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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Identification of a small molecule that selectively activates alpha-synuclein translational expression

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Author Information

,1 ,1 ,1 ,1 ,2 ,1 ,1 ,1 ,1 ,2 and 1,3.

1 The Broad Institute Probe Development Center, Cambridge, MA
2 Massachussetts General Hospital, Charlestown, MA
3 Howard Hughes Medical Institute, Chemistry & Chemical Biology, Harvard University, Cambridge, MA

Received: ; Last Update: February 15, 2011.

Alpha-synuclein is a protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies including Parkinson’s disease (PD), the most prevalent movement disorder in humans. Evidence suggests that misregulation of alpha-synuclein translation plays a clear role in the pathology of this disease. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, and selective small-molecule activators of alpha-synuclein expression, which will aid understanding of the role of alpha-synuclein in PD and other neurodegenerative diseases. Of the 303,224-screened compounds, 22 hits were identified as activators of alpha-synuclein expression and subsequently validated to confirm their activation activity and selectivity. One of these compounds (ML163) displayed greater than 100-fold selective activation of alpha-synuclein expression compared with a related system and is inactive in a control system at the highest tested dose. Our results indicate that ML163 has a drug-like structure with no obvious chemical liabilities, excellent solubility, and stability in aqueous conditions. This new probe should be very useful in future cell-based investigations and in vivo studies of alpha-synuclein expression.

Assigned Assay Grant #: 1 R21 NS059434-01

Screening Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber, PhD

Chemistry Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber, PhD

Assay Submitter & Institution: Jack Rogers, PhD. Massachusetts General Hospital

PubChem Summary Bioassay Identifier (AID): AID 1829

Probe Structure & Characteristics

Image ml163fu1
IUPAC Chemical NameN-naphthalen-1-yl-5-pyridin-4-yl-1,3,4-thiadiazol-2-amine
PubChem CID/ML#CID 1090779/ML163
Molecular Weight304.37
Molecular FormulaC17H12N4S
XlogP4.1
H-Bond Donor1
H-Bond Acceptor4
Rotatable Bond Count3
Exact Mass304.08
Topological Polar Surface Area78.9
CID/ML#Target NameEC50 (μM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (μM) [SID, AID]
1090779/163α-synuclein
5′-UTR
2 [SID 87218802, AID 2442]Prion 5′-UTR (H4-PRP)>200 [SID 17410558, AID 2446]>100H4-C: 40 [SID 17410558, AID 2453]

Recommendations for scientific use of the probe

The goal of this project is to identify novel small molecule probes that activate alpha-synuclein translational expression in dopaminergic neurons by targeting the 5′-untranslated region (5′UTR) stem-loop of alpha-synuclein as a novel probe for alpha-synuclein translational expression. The 5′UTR stem-loop of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Probes that can successfully increase alpha-synuclein expression levels as measured in a luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5′UTR stem-loop (H4-C) to confirm that the probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein (PRP) 5′UTR mRNA stem-loop, which is also fused to a luciferase reporter. Finally, probe candidates will be verified using Western blot analysis in untransfected H4 neuroglioblastoma cells.

The probe (CID 1090779/ML163) described in this report selectively increases alpha-synuclein expression in transfected H4 neuroglioblastoma cells and also increases protein levels in native H4 cells without affecting actin levels. The probe has an EC50 of 2 μM in the H4 primary screening cell line (H4-2a) and was found to be over 20-fold selective versus the H4-C cell line and over 95-fold versus the H4-PRP counterscreen cell lines. Additionally, a dose-response increase of alpha-synuclein levels was observed in H4 cells by Western blotting.

This probe is a first-in-class compound; there are no other published examples of compounds that act via this mechanism. In the future, this probe will be used to examine the effect of increasing alpha-synuclein levels in neurons and mouse models of Parkinson’s disease (PD). The probe may also serve as a positive control in future cell-based and animal-based experiments.

1. Introduction

Scientific Rationale

Alpha-synuclein is an approximately 15 KDa protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies (1), including Parkinson’s disease (PD), the most prevalent movement disorder in humans. In these disorders, alpha-synuclein undergoes a conformational change and subsequent oligomerization, which causes a toxic gain of function, neurodegeneration, and deposition of alpha-synuclein aggregates in the form of Lewy bodies. Increased alpha-synuclein levels caused by gene duplication causes familial PD (2), and GATA transcription factors (3) directly regulate the PD-linked alpha-synuclein gene. In this regard, regulation of alpha-synuclein translation is physiologically relevant to Lewy body dementia brains, which exhibit lowered alpha-synuclein mRNA but higher insoluble protein. This suggests misregulation of alpha-synuclein translation in addition to protein clearance by chaperones (3, 4). Consequently, our therapeutic strategy is to limit alpha-synuclein translation.

Alpha-synuclein translation is governed, at least in part, by an iron-responsive element (IRE) in the 5′ untranslated region (5′-UTR) stem-loop of alpha-synuclein mRNA. Iron-responsive elements (IREs) are RNA stem loops in the UTRs of ferritin and transferring-receptor mRNAs that are critical to iron homeostasis (5, 6). However, the protein binding profile of the alpha-synuclein IRE is different from the canonical IREs of iron homeostasis. These findings reflect the sensitivity of this RNA sequence as a probe target. The IRE of alpha-synuclein is known to interact with iron regulatory protein-1 (IRP-1), which causes an increase in alpha-synuclein translation. There are currently no small molecules which activate the expression of alpha-synuclein by this mechanism (see Section 4.1, Table 2).

Table 2. Search Strings and Databases Employed in the Prior Art Search.

Table 2

Search Strings and Databases Employed in the Prior Art Search.

Through this screening campaign, we have discovered a probe that specifically activates this RNA-protein interaction and increases alpha-synuclein translation. This probe, N-naphthalen-1-yl-5-pyridin-4-yl-1,3,4-thiadiazol-2 amine, inhibits IRE driven alpha-synuclein translation with an EC50 of 2 μM and is over 20-fold selective over cells lacking an IRE (H4-C) and over 95-fold selective over cells with an IRE from Scrapies prion protein (H4-PRP).

2. Materials and Methods

Materials and Reagents

Dulbeccos modified essential medium (DMEM, catalog no. 12-614Q); FBS (catalog no.14-503E), L-glutamine (catalog no. 17-605E), penicillin/streptomycin (catalog no. 17-602E); phenol red free media (phenol red-free DMEM with 4.5 g/L D-glucose (catalog no. 12-917F) were purchased from Lonza (Portsmouth, NH). Geneticin (catalog no. 10131-027)) and alpha-synuclein ELISA kit KHB0061 were acquired from Invitrogen. Trypsin/EDTA (catalog no. 25-053-Cl) was purchased from Cellgro, and strophanthidine (catalog no. S6626-250MG, Lot 038K1036) was purchased from Sigma. Steady-Glo (catalog no. E2250) was purchased from Promega (Madison, WI). For the Western Blot secondary screen, penicillin/streptomycin was acquired from Bio-Whittaker (Walkerville, MD), mouse monoclonal anti-alpha-synuclein was purchased from BD Transduction Laboratories, and anti-beta-actin was acquired from Chemicon. 384-well plates (catalog no. 3570) were purchased from Corning and Falcon TC flasks (catalog no. 353112) were purchased from Becton Dickison (Waltham, MA).

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

2.1.1. Primary Screen for ASYN 5′-UTR IRE-driven Luciferase Expression in H4 Cells

Stably transfected H4 neuroglioblastoma cells were grown to confluency in 35 mL complete DMEM with 4.5 g/L D-glucose supplemented with 10% FBS, 200 μM L-glutamine, 100 μM penicillin/streptomycin, and 200 μg/mL geneticin in a T175 TC flask in a TC incubator (37°C, 95% humidity, 5% CO2) (doubling time = 24 h). Cells were harvested by washing the monolayer quickly with 5 mL trypsin/EDTA (1X), aspirating, then adding 5 mL trypsin/EDTA and incubating for 5 min at 37°C, 95% humidity, 5% CO2. Then, 5 mL of complete media was added and mixed by pipetting up and down. Then, 10 mL of cell solution was aspirated and added to a conical tube; cells were pelleted for 3 min at 1000 rpm in a swinging bucket centrifuge. The media was aspirated, and the cell pellet was resuspended in 10 mL fresh complete media. Cells were then counted and expanded by plating 1×106 cells in 35 mL complete media per T175 flask (20×T175 flasks per 200 assay plate run) and allowing 72 h for cells to grow to confluency. Cells were harvested as above, but were resuspended in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose supplemented with 10% FBS, 200 μM L-glutamine, 100 μM penstrep, and 200 μg/mL geneticin, and then were diluted to 60,000 cells/mL in phenol-red free complete media prewarmed to 37°C in 1-liter sterile plastic bottles. Cells were plated into white TC treated 384-well plates (batch size = 200–220 assay plates) at 3,000 cells/well in 50 μL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser and a sterilized dispensing cassette and stir bar, in a TC hood. Assay plates were loaded into 22 slot holders which were then placed into an online Liconic (STX 2201C) incubator set to 37°C, 95 % humidity, 5% CO2, and were incubated overnight.

Screening was performed using an open system (HiRes Biosolutions). Each run was initiated in CBIP (Broad Chemical Biology Informatics Platform) and scheduled with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system. Replicate assay plates were delidded and then pinned with 100 nL of 3.75 mM compound (final concentration = 7.5 μM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions). The positive control used in this assay was strophanthidine (Sigma, Cat. S6626-250MG, Lot038K1036), which was used in dose (12-point, 2-fold dilution, from 20 μM final [compound]), and was incorporated at the beginning and end of each run. Plates with compound were then relidded and returned to the Liconic incubator (STX 2201C) and incubated for 48 h at 37°C, 95% humidity, 5% CO2. Plates were then individually moved to a room temperature Liconic Carousel (LPX 220) and were allowed to temperature equilibrate for 30 min. Plates were then delidded and moved to a MultiDrop Combi liquid dispenser where 30 μL of 0.5X Steady-Glo was added (Steady-Glo was maintained at 4°C and warmed to room temperature using a Combi cassette with a 6-foot input line submerged in a room temperature water bath). Plates were then relidded, returned to the Liconic Carousel, and incubated at room temperature for 30 min. Plates were then delidded and moved to an Envision plate reader (Perkin Elmer); luminescence values were collected using with 100 ms read time per well. Plates were then relidded and discarded.

2.1.2. Primary ReTest in H4 Cells

As described in the primary screen in H4 cells but the retest samples were cherry picked from the central library by Discovery Partners Inc.

2.1.3. Secondary Assay in H4-C Cells

As described in the primary retest in H4 cells but using the H4-C cell line.

2.1.4. Secondary Assay in H4-PRP Cells

As described in the primary retest in H4 cells but using the H4-PRP cell line.

2.1.5. Western Blot Secondary Screen in H4 Cells

Human H4 neuroglioblastoma cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Cells were exposed to increasing concentrations of compound (0, 0.1, 1, 10, 100 μM) for 48 hours. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor, and 10 mM DTT). Western blotting for alpha-synuclein was performed using mouse monoclonal anti-alpha-synuclein, and anti-beta-actin. The blots were developed using chemiluminescence (Pierce), visualized with a PhosphoImager (BioRad, Hercules, CA), and the bands were quantified using QuantityOne software (BioRad).

2.2. Probe Chemical Characterization

The probe 1 (CID 1090779/ML163) was purchased from ASINEX (ID BAS 07778946), however it could be synthesized starting from known hydrazide 2 in one step as shown in Scheme 1, based on literature precedent (7). Treatment of 2 with 1-isothiocyanatonapthalene 3 would form the thiosemicarbazide 4. Subsequent treatment with concentrated sulfuric acid at room temperature would result in closure to the probe compound (CID 1090779, CID 17410558, ML163). The probe was determined to have a solubility of <1 μM in PBS at room temperature. The probe and five analogs were submitted to the SMR collection (MLS002825686, MLS002725688, MLS002725689, MLS002729069, MLS002825679).

Scheme 1. Synthesis of Probe.

Scheme 1

Synthesis of Probe.

The 1H NMR spectra and LC-MS chromatograms of the probe (CID 1090779/ML163) and analogs are provided in Appendix C.

2.3. Probe Preparation

Not applicable. The probe (CID 1090779/ML163) was purchased from ASINEX (ID BAS 07778946).

3. Results

Probe Attributes

  • EC50 ≤ 10 μM in the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (H4-2a).
  • Greater than 10-fold selectivity as defined by: EC50 ratios of the primary screening cell line (H4-2a) and both the non-IRE containing H4 cell line (H4-C) and the prion protein IRE containing H4 cell line (H4-PRP).
  • Compound has a dose-response activation in native non-transfected H4 cells as observed by Western blot.

3.1. Summary of Screening Results

A high-throughput screen of 303,811 compounds (AID 1814) was performed in duplicate in the alpha-synuclein IRE containing the H4 neuroglioblastoma primary screening cell line (H4-2a). Using a screening “hit” cutoff of ≥ 62% activation at a screening concentration of 7.5 μM, 21,690 compounds were identified as activators of IRE-driven luciferase expression (Figure 1). These active compounds were then clustered based upon chemical structure, which resulted in 2,600 clusters. The most active compound from each cluster was then cherry picked. Out of these 2,600 compounds, 2,336 were available and these were retested in eight-point dose (20 to 0.16 μM) in the primary screening cell line (AID 2003). Compounds that had EC50 values ≤ 20 μM were considered to be successful primary retests (1272/2336, 54% retest rate).

Figure 1. Critical Path for Probe Development.

Figure 1

Critical Path for Probe Development.

Compounds were also tested in two counterscreens, the first of which was an H4 neuroglioblastoma cell line transfected with the same plasmid as the primary screening line, except the alpha-synuclein IRE stem-loop was removed from the 5′UTR (H4-C). The purpose of this counterscreen was to determine if active compounds were either cytotoxic, inhibiting luciferase activity, or causing global reductions in transcription/translation. Compounds were tested in dose, and hit criteria were the same as the primary retest. Of these, 1720 compounds were active (AID 2002). The second counterscreen was with H4 neuroglioblastoma cells transfected with a construct containing the prion protein IRE in the 5′UTR stem-loop upstream of luciferase (H4-PRP). The purpose of this counterscreen was to determine if compounds active in the primary retest were specific for the alpha-synuclein IRE stem-loop or if they were capable of binding a closely related IRE stem-loop. Compounds were tested in the same means as in the previous confirmatory screens using the same activity criteria. Of these, 1353 compounds were active (AID 1999).

Compounds with at least 10-fold selectivity for the primary H4-2a screening line over the H4-C cell line and 10-fold selectivity for H4-2a over the H4-PRP cell line were prioritized for further development. Of these 22 compounds 13 were obtained as dry powders and were then retested in the primary and counterscreen assays (AID 2442, AID 2453, AID 2446). Three compounds met the selectivity criteria using dry powder samples. These compounds were then tested in non-transfected H4 neuroglioblastoma cells. Western blot analysis of alpha-synuclein protein levels in H4 cells showed a dose dependent increase corresponding to increasing doses of compound CID 1090779/ML163 (AID 2627). This compound became probe series 1.

3.2. Dose Response Curves for Probe

The probe (CID 1090779/ML163) met the defined probe criteria and has acceptable solubility and stability in aqueous conditions. The activity curves of the probe in the aforementioned cell lines and alpha-synuclein Western blot are depicted in Figure 2 and Figure 3, respectively.

Figure 2. Concentration-dependent Activities of the Probe (CID 1090779/ML163) in Target and Counterscreen Assays.

Figure 2

Concentration-dependent Activities of the Probe (CID 1090779/ML163) in Target and Counterscreen Assays. Activity curves for probe (CID 1090779/ML163): the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (A); the H4 neuroglioblastoma (more...)

Figure 3. Western Blot Results From Probe (CID 1090779/ML163) in H4 Cells (AID 2627).

Figure 3

Western Blot Results From Probe (CID 1090779/ML163) in H4 Cells (AID 2627).

3.3. Scaffold/Moiety Chemical Liabilities

A search of PubChem for the probe compound (CID 1090779) revealed that the probe has been tested in 413 BioAssays and was confirmed as active in only four other assays. Therefore, the compound is not promiscuous. The compound has a drug-like structure with no obvious chemical liabilities that would be a concern.

3.4. SAR Tables

The structures and the activities of the probe and analogs in the primary assay and the suite of secondary assays are summarized in Table 1.

Table 1. SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs.

Table 1

SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs.

Analogs to the probe were generated and tested in the H4-2a primary screening cell line in dose format. Screening results for the primary assay are shown in Figure 4 and Table 2. The highest level of activation was measured with compound 1 (CID 1090779/ML163), which has been designated as the probe for this series. Replacement of the napthalene with other aromatic groups results in a decrease in activity (CID 44826047). Several other aromatic groups were tested with various substituents and showed a range of activity (CID 1257201, CID 1864426, and CID 1294885). This probe will serve quite well in further cell-based investigations, but additional probes will be sought with good potency and improved physical properties.

Figure 4. Concentration-dependent Activity of Alpha-synuclein Analogs.

Figure 4

Concentration-dependent Activity of Alpha-synuclein Analogs. Dose-response curves of five probe analogs: CID 1090779/ML163 (A), CID 44826047(B), CID 1257201(C), CID 1864426(D), CID 1294885(E).

3.5. Cellular Activity

No specific assays were conducted to measure cellular toxicity or permeability of the probe or analogs. Since, however, the probe and analogs cause an increased readout of the reporter enzyme luciferase in the screening cell line, these compounds do not appear to be toxic at the concentrations measured over the 48-hour incubation time of the assays.

3.6. Profiling Assays

No profiling assays were conducted.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

The literature searches described in Table 2 were unable to find any small molecules that activated the 5′UTR of alpha synuclein mRNA.

4.2. Mechanism of Action Studies

No mechanism of action studies were conducted.

4.3. Planned Future Studies

The probe and analogs will be tested in primary neuronal cells to confirm their activity. Furthermore, they will be used in mouse models of Parkinson’s disease

5. References

1.
Uéda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DA, Kondo J, Ihara Y, Saitoh T. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci USA. 1993 Dec 1;90:12282–12286. [PMC free article: PMC47966] [PubMed: 8248242]
2.
Singleton A, Gwinn-Hardy K. Parkinson’s disease and dementia with Lewy bodies: a difference in dose. Lancet. 2004 Sep 25-Oct 1;364(9440):1105–1107. [PubMed: 15451205]
3.
Scherzer CR, Grass JA, Liao Z, Pepivani I, Zheng B, Eklund AC, Ney PA, Ng J, McGoldrick M, Mollenhauer B, et al. GATA transcription factors directly regulate the Parkinson’s disease-linked gene alpha-synuclein. Proc Natl Acad Sci USA. 2008 Aug 5;105(31):10907–10912. Epub 2008 Jul 31. [PMC free article: PMC2504800] [PubMed: 18669654]
4.
Cantuti-Castelvetri I, Klucken J, Ingelsson M, Ramasamy K, McLean PJ, Frosch MP, Hyman BT, Standaert DG. Alpha-synuclein and chaperones in dementia with Lewy bodies. J Neuropathol Exp Neurol. 2005 Dec;64(12):1058–1066. [PubMed: 16319716]
5.
Thomson AM, Rogers JT, Leedman PJ. Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation. Int J Biochem Cell Biol. 1999 Oct;31(10):1139–1152. [PubMed: 10582343]
6.
Thomson AM, Cahill CM, Cho HH, Kassachau KD, Epis MR, Bridges KR, Leedman PJ, Rogers JT. The acute box cis-element in human heavy ferritin mRNA 5′-untranslated region is a unique translation enhancer that binds poly(C)-binding proteins. J Biol Chem. 2005 Aug 26;280(34):30032–30045. Epub 2005 Jun 20. [PubMed: 15967798]
7.
Oruç EE, Rollas S, Kandemirli F, Shvets N, Dimoglo AS. 1,3,4-thiadiazole derivatives: Synthesis, structure elucidation, and structure-antituberculosis activity relationship investigation. J Med Chem. 2004 Dec 30;47(27):6760–6767. [PubMed: 15615525]

6. Appendices

Appendix A. Compound Characterization

Table A1Summary of Completed Assays and AIDs

PubChem AID No.TypeTargetConcentration Range (μM)Samples Tested
1829SummaryNANANA
1814PrimaryASYN 5′UTR7.5303811
2003ConfirmatoryASYN 5′UTR20 - 0.162336
2002CounterscreenH4-C20 - 0.162336
1999Confirmatory-powderH4-PRP20 - 0.162336
2442Confirmatory-powderASYN 5′UTR20 - 0.1613
2453Counterscreen-powderH4-C20 - 0.1613
2446Counterscreen-powderH4-PRP20 - 0.1613
2627Western BlotASYN100 - 0.11
2670AnalogsASYN 5′UTR20 - 0.164

Appendix B. Detailed Assay Protocols

Primary Screen in H4 Cells

  1. Grow stably transfected H4 neuroglioblastoma cells to confluency in 35 mL complete media (DMEM with 4.5 g/L D-glucose [Lonza, 12-614Q] supplemented with 10% FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E] and 200 μg/mL geneticin [Invitrogen, 10131-027]) in a T175 TC flask (BD Falcon, 353112).
  2. Incubate in a TC incubator (37°C, 95% humidity, 5 % CO2) (doubling time = 24 h).
  3. Harvest cells by washing the monolayer quickly with 5 mL trypsin/EDTA (1X, Cellgro, 25-053-Cl). Aspirate, then add 5 mL trypsin/EDTA.
  4. Incubate for 5 min (37°C, 95% humidity, 5% CO2).
  5. Add 5 mL of complete media and mix by pipetting up and down. Aspirate 10 mL of cell solution and add to a conical tube.
  6. Pellet the cells for 3 min at 1000 rpm in a swinging bucket centrifuge.
  7. Aspirate the media and resuspend the cell pellet in 10 mL fresh complete media.
  8. Count the cells and expand by plating 1×106 cells in 35 mL complete media per T175 flask (20×T175 flasks per 200 assay plate run). Allowing 72 h for cells to grow to confluency.
  9. Harvest the cells as above, but resuspend cells in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose [Lonza, 12-917F] supplemented with 10% FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E], and 200 μg/mL geneticin [Invitrogen, 10131-027]).
  10. Dilute resuspended cells to 60,000 cells/mL in phenol-red free complete media prewarmed to 37°C in 1-L sterile plastic bottles.
  11. Plate cells into white TC-treated 384-well plates (Corning, 3570; batch size = 200–220 assay plates) at 3,000 cells/well in 50-μL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser, a sterilized dispensing cassette, stir bar in a TC hood.
  12. Load assay plates into 22 slot holder and place into an online Liconic (STX 2201C) incubator set to 37°C, 95% humidity, 5% CO2. Incubate plates overnight.
  13. Perform screening using an open system (HiRes Biosolutions). Initiate each run in Broad Chemical Biology Informatics Platform (CBIP) and schedule with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system.
  14. Delid replicate assay plates and pin with 100 nL of 3.75 μM compound (final concentration = 7.5 μM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions).
  15. Use strophanthidine (Sigma, Cat. No. S6626-250MG, Lot No.038K1036) as the positive control in dose (12-point, 2-fold dilution, from 20 μM final [compound]) at the beginning and end of each run. Relid plates with compound and return to the Liconic incubator (STX 2201C).
  16. Incubate plates for 48h at 37°C, 95% humidity, 5% CO2. Move plates individually to a room temperature Liconic Carousel (LPX 220) and allow to temperature equilibrate for 30 min.
  17. Delid plates and move to a MultiDrop Combi liquid dispenser and add 30 μL of 0.5X Steady-Glo (Promega, E2250). Maintain Steady-Glo at 4°C and warm to room temperature using a Combi cassette with a 6-foot input line submerged in a room temperature water bath.
  18. Relid plates and return to the Liconic Carousel. Incubate at room temperature for 30 min.
  19. Delid plates and move to an Envision plate reader (Perkin Elmer). Collect luminescence values using with 100 ms read time per well. Relid the plates and discard.

Western Blot Secondary Screen in H4 Cells

  1. Culture human H4 neuroglioblastoma cells in Dulbeccos modified essential medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Bio-Whittaker, Walkerville, MD).
  2. Expose cells to increasing concentrations of compound (0, 0.1, 1, 10, 100 μM) for 48 hours.
  3. Prepare cytoplasmic protein lysates by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT).
  4. Perform Western blotting for alpha-synuclein using mouse monoclonal anti-alpha-synuclein (BD Transduction Laboratories), and anti-beta-actin (Chemicon). Develop the blots using chemiluminescence (PIERCE) and visualize with a PhosphoImager (BioRad, Hercules, CA). Quantify the bands using QuantityOne software (BioRad).

Appendix C. NMR and LC Data of Probe and Analogs

1H NMR Spectra of Probe (CID 1090779/ML163).

1H NMR Spectra of Probe (CID 1090779/ML163)

LC/MS Chromatogram of Probe (CID 1090779/ML163).

LC/MS Chromatogram of Probe (CID 1090779/ML163)

1H NMR Spectra of Analog CID 44826047.

1H NMR Spectra of Analog CID 44826047

LC/MS Chromatogram of Analog CID 44826047.

LC/MS Chromatogram of Analog CID 44826047

1H NMR Spectra of Analog CID 1257201.

1H NMR Spectra of Analog CID 1257201

LC/MS Chromatogram of Analog CID 1257201.

LC/MS Chromatogram of Analog CID 1257201

1H NMR Spectra of Analog CID 1864426.

1H NMR Spectra of Analog CID 1864426

LC/MS Chromatogram of Analog CID 1864426.

LC/MS Chromatogram of Analog CID 1864426

1H NMR Spectra of Analog CID 1294885.

1H NMR Spectra of Analog CID 1294885

LC/MS Chromatogram of Analog CID 1294885.

LC/MS Chromatogram of Analog CID 1294885

Appendix D. Compounds Submitted to BioFocus

Table A2Probe and Analog Information

BRDSIDCIDP/AMLSIDML
BRD-K32644160994958081090779PMLS002725686163
BRD-K448935438985617444826047AMLS002725688NA
BRD-K53011428874571661257201AMLS002725689NA
BRD-K07530279874571671864426AMLS002729069NA
BRD-K06020002874571651294885AMLS002825679NA

P = probe, A = analog

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