Intrinsically disordered protein ELN484228 targets α-synuclein [1]

In vitro activity: ELN484228, a phenyl-sulfonamide compound, is a cell-permeable blocker of α-synuclein which is a key protein in Parkinson’s disease. It was identified by a combination of computational and experimental techniques. ELN484228 has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules such as ELN484228 represents a promising approach to the development of therapeutic treatments of Parkinsons disease and related conditions.

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Kinase Assay: Aggregation of αSyn was assayed in triplicates at 37°C under shaking (300 rpm) in solutions containing 50 µM protein in the absence and presence of tenfold higher concentration of compound ELN484228 in 25 mM Tris buffer pH 7.4, 100 mM NaCl with the addition of 0.01% NaN3. Aliquots were withdrawn on a daily basis and the thioflavin T (ThT) fluorescence signal was recorded after addition of 20 µM of ThT to each aliquot. Fluorescence emission spectra from 460 to 600 nm were then recorded at an excitation wavelength of 446 nm employing a Cary-Eclipse spectrofluorimeter (Varian, Palo Alto CA). Quenching of the ThT fluorescence by the addition of ELN484228 was assayed by incubating pre-formed fibrils with the compound and by comparison of the ThT fluorescence signal before and after the addition of ELN484228, but no significant change in signal was found. The aggregation of αSyn in the presence of ELN484228 was also characterized in the presence of low concentrations of SDS (200 µM) under the same experimental conditions as described above. The time-dependences of the ThT fluorescence signal were fitted to a nucleation-elongation model as previously described. TEM images were obtained using a Philips CEM100 transmission electron microscope. The samples were applied on Formvar-carbon coated nickel grids and stained with 2% (w/v) uranyl acetate.

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Cell Assay: Microglia were obtained from cerebral cortices of 1–3 day old neonate mice. A full description of microglia culture methods is provided in the Supporting Information text. Hippocampal neurons were isolated from embryonic day 18 prenatal rat hippocampi and cultured in antibiotic- and serum-free NbActiv4 medium (both from BrainBits, Springfield IL) at 37°C in an atmosphere of 5% CO2, 9% O2 and on glass coverslips coated with poly-lysine. Half of the medium was replaced every 3 to 4 days. Cells were used for the experiments after 21–28 days in vitro.

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1. Alleviates α-synuclein-mediated impairment of vesicular dynamics: H4 neuroglioma cells with tetracycline-inducible α-synuclein overexpression were cultured for 24 hours in the presence or absence of 1 µg/ml tetracycline (to induce α-synuclein expression) and ELN484228 or control compound ELN484217. After adding 4 μ beads for 90 minutes, microscopic visualization was used to calculate the phagocytic index. Statistical analysis (t-test) of the combined averages from three independent experiments (each sample run in triplicate) showed a significant difference in phagocytosis between tetracycline-induced samples treated with and without ELN484228 (n = 3 ± s.e.m, p≤0.001 versus no compound tet-induced sample) [1]
2. Reduces translocation of α-synuclein to the phagocytic cup: H4 cells were treated with 100 µM ELN484228 and 1 µg/ml tetracycline for 24 hours, then stimulated with 4 μ beads for 90 minutes. After fixation, α-synuclein was detected by staining with 5C12 antibody (red), and cells were counterstained with 488-phalloidin (green) and Hoechts (blue). The results showed that ELN484228 reduced the translocation of α-synuclein to the phagocytic cup [1]
3. Reduces synaptic α-synuclein levels in rat hippocampal neurons: Rat hippocampal neurons (~21 DIV) grown in serum-free conditions were treated with 1 µM ELN484228 or 0.01% DMSO vehicle for 24 hours. Confocal microscopic imaging was performed to detect α-synuclein (red, 5C12 antibody) and the presynaptic marker synaptophysin (green). Quantitative analysis using Metamorph imaging analysis software (measuring integrated intensity for synaptic α-synuclein and pixel area for synaptophysin) demonstrated that ELN484228 reduced synaptic α-synuclein levels. The analysis included 1000 terminals (for α-synuclein) or 18 optical fields (for synaptophysin) per condition, derived from 2–3 independent cultures (values represent mean ± SEM) [1]
4. Alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of α-synuclein: Primary rat embryonic midbrain cultures were non-transduced (control) or transduced with adenovirus encoding A53T α-synuclein, in the absence or presence of 10 µM ELN484228. Immunocytochemical staining for MAP2 and TH was performed. The percentage of MAP2-positive cells that were also TH-positive was evaluated to assess preferential dopaminergic cell death, and the lengths of neurites positive for both MAP2 and TH were measured using NIS-Elements software. The results showed that ELN484228 alleviated dopaminergic neuronal loss (p-value≤0.05) and neurite retraction (p-value≤0.001 versus A53T α-synuclein virus without compound; one-way ANOVA with Newman-Keuls post-test). For neuron viability analysis, n = 3; for neurite length analysis, n = 160–206 (data plotted as mean ± s.e.m) [1]
5. Protects microglia from α-synuclein-mediated dysfunction: Microglia isolated from postnatal day 1 to 3 pups of hSNCA^E46K transgenic (α-synuclein-overexpressing) or non-transgenic littermates were incubated with 100 µM ELN484228 or control compound ELN484217 for 24 hours, followed by addition of 10 µm beads for 90 minutes. The phagocytic index was calculated by microscopic visualization, showing significant protection by ELN484228 (n = 3 ± s.e.m, p≤0.001) [1]

A full description of the generation of transgenic animals is provided in the Supporting Information text. Briefly, Bacterial Artificial Chromosome (BAC) clone RP11-458H10, containing the human SNCA gene sequence (Life Technologies, Carlsbad, CA) was modified to generate both the Rep1 mutation and E46K mutation by BAC recombineering methods as described. Circular BAC constructs containing the hSNCA transgene (∼168 Kb) were used to perform pronuclear microinjection into B6SJL F2 mouse strains (The Jackson Laboratories, Bar Harbor, ME) in the concentration of 1–3 µg/µl followed by implantation into pseudo pregnant females (Xenogen Biosciences, Cranbury, NJ). Founder animals were bred with B6D2F1 mice and maintained as heterozygotes on this background with non-transgenic littermates as controls. Line BAC-Tg3(SNCAE46K) animals were bred in sufficient numbers for a 3, 8–9, 12–14, 18–20 month old (MO) characterization cohorts and were 3–8 generations from founders. All mice were housed in a pathogen-free, climate controlled and given food and water ad libitum. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at Elan pharmaceuticals in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Fragment probe mapping to identify small-molecule binding pockets in monomeric α-synuclein: First, 100 conformations from the NMR-derived ensemble structure of α-synuclein were analyzed, including correlations between radius of gyration (Rg), solvent accessible surface area (SASA), and the number of non-bonded contacts. Then, specific small molecules were used as fragment probes, and fragment probe mapping calculations were performed to identify potential binding pockets in α-synuclein conformations. Eight pockets filled with clusters of small molecules were identified, with α-synuclein residues colored according to the amino acid sequence [1]

1. Phagocytic activity assay for H4 neuroglioma cells: H4 neuroglioma cells with tetracycline-inducible α-synuclein overexpression were seeded and cultured. The cells were divided into groups: non-tetracycline-induced (without α-synuclein overexpression) and tetracycline-induced (1 µg/ml, to induce α-synuclein overexpression), with or without ELN484228 or control compound ELN484217. After 24 hours of culture, 4 μ beads were added to the cells and incubated for 90 minutes. The cells were then observed under a microscope, and the phagocytic index was calculated to evaluate α-synuclein-mediated impairment of vesicular function. Each sample was run in triplicate, and the experiment was repeated three times independently. The phagocytic indices of each experiment were averaged, and statistical analysis was performed on the final averages [1]
2. α-synuclein translocation assay for H4 cells: H4 cells were treated with 100 µM ELN484228 and 1 µg/ml tetracycline simultaneously and cultured for 24 hours to induce α-synuclein expression and allow drug action. Then, 4 μ beads were added to stimulate the cells for 90 minutes. After stimulation, the cells were fixed, and α-synuclein was stained with 5C12 antibody (red). 488-phalloidin (green) was used to stain the cytoskeleton, and Hoechts (blue) was used to stain the nucleus. Confocal microscopy was used to observe the localization of α-synuclein, particularly its translocation to the phagocytic cup (marked by a dotted circle indicating the bead position) [1]
3. Synaptic α-synuclein level detection assay for rat hippocampal neurons: Rat hippocampal neurons were cultured in serum-free medium until ~21 DIV. The neurons were divided into two groups: one treated with 1 µM ELN484228 and the other with 0.01% DMSO as a vehicle control. After 24 hours of treatment, the cells were fixed and immunostained with 5C12 antibody (to detect α-synuclein, red) and an antibody against synaptophysin (a presynaptic marker, green). Confocal microscopic images were acquired, and Metamorph imaging analysis software was used for quantitative analysis. The integrated intensity was measured to determine synaptic α-synuclein levels, and the pixel area was measured to determine synaptophysin levels. A total of 1000 terminals (for α-synuclein) or 18 optical fields (for synaptophysin) per condition were analyzed, with samples derived from 2–3 independent cultures [1]
4. Dopaminergic neuron protection assay in primary rat embryonic midbrain cultures: Primary rat embryonic midbrain cultures were prepared and divided into three groups: non-transduced (control), transduced with A53T α-synuclein-encoding adenovirus, and transduced with A53T α-synuclein-encoding adenovirus plus 10 µM ELN484228 treatment. After incubation, the cells were subjected to immunocytochemical staining for MAP2 (a neuronal marker) and TH (a dopaminergic neuron marker). The percentage of MAP2-positive cells that were also TH-positive was calculated to assess dopaminergic neuron survival. The lengths of neurites positive for both MAP2 and TH were measured using NIS-Elements software. Statistical analysis was performed using one-way ANOVA with Newman-Keuls post-test, with n = 3 for neuron viability analysis and n = 160–206 for neurite length analysis [1]
5. Phagocytic activity assay for microglia from transgenic mice: Microglia were isolated from postnatal day 1 to 3 pups of hSNCA^E46K transgenic mice (α-synuclein-overexpressing) or non-transgenic littermates. The isolated microglia were plated and incubated with 100 µM ELN484228 or control compound ELN484217 for 24 hours. Then, 10 µm beads were added to the microglia and incubated for 90 minutes. The cells were observed under a microscope, and the phagocytic index was calculated to evaluate the protective effect of ELN484228 on microglial function. The experiment was performed in triplicate (n = 3 ± s.e.m) [1]

Mouse

[1]. Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease. PLoS One. 2014 Feb 14;9(2):e87133.

1. ELN484228 is a drug-like benzenesulfonamide compound that, through a combination of computational and experimental techniques, is identified as targeting α-synuclein, a key protein in Parkinson's disease [1]. 2. The bioactivity of ELN484228 is likely achieved by reducing the amount of α-synuclein targeting vesicle mobilization sites, such as neuronal synapses or microbead phagocytic sites in microglia [1]. 3. Misfolding of intrinsically disordered proteins such as α-synuclein is associated with neurodegenerative diseases such as Parkinson's disease, and targeting the monomeric state of these proteins with small molecules is a potential therapeutic strategy [1].

C12H10FNO2S

251.28

251.042

312-63-0

295229

White to off-white solid powder

1.374g/cm3

379.1ºC at 760 mmHg

183ºC

1.618

3.78

1

4

3

17

325

0

AZORQGXJAXEIGC-UHFFFAOYSA-N

InChI=1S/C12H10FNO2S/c13-10-6-8-11(9-7-10)14-17(15,16)12-4-2-1-3-5-12/h1-9,14H

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)