carbamate compound, can block effects of increased vascular endothelial growth factor (VEGF).

[0013] U.S. Patent Publication No. 2005/0038022 describes that albendazole, a benzimidazole carbamate compound, can help in treatment of certain tumors.

DISCLOSURE OF INVENTION

[0014] We have discovered that triclabendazole (TCBZ) and fenbendazole (FBDZ) are effective in extending the lifespan of yeast and mammalian cells (a model for apoptosis), in increasing protection for cells from oxidative stress, and in protecting against diseases based on toxic protein aggregates or "proteinopathies," for example, Parkinson's disease and the related protein alpha-synuclein. Both triclabendazole and fenbendazole can be used to extend the lifespan of eukaryotic cells, protect cells from oxidative stress, and treat various neurodegenerative diseases that are known to involve toxic protein aggregates, for example, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, polyglutamine expansion diseases (e.g., Huntington's disease (HD)) and tauopathies (e.g., AD, frontotemporal dementia associated with tau-immunoreactive inclusions (FTD-tau), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD)). We have shown that that TCBZ and FBDZ increase the yeast chronological lifespan and protect yeast and mammalian cells from various stresses. In addition, both TCBZ and FBDZ decreased the level of cAMP in yeast and human cells. We believe the mechanism is by inhibiting the enzyme adenylate cyclase. In addition, we discovered that the human Parkinson's disease-related protein alpha-synuclein increased cAMP in yeast cells, and this increase could be inhibited by TCBZ. Thus both TCBZ and FBDZ can be used to treat Parkinson's disease. In addition, TCBZ and FBDZ can be used to treat symptoms of other diseases caused by protein aggregation (e.g., AD, HD, ALS, etc.). We believe that close derivatives or metabolites of either TCBZ or FBDZ would have similar effects. Known metabolites of TCBZ include a sulfoxide (TCBZ-SO) and a sulfone (TCBZ-S02). Similar metabolites of FBDZ include a sulfoxide (FBDZ-SO) and a sulfone (FBDZ-S02). In addition, since both TCBZ and FDBZ are low molecular mass compounds and hydrophobic, it is believed that both would cross the blood-brain barrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 illustrates the chemical structures for the benzimidazoles of triclabendazole (TCBZ), fenbendazole (FBDZ), flubendazole (FLBZ), nocodozole (NCDZ) and albendazole (ALBZ) and of benomyl (BEN).

[0016] Fig. 2 illustrates the growth of Saccharomyces cerevisiae (baker's yeast) under three conditions (liquid YPD, 30°C (top curve); water, 37°C (middle curve); and 2% glucose/water, 37°C (bottom curve)). Data shown are mean ± s.e.m. (n=3).

[0017] Fig. 3A illustrates the growth of Saccharomyces cerevisiae (baker's yeast) at 30°C in liquid YPD with added dimethylsulfoxide (DMSO; control), TCBZ (triclabendazole), or BEN (benomyl). Data shown are mean ± s.e.m. (n = 3)

[0018] Fig. 3B illustrates the growth of Saccharomyces cerevisiae (baker's yeast) at 30°C in liquid YPG with the carbon source galactose with added DMSO (control), TCBZ

(triclabendazole), or BEN (benomyl). Data shown are means ± s.e.m. (n = 3).

[0019] Fig. 3C illustrates the growth of Saccharomyces cerevisiae (baker's yeast) at 30°C in liquid YPGly with the carbon source glycerol with added DMSO (control), TCBZ

(triclabendazole), or BEN (benomyl). Data shown are means ± s.e.m. (n = 3).

[0020] Fig. 4A illustrates the growth curve of Saccharomyces cerevisiae (baker's yeast) incubated in liquid YPD at 30°C until reaching stationary phase (48 h).

[0021] Fig. 4B illustrates the survival of stationary-phase Saccharomyces cerevisiae

(baker's yeast) cells at 30°C in 2% glucose/water (conditions for sugar-induced cell death

(SICD)), and supplemented with 5 μΜ triclabenzadole (TCBZ), albenzadole (ALBZ), bendomyl (BEN), or vehicle (DMSO).

[0022] Fig. 5A shows differential interference contrast microscopy images of Saccharomyces cerevisiae (baker's yeast) cells grown under conditions for SICD with and without triclabenzadole (TCBZ).

[0023] Fig. 5B illustrates the percent survival as measured by the colony forming unit (CFU) assay of four strains of Saccharomyces cerevisiae (baker's yeast) cells (wild type (WT), mutant strain atglA, mutant strain atgSA, and mutant strain atgllA) pre-grown in liquid YPD media for 2 d at 30°C, washed twice in water and inoculated into SC-glucose medium with 5 μΜ TCBZ or drug vehicle (control), and then incubated at 30°C for 10 days. Data represent means ± s.e.m (n = 3).

[0024] Fig. 5C shows fluorescence microscopy images using that reveal the location of the key autophagy protein Atg8 tagged with the green fluorescent protein (EGFP-Atg8) in wild type yeast cells carrying the plasmid (pAG426GAL-EGFP-ATG8 or pAG426GAL- EGFP) grown in liquid SC-glucose-URA medium with 5 μΜ of TCBZ, BEN or drug vehicle (DMSO (control)) for 1 d.

[0025] Fig. 6A illustrates chronological aging curves measured in a colony formation assay of a wild type yeast strain (control) and the same yeast strain engineered to express human -synuclein (a-syn) incubated with 5 μΜ of DMSO, TCBZ, ALBZ, or BEN. The arrow indicates when the various drugs were added.

[0026] Fig. 6B shows the detection of reactive oxygen species (ROS) by fluorescence microscopy of yeast cells. A wild type strain (control), an a-syn expressing strain (a-syn), and an a-syn expressing strain treated with 5 μΜ TCBZ (a-syn + TCBZ). Each strain was incubated with the ROS-sensitive dye (2',7'-dichlorfluorescin-diacetate (DCFH-DA)) at 10 μg ml for 1 h, and visualized by fluorescence (DCF) and differential interface contrast (DIC) microscopy.

[0027] Fig. 6C shows the percent cells staining for DCF in stationary-phase yeast cells of a wild type strain (control), an a-syn expressing strain (a-syn), and an a-syn expressing strain treated with 5 μΜ TCBZ (a-syn + TCBZ), and each strain incubated with ROS-sensitive dye (2',7'-dichlorfluorescin-diacetate), and visualized by fluorescence (DCF) and differential interface contrast (DIC) microscopy. Values were obtained from two independent experiments, where the total number of cells counted was 500. Error bars in are means ± s.e.m., with an "*" indicating P<0.01 (two-tailed Student's t test, versus a-syn).

[0028] Fig. 7 illustrates the percent survival as measured by the colony formation unit assay of yeast cells pre-grown in liquid YPD for 2 d at 30°C, washed twice in water, and then inoculated in SC-glucose medium with 5 μΜ of TCBZ, benzimidazole (BMDZ), imidazole (IMDZ), or nocodazole (NCDZ), and then incubated at 30°C for 10 d before checking viability. Data shown are mean ± s.e.m. (n = 3).

[0029] Fig. 8A illustrates the percent survival of stationary-phase yeast cells as measured by the colony formation assay that were first diluted to a low optical density in liquid YPD with 5 μΜ DMSO, TCBZ, or ALBZ, and then cultured until stationary-phase was reached (48 h at 30 °C). H₂O₂ was then added for 1 h, and viability was measured. Values are the mean + s.e.m. of three independent experiments. The "*" indicates P<0.005 (two-tailed Student's t test, versus DMSO).

[0030] Fig. 8B illustrates the percent survival of rat PC12 cells pretreated with 50 μΜ of DMSO, TCBZ, or ALBZ for 3 h, and then with H₂0₂ (1 mM) for 21 h at 37 °C. Viability was determined by a colorimetric assay. Values are the mean + s.e.m. of three independent experiments. The "*" indicates P<0.005 (two-tailed Student's t test, versus DMSO).

[0031] Fig. 9A shows the growth curves yeast cells (strain JB289-1A) expressing Tubl- GFP incubated with 5 μΜ triclabenzadole (TCBZ), nocodazole (NCDZ), or benomyl (BEN) cultured in liquid SC-glucose medium at 30°C. The arrow denotes when the drug was added.

[0032] Fig. 9B illustrated chronological aging curves of JB289-1A yeast cells which express Tubl-GFP (green fluorescent protein). Plots show survival of cells as a function of time as determined by a colony forming assay. At the zero point, cells had been incubated with the indicated drug (5 μΜ triclabenzadole (TCBZ), nocodazole (NCDZ), benomyl (BEN) or DMSO) at 30°C for 48 h. Values are the mean ± s.e.m. of three independent experiments.

[0033] Fig. 9C are fluorescence microscopy images of yeast cells expressing Tubl-GFP. Cells were inoculated into liquid SC-glucose medium, incubated until mid-log phase, and then incubated for 1 h with 5 μΜ triclabenzadole (TCBZ), nocodazole (NCDZ), benomyl (BEN) or DMSO at 30°C before observing.

[0034] Fig. 9D is a plot of the number of yeast cells expressing Tubl-GFP exhibiting different spindle characteristics as depicted in the figure. Each value was obtained from three independent experiments, where the total number of cells counted was 200-300. Error bars are ± s.e.m.

[0035] Fig. 10A illustrates chronological aging curves of four yeast strains (wild type (WT), a msn2A mutant, a msn4A mutant, and a msn2Amsn4A double mutant). Plots show survival of cells as a function of time as determined by colony forming assay. At the time zero point, each sample of cells was incubated with TCBZ or DMSO for 48 h. Values shown in are the means ± s.e.m. of three independent experiments.

[0036] Fig. 10B are fluorescence microscopy images of yeast cells expressing Msn2- green fluorescent protein (GFP) inoculated into liquid SC-glucose medium, incubated until mid-log phase, and then incubated at 30°C for 2 h with 5 μΜ triclabenzadole (TCBZ), nocodazole (NCDZ), or DMSO or with 100 nM rapamycin (RAP) at 30°C before observing. [0037] Fig. IOC shows a plot of the percentage of yeast cells Msn2-GFP in the nucleus. Cells were incubated as in Fig. 10B, and then analyzed by fluorescence microscopy using a dye DAPI (stains the nucleus). Values are means ± s.e.m. from four independent experiments, where the total number of cells counted was 300-350. The "*" indicates P < 0.001 (two-tailed Student's t test, versus DMSO).

[0038] Fig. 10D shows a growth assay of yeast cells subjected to different stresses. Wild type yeast cells were initially inoculated into SC-glucose medium with the drug (TCBZ) or vehicle (DMSO), and incubated at 30°C for 4 d. The two cultures were normalized to the same ΟΌβ ο, serially diluted in 1-fold increments, and subjected to three different stresses (100 mM H2O2 or 300 μΜ menadione or heat shock (50°C) for 60 min, and then spotted onto YDP plates.

[0039] Fig. 11A illustrates the amount of intracellular cAMP in two yeast strains (wild type strain or ras2A strain). Strains were incubated in liquid SC-glucose medium with TCBZ (5 μΜ), RAP (100 nM), or vehicle (DMSO) for 15 h at 30°C prior to determining the cAMP content. Values are the means ± s.e.m. of the four independent experiments. The "*" indicates P < 0.005 (two-tailed Student's t test, versus DMSO).

[0040] Fig. 11B illustrates plots of the doubling time for wild type yeast cells inoculated into SC-glucose medium containing 5 μΜ drug (DMSO or TCBZ), supplemented with 5 mM of either cAMP or ATP, and incubated at 30°C. The "*" indicates P < 0.005 (two-tailed Student's t test, versus DMSO).

[0041] Fig. l lC shows fluorescence microscopy images of yeast cells expressing Msn2- GFP incubated in SC-glucose medium until mid-log phase, and then 5 μΜ TCBZ with or without 5 mM cAMP was added, and cells were incubated for 2 h before observing.

[0042] Fig. 12A shows the doubling time of wild type yeast cells incubated in SC-glucose at 30°C, and with an addition of DMSO or of FBDZ or TCBZ at two concentrations (2 μΜ or 5 μΜ).

[0043] Fig. 12B shows fluorescence microscopy images of yeast cells expressing Msn2- GFP incubated in SC-glucose medium until mid-log phase. Then 5 μΜ TCBZ, FBDZ, or DMSO was added, and cultures incubated for 2 h before observing. DAPI is a dye that stains the nucleus.

[0044] Fig. 12C shows a plot of the percent yeast cells containing nuclear localized Msn2-GFP. Cells were incubated in SC-glucose medium until mid-log phase, and then incubated for 2 h with 5μΜ triclabenzadole (TCBZ), fenbenzadole (FBDZ), or DMSO at 30°C. Vales were obtained from four independent experiments, where the total number of cells counted was 200-250. Error bars are ± s.e.m., and an "*" indicates P < 0.001 (two- tailed Student's t test, versus DMSO).

[0045] Fig. 13 illustrates the amount of intracellular cAMP from human neuroblastoma cells (SH-SY5Y) inoculated in 96-well plates containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% horse serum and 5% fetal bovine, incubated overnight at 37°C, rinsed with warm PBS, and then incubated with DMSO, TCBZ (5 μΜ), FBDZ (5 μΜ), ALBZ(5 uM), or 2,5-DDA (300 uM), for 3 h before extraction for the cAMP assay. Values are the means ± s.e.m. (n = 5); and "*" indicates P < 0.01 and indicates P < 0.05 (two- tailed Student's t test, versus DMSO).

[0046] Fig. 14 illustrates the amount of intracellular cAMP from wild-type yeast cells expressing wild type alpha-synuclein (WT a-syn), the point mutant A30P (A30P a-syn), or the point mutant A53T (A53T a-syn) made using plasmids pAG415GPD-WT (a-syn), pAG415GPD-A30P (A30P), and pAG415GPD-A53T (A53T). Strains were incubated in SC- glucose-LEU medium with TCBZ (2 μΜ) or vehicle (DMSO) for 15 h at 30°C prior to extraction for the cAMP assay. Values are the mean ± s.e.m. of two independent experiments, each in quadruplicate, and "*" indicates P < 0.005 (two-tailed Student's t test, versus vector).

MODES FOR CARRYING OUT THE INVENTION

[0047] We have discovered that both triclabendazole and fenbendazole can be used to protect cells from death due to stress. Baker's yeast (Saccharomyces cerevisiae) has been used to model numerous human diseases, from asthma to various neurodegenerative diseases [1,18,21]. Drugs were screened based on the ability to protect baker's yeast from sugar- induced cell death (SICD), a model for apoptosis or cell death due to the accumulation of reactive oxygen species ( OS) [6]. Triclabendazole was found to also protect yeast cells from death induced by the Parkinson's disease-related protein alpha-synuclein, which is known to induce the accumulation of ROS. As shown below, triclabendazole and fenbendazole protected both yeast and mammalian cells from oxidative stress. Two other benzimidazole antihelminthics, albendazol and flubendazol, were not effective in protecting cells from death due to stress.

[0048] Sugar-induced cell death (SICD) is produced in yeast cells based on the growth media and phase of growth. S. cerevisiae cells grown in glucose-containing liquid media ferment glucose and produce ethanol. When glucose becomes limiting, the cells utilize ethanol and switch to a respiratory mode for energy production. After these two carbon sources are consumed, cells stop dividing and enter a quiescent or stationary state. Stationary-phase yeast cells maintained in spent liquid medium, or that are even washed and resuspended in pure water, can live for weeks, whereas the same cells when washed and resuspended in water with 2% glucose and no other nutrients die within hours. This death is called sugar-induced cell death (SICD), and is an apoptotic form of cell death that occurs because of the accumulation of ROS [6].

[0049] We have shown that TCBZ and FBDZ are effective in protecting the yeast cells from SICD, or cell death due to glucose in the media. Glucose in the absence of other nutrients is extremely toxic to stationary-phase yeast cells [5]. Stationary phase yeast cells do not divide; they are quiet like the majority of non-dividing or "post-mitotic" cells in the human body. The screen for drugs that protect against SICD was conducted because of the similarity of SICD to the damage to human cells that occurs due to excess glucose in individuals with diabetes. In humans, elevated levels of glucose often occur in individuals with age, and such elevated levels of glucose are indicative of the disease type 2 diabetes. This damage produces a wide range of complications in humans, including circulation problems, diabetic retinopathy, heart disease, renal failure, to name a few. The mechanism by which excess glucose harms cells is not fully understood. TCBZ and FBDZ can have clinical use in protecting human cells from excess glucose, and may be an effective treatment for complications from type 2 diabetes or from high serum glucose. It is also intriguing that insulin often aggregates in diabetic patients. Thus, even a disease like diabetes (type 1 or 2) may be considered a proteinopathy [28], and insulin signaling is known to be important to aging [29]. We believe that TCBZ and FDBZ can be used as cell protective agents for diabetic patients.

[0050] TCBZ protects yeast and mammalian cells from various stresses by decreasing the level of cAMP. Decreased cAMP triggers activation of a protective stress response, i.e., the transcription of genes whose protein products protect cells in a variety of ways. TCBZ by decreasing cAMP likely activates the expression of proteins that protect against oxidative stress. Because proteins often aggregate more when they are oxidized, by decreasing the oxidative load in cells, TCBZ can help prevent the formation of toxic protein aggregates. Given the ability of TCBZ to protect cells from the toxic effects of high doses of a-syn, we believe that TCBZ will also protect cells from aggregation of the polyglutamine-expanded huntingtin protein, and other protein aggregation. The idea is that TCBZ up-regulates the response to oxidative stress in cells, and this up-regulated response prevents or lessens protein aggregation and thus protects cells. Thus, TCBZ and FBDZ can be used to treat the proteinopathies of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, polyglutamine expansion diseases including Huntington's disease (HD) and tauopathies, which include AD, frontotemporal dementia associated with tau-immunoreactive inclusions (FTD-tau), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD).

[0051] In addition, TCBZ and FBDZ may be used on Parkinson's disease patients as a neuroprotective drug that prevents degeneration of dopaminergic neurons. Additional uses for TCBZ and FBDZ are for treatment for Alzheimer's and Huntington's diseases, which are also based on protein pathology.

[0052] We have also shown that the protective action of TCBZ is not disruption of microtubule formation, the mechanism thought to convey the antihelmintic activity. We have shown that the protection of TCBZ and FBDZ of yeast cells is dependent on Msn2/4 transcription factors. This means that TCBZ protects yeast cells by triggering a stress protective response, and we believe that TCBZ will also trigger a stress response in human cells. This stress response in yeast cells was shown to be triggered by inhibiting the synthesis of cyclic AMP. We have also shown that TCBZ up-regulates a stress response in rat cells which serves to protect these cells from the toxic oxidant hydrogen peroxide. Thus TCBZ and FBDZ are effective for both yeast cells and mammalian cells.

Miscellaneous

[0053] The term "triclabendazole" or TCBZ is defined as 5-Chloro-6(2,3- dichlorophenoxy)-2-methylthio-lH-benzimidazole and its metabolites and derivatives that show the same functional effect as 5-Chloro-6(2,3-dichlorophenoxy)-2-methylthio-lH- benzimidazole, for example the protection of cells against oxidative stress or causes a decrease in the amount of cAMP in cells. Known metabolites of TCBZ include a sulfoxide (TCBZ-SO) and a sulfone (TCBZ-S02).

[0054] The term "fenbendazole" or FBDZ is defined as methyl N-(6-phenylsulfanyl-lH- bensoimidazol-2-yl)carbamate and its metabolites and derivatives that show the same functional effect as methyl N-(6-phenylsulfanyl-lH-bensoimidazol-2-yl)carbamate, for example the protection of cells against oxidative stress or causes a decrease in the amount of cAMP in cells. Metabolites of FBDZ include a sulfoxide (FBDZ-SO) and a sulfone (FBDZ- S02).

[0055] The term "effective amount" as used herein refers to an amount of TCBZ or FBDZ or both sufficient to decrease the amount of cAMP in cells or to protect cells from death due to oxidative stress or to an increase in reactive oxygen species (ROS) to a statistically significant degree (p<0.05). The term "effective amount" therefore includes, for example, an amount sufficient to promote the increase in cell survival in cells exposed to stress or to toxic protein aggregates, delay cell death, inhibit adenylate cyclase, or improve symptoms due to a neurodegenerative disease or a disease due to a proteinopathy (e.g., Parkinson's, Alzheimer's or Huntington's diseases), preferably by at least 50%, and more preferably by at least 90%. The dosage ranges for the administration of triclabendazole or fenbendazole are those that produce the desired effect. Generally, the dosage will vary with the age and condition of the patient, and with the manner of administration. A person of ordinary skill in the art, given the teachings of the present specification, may readily determine suitable dosage ranges. The dose of triclabendazole or fenbendazole given orally may be from 500 nM to 20 μΜ, but more preferably from 1 μΜ to 10 μΜ. In any event, the effectiveness of treatment can be determined by monitoring symptoms by methods well known to those in the field, for example, monitoring the tremor frequency in Parkinson's patients. Moreover, triclabendazole or fenbendazole can be applied in pharmaceutically acceptable carriers known in the art, or with other drugs known to be neuroprotective or known to treat Parkinson's or other neurodegenerative diseases. The manner of administration will usually be orally or by injection.

[0056] TCBZ or FBDZ may be administered to a patient by any suitable means, including oral, parenteral, subcutaneous, intrapulmonary, topically, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or intravitreal administration. Additionally, the infusion could be into an organ or site of cell damage. Injection of TCBZ or FBDZ may include the above infusions or may include intraperitonieal, intravitreal, or direct injection into brain tissue.

[0057] Pharmaceutically acceptable carrier preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. TCBZ or FBDZ may be mixed with excipients that are pharmaceutically acceptable and compatible. Suitable excipients include water, saline, dextrose, and glycerol, or combinations thereof. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like.

[0058] The form may vary depending upon the route of administration. For example, compositions for injection may be provided in the form of an ampule, each containing a unit dose amount, or in the form of a container containing multiple doses.

[0059] Controlled delivery may be achieved by admixing the active ingredient with appropriate macromolecules, for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, prolamine sulfate, or lactide/glycolide copolymers.

[0060] The present invention provides a method of using TCBZ or FBDZ in preventing, treating, or ameliorating the symptoms of a disease based on neurodegeneration or cell death produced by oxidative stress or based on protein pathology, for example, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, polyglutamine expansion diseases including Huntington's disease (HD) and tauopathies, which include AD, frontotemporal dementia associated with tau-immunoreactive inclusions (FTD-tau), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD. The term "ameliorate" refers to a decrease or lessening of the symptoms or signs of the disease being treated. The present invention provides a method of using TCBZ and FBDZ in promoting the survival of cells exposed to stress or toxic chemicals or proteins. The method would further comprise administering other known drugs for treating the disease.

Example 1

Materials and Methods

[0061] Reagents: Unless stated otherwise, all chemicals and media were purchased from Sigma-Aldrich (St. Louis, Missouri). Adenosine 3', 5 '-cyclic monophosphate, sodium salt (Cyclic AMP) was purchased from Enzo Life Science (Farmingdale, New York). Some yeast drop-out mixtures were purchased from United States Biological (Swampscott, Massachusetts). Restriction enzymes were purchased from Promega (Madison, Wisconsin). Primers were synthesized by Integrated DNA Technologies (Coralville, Iowa). Plasmids were purchased from Addgene (Cambridge, Massachusetts), Invitrogen (Carlsbad, California) and Open Biosystems (Thermo Fisher, Lafayette, Colorado). Yeast strains were purchased from Open Biosystems and Invitrogen unless stated otherwise, whereas rat cells PC 12 and human SH-SY5Y cells were purchased from Invitrogen. Sources of strains and plasmids are given in Table 2.

[0062] Yeast media: All media and deionized water was sterilized by autoclaving. Liquid YPD was composed of 1% (weight/volume) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose (dextrose) in water. Solid YPD plates were made with_l% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, and 2% (w/v) agar in water. After autoclaving this media was poured into standard sterile petri dishes and allowed to solidify. YPG and YPGlv were prepared as YPD but with 2% (w/v) galactose and 2% (v/v) glycerol replacing the glucose, respectively. Liquid synthetic complete medium with lucose (SC-zlucose) was composed of Yeast Nitrogen Base without amino acids (Y0626, Sigma-Aldrich; YNB) (0.67% w/v) and Yeast Synthetic Drop-Out Media Supplement without leucine (Y 1376; Sigma-Aldrich) (0.16% w/v) supplemented with L-leucine (0.02% w/v) in water. Liquid SC- slucose - leucine (SC-slucose-LEU) was composed of YNB (0.67% w/v) and Yeast Synthetic Drop-Out Media Supplement without leucine (0.16% w/v) in water. Liquid SC-zlucose - uracil (SC-glucose-URA) was composed of YNB (0.67% w/v) and Drop-out Mix Synthetic Minus Uracil w/o Yeast Nitrogen Base (D9535; United States Biological, Swampscotts, Massachusetts) (0.2% w/v) in water. For the SICD experiments, 2% slucose/water was used as the liquid medium and composed of (20 g glucose/liter of water).

[0063] Yeast growth assays: When grown in liquid media, the doubling time analysis was based on the optical density (ODgoo) as measured every 3 h. The doubling time was determined using the formula (logio (N No)) / 0.3 = g, where doubling time = t/g, t = time cultured, o = initial OD₆₀o and N_t = OD₆oo at time t. When grown on solid plates, the yeast cells were grown overnight in liquid medium and then diluted (to ΟΌ ο = 0.2) into fresh liquid medium and incubated for 6-8 hours. Cultures were normalized to the same OD, serially diluted in 10-fold increments, spotted (10 μί) onto solid plates and incubated for 2-3 days at 30 °C.

[0064] SICD Yeast Assay: Detailed protocol for high-throughput screening of the Prestwick and NIH chemical libraries: Yeast cells (pdrlA) (Table 2) were grown in 3 ml of liquid YPD in standard sterile, capped culture tubes with shaking for 2 days at 30 °C to yield a cell density of 2 x 10⁸ cells/ml. The cells were then pelleted at 4000 x g, washed twice with 3 ml sterile water, and resuspended in 1 ml of sterile water, using standard microbiological practices. 100 μΐ aliquots were diluted into 15 ml tubes containing 3 ml of water or 3 ml 2% glucose (w/v)/water to a concentration 2 x 10⁷ cells/ml. These cultures were used to fill the wells of 96-well plates for the drug screening. 135 μΐ aliquots of the cultures were deposited into wells of a sterile, flat bottomed 96-well plate. 15 μΐ of each drug (50 μΜ) was added to each well to yield a final drug concentration of 5 μΜ per well. The 96-well plates were incubated for 2 days in a humid incubator at 37°C with gentle rocking. If a drug protected cells in 2% glucose/water from sugar-induced cells death (SICD), then during this incubation the cells remained alive. If the drug failed to protect cells from SICD, then during this incubation the cells died. After this 2 d incubation, a small aliquot of cells in the wells of each plate were transferred to the corresponding wells of new plates, with fresh media in each well. 10 μΐ of each well was transferred into a well of a new 96-well plate, which contained 200 μΐ of liquid YPD medium per well, and then the plate was incubated with gentle rocking for 18 h at 30°C . This step permitted regrowth of the cells. If a drug protected cells from SICD, then the cell regrowth caused an increase in OD. On the other hand, if a drug failed to protect cells from SICD, then little or no regrowth occurred, and the OD failed to increase. An automatic plate reader was used to measure the optical density of the wells in each plate. Typical values for wells without regrowth ranged from 0.05 to 0.1 absorbance at 600 nm; whereas, for positive hits the absorbance at 600 nm ranged from 0.5 to 1.0. The drugs being tested were from the Prestwick chemical library (Prestwick Chemical, Washington, D.C.) and the NIHCC chemical library of 447 FDA-approved drugs (National Institutes of Health, Besthesda, Maryland). The 96-well sterile, flat-bottom, polystyrene plates with lid were made by (Becton Dickinson Biosciences (Bedford, Massachusetts), and ordered plates through VWR (Radnor, Pennsylvania).

[0065] SICD Assay: The SICD assay was performed as described previously [5]. Cells were pre-grown (with vehicle or TCBZ, each at 2 μΜ) in 4 ml of liquid YPD media in 16 mm sterilized glass tubes with plastic caps for 2 d at 30°C. Cells were then pelleted by centrifugation (7000 x g), washed twice with 5 ml of water, resuspended in 2 ml of water, and aliquots of the cells were transferred to 3 ml 2 % glucose/water (with vehicle or TCBZ) to yield a concentration of 2.0^χ 10⁷ cells/ml [6]. The cultures were incubated with shaking at 37°C. For the viability assay, aliquots were taken at the indicated times, diluted and plated on YPD plates; the plates were incubated for 3 days at 30°C and then colony-forming units (CFUs) were counted.

[0066] Chronological aging assay: The chronological aging assay was performed as previously described [10]. Cells were pre-grown (with vehicle or TCBZ, 2 μΜ) in 4 ml of SC-glucose media in 16 mm sterilized glass tubes with plastic caps for 2 days at 30°C with shaking; the aging experiment then started by continuing to incubate the cells with shaking at 30°C. To measure survival, aliquots were removed at the indicated times, diluted and plated on YPD plates. The plates were incubated for 3 days at 30°C, and then colony-forming units (CFUs) were counted. The CFUs at day 2 is the zero point in the aging experiment (= CFU (t=0)). The percent survival at time t was calculated using the following formula: [CFU (t) / CFU (t=0)] x 100.

[0067] cAMP detection in human SH-SY5Y cells: Human neuroblastoma SH-SY5Y cells were inoculated in 96-well plates containing Dulbecco's Modified Eagle Medium (DMEM) (ATCC, Manassas, Virginia) supplemented with 10% fetal bovine serum (Biowest, Miami, Florida), and the cells were grown overnight in an incubator at 37°C with 5% CO2. The cells were rinsed with 200 μΐ warm phosphate buffered saline (PBS) buffer (pH 7.4) and resuspended in serum- free media with the indicated drug and incubated for 3 h at 37°C. Cells in each well were washed twice with 200 μΐ cold PBS buffer, and then lyzed with the buffer (100 μΐ) was included in the cAMP assay kit. Cells were then kept on ice for 5 to 10 min. Cyclic AMP was determined using an enzyme immunoassay system (Cyclic AMP XP® Assay kit, Cell Signaling Technology) following the manufacturer's instructions. Protein was determined with Bio-Rad protein assay kit.

[0068] Cyclic AMP (cAMP) detection in yeast: Yeast cells (WT, BY4741) were inoculated in SC-glucose (SCD) medium and incubated at 30°C with shaking until mid-log phase (OD600 = 0.5-0.6), and then a selected drug was added and the cultures were incubated for the indicated times. Cells were washed three times with phosphate buffered saline buffer (PBS) (pH 7.4) (Gibco, Grand Island, New York) and were homogenized (lyzed) in 5% trichloroacetic acid (TCA) with glass beads (Sigma-Aldrich; ST. Louis, Missouri). Supernatants were collected and washed with water-saturated diethyl ether (Sigma-Aldrich). Cyclic AMP was determined using an enzyme immunoassay system (Cyclic AMP XP® Assay kit; Cell Signaling Technology; Danvers, Massachusetts) following the manufacturer's instructions. The cells were boiled in 0.2 M NaOH for 10 min, and the protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, California).

[0069] Fluorescence microscopy: Fluorescent images of cells were acquired with an Olympus AX70 microscope equipped with an Olympus UPlanFl 100 x/ 1.35 NA objective and a CoolSNAP HQ CCD camera. For green fluorescent protein (GFP) detection, a Chroma 41001 filter was used (excitation 480/40 nm, emission 535/50 nm, Chroma Technology, Brattleboro, Vermont). Image analysis, filter wheels, shutters, and Z axis stepping motor were under the control of imaging software Slidebook 4.0 (Intelligent Imaging Innovations, Inc., Denver, Colorado). Data were acquired at room temperature. [0070] Mammalian cell culture: Human SH-SY5Y cells (ATCC, Manassas, Virginia) were grown in a 1 : 1 mixture of Dulbecco's Modified Eagle Medium (DMEM) (ATCC) and Ham's F12 medium (Invitrogen) supplemented with 10% fetal bovine serum (Biowest) and 1% penicillin- streptomycin solution (Sigma-Aldrich); and were then maintained in a humid incubator (37°C, 5% C0₂). Rat PC12 cells were grown in antibiotic-free DMEM supplemented with 10% horse serum and 5% fetal bovine serum and were maintained in a humid incubator (37°C, 5% C02). A colorometric assay was used to measure viability of the PC 12 cells. After the various incubations, 20 μΐ CellTiter 96® AQ_ue0us One Solution reagent (Promega, Madison, WI) was added to each well, and the plates were incubated for 3 h. Cell viability was determined by measuring the optical density (OD) at 490 nm using a Wallac 1420 Multilabel Counter.

[0071] Adenylate Cyclase (AC) activity assay in yeast: Adenylate cyclase activity was determined by a nonradioactive enzymatic method [30]. Plasma membrane fractions were prepared as previously described [31]. Yeast cells (WT, BY4741) (American Type Culture Collection (ATCC); Manassas, Virginia) were inoculated in SC-glucose medium and incubated at 30°C with shaking for overnight (OD600 = 3.5-4.0). Cells were harvested by centrifugation, washed with 1 M sorbitol containing 20 mM potassium phosphate buffer (pH 7.0) (buffer A) and resuspended in 1 ml of buffer A. Zymolase (2000 units) (Sigma-Aldrich) was added, and the mixture was incubated at 30°C for 1.5 h before adding 4 ml of chilled buffer A. The spheroplasts were collected by centrifugation and resuspended gently in 1 ml of 0.8 M sorbitol solution containing 10 mM MgCl₂, 1 mM CaCl₂, 1 mM MnCl₂, 0.1 mM EDTA, and 50 mM Tris-HCl (pH 7.5) (buffer B). An equal volume of concanavaline A (0.5 mg/ml in buffer B) (Sigma-Aldrich) was added, the mixture was incubated at 30°C for 10 min, and the spheroplasts were collected by centrifugation. The spheroplasts were lysed by the addition of 5.5 ml of 25 mM PIPES buffer (pH 6.2; Sigma-Aldrich) containing 1 mM MnCl₂, 0.1 mM EDTA, and 1 mM phenylmethylsulfonylfluoride (buffer C), followed by homogenizing in a Dounce glass homogenizer (Pyrex; Lowell, Massachusetts). The crude plasma membrane fraction was collected by centrifugation at 20,000 x g for 45 min and resuspended gently in 1 ml buffer C. To solubilize the membrane-bound adenylate cyclase, Polyethylene glycol ether W- 1 was added to the crude plasma membrane fraction (about 5 mg of protein/ml) at a final concentration of 1.0% and kept for 60 min at 4°C. The extract was used for adenylate cyclase activity assay. [0072] For the adenylate cyclase activity assay, a volume of 1.0 μΐ of 0.1 mM Guanosine 5'-[ ,y-imido]triphosphate trisodium salt hydrate (GppNHp) (Sigma-Aldrich) was added to each reaction tube and maintained in ice. Next, 25 μΐ of reaction mixture (100 mM Tris- acetate (pH 7.4), 20 mM KC1, 10 mM MgCl₂, 20 mM phosphoenolpyruvate, 2 mM ATP, 0.02 mM GTP, 2 mM dithiothreitol, 0.04% bovine serum albumin, 0.2 mM theophylline, 1.0 mg/ml pyruvate kinase, and a drug (TCBZ, FBDZ, ALBZ, or 2,5-dideoxyadenosine (Enzo Life Science; Farmingdale, New York)) was added to each reaction tube. Finally, 25 μΐ of the extract was added to each reaction tube, and the reaction was initiated by placing tube in a water bath at 37°C. After 30 min, the reaction was stopped by the addition of 50 μΐ of 50 mM NaOH, and the mixture was heated for 5 min at 95°C. Newly synthesized cAMP was measured using the Cyclic AMP XP® Assay kit (Cell Signaling Technology). Protein was determined with Bio-Rad protein assay kit (Hercules, California). The adenylate cyclase activity was reported as pmol cAMP/mg protein/min (pmol = picomole = 1 x 10 -^"12 mole; 30 min was the reaction time).

Table 2. Strains and plasmids

Strain / plasmid Description Source

WT (BY4741) MATa his3 l leu2A0 met 15 AO ura3A0 ATCC

MATa his3Al leu2A0 met 15 AO ura3A0

atglA Kelly Tatchell¹ atglAr.KanMX

MATa his3Al leu2A0 met 15 AO ura3A0

atg8A Kelly Tatchell¹ atg8A::KanMX

MATa his3Al leu2A0 met 15 AO ura3A0

atgll Kelly Tatchell¹ atgllA::KanMX

MATa his3Al leu2 0 lysAO ura3A0

msn2A Su-Ju Lin²

msn2A: :KanMX

MATa his3Al leu2A0 lysAO ura3A0

msn4A Su-Ju Lin

msn4A::KanMX

MATa his3Al leu2A0 lysAO ura3A0

msn2Amsn4A Su-Ju Lm

msn2Amsn4A: :KanMX

MATa his3Al leu2A0 metl5A0 ura3A0 Msn2-

Msn2-GFP Invitrogen

GFP::HIS3

JB289-1A MATa leu2 his3 ura3::GFP-TUBl:URA3 Kelly Tatchell

MATa. his3Al leu2A0 met 15 AO ura3A0

pdrlA pdrlAr.KanMX Open Biosystems

MATa his3Al leu2A0 met 15 AO ura3A0

ras2A Open Biosystems ras2A::KanMX

pAG426GAL-

2 μ URA3 Amp^r GAL1 promoter Addgene

EGFP pAG426GAL-

ATG8 in pAG426GAL-EGFP Made

EGFP-ATG8

pAG415GPD Low copy CENLEU2 ARS Amp GPD1 promoter Addgene pAG415GPD-WT WT a-syn in pAG415GPD Made

pAG415GPD-

A3 OP a-syn in pAG415GPD Made

A30P

pAG415GPD-

A53T a-syn in pAG415GPD Made

A53T

Department of Biochemistry & Molecular Biology, Louisiana State University Health Sciences Center at Shreveport, Louisiana.

² Department of Microbiology, University of California, Davis, California.

Abbreviations:

ALBZ, albendazole

cAMP, cyclic adenosine monophosphate

BEN, benomyl

BMDZ, benzimidazole

CLS, yeast chronological lifespan

CFU, colony forming unit

DCFH-DA, dichlorofluorescein-diacetate

DIC, differential interference contrast microscopy

DMSO, dimethylsulfoxide

FBDZ, fenbendazole

GFP, green fluorescent protein

H2O2, hydrogen peroxide

IMDZ, imidazole

NCDZ, nocodazole

OD, optical density

RAP, rapamycin

SC, refers to synthetic complete media

SICD, sugar-induced cell death

TCBZ, triclabendazole

YPD, "Y" yeast extract, "P" peptone and "D" dextrose (same as glucose);

Example 2

Induction of Sugar-Induced Cell Death (SICD)

[0073] SICD, a form of apoptosis, is an unusual form of cell death in yeast. This type of cell death has pertinence to cancer and neurodegenerative diseases like Parkinson's and Alzheimer's. Yeast cells divide and increase in numbers when placed in rich media. After the nutrients are depleted from the media, the yeast cells stop growing, but they are still alive. These non-dividing cells are called "stationary-phase" cells. Another name for them might be "post-mitotic" cells. Stationary phase cells normally can live for weeks in nutrient-depleted medium. "Sugar-induced cell death" (SICD) of yeast occurs when stationary-phase yeast cells are washed with water a couple of times and then resuspended in water and 2% glucose. Resuspending the stationary phase cells in water plus glucose is extremely toxic to these cells. Instead of the cells living for weeks, stationary phase cells transferred into water and glucose have a 50% survival time of -0.5 days, whereas the same strain would have a 50% survival time of about 12 days in depleted medium.

[0074] To induce SICD, Baker's yeast cells {Saccharomyces cerevisia) are diluted into rich media and grown for several days until the media is depleted and the cells are at stationary phase (Go). When a dense culture of stationary phase cells is washed and resuspended in water with 2% glucose (and no other nutrients) at 37°C, the cells generate large amounts of reactive oxygen species (ROS) and die an apoptotic death (Fig. 2, bottom curve). In Fig. 2, yeast cells were pre-grown in liquid YPD media for 2 d at 30°C, washed twice in water, and transferred at 1 x 10⁷ cells/ml in YPD (top curve), water (middle), and 2% glucose/water (bottom curve). Cells were then incubated at 37°C for up to 24 hr, and viable cell number was measured by colony forming assay. The data shown are mean ± s.e.m. (n = 3). As shown in Fig. 2, when transferred to a media with other nutrients, the yeast continues to grow. When transferred to just water (depleted media), the cells are in stationary phase and remain alive. However, when transferred to glucose/water media, the cells die an apoptotic death (bottom curve, Fig. 2). SICD was thus used as a model of apoptosis to screen for drugs that might inhibit apoptosis.

Example 3

Screening for SICD Inhibitor

[0075] The Prestwick and NIHCC chemical libraries were screened to identify those drugs that inhibit SICD as described above in Example 1. The abbreviated protocol is described below. Yeast cells (pdrl ) (Table 2) were grown in 3 ml liquid YPD in standard sterile, capped culture tubes with shaking for 2 d at 30°C. The cells were pelleted at 4000 ^χ g, washed and resuspended in 1 ml sterile water, using standard microbiological practices. 100 μΐ aliquots were diluted into 15 ml tubes containing 3 ml water or 3 ml 2% glucose (w/v)/water to a concentration of 2 10⁷ cells/ml. 135 μΐ aliquots of the cultures were deposited into wells of a sterile, flat bottomed 96-well plate, and 15 μΐ of each drug (50 μΜ) was added to each well to yield a final drug concentration of 5 μΜ per well. The plates were incubated with for 2 d at 37°C with gentle rocking. After this 2 d incubation, a 10 μΐ aliquot of cells was transferred from each well into the corresponding well of a new 96-well plate, in which each well contained 200 μΐ fresh liquid YPD medium, and then the plate was incubated with gentle rocking for 18 h at 30°C. If a drug protected cells from SICD, then the cells grew and an increase in the OD occurred. On the other hand, if a drug failed to protect cells from SICD, then the cells failed to grow and no change in OD occurred. The OD of the wells in each plate was measured. For wells without regrowth, OD values ranged from 0.05 to 0.1 absorbance units at 600 nm; whereas, for wells with regrowth, which indicated a "hit", OD values ranged from 0.5 to 1.0. Only two drugs out of approximately 2000 drugs tested protected yeast cells from SICD. One of these drugs was triclabendazole (TCBZ).

Example 4

TCBZ Effects on Carbon Source Utilization

[0076] After identifying TCBZ as an inhibitor of SICD, additional experiments were performed to test for other effects. The triclabendazole (TCBZ) was purchased (32802- lOOmg; Sigma- Aldrich, St. Louis, Missouri). TCBZ was tested for its effect on growth of wild type yeast in liquid YPD. Wild type (WT) (BY4741) cells were pre-grown in YPD for 2 d at 30°C at which time the optical density was about 12 at 600 nm. The cells were then washed twice in sterile distilled water (DW) and inoculated in the various media with a drug vehicle (control), 5 μΜ triclabendazole (TCBZ), or 5 μΜ benomyl (BEN). Cell growth was measured by optical density (OD) readings at 12 h intervals up to 72 h. Data shown in Fig. 3A-3C represent means ± s.e.m. (n = 3).

[0077] As shown in Fig. 3A, TCBZ (5 uM) had no appreciable effect on the growth of yeast cells cultivated in liquid YPD. In contrast, TCBZ inhibited the growth of yeast cells in medium containing galactose (YPG) or glycerol (YPGly) as a carbon source (Figs. 3B and 3C). For comparison purposes, all experiments were also conducted with benomyl (BEN), a known tubulin inhibitor [7]. If TCBZ and BEN each targeted tubulin, these two drugs would be expected to have the same effect on carbon source utilization. However, as shown in Figs. 3A-3C, they do not have the same effect. Cells grown in TCBZ can only utilize glucose; whereas, cells grown in BEN can use utilize glucose, galactose or glycerol. The data shown in Figs. 3A-3B indicate that TCBZ has a profound effect on cellular metabolism (carbon source utilization) and prevents cells from using two important carbon sources. Example 5

TCBZ Protects Yeast Cells from SICD

[0078] SICD Assay: The SICD assay was performed as described previously [5]. Cells were pre-grown (with vehicle or TCBZ, each at 2 μΜ) in 4 ml of liquid YPD media in 16 mm sterilized glass tubes with plastic caps for 2 d at 30°C. Cells were then pelleted by centrifugation (7000 x g), washed twice with 5 ml of water, resuspended in 2 ml of water, and aliquots of the cells were transferred to 3 ml 2 % glucose/water (with vehicle or TCBZ) to yield a concentration of 2.0^χ 10⁷ cells/ml [6]. The cultures were incubated with shaking at 37°C. For the viability assay, aliquots were taken at the indicated times, diluted and plated on YPD plates; the plates were incubated for 3 days at 30°C and then colony-forming units (CFUs) were counted.

[0079] Chronological aging assay. The chronological aging assay was performed as previously described [10]. Cells were pre-grown (with vehicle or TCBZ, 2 μΜ) in 4 ml liquid SC-glucose in 16 mm sterilized glass tubes with plastic caps for 2 days at 30°C with shaking; the aging experiment then started by continuing to incubate the cells with shaking at 30°C. To measure survival, aliquots were removed at the indicated times, diluted and plated on YPD plates. The plates were incubated for 3 days at 30°C, and then colony-forming units (CFUs) were counted. The CFUs at day 2 is the zero point in the aging experiment (= CFU (t=0)). The percent survival at time t was calculated using the following formula: [CFU (t) / CFU (t=0)] x 100.

[0080] In the SICD experiments, yeast cells were diluted to a low optical density in liquid YPD and then cultured until stationary phase was reached as described in Example 1 (48 h at 30 °C). Fig. 4A shows the growth curve of yeast pre-incubated in liquid YPD until reaching stationary phase (48 h). The stationary-phase cells were then washed and resuspended in 2% glucose/water with the indicated drug (TCBZ, albenzadole (ALBZ) or BEN; each at 5 μΜ) or vehicle (DMSO). Fig. 4B illustrates the survival of stationary-phase Saccharomyces cerevisiae (baker's yeast) cells at 30°C in 2% glucose/water (conditions for sugar-induced cell death (SICD)), and supplemented with 5 μΜ triclabenzadole (TCBZ), albenzadole (ALBZ), benomyl (BEN), or vehicle (DMSO). Data points shown in Fig. 4B are the mean ± s.e.m. of three independent experiments. TCBZ partially protected yeast from SICD, increasing the mean survival time (tm) from 0.3 ± 0.1 d to 0.9 ± 0.3 d (Fig. 4B). In contrast, albendazole and benomyl had no effect. Example 6

TCBZ Does Not Induce Autophagy

[0081] Yeast cells were pre-grown in liquid YPD liquid media for 2 d at 30°C, washed twice in water, and then inoculated into SC-glucose. After 1 day, cells were observed using differential interference contrast microscopy. Fig. 5A shows differential interference contrast microscopy images of Saccharomyces cerevisiae (baker's yeast) cells grown under conditions for sugar-induced cell death with and without triclabenzadole (TCBZ). Yeast cells incubated with TCBZ displayed enlarged vacuoles compared to untreated cells (Fig. 5A). Enlarged vacuoles could mean that TCBZ induces macroautophagy. Macroautophagy is an important cellular process whereby contents of the cytosol, or even whole organelles, are encapsulated by an autophagosome double bilayer structure, and the autophagosome then merges with the vacuole [8]. Contents of the autophagosome are then degraded in the vacuole. The yeast vacuole is the functional equivalent of the mammalian lysosome.

[0082] To test the effect of ATGs-deletion on the chronological aging of cells, four strains of yeast cells were used. Fig. 5B illustrates the percent survival as measured by the colony forming unit (CFU) assay of four strains of Saccharomyces cerevisiae (baker's yeast) cells (wild type (WT), mutant strain atglA, mutant strain atg8A, and mutant strain atgllA) pre- grown in YPD media for 2 d at 30°C, washed twice in water and inoculated into SC-glucose with 5 μΜ TCBZ or drug vehicle (control), and then incubated at 30°C for 10 days. Data represent means ± s.e.m (n = 3). Survival rate was measured by the colony forming unit (CFU) assay.

[0083] As shown in Fig. 5B, TCBZ significantly increased the survival of stationary phase cells in which key autophagy genes were deleted (Fig. 5B). In the mutant strains atglA, atg8A, and atgllA, the ATG1 , ATG8, and ATGl l genes are deleted, respectively. Loss of any one of these genes blocks autophagy. Because TCBZ increased the survival of each of these strains in the chronological life span assay (Fig. 5B), the TCBZ mechanism of protection probably does not involve autophagy.

[0084] In addition, WT cells transformed with the pAG426GAL-EGFP-ATG8 or pAG426GAL-EGFP plasmid (Table 2; Addgene, Cambridge, Massachusetts) were grown in liquid SC-glucose with 5 μΜ TCBZ, BEN or drug vehicle (DMSO (control)) for 1 day, and then were visualized by fluorescence microscopy. Fig. 5C shows fluorescence microscopy images that reveal the location of the key autophagy protein Atg8 tagged with the green fluorescent protein (EGFP-Atg8) in wild type yeast cells carrying the plasmids (pAG426GAL-EGFP-ATG8 or pAG426GAL-EGFP. Fig. 5C indicates that TCBZ and BEN do not affect the localization of the key autophagy protein Atg8 that is tagged with the green fluorescent protein (EGFP-Atg8). This is further evidence that TCBZ does not induce autophagy.

Example 7

TCBC Protects Yeast Cells From the Toxicity of the Human Parkinson 's Disease Protein Alpha-synuclein (a-syn)

[0085] Given that TCBZ partially protects cells from SICD, experiments were conducted to determine whether this drug could protect yeast cells from death induced by the human Parkinson's disease-related protein alpha-synuclein (a-syn). This protein is known to induce reactive oxygen species, such as hydrogen peroxide and superoxide, to accumulate in a variety of cells, including yeast [4,22]. A wild-type yeast strain was engineered that contains three chromosomally integrated copies of a-syn. The protein a-syn has been implicated in the etiology of Parkinson's disease [17]. Each a-syn gene is under control of a fatty acid promoter, and western blotting was used to verify that a-syn is robustly expressed when this strain is grown in standard yeast media. To determine the effect of a-syn on the chronological lifespan, the survival of stationary phase cells (with or without a-syn expression) was analyzed over the course of several days.

[0086] To construct the yeast strains, DNA manipulations followed standard protocols [15]. A BY4741 strain containing three integrated copies of human a-syn (ΜΑΤΆ, his3Al, leu2A0, metl5A0, ura3A0 Iys2::a-SYN(3)) was constructed in several steps as described below. Table 3 gives the primers used in constructing of the strains.

[0087] Step (i): Amplification of the FAA2 terminator region. The 400 base pair (bp) terminator region of the FAA2 gene was PCR (polymerase chain reaction) amplified from BY4741 genomic DNA (Table 2) using forward and reverse primers Fl and Rl (Table 2; Integrated DNA Technologies, Coraville, Iowa, respectively. After digestion of the product with Smal and Xhol (Table 2), the purified DNA fragment was inserted into the same sites on the yeast integrating plasmid pRS306 (Table 2, Invitrogen) [20].

[0088] Step (ii): Construction of the FAA2 promoter and a-syn gene. For construction of the FAA2 promoter and a-syn gene, first a 400 bp sequence of the FAA2 promoter and a 420 bp sequence of the a-syn gene (Neil Mathias, Louisiana State University Health Sciences Center, Shreveport, Louisiana) were amplified from BY4741 genomic DNA (primers F2/R2; Table 3) and the plasmid pTF201 [4] (primer F3/R3), respectively. Second, because the reverse primer of the FAA2 promoter and the forward primer of a-syn were designed to contain complementary sequences, the mixture of these two PCR products was used as the template for the second round of amplification. The resultant product, which contained the fatty acid promoter followed by the a-syn gene, was ligated into the Xbal and Smal restriction sites of the pRS306 plasmid generated in Step (i).

[0089] Step (Hi): Construction of the pRS306 integrating plasmid with three copies of a- syn. The fatty acid promoter/a-syn cassette from Step (ii) was amplified using forward and reverse primers F4 and R4, respectively, and this second cassette was ligated into the linearized pR306 plasmid from Step (ii) at the Xbal site. The third cassette, generated with primers F5 and Rl, was ligated into the linearized plasmid containing two FAA2 promoter/a- syn cassettes at the Xhol site. To enable homologous recombination at the LYS2 locus, a fragment of the LYS2 gene (bp 749 to 1891), amplified from genomic DNA using primers F6 and R6, was ligated into the Sad site on pRS306. ^g of the resultant plasmid was linearized with Bsu36l and integrated into BY4741 genomic DNA at the LYS2 locus [16]. Western blot analysis confirmed that this strain synthesizes a-syn when cells are cultured in a variety of different media.

Table 3. Primers for a-syn Yeast Strain Construction.

Fl (SEQ ID O:l) 5 '-CTATCCCGGGGTACTTATGACGATTTGGAACAC-3 '

Rl (SEQ ID NO:2) 5 ' -TTCCTCGAGCCGTTTTC AATCATCTTGACC-3 '

F2 (SEQ ID O:3) 5 ' -TGGGATTTTCTAGAAGTCC-3 '

R2 (SEQ ID NO:4) 5 ' -GAATACATCCATATTTCGAAACAAGTTTGT-3 '

F3 (SEQ ID O:5) 5 ' -TGTTTCGAAATATGGATGTATTCATGAAAGGAC-3 '

R3 (SEQ ID NO:6) 5 ' -CTTACCCGGGTTAGGCTTCAGGTTCGTAGTCTTG-3 '

F4 (SEQ ID O:7) 5'-GGATTTTCTAGAAGTCCCGGTGTC-3'

R4 (SEQ ID NO:8) 5 ' -CCTTCTAGACCGTTTTC AATCATCTTGACC-3 '

F5 (SEQ ID O:9) 5'-CTTCTCGAGTCTAGAAGTCCCGGTGTCCCTGAC-3'

F6 (SEQ ID NO:10) 5'-CGCGAGCTCGCGCCAGAGAGAACCTGTGTTGTG-3'

R6 (SEQ ID NO:l l) 5'-CGCGAGCTCGCGTCGGCCAAACCACCTGCACGAA-3' [0090] The ability of TCBZ to protect yeast cells from a-syn-induced death was conducted in a chronological aging experiment. The chronological lifespan (CLS) refers to the length of time stationary-phase cells that can survive in culture. In the CLS experiments, yeast cells were diluted to a low optical density in liquid SC-glucose medium, and cultured until stationary phase (48 h at 30°C); this was the zero time for the CLS experiment. Cells were periodically removed to determine the number of viable cells in culture. Previous studies have shown that a-syn significantly shortened the CLS of yeast [2] and induced reactive oxygen species (ROS) to accumulate [12].

[0091] Fig. 6A illustrates chronological aging curves of a wild type yeast strain (control) and the same yeast strain engineered to express human a-synuclein (a-syn) incubated with 5 μΜ of DMSO, TCBZ, ALBZ, or BEN. The arrow indicates when the various drugs were added. Viability was measured in a colony formation assay, as described in Example 1. As shown in Fig. 6A, TCBZ protects cells from a-syn-induced cell death. Yeast cells expressing a-syn exhibited decreased survival (tl/2 = 5.0 ± 0.4 d) compared to cells without a-syn expression (tl/2 = 8.2 ± 0.1 d) (Fig. 6A). Notably, TCBZ reversed the effect of a-syn. For cells expressing a-syn, TCBZ increased mean survival from 5.0 d to 9.7 d, whereas ALBZ and BEN had no effect.

[0092] To test whether TCBZ decreases the ROS burden in cells expressing a-syn, stationary-phase cells expressing a-syn were incubated with the ROS-sensitive dye ~ 2',7'- dichlorfluorescin-diacetate (DCFH-DA) (Sigma-Aldrich). DCFH-DA is a non-fluorescent, cell permeant dye that becomes trapped inside cells once it is oxidized by cytoplasmic ROS to the polar, fluorescent compound 2',7'-dichlorfluorescein (DCF). Fig. 6B shows the detection of reactive oxygen species (ROS) by fluorescence microscopy of stationary-phase yeast cells incubated for 7 d at 30 °C. A wild type strain (control), an a-syn expressing strain (a-syn), and an a-syn expressing strain treated with 5 μΜ TCBZ (a-syn + TCBZ). Each strain was incubated with the ROS-sensitive dye (2',7'-dichlorfluorescin-diacetate (DCFH- DA)) at 10 μg/ml for 1 h, washed, and visualized by fluorescence (DCF) and differential interface contrast (DIC) microscopy. Fig. 6C shows the percent cells staining for DCF. Values were obtained from two independent experiments, where the total number of cells counted was 500. Error bars in Fig. 6C are means ± s.e.m., with an "*" indicating P<0.01 (two-tailed Student's t test, versus a-syn).

[0093] TCBZ-treated cells expressing a-syn showed significantly fewer cells (45 ± 6 %) staining for ROS compared with the same cells without drug (74 ± 14%) (Figs. 6B and 6C). Note that no further decrease in ROS occurred when TCBZ was added to stationary-phase cells. This suggests that the first addition of TCBZ to exponential phase cells may up-regulate genes that protect cells from oxidative stress, and that subsequent additions cannot enhance the response.

Example 8

Effect of TCBZ and Other Drugs on Life Span

[0094] TCBZ is known to inhibit tubulin, and yeast cells express tubulin. To determine whether nocodazole (NCDZ) (Sigma-Aldrich), which is known inhibitor of tubulin, increases survival of cells in the chronological aging assay, the following experiment was conducted and results shown in Fig. 7. Benzimidazole (BMDZ) and imidazole (IMDZ) were also tested because TCBZ is composed of these structural units. Fig. 7 illustrates the percent survival as measured by the colony formation assay of yeast cells pre-grown in liquid YPD for 2 d at 30°C, washed twice in water, and then inoculated in SC-glucose with 5 μΜ of TCBZ, benzimidazole (BMDZ), imidazole (IMDZ), or nocodazole (NCDZ), and then incubated at 30°C for 10 d before checking viability. Data shown are mean ± s.e.m. (n = 3). NCDZ produced a modest increase in survival compared to untreated cells (20% survival as compared to 33 % survival, respectfully) after 10 d. BMDZ and IMDZ had no effect. By far, the greatest effect was due to TCBZ (20% survival as compared to 80 % survival). The results show that TCBZ increases survival much better than the known inhibitor of tubulin, i.e., NCDZ. We have also tested albendazole (ALBZ) using similar survival experiments, and have shown that it was not as effective as TCBZ.

Example 9

TCBZ Induces a Protective Stress Response

[0095] Experiments were conducted to determine whether TCBZ protects cells from an exogenous oxidant (hydrogen peroxide). Fig. 8A shows the effect of various drugs (5 μΜ; DMSO, TCBZ, and ALBZ) on the survival of stationary-phase yeast cells incubated with H2O2 (10 mM) for 1 h at room temperature. In this experiment, yeast cells were first diluted to a low optical density in synthetic complete media with added drug, and then cultured until stationary phase was reached (48 h at 30°C). H2O2 was then added for 1 h, and viability was measured. The results for the yeast cells are shown in Fig. 8A. Values are the mean ± s.e.m. of three independent experiments. The "*" indicates P<0.005 (two-tailed Student's t test, versus DMSO). TCBZ protected cells from death induced by such a high concentration of H₂0₂, i.e., 54 ± 7% of the TCBZ-treated cells survived whereas only -30% of DMSO or ALBZ-treated cells survived.

[0096] Fig. 8B shows the effect of various drugs (50 μΜ) on the survival of rat PC12 cells (ATCC) incubated with H₂0₂ (1 mM) for 21 h at 37 °C. In this experiment, PC12 cells were seeded at a density of 1 x 10⁴ cells/well in a flat-bottomed 96- well plate with poly D- lysine (0.2 μg/ml; Sigma-Aldrich). The next day, cells were pre-treated with drug (50 μΜ TCBZ, ALBZ, or DMSO) for 3 h, incubated with 1 mM H₂0₂ for 21 h and then viability was measured using a colorometric assay as described in Example 1. The results are shown in Fig. 8B. Values are the mean ± s.e.m. of three independent experiments. The "*" indicates P<0.005 (two-tailed Student's t test, versus DMSO). As shown in Fig. 8B, TCBZ protected cells from H20₂-induced death, i.e., 62 ± 6% of the TCBZ-treated cells survived, whereas only 35-41% of the DMSO or ALBZ-treated cells survived. No evidence was found that any of these drugs decomposed H₂0₂.

[0097] The results in Figs. 8A and 8B indicate that TCBZ protects yeast cells from SICD and death induced by -syn or H₂0₂, and at a 10-fold higher concentration TCBZ also protects rat PC 12 cells from H₂02-induced death. This indicates that TCBZ protects cells from stress-related death, and that TCBZ can be used for treatment of neurodegenerative diseases that involve oxidative stress such as Parkinson's disease.

Example 10

TCBZ Does Not Inhibit Microtubule Formation

[0098] In yeast, TCBZ was shown to inhibit growth of cells and dramatically increase the chronological lifespan (CLS), but not to inhibit microtubule formation at 5 μΜ. As shown in Fig. 9A-9E, TCBZ-induced CLS extension is not due to microtubule destabilization. Fig. 1 shows the structures of TCBZ and known microtubule inhibitors in yeast, nocodazole (NCDZ) and benomyl (BEN). Fig. 9A shows the growth curves of yeast cells (strain JB289- 1A) expressing Tubl-GFP incubated with 5 μΜ of triclabenzadole (TCBZ), nocodazole (NCDZ), or benomyl (BEN). The arrow denotes when the drug was added. Cells were cultured in liquid SC-glucose at 30°C. Cells were inoculated into liquid SC-glucose medium and incubated at 30°C until mid-log phase (about 5 h, ΟΌβοο = 0.5-0.6). The drug or vehicle was added to the culture at the mid-log phase, and the cells then re-incubated at 30°C.

[0099] Fig. 9B illustrates chronological aging curves of JB289-1A cells which express Tubl-GFP. Plots show survival of cells as a function of time as determined by a colony forming assay as described in Example 1. At the zero point, cells had been incubated with the indicated drug (5 μΜ triclabenzadole (TCBZ), nocodazole (NCDZ), benomyl (BEN) or DMSO) for 48 h. Values are the mean ± S.E. of three independent experiments. As shown in Fig. 9B, TCBZ, but not BEN or NCDZ, dramatically increased the CLS.

[0100] Fig. 9C is fluorescence microscopy images of yeast cells expressing Tubl-GFP. Cells were inoculated into liquid SC-glucose medium, incubated until mid-log phase and then incubated for 1 h with 5 μΜ of triclabenzadole (TCBZ), nocodazole (NCDZ), benomyl (BEN) or DMSO at 30°C before observing with fluorescence microscopy. Fig. 9C indicates that TCBZ had no effect on spindle formation. The Tubl-GFP spindle, which appears as a line that spans a mother and daughter cell, was the same size in TCBZ-treated cells as in control cells (DMSO), indicating TCBZ did not affect spindle formation. In contrast, the spindles appeared as a punctuate pattern in cells treated with NCDZ and BEN, indicating that these two compounds disrupt spindle formation. Fig. 9D is a plot of the number of yeast cells expressing Tubl-GFP exhibiting different spindle characteristics as depicted in the figure. Each value was obtained from three independent experiments, where the total number of cells counted was 200-300. Error bars are ± s.e.m.

[0101] The data in Figs. 9A-9D indicate that TCBZ (5 μΜ), but not NCDZ or BEN, inhibited growth of yeast cells (Fig. 9A), dramatically increased CLS (Fig. 9B), and had no effect on microtubule formation (Figs. 9C and 9D).

Example 11

TCBZ-induced CLS Extension Depends on Msn2/4 Transcription Factors

[0102] Chronological aging experiments were conducted using wild-type cells or various deletion strains. Msn2 and Msn4 are transcription factors that control the response to stress in yeast. Strains of yeast cells were obtained with either single deletions in Msn2 or Msn4 or a double deletion in both (see Table 2). TCBZ failed to increase the CLS in the double deletion strain msn2Amsti4A indicating that these two genes are necessary for the TCBZ- induced increase in CLS. Four yeast strains (wild type, msn2A, msn4A, and msn2Amsn4A) were inoculated in liquid SC-glucose medium with the drug or vehicle, and incubated at 30°C. At the zero point, cells had been incubated with the drug for 48 h. The number of viable cell number was conducted by colony forming units assay as described in Example 1. The results are shown in Fig. 10A. Since Msn2 and Msn4 have overlapping functions, deletion of both genes was necessary to see the TCBZ effect. Values shown in are the means ± s.e.m. of the three independent experiments.

[0103] Fig. 10B are fluorescence images of yeast cells expressing Msn2-green fluorescent protein (GFP) inoculated into SC-glucose medium, incubated until mid-log phase, and then incubated for 2 h with 5 μΜ triclabenzadole (TCBZ), nocodazole (NCDZ), or DMSO or 100 nM rapamycin (RAP) at 30°C before observing with fluorescence microscopy. TCBZ triggered the translocation of a green fluorescent protein-Msn2 fusion (Msn2-GFP) into the nucleus of cells (Fig. 10B). Fig. IOC shows a plot of the percentage of yeast cells containing Msn2-GFP in the nucleus. Cells were incubated as described in Fig. 10B, and then cells were analyzed by fluorescence microscopy to determine the percent of cells with nuclear Msn2-GFP. DAPI is a dye that stains the nucleus. Values are means ± s.e.m from four independent experiments, where the total number of cells counted was 300-350. "*" indicates P < 0.001 (two-tailed Student's t test, versus DMSO).

[0104] Fig. 10D shows a growth assay of yeast cells subjected to different stresses. Wild type yeast cells were initially inoculated into SC-glucose medium with the drug (TCBZ) or vehicle (DMSO) and incubated at 30°C for 4 d. The two cultures were normalized to the same OD600, serially diluted in 1-fol dincrements and subjected to three different stresses (100 mM H2O2 or 300 μΜ menadione or heat shock (50°C) for 60 min), and then spotted onto YPD plates. As shown in Fig. 10D, TCBZ protected the cells from the stresses. The combined results of Figs. 10A-10D demonstrate that TCBZ protected yeast cells by activating a stress response mediated by Msn2 and Msn4.

Example 12

TCBZ Decreases Intracellular cAMP level

[0105] TCBZ has been thought to bind to beta-tubulin in liver flukes and kills the cells because of its disruptive effect on microtubules, which are made of tubulin. Experiments were conducted to measure the changes in intracellular cAMP levels due to TCBZ. [0106] As described below, TCBZ was shown to decrease intracellular cAMP in yeast cells. Cells (wild-type or ras2A; Table 2) were inoculated in liquid SC-glucose medium with indicated drug (TCBZ or RAP) or vehicle (DMSO) and incubated for 15 h at 30°C. Cells were then washed three times with water, resuspended in 5% trichloroacetic acid (TCA), and incubated at 4°C for 1 h. The TCA extract was used for cAMP assay after extraction with diethyl ether and neutralization with 0.5 M Tris-HCl (pH 7.5). cAMP was determined with kit from Cell Signaling Technology (Danvers, Massachusetts) as described in Example 1. The results are shown in Fig. 11 A. Values are the means ± s.e.m. of the four independent experiments. The "*" indicates a P < 0.005 (two-tailed Student's t test, versus DMSO). The data in Fig. 11 A shows that TCBZ inhibited the synthesis of cyclic AMP (cAMP).

[0107] Experiments were run to measure the effect of added cAMP on TCBZ-induced growth inhibition. Wild-type yeast cells were inoculated into SC-glucose containing 5 μΜ drug (DMSO or TCBZ) supplemented with 5 mM cAMP or ATP and incubated at 30°C. The results are shown in Fig. 11B which plots the doubling time for the cells. The "*" indicates P < 0.005 (two-tailed Student's t test, versus DMSO). As shown in Fig. 11B, the addition of cAMP to cells reversed the effect of TCBZ on cells. Specifically, TCBZ increased the yeast doubling time to 3 h (from 2 h), and added cAMP abolished this effect (Fig. 1 IB).

[0108] In addition, cAMP reversed TCBZ-induced Msn2-GFP nuclear localization. Fig. l lC shows fluorescence microscopy images of yeast cells expressing Msn2-GFP incubated in SC-glucose medium until mid-log phase, and then TCBZ (5 μΜ) with or without 5 mM cAMP was added and cells were incubated for 2 h before observing with fluorescence microscopy. The images show that added cAMP inhibits the ability of TCBZ to induce the nuclear localization of Msn2-GFP. Overall, the data in Figs. 1 lA-11C indicate that TCBZ lowered the intracellular level of cAMP, which is a key second messenger molecule in eukaryotic cells.

Example 13

Fenbendazole (FBDZ) Slows Growth and Induces the Nuclear Localization of Msn2

[0109] Experiments were conducted to see if another benzimidazole, fenbendazole (FBDZ), might have similar effects as TCBZ. Fig. 1 shows the structures of TCBZ and FBDZ. Fig. 12A shows the effect of TCBZ and FBDZ on the doubling time of WT BY4741 yeast cells in liquid YPD. Both compounds significantly increased the yeast doubling time compared to control cells (DMSO). As shown in Fig. 12A, FBDZ slowed down growth at 2 μΜ much better than TCBZ at the same concentration. Fig. 12B shows fluorescence images of yeast cells expressing Msn2-GFP incubated in SC-glucose medium until mid-log phase, drug (5 μΜ TCBZ or 5 μΜ FBDZ) or vehicle (5 μΜ DMSO) was added and cultures were incubated for 2 h before observing with fluorescence microscopy. DAPI is a dye that stains the nucleus. Fig. 12B indicates that FBDZ induced Msn2 nuclear localization. Fig. 12C is a plot of the percent of yeast cells containing nuclear localized Msn2-GFP. Each value was obtained from four independent experiments, where the total number of cells counted was 200-250. Error bars are ± s.e.m., and an "*" indicates P < 0.001 (two-tailed Student's t test, versus DMSO).

[0110] As shown in Fig. 12A, FBDZ, a benzimidazole like TCBZ, behaved like TCBZ. FBDZ significantly slowed down the growth of yeast cells; and only 2 μΜ FBDZ was required to increase the doubling time from 2 h to 3 h (Fig. 12A). Based on the doubling time data, FBDZ may be more potent than TCBZ since 2 μΜ FBDZ increased the doubling time from 2 to 3 h, while 2 μΜ TCBZ had no appreciable effect on the doubling time. Similar to TCBZ, FBDZ also induced Msn2-GFP to localize to the nucleus (Figs. 12B and 12C). These data indicate that FBDZ would also trigger a stress response in yeast, and would protect cells from the human Parkinson's disease-related protein alpha-synuclein similar to TCBZ.

Example 14

TCBZ and FBDZ Inhibit Adenylate Cyclase

[0111] The effect of TCBZ and other drugs on on adenylate cyclase activity was evaluated. The adenylate cyclase assay was conducted on yeast lysates as described above in Example 1. For comparison, a known inhibitor of adenylate cyclase was used (2',5 '- dideoxyadenosine, 2,5-DDA ). As shown in Table 4, TCBZ and FBDZ inhibited adenylate cyclase activity much more effectively than 2,5-DDA. For example, only 1.0 μΜ TCBZ or FBDZ caused an approximate 70% inhibition of adenylate cyclase, whereas 300 μΜ of 2,5- DDA caused only a 10% inhibition. Table 4. Effect of drugs on adenylate cyclase activity in yeast.

Values are the mean ± s.e.m. of the two independent experiments (n = 3).

" The percentage change was determined by comparing the experimental treatments TCBZ,

FBDZ, and 2,5-DDA to the DMSO control (dimethylsulfoxide).

Example 15

TCBZ and FBDZ Decrease Intracellular cAMP Level in Human Cells.

[0112] Human neuroblastoma cells (SH-SY5Y; Table 2) were inoculated into 96-well plates containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% horse serum and 5% fetal bovine serum, and then incubated overnight at 37°C (see Example 1). Cells were rinsed with warm PBS, then the drug was added in serum free medium and incubated for 3 hrs. Cells were lysed, and the amount of cAMP was determined using the cAMP assay kit (Cell Signaling Technologies). Fig. 13 illustrates the amount of intracellular cAMP from human neuroblastoma cells (SH-SY5Y) incubated with DMSO, TCBZ (5 uM), FBDZ (5 μΜ), ALBZ (5 μΜ), or 2,5-DDA (300 μΜ), for 3 h before extraction for the cAMP assay. Protein concentration was determined with the Bio-Rad protein assay kit. Values are the means ± s.e.m. (n = 5); "*" indicates P < 0.05 and indicates P < 0.01 (two-tailed Student's t test, versus DMSO). As shown in Fig. 13, both TCBZ and FBDZ decreased the intracellular cAMP in human cells. Example 16

The human Parkinson 's Disease-related Protein Alpha-synuclein Increase cAMP in Yeast Cells.

[0113] Alpha-synuclein is a human protein that is associated with Parkinson's disease. Age-related modifications of a-syn, or age-related increases in the concentration of this protein, are thought to trigger Parkinson's disease. Fig. 14 illustrates the amount of intracellular cAMP from wildtype yeast cells expressing wild type alpha-synuclein (WT a- syn), the point mutant A30P (A30P a-syn) or the point mutant A53T (A53T a-syn). Cells were incubated in SC-glucose medium with TCBZ (5 μΜ) or vehicle (DMSO) for 15 h at 30°C prior to extraction for the cAMP assay. Values are the mean ± s.e.m. of the two independent experiments (each in quadruplicate); "*" indicates P < 0.005 (two-tailed Student's t test, versus vector). The two a-syn mutants A30P and A53T were made using the wild type a-syn gene as template as previously described [4]. cAMP was measured in yeast cell lysates using the cAMP detection kit from Cell Signaling Technology as described in Example 1. When TCBZ was added, the effect was reversed as shown in Fig. 14. This is the first report of a-syn increasing cAMP, and of TCBZ reversing that effect.

Example 17

TCBZ and FBDZ Inhibit Formation of Toxic Protein Aggregates [0114] The following experiments will be conducted:

[0115] Yeast: We will test whether TCBZ or FBDZ inhibit the formation of toxic aggregates of three disease-associated proteins in yeast using fluorescence microscopy and western blotting. The three proteins that we will analyze are (i) human TDP-43 (to model amyotropic lateral scelerosis), (ii) an expanded glutamine segment of exon 1 of the human huntingtin protein or an expanded glutamine segment of GFP (to model Huntington's disease), and (iii) human a-syn (to model PD). Two sets of plasmids will be constructed, i.e., one that contains the untagged gene of interest and the other that contains the gene of interest fused to a segment of DNA which codes for the green fluorescent protein (GFP). Tagging the respective proteins with GFP will permit aggregation of these proteins in live cells to be monitored by fluorescence microscopy. We already have various plasmids that contain the gene for a-syn and GFP-a-syn; thus, we will first test the ability of TCBZ and FBDZ to inhibit the formation of aggregates of a-syn in yeast. Genes will be harbored on well- characterized plasmids, driven by the GAL1 or GAL 10 promoters.

[0116] Monitoring the aggregation of GFP-a-syn by fluorescence microscopy in yeast: Wild type yeast cells will be transformed with a plasmid harboring human wild type a-syn (or one of its disease-associated mutants such as A30P, A53T or E46K tagged with GFP). Cells will be incubated for varying lengths of time to promote aggregate formation, which can be readily detected by fluorescence microscopy. By including TCBZ or FBDZ in the cell culture medium, we can determine whether these drugs decrease inclusion formation. It is expected that TCBZ and FBDZ will eliminate detectable aggregates of a-syn or TDP-43 or expanded glutamine protein from yeast cells.

[0117] Monitoring the aggregation of a-syn by western blot analysis: Another way to detect a-syn in cells is to prepare a cell lysate and subject an aliquot of the lysate to SDS- PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), followed by transfer of the protein from the gel to a polyvinyl membrane for immunoblotting using standard procedures. The a-syn protein will be detected in cell lysates by blotting the membrane with an antibody specific for human a-syn with such antibodies purchased commercially (e.g., Sigma-Aldrich or Santa-Cruz Biotechnology, Santa Cruz, California). To ensure the same amount of protein is loaded in each well of the SDS-PAGE gel, the protein concentration of lysates will be determined by a standard colorometric assay. It is expected that TCBZ and FBDZ will significantly decrease the amount of a-syn, TDP-32 or expanded glutamine protein compared to control cells that lacked drug. This will confirm that TCBZ and FBDZ are effective in decreasing the amount of protein aggregate in Parkinson's, Huntington's, and ALS.

[0118] (ii) Human or rat cells in culture: We will conduct similar experiments as described above in mammalian (e.g., rat or human) cells in culture. As in the yeast experiments, plasmids will be constructed that contain the gene of interest with or without GFP. The plasmids are transiently transfected into the mammalian cells and then cultured by standard procedures with or without the drug of interest (e.g., procedures described in Example 1). Similar to the yeast experiments, we will monitor aggregate formation by fluorescence microscopy and western blotting. It is expected that TCBZ and FBDZ will block the formation of toxic aggregates of a-syn, TDP-43 and expanded glutamine proteins compared to identical cells without drug, confirming the use of these two drugs in treating proteinopathies.

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[0119] The complete disclosures of all references cited in this application are hereby incorporated by reference. Also, incorporated by reference is the following: Lee, W.J., Burlet, E., Wang, S., Xu, B., Huang, S., Galiano, F.J., Witt, S.N. (2011), "Triclabendazole protects yearst and mammalian cells from oxidative stress: Identification of a potential neuroprotective compound," Biochem. Biophys. Res. Commun., vol. 414, 205-208 (2011 ; epub 16 September 2011). In the event of an otherwise irreconcilable conflict, however, the present specification shall control.

Claims

What is claimed:

1. A method to decrease symptoms in a mammal due to a neurodegenerative disease, said method comprising administering to the mammal an effective amount of at least one neuroprotective compound selected from the group consisting of triclabendazole and fenbendazole.

2. The method of Claim 1, further comprising administering at least one neuroprotective drug selected from the group consisting of insulin- like growth factor 1 (IGF-1), tripeptide Gly-Pro-Glu (GPE), cyclic Pro-Gly ("cPG"), diketopiperazine analogues of thyrotropin-releasing hormone (TRH), insulin-like growth factor-II (IGF-II), transforming growth factor-.beta.l, activin, growth hormone, nerve growth factor, growth hormone binding protein, and IGF-binding proteins.

3. The method of Claim 1 , wherein the symptoms of the neurodegenerative disease are due to toxic protein aggregates.

4. The method of Claim 1, wherein the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, polyglutamine expansion diseases, Huntington's disease (HD), tauopathies, frontotemporal dementia associated with tau- immunoreactive inclusions (FTD-tau), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD).

5. The method of Claim 1, wherein the neurodegenerative disease is Parkinson's disease.

6. The method of Claim 5, further comprising administering at least one compound selected from the group consisting of adenylate cyclase inhibitor, carbidopa, levodopa; dopamine agonists, anticholinergics, MAO-B inhibitors, L-dopa (levo- dihydroxy-phenylalanine), pramipexole, ropinirole, rotigotine, bromocriptine, apomorphine, trihexyphenidyl, benztropine, benzhexol, orphenedrine, procyclidine, selegiline, rasagiline, amantadine, and rivastigmine.

7. A method to decrease cell damage in a mammal due to oxidative stress, said method comprising administering to the mammal an effective amount of at least one protective compound selected from the group consisting of triclabendazole and fenbendazole.

8. A method to decrease symptoms in a mammal due to diabetes, said method comprising administering to the mammal an effective amount of at least one compound selected from the group consisting of triclabendazole and fenbendazole.

9. A method to decrease symptoms in a mammal due to high glucose, said method comprising administering to the mammal an effective amount of at least one compound selected from the group consisting of triclabendazole and fenbendazole.

10. A composition for treating a neurodegenerative disease, said composition comprising at least one compound selected from the group consisting of triclabendazole and fenbendazole; and at least one compound selected from the group consisting of insulin- like growth factor 1 (IGF-1), tripeptide Gly-Pro-Glu (GPE), cyclic Pro-Gly ("cPG"), diketopiperazine analogues of thyrotropin-releasing hormone (TRH), insulin-like growth factor-II (IGF-II), transforming growth factor-.beta.l, activin, growth hormone, nerve growth factor, growth hormone binding protein, and IGF- binding proteins.

11. A composition for treating a patient with Parkinson's disease, said composition comprising at least one compound selected from the group consisting of triclabendazole and fenbendazole; and at least one compound selected from the group consisting of carbidopa, levodopa; dopamine agonists, anticholinergics, MAO-B inhibitors, L-dopa (levo-dihydroxy-phenylalanine), pramipexole, ropinirole, rotigotine, bromocriptine, apomorphine, trihexyphenidyl, benztropine, benzhexol, orphenedrine, procyclidine, selegiline, rasagiline, amantadine, and rivastigmine.